Chapter 14

Moving Out: Invasion and Metastasis

The fact of cells identical with those of the cancer itself being seen in the blood may tend to throw some light upon the mode of ori­gin of multiple tumors existing in the same person.

T.R. Ashworth, physician,1869

It is not birth, marriage, or death, but gastrulation, which is truly the most important time in your life.

Lewis Wolpert, embryologist, 1986

I n the early phases of multi-step tumor progression, cancer cells multiply near the site where their ancestors first began uncontrolled proliferation. The

result, usually apparent only after many years' time, is a primary tumor mass. Given the fact that a cubic centimeter oftissue may contain as many as 109 cells, we can easily imagine that tumors may often reach a size of 10lD or lOll cells before they make themselves apparent to the individual carrying them or to the clinician in search of them.

Primary tumors in some organ sites-specifically those arising within the peri­toneal or pleural space-may well expand without causing any discomfort to the patient, simply because these cavities are expansible and their contents are quite plastic; in other sites, such as the brain, the presence of a tumor is often apparent when it is still relatively small. Sooner or later, however, in all sites throughout the body, tumors of substantial size compromise the functioning of the organs in which they have arisen and begin to evoke symptoms.

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Chapter 14: Moving Out: Invasion and Metastasis

Figure 14.1 Disseminated tumors The diagnosis of metastatic disease often represents a death sentence for a cancer patient, yet the mechanisms by which cancer cells metastasize from a primary tumor to distant sites in the body remain poorly understood. Seen here is a whole­body scan of a patient w ith metastatic non-Hodgkin's lymphoma (NHL) This is a fusion image of a CT (computed X-ray tomography) scan of the body's tissues (gray, blue) and a PET (positron-emission tomography) scan in w hich the uptake of radioactively labeled fluorodeoxyglucose (FDG) in various tissues (yellow) has been detected . FDG uptake indicates regions of highly active cell metabolism throughout the body. The activity associated with the brain is normal. However, the presence of numerous yellow spots in the abdominal regions indica tes multiple NHL metastases. (Courtesy of S.S. Gambhir.)

In many cases, the effects on normal tissue function come from the physical pressure exerted by the expanding tumor masses. In others, cells from the pri­mary tumor mass invade adjacent normal tissues and, in so doing, begin to compromise vital functions. Large tumors in the colon may obstruct passage of digestion products through the lumen, and in tissues such as the liver and pan­creas, cancer cells may obstruct the flow of bile through critical ducts. In the lungs, airways may be compromised.

As insidious and corrosive as these primary tumors are, they ultimately are responsible for only about 10% of deaths from cancer. The remaining approxi­mately 90% of patients are struck down by cancerous growths that are discov­ered at sites far removed from the locations in their bodies where their primary tumors first arose (Figure 14.1; see also Figure 2.2). These metastases are formed by cancer cells that have left the primary tumor mass and traveled by the body's highways-blood and lymphatic vessels-to seek out new sites throughout the body where they may found new colonies (Figure 14.2). Breast cancers often spawn metastatic colonies promiscuously in many tissues throughout the body, including the brain, liver, bones, and lungs. Prostate tumors are most often seeded to the bones, while colon carcinomas preferentially form new colonies in the liver.

Such wandering cancer cells are the most dangerous manifestations of the can­cer process. vVhen they succeed in founding colonies in distant sites, they often wreak great havoc. The female body can dispense with its mammary glands without losing vital physiologic functions, and so almost all primary breast car­cinomas do not compromise survival while they are confined to the breast. However, the metastatic colonies that breast cancer cells initiate in the bone can cause localized erosion of bone tissue, resulting in agonizing pain and skeletal collapse. Metastases in the brain may rapidly compromise central nervous sys­tem function, while those in the lung or liver are similarly threatening to life because of the vital functions ofthese organs.

For reasons that remain obscure, tumors in certain tissues have a high probability of metastasizing, while those arising in other tissues almost never do so. After pri­mary melanomas penetrate a certain distance downward into the tissue underly­ing the skin, the presence of metastases at distant sites in the body is almost a cer­tainty. In contrast, squamous cell carcinomas of the skin and astrocytomas-pri­mary tumors of the glial cells in the brain-rarely spawn metastases.

In a variety of human tumor types, the dissemination of cancer cells throughout the body has already occurred by the time a primary tumor is first detected; at the time of initial diagnosis, these scattered cells will be inapparent because they only form minute tumor colonies-micrometastases. Such behavior provokes a question that we will confront in this chapter and again in Chapter 16: Do the properties of a primary tumor (Sidebar 14.1) reveal whether it has broadcast

588

Metastases contribute to cancer mortality

(A) (8)

(C)

cancer cells throughout the body that will eventually create life-threatening metastatic disease long after the primary tumor has been surgically removed?

In this chapter, we confront the processes that create these most aggressive prod­ucts of tumor progression. These processes depend on complex biochemical and biological changes in cancer cells and in the associated stroma. Most of the steps of cancer formation, as described in earlier chapters, are understood in consid­erable detail. In contrast, our understanding of invasion and metastasis is still quite incomplete, explaining why these late steps of tumor progression represent the major unsolved problems of cancer pathogenesis.

14.1 Travel of cancer cells from a primary tumor to a site of potential metastasis depends on a series of complex biological steps

Figure 14.2 Histology of metastases in various tissues throughout the body (A) In the Rip-Tag transgenic mouse model of pancreatic islet cell tumorigenesis (see Figure 13.37), metastasis via the lymph nodes can be encouraged through the forced expression of VEGF-C-a Iymphangio­genic factor-in the islet tumor cells. Seen here is a small metastasis of islet cells (dark pink) to a lymphatic vessel that is lined with endothelial cells (brown). (B) A small metastasis of a human breast cancer is seen growing within a lymph node associated with one of the lymphatic ducts draining the breast. Note the fact that this metastasis exhibits the detailed structure, including ducts and stroma, that is characteristic of many primary tumors of the breast, as well as the numerous lymphocytes in the surrounding lymph duct (dark nUclei). (C) The presence of metastatic carcinoma cells (blue) in the bone marrow can be revealed using specific immunohistochemistry to detect cells displaying epithelial markers, which sets them apart from the mesenchymal cells naturally present in the marrow; or, as seen here, through use of the Wright­Giemsa stain, which has rendered them blue. (A, from S.J. Mandriota, L. Jussila, M. Jeltsch et aI., EMBO J. 20:672-682, 2001; B, courtesy of T.A. Ince; C, from A.T. Skarin, Atlas of Diagnostic Oncology, 3rd ed. Philadelphia: Elsevier Science Ltd., 2003)

The great majority (>80%) of life-threatening cancers occur in epithelial tissues, yielding carcinomas. Consequently, most of our discussions in this chapter, as in the last, will refer to this class of tumors, with the understanding that cancers

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Chapter 14: Moving Out: Invasion and Metastasis

Sidebar 14.1 The connection between tumor size and dis­ease prognosis is unclear The crude measurement of pri­mary tumor size can often be correlated with the likelihood that the tumor has already seeded metastases in distant sites in the body. For example, as indicated in Figure 14.3~ in one study 22% of women With primary breast cancers of less than 1 cm diameter (at initial diagnosis) eventually developed metastatic disease. In contrast, 77% of those women whpse primary tumors were more than 8 cm in diameter progressed to metastatic disease. Independent of these measurements are others indicating that, in general, larger tumors have passed through more of the genetic (and epigenetic) steps of tumor progression. Thus, 4% of breast cancers ofless than 1 cm diameter have been found to carry mutant p53 alleles, while 42% of tumors greater than 3 cm bear cells with these mutations. We might con­clude from such observations that the ability to metastasize is acquired by cancer cells as a relatively late step in tumor progression when a primary tumor is expanding from a small to a large size. Moreover, acquisition of this ability might well be due to the loss of p53 function.

Actually, the true meaning of these statistics is very dif­ficult to assess and can be explained by a number of alter­

~ 80 VI.;;;

Figure 14.3 Primary tumor size tl 70and risk of metastasis This bar co graph reveals that as the diameter ~

E 60 of an initially diagnosed primary 0\

breast cancer increases, the C

.~ 50probability increases that distant Qi

metastases will arise in a patient's Q) >

body. This is indicated here as the 1:l .40 ~

percentage of women bearing co primary tumors of a given size who ~ 30 27 c eventually develop distant Q)

macroscopic metastases. In this Q) 20 '0study, 1589 breast cancer patients

were followed for periods as long .~ :0 10

as 46 years after initial diagnosis co and treatment. (From R. Heimann

.D

>

I I ~ 0and S. Hellman, 1. Clin. Onco/. 0.0-1.0 1.1-2.0

16:2686-2692, 1998)

native mechanisms: (1) The trait of metastasis may be acquired only relatively late in the growth of the primary tumor, thereby limiting this ability to cells present in the largest primary tumors. (2) Individual cancer cells in small and large tumors may be equally capable of metastasizing. However, the larger tumors may dispatch proportionately . greater numbers of metastasizing cells (per unit of time) to the rest of the body simply b.ecause they contain more cells. (3) At some point· during multi-step tumor progression, those cells showing an enhanced abflity to proliferate in the primary tumor also exhibit a closely associated trait-the ability to metastasize. Such cancer cells generate larger pri­mary tumors more rapidly than other cells that proliferate slowly, and these rapidly mUltiplying cells' also serve as sources of widely disseminated metastases.

Siinilar arguments apply to mutant genes present in advanced tumors: The greater frequency of mutant p53 alleles in large tumors may not mean that this mutant allele is acquired late in primary tumor progression. Instead, tumor cell populations that acquire mutant p53 alleles relatively early in multi-step tumor progression may be able to grow more rapidly and generate tumors of a larger size by the time of diagnosis.

77 77

66

61

54

44 40

2.1-3.0 3 .1-4.0 4.0-5.1 5.1-6.0 6.1-8.0 8.1-10.0 >10.1

diameter (em) of primary tumor

arising in other tissue types, such as connective and nervous tissues, often fol­low similar paths when they become invasive and metastatic. Even certain hematopoietic tumors, notably lymphomas, often have an early, localized phase and a later phase during which they become disseminated to distant anatomi­cal sites (see Figure 14.1). This complex sequence of steps, sometimes called "the invasion-metastasis cascade," is illustrated in Figure 14.4.

Our focus on carcinomas requires us to draw from earlier cliscussions of these tumors and the epithelial tissues in which they arise (see, for example, Sections 2.2 and 13.1). To recap briefly, the great majority of epithelial tissues are con­structed according to a common set of architectural principles; in most cases,

590

~~

The invasion- metastasis cascade

primary tumor localized intravasation transport arrest in microvessels extravasation formation invasion through of various organs

circulation

-~-~ interaction with 1\if !

platelets, lymphocytes, V and other blood ~ formation of a

components ~ micrometastaSIS

Figure 14.4 The invasion-metastasis cascade This depiction of the invasion-metastasis cascade ascribes six distinct steps to the overall process. The initial step of localized invasiveness enables in situ carcinoma cells to breach the basement membrane. Thereafter, they may intravasate into either lymphatic or blood microvessels. The latter may then transport these cancer cells to distant anatomical sites, where they may be trapped and subsequently extravasate and form dormant micrometastases. Eventually some of the micro metastases may acquire the ability to colonize the tissue in which they have landed, enabling them to form a macroscopic metastasis. The last step-colonization-seems to be the most inefficient of all. The small probability of successfully completing all steps of this cascade explains the low likelihood that any single cancer cell leaving a primary tumor will succeed in becoming the founder of a distant, macroscopic metastasis. (Adapted from I.J. Fidler, Nat. Rev Cancer 3453-458, 2003)

relatively thin sheets of epithelial cells sit atop deep, complex layers of stroma. Separating the two is the specialized type of extracellular matrix (ECM) known as the basement membrane (see Figures 2.3 and 13.5). This proteinaceous meshwork is constructed collaboratively by proteins secreted by both epithelial and stromal cells.

By definition, carcinomas begin on the epithelial side of the basement mem­brane and are considered to be benign as long as the cells forming them remain on this side. Sooner or later, however, many carcinomas acquire the ability to breach the basement membrane, and individual cancer cells or groups of can­cer cells begin to invade the nearby stroma (Figure 14.5). This mass of neoplas­tic cells is now reclassified as malignant. In fact, as mentioned in an earlier chapter, many pathologists and surgeons reserve the word "cancer" for those epithelial tumors that have acquired this invasive ability. To be sure, this disso­lution of the basement membrane by invading carcinoma cells removes an important physical barrier to the further expansion of tumor cell populations. But in addition, as we learned in the last chapter, by degrading various compo­nents of the basement membrane, invasive cells harvest growth and survival factors that have been sequestered by attachment to this specialized extracellu­lar matrix.

Recall that even before carcinoma cells breach the basement membrane, they often succeed in stimulating angiogenesis on the stromal side ofthe membrane, apparently by dispatching angiogenic factors through this porous barrier to endothelial cells within the stroma (see Figure 13.39). However, invasion through the basement membrane places them in a far better position for exe­cuting subsequent steps of the invasion-metastasis cascade. Once present in the stromal compartment, carcinoma cells can gain direct access to the blood and lymphatic vessels (see Figure 13.41), which are normally found only on the stromal side of the basement membrane (see, however, Sidebar 14.2). Close con­tact with the capillaries affords tumor cells improved access to the nutrients and

~ colonization ­formation of a macrometastasis

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Chapter 14: Moving Out: Invasion and Metastasis

Figure 14.5 Patterns of invasion (A) These invasive lobular mammary carcinoma cells (brown) have left the primary tumor (not shown to the left) and are proceeding rightward, one-by­one in single file, through channels they have carved in the adjacent stroma (B) However, far more typical is the behavior of this cohort of 5-10 melanoma cells, view ed in a confocal micrograph, which are moving together through a collagen matrix (blue); like normal melanocytes, they continue to adhere to one another through adherens junctions that are formed by E-cadherin molecules (red). Large gaps in the matrix (black) indicate areas that have been degraded by the advancing cancer cells. At the leading invasive edge (white arrow), the melanoma cells are displaying ~1 integrins (green), which enable them to attach to the extracellular matrix ahead of them. (C) On a far larger scale, such coordinated invasion is reflected by the cells in this squamous ce ll carc inoma of the cervix, in w hich a large finger or tongue of many hundreds of cancer cells (pink, brown) has breached the basement membrane and is invading the stroma. The latter being characterized by both stromal fibroblasts and inflammatory cells (dark green). The basement membrane is seen here as a dark brown, horizontal line (pink arrows, top right) that separates the bulk of the carcinoma cells (above) from the stromal cells (below) and is uninterrupted except for a capillary and the tongue of invasive cancer cells. (A, courtesy of J. Jonkers; B, from P. Friedl, Y Hegerfeldt and M. Tusch, Int. I Dev. BioI. 48:441-449, 2004; C, courtesy of lA Ince)

(A)

(B)

(C)

oxygen carried by the blood. In addition, their invasive properties enable these cancer cells to move through the walls and into the lumina (i.e. , the bores) of blood and lymphatic vessels. This invasion into vessels is often termed intrava­sation.

Once they have arrived within the lumen of a blood or lymphatic vessel, individ­ual cancer cells may travel with the blood or lymph to other areas in the body. These long-range migrations are fraught with great danger for the wanderers. Like normal cells, the cancer cells may continue to depend on anchorage to solid substrates; without such attachment, the migrating cells may die rapidly from anoikis, the form of apoptosis that is triggered by detachment of a cell from a solid substrate such as an extracellular matrix (Sections 5.8 and 9.13). Also, like their forebears in the primary tumor, these pioneers may depend on various types of stromal support, whlch will be lacking the moment they leave the pri­mary tumor mass. Recall that the stroma can benefit carcinoma cells in multi­ple ways by supplying both mitogenic and trophic (survival) factors .

592

ventricle

pulmonary circuit

• oxygenated blood

The blood represents an actively hostile environment for metastasizing cancer cells. Hydrodynamic shear forces in the circulation, which are often substantial in smaller vessels, may tear the wandering cancer cells apart. Some experimen­tal models of metastasis in the mouse indicate that survival of metastasizing cancer cells in the general circulation is greatly enhanced if they can attract an entourage of blood platelets to escort them through the rapids into safe pools within tissues (Sidebar 27 . ).

If metastasizing cells survive these initial dangers and gain access to the larger vessels in the venous system, they will travel with the blood through the heart and then lodge, with high probability, in the first set of capillaries that they encounter after their initial passage through the heart-the capillary beds of the lungs (Figure 14.6). Unlike red and white blood cells, carcinoma cells are ill suited for passage through most capillaries, whose internal diameters are far too small to accommodate them. Capillaries usually have internal diameters in the range of 3 to 8 !lm, and the blood cells that must pass through them are well adapted to do so. Erythrocytes, for example, are only about 7 ,llm in diameter and are easily deformed, facilitating their passage through capillaries (Figure 14.7). Most cancer cells, in contrast, are more than 20 !lm in diameter and are not especially deformable. (Moreover, if cancer cells in the blood are coated with platelets, as discussed in Sidebar 27 . , their effective diameters are greatly increased, causing them to be trapped in vessels far larger than capillaries, e.g., the small arteries known as arterioles.)

Once trapped within the lungs, some metastasizing cancer cells may attempt to found metastases there. However, the metastases of many types of human tumors are often found elsewhere in the body, indicating that cancer cells fre­quently succeed in escaping from the lungs and travel further to other sites in the body. How they do so is unclear. In some experiments, cancer cells trapped in capillaries have been observed to pinch off large amounts of cytoplasm, leaving

tissue capillaries

• deoxygenated blood

tissue capillaries

The invasion- metastasis cascade

Sidebar 14.2 Cancer cells usu­ally invade as a unified phalanx The language used here and elsewhere to describe cancer cell invasiveness attributes this property to individual cells that venture outside the perimeter of a primary tumor, make their way into nearby stroma, and eventually intravasate. In fact, there are such cancers-for example, invasive lobular carci-" . nomas of the breast (see Figme 14.5A)-in which individual cancer cells leave the primary tumor mass, one by one, and proceed in single file (some­times called "Indian file") through channels that they have carved in the nearby stroma. However, a far more typical . behaVior of invasive carcinoma cells is shown in Figure 14.5C, in which a phalanx (Le., a well­organized cohort) of these cells invades nearby stroma. As we will see later in this chapter, the carcinoma cells in direct con­tact with the stroma may pave the way for others to follow behind them. Sooner or later, however, as these invasive tongues approach nearby vas­culature, any intravasation that occurs must depend on the abil­ity of individual cancer cells or small clumps of these cells to break away from their neoplas­tic neighbors and enter on their ovm into the circulation.

Figure 14.6 Major routes of blood circulation through the body This schematic diagram of the mammalian circulation indicates that venous blood (blue) leaving a tissue (and thus cancer cells that have escaped from a primary tumor and intravasated) must first pass through the right ventricle of the heart and thence through the lungs before it passes th rough the left ventricle and is pumped into the general arteria l circulation. Since passage through the pulmonary circulation of the lung requires passage through its capilla ries, almost all metastasizing cells entering into the venous circulation are rapidly trapped in the pulmonary capillary beds . (From PH Raven, G.B. Johnson, S. Singer et ai, Biology, 7th ed . New York McGraw-Hili, 2005.)

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Chapter 14: Moving Out: Invasion and Metastasis

Figure 14.7 Passage through capillaries (A) Intra-vital microscopy reveals the tw o endothelial walls of a capillary (E), erythrocytes (R), and some leukocytes (W) The small size and deformability of these cells allow them to pass through capillaries without becoming trapped. (8) The vessels in this intra-vital fluorescence micrograph are slightly larger than capillaries. The plasma has been stained w ith a green dye, w hile the erythrocytes have been colorized red . The high deformability of these red blood cells is apparent. Since most cancer cells have more than twice the diameter of erythrocytes and are not deformable, they are unable to negotiate narrow passages, such as the lumina of capillaries. (From I.C MacDonald, AC Groom and AF Chambers, BioEssays 24885-893, 2002.)

Figure 14.8 Cancer cells growing in the lumen of a microvessel Confocal microscopy reveals a colony of rat fibrosarcoma cells, w hich express green fluorescent protein (GFP), proliferating within an arteriole whose walls (red) are labeled through use of a dye bound to low-density lipoprotein (LDL). (LDL binds to the LDL receptor displayed on the luminal surfaces of endothelial cells.) These cells w ere visualized 5 days after single cells w ere introduced into the venous circulation of a mouse. (Courtesy of R.J. Muschel.)

(8)

behind cells that, while greatly reduced in size, seem to be vjable; once they have undergone this amputation, such slenderized cells may succeed in negotiating passage through the narrow straits of the lung capillaries. A more plausible explanation is that wandering cancer cells may avoid being trapped altogether: in many organs, including the lung, metastasizing cells can bypass capillaries by traveling through arterial-venous shunts, which form large-bore, direct connec­tions between the two parts of the circulatory system.

Having snaked their way through the lungs and arrived in the general arterial circulation (see Figure 14.6), roaming cancer cells can then scatter to all tissues in the body. Some experiments suggest that cancer cells use specific cell surface receptors, such as integrins, to initially adhere to the luminal walls of arterioles and capillaries in certain tissues. However, far more extensive evidence indi­cates that simple physical trapping within small vessels, as discussed above in the context of the lung, provides most wandering cancer cells with their first foothold within a tissue.

Once lodged in the blood vessels of various tissues, cancer cells must escape from the lumina of these vessels and penetrate into the surrounding tissue­the step termed extravasation. The process of extravasation depends on com­plex interactions between cancer cells and the walls of the vessels in which they have become trapped. Cancer cells can use several alternative strategies to extravasate. They may begin to proliferate within the lumen of the vessel, cre­ating a small tumor that grows and eventually obliterates the adjacent vessel wall (Figures 14.8 and 14.9). In doing so, they push aside endothelial cells, per­icytes, and smooth muscle cells that previously separated the vessel lumen from the surrounding tissue, the latter often being called the tissue parenchyma. Alternatively, cancer cells may proceed immediately to elbow their way through the vessel wall. Their ability to do so may depend on the same biochemical and cell-biological mechanisms that previously enabled them or their immediate ancestors to invade from the primary tumor and to intravasate (see Sidebar 14.3).

14.2 Colonization represents the most complex and challenging step of the invasion-metastasis cascade

Once they have arrived within the parenchyma of a tissue, metastasizing cancer cells may begin forming a tumor mass in their newfound homes, the process often termed colonization. This step is also a challenging one-perhaps the

594

Colonization of site of dissemination

tissue endothelial (A) cancer cell parenchyma (B) platelets cells (e) capillary basement membrane

1j. .. .. ~.

(0) (E)

Figure 14.9 Steps leading to extravasation Electron-microscopic capillary basement membrane (orange). (D) Within a day, the observations of metastasizing lung cancer cells injected into the microthrombus is dissolved by the proteases in the blood that are venous circulation of mice have suggested that the process of usually responsible for dissolving clots. (E) The cancer cell begins to extravasation often proceeds through the following sequence of proliferate in the lumen of the capillary. (F) Within severa l days, steps. (A) A metastasizing cell (light brown cytoplasm, dark brown sometimes earlier, the cancer cells break through the capillary nucleus) is trapped physically in a capillary. (8) Within minutes, a basement membrane and invade the surrounding tissue large number of platelets (blue) become attached to the cancer cell, parenchyma (gray area). (Note that in this scenario, the forming a microthrombus. Some of these have not yet microthrombus forms only after the cancer cell has become trapped degranulated and released growth factors, proteases, etc. (C) The in a capillary.) (From JD. Crissman, J.S. Hatfield, D.G. Menter et ai, cancer cell pushes aside an endothelial cell (green) on one wall of Cancer Res. 484065-4072, 1988.) the capillary, thereby achieving direct contact with the underlying

most difficult step of all, ostensibly because the foreign tissue environments do not provide cancer cells with the collection of familiar growth and survival fac­tors that allowed their progenitors to thrive in the primary tumor. Without these various types of physiologic support, the metastasizing cells may rapidly die or, at best, survive for extended periods of time as single cells or small clumps of cancer cells-so-called micrometastases-that can only be detected microscop­ically and rarely increase beyond this size. In general, the number of micrometastases in the body of a cancer patient vastly exceeds those that will eventually grow large enough (several millimeters or more in diameter) to be clinically detectable. These micrometastases may be widely disseminated throughout the tissues of a cancer patient, occasionally leading to disastrous outcomes (Sidebar 28 . ).

Sidebar 14.3 Cancer cells are clumsy escape artists The complex task of escaping from the circulation into the surrounding tissue parenchyma is accomplished routinely by leukocytes, which must be able to enter into the parenchyma in response to certain inflammatory stimuli, including the presence of infectious agents. Through a sequence of steps known as diapedesis, leukocytes are able to induce endothelial cells in post-capil­lary venules to retract and provide a portal into the underlying tissue. The entire process from attachment to the

endothelial wall to entrance into the tissue parenchyma takes less than a minute and involves an elaborately choreographed program of biochemi­cal and cell-biological changes!

In contrast, the vast majority of metastasizing cancer cells are not endowed with the receptors and bio­chemical response mechanisms required to execute diapedesis. Accordingly, if neoplastic cells do suc­ceed in penetrating through the wall of a capillary or slightly larger vessel, they seem to do so by brute force, per­haps by degrading patches of

endothelium in a process that requires many hours, or even a day, rather than a minute to complete. Of additional interest here is the fact that the throm­bin produced during the formation of micro thrombi (see Sidebar 27 . ) is quite effective in cleaving the various proteins used by endothelial cells to attach to the underlying vascular basement membrane; this may cause endothelial cells to retract from microemboli, thereby exposing the capillary basement membrane to direct attack by invasive cancer cells and the proteases that they generate.

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Chapter 14: Moving Out: Invasion and Metastasis

Antibodies reactive with cytokeratins are useful for detecting the micrometas­tases that primary carcinomas spawn in the bone marrow and blood, while an antibody against the epithelial cell adhesion molecule (EpCAM) is often used to detect micrometastases in the lymph nodes. In all these cases, the presence of isolated cytokeratin-positive (and thus epithelial) cells in otherwise fully mes­enchymal tissues represents a clear sign that metastatic seeding has taken place. Current microscopic techniques using cytokeratin-specific antibodies make it possible to detect a single-cell micro metastasis among 105 or even 106

surrounding mesenchymal cells in the blood, bone marrow, or lymph node (Figure 14.10) .

Many of the steps proceed inefficiently, and the probability of an individual cell successfully completing all of them is very low. For example, in some mice car­rying primary tumors of about 1 gram mass (-109 cells), as many as a million cells may be seeded into the circulation each day, yet the visible metastases formed in such animals may be counted on the fingers of one hand.

This low rate of success in forming metastases is sometimes termed metastatic inefficiency and is the end product of the sequence of inefficient steps that together make up the invasion-metastasis cascade. Some experiments indicate that the earlier steps in this cascade are executed quite efficiently by metastasiz­ing cells, while the last step, involving colonization, succeeds only rarely and therefore is the rate-limiting determinant of the process as a whole. Consequently, vast numbers of micro metastases may be seeded throughout the body and many may persist for extended periods of time before one or another

(A)

(8)

Figure 14.10 Detection of a micro-metastasis in a lymph node or in the marrow (A) The presence of metastatic cancer cells in the bone marrow is usually analyzed by withdrawing marrow from the iliac crest of the pelvis. In this micrograph, the presence of a micrometastasis containing several cancer cells in the bone marrow of a colon cancer patient has been detected by staining with an anti-cytokeratin antibody. (B) Essentially identical images can be detected in the bone marrow extracted from breast cancer patients. In this case, a cluster of eight mammary carcinoma cells, viewed at slightly lower magnification, form a micrometastasis. (e) The presence of micro metastases in a lymph node is indicated by their appearance, which contrasts strongly with that of the surrounding lymphocytes . Here two metastases of a mouse lung adenocarcinoma (arrows) to a lymph node are seen amid a sea of lymphocytes. Note the formation of a ductlike structure by the right micrometastasis. (A, courtesy of I. Funke and G. Riethmuller; B, from S. Braun, K. Pantel, P. Muller et aI., N. Eng/. 1. Med. 342:525-533, 2000; C, courtesy of K. P. Olive and T Jacks.)

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Micrometastases at sites of dissemination

of these migrants or its lineal descendants finally acquires the ability to grow into a clinically detectable mass (Sidebar 14.4).

Support for the existence of dormant micrometastases, which persist in a non­growing state for extended periods of time, comes from experiments in which living cancer cells were initially marked by brief exposure to fluorescence label-containing particles, which persist for extended periods of time within cells but do not affect their viability. Following such labeling, the intracellular concentration-and thus the fluorescence intensity of the dye particles­decreases by a factor of 2 each time a cell divides; hence, the residual fluores­cence intensity in cells after an extended period of time allows the experimenter to estimate how many cell divisions occurred since the time when the cells were initially marked.

Such dye-labeled cancer cells were introduced via the portal circulation into mouse livers, in which they formed large numbers of single-cell micrometas­tases. Eleven weeks later, cancer cells were recovered from these livers, and many of these still possessed full fluorescence intensity (Figure 14.12), indicat­ing that they had not divided even once since their arrival in the liver. Importantly, these recovered cancer cells remained capable of proliferating in vitro and were able to generate new tumors when injected subcutaneously into other host mice.

The above experiment shows dramatically that metastatic cancer cells can remain viable for extended periods of time in a nondividing, dormant state within foreign tissue sites. (Note that a different type of micro metastasis arises when disseminated cancer cells succeed in proliferating and forming colonies of a very small size within a foreign tissue; however, these micro metastases never increase in size, since the rate of cell proliferation in these clumps is coun­terbalanced by an equal rate of apoptosis, perhaps because of the failure of these cells to execute the angiogenic switch. See Section 13.7.) Whatever their nature, micro metastases represent an imminent threat, since they are often present in vast numbers throughout the body and may erupt years after the can­cer has been judged to be cured.

In summary, the multiple steps of the invasion-metastasis cascade encompass as many distinct biological changes as all the steps that preceded them during the course of primary tumor formation. The complexity of this cascade, as out­lined in these two sections, raises questions that will motivate many of our dis­cussions in the rest of this chapter: How do cancer cells learn to become metastatic? Does each of the steps in this cascade require the actions of a spe­cific gene that becomes altered during tumor progression? Or are many of the individual steps of invasion and metastasis orchestrated by a single master con­trol gene or a small group of such genes?

We will also touch on another simple but profoundly important issue: Do highly malignant cells carry genes that in mutant form are specialized to induce inva­siveness or metastasis? Or do these late steps in tumor progression depend on the actions of familiar actors, specifically the oncogenes and tumor suppressor genes that we have encountered repeatedly throughout this book?

14.3 The epithelial-mesenchymal transition and associated loss of E-cadherin expression enable carcinoma cells to become invasive

The first of the many steps leading to metastasis-the acquisition of local inva­siveness-involves major changes in the phenotype of cancer cells within the primary tumor. As before, we will focus this discussion on epithelial tissues and

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Chapter 14: Moving Out: Invasion and Metastasis

Sidebar 14.4 Genetic analyses suggest that the evolution of metastatic ability can occur outside of the primary tumor In perhaps 30% of breast, prostate, and colon cancer patients whose primary carcinomas have been surgically removed, one can still detect micrometastases in the mar­row, lymph nodes, or blood; these patients are considered to have "minimal residual disease." At this stage, genetic analysis of the micro metastases indicates that they are genetically heterogeneous. However, years later, when a patient develops disease relapse and manifests readily detectable metastatic masses, the patient's single-cell micrometastases are now much more similar to one another genetically (Figure 14.11A).

This suggests a stage of tumor progression in which the ability to colonize is acquired separately from the ability to disseminate to distant organ sites. Initially, genetically diverse cancer cells are seeded by a primary tumor throughout the body, but none of these succeeds in estab­lishing a macroscopic metastasis simply because none is capable of doing so. After a period of time,however,

genetic evolution occurring in a micrometastasis some­where in the body yields a clone of cells with the newly acquired ability to colonize efficiently. As this clone expands, it also begins to seed cancer cells throughout the body, and therefore generates a new, second wave of metastatic dissemination. The individual cancer cells that are released by this clone soon constitute the majority of the single-cell micrometastases in the marrow of the patient, and these micro metastases are genetically very similar to one another because of their shared descent from the same clonal cell population. Importantly, because the cells in these new micrometastases all have inherited the ability to colonize, many grow rapidly into macro­scopic metastases, creating a life-threatening burden of disseminated cancer cells in the patient. This model (Figure 14.11 B) suggests that the evolution toward advanced malignancy often occurs at anatomical sites far removed from the primary tumor. While suggested by these observations, the scheme of Figure 14.11B is not yet vali­dated by large, statistically robust sets of observations.

Figure 14.11 Genetic heterogeneity of micrometastases and the evolution of colonizing ability (A) Single-cell micrometastases of primary carcinomas can be identified in bone marrow biopsies because of their display of epithelial cell markers, such as cytokeratins or EpCAM (see Figure 14.10), and then isolated using a micropipette. (B) Adaptation of thecomparative genomic hybridization (CGH;see Figure 11.20) procedure is used to analyze Individual micrometastatic cells for the gain or loss of various chromosomal arms. This, in turn, has made it possible to relate the resulting" genetic profile" of each micrometastasis with the profiles of several other micrometastases isolated from the same patient. Those profiles that are similar to one another are placed closely to one another on a branch of a tree termed a dendrogram; conversely, those that have very different genetic profiles are located far away from one another on the tree. In the event that multiple micrometastases from a single patient are clustered next to one another on a branch of the dendrogram (and thus are closely related to one another genetically), this is indicated by a blue horizontal bar associated with this pair or with a larger group of micrometastases. Micrometastases in the bones of patients (identified by labels) carrying breast, prostate, and colorectal tumors were obtained atthe time of surgical removal of their primary tumors, a stage termed "minimal residual disease." As seen in the upper dendrogram, only a minority of micrometastases in patients with minimal residual disease are clustered together on the dendrogram, indicating substantial genetic heterogeneity of micrometastases within each patient at this stage of disease. However, months or years later, when disease relapse with macroscopic metastases occurs, the several micrometastases detected in almost every patient are located close to one another on the lower dendrogram, indicating close genetic relationship to one another. (C) The analyses shown in panel (B) suggest the following model. An initially formed, genetically heterogeneous primary tumor cell population (see Figure 11.18) seeds equally heterogeneous micrometastases throughout the body of the cancer patient. The primary tumor is then removed surgically, leaving behind only the micrometastases and creating the state of minimal residual disease. Over a period of years, in one or another site in the body, one of these micrometastatic cell clones (aquamarine) acquires the ability to colonize, i.e., to grow into a macroscopic metastasis. The latter now acts as a source of cells that generate a new cascade of metastatic dissemination throughout the body. Because these newly dispersed cells all share a common clonal origin, the micrometastases that they form are genetically very similar to one another. Moreover, because the cells in each of these secondary micrometastases are endowed with the ability to colonize (since they all derive from a cell clone in a macrometastasis that has previously acquired this ability), many of these micrometastases can rapidly grow into macroscopic, clinically detectable metastases that result in disease relapse. (A and B, from c.A. Klein, 1J. Blankenstein, O. Schmidt-Kittler et al., Lancet 360:683-689, 2002.)

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599

Chapter 14: Moving Out: Invasion and Metastasis

Figure 14.12 Persistence of solitary dormant tumor cells many weeks after introduction into the liver Tumorigenic mouse breast cancer cells were labeled by inducing them to take up styrene nanoparticles (48 nm diameter) that had been tagged with a fluorescent dye. These cells were then injected into a mesenteric vein, which carried them via the portal vein into the liver. Eleven weeks later, tumor cells could still be detected in the liver (white arrows). Importantly, the fluorescence intensity of many of these cells did not differ significantly from the intensity of cells shortly after labeling, indicating that these cells had not divided even once following labeling. Following isolation and in vitro culturing, the descendants of many of these cells were tumorigenic, i.e., capable of fo rming tumors when injected into the mammary fat pads of host mice. (Moreover, the dormant cancer cells were found to be fully resista nt to a chemotherapy that reduced by 75% the size of metastases that were growing rapidly in the same mice.) (From G.N. Naumov, I. e. MacDonald, P.M. Weinmeister et ai, Cancer Res. 62:2162-2168, 2002)

the carcinomas that they spawn. The organization of the epithelial cell layers in normal tissues is incompatible \Nith the motility and the invasiveness displayed by malignant carcinoma cells, yet this epithelial organization plan continues to be respected in many primary carcinomas. In these tumors, well-organized sheets of epithelial cells are present, although their overall topology may be quite different from that of comparable normal epithelia (see, for example, Figure 2.6).

In order to acquire motility and invasiveness, carcinoma cells must shed many of their epithelial phenotypes, detach from epithelial sheets, and undergo a drastic alteration-the epithelial-mesenchymal transition (EMT), which we mentioned in the context of wound healing (Section 13.3). Recall that the EMT involves a shedding by epithelial cells of their characteristic morphology and gene expression pattern and the assumption of a shape and transcriptional pro­gram characteristic of mesenchymal cells. This major shift in epithelial cell phe­notype is necessary for the reconstruction of epithelial cell layers after wound­ing (see Figure 13.13C). The EMT is used widely in certain morphogenetic steps occurring during embryogenesis, when tissue remodeling depends on EMTs executed by various types of epithelial cells (Table 14.1). It is plausible, though hardly proven, that all types of carcinoma cells must undergo a partial or com­plete EMT in order to become motile and invasive.

During one of the steps of gastrulation in early embryogenesis, individual cells peel away from the ectoderm and migrate inward toward the center of the embryo to form the mesoderm, the precursor of mesenchymal tissues, including

Table 14.1 Examples of EMTs during mouse embryonic development

Process Transition From To

Gastrulation epiblast mesoderm Prevalvular mesenchyme endothelium atrial and ventricular septum

inthe heart Neural crest cells neural plate neural crest cells, which can yield

bone, muscle, peripheral nervous system

Somitogenesis somite walls sclerotome ··Palate formation oral epithelium mesenchymal cells

Mullerian duct regression MUllerian tract mesenchymal cells

Adapted from P. Savagner, BioEssays 23:912-923, 2001.

600

The EMT enables invasiveness and dissemination

(A) (B) migrating neural crest cells

neural tube

fibroblasts and hematopoietic cells (in chordates). This conversion of ectoder­mal cells, which at this stage are arrayed in an epithelial cell layer, to those hav­ing a mesodermal phenotype involves an EMT (Figure 14.13A). At the same time, these cells undergoing an EMT acquire the ability to translocate from one location (the outer cell layer) to another (the interior) within the embryo.

The migration of neuroepithelial cells from the neural crest into the mes­enchyme of early vertebrate embryos also depends on a transformation of cell phenotype that can best be described as an EMT (Figure 14.13B) . Similarly, the migration of myogenic precursor cells (the progenitors of muscle cells) from the dermomyotome of the early embryo to the limb buds depends on an EMT-like transformation of cell phenotype. All of these processes, in turn, bear a striking resemblance to the EMT undertaken by the cells at the edge of a wound within an epithelium; these cells must undergo a transient EMT in order to migrate into the wound site and close the gaps in the epithelial cell sheet that were created by the wounding process (see Figure 13.14) .

An EMT can also be seen at the edges of carcinomas that are invading adjacent tissues (Figure 14.14). This pathological process is strikingly similar to the EMTs occurring during early embryogenesis and wound healing. Once again, epithe­lial cells cease expressing epithelial protein markers and express mesenchymal ones in their stead; at the same time, these cells lose their epithelial morphology and take on the appearance of fibroblasts (see Figure 13.13).

The strong resemblance between the pathological process of tumor invasive­ness and normal steps of embryogenesis and wound healing suggests a plausi­ble mechanistic model: according to this modeL which is supported by exten­sive evidence gathered in recent years, the complex program of cellular reorgan­ization exhibited by invasive carcinoma cells depends on the reactivation of latent behavioral programs whose expression is usually confined to early embryogenesis and to damaged adult tissues. According to this thinking, once carcinoma cells acquire access to the EMT program, they can exploit it to pro­foundly change their own morphology, motility, and ability to invade nearby cell layers. This model implies that the mUltiple changes in cell phenotype associ­ated with invasiveness, some of which are described below, need not be acquired piecemeal by carcinoma cells. Instead, these cells simply activate a morphogenetic program that is already encoded in their genomes. (This logic echoes our earlier discussion of epithelial-stromal interactions in Section 13.3, where we argued that cancer cells exploit the wound-healing program in order to acquire an activated stroma.)

The normal and pathological versions of the EMT involve, in addition to changes in shape and the acquisition of motility, fundamental alterations in the gene expression profiles of cells (Table 14.2). Expression of E-cadherin and

notochord

Figure 14.13 Embryogenesis and the epithelial-mesenchymal transition (A) This scann ing electron micrograph shows the delamination of cells from the primitive ectoderm of a sea urchin embryo (white arrows) and their migration into the interior of the embryo. The cells have become round and acqu ired motility in anticipation of their forming the rudiments of the mesoderm-changes associated w ith an epithelial-mesenchymal transition (EMT) They are migrating along strands of extracellular matrix in the lumen of this early embryo. (B) The delamination of neuroepithelial cells from the neural tube also requires cells to undergo an EMT. Cells of the embryonic neural crest (orange), w hich derive from the upper region of the initially epithelial neural tube (gray), delaminate from this epithelium, acquire motility and invasiveness, and disperse throughout the embryo and eventually throughout the body of the resu lting organism, w here they form melanocytes, much of the peripheral nervous system, and much of the skeleton of the face. (A, courtesy of G. Cherr; B, from A.E. Vernon and C. LaBonne, Curr Bioi. 14R719-R721,2004)

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plasma membrane

a-caten in

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Chapter 14: Moving Out: Invasion and Metastasis

cytokeratins-hallmarks of epithelial cell protein expression-is repressed, while the expression of vimentin, an intermediate filament component of the mesenchymal cell cytoskeleton, is induced. Epithelial cells that have undergone an EMT often begin to make fibronectin, an extracellular matrix protein that is normally secreted only by mesenchymal cells such as fibroblasts. At the same time, expression of a typical fibroblastic marker-N-cadherin-is often acquired in place of E-cadherin (Figure 14.15).

(C) EPITHELIAL MESENCHYMAL STATE STATE

adherens EMT junctions

I .• J.

i l ­I

""\ r binding to \ Tcf/LEF TFs

loss of E-cadherin to nucleus

linkage to actin free p-catenin cytoskeleton in cytosol

Figure 14.14 Epithelial-mesenchymal transition at the invasive edge of a tumor Colon carcinoma cells at the invasive edge of a primary human tumor undergo changes in gene expression and the localization of certain proteins. (A) While E-cadherin (brown) is strongly expressed on the plasma membranes of cells in the core of a primary tumor, where it forms adherens Junctions (left panel), its expression decreases substantially in individual invasive cells at the edge of this tumor (red arrows, right panel) and is no longer localized to their plasma membranes. (B) At the same time, cells in the core of this tumor express ~-catenin (dark red, left side) under their plasma membranes and diffusely throughout the cytoplasm, while tumor cells at the invasive edge of this tumor show intense staining ~-catenin in their nuclei

(right side). (C) In normal epithelial cells, ~-catenin serves to link the cytoplasmic tail of E-cadherin, w hich forms adherens junctions w ith neighboring cells (see Figure 13 .12), to the actin cytoskeleton . It also functions in the cytoplasm as a key intermediary in the Wnt signaling pathway (Section 6.10) Loss of E-cadherin from the plasma membrane liberates ~-catenin molecules, which may then migrate to the nucleus and associate with Tef/Lef transcription factors, thereby inducing expression of genes orchestrating the EMT program. (A, from T. Brabletz, A. Jung, S. Reu et al., Proc. Natl. Acad Sci. USA 98:10356-10361,2001; B, courtesy of T. Brabletz and T. Kirchner; C, from TA Graham, C. Weaver, F. Mao et aI., Cell 103885-896, 2000, courtesy of H.J. Choi and WL Weis)

translocation

602

The EMT and loss of E-cadherin expression

Table 14.2 Cellular changes associated with the epithelial-mesenchymal transition

Loss of Cytokeratin (intermediatefHament) expression Epithelial adherensjunction protein (E-cadherin) Epithelial Cell polarity

.Acquisition of Fibroblast-like shape Motility Invasiveness Mesenchymal gene expression program Mesenchymal adherens junction protein (Nccadherin) Protease secretion (MMP-2, MMP-9) Vimentin (intermediate filament) expression Fibronectin secretion PDGF receptor expression av~6 integrin expression

Of all these proteins, the transmembrane E-cadherin molecule plays the domi­nant role in influencing epithelial versus mesenchymal cell phenotypes. Recall our earlier encounters v\Tith E-cadherin and its role in enabling epithelial cells to adhere to one another (see Figures6.26A and 13.12) . In normal epithelia, the ectodomains of E-cadherin molecules extend from the plasma membrane of one epithelial cell to form complexes with other E-cadherin molecules protrud­ing from the surface of an adjacent epithelial cell. This enables homodimeric (and higher-order) bridges to be built between adjacent cells in an epithelial cell layer, resulting in the adherens junctions that are so important to the structural integrity of epithelial cell sheets.

The cytoplasmic domains of individual E-cadherin molecules are tethered to the actin fibers of the cytoskeleton via a complex of a- and ~-catenins (see Figures 13.12 and 14.14) and other ancillary proteins. The actin cytoskeleton, for

(A) epithelial markers

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Figure 14.15 Biochemical changes accompanying the EMT As discussed later, the EMT can be induced by several transcription factors. Shown here are the effects of expressing the Twist transcription factor in MOCK (Maden-Darby canine kidney) cells, which are widely used to study epithelial cell biology. (A) These immuno­fluorescence analyses indicate that expression of epithelial markers, specifically E-cadherin, ~-caten in, and y-catenin, is depressed, while expression of mesenchymal markers, specifically vimentin and fibronectin, is induced by ectopic expression of Tw ist. (B) Immunoblots conf irm the results of immunofluorescence, but in a more quantitative fashion. Lysates of control cells are analyzed in the left channels, while Iysates of MOCK cells forced to express Tw ist are analyzed in the right channels. ~-actin, whose expression is unaffected by the EMT, is used here as a control to ensure that equal amounts of cell lysate have been analyzed in all cases. sm-actin, a-smooth muscle actin. (A and B, from J. Yang, SA Mani, J.L Donaher et aI. , Cell 117927-939, 2004)

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Chapter 14: Moving Out: Invasion and Metastasis

its part, provides tensile strength to the cell. Hence, by knitting together the actin cytoskeletons of adjacent cells, E-cadherin molecules help an epithelial cell sheet resist mechanical forces that might otherwise tear it apart. Once E­cadherin expression is suppressed, many of the other cell-physiologic changes associated with the EMT seem to follow suit. Some experiments indicate that simply by suppressing the expression of the E-cadherin protein, cells acquire a mesenchymal morphology and increased motility.

The pivotal role ofE-cadherin in the acquisition of malignant cell phenotypes is further supported by observations indicating that the CDHl gene, which speci­fies E-cadherin, is repressed by promoter methylation in many types of invasive human carcinomas (see Table 7.2) and in others by certain transcriptional repressors; this gene can also be inactivated by reading-frame mutations. For example, an analysis of 26 human breast cancer cell lines indicated that 8 had mutations that led to inactivation of E-cadherin gene expression, 5 had truncat­ing mutations in the E-cadherin reading frame, while 3 had in-frame deletions resulting in the expression of mutant E-cadherin molecules at the cell surface. By now, loss of E-cadherin expression or expression of mutant E-cadherin pro­teins has been documented in advanced carcinomas of the breast, colon, prostate, stomach, liver, esophagus, skin, kidney, and lung. And mutant germ­line alleles of the CDHl gene result in familial gastric cancer (see Table 7.1).

Additionally, in studies of several types of carcinoma cells that had lost E-cad­herin expression, re-expression of this protein (achieved experimentally by introduction of an E-cadherin expression vector) strongly suppressed the inva­siveness and metastatic dissemination of these cancer cells. Together, these diverse observations indicate that E-cadherin levels are key determinants of the biological behavior of epithelial cancer cells and that the cell-to-cell contacts constructed by E-cadherins impede invasiveness and hence metastasis.

The replacement of E-cadherin by N-cadherin during the EMT (see Figure 14.15B) is also seen during gastrulation in early embryogenesis. Moreover, hepa­tocyte growth factor (HGF) promotes the E- to N-cadherin switch in cultured epi­blast cells (which derive from the embryonic ectodermal cell layer) ; in this way, it induces an EMT and enables emigration of muscle and dermal precursor cells from the primitive dermomyotome-the collection of epithelial-like cells located in the somites of early vertebrate embryos. In the context of cancer pathogene­sis, HGF potently induces motile and invasive behavior in carcinoma cells.

Like E-cadherin, the N-cadherin that is produced in its stead participates in homophilic interactions, that is, binds to other molecules of the same type dis­played by nearby cells. Consequently, the N-cadherin molecules expressed on the surface of a carcinoma cell that has undergone an EMT increase the affinity of this cancer cell for the stromal cells that normally display N -cadherin, notably the fibro blasts in the stroma underlying the epithelial cell layer. This association seems to help invading carcinoma cells insert themselves amid stromal cell populations. Precisely the same dynamiCS have been proposed to explain how melanomas develop: normal melanocytes express E-cadherin, which binds them to the keratinocytes around them; melanoma cells-the transformed derivatives of melanocytes-express N-cadherin, which facilitates their inva­sion of the dermal stroma of the skin and their association with its fibroblasts and endothelial cells (Figure 14.16) .

Importantly, the acquisition of N-cadherin expression does not result in the assembly of large sheets of cancer cells that might, in principle, be created by the formation of cell-cell N-cadherin bridges. It seems that the intermolecular bonds formed between pairs of N -cadherin molecules are far weaker than those formed by E-cadherin homodimers. This helps to explain why cell-surface N­cadherin molecules actively favor cell motility, and thus behave very differently from their E-cadherin cousins, which function to immobilize cells within

604

The EMT and the mesenchymal gene expression program

gap junction

(A) (B)E-cadherin

N-cadherin

melanoma cell

endothelial cell (mesenchymal)

epithelial cell layers. This point is driven home by experiments in which expres­sion vectors are used to force high levels of N -cadherin expression in otherwise normal cultured epithelial cells. Such ectopic expression causes the epithelial cells to acquire motility and invasiveness, as indicated by their ability to break through reconstructed extracellular matrix (ECM) introduced into a culture dish.

14.4 The epithelial-mesenchymal transition is often induced by stromal signals

As described above, the epithelial-mesenchymal transition (EMT) seems to be an irreversible change that carcinoma cells acquire as they advance down the road toward a highly malignant growth state. Actually, there are reasons to believe that during the development of many carcinomas, the EMT phenotype is acquired reversibly, and that once carcinoma cells have completed the multi­ple steps of invasion and metastasis, they often revert back to a more epithelial phenotype by passing through the mesenchymal-epithelial transition (MET) mentioned in the last chapter.

This reversion suggests that the EMT is often triggered by signals that cancer cells experience in one environment but no longer experience in another. Thus, carcinoma cells at the invasive front of a primary tumor may receive specific sig­nals from the nearby reactive stroma (Section 13.3) that has developed during the formation of this tumor. However, once these cancer cells have left the pri­mary tumor and settled at a distant site, they may experience a stromal environ­ment that does not release EMT-inducing signals. In the absence of these con­textual signals, some of which are described below, the carcinoma cells may then undergo a mesenchymal-epithelial transition (MET) and revert to the phe­notype of their ancestors in the heart of the primary tumor (Figure 14.17; Sidebar 14.5).

In fact, traditional histopathological techniques have failed to demonstrate the EMT at the invasive edges of primary carcinomas for a simple reason: once tumor cells undergo a full EMT (Le., shed all epithelial traits and acquire mes­enchymal ones instead), they are essentially indistinguishable from the mes­enchymal cells in the surrounding stroma. For this reason, demonstrations of the EMT at the invasive edges of tumors has required the use of antibodies and cel­lular reagents that are not normally used in diagnostic pathology laboratories. A fluorescent-coupled antibody that can detect intracellular ~-catenin localization

Figure 14.16 Cadherin shifts and melanoma cell invasiveness Melanomas are among the most malignant tumors because of their tendency to metastasize w idely once they reach a certain stage of tumor progression. This behavior is attributable, in part, to the reactivation of a cell-biological program that enabled the migratory behavior of their neural crest ancestors. The shift from E- to N-cadhenn expression, which occurs when melanocytes (A) become transformed into melanoma cells (B), has been proposed to facilitate invasion of the stroma, since the shutdown of E-cadherin expression (blue) enables these cancer cells to ext ricate themselves from their keratinocyte neighbors in the epidermis, while its replacement by expression of N-cadherin (red) allows these tumor cells to form homotypic interactions with various types of mesenchymal ce lls, such as fibroblasts and endothelial cells, that reside in the stroma of the skin (i.e., the dermis). (From N.K Haass, K.S.M Smalley and M. Herlyn, J. Mol. Histol. 35309-318, 2004)

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Chapter 14: Moving Out: Invasion and Metastasis

(see Figure 14.14B) is one example of such a reagent. Another demonstration comes from xenografted human tumor cells, in which expression of the (Xv~6 integrin has been detected, once again through immunostaining (Figure 14.19A). In this instance, (Xv~6 expression, which is another marker of the EMT, is seen only in a thin outer layer of tumor cells that are in contact with the tumor-associated stroma.

(A)

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primary tumor invasive edge liver metastasis

(8)

carcinoma in situ invasive carcinoma

PROGRESSION

EMT -INVASION

INTRAVASATION• •

basement membrane

COLONIZATION MET

~ micrometastasis

_ EXTRAVASATION

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macrometastasis

JFigure 14.17 Reversibility of EMT While cells at the invasive edge of a primary carcinoma often give evidence of an EMT, derived metastases may exhibit a histology typical of the center of the primary tumor. (A) Release of degradative enzymes, notably matrix metalloproteinases (MMPs), is one of the many manifestations of the EMT. These cells in a primary colorectal carcinoma show expression of both cytokeratin 18 (red) and a basement membrane protein (green). How ever, at the invasive edge of this tumor, the cells have undergone a partial EMT, in that they have degraded the adjacent basement membranes while still expressing cytokeratin 18, a key epithelial marker. In a subsequently arising metastasis in this patient, which presumably descends from cells that acquired invasiveness en route to metastatic dissemination, the cancer cells form a grow th having, once again, the histological appearance of cells in the heart of the primary tumor. (B) Observations such as

those of panel A have suggested the scheme depicted here. Thus, epithelial cancer cells at the edge of a primary carcinoma (pink) undergo an EMT as they invade into the stroma and become mesenchymal (red cells). This change would seem to be triggered by signals that these carcinoma cells receive from the tumor­associated stroma. The newly acquired mesenchymal state enables these cells to invade locally, intravasate, and subsequently to extravasate into the parenchyma of a distant tissue . Once they have become established in this tissue, these cells find themselves in a new type of stromal environment that lacks the signals that previously induced their ancestors to undergo an EMT. This allows these cells to revert to an epithelial phenotype via a mesenchymal­epithelial transition (MET) (The regeneration of a basement membrane is not indicated here) (A, courtesy of T. Brabletz; B, adapted from JP. Thiery, Nat. Rev. Cancer 2:442-454,2002 .)

606

The EMT and contextual signals

Yet another marker of the EMT is provided by laminin 2y. Normally, this protein is one of three subunits of laminin-5, an important constituent of the basement membrane to which epithelial ceUs attach via specific integrins. However, cells that have undergone an EMT and become invasive now release only laminin 2y (and not the other two subunits of laminin-5), and cleavage of laminin-2y by matrix metalloproteinases (Section 14.6) releases a fragment that functions as an EGF-receptor ligand, facilitating cell survival and motility (Figure 14.19B).

Possibly the most vivid demonstration of the phenotypic conversion of carci­noma cells at the invasive edge of a tumor has come from the use of a human­specific anti-vimentin antibody, which demonstrates the EMT at the invasive edge of a tumor formed by experimentally transformed human mammary epithelial cells growing as a xenograft in an immunocompromised mouse

Sidebar 14.5 The reversibility of the EMT explains one peculiarity of many metastases All carcinoma cells that become invasive and metastatic may need to undergo an EMT in order to acquire these complex phenotypes. (Such cells may undergo a full EMT, during which they shed all epithelial characteristics, or, alternatively, may enter only partially into an EMT, where some epithelial characteristics are retained together with newly acquired mesenchymal traits.) This behavior would seem to be incompatible with one frequently observed aspect of human metastases: these secondary growths often closely resemble, at the

Figure 14.18 Appearance of a primary tumor and derived metastasis These micrographs illustrate the striking similarity in the appearance and expression patterns of a resected primary breast tumor (left images) and a brain metastasis that was detected two years later (right images). (A) Immuno­histochemistry using an anti-estrogen receptor (ER) antibody reveals the presence of this protein (brown) in the nuclei of both·primary tumor cells and the cells of the derived metastasis. (8) Immunohistochemistry using an anti­HER2/Neu antibody reveals the expression of this receptor at the surfaces of tumor celis in both the primary tumor (rimmed in brown) and the derived brain metastasis. In both cases, the appearance of the epithelial celis and that of the recruited stromal cells are remarkably similar. Indeed, such resemblances often help a pathologist to ideritify the primary tumor origin from which a metastasis derives and provide support for the model depicted in Figure 14.178. (Courtesy of TA. Ince.)

histopathological level, the primary tumors from which they originated (see, for example, Figures 14.17A and 14.18). Indeed, the cells in such metastases seem to be as epithelial in their behavior as the bulk of the cells in the pri­mary tumor, yet they are descended from invasive cells that supposedly underwent an EMT-ih order to initiate metasta­tic spread. This inconsistency is resolved if one assumes ' that the EMT is fully reversible, and that lineages -of highly malignant cells often pass only transiently through a mes­enchymal state while traveling from the primary tumor to the site of metastasis formation.

(A)

estrogen receptor

primary breast !brain tumor metastasis

(B) !.

HER2/Neu

607

carcinoma cells

:- invasive edge

•

Chapter 14: Moving Out: Invasion and Metastasis

(A) (B)

CXy~6 integrin

Figure 14.19 Manifestations of the EMT at the interface between tumor epithelium and stroma (A) The expression of the C(v~6 integrin is associated with the EMl This integrin is expressed in epithel ia l tissues that are undergoing wound healing or suffering chronic Inflammation; it is also seen at the invasive edge of carcinomas. In a xenografted tumor formed by SC C -14 human pharyngeal carcinoma cells, expression of the C(v~6 integrin is exhibited by carcinoma cells at the invasive edge of the tumor (dark brown) that are in direct contact with the tumor-associated stroma, suggesting that stromal signals are responsible for its expression in epithelial cells. (B) Laminin 2y normally serves as as one of the subunits of the heterotrimeric laminin-5 molecule of the basement membrane (see Figure 13.5). However, carcinoma cells that have undergone an EMT at the invasive edge of the tumor secrete this protein on its own, whereupon it becomes cleaved in the extracellular space by a matrix metalloproteinase (MT1-MMP) into an EGF receptor ligand that encourages cancer cell survival and motility In this micrograph, cells from the invasive edge of a human colorectal carcinoma (dotted line, left) have begun to invade into the stroma (right). As indicated by immunohistochemistry, individual carcinoma cells that have already invaded deep into the stroma express high leve ls of laminin 2y (brown, arrows). (C) Experimentally transformed human mammary epithelial cells (MECs) w ere implanted

608 in an immunocompromised mouse host. The cytokeratin-positive

(C)

(human) vimentin

200x40x

human carcinoma cells (red) toward the center of the tumor mass are not in direct contact w ith the surrounding mouse stromal cells, whose presence is indicated only by their DAPI-stained nuclei (blue) However, many of the human MECs that are in direct contact with the stroma have undergone an EMT, as indicated by their loss of cytokeratin staining and their display instead of human-specific vimentin (green). [The use of antibody that specifically recognizes human (and not mouse) vimentin ensures that the green cells at the invasive edge derive from the engrafted human cells rather than from the mouse host.) Moreover, some of these cancer cells at the invasive edge have lost the cuboidal shape of the epithelial cancer cells and have assumed, instead, a more elongated, fibroblastic shape. (D) The preferential display of vimentin at the edge of these carcinoma cell islands is revealed here. A tumor formed by the same strain of transformed human MECs described in panel C is seen here at lower magnification. This image makes it apparent that the carcinoma cells that are in direct contact with the surrounding stroma have undergone an EMT, as judged by the criterion of human vimentin display (brown), w hile tumor cells at the interior of these islands do not display human vimentin and have presumably remained in an epithelial state. (A, courtesy of D.R, Leone, B.M. Dolinski, and S.M. Violette, Biogenldec; B, from E. Shinto, H. Tsuda, H. Ueno et ai, Lab. Invest. 85:257-266, 2005; C, courtesy of K. Hartwell and lA. Ince; D, courtesy of lA. Ince )

The EMT and contextual signals

(Figure 14.19C and D). (The use of a human-specific antibody guarantees, in this instance, that the vimentin-expressing cells derive from keratin-positive human carcinoma cells rather than from the surrounding stroma produced by the mouse host.)

These observations and others like them indicate the involvement of certain heterotypic signals that originate in the reactive stroma of primary carcinomas, impinge on neoplastic cells located at the outer edges ofthe epithelial cell mass, and induce these cells to undergo an EMT. Abundant evidence indicates that TGF-~ is an important agent for conveying these stromal signals. Other observa­tions implicate a variety of other factors, including TNF-a (tumor necrosis fac­tor-a), epidermal growth factor (EGF), HGF (hepatocyte growth factor), and IGF-l (insulin-like growth factor-l). It appears likely that these stromal signals act in various combinations with one another and with mutant alleles, such as ras oncogenes, that reside in transformed epithelial cells in order to activate their EMT program. (Presumably the diversity of EMT-inducing factors reflects the multitude of steps in normal embryogenesis during which EMTs occur, these being induced at various sites throughout the developing embryo by diverse heterotypic signals.)

In one set of influential experiments, exposure of ras-transformed EpRas mouse mammary epithelial cells (MECs) to TGF-~ resulted in the progressive loss of epithelial morphology and a reduction of epithelial markers, including cytoker­atins and E-cadherin. At the same time, these transformed cells acquired mes­enchymal protein markers, such as vimentin, and assumed a morphology resembling that of fibroblasts-all the hallmarks of an EMT. Provocatively, once these ras-transformed cells underwent an EMT, they began to produce their own TGF-~l; this TGF-~l, acting via an autocrine Signaling loop, allowed them to maintain their mesenchymal phenotype for extended periods of time, long after the inciting TGF-~ was withdrawn from their culture medium (Figure 14.20A-D). These studies suggest that TGF-~ signaling can conspire with a ras oncogene to cause epithelial cancer cells to undergo an EMT. Similarly, mainte­nance ofTGF-~ signaling through a positive-feedback loop may play an impor­tant role in maintaining av~6 integrin expression and the EMT in human carci­noma cells. This figure also provides indication that TGF-~ may often be pro­duced in abundance by the tumor-associated stroma (Figure 14.20E).

Two of the downstream effectors ofRas function (see Sections 6.5 and 6.6) seem to be responsible for the collaboration between Ras and TGF-~ signaling. The Raf oncoprotein, which functions immediately downstream of Ras, can elicit an EMT on its own: it causes transformed EpRas cells to secrete TGF-~, which then acts in an autocrine fashion to induce the EMT in these cells. PI3K, another effector of the Ras oncoprotein, protects Ras-transformed cells from the cytosta­tic and pro-apoptotic effects ofTGF-~.

A related mechanism also seems to explain the progression of mouse skin tumors that have been initiated by ras oncogene activation. The cells in these tumors evolve from having a highly differentiated, squamous cell phenotype (see Figure 2.6A,B) into cancer cells that are motile, spindle-shaped, and metastatic. (Spindle-shaped cells exhibit a morphology similar to that of transformed fibroblasts.) This progression to a highly malignant state is accompanied by and likely caused by progressively increasing levels of the Ras oncoprotein as well as increasing nuclear localization of the Smad2 transcrip­tion factor, the latter indicating intense signaling through the TGF-~ pathway (see Figure 6.29D).

TGF-Ws role in actively promoting the aggressiveness of malignant cancer cells contrasts starkly with our earlier discussions of its anti-proliferative effects (see Sidebar 29 0 ). Strong support that TGF-~ can favor malignant cell behavior is provided by numerous studies in which the levels of tumor-associated TGF-~ (often TGF-~l) were found to rise in parallel with increasing degrees of tumor 609

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Chapter 14: Moving Out: Invasion and Metastasis

(8)

TGF-~ for 7 days ---

EpRas cells

remove TGF-~ + 3 days + 5 days + 10 days

(D)

in vitro

EpRas cells + EpRas cells EpRas cells expressing dn TGF-~RII anti-TGF-~ serum

(E) TGF-~ (stromal cells)

Figure 14.20 Control of the EMT by TGF-~ and its effects on tumorigenic cells Mouse mammary epithelial cells (MECs) of the EpH4 cell line were transformed into tumor cells through the introduction of a ras oncogene, yielding EpRas cells. (A) EpRas cells (left) usually have an epithelial, cobblestone-like appearance and express E-cadherin (green) at their cell-cell junctions. However, when they are cultured for 7 days in the presence of TGF-~l (right), they undergo an EMT and assume an elongated fibroblastic appearance (not visible here) . In addition, they suppress expression of E-cadherin and express instead vimentin (red), thereby shifting from an epithelial to a mesenchymal gene expression program. Nuclei are stained blue w ith the DAPI dye. (B) After the EpRas cells undergo an EMT (panel A), they maintain the resulting mesenchymal, fibroblast-like state through their ow n production of and response to TGF-~l (ie., via TGF-~l autocrine signaling). However, when these cells are cultured in a medium that lacks added TGF-~l and their growth medium is changed on a daily basis (to remove any TGF-~ that they may have secreted into the medium), their appearance gradually reverts to an epithelial cobblestone phenotype, as shown after 3, 5, and 10 days of culture (left to right), indicating that they have undergone a mesenchymal­epithelial transition (MET). (C) When EpRas cells that have been grow ing in a tumor in the presence of high concentrations of autocrine TGF-131 are placed into collagen gels in vitro, they form the highly invasive structures seen here (left panel). How ever, if these cells are propagated in vitro under identical conditions in the presence of antiserum that neutralizes TGF-~1 (to sequester any TGF-~ produced by these cells), they revert to an epithelial phenotype and now form ductal structures in collagen gels that are typical of those formed by cultured normal MECs (right panel). (D) Use of a dominant-negative (dn) type II TGF-~ receptor (which effectively blocks autocrine TGF-~ signaling) provides further proof that autocrine TGF-~ signaling by EpRas cells is required to maintain their mesenchymal state. When this signaling is blocked by expression of this receptor, the mesenchymal appearance of the EpRas cells (Jeft) disappears and they assume an epithelial appearance (nght), indicating, as before, that they have undergone a mesenchymal-epithelial transition (MET). (E) Detroit 562 carcinoma cells growing in a tumor express the av~6 integrin (red, see Figure 14.19A), which is indicative of their having undergone an EMT; while TGF-~ (green) is being produced by cells in the nearby tumor-associated stroma. The av~6 displayed by the tumor cells can activate the latent form of TGF-~ produced by the stromal cells, thereby creating a factor that is a strong inducer of the EMT and additional av~6 integrin expression by epithelial cells, yielding a self-sustaining, positive-feedback loop. (A, from E. Janda, K. Lehmann, I. Killisch et ai. , I Cell BioI. 156299-314, 2002; Band C, from M. Oft, J. Peli, C. Rudaz et ai. , Genes Dev. 10:2462-2477,1996; 0 and E, courtesy of D.R. Leone, B.M. Dolinski and S.M. Violette, Biogenldec.)

610

The EMT and its induction byTGF-~

invasiveness and general aggressiveness. Indeed, high levels of TGF-p, both in the tumor mass and in the general circulation, augur poorly for the long-term survival of the cancer patient.

To summarize these and other observations, TGF-p can contribute to cancer cell invasiveness for at least four reasons. First, most human carcinomas arising out­side the intestine retain at least some functional TGF-p receptor signaling, allowing them to continue to respond to TGF-p during the entire course of tumor progression. (This contrasts with the situation observed in a subset of colon carcinomas in which receptor function may be lost entirely through mutations in the receptor-encoding genes; see Section 12.9.) Second, the inacti­vation of the pRb pathway, which occurs in most if not all human cancers, causes malignant cells to lose their responsiveness to the cytostatic effects of TGF-p (see Section 8.10); this loss allows these cells to respond to other types of downstream signals that are released by ligand-activated TGF-p receptors. Third, in the absence of the cytostatic effects ofTGF-p, exposure of cancer cells to this factor may actually favor their proliferation. For example, glioblastoma Figure 14.21 The NF-ICB signalingand osteosarcoma cells that are treated with TGF-p respond by producing and pathway and induction of the EMT secreting PDGF; once released, the latter acts in an autocrine fashion to stimu­ Cells of the EpRas line of transformed late the proliferation of these cancer cells via the PDGF receptors that they dis­ mouse mammary epithelial cells (see play. Finally, exposure of breast cancer cells to TGF-p causes them to release Figure 1420) were treated with TGF-~

other factors that accelerate the breakdown of mineralized bone-a critical step (2nd and 4th micrographs, upper row)

in the formation of osteolytic metastases; as we will learn later, this breakdown This ca used a suppression of E-cadherin expression (pink, 7st and 2ndliberates additional mitogens that drive cancer cell proliferation. micrographs, upper row) as well as an induction of vimentin (pink, 3rd and 4thTNF-cx, acting on its own or in concert with TGF-p, also appears to be an impor­micrographs, upper row). How ever,

tant agent for inducing the EMT. Early in tumor progression, TNF-cx is often pro­w hen a dominant-negative form of IKBcx,

duced by inflammatory cells, such as macrophages (see Section 11.16) . At this which blocks NF-KB signaling (see Figurestage, it functions via its receptor to activate the NF-KB signaling pathway in 6.29A), was expressed in these cells epithelial cells. TGF-p also activates the NF-KB pathway in epithelial cells, such (lower row), TGF-~ treatment failed to as the immortalized mouse mammary epithelial cells discussed above. In vari­ suppress E-cadherin expression (pink, ous tumors, TNF-cx and TGF-p may contribute, to differing extents, to the long­ 7st and 2nd micrographs) and failed to

term maintenance of active NF-KB signaling. This signaling seems to be critical induce vimentin expression (pink, 3rd and 4th micrographs). This indicated for the induction and maintenance of the EMT, as indicated by work with the that NF-KB signaling w as necessary forEpRas mouse mammary epithelial cells. Thus, blockage of NF-ICB signaling pre­induction of the EMT in these cell s. vents expression of their EMT program (Figure 14.21). Other experiments (not shown) indicated that constitutive activa tion of NF-KB

The influence of stromal macrophages on the invasive and metastatic behavior signaling w as sufficient to induce the of primary cancer cells can be demonstrated by studying genetically altered EMT in these cells . (From MA Huber, mice that lack the ability to make colony-stimulating factor-l (eSF-l). As was N. Azoitei, B. Baumann et al., I Clin. discussed in the last chapter, mammary carcinomas arising in cancer-prone Invest. 114:569-581, 2004.)

-TGF-p +TGF-p - TGF-p +TGF-p

control vector

DN IKBcx vector

611

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Chapter 14: Moving Out: Invasion and Metastasis

transgenic mice usually recruit large numbers of tumor-associated macro­phages (TAMs). However, when the tumor cells in such mice lack the ability to make CSF-l , TAMs are virtually absent (see Figure 13.24). The absence of CSF-l and TAMs has no effect on primary tumor growth (Figure 14.22A), but such tumors show a benign, noninvasive behavior, in contrast to tumors that succeed in recruiting TAMs (Figure 14.22B). The influence of these macrophages on metastatic behavior is striking: without TAMs, these breast tumors fail to seed metastases to the lungs (Figure 14.22C).

This experiment provides compelling evidence that the invasive and metastatic behavior of these mouse breast carcinoma cells is strongly influenced by signals that these carcinoma cells receive from stromal cells, in this case macro phages. It fails, however, to reveal the precise nature of these signals. Macrophage­derived TNF-cx, as argued above, is likely to contribute to induction of the EMT by cancer cells, and therefore to the invasive and metastatic behavior described in Figure 14.22. Another key macrophage-derived signal is likely to be conveyed by EGF.

Some of the evidence favoring EGF as a key inducer of cancer cell invasiveness comes from studies of mouse breast cancer cells both in vivo and in vitro. Like most epithelial cells, these carcinoma cells express the EGF receptor, and activa­tion of this receptor by EGF causes them to acquire both motility and invasiveness

(A) (B)

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Figure 14.22 Effects of macrophages on invasion and metastasis Mice of a transgenic strain develop mammary carcinomas through the expression of a transgene in which the MMTV promoter drives expression of the polyoma middle T (PyMT) oncogene. This transgene has been introduced, through breeding, into mice that can (Csf+IOP) or cannot (CsfOPIOP) make colony­stimulati ng factor-1 (CSF-1 ), which is needed to recruit macrophages to the tumor mass (see Figure 13.24). (A) The presence (in Csf+1op mice) or absence (in CSfOplop mice) of recruited tumor-associated macrophages (TAMs) has no effect on the ability of the primary breast tumors to grow in these transgenic mice. (B) Such mammary tumors arising in Csf+1op mice (whose tumors contain many TAMs, not shown) develop a highly invasive phenotype, in which individual carcinoma cells invade the nearby

stroma in large numbers (left panel, arrows). How ever, if tumors develop in CsfOplop mice, in which macrophages cannot be recruited into the tumor-associated stroma (right panel), the tumor cells do not break through the basement membrane and the tumor as a w hole remains encapsulated and benign, indicating that the macrophages contribute in essential ways to tumor invasiveness. (C ) In the Csf+1op mice, metastases in the lungs begin to appear at 18 weeks of age and increase progressively thereafter (blue bars), as gauged by the amount of polyomavirus middle T RNA (expressed in tumor cells) present in the lungs (ordinate). However, in the CsfOplop TAM-negative mice (orange bars), metastases are virtually absent. (From EY Lin , A. v. Nguyen, R.G . Russell and J.W Pollard, I Exp. Med 193:727-740, 2001 .)

612

Contributions of macrophages to invasiveness

(A) (B)recruited carcinoma

macro phages cells I

'" 1~-actin carcinoma cells ... .. _... .-.'.,.. ..,! .....

EGF receptor overlayed

} collagen

macrophages EGF

J"carcinoma cells . .\ CSF-l receptor + macrophages ta· ~'~: . . ... 1

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middle T antigen •

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blood vessel

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Figure 14.23 Reciprocal stimulation by breast cancer cells and where they remained. Similarly, macrophages (red) also remained macrophages A variety of experiments indicate that macrophages where they were initially placed at the bottom of the Petri dish are the major source of EGF in breast cancers. EGF is known to be below a collagen gel (middle image). However, when the two able to st imulate epithelial cancer cells to proliferate and invade populations were co-cultured at this location, the breast cancer cells through extracellular matrix. In addition, EGF exposure causes (green) were now induced to move upward and invade the breast cancer cells to release eSF-1, which allows them to recruit overlying collagen gel (lower image). (e) The reciprocal interactions macrophages and stimulates production by the macrophages of between breast cancer cells and macrophages are illustrated more EGF, resulting in a positive-feedback loop between these two schematically here. Because macrophages are often found in close cell types. (A) Using peR analysis, the mRNA levels of these two proximity to microvessels, the stimulation by tumor-associated growth factors and their receptors are found to be reciprocally macrophages (TAMs) of breast cancer cell motility and invasiveness expressed in mammary carcinoma cells arising in tumor-prone may also contribute to cancer cell intravasation, as depicted here . transgenic mice (see Figure 14.22) and in recruited stromal (A, from J. Wyckoff, W Wang, EY Lin et ai, Cancer Res. macrophages. (B) Breast cancer cells (labeled here w ith green 647022-7029, 2004; B, from S. Goswami, E. Sahai, LB. Wyckoff fluorescent protein, GFP) were placed at the bottom of a Petri dish et ai, Cancer Res 65:5278-5283, 2005; C. from W Wang, (seen here in side view) below a layer of collagen gel (top image), S Goswami, E. Sahai et ai, Trends Cell BioI. 15: 138-145, 2005.)

and to secrete eSF-I, the attractant and stimulant of macrophages (Figure 14.23). Macrophages respond to eSF-1 by proliferating and releasing EGF, which activates the cancer cells. These effects all proceed through paracrine rather than autocrine signaling, since the breast cancer cells do not express the eSF-1 reCeptor and the macrophages do not express the EGF receptor (see Figure 14.23A) . Accordingly, these two cell types collaborate by reciprocally stimulating one another, yielding another type of positive-feedback loop. While cancer cell motility and invasiveness are clearly demonstrated in these experiments, the

613

Chapter 14: Moving Out: Invasion and Metastasis

Figure 14.24 Cell scattering and invasive behavior induced by HGF Hepatocyte growth factor (HGF), also known as scatter factor (SF), is produced by a variety of stromal cell types. It has profound effects on epithelial cells that display its cognate receptor, Met. (A) Shown here are MDCK (Maden-Darby canine kidney) cells (red cytoplasms), which are used widely to study epithelial cell biology. The cells in the left panel were grow n in a normal medium, while those in the right panel were grown in medium to which HGF/SF was added. In monolayer culture, these epithelial cells normally form clusters of cobblestone-like cells . However, after treatment by HGF/SF, they become motile and scatter in many directions. (B) When introduced into collagen gels, MDCK cells normally form small spherical clumps (left panel). How ever, follow ing exposure to HGF/SF, these cells grow in long processes that invade the surrounding collagen gel (right panel). (A, courtesy of J.H. Resau; B, courtesy of U. Schaeper, from M. Rosario and W Birchmeier, Trends Cell Bioi. 13:328-335, 2003)

(A)

monolayer culture

!normal !+ HGF/SFmedium (B)

collagen gel

induction of an EMT in the cancer cells can only be inferred from their acquisi­tion of motile, invasive behavior. HGF, another ligand of stromal origin, is also capable of inducing many of the attributes of the EMT in epithelial cells, which generally display Met, its cognate receptor, on their surface (Figure 14.24).

These diverse lines of evidence strongly suggest that the acquisition of malig­nant traits by cancer cells, including induction of the EMT, is not governed solely by the genomes of these cells. Instead, these profound shifts in cell phenotype are often initiated by a collaboration between specific mutant alleles harbored in cancer cell genomes (e.g., a ras oncogene) and the signals that these cancer cells receive in some tissue micro environments, specifically at the boundary between a tumor epithelium and reactive stroma. In many tumors, these con­textual signals are conveyed by certain factors, such as TGF-~ and TNF-a, that are released by cells in the reactive stroma (Figure 14.25) . Yet other stromal sig­nals, such as those carried by EGF and HGF, may also help to elicit many of the changes that we associate with cancer cell invasiveness and the EMT. While this scenario depicts the behavior of many carcinomas, it does not accurately describe all of them (Sidebar 30 . ).

Throughout much of this text, our thinking has been driven by the notion that the phenotypes of cancer cells are dictated by their genotype and that tumori­genic grovvth is essentially a cell-autonomous phenomenon. Our encounters with heterotypic interactions (Chapter 13) revised this notion slightly, by indi­cating that cancer cells show a surprising degree of dependence on normal neighbors for various types of sustenance and support. Now, we must come to terms with the idea that the microenvironment of the cancer cell can also fun­damentally reshape that cell's phenotype, specifically by inducing the profound changes in cell behavior that comprise the EMT.

614

Induction of the EMT by embryonic TFs

Figure 14.25 Signals that trigger the STROMA EMT This diagram presents a highly

simp lified view of the signaling channels

inflammatory cells that originate in the stroma and fibroblasts l maCrO~hages myofibroblasts

~ - EGF ~ ~ TNF-aFGF, HGF, ~ - - TGF-~

TGF-a II

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'popto';' \ T PI3K -~ invasiveness

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Ras

t -nInIF ras ~ EPITHELIAL

.IUIUIoncogene~'" CELL

14.5 EMTs are programmed by transcription factors that orchestrate key steps of embryogenesis

Execution of the EMT program depends on changes in the expression of dozens, possibly hundreds of distinct genes. These changes affect many aspec ts of cell biology, not all of which have been enumerated here. The changes include the organization of a cell's intermediate filament cytoskeleton, its motility, its asso­ciation with neighboring cells, its release of proteases, and even its disp lay of cell surface integrins and growth factor receptors (see Table 14.2). \\bile exten­sive evidence implicates stromal signals as key elements in triggering the EMT of carcinoma cells, none of this evidence, on its own, reveals how the complex EMT program is actually coordinated within the responding epi thelial cells.

The genetics of early development has provided much of the answer to this question. A number of genes that specify pleiotropically acting, D1T-inducing transcription factors have been identified, largely in Drosophila melanogastel: Many of these genes and the transcription factors that they encode are con­served in chordates and have been found to control key steps in early embryo­genesis in frog and mouse embryos; these steps involve \'arious types of EMT. (The strong conservation of these genes indicates that the E\IT and key steps of early embryogenesis were developed early in metazoan e\'olution, long before the radiation of the various metazoan phyla. ) By activating these transcription factors, cancer cells gain access to the complex, multi -component EMT pro­grams that they orchestrate.

More than half a dozen such transcription factors have been described, each of which is capable of inducing an EMT when ectopically expressed in certain epithelial cells (Table 14.3). For example, Snail is a transcription factor that was first described in Drosophila (Figure 14.26). Since its initial description, Snail has been discovered in a wide range of metazoa, including vertebrates, insects, worms, and mollusks. In early vertebrate embryos, Snail is first expressed in the portion of the ectoderm that is destined to become mesoderm following gastru­lation. When operating during embryogenesis, Snail, Slug, and Twist convert

influence epithelial cancer cells to undergo a partial or complete EMT. It is likely that the EMT is normally triggered in response to a mixture of signals that ca rcinoma cells receive from the stroma together with intrace llular signals, such as those released by a (as oncogene, as indicated here. The precise identities of these stromal signals and their combinatorial mechanisms of action remain to be elucidated .

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Chapter 14: Moving Out: Invasion and Metastasis

Table 14.3 Transcription factors orchestrating the EMT

Name Where first identified Type of transcription factor Cancer association . .

E47/E2A associated with E-cadherin bHLH promoter

FOXC2 mesenchyme formation winged helixlforkhead basal-like breast cancer Goosecoid gastru~ation in frog paired homeodomain various carcinomas SIP1 neurogenesis 2-handed zinc finger/homeodomain ovarian, breast, liver carcinomas " Slug delamination of the neural crest C2H2-type zinc finger " breast cancer cell lines, melanoma

and early mesoderm in chicken Snail mesoderm induction in Drosophila; C2H2-type zincfinger invasive ductal carcinoma

neural crest migration in vertebrates Twist mesoderm induction in Drosophila; bHLH invasive lobular breast cancer,

" emigration from neural crest diffuse-type gastri.c carcinoma, high-grade melanoma and "neuroblastoma

epithelial cells into the migratory mesenchymal cells that form the mesoderm. Snail and its relative Slug are involved in yet other embryonic steps in which one type of tissue is transformed into another.

Moreover, when epithelial monolayers are wounded experimentally, Slug expression is induced in the surviving epithelial cells at the edge of the wound in order to enable these cells to acquire motility and migrate into the wound site (Figure 14.27). Expression of Slug helps to explain how epithelial cells at the edges of wound sites undergo a transient EMT in order to reconstruct epithelial cell sheets (see Figure 13.13C). Observations like these broaden our perspective on these transcription factors (TFs) and their normal biological roles: in addi­tion to programming key steps in early embryogenesis, the expression of some of these TFs may be resurrected transiently in adults in order to reconstruct damaged tissues.

Snail and Slug are members of the C2H2-type zinc fingerTFs. The Snail-SlugTFs seem to operate largely as repressors of transcription. Thus, both have been found to be able to repress transcription of the E-cadherin gene. As we read ear­lier, the loss of E-cadherin expression can, on its own, cause epithelial cells to assume many of the phenotypic changes associated with the EMT.

The Snail TF has been found to be expressed in the invasive fronts of chemically induced mouse skin carcinomas, and its expression is associated with the degree of lymph node metastasis of human breast cancers. Moreover, embry­onic expression of Snail, the related Slug TF, and Goosecoid is induced by con­textual signals, such as TGF-~ and Wnts, that are known to be responsible for inducing the EMT conversion of mouse tumor cells. Twist is expressed during the gastrulation of Drosophila embryos (see Figure 14.26B) and the out-migra­tion of neuroepithelial cells from the neural crest of chordate embryos. Its expression is also induced by exposure to TGF-~.

Rapidly accumulating evidence associates some of these transcription factors with various types of human malignancy. For example, Snail has been found to be expressed in islands of human mammary ductal carcinoma cells that lack E­cadherin expression. Slug has also been implicated in the repression of E-cad­herin expression in human breast cancers (Figure 14.28A) . Twist is found to be expressed at elevated levels in many invasive mammary lobular carcinomas and, to a much lesser extent, in invasive ductal carcinoma cells (Figure 14.28B). Similarly, Twist is expressed preferentially in the "diffuse" subtype of gastric car­cinomas and to a lesser extent in the "intestinal" subtype of gastric carcinomas, in which expression of Sip1-another EMT-inducing TF-is elevated (Figure 14.28C). And significantly, both Twist and Slug enable cells to resist apoptosis

616

Induction of the EMT by embryonic TFs

and anoikis, and therefore can protect metastasizing cells from some of the physiologic stresses that would normally cause their death long before they suc­ceed in reaching distant tissue sites and forming micro metastases.

These associations have not been studied systematically, and in any event, even when they are observed, they do not prove definitively that the various TFs are causally involved in programming the invasive and metastatic traits of human

(A) Snail (B) Twist (C) Slug

amphioxus Xenopus laevis

(F) SI Pl neural crest

Drosophila

(0) Goosecoid

(E) FOXC2 I --------------~

amphioxus mouse Xenopus

Figure 14.26 Embryonic transcription factors programming epithelial-mesenchymal transitions (A) The Snail transcription factor (TF), whICh can program the EMT, is shown being expressed in Amphioxus, a primitive chordate, in the cells (dark areas) that are counterparts of the cells in higher chordates that will form the neural crest. (B) The Twist TF, which also can program an EMT, is shown here (brown) in an early Drosophila embryo at the site of gastrulation, which it helps to orchestrate. (C) The Slug TF, a close relative of Snail, is also expressed in the embryonic neural crest. Here its expression is visualized (dark blue) in the neural crest of an embryo of Xenopus laevis, the African clawed toad . (D) The Goosecoid TF is expressed at the blastopore lip in gastrulating chordate embryos. Here its expression, which is inducible by the TGF-~ signaling pathway, is shown adjacent to the blastopore 8 hours after fertilization of an amphioxus egg. (E) The FOXC2 TF,

previously known as Mesenchyme Forkhead-l, is expressed in important mesodermal structures in this day 9.5 mouse embryo, including the mesoderm around the developing spinal column as well as the somites, which are precursors to many of the body's muscles. (F) In Xenopus laevis, expression of the SIP1 TF is see n in the neural crest and neural tube, where it appears to be responsible for the cell movements that lead to closure of the neural tube and emigration of cells from the neural crest to other parts of the body. (A, courtesy of J. Langeland; B, from M . Leptin, J. Casal, B. Grunewald and R. Reuter Dev. Suppl. 22-31, 1992. © The Company of Biologists; C. courtesy of C. LaBonne; D, from A.H. Neidert, G. Panopoulou and JA Langeland, Evo/. Dev. 2:303-310,2000; E, from T. Furumoto, N. Miura, T. Akasaka et al., Dev. Bioi. 210 15-29,1999; F, from L. van Grunsven, C. Papin, B. Avalosse et ai, Mech. Dev. 94189-193, 2000)

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Chapter 14: Moving Out: Invasion and Metastasis

24 hours 48 hours Figure 14.27 Transient expression of an EMT-inducing transcription factor in wound healing Expression of the Slug transcription factor is induced transiently in a monolayer of keratinocytes that have been wounded by scraping away a swath of cells. As seen here, 48 hours after wounding, keratinocytes at the edge of the wound induce expression of Slug (dark brown) as they separate from the monolayer and begin to make their way into the wound site (bottom of each panel) in order to reconstruct an intact monolayer. By 96 hours, most of these cells cease expressing Slug and become integrated into a continuous monolayer. (From P. Savagner, D.F Kusewitt, EA Carver et ai, I Cell Physioi. 202:858-866, 2004) 72 hours 96 hours

tumor cells. Still, it seems increasingly likely that once some of these TFs are expressed in cancer cells, they act singly or collaboratively to orchestrate the complex cellular changes associated with invasion and metastasis (Figure 14.29; see also Figure 14.15). Finally, we should note that the close parallels between embryonic EMTs and those contributing to cancer pathogenesis are extended by the similarities in the signaling pathways that are active in these two situa­tions (Figure 14.30).

These descriptions of the molecules that contribute in key ways to carcinoma cell invasiveness are reminiscent of our cliscussion' in the previous chapter about the activated stroma and its contributions to the tumorigenicity of carci­noma cells. In both places, for example, we encounter cadherins, the EMT, and TGF-~. Such connections hint at an interesting but still speculative idea: per­haps the formation of primary carcinomas and the acquisition of invasiveness are not as separate and distinct as most descriptions of cancer would suggest. Although it is convenient to place them in separate conceptual boxes, the bio­logical reality may be quite different. Quite possibly, cancer cell invasiveness is a natural extension-an exaggerated form-ofthe cell transformation processes that lead initially to the formation of many types of primary tumors. Because transformation and invasiveness depend on many of the same regulatory cir­cuits and effector proteins, they may lie on a continuum in which one process blends seamlessly into the next.

To summarize, the discovery of the EMT-inducing TFs suggests at least three important ideas about malignant progression. First, many malignant cell pheno­

618

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types may be induced by non-genetic changes-heterotypic signals of stromal origin-rather than genetic changes occurring within carcinoma cells. Second, cancer cells do not need to cobble together all of the phenotypes associated with highly malignant cells; instead, a multitude of malignancy-associated traits may be acquired concomitantly because these TFs, once expressed, act in a highly pleiotropic fashion. Third, because expression of these TFs and the resulting EMT is dependent on heterotypic signaling from the stroma of the primary tumors, carcinoma cells may revert from the mesenchymal state to an epithelial state once they have left the primary tumor and landed in new stromal microen­vironments, such as those present in sites of metastasis.

Figure 14.28 Expression of EMT­inducing embryonic transcription factors in human tumors Still­fragmentary evidence implicates a number of embryonic transcription factors (TFs) in the induction of the EMT (and associated loss of E-cadherin) in human cancer cells. (A) This analysis of the mRNA transcripts expressed in a panel of human breast cancer lines indicates that Slug is present in tumor cell lines lacking E-cadherin expression; conversely, E-cadherin is generally present in tumor cell lines lacking significant expression of this TF. These inverse patterns of expression are likely due to the ability of Slug to bind an E-box sequence in the E-cadherin promoter and to potently repress transcription of this key epithelial gene. (B) Expression array analysis demonstrates that Twist expression varies inversely with E-cadherin expression in these human breast carcinomas. Since Twist can repress expression of the encoding CDHI gene 250-fold in cultured cells, Twist is likely to be responsible for the loss of the E-cadherin mRNA, which is lost preferentially in the more aggressive invasive lobular carcinomas of the breast but is present at significant levels in the less aggressive invasive ductal carcinomas. (C) Analysis of mRNA levels of three EMT-inducing TFs-SIP1, Snail, and Twist-reveals that in the "diffuse" subtype of gastric cancer, expression of Snail and Twist is significantly elevated, as is expression of N-cadherin, a mesenchymal marker. In contrast, in the" intestinal" subtype of gastric cancer (which is very different in its etiology and biological behavior), SIP1 expression is elevated and the mRNAs encoding the other TFs are either repressed or expressed at the basal level seen in the normal gastric mucosa. Observations like these indicate that EMT-inducing TFs act in various combinations in different types of human cancers to program invasive growth. Note the logarithmic scale of the ordinate. (A, from K.M. Hajra, DY Chen and E.R. Fearon, Cancer Res. 62:1613-1618,2002; B, from J. Yang, SA Mani, H. Donaher et ai, Cell 117:927-939, 2004; C. from E. Rosivatz, I. Becker, K. Specht et ai, Am. I Pathol. 161:1881-1891,2002)

619

Twist expression in melanomas

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Chapter 14: Moving Out: Invasion and Metastasis

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Figure 14.30 Similarities between Ras EMT signaling during embryogenesis and tumor progression The signal t ransduction cascades that are ! responsible for activating the 8epithelial-mesenchymal transition (EMT) in a rat bladder carcinoma model (left) and during gastrulat ion ea rly in mouse ! embryogenesis (right) have st riking ~ para llels. These similar ities provide fu rther support for the notion that the EMT program expressed by invasive 1 carcinoma cells represents a reactivation of latent cell-biological programs, many of which are normally active in early mammal ian embryonic development. (From J P Thi ery, Nat. Rev. Cancer 1

EMT2442-454,2002.)

Figure 14.29 Embryonic transcr iption factors and tumor progression The evidence that the various embryonic transcription factors act to cause malignant progression in humans is st ili fragmentary and indirect. (A) Mouse 4T1 breast cancer cells, when implanted subcutaneously in a mouse host, generate large numbers of metastases in the lungs (purple bar, far right). This number is unaffected when these cancer ce lls are infected by a control retrovirus vector (blue bars) Ho ever, when the cancer cells were deprived of Twist expression through infection with an siRNA­expressing viral vector (which causes degradation of Twist mRNA), growth of the resulting prima tumors was unaffected, but the number of metastases that the generated was strongly (-85%)

reduced (red bars). Significantly, those few metastases that did form (red bars) ali continued to express high levels of Twist, indicating that they derived from primary tumor ce lls in w hich Twist mRNA levels had never been properly suppressed. Each bar represents one tumor-bearing mouse. Each bar represents the metastases counted in a single tumor-bearing mouse. (B) Twist expression was gauged in a large group of melanomas by immunohistochemistry As seen in this Kaplan-Meier plot, those patients whose tumors expressed elevated levels of Twist fared far worse than those whose tumors showed low, basal levels of Twist. (A. from J. Yang, SA Mani, J.L. Donaher et al., Cell 117:927-939, 2004; B, from K. Hoek, D.L. Rimm, K.R. Williams et ai, Cancer Res. 645270-5282, 2004.)

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MMPs and cancer cell invasiveness

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Figure 14.31 Matrix metalloproteinases produced by tumor-associated cells MMPs are produced large ly by inflammatory cells and fibroblasts in the stroma. (A) In this mammary carcinoma arising in a tran sgenic MMTV-polyoma middle T mouse, the presen ce of MMPs ca n be detected by their ab ility to cleave a synthetic substrate, which releases a polycationi c fluorescent tag that migrates into nearby cells, generating a fluorescent signal. Th e tumor stained w ith hematoxylin-eosin (left pane!) is seen to generate a halo of proteolysis (right pane!), suggesting the involvement of surrounding stroma l cells in protease production. (B) The abil ity of tumors to degrade collagen IV, a major component of the basement membrane (see Figure 13.5), can be measured by generating a modif ied col lagen IV substrate that creates a fluorescent green color upon cleavage. In this experiment, both human mammary carc inoma cells (not seen) and human mammary fibroblasts (red) showed relatively weak ability to degrade the collagen IV substrate. How ever, w hen these tw o ce ll populations were co-cultured, regions of collagen IV cleavage (green) were evident, often in areas where fibroblasts w ere also present (yellow: overlap of green and red). This cleavage was essentially el iminated in the presence of MMP inhibitors. (C) An even more important source of MMPs is t he populations of macrophages (M<I>s) that are recruited into the tumor stroma . In this in vitro culture experiment, the presence of MMP-2 w as measured in the culture medium of M0S that w ere either cultured alone (right) or co-cultured in the presence of two human breast cancer celilines-MCF7 or SK-BR-3 (left, middle). Neither o f these cancer cell types made signif icant levels of MMP-2 on its own, but in the presence of M<I>s, both caused the M<I>s to increase MMP-2 production 4- to 5-fold; the increase cou ld be traced to the induction of MM P-2 mRNA expression by the M<I>s (not shown) The released MMP-2 imparted increased invasiveness to these breast cancer cells (not shown). (A, courtesy of E. Olson, T Jiang, L Ellies, and R. Tsien; B, from M. Sameni , J. Dosescu, K. Moin and B.F Sloane Mol. Imaging 2: 159-175, 2003; C, from T Hagemann , S.CRobinson, M. Schu lz et ai, Carcinogenesis 25: 1543-1549, 2004)

14.6 Extracellular proteases play key roles in invasiveness

The epithelial-mesenchymal transition (EMT) represents a complex biological program that enables cancer cells to acquire the attributes of invasiveness and motility. In order to properly appreciate the processes that together constitute the EMT, we need to examine the roles of some of its key effectors-the proteins that work to create the phenotypes associated with the EMT. To begin, we exam­ine the most obvious trait of malignant cells-their ability to invade adjacent cell layers. This burrowing requires that cancer cells remodel the nearby tissue environment by excavating passageways through the extracellular matrix (ECM) and pushing aside any cells that stand in their path.

The most important effectors of these complex changes are the matrix metallo­proteinases (MMPs; see Table 13.1). In carcinomas, the great bulk of these pro­teases are secreted by recruited stromal cells, notably macrophages, mast celis, and fibroblasts, rather than by the neoplastic epithelial cells (Figure 14.31). By

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621

Chapter 14: Moving Out: Invasion and Metastasis

dissolving the dense thickets of ECM molecules that surround and confine indi­vidual cells within tissues, MMPs create spaces for these cells to move. Included among the ECM components that are cleaved by MMPs are fibronectin , tenascin, laminin, collagens, and proteoglycans. During the course of degrading ECM components, MMPs also mobilize and activate certain growth factors that have been tethered in inactive form to the ECM or to the surfaces of cells.

MTl-MMP (membrane type-1 MMP) is one of six MMP types that are mem­brane-anchored and therefore limited to cleaving substrate proteins in the immediate vicinity of the cells that have produced them. Of the 187 distinct metalloproteinases known to be encoded in the human genome, 28 are secreted MMPs. In contrast to MTl-MMP, which acts as a plasma membrane- bound enzyme, most of the MMPs function as soluble enzymes in the spaces between cells. MTl-MMP may attack and cleave cell surface adhesion molecules, such as cadherins and integrins, as well as growth factor receptors and chemokines. It can also cleave inactive pro-enzymes, such as pro-MMP-2, into enzymatically active MMPs.

These secreted proteases clearly play important roles in normal cell survival and proliferation. After all, each time a cell within a normal tissue goes through a cycle of growth and division, space within the ECM must be carved out for its future daughters, and once these two are formed, each of these cells must, in turn, reconstruct new ECM around itself. Hence, the remodeling of the ECM takes place continuously in mitotically active tissues. Consequently, rather than being aberrations of invasive cancer cells, the activities of MMPs and other extracellular proteases, such as urokinase plasminogen activator (uPA) , accom­pany and participate in normal cell proliferation. Of relevance here are clinical trials of certain MMP-inhibitory drugs, which have been terminated due to the effects of these inhibitors on a variety of normal tissues; because these agents suppress the normal turnover of cartilage and other joint components, they cre­ated unacceptable levels of joint stiffness and pain.

Each type of MMP usuaJly acts on a well-defined set of substrates (see Table 13.1), doing so in a highly regulated and localized fashion. It is likely that these enzymes continue to show such substrate specificity during the process of can­cer cell invasion. However, in this instance, the proteolysis se~ms to proceed continuously rather than in the brief spurts that accompany normal cell growth and division.

One of the consequences of the EMT programmed by several well-studied embryonic transcription factors (Section 14.5) is the induced synthesis and release by carcinoma cells of MMPs, notably MMP-2 and -9. However, it is clear that the bulk of the MMPs found in tumors originate in the stroma. For example, the best-studied of the matrix metalloproteinases, MMP-9, is expressed largely by macrophages (Section 13.5), neutrophils, and fibroblasts at the invasive fronts of tumors. MMP-9 expression at these fronts correlates positively with the metastatic ability of a primary tumor, suggesting that MMPs like this one can act at several stages of the invasion-metastasis cascade, including local invasion of the primary tumor stroma, intravasation, and extravasation. In vitro assays indi­cate that MMP-9 can degrade collagens that are prominent components of the EG.! including basement membranes, specifically collagen types IV; V, XI, and XIV Other targets of MMP-9 include laminin (another important constituent of the basement membrane; see Figure 13.5), chemokines, fibrinogen, and latent TGF-~. In the case ofthe latter two, cleavage by MMPs converts them from latent into activated forms.

These widely ranging functions of MMPs indicate that their enzymatic activity must be tightly controlled, at least in normal tissues. Reflecting this requirement is the fact that the soluble MMPs, such as MMP-9, are all initially synthesized as inactive pro-enzymes that can only fu nction, like the caspases (Section 9.13),

622

MMPs and cancer cell invasiveness

following activation by other proteases. Negative regulation is also provided by a class of proteins termed tissue inhibitors of metalloproteinases (TIMPs)' which bind MMPs and put them in an inactive configuration (Section 13.9). Moreover, the activities of some of these extracellular proteases seem to be fur­ther confined through their concentration at discrete foci, termed podosomes, where membrane-tethered MMPs, notably MT1-MMp, are active in creating highly localized areas of proteolysis (Figure 14.32).

While MMPs have been depicted as the direct effectors of specific steps in inva­sion and metastasis, it is clear that the deregulation of MMPs can, through unknown mechanisms, drive the progression of cells through all of the stages of multi-step tumorigenesis including completion of the invasion-metastasis cas­cade. Thus, when expression of MMP-3 (also known as stromelysin-1) is forced in the mammary gland of transgenic mice, these mice initially develop mam­mary hyperplasias (Figure 14.33). Some ofthese growths progress to carcinomas that eventually become invasive and metastatic. These mice reveal how critical the regulation of MMP action is and why it must be kept under control in nor­mal tissues.

The complexity of the regulatory network governing MMP activation is further illustrated by the behavior of urokinase plasminogen activator (uPA) , a non­MMP protease. uPA is secreted by stromal cells as an inactive pro-enzyme. This form of uPA proceeds to bind to its own cell surface receptor (termed uPAR), which is displayed by epithelial cells, including malignant ones, and thereby becomes catalytically active (Figure 14.34). The tethered, activated uPA can then be wielded by the epithelial cells to cleave a variety of extracellular substrates in their immediate vicinity. Prominent among these are a series of pro-enzymes of other extracellular proteases that are activated by this cleavage. For example, by cleaving plasminogen into plasmin, uPA creates an activated protease that, in turn, proceeds to cleave and thereby activate the pro-enzyme forms of yet other extracellular proteases, notably, the matrix metalloproteinase (MMP) types 1,2, 3,9, and 14. Alternatively, uPA may cleave and activate some MMPs directly. Not surprisingly, inhibitors of the uPA-uPAR complex have been found to block both tumor growth and metastasis in animal models of cancer pathogenesis. Moreover, high levels of solubilized uPAR in the serum represent a bad progno­sis for cancer patients.

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Figure 14.32 Podosomes and focal degradation of the extracellular matrix Podosomes are small, focal protrusions from the cell surface that are displayed by many cell types and used by these cells to degrade highly localized areas of extracellular matrix (ECM) in their immediate vicinity. Podosomes are thought to be used by invasive cancer cells as well, to direct controlled degradation of the ECM near the leading edge of an invading cell; when operat ing in this context, they are sometimes called" invadosomes." Here, cells were stained for actin fibers with the dye phalloidin (red, left micrograph), revealing a number of discrete clusters of actin that are associated with the podosomes, which are located on the ventral (underside) surface of these src-transformed rat fibroblasts. These cells have been growing on a layer of the ECM protein fibronectin coupled to the FITC dye (green, middle), revealing that directly below the podosomes, discrete holes (black) have been eroded in the fibronectin sheet, ostensibly by the actions of podosome-associated proteases, such as MT1-MMP. Direct overlap between the actin clusters of the podosomes and the eroded holes in the fibronectin are indicated by white arrowheads in the right panel. (From K. Mizutani, H. Miki, H. He et ai, Cancer Res. 62:669-674, 2002)

Figure 14.33 Ectopic expression of MMP-3 and l11ammary tumor progression The normal mouse mammary gland (left panel) is composed of resting ducts (purple) and abundant adipose tissue (white, lipid-filled cells), as well as collagen (light blue) However, w hen the gene encoding MMP-3 (also known as stromelysin-1 ) is expressed constitutively as a transgene that directs its expression to the mammary epithelium, the mice develop abundant hyperplasia (right panel), including extensive islands of hyperplastic epithelial cells (purple) forming ducts, as well as a fibrotic, collagen-rich stroma (light blue) and abnormal adipocytes (white oval structures, lower right) . Many of these areas subsequently progress to invasive, metastatic tumors (not shown) (From M.D. Stern licht, A. Lochter, c.J. Sympson et ai, Cell 98: 137- 146, 1999)

623

• • ••• • ••

ca ncer cell

'

Chapter 14: Moving Out: Invasion and Metastasis

stromal cellFigure 14.34 uPA, uPAR, and the activation of extracellular proteins The inactive, pro-enzyme form of urokinase plasminogen activator (pro-uPA) is released by stromal cells. Once released, it binds to its cognate receptor, uPAR, w hich is displayed on the surface of epithelial cells; this binding converts uPA into a catalytically active protease. Active, receptor-bound uPA can then convert inactive, soluble plasminogen to the active plasmin form; the latter functions as a protease to cleave pro-enzyme forms of matrix metalloproteinases (pro-MM Ps) into active MMPs and latent TGF-~1 into its act ive form . At the same time, there is evidence that uPA can also act directly on pro-MMPs to convert them into active proteases. (From F. Blasi and P. Carmeliet, Nat. Rev. Mol. Cell Bioi. 3:932-943, 2002.)

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These brief vignettes of protease and their contributions to cancer cell inva­siveness describe only mall pans oh\"hat are surely highly complex networks of interacting proteases and ub crates. The total number of proteases made by mammalian cells is vast and rh'aJ the number of proteins that form the highly complex intracellular signal -proce ing circuits described in Chapter 6. To date, the actions of only a small proportion of these enzymes have been studied in the context of cancer pathogene i idebar 31 0 ).

14.7 Small Ras-like GTPa es control cellular processes including adhesion, cell shape, and cell motility

The actions of extracellular proteases, notably the MMPs, explain at the bio­chemical level how paths are cleared for the advance of invasive cancer cells through the extracellular matrix and thus through tissues.They fail, however, to tell us how individual cancer cells take advantage of these cleared paths to move ahead-the trait of cell motility. The motile behavior of cells has been studied extensively with cultured cells, and it is presumed that their crawling on solid substrates in vitro reflects the in vivo behavior of cancer cells as they invade nearby cell layers and intravasate. Such motility is also presumed to be impor­tant for cancer cells' escape from blood vessels or lymph ducts-the process of extravasation.

624

Cancer cell invasiveness and motility

Motile behavior can be induced in cultured cells by exposing them to a variety of growth factors. (The ability of these factors to induce such locomotion is sometimes recognized by designating them as being motogenic in addition to being mitogenic.) In the case of epithelial cells, the best inducer of motility is usually hepatocyte growth factor (HGF); this protein is also called scatter factor (SF) in recognition of its ability to induce multidirectional movement of cells in monolayer culture. Most and perhaps all types of epithelial cells express Met, the receptor for HGF, and many of these cell types have been found to acquire motility in response to HGF treatment (see Figure 14.24A). Similarly, EGF is clearly able to induce motility of breast cancer cells (see Figure 14.23B) .

The cellular machinery that responds to motogenic signals and operates as the engine of motility is extraordinarily complex at the molecular level. Cell motility involves continuous restructuring of the actin cytoskeleton in different parts of a cell (Figure 14.35), as well as the making and breaking of attachments between the migrating cell and the e:>.1:racellular matrix (ECM). (In the case of cultured cells, the ECM in question is the network of proteins that has previously been laid down by these cells on the surface of the Petri dish.)

The process of cellular movement can be broken down into several distinct steps. To begin, a cell will extend its cytoplasm in the direction of intended movement. This extension involves the protrusion from the cell surface of lamellipodia-broad, flat, sheetlike structures that may be tens of microns in width but only 0.1 to 0.2!lm thick (Figure 14.36). At the same time, cell surface proteases, such as those described earlier, are used to selectively degrade ECM proteins that stand in the way of the "leading edge" of the migrating cell. \l\Ihile this is going on, the cell deploys integrins to construct new points of attachment between the lamellipodia and the ECM at its leading edge and breaks such adhesions at its "trailing edge," thereby liberating cytoplasm and plasma mem­brane for redeployment to the leading edge.

Protruding from the lamellipodia are spikelike structures termed filopodia that are thought to enable an advancing cell to explore the territory that lies ahead (Figure 14.37). Like the lamellipodia, these are assembled through the

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625

Chapter 14: Moving Out: Invasion and Metastasis

(A) (B)

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Figure 14.36 Lamellipodia (A) This scanning electron micrograph (SEM) of a spontaneously transformed rat liver cell shows the elaborate ruffles-Iamellipodia-that migrating cells extend at their leading edge during forward locomotion (arrow) Lame ll ipodia are presumed to playa key role in the advance of invasive cancer ce lls in vivo, but such a role has not yet been directly demonstrated. Lamellipodia that are extended on the leading edge of the ce ll but fail to attach to the substrate seem to be swept back as ru ffl es along the dorsal (top) side of the cell; their component parts are then reintegrated into the larger plasma membrane and cytoskeleton. (B) This fluorescence micrograph image shows a lamellipodium being extended by a fibroblast in anticipation of locomotion. As seen here, the actin fibers are labeled with phalloidin (red), while the Ena protein, which programs the advance of the lamellipodium by organizing its focal adhesions and its outer edge, is labeled here with green fluorescent protein (GFP), to which it has been fused. (e) Phase microscopy of a fish keratocyte, studied because of its prominent lamellipodia, indicates the presence of substantial actin in this protrusion (cyan, top left figure). Electron microscopy of such a cell (top right image) and its cytoskeleton (bottom image) at much higher magnification indicates a densely

woven network of actin filaments that are organized in order to extend the lamellipodium in the direction of cell movement. (D) Lamellipodium formation and resulting cell motility are strongly stimulated by a number of growth factors and their cognate tyrosine kinase receptors. Seen here are the effects of adding heregulin, a ligand of the erbB2/erbB3 family of receptors, to a human breast cancer cell. An untreated cancer cell is to the left, while a cell exposed to heregulin for 20 minutes is to the right. The actin cytoskeletons have been stained green (using phalloidin coupled to a fluor), while the nuclei are stained blue. (Because the heregulin is present uniformly in the surrounding medium, this cell has been induced to develop a lamellipodium that faces in all diretions rather than toward a single, localized source of this motogen.) Elevated signaling by erbB2 (= HER2/Neu) is correlated with increased metastatic progression of human breast cancer cells, which may be explained in part by the receptor-mediated induction of lamellipodium formation and associated cell motility. (A, courtesy of Julian Heath; B, from J.J. Loureiro, DA Rubinson, JE. Bear et al., Mol. Bioi. Cell 13:2533-2546, 2002; C, from 1M. Svitkina, ABVerkhovsky, K.M. McQuade and G.G. Borisy J. Cell Bioi. 139397-415, 1997; D, courtesy of A Badache and N.E. Hynes.)

626

Cancer cell invasiveness and motility

(A) (8)

(C)

reorganization of actin fibers, in this case fibers that are tightly bundled together beneath the plasma membrane of each filopodium. The precise role played by filopodia in cancer cell invasiveness remains to be elucidated.

The detailed management of cell shape and motility is under the control of members of a group of Ras-related proteins belonging to the Rho family. As dis­cussed briefly in Chapter 6, the Rho proteins, like Ras, operate as binary switches, being in a functionally active state while binding GTP and in an inac­tive state once they hydrolyze their bound GTP to GDP. More than 20 members of the Rho family of proteins have been discovered in human cells. They are divided into three subfamilies-the Rho proteins proper, the Rac proteins, and Cdc42. Like the Ras proteins, most members of the Rho protein family bear lipid groups at their C-termini that enable anchoring to intracellular membranes. Each of these has specialized functions in reorganizing cell shape and enabling cell motility (Figure 14.38).

Figure 14.38 actually misrepresents the actions of these various Rho-like pro­teins in one important respect: it implies that each of them acts globally throughout the cell to organize certain changes in the configuration of the actin cytoskeleton. In reality, they act quite differently. The complex program of cell motility depends on the localized activation of each of these proteins in very

Figure 14.37 Filopodia (A) Filopodia (orange spikes) extend from the cell surface and are presumed to allow ar invading cell to explore its extracellular environment. In this case the actin fibers that form within filopodia have been induced to assemble through expression of a Cdc42-interacting protein termed Zizimin. (B) The leading edges of lamellipodia are often interspersed with filopodia, which serve, in some poorly understood fashion, as sensors for the advancing cell. The cell shown here has extended lamellipodia and filopodia in all directions and therefore does not have a "leading edge." (C) The actin fibers within a filopodium are bundled tightly together and extend forward in the direction of cell migration in order to enable protrusion of the filopodium from the leading edge of a lamellipodium. (A, courtesy of W.B. Kiosses and MA Schwartz; B, courtesy of M. Cayer, L. Lim and C.A. Heckman; C, courtesy of E. Bulanova; see also TM, Svitkina, EA Bulanova, OY Chaga et al., J. Cell Bioi. 160409-421, 2003.)

627

Chapter 14: Moving Out: Invasion and Metastasis

actin staining vincul in stain ing actin sta ining vinculin staining

(A) QUIESCENT CELLS (8) Rho ACTIVATION

(C) Rac ACTIVATION

Figure 14.38 Effects of Rho-like proteins on the actin cytoskeleton and cell adhesion Members of the Rho family of sma ll GTPases, wh ich consists of the Rho, Rac, and Cdc42 subfamilies, control both the actin cytoske leton and the formation of focal adhesions, seen here through staining with an antibody reacti ve with vinculin, one of the proteins that link integrins at focal adhesions to this cytoskeleton (see Figure 528) The actin fibers were labeled by fluorescent phalloidin, a compound w ith high affinity for actin . (A) Quiescent, serum-starved 3T3 fibroblasts, w hich serve as controls for the panels that follow. (8) Exposure of cells to lysophosphatidic acid, wh ich specifically activates Rho subfamily proteins, causes a cell to construct large numbers of stress fibers and focal adhesions. (C) Micro-injection of a constitutively activa ted form of a Rac protein into a cell causes it to construct a single enormous lamellipod ium around its entire circumference. (In contrast, a focal source of a Rac-activating signal is likely to induce lamellipodia only on t he side of the cell facing this source .) (D) Micro-injection of a guanine nucleotide exchange factor (GEF) of Cdc42 into these cells causes a ce ll to extend hundreds of filopodia in all directions. (From A. Hall, Science 279 509-514, 1998 )

(0) Cdc42 ACTIVATION

small domain- of the cytoplasm, which in turn enables the cell as a whole to mm'e in one direction or another. The alternative-global activation-would lead ro anempt by a cell to move simultaneously in all directions, a scenario sugge red b\' me lamellipodia of Figures 14.36D and 14.38C, which form a con­tinuou rino around the entire perimeter of the cytoplasm, (The global activa­tion of Rae functio n in this cell is an artifact of introducing mutant, constitu­ti\'ely acti\-ared Rae protein into the cell by micro-injection.)

GrO\nh facror acti\'atlon of tyrosine kinase receptors leads to the activation of many rnembe of the Rho family of G proteins (which includes proteins belonging to three subfamilie Rho proper, Rac and Cdc42 proteins), For example, treatment of culrured. fibroblasts with platelet-derived growth factor (PDGF), a potent mito­gen for these cells. activates a number of Rho proteins and stimulates these fibrob­lasts to rno 'e aero s the bottom of a Petri dish. Alternatively, when fibroblasts are placed in three-dimensional culture by being suspended in a collagen gel, PDGF induces them to in 'ade through this gel. All three subfamilies of Rho proteins appear to contribute to this invasion, while only Rac may be needed for the move­ment offibrobl ts across a solid substrate in culture. These various behaviors also illusrrate an imponant distinction between the Ras proteins and their distant Rho family Cali ins: in cancer celis, a Ras protein is often activated by alterations in its structure (more pecificaliy, amino acid substitutions), while the various Rho pro­teins are functionally activated by their upstream physiologic regulators,

All of the signaling connections between the PDGF receptor and these Rho fam­ily proreins are nor known , However, it is clear that this receptor, by activating Ras, stimulates at lea t three dovmstream signaling pathways involving the Raf, Ral-GEF, and PI3K (phosphatidylinositol-3 kinase) effectors (see Sections 6.5, 6.6, and 6.7), In addition, activated Ras binds and appears to activate Tiam1, which functions as a guanine nucleotide exchange factor (GEF) for Rac. (Recall that GEFs are responsible for causing small G proteins, such as Ras and Rho, to jettison bound GDP and take on GTP, thereby activating signaling by these G proteins,) Hence, Tiam1 should also be considered to be another effector ofRas.

In fact, from the perspective of celi motility, PI3K is clearly the most important of the Ras effectors, By generating PIP3 [phosphatidylinositol (3,4,5) triphosphate],

628

Cell motility controlled by small GTPases

the PI3K enzyme creates a chemical structure on the cytoplasmic face of the plasma membrane to which a variety of cytosolic proteins can attach via their PH domains (Section 6.6). Among these proteins are a number of guanine nucleotide exchange factors (GEFs) that are responsible for activating members of the Rho family of G proteins. These Rho-GEFs become activated following their tethering to the plasma membrane.

(The overarching role of PI3K and PIP3 in choreographing cell motility is illus­trated by studies of the motility of the slime mold Dictyostelium discoideum. PI3K, and thus its product, PIP3, is localized at the leading edge of an advancing slime mold cell. Conversely, PTEN, the enzyme that destroys PIP3 and thereby antagonizes PI3K, is localized to the sides-and the rear, lagging edge of such a cell. This introduces another element into our thinking: while growth factor receptors, such as the PDGF-R, may release signals encouraging PI3K activation throughout the cell, the actual signaling by this enzyme may also be influenced by its localization within a cell.)

Tiam1 was originally identified as the product of a T-cell lymphoma invasion and metastasis gene, indicating the importance of its encoded protein to these late steps of tumor progression. Tiam1 function appears to be stimulated both by its association with GTP-bound, active Ras and by its binding to PIP3. By acti­vating Rac proteins, the Tiam1 GEF encourages the localized polymerization of actin at the leading edge of migrating cells, thereby yielding the lamellipodia that are so critical to cell locomotion (see Figures 14.36C and 14.38C).

The other Rho-like proteins that are activated by Rho-GEFs are responsible fo r a number of distinct components of the cell motility program. For example, Rho proteins like RhoA and RhoB, acting in concert with Rac proteins, promote the establishment of new points of adhesion between the leading edge of the cell and the extracellular matrix. The reverse is also true: the forging of new focal adhesions (see Figure 14.38B) also encourages Rac activation, suggesting the operation of some self-sustaining, positive-feedback loop that ensures the con­tinuity of forward motion. Rac and Cdc42 proteins also appear able to induce expression of certain secreted proteases, notably the matrix metalloproteinases described in the last section. By doing so, they may coordinate localized remod ­eling of the extracellular matrix with extension of lamellipodia at the leading edge of a motile cell.

The contraction of the cell body (which helps to pull the lagging edge of the cell forward toward the leading edge) is equally important for a cell's directed move­ment. This contraction is also governed largely by members of the Rho subfam­ily of proteins. By encouraging the formation of actin bundles in the cytoplasm, Rho proteins are able to create the structures known as "stress fibers" (see Figures 14.35 and 14.38B) and thus contribute to the regulation of the contractility of the cytoplasm. As many as 22 distinct guanine nucleotide exchange factors (GEFs) for the Rho subfamily of proteins have been discovered. Much of this complexity is likely attributable to the need to activate certain Rho proteins in micro do mains of the cytoplasm in response to specific cell-physiologic signals. Stated differ­ently, the activation of these Rho proteins needs to be tightly coordinated in time and space in order for the cell motility program to perform properly.

Cdc42, which represents the third subfamily of Rho-like proteins, has its own specialized function: it is able to induce the extension of the finger-like filopo­dia (see Figures 14.37 and 14.38D) . Precisely how filopodia contribute to cell motility remains unclear. In addition, activated Cdc42 is able to stimulate gen­eralized cell motility, independent of its specific effects on filopodia.

To complicate things even more, the actions of Rho, Rac, and Cdc42 differ in dif­ferent cell types. For example, in normal epithelial cells (rather than the fibro­blasts discussed above), the Rac and Rho subfamily proteins are responsible for

629

Chapter 14: Moving Out: Invasion and Metastasis

Figure 14.39 The circuitry mediating EGF-induced cell motility Many of the signal-transducing proteins that lie downstream of the EGF receptor and are responsible for mediating EGF-induced cell motility are indicated here. The relative proportions of these proteins change as breast cancer cells acquire increased motility and invasiveness: the extent of overexpression of certain proteins in the cancer cells is indicated by the pink numbers, w hile the extent of reduced expression is indicated by the green number. Some proteins, such as cofilin, sever existing actin fibers in certain regions of the cell, while capping protein prevents further extension of existing fibers. The actions of some of these signaling proteins are discussed in Section 6.6 . (From W Wang, S. Goswami, E. Sahai et ai, Trends Cell BioI. 15138-145, 2005)

++EGF '0"'''

~ EGF-R + EGF

'-------"PLC-y --_a PI(4,5)P2 -. DAG

2X RhoA •• ______ 2: 1:0 _ 3X ROCK _ + 3X LlMK COfjilin ) + IP3

N-WASP..-- 4X Cdc42 ~+ SSH cofilinT

I Rae - PAKl 3X PKG~ )

j 3X Pl14.5lK '-TIiC ZBPl

I 6X Arp213 PI(4,5)P2 cofilin (total) 2X -lOx complex ! J

5X npp;cg pmte;c I- 4X MENA

~-actin mRNA ba b± ends - F-actin severing

targeting ~ ~

L dendritic ··----G-actin nucleation

chemotaxIs ~toward EGF

maintaining the E-cadbenn-dependent cell-cell adherens junctions; as we have read, these junctions are \i tal for preserving the epithelial cell sheet and there­fore immobilize participating epithelial cells. However, in transformed epithelial cells, such as colon carcinoma cells that have undergone a partial or complete EMT, Rac clearly contribures to increased motility. In nonmotile cells, Tiam-1 (the Rac exchange facmr) i found in these adherens junctions, while in migrat­ing cells, Tiam-110calize to lameUipodia and related membrane ruffles.

The task of integrating these disparate observations into a single scheme has only begun. An initial anempt is seen in Figure 14.39, which depicts part of the signaling circuitry tha t lie dovmstream of the EGF receptor and is responsible for coordinating EGF-stimulated cell motility. Virtually all of the sub circuits depicted in this scheme are likely to participate in organizing the motility stim­ulated by other motogenic growth factors, such as HGF and PDGF, as well.

The relevance of the Rho family proteins to cancer metastasis has been high­lighted by searches for genes that are specifically expressed in metastatic cells but are expressed to a much lesser extent in nonmetastatic cells. In one set of experiments, strongly metastatic variants of mouse and human melanoma tumor cell lines were selected and the gene expression patterns of the cell lines were then compared with weakly metastatic cells. Prominent among the genes whose expression was elevated in the metastatic variants was the gene encoding the RhoC protein (one of the Rho subfamily). Indeed, introduction of a RhoC­expression vector into the poorly metastatic melanoma cells caused them to become highly metastatic, while ectopic expression of a dominant-interfering

630

Metastatic dissemination via lymph ducts

form of RhoC reduced the metastatic powers of the usually metastatic cells (Figure 14.40). RhoC has also been found to be strongly expressed in cells of inflammatory breast cancers, a particularly aggressive form of this disease, and in pancreatic carcinomas, which are almost always highly aggressive.

Forced expression of the RhoA gene in normally noninvasive rat hepa toma cells causes them to exhibit invasive behavior; it also enhances the metastatic behav­ior of transformed NIH 3T3 cells and of poorly metastatic melanoma cells. And a comparison of the gene expression patterns of aggressive (i.e., invasive and metastatic) and nonaggressive variants of the human T24 bladder carcinoma cell line indicates that one of the genes whose expression is most diminished in the aggressive cells specifies the RhoGDI-2 protein, a known inhibitor of Rho activa tion.

We still do not know how accurately the studies of the motility of cancer cells in culture reflect their behavior in living tissues. Nonetheless, it seems likely that both the in vitro and in vivo motile behaviors of cells are coordinated by a net­work of Rho-like proteins that modulate cell shape, adhesion, and the localized proteolysis of nearby extracellular matrix. These cellular functions are clearly critical to the invasive and metastatic behaviors of malignant cells. Perhaps most disconcerting is the potential complexity of this signaling machinery: analyses of the human genome sequence indicate that the approximately 20 Rho family proteins are regulated by as many as 80 distinct guanine nucleotide exchange factors (i.e., Rho-GEFs).

14.8 Metastasizing cells can use lymphatic vessels to disperse from the primary tumor

After invasive, motile cells enter into the vessels of blood or lymphatic systems­the process of intravasation-they disperse and, should they survive the rigors of the voyage, eventually settle in a tissue site that lies at some distance from the

Figure 14.40 Influence of RhoC onprimary tumor. Travel via the blood circulation is often called hematogenous metastasis Variants of a human melanoma cell line were derived that were either weakly or potently metastatic; the latter were far more motile and invasive in vitro . Metastatic ability was measured by injecting tumor

wt RhoC cells into the mouse tail vein and expression

counting resulting lung metastases; this vector assay ga uges some of the steps of the .. invasion-metastasis cascade (e.g., extravasation) but not all of them. The weakly metastatic cells (top left) showed an epithelial morphology, while the highly metastatic cells (bottom left) appeared fibroblastic. Introduction of a retrovirus vector expressing high levels of wild-type (wt) RhoC into the w eakly metastatic cells yielded cells (top right) that showed an elongated, fi broblast ic appearance and were hig hly metastatic. Conversely, introduction of a dominant­

expression dn RhoC

negative (dn) RhoC protein into the vector potently metastatic cells yielded cells .. that were more epi thelial in appearance

(bottom right) and lost almost all of their metastatic ability as well as their motility and invasiveness in vitro. (From EA Clark, TR. Golub, E.S. Lander and R.O .Hynes Nature 406:532-535, 2000.)

weakly metastatic

~ select variants

highly metastatic

highly metastatic weakly metastatic

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Chapter 14: Moving Out: Invasion and Metastasis

spread. and it depends on prior successful angiogenesis by the tumor. This emphasizes the fact that angiogenesis benefits cancer cells in two distinct ways. On the one hand, it supports the metabolic activity required in order for these cells to survive and proliferate. On the other. it provides tumor cells with direct access to avenues through which they can disperse throughout the body.

The extended discussion of hematogenous spread in Section 14.1 reflects the clearly important role of the blood circulation in metastatic dissemination. The contribution of the lymphatic vessels to the dispersion of cancer cells is, how­ever, less obvious. Almost all tissues in the body carry networks of lymphatic vessels that are responsible for continuously draining the interstitial fluid that accumulates in the space between cells (see Sidebars 13.5 and 26 0 ). Most of these vessels converge on a major abdominal vessel that empties its lymph into the left subclavian vein near the heart and thence into the general circulation. Consequently. cancer cells present in lymphatic vessels may occasionally enter through this cross connecrion into the general circulation.

Tumor cells and recruited rrornal companions may secrete VEGF-C, which drives lymphangiogenesis- rhe fo rmation of new lymphatic vessels (Section 13.6). Moreover, experimemal rumors forced to secrete increased levels of VEGF-C will seed larger number of metastatic cells in nearby "draining" lymph nodes-the lymph nodes associated with the lymphatic ducts that drain the tis­sue in which the tumor lie (Figure 14.41A). However, detailed histological analyses of spontaneously ari ing rumors indicate that functional lymphatic vessels are rarely fOlmd throughout rumor masses. Instead. they are largely pres­ent in a zone at the periphery of olid tumors. Those few lymphatic vessels dis­covered in the central regions of rumor masses are usually collapsed (see Figure 13.36). As discussed in the pre\;ous chapter, it seems that the expanding masses of cancer cells vvithin a rumor press on these vessels; because the lymphatic ducts have little internal hydro ratic pressure. they cannot resist these forces and collapse.

The absence of functionallnnphatic vessels within tumor masses must influ­ence the paths used by metasra izing cancer cells to leave the primary tumor. Without ready access to lymph ducts. most motile cancer cells are forced to use the far more numerous, functional capiJIaries. which are threaded throughout the tumor mass, as their route of escape. In spite of such limited access, some cancer cells do indeed succeed in entering the lymphatic system. In the specific case of mammary carcinomas, some metastasizing cancer cells enter into the lymphatic vessels that directly dra in the mammary gland and collect in the nearby downstream lymph node (see Figure 14.41A). These wandering carci­noma cells are readily detected in the lymphatic ducts and lymph nodes. because their appearance differ so strongly from the surrounding lymphoid cells (Figure 14.41C) and they e:-"'P ress epithelial proteins. such as cytokeratins. that are otherwise absent from lvm phatic tissues (Figure 14.41D). Histological examination of draining lymph nodes is routinely used to determine whether a primary breast cancer has begun to dispatch metastatic pioneer cells to distant sites in the body (Sidebar 14.6).

The lymph nodes draining a primary tumor might well function as staging areas. Thus. once cancer cells multiply and form small metastases within these nodes. they may disperse further by dispatching metastatic pioneers to more distant sites in the body. In fact, through much of the twentieth century. surgeons believed that the draining lymph nodes of a tissue function as filters, and that once these nodes become fiJIed with metastasizing cancer cells, these cells spill over into other lymphatic vessels, through which they travel in order to dissem­inate widely throughout the body.

The alternative notion is that draining lymph nodes represent dead ends for dis­seminated cancer cells. that is, that those cancer cells that proliferate within

632

Metastatic dissemination via lymph ducts

these nodes rarely move on to more distant sites in the body. Indeed, studies of patients carrying breast, head and neck, gastric, and colorectal carcinomas indi­cate that surgical removal of draining lymph nodes has no effect on long-term patient survival. Such observations suggest that metastasis through the lymph and through the blood operate in parallel, and that the cancer cells that arrive in lymph nodes usually venture no farther. Accordingly, lymph nodes represent useful "surrogate markers" of metastasis by providing useful diagnostic and prognostic data without being directly involved in the processes that lead to widespread cancer cell dissemination and metastatic disease.

(A)

central (middle) axillary nodes

I >0. 1 I : ~~ proximal (lower) : ~ axi llary nodes I

/ int ern{l l \"" I

..--/ mammary chain --q

(8)

(tumor)

medial lateral

(C) (D)

Figure 14.41 Draining lymph nodes of the mammary gland (A) The lymphatic ducts (red) and the lymph nodes draining the breast (swellings along ducts) are initial sites of metastatic spread, being ca rried there by the flow of lymph (arrows) leaving various sectors of the breast Carcinomas arising in these different sectors tend to deposit metastatic cells in different sets of draining lymph nodes. Discovery of carcinoma cells in these lymph nodes, which is observed in more than 30% of human breast carcinomas at the time of initial diagnosis, suggests the possibility of deposits of metastatic cells in more distant sites in the body, particularly if large numbers of draining nodes are found to carry breast cancer cells. (B) The lymph node that serves as the sentinel node of a tumor can usually be identified among all of the lymph nodes draining the breast (panel A) by injecting a blue dye into the tumor (outside photographic field to right) and follow ing the trail of the dye via

the lymphatic duct (arrows) to the draining node (outlined in dashed line, left) (C) Hematoxylin-eosin (H&E) staining of a sect ion of an axillary lymph node reveals that three micrometastases arising from a primary breast tumor (arrows) have grown in the space between the capsule surrounding this node (not seen, below) and the mass of lymphocytes w ithin the node (small cells, dark nuclei, above), displacing the latter upward . (D) Immunohistochemistry using an antibody specific for cytokeratins (brown) reveals this small micrometastasis in a sentinel node. This procedure is far more sensitive than the H&E staining of panel C in detecting small micrometastases, since the mesenchymal cells of the lymph node do not expressed cytokeratin, which is made by epithelial cells. (A, B, and C, from AT Skarin, Atlas of Diagnostic Oncology, 3rd ed. Philadelphia : Elsevier Science Ltd, 2003; D, from J.P. Leikola, TS. Toivonen, LA Krogerus et ai, Cancer 104: 14-19, 2005.)

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Chapter 14: Moving Out: Invasion and Metastasis

Sidebar 14.6 Lymph nodes are sen­tinels that carry important prognos­tic information The lymph nodes associated with the lymphatic ducts that drain various tissues operate as collection points for subcellular debris and cells shed by these tissues; in addition, cells of the immune system become activated in response to anti­gens that they first confront in these nodes. This explains why draining nodes associated with a variety of organs (e.g., the mammary gland; Figure 14.41A) are routinely examined to determine. whether cancer cells have been released by primary tumors

in those organs. For example, upon initial diagnosis, about one-third of breast, colorectal, cervjca\' arid oral carcinoma patients have metastasized cancer cells in the lymph nodes near their primary tumors (see Figure 14.41C and OJ.

These draining lymph nodes serve as proverbial "canaries in the mine," by providing early warning of the pres­ence of metastasizing cells in the body. Among these regional lymph nodes, the single node that directly drains the primary tumor is often termed the "sentinel" node (Figure 14.418) . Patients with small numbers ' of

affected nodes often have only local­ized spread of the breast cancer and may never develop metastatic disease, while those with many affected nodes are far more likely to harbor other deposits of metastatic cells in distant sites in the body. For example, in one study 90% of long-term survivors of a variety of carcinomas had one, two, or occasionally three "positive" lymph nodes at diagnosis. Conversely, fewer than 5% of patients with more than five positive lymph nodes when their primary tumors were removed enjoyed long-term, disease-free sur­vival.

14.9 A variety of factors govern the organ sites in which disseminated cancer cells form metastases

The descriptions in the previous sections of the mechanisms of invasion and metastatic dissemination seem to explain, at least in outline, how most of the steps of the invasion-metastasis cascade proceed. Moreover, it is plausible that the dispersion strategies used by a wide variety of invasive, metastatic cancer cell types will one day be found to be governed by a common set of mechanistic principles, such as those discussed here. Importantly, however, our discussions did not address the last step of the invasion-metastasis cascade-colonization.

The growth of micrometastases «2 mm cliameter) into macro metastases (>2 mm diameter) is clearly the key step in determining whether or not metastatic disease will ever develop. For example, 30% of the women diagnosed with pri­mary breast carcinomas have thousands of micrometastases in their bone mar­row, many composed of single cells or tiny clusters of cells (see, for example, Figure 14.2C). Yet only half of these women will ever suffer a disease relapse trig­gered by the appearance of macroscopic metastases. Clearly colonization is an extremely inefficient process, and the vast majority of cells that end up forming small micro metastases never succeed in properly adapting to the tissue in which they have landed by spawning a macrometastasis.

In addition, while a variety of cancer cell types may execute the earlier steps of the invasion~metastasis cascade in a very similar fashion, it is likely that colo­nization by each type of cancer cell proceeds quite differently. Thus, successful adaptation of metastasized breast cancer cells to the bone marrow (which, by definition, enables these cells to colonize the marrow) is likely to involve a quite different set of cellular changes from those required for successful bone marrow colonization by prostate cancer cells. In addition, the changes required for a breast cancer cell to colonize the bone marrow are likely to be quite different from those needed for it to succeed in brain or lung colonization.

Abundant evidence supports the notion that metastatic cancer cells that have colonized a certain target organ have become highly specialized to do so. Here are some indications of this. (1) 75% of young patients with papillary thyroid carci­nomas have significant lymph node metastases, but only 3% will ever develop distant metastases. Hence, adaptation to the lymph nodes by metastasizing thy­roid carcinoma cells does not allow them to colonize other tissues in the body. (2) Similarly, duodenal carcinoid tumors greater than 1 cm in diameter (containing >109 cells) have a high rate of lymph node metastasis, yet they rarely metastasize

634

Metastatic tropisms of disseminating cancer cells

Sidebar 14.7 Cancer cells must "learn" to colonize sites of target organ. Some subclones of cells within the primary metastasis Implicit in these descriptions of colonization is tumor mass may, through happenstance, acquire this abil­the notion that the neoplastic cells within a primary tumor ity as an unselected side product of the forces that led ini­do not gain the ability to colonize one or another target tially to their development of tumorigenic potential. More organ the moment they become tumorigenic. Instead, for frequently, however, the acquisition of colonizing ability is most kinds of cancer celis, colonizing ability is a trait that likely to occur at the site of eventual metastasis formation, seems to be acquired independently of the acquisition of where large numbers of micro metastases reside for ex­tumorigenicity. This makes sense, since the biological tended periods of time until one of the dispersed cancer forces operating within the primary tumor site select for cells happens to acquire colonizing ability. In this case, the the ability of variant cells to proliferate in that particular Darwinian selection favoring such a variant cell is clear and microenvironment and not in the microenvironment of a obvious, since its descendants will thrive and proliferate distant, biologically unrelated tissue. strongly in this distant site, while all the other micro­

Unanswered by these arguments is the question of metastatic neighbors in this tissue will stagnate and even­where and when tumor cells learn to colonize a particular tually disappear.

to the liver, which is the common site of metastasis of the tumors that arise in the nearby colon. (3) Cancer cells isolated from human lymph node metastases have been found, after injection into the venous system of mice, to grow preferentially in the lymph nodes of their mouse hosts rather than other possible sites of colo­nization. (4) Surgical removal of isolated, relatively large colorectal carcinoma metastases present in the liver or lung often results in disease-free survival for a number of years, in spite of the fact that the circulation of patients with these metastases clearly carries large numbers of metastasizing cells, including some that already possess colonizing ability in one or several organs. (5) Mouse melanoma cells can be selected that metastasize preferentially to lungs, or breast cancer cells that metastasize to the lungs or, alternatively, to the bone. These dis­parate observations reinforce the notion that the ability to colonize a certain organ represents an acquired specialization (Sidebar 14.7).

Yet another factor affects these dynamics: different types of cancer cells acquire the ability to colonize a given tissue more or less readily. Thus, the ability of metastasizing prostate cancer cells to colonize the bone marrow seems to be much more readily acquired than their ability to colonize the liver or the pan­creas. This suggests that the differentiation program of normal prostatic epithe­lial cells exerts a strong influence on the ability of derived carcinoma cells to metastasize to specific organs. If we were to place prostate carcinoma cells and Figure 14.42 Primary tumors and potential target organs on a map that depicts metastatic tendencies (Figure their metastatic tropisms In this 14.42), we would indicate that the prostatic cells have relatively easy "access" to diagram, th e relative width of each the bone marrow, implying that they need to undergo fewer changes in order to arrow indicates the relative proportion of adapt to this site. Conversely, they have more limited access to other organs, clinically apparent metastases that are

such as the liver or pancreas, in which they rarely form macroscopic metastases. generated by a primary adenocarcinoma. Four types are indicated here: prostate, breast, pancreas, and colon. In some cases, the tendencies of a tumor to spawn metastases in one or another tissue reflect the abilities of the cancer cells from the primary tumor cells to adapt to (and thus colonize) the microenvironment of distant tissues; this

brain is likely to explain the strong tendencies of prostate and breast cancers to generate metastases in the bone marrow. In other cases, the layout of the circulation may strongly influence the site of metastasis. For example, the high proportion of liver metastases deriving from primary colon cancers may reflect the drainage via the portal vein of blood from the colon directly into the liver (see

pancreas 'breast Figure 14.44).

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Chapter 14: Moving Out: Invasion and Metastasis

Figure 14.43 Stephen Paget The British physician Stephen Paget was the first to enunciate the "seed and soil" hypothesis, which states that the abil ity of a disseminated cancer cell to successfully found a metastasis depends on whether a distant tissue offers it a hospitable environment to survive and proliferate. (From I.J . Fidler, Nat. Rev. Cancer 3:453-458, 2003 .)

Sidebar 14.8 Contralateral metastases are relatively rare Possibly the greatest embarrass­ment for the seed and soil hypothesis comes from its fail­ure to explain the rarity of con­tralateral metastases. For exam­ple, cancer cells disseminated from a primary tumor in one breast should find that the con­tralateral (Le., opposite) breast provides the most hospitable environment for colonization. In fact, only about 2% of breast cancer cases result in contralat­eral metastases,. comparable to the frequency of tumors in the breast that arise as metastases of primary tumors located out­side of the breast. Similarly, pri­mary kidney cancers metasta­size infrequently to contralat­eral kidneys. These behaviors are clearly incompatible with the seed and soil hypothesis and still require explanation.

This predilection to form macro metastases in one or another organ site was noted as early as 1889 by the British pathologist Stephen Paget (Figure 14.43). He proposed the "seed and soil" hypothesis, in which he analogized the seeding of cancer cells to the dispersal of the seeds of plants. After studying the clinical course of 735 breast cancer patients, Paget concluded that the patterns of metastasis formation in these patients could not be explained either by random scattering throughout the body or by the patterns of dispersal from the breast through the general circulation. He therefore proposed that the metastasizing cancer cells (the seed) find a compatible home only in certain especially hos­pitable tissues (the soil). He wrote, "a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil." This ability to form macroscopic metastases in some sites but not others has been highlighted by certain clinical procedures (Sidebar 32 8 ).

The seed and soil model states quite explicitly that disseminating cancer cells are dispersed "in all directions," that is, throughout the body. This phrase injects another idea into our thinking: the reason why many kinds of cancer cells form metastases in a specific target organ is not attributable to their directed migra­tion or homing to this organ. Instead, they are scattered randomly, and only those cancer cells that happen to land in a reasonably hospitable tissue succeed in surviving, forming micro metastases, and occasionally, having learned how to colonize, forming macroscopic metastases. Conversely, essentially identical dis­seminating cells may land, with an even higher frequency, in other organs where they perish immediately or survive as micrometastases without ever succeeding to colonize these sites.

The seed and soil hypothesis cannot, however, explain the metastatic patterns of all types of human cancers (see Sidebar 14.8). Instead, in certain cases, the predilection to metastasize to a certain target organ is likely to be dictated by the layout of the vessels connecting the site of a primary tumor and the site of metas­tasis. For example, the strong tendency of colon carcinoma cells to metastasize to the liver may simply reflect the fact that these cancer cells leave the gut via the ponal vein (which drains the lower gastrointestinal tract and the spleen) and, after a very brief trip, almost inevitably become lodged in the capillary beds of the li\-er that are fed by this vein (Figure 14.44). Even if individual metastasizing colon cancer cells colonize the liver with an extremely low efficiency, the sheer num­bers of the cancer cells trapped in the liver guarantee that, with the passage of enough time, substantial numbers of metastases will arise in this target organ.

The same logic may explain why breast cancer cells often form metastases in the lungs. As is the case with metastasizing colorectal carcinoma celis, wandering mammary carcinoma cells may not find that the lungs provide them with an especially hospitable environment, and individual cancer cells will have a low probability of successfully colonizing the lungs. Nonetheless, some metastases vvili eventually form there, simply because so many of these cells become phys­ically trapped in this tissue (see Figure 14.44). This logic suggests that, in gen­eral, the frequency of metastases to an organ is governed by two parameters­the frequency with which metastasizing cells are physically trapped in an organ, and the ease vvith which they can adapt to the microenvironment of that organ, thereby colonizing it.

There are also indications that tissues that are normally not hospitable sites for colonization may become so through specific pathological processes, such as localized wounding (Sidebar 330). This suggests that areas of chronic inflam­mation \-vithin the body of a cancer patient may occasionally become congenial environments for metastasizing cancer cells, simply because they provide a spectrum of mitogenic and trophic signals, as discussed in Chapter 13.

Yet other mechanisms have been proposed to explain the tissue tropisms of metastasizing cells. For example, target organs may release specific chemical

636

Metastatic tropisms of disseminating cancer cells

portal system

I lungs l

arterial circulation

venous circulation

messages-the chemoattractants sometimes termed chemokines-that might actively recruit wandering cancer cells to enter into them from the drculation. Such chemoattraction clearly operates to ensure the homing of a variety of cir­culating immune cells to specific tissues as part of the normal operations of the immune system. In one study, when B16 mouse melanoma cells were forced to express the CXCR4 chemokine receptor, their metastases to the lung increased by a factor of 10. However, when an expression vector specifying the CXCR7 receptor was introduced into these melanoma cells, they then showed substan­tially increased metastasis to the lymph nodes, thereby appropriating a mecha­nism normally used by lymphocytes for homing to these nodes. (In truth, since these chemokine-activated receptors often provide mitogenic and survival sig­nals to cancer celis, it is difficult to know whether these receptors, when acti­vated by their ligands, induce metastasizing cells to migrate into a tissue or sim­ply encourage the survival and proliferation of these cells after they have landed in the tissue.)

According to another mechanistic model of metastatic tropism, the capillaries forming the vascular beds (i.e., the networks of blood vessels) in various tissues express tissue-specific molecules on their luminal surfaces. These molecules may offer specialized docking sites for cancer cells that express certain adhesion molecules, such as integrins, on their surfaces. This model is sometimes termed the "vascular ZIP code" theory, because it implies that the luminal surfaces of vessels in different tissues carry, in chemical form, specific homing addresses, much like those used by a postal system. This model fails to take into account the fact that cancer cells are often surrounded by clouds of platelets that are capable of blocking direct association between the cancer cell and the luminal surfaces of endothelial cells.

One detailed study of the behavior of metastasizing human cancers calculated that 66% of metastases could be explained simply by the blood flow patterns between the primary tumor and the sites of observed metastases. In 20% of the cases, the specialized micro environments of target tissues (rather than blood flow patterns) seemed to provide the explanation of the tendency of certain can­cer types to form macroscopic metastases in these tissues. And finally, in 14% of cases, negative interactions (in which tissues seemed to actively repel wander­ing cancer cells) seemed to explain smaller-than-expected numbers of metas­tases predicted by blood flow patterns.

To summarize, these diverse observations suggest that metastasizing cells dis­perse to many organ sites in the body and that their dispersion is affected by the layout of the vasculature. Once arrived in these various sites, the cancer cells will usually survive and eventually colonize only those tissues that provide them with specific chemokines, trophic factors, and mitogens. On occasion, however, these cells may succeed in founding macroscopic metastases in relatively inhos­pitable organ sites, only because the routing of the blood circulation introduces these cells in vast numbers into such sites.

Figure 14.44 Portal circulation and liver metastasis While the venous systems of most tissues drain to the right side of the heart and thereafter into the capillary beds of the lungs, the veins draining the spleen and gut are organized differently, in that they empty directly Into the liver via the portal circulation. (After passing through the liver, venous blood is only then dispatched back to the heart.) Consequently, vast numbers of metastasizing colorectal carcinoma cells are trapped in capillary beds of the liver within seconds of leaving the colon. (Adapted from I.e. MacDonald, G.e. Groom and A.F. Chambers, BioEssays 24:885-893, 2002.)

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14.10 Metastasis to bone requires the subversion of osteoblasts and osteoc1asts

The development of bony metastases represents one instance in which we understand in some detail the biochemical and biological mechanisms that per­mit metastasized cancer cells to thrive in a specific tissue microenvironment. This fact, on its own, justifies a detailed discussion of osteotropic metastasis. In addition, and as mentioned repeatedly, several of the most common types of cancer occurring in the Western world-carcinomas of the lung, breast, and prostate-show a strong tendency to metastasize to the bone. In fact, patients with advanced breast and prostate cancer almost always develop bone metas­tases. And in those patients who succumb to these cancers, the bulk of the tumor cells in their bodies at the time of death are usually found among the metastases scattered throughout their bones.

We usually think of bone as being a static tissue which, once formed, retains its structure throughout life. The truth is far more interesting. In mammals, about 10% of skeletal bone mass is replaced each year, resulting in an essentially com­plete replacement over the course of a decade. This continuous remodeling enables the bones to respond to mechanical stresses by compensatory reinforc­ing of stressed regions. For example, the bones of the legs are continuously being remodeled in response to the weight-bearing signals that different por­tions of each leg bone receives.

The turnover of bone is the work of osteoclasts, which break down mineralized bone, and of osteoblasts, which reconstruct it. The osteoclasts function first to demineralize the bone (by dissolving its calcium phosphate crystals) and then to degrade the now-exposed extracellular matrix, which previously formed the organic scaffolding for the calcium phosphate crystals (Figure 14.45). Osteoblasts move in soon after to carry out reconstruction, which involves both the assembly of new ECM and the deposition of calcium phosphate crystals in the interstices of this matrix. As can be deduced from this description, the two cell types normally work in close coordination.

Most kinds of metastasizing cancer cells are, on their own, incapable of remod­eling bone structure. Instead, they manipulate and exploit these two types of cells present in the bone in order to change its shape. Thus, breast cancer cells activate the osteoclasts, resulting in osteolytic metastases-literally, metastases that dissol\'e bone. Prostate cancer cells tend, on the other hand, to activate osteoblasts, "ielding osteoblastic lesions, in which mineralized bone actually accumulates in the vicinity of the metastases (Figure 14.46).

In fact, these two behaviors represent the extremes of a continuum, since both types of cancers acti ate both osteoblasts and osteoclasts to a greater or lesser extent. For example. \ hile osteolytic metastases predominate in advanced breast cancer patients, as many as one-quarter of these women also have clearly defined osteoblastic lesions in their bones. Similarly, prostate carcinomas also generate occasional osteolytic metastases scattered among the many osteoblas­tic growths spavvned by these tumors. One exception to this rule of a mingling of both types of bone metastases is provided by myeloma cells (tumors of the B-cell, antibody-secreting lineage)' which create exclusively osteolytic lesions in bone.

The close coordination between osteoblasts and osteoclasts is mediated, at least in part, by the exchange of growth factor signals. An important inducer of osteo­clast differentiation is RANK (receptor activator of NF-KB) ligand, or simply RAl'-JKL. RANKL is produced by and displayed on the surface of osteoblasts. When an osteoclast precursor displaying the RANK receptor comes into contact with an osteoblast and its cell surface RANKL molecules, this results in activa­tion of the RANK receptors of the osteoclast precursor and its maturation into a

638

Mechanisms of osteotropic metastasis

( (8) A) ':.-. LIi, ... .~.iZ P ~ ZI .,<, _ , . A•• _

functional osteoclast (Figure 14.47). At the same time, osteoblasts produce a sol­uble decoy receptor, termed osteoprotegerin (OPG), which can bind RANKL and ambush it before it succeeds in activating the RANK receptor on the surface of osteoclast precursors. The result is a blockage of the RANKL-RANK Signaling and the inhibition of osteoclast maturation. Hence, the balance between the RANKL (stimulatory) and OPG (inhibitory) signals determines the state of acti­vation of osteoclasts.

This dynamic interaction of osteoblasts and osteoclasts provides the back­ground for the actions of cancer cells that metastasize to bones. Their attraction to the bone derives ultimately from the nonmineralized, collagenous extracellu­lar matrix (ECM) that forms the organic scaffolding in which calcium phosphate crystals are deposited (see Figure 14.45B). As it happens, bone ECM is an unusu­ally rich source of the mitogenic and trophic factors that allow several types of carcinoma cells to thrive. Consequently, by provoking the demineralization of bone, cancer cells gain access to the storehouse of factors sequestered in the bone ECM and use them to support their own proliferation and survival.

Metastasizing cancer cells reach the bone through the vessels feeding the mar­row. Once there, they adhere to specialized stromal cells coating the surfaces of the bone facing the marrow. Metastasizing breast cancer cells, in particular, upon arrival in bone, revert to a behavior characteristic of their normal precur­sors (mammary epithelial cells, or MECs). During lactation, when producing

Figure 14.45 Bone degradation by osteoclasts (A) This micrograph shows osteoclasts (purple, arrows) excavat ing small pits in the surface of a mouse jaw bone (pink). (B) At far higher magnification, this scanning electron micrograph shows a rat osteoclast that has excavated a shallow pit in the surface of mineralized bone. The calcium apatite crystals in the bone have been dissolved away by acid secreted by the osteoclast, revealing the complex meshwork of collagen-rich extracellular matrix (ECM) at the bottom of the pit. Associated with this ECM are mitogens and survival factors that become available to cancer cells after osteoclasts break down the ECM. (C) This scanning electron micrograph reveals how devastating the osteolytic lesions (arrows) can be in terms of compromising bone structure in a patient w ith metastatic osteolytic lesions. (A and B, from TR. Arnett and D.W Dempster, Endocrinol. 119: 199-124, 1986; C, courtesy of G.R. Mundy.)

639

Chapter 14: Moving Out: Invasion and Metastasis

(C) osteoblastic metastasis

muscle marrow mineralized bone

Figure 14.46 Osteolytic and osteo­blastic metastases These micrographs present sections of vertebrae and femurs in which the mineralized bone (orange), surrounding muscle (bright red), and bone marrow (dark purple) are clearly delineated. (A) This vertebra of a control mouse injected only with buffer is seen to be composed of extensive marrow with ribbons of mineralized bone running through the marrow. (B) In a mouse bearing a human breast cancer cell line (MDA-MB-231) that creates osteolytic lesions, much of the mineralized bone is seen to be missing, and the marrow has been displaced by tumor cells (dark red) (C) In a mouse bearing a human breast cancer cell line (ZR-75-1) that creates osteoblastic lesions, much of the marrow space is now filled with mineralized bone (orange) with tumor masses evident to the left and right. (From J.J. Yin, KS Mohammad, S.M. Kakonen et al., Proc. Natl. Acad Sci. USA 100 10954-10959, 2003)

tumor tumor

milk, MEC forming the small sacs (alveoli) of the mammary gland release parathFoid hormone-related peptide (PTHrP). PTHrP then travels through the circulation to the bo nes, where it triggers a chain of events that encourages the disso lution of bone minerals by osteoclasts. This results in the mobilization of calcium ion . duch travel back via the circulation to the mammary gland, where the\- are incorporated into the milk by the MECs.

This normal calcium-mobilizing mechanism is subverted by metastaslZlng breast cancer cell that become established in bones (Figure 14.48). Having attached (0 £he llomal cells covering the surfaces of mineralized bone, the breast cancer cell , re,-erting to the habit of normal MECs, release PTHrP. The PTHrP, in rum, im!Jinges directly on its receptors displayed by osteoblasts, caus­ing the e cells 0 release RANKL. RANI(L then induces the differentiation of osteoclast precur-ors into active osteoclasts. The activated osteoclasts degrade nearby mineralized bone, £hereby liberating the rich supply of growth factors attached to me exr:acellular matrix of the bone.

The grO\\lb factO[- uberated from the bone ECM, including PDGF, bone mor­phogenetic proreiP- _IPs), fibroblast growth factors (FGFs), insulin-like groV\rth factor-1 (lGF -J). and TGF-~, fuel the further growth of the breast cancer cells , in ducing mem (0 ~ec:re[e more PTHrP. This PTHrP engenders more osteol­ysis bv the osteocla (-.leading to a self-perpetuating Signaling system that has been called a '\ iciou c'.-cJe~ ee Figure 14.48) in which TGF-~ also plays a key role (Sidebar 14.9).

This cycle suggests pos ible pointS of therapeutic intervention. Most promising are drug compounds such a bisphosphonates, which are taken orally and become adsorbed to the apatite crystals that constitute the mineral portion of bone; the drug molecules can persist there for extended periods of time as long as a decade or more. \tI,Then bisphosphonate-containing bone is later dissolved by osteoclasts, the latter are poisoned by the liberated bisphosphonates, leading to their apoptosis. Hence, bisphosphonates are useful for reducing the burden of osteolytic lesions in patients with various types of metastatic cancer.

Wilen immunocompromised mice carrying human breast cancer cells are treated with bisphosphonates, the number of osteolytic lesions is reduced and, at the same time, the total burden of tumor cells in these animals is decreased. This observation provides additional indication that late in tumor progression, 640

Mechanisms of osteotropic metast asis

mineralized bone /

osteoclast ( OPG)

-m...·· - , 1 ~ osteoclast osteoblasts precursors

the proliferation of these breast cancer cells depends greatly on osteolysis and the resulting liberation of growth factors from dissolved bone. Moreover, bis­phosphonate therapy can provide additional benefits to patients suffering from metastatic breast cancer (£ee Sidebar 14.10). Recently, derivatives of osteoprote­gerin (OPG)' which should also block the "vicious cycle," have also been found, in early-phase clinical trials, to substantially reduce the rate of bone dissolution in patients with myelomas and metastatic breast cancers.

As might be predicted, osteoblastic lesions depend on other Signals-ones that activate osteoblasts rather than osteoclasts. In this case, the release by metasta­tic cancer cells of the grovvth factor termed "endothelin-l" (ET-l) plays a domi­nant role in stimulating osteoblasts and, at the same time, suppressing osteo­clast activity. Thus, prostate cancer cells in primary tumors release endothelin; since its cognate receptor is also expressed by these cancer cells, an autocrine growth-stimulatory loop results. However, when these cancer cells arrive in the marrow, the endothelin that they release also acts via heterotypic signaling to stimulate osteoblasts, creating the osteoblastic lesions characteristic of this malignancy. (Precisely how osteoblast activation benefits the prostate cancer cells is less well understood. It is plausible that activated osteoblasts secrete large amounts of growth factors during the construction of mineralized bone, and that some of these factors are diverted by the cancer cells in osteoblastic metastases.)

So, Paget's seed and soil metaphor is useful, but it does not go far enough. Like seeds, metastatic cells are cast in many directions, but once they fallon certain ground, they can hardly be portrayed as being passive participants in their

Ca2+

osteolysis

~W - L.,_........ osteoclast precursors

Figure 14.47 Osteoblasts versus osteoclasts The physiologic balance between bone formation and resorption is created by signaling betvveen osteoblasts, w hich assemble bone, and osteoclasts, w hich dissolve it. In an ongoing cycle, osteoclasts remove mineralized bone by covering and sealing off a section of bone and secreting digestive acid into the bone below them; this is followed by osteoblastic filling of resulting cavities w ith new bone. The osteoblasts release RANKL, w hich acts via the RANK receptor (not shown) displayed by osteoclast precursors to induce the latter to mature into functional osteoclasts. The osteoblasts may al so secrete osteoprotegerin (OPG), w hich acts as a decoy receptor to ambush RANKL before it has had a chance to activate osteoclast precursors. Hence, the balance between RANKL and OPG determines the net rate of bone growth/loss.

Figure 14.48 The vicious cycle of osteolytic metastasis Release by a breast cancer cell (right. gray) of PTHrP (parathyroid hormone-related peptide) causes nearby osteoblasts to change the mix of signals that they release: they increase RANKL synthesis (red arrow) and decrease OPG (osteoprotegerin) synthesis (blue line). RANKL induces osteoclast precursors to mature into functional osteoclasts (see Figure 14.47). The latter undertake osteolysis, which causes bone demineralization, exposes the extracellular matrix within the bone (Figure 14.458), and results in liberation of TGF-~, Ca2+, and IGF-1 (upper left and middle). IGF-l and Ca 2+ cause cancer cell proli feration and survival , and the additional presence of TGF-~ induces the cancer cell to release more PTHrP, resulting in a self-sustaining positive­feedback loop that has been termed the "vicious cycle " of osteolytic metastasis . (From G.R. Mundy, Nat. Rev. Cancer 2584-593, 2002) 64

Chapter 14: Moving Out: Invasion and Metastasis

future fate. Instead, these cancer cells begin to actively till the soil in which they have landed, cultivating it so that it is guaranteed to become fertile ground for their own prolifera tion and that of their descendants.

14.11 Metastasis suppressor genes contribute to regulating the metastatic phenotype

We have read here of a nwnber of genes that actively promote some of the steps in the invasion-metastasis cascade. Many of these encode familiar growth fac­tors, growth factor receptors, or signal-transducing proteins that we encountered

Sidebar 14.9 TGF-~ and PTHrP play pivotal roles in the vicious cycle of breast cancer osteolytic metastases Breast cancer cells that have metastasized to the bone produce far more PTHrP than do others in the same animal that have not-a reflection of the fact that certain grO\,vth factors lib­erated from the bone ECM stimulate PTHrP production by the metastatic cancer cells. The most important of these bone-derived factors is TGF·~, as illustrated by some sim­ple experiments. In one of these, a dominant-negative TGF­

.~ receptor (which blocks a cell's ability to respond to TGF­~) has been expressed in human breast cancer cells. Such cells now cease producing PTHrP and lose the ability to

efficiently produce osteolytic metastases in the bone (Figure 14.49). In another experiment, breast cancer cells that usually lack the ability to metastasize to bone and fail to secrete TGF-~can be forced (through the use of an expression vector) to secrete TGF-~. The latter then acts in an autocrine fashion to stimulate these cells to produce their own PTHrp, allowing them to form large numbers of bone metastases. Finally; antibodies that bind and neutral­ize PTHrP are able to block the ability ofhuman breast can­cer cells to generate osteolytic lesions in mice. These are some of the disparate observations that have inspired the "vicious cycle" model depicted in Figure 14.48.

dn TGF-~RII dn TGF~-R II + ca TGF~-RI

MDA-MB-231cells transfected with:

dn TGF~-RII + PTHrP

Fig.ure 14.49 TGF-13 and the formation of osteolytic metastases The evidence supporting the "vicious cycle" model of osteolytic metastasis (Figure 14.48) comes in part from experiments involving the use of MDA-MB-231 cells, a line of human breast cancer cells that usually show a high tendency to produce osteolytic metastases(see Figure 14.46B). The ability of these cells to do so is gauged here by X-ray analyses of the hind limbs of mice that have borne MDA-MB-231 tumor xenografts. When a dominant-negative type II TGF-p receptor (dn TGF-pRII) expression construct is introduced into these cancer cells, this mutant protein blocks their ability to respond to TGF-p, specifically the TGF-~ that they would otherwise be liberated from the extracellular matrix of the osteolytic lesions that they may have induced. Without TGF-p stimulation, these cancer cells fail to form osteolytic metastases (left panel). However, if this

inablli -0 respond to TGF-p is overridden by introducing additional! into these cells an expression construct specifying a constl uti ely act ive type I TGF-p receptor (ca TGF-PRI}, then the powers of these breast cancer cells to induce osteolytic lesions are restored (arrows, center panel). This observation, on its own, does not indicate precisely how the ca TGF-pRI succeeds in restoring the osteolytic activity to these cells. The explanation comes from an experiment in which a vector causing PTHrP expression (instead of ca TGF-pRI expression) is introduced into the dn TGF-PRII. Now, they regain the ability to form osteolytic metastases (arrows, right panel). This demonstrates that TGF-p stimulates osteolytic activity by forcing the breast cancer cells to release PTHrp, which proceeds to activate osteoblasts and, in turn, osteoclasts (see Figure 14.48). (From J.J. Yin, K. Selander, J.M. Chirgwin et ai, I Clin In vest. 1 03: 197-206, 1999.)

642

Metastasis and its regulation by suppressor genes

in our earlier discussions of oncogenes and their mechanisms of action. When introduced into a variety of epithelial cells, these genes are able to encourage changes such as the epithelial-mesenchymal transition (EMT), the acquisition of cell motility, and even invasiveness. Indeed, it seems increasingly likely that deregulated versions of these genes are the primary forces driving many of the steps of invasion and metastasis.

Importantly, the protein products of these various genes operate as components of the complex regulatory circuits that govern many aspects of cell physiology. And like all well-designed circuits, these have both positive regulators and coun­terbalancing negative regulators in order to ensure finely tuned outputs. This logic leads to the conclusion that there must be a number of control elements operat­ing in cells that counteract and balance the invasive and metastatic actions of the positive effectors of advanced malignancy. Such negative regulators, in analogy with the tumor suppressors, have been called metastasis suppressor genes.

As one might anticipate, these metastasis suppressors operate at various levels in regulating the steps of invasion and metastasis, ranging from master, pleiotropi­cally acting regulators and signal-transducing proteins to the ultimate effectors of the various biochemical changes (Table 14.4). These genes have been identi­fied through a variety of experimental strategies. Quite often, their expression in primary tumors and their far lower expression in derived metastases have sug­gested important roles in blocking the late steps of malignant progression.

Such observations, being only correlations, do not prove causal roles in prevent­ing metastasis, which can only be demonstrated through other types of experi­ments. For example, the role of a candidate gene as a bona fide metastasis sup­pressor gene can be tested by a simple operational criterion: when the gene's expression is forced in the cells of a primary tumor, does this expression permit the continued expansion of this tumor mass while, at the same time, block the appearance of distant metastases that are usually seeded by this tumor and oth­ers like it? Some of these genes have passed such a test, while others act in a less specific way by inhibiting proliferation by all types of cells, including some that lack invasive and metastatic properties. Yet other candidate tumor suppressor genes have been found able to suppress metastasis in only a small subset of malignant tumor types

The first of these genes to be discovered-NM23-was reported in 1988 to reduce the metastatic powers of mouse melanoma cells. Subsequent research indicates that it suppresses the motility and invasiveness of cells in vitro. Its ability to sup­press the metastatic powers of experimental tumors has also been reported. The biochemical mechanism of action of the NM23 protein may be related to its abil­ity to form physical complexes with the KSR protein, which serves as a scaffold­ing protein that brings together several of the kinases in the ERK-MAPK cascade

Table 14.4 Candidate metastasis suppressor genes

Sidebar 14.10 Osteolytic les­ions wreak havoc in other ways In addition to the corrosive defects of osteolytic lesions on the skeleton, including severe bone pain and fractures, osteol­ysis has another, more subtle effect: The large-scale degrada­tion of bone mineral leads to hypercalcemia-elevated con­centrations of calcium in the circulation. At the same time, the PTHrP (parathyroid hor­mone-related peptide) released into the circulation by bone metastases causes kidneys to secrete less calcium into the urine, further increasing cal­cium levels in the blood. Such hypercalcemia, which usually signals the final stages of malig­nant disease, causes a wide vari­ety of symptoms, including gas­trointestinal, urinary tract, car­diovascular, and neuropsychi­atric problems. Hypercalcemia can be substantially reduced by treating patients with bisphos­phonates (to reduce osteolysis) and with antibody to PTHrP (to increase calcium excretion by the kidneys).

Name

BRMsl CRsP3 KAI1iCD82 KISS 1 NM23 RhoGDI-2 SseCKs VDUPl

Cellular location Mechanism of action

nuclear protein involved in chromatin· remodeling nuclear protein transcription factor tranmlembrane protein · cell-cell association (?) secreted protein ligand of G-protein-coupled receptor cytoplasmic kinase regulator of MAPK cascade(?) cytoplasmic protein negative regulator of Rho action cytoplasm cytoskeleton-associated protein cytoplasm regulator of MAPKcascade (?)

CDHl (=E-cadherin) cell surface adhesion protein favors formation of epithelial cell sheets TlMPs secreted protein . inhibitor of metalloproteinases MKK4 . cytoplasm protein kinase. component of MAPK cascade

Adapted in part from p.s. Steeg, Nat. Rev. Cancer 3:55- 63, 2003.

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Chapter 14: Moving Out: Invasion and Metastasis

(Section 6.5) in order to facilitate rapid and efficient transfer of signals be~een them. Hence, NM23 may affect the metastatic powers of cells through its ability to modulate the flux of signals passing through this critical survival and mito­genic pathway. NM23 has also been reported to function variously as a nucleo­side diphosphate kinase (which converts nucleoside diphosphates to triphos­phates), a histidine kinase, or a serine kinase. Studies ofthe Drosophila homolog of NM23, termed awd, indicate that it acts during development to suppress unwanted cell migration. Its candidacy as a metastasis suppressor is complicated by the fact that it has been reported to be overexpressed in advanced ovarian, gastric, and colon carcinomas, as well as in sarcomas.

The E-cadherin molecule, about which much has been said in this chapter, is also considered to be the product of a metastasis suppressor gene. Its role in sta­bilizing cell-cell contacts in epithelial sheets and in preventing the epithelial-mesenchymal transition (Section 14.3) clearly places it among the major molecular obstacles that block acquisition ofthe invasive cell phenotype. As we have read, its expression is lost in invasive carcinomas through a variety of mechanisms. Similarly, we can easily imagine how tissue inhibitors of metal­loproteinases (TIMPs), which bind and inactivate MMPs in the intercellular space, might block a number of steps of invasion and metastasis.

Another metastasis suppressor gene encodes the KAIl/CD82 protein, which weaves its way back and forth four times through the plasma membrane. Its expression has been found to be substantially repressed in many advanced lung, pancreatic, prostate, colon, and gastric carcinomas. A poor prognosis for breast cancer patients is associated with low KAIl expression in their cancer cells. In cultured cells, KAII suppresses migration and invasiveness and, at the same time, enhances their aggregation with one another. Its location near adherens junctions is compatible with its playing a role in cell-cell adhesion. KAII has also been reported to act as an antagonist of EGF receptor signaling.

Another gene of interest encodes the KISSI protein, which has been identified tentatively as a ligand of a cell-surface G-protein-coupled receptor (GPCR; see Section 5.7) . Ectopic expression of the KISSl gene in tumor cells suppressed their metastatic tendencies without affecting the growth of these cells in pri­mary tumors. Like several others in this class of genes, its precise biochemical role in metastasis suppression is poorly understood.

There are yet more candidates for suppressors of metastasis. The Rho guanine nucleotide dissociation inhibitor-2 (RhoGDI-2) is a negative regulator of Rho proteins, which acts by sequestering the GDP-bound forms of these proteins in the cytoplasm, thereby preventing them from remodeling the actin cytoskeleton (Section14.7). The expression of its encoding gene was found to be correlated inversely with the invasive tendencies of a large group (105) of bladder carcino­mas. Given the critical roles of Rho-like proteins in -cell motility and invasive­ness, RhoGDI-2 becomes an attractive candidate for being an inhibitor of can­cer cell invasiveness and metastasis.

The breast cancer metastasis suppressor-l (BRMS-l) gene was identified because of its decreased expression in breast cancer metastases. Its ectopic expression in breast carcinoma and melanoma cells suppressed their metasta­tic tendencies without affecting their tumorigenicity. It has been reported to increase the gap-junctional communication between cells, which involves channels that allow adjacent cells to exchange molecules of molecular weight less than about 103. At the same time, the BRMS-l protein has been found in the nucleus as part of a complex of proteins involved in chromatin remodeling. Clearly, these disparate roles will need to be reconciled in the future .

Research on metastasis suppressor genes is still in its infancy, and in most cases, clear and definitive molecular mechanisms have yet to emerge. Some of the genes in this category, including those specifying E-cadherin, RhoGDI-2, and 644

Micrometastases and clinical relapse

TIMPs, produce proteins that are part of the known biochemical mechanisms of invasion and metastasis. The biochemical connections between many of the other candidate metastasis suppressor proteins and malignant cell phenotypes are less apparent. Until these genes have been found to be inactivated in human tumor cell genomes, either by mutation or promoter methylation, their involve­ment in regulating the malignant phenotypes of these cells will remain unclear.

14.12 Occult micrometastases threaten the long-term survival of cancer patients

Throughout this chapter, we have read repeatedly about the extraordinary inef­ficiency with which metastases are produced. Some of this metastatic ineffi­ciency is created by the profound difficulties that cancer cells experience as they undertake the initial steps of the invasion-metastasis cascade. Most of those that do manage to reach distant sites and survive in their newfound homes fail to form clinically detectable metastases. The result is the presence of myriad micro metastases seeded throughout the tissues of many cancer patients.

While micrometastases are, with rare exception, unable to expand to form clin­ically detectable metastases, they do provide clear indication that a primary tumor has seeded cells throughout the body. These micro metastases represent a threat to the long-term survival of cancer patients, if only because some of them may erupt into full-fledged, clinically significant macroscopic metastases years after they become implanted in some distant tissue site. Breast cancers are notorious for yielding relapses one and even two decades after the primary tumor has been removed and the patient has been declared to be free of cancer.

In one study of breast cancer patients, micrometastases were detected by sam­pling the bone marrow of the iliac crest of the pelvis. About 1 % of a population of patients suffering from nonmalignant conditions showed cytokeratin-positive cells (i.e., epithelial cells) in their marrow. In contrast, 36% of breast cancer patients carrying tumors of stages I, II, or III had such micro metastases in their marrow. The presence of these micro metastases in the marrow proved to be a highly useful prognostic marker for the risk of relapsing with clinically detectable metastasis (Figure 14.50A). Thus, within four years, one-quarter of the marrow­positive patients had died from cancer, while only 6% of those lacking cancer cells in their marrow had died from this disease. Overall, the presence of micro metastases in these patients represented about a 4-fold increased risk of eventual relapse or death from this disease. Another study found a more than 10­fold increased risk of death from breast cancer among those whose marrow car­ried micro metastases composed of single cells or small clumps of cancer cells.

Colon cancer patients who have undergone resection (surgical excision) oftheir primary tumor will often appear in the cancer clinic a year or two later with a small number of metastases in their liver but none elsewhere; these can then be removed surgically, often with significant clinical benefit. Once again, micro metastases in the marrow of the pelvis can be scored. About 90% of those who lack these micro metastases are still alive 15 months later, while only 30% of those who carry such micrometastases survive to this point (Figure 14.50B).

A procedure used to treat cancer of the esophagus provides yet another insight into metastatic spread. These tumors are often treated surgically, which neces­sitates the removal of one or more rib segments, from which marrow can be easily flushed. Two independent studies reported that 79% and 88% of these patients, respectively, harbored carcinoma micro metastases in their rib mar­row at the time of their surgery. These numbers, which contrast with the approximately 30% of initially diagnosed breast cancer patients with micro metastases, correlate with the far grimmer prognosis for the patient suf­fering this type of cancer.

(A)

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breast cancer

micrometastases

12 24 36 48 months after surgery

colon cancer

no I micrometastases

micrometastases

o 0.2 0.6 1.0 1.4 years since entry

Figure 14.50 Micrometastases and clinical prognosis (A) This Kaplan­Meier plot presents the proportion of breast cancer patients who survive free of distant macroscopic metastases as a function of the elapsed time following initial surgical treatment of their primary tumors. The patients w ith micrometastases in the bone marrow at the time of surgery (red line) suffered from a far higher relapse rate than those who lacked such micrometastases (blue line). (B) Strikingly similar patterns characterize the probability of survival of a group of 54 colon cancer patients, all of w hom exhibited no apparent metastases outside of the liver at the time of preoperative diagnosis; all were treated surgically to remove large metastases from the liver. Those whose bone marrow showed no cytokeratin­positive metastatic cells had a far more favorable clinical course (green curve) than those whose marrow did indeed carry such cancer cells (red curve). In this case the fraction of patients surviving (ordinate) is plotted versus the time in years since they were initially treated and entered into a clinical study. (A, from S. Braun, K. Pantel, P. Muller et aI., N. Eng!. J. Med. 342:525-533, 2000; B, courtesy of RA Tollenaar.) 645

Chapter 14: Moving Out: Invasion and Metastasis

Sidebar 14.11 Are all micro­metastases truly dormant? The dormancy of micrometastatic disease may often be an illu­sion. Thus, we can imagine that in many patients with "minimal residual disease" following removal of their primary tu­mors, micrometastatic clones occasionally acquire the ability to colonize and thereby create a new cascade of metastatic dis­semination and disease relapse (see Figure 14.11). Importantly, such a Ipajor alteration of cell phenotype (and possibly an underlying change of genotype) has a low probability per cell generation of occurring in pop­ulations of nongrowing, dor­mant cells. Instead, extensive observations made over many years' time indicate that such changes happen spontaneously only in proliferating cell popula­tions. Hence; in many patients with minimal residual disease, some clones of micro metastatic cancer cells must be passing through repeated growth-and­division cycles and occasionally spawning variants that have, through some random accident, acquired colonizing ability. (These micrometastases may remain clinically inapparent for many years simply because the rate of cell proliferation in these growths is balanced by an equal rate of cell attrition by apopto­sis.)

The melanoma literature provides equally dramatic testimony of the long-term dangers posed by occult, dormant micro metastases (i.e. , those that are hidden' and apparently not growing). In one particularly well documented case, kidneys were prepared for organ transplantation from the cadaver of a patient who had undergone resection of a small melanoma 16 years earlier. The patient had been followed closely for 15 years after removal of this small primary tumor and had remained symptom-free. However, soon after transplantation, the two recipi­ents of his kidneys de\'eloped aggressive melanomas that were directly traceable to this donor.

The mechanism that prevent micrometastases from erupting into clinically threatening grmnhs are poorly understood. In some instances, one can observe micrometasta e growing as cuffs around small vessels; this suggests that they lack the ir m\TI angiogenic capabilities but are nonetheless able to take limited advantage of host capillaries that happen to be nearby. In the great majority of micromerasta e'5 found in the marrow, the involved cells lack any indication of cell proliferation markers and thus are in a nongrowing, Go-like state for extended periods of time (see Figure 14.12), perhaps for months and even years (see, howe 'er, idebar 14.11). (Because such cells are nongrowing, they may be especially re i tant to chemotherapeutic treatment designed to eliminate the residual disea e that persists following surgical removal of a primary tumor.)

Inun .e mechanisms may also contribute to suppressing the growth of microme:asrases, thereby preventing metastatic disease relapse. This is sug­gested _- the occasionally observed explosive growth of aggressive metastatic tumor- - immunosuppressed organ transplant recipients. In addition, the phe­nomenon of tumor stem cells may help to explain the inability. of the great majorj~- of initially seeded micro metastases to generate macrometastases ( 'de .., - 3-1 . ). Beyond this, relatively little is known about the mechanisms mat preclude most micrometastases from successfully colonizing the tissues in \\-hicb ibey ha e landed.

1 .1 ~ Synopsis and prospects

Like all orner biological phenotypes, those contributing to invasion and metas­tasi musr be directed by the actions of genes. Several major issues have compli­cated me earch for the genetic determinants of these aggressive phenotypes of cancer: Are the e phenotypes programmed by a small number of pleiotropically actina, ill rer control genes, or do the actions of multiple genes collaborate to create each of these phenotypes? Do these genes undergo mutation during tumor pro!!Ie!>~ ion, or do they become involved in the late steps of tumor pro­gres ion through epigenetic mechanisms that control their expression? Are there rna rer conITol genes that are specialized to program the phenotypes of im -asion and mera tasis, or are these behaviors the by-products of the actions of familiar oncogene and tumor suppressor genes?

\Vhile the genetic mechanisms involved in metastasis remain unclear, some progress is being made in solving another puzzle: Are the cells within a primary human tumor that undertake invasion and metastasis rare variants (among the larger population of tunlor cells) that have, through some genetic or epigenetic accident. acquired the abili ty to execute these steps? Or are all the cancer cells within certain primary tumors equally capable of invading and metastasizing (albeit vvith extraordinarily low efficiency), while the great majority of the can­cer cells in other tumors lack these abilities? Recent analyses of the gene expres­sion patterns of various human tumors have lent some weight to the second mechanism. Still, this issue remains a bone of much contention.

One such study indicates that the tendency to metastasize is associated with a particular pattern of gene expression in some but not other human breast can­cers. Moreover, this expression pattern is manifested by the bulk of the cells in 646

Synopsis and prospects

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Figure 14.51 Use of expression arrays to predict disease progression Gene expression analyses with microarrays make possible the simultaneous monitoring of the expression of thousands of genes to determine a specific pattern or expression signature that IS correlated with a specific phenotype or set of phenotypes. (A) In this expression array analysis, genes that are expressed at high levels are in red, while those that are expressed at low levels are in blue. RNAs prepared from 64 primary adenocarcinomas (from various tissues) and 12 metastatic nodules of adenocarcinomas (arrayed across the top) were analyzed (black, red bars, respectively). Of the thousands of genes analyzed in an initial gene expression array (not shown), the expression of 128 genes (arrayed vertically here) was found to be associated­because of over- or under-expression-with metastasis (vertical red, black bars, respectively). Further distillation of the data yielded a set of 17 genes whose expression was as useful as that of the larger 128 gene set in distinguishing metastases from primary tumors. Importantly, this metastasis-specific expression signature was found to be exhibited by a small subset of the initially analyzed primary tumors, suggesting that it could be used to predict the metastatic tendencies of other groups of human tumors. (B) When researchers used the metastasis expression signature of panel A to analyze the gene expression patterns of other groups of primary tumors, they w ere able to separate the patients bearing adenocarcinomas of the breast (I ) and prostate (II) as well as medulloblastomas (III) into two groups (blue, red lines) having markedly different times to clinical progression or relapse following initial surgery. However, the clinical progression of lymphomas (IV) was not predicted using the metastasis­specific expression signature, suggesting that a common set of genes is involved in mediating metastasis by solid tumors and other, unknown genes mediate malignant progression and metastasis by hematopoietic tumors. (From S. Ramasw amy, K.N . Ross, E.S. Lander and TR. Golub, Nat. Genet. 3349-54, 2002 .)

each of a group of primary tumors, rather than by a small subset of cells within each tumor (Figure 14.51). This suggests that the proclivity to metastasize was developed during the course of the multi-step progression that culminated in

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647

Chapter 14: Moving Out: Invasion and Metastasis

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ILNmeta 17 / Prim S

derived metastases Gene expression Prim 12 LNmeta 14 arrays can be used to classify different 0.4 LNmetaS ~ primary tumors and derived metastases Prim 17 _ Prim 16 according to their respective gene expression profiles (e.g., see Figure 0.2

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1451 ) If this is done, the degree of Prim 6 Prim 3 LNmeta 12 Prim 14 similarity between pairs of biopsies can be calculated using statistical methods. (A) As seen here, the biopsies of primary tumors and derived lymph nodes from a group of patients (each patient identified by a number) have been placed on a two-di mensional map, in which proximity indicates similarity in gene expression patterns. This revea ls that the great majority of primary tumors (Prim) map closely to their derived lymph node metastases (LNmeta), indicating similarity in gene expression patterns. (B) Alternatively, gene expression patterns can be used to map the relatedness of primaries and deri' ed metastases, where the degree of relationship is placed on a dendrogram-i.e ., the most closel related tumor samples are placed near one another on the sa me or neighboring branches. Once again , the gene expression pattern of a metastasis 5,

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the parental primary tumor from ./ ·co it arose. (Courtesy of B. Weigelt and L.J. van't Veer.)

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primary tumor formation, not afterward by a small, specialized subpopulation of cancer cells within a primary tumor. (lfthe ability to metastasize were limited to only a small minority of cancer cells in the primary tumor, their gene expres­sion pattern would not significantly influence the expression pattern of the rumor cell population as a whole, and this larger population would therefore not manifest a metastasis-prone gene expression signature.)

:illother analysis indicates strong resemblance between the gene expression panern ofthe bulk of the cells in a primary tumor and that present in its derived me[as(ases; this held true for 15 pairs of primary tumors and associated metas­[ase- (Figure 14.52). If further validated, this means that cancer cells in the pri­mary rumor do not need to undergo major changes in gene expression in order to me[as(a ize and colonize distant sites.

The idea tha[ the great bulk of cells in certain primary tumors are capable of metastasizing recei es further support from the phenomenon of "cancer of unkno\\11 primary" (CUP) , which constitutes as much as 5% of total cases in the oncology clinic. In these cases, metastatic growths represent the first clinical inclication that a patient is harboring cancer. In about 30% of these patients, the primary tumors are never detected, while in the remaining 70%, these primaries are found only upon autopsy.

In the great majority of these CUP cases, the primary tumors are likely to have been very small when they began seeding metastatic cells. Given the profound 648

inefficiency of metastasis (possibly as low as a 10-6 to 10-8 probability of success) and the small sizes of these primary tumor cell populations, it seems plausible that the great bulk of cells in these primaries were capable of seeding metasta­tic progeny. And, as before, it seems likely that the genetic evolution that yielded the tumorigenic cells in these primary tumors simultaneously created cell pop­ulations, all of whose members had the ability to disseminate their progeny to distant sites in the body.

While none of these studies represents an unassailable proof, they provide sup­port for the argument that metastatic cells are drav\TI1 from the general popula­tion of cells in a primary tumor rather than from small, specialized, genetically unrepresentative subclones of cells. If this thinking is eventually validated, it would suggest that the reason why relatively few macroscopic metastases are formed is not because there are few cells within primary tumors that are intrin­sically competent or qualified to metastasize. Instead, the majority of cells in certain primary tumors may be endowed with metastatic ability, and many of these cells may journey to distC!nt anatomical sites. However, the actual number of macroscopic metastases that are eventually formed may be severely limited by metastatic inefficiency. Some speculations about the genetic evolution of metastatic competence further reinforce this notion (see Sidebar 350 ).

Contrary results derive, however, from experiments in mice in which primary tumors have been allowed to metastasize, for example, to the lungs. As described in this chapter, the cells isolated from these pulmonary metastases have been found, time and again, to have greater powers to metastasize to the lungs than do the bulk of cells in the primary tumor. Such observations argue strongly that these metastatic cells are at least marginally different from most cells in the primary tumor and that their genotypes or gene expression pro­grams facilitate greater metastatic ability. This suggests, in turn, that these metastatic cells are indeed representative of only a minority subpopulation in the primary tumor and are likely to be variants that arise relatively late in tumor progression.

Whether one or the other model of metastasis is ultimately validated, it is clear that the identities of the genes that are specifically involved in programming metastasis have been elusive. In addition, it has been difficult to learn precisely how certain genes become functionally activated during the multi-step cascade of invasion and metastasis. Experimental resolution of these problems is con­founded by complications at every level.

1. The first dimension of difficulty derives from the complexity of the inva­sive-metastatic process. Given the distinct steps that are involved in this process, are there a comparable number of genes involved? Or do metas­tasizing cancer cells activate and exploit latent, preexisting developmental programs (Section 14.3), such as the epithelial-mesenchymal transition (EMT), which simultaneously impart an ability to execute mUltiple steps, including breaching of the basement membrane, invasion into the stroma, intravasation, and extravasation (Sidebar 14.12)?

2. Experimental analyses are complicated by the inefficiencies of the metastatic process when compared with other steps in tumor pathogene­sis. Even when cancer cells have purportedly acquired the genotype and phenotype enabling metastasis, they succeed in metastasizing with extraordinarily low efficiency. Such a weak connection between genotype and measurable phenotype derails most currently available experimental strategies.

3. Yet another complexity arises from the apparent collaboration of genetic and epigenetic factors in creating the metastatic trait. Recall , for example, that in certain experimental models of cancer, the epithelial-mesenchy­mal transition (EMT) is achieved when ras-transformed cells are exposed to TGF- ~ (Section 14.4). This transition, which may operate in many

Synopsis and prospects

Sidebar 14.12 Does the inva­sion-metastasis cascade need to be reconfigured? The inva­sion-metastasis cascade is often portrayed as a succession of six distinct steps (localized inva­siveness' intravasation, translo­cation, extravasation, micro­metastasis formation, and colo­nization; see Figure 14.4). This scheme might imply that cancer cells must acquire the ability to execute each of these steps through a comparable number of genetic or epigenetic alter­ations of their genomes. How­ever, the pleiotropic powers of EMT-inducing transcription fac­tors (TFs) and evidence gath­ered on the genetic evolution of micrometastases (see, for exam­ple, Sidebar 14.4) may soon

. reshape the depiction of the invasion-metastasis cascade. In many tumors, a three-step process may be a far more appropriate description. The first step would involve the acti­vation of expression of one or several ofthese TFs and the con­comitant acquisition of an abil­ity to invade, intravasate, translocate to a distant site, extravasate, and form a micrometastasis. Thus, many primary tumors (e.g., 30% of human breast cancers) may be able to seed micr6metastases even when these tumors are quite small, because the great majority of the cells in these tumors express one or more of the EMT-inducing TFs. The sec­ond step would involve acquisi­tion of colonizing ability, which may be achieved only by rare micrometastases, often years after they were seeded in various tissue sites throughout the body. The third step would occur when such a micrometastasis, no\\" grown into . a macrometastasis, showers the body ,;vith metasta­

. tic cells that are capable of colo­nization, thereby generating clinical relapse with widely dis­seminated metastatic disease.

649

Chapter 14: Moving Out: Invasion and Metastasis

human carcinomas and enable their invasiveness, can be triggered by spe­cific signals that genetically altered cells encounter in some tissue micro environments but not in others. Hence, in these cases, invasion and subsequent metastasis can hardly be portrayed as genetically templated traits and, for this reason, are not readily studied by commonly used exper­imental techniques.

4. In many tumors, the genes and proteins that participate directly in pro­gramming invasion and metastasis may be expressed only at the invasive edges of primary tumors (see Figure 14.19), and the cancer cells in these invasive edges may represent only a tiny fraction of the neoplastic cell pop­ulations in these tumors. This enormously complicates experiments designed to reveal the biochemical and genetic bases of invasiveness and metastatic ability, which often rely on analyzing bulk populations of can­cer cells prepared from large chunks of surgically resected tumors.

5. Carcinomas constitute the most common class of human cancers, and the neoplastic epithelial cells within these tumors may need to undergo an EMT in order to become invasive and metastatic. However, if invading car­cinoma cells pass through a complete EMT and shed all epithelial traits, they become the proverbial "wolves in sheep's clothing," since most com­monly used histological analyses are unable to distinguish these cells from the non-neoplastic mesenchymal cells of the tumor-associated stroma. (Indeed, this difficulty explains why many tumor pathologists deny the very existence of the EMT as the key process in the development of carci­noma invasiveness.)

6. Metastatic dormancy creates another experimental problem. In the case of breast cancers, for example, metastases may suddenly appear as long as 20 years after the initial primary tumor has been removed. Because of this long latency period and the sheer number of micrometastases carried by many patients, it has been difficult to learn how only a few of them sud­denly acquire the ability to mushroom into macroscopic, life-threatening tumors.

These experimental difficulties have greatly retarded the progress of invasion-metastasis research, leaving many simple yet fundamental questions unanswered. For example, are there really genes that are specialized to impart an invasive or metastatic phenotype to cancer cells (see Sidebar 35 . )? And in the same vein, are there specialized metastasis suppressor genes (Section 14.11) that must be inactivated before a population of tumor cells can acquire invasive and/ or metastatic ability? Or do the genes and proteins that affect metastasis operate as components of the regulatory circuits that we have repeatedly encountered throughout this book, namely, the circuits governed by the prod­ucts of oncogenes and tumor suppressor genes?

The tissue tropisms of metastasizing cancer cells-their tendencies to colonize some but not other organs-represent another class of unsolved problems. Some insights have been gained from the substantial advances in understand­ing the detailed mechanisms of osteotropic metastasis, as described in Section 14.10. However, this class of metastases represents a rare exception, in that, in general, we know almost nothing about the functionally important interactions of disseminated cancer cells with the other tissues in which they settle and col­onize. This is beginning to change (Sidebar 14.13).

The existence of micro metastases represents a major challenge for clinicians who would like to prevent disease relapse years after the primary tumor has been eliminated. Micrometastases of less than 0.2 mm diameter may carry sev­eral hundred to several thousand cells, and their detection in an organism car­rying approximately 5 x 1013 cells represents a daunting undertaking. Without eradication, these micro metastases represent an ongoing threat, since some of them may erupt at an unpredictable future time into a lethal growth. 650

Synopsis and prospects

Sidebar 14.13 Some expressed genes within tumor cells facilitate specific types of metastasis One strategy for dis­covering the genes and proteins responsible for specific organ tropisms involves the isolation of tumor cells that show preference for colonizing one t arget organ but not another. By retrieving resulting metastatic cells from that organ, propagating them in uitro, and re-injecting them into host mice, followed, once again, by isolating metasta­tic cells from that organ, it is possible to select clones of cancer cells that stably express a highly specific tropism for that organ.

Alternatively, single-cell clones (i.e., clo'nal cell popula­tions each descended from an isolated cell) can be pre­pared from a heterogeneous population of cells present in

a human cancer cell line. The gene expression profile of each can then be analyzed, and independently, its ten­dency to form metastases in one or another target organ can be determined. This can lead to the identification of certain genes whose expression in a cancer cell is corre­lated with the metastatic tropism of that cell and may even contribute causally to this behavior (Figure 14.53). Indeed, ectopic expression of a group of such genes in otherwise poorly metastatic clonal cell populations can induce these cells to exhibit potent osteotropic metastasis. Such experi­ments also indicate that within a heterogeneous tumor cell population, various preexisting gene expression patterns can influence individual cells to exhibit a variety of metastatic behaviors.

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Figure 14.53 Gene expression patterns and metastatic tropism Thirty-three single cells were picked from a large population of human MDA-MB-231 cells (see Figure 14.49), and each was expanded into a clonal population in culture. The mRNA expression pattern of each subclone was analyzed (columns, arrayed left to right) using probes for the mRNAs of a series of five genes- IL II (interleukin-11), OPN (osteopontin), CTGF (connective tissue growth factor), CXCR4 (chemokine receptor 4), and MMPI (matrix metalloproteinase-1 )-and, a sixth, as loading control, GAPDH (g lyceraldehyde-3-phosphate dehydrogenase) mRNA. In addition, the expression patterns of the original tumor cell population (ATCC, left column) and a subcloned cancer cell population termed 2287 (which was selected for its ability to generate osteolytic metastases; 2nd column) were analyzed. The five experimental genes were chosen because of their overexpression in osteotropic metastatic cell s and their known biological properties in promoting

osteolytic metastases. As seen here, clone 2 cells (red box), when injected into the arterial circulation, showed a tendency to produce osteotropic metastases, as indicated by in vivo imaging of the tumors developed by these mice; these cells expressed high levels of all five experimental mRNAs. Clone 3 cells (yellow box), in contrast, expressed low levels of all five mRNAs and preferentially formed lung metastases. Af1d clone 26 genes (yellow box), which expressed almost none of these mRNAs, formed no metastases at all. Moreover, when otherwise poorly metastatic cells were forced to express combinations of three of these genes, they acquired the ability to form bone metastases efficiently (not shown), pointing to their causal role in forming these metastases. Metastases were visualized through the presence of a luciferase gene in the tumor cells, which causes these cells to release a bioluminescent signal. (From Y. Kang, P.M. Siegel, W. Shu etal., Cancer Cell 3:537-549, 2003.)

651

Chapter 14: Moving Out: Invasion and Metastasis

This issue leads directly to another: Can the therapies used to treat primary tumors also be used to treat their metastatic derivatives? Or are metastatic cells so different from their progenitors in the primary tumor that they require their own customized therapies? In fact, the expression array analyses indicating sub­stantial similarity betvveen the gene expression profiles of primary tumors and their metastatic offshoots (see Figure 14.52) provide some hope. Such similari­ties make it plausible that metastatic cells will often be found to respond to the same therapies that succeed in attacking and destroying the primary tumors from which they derive.

A deep understanding of the processes leading to the formation of metastases is surely critical for the future development of cancer cures. For the moment , how­ever, the questions and unresolved issues listed above give us pause, because they shQ\;\ how little we understand about the details of the metastatic cascade and why, in the eyes of many, invasion and metastasis represent the remaining major challenges of basic cancer research.

To end, we go back to the beginning of this chapter: if, as experimental evidence increasingly hows, the epithelial-mesenchymal transition is a critical event in the acquisition of invasiveness, and if cancer cells resurrect embryonic tran­scription factors to acquire these traits, then Lewis Wolpert's statement might require red ion, in that gastrulation and the associated EMT might well loom as one of the most dangerous events in our lives!

Key concepts • Invasion and metastasis are responsible for 90% of cancer-associated mortal­

ity, and the majority of cancer cells at the time of death may often be found in metastases rather than the primary tumor.

• The in\'asion- metastasis cascade involves local invasion, intravasation, trans­port, extra\'asation, formation of micro metastases, and colonization .

• The sequence of steps in this cascade is completed only infrequently, result­ing in metastatic inefficiency. The least efficient of these steps appears to be colonization.

• Many ofthe e steps can be executed by carcinoma cells that activate a cell-bio­logical program called the epithelial-mesenchymal transition (EMT) , which is normally used by cells early in embryogenesis and during wound healing.

• The EMT can be programmed by pleiotropically acting transcription factors that are normally involved in various steps of early embryogenesis.

• Signals released by the stromal microenvironment of a cancer cell, operating together \.vith genetic and epigenetic alterations of the cancer cell genome, are often responsible for inducing expression of the EMT-inducing transcription factors in the cancer cell and thus the EMT.

• The EMT involves 10 s of an epithelial cell gene expression program and acquisition of mesenchymal gene expression. The latter enables cells to acquire invasiveness and motility.

• Cell motility is regulated by a series of small G proteins of the Rho family that are activated by cytoplasmic signal-transducing pathways and control the assembly of the actin cytoskeleton.

• Cell invasiveness is controlled in large part by various matrix metallopro­teinases (MMPs) that function to degrade components of the extracellular matrix. These enzymes are often manufactured by cells within the tumor­associated stroma.

• Metastatic cancer cells may travel via the lymph ducts to nodes. However, their spread via the blood circulation is responsible for the great majority of distant metastases. 652

Additional reading

• Many cancer cells that are carried through the circulation form micro thrombi that lodge in the arterioles and capillaries of various tissues.

• The ability of cancer cells to extravasate may depend on many of the same activities that were used earlier to execute invasiveness and intravasation.

• While the great majority of earlier steps of the invasion-metastasis cascade are likely to be similar in various types of human tumors, the last step-colo­nization-is likely to depend on complex interactions between various types of metastasizing cells and the microenvironments of the host tissues in which they land.

• The details of colonization are well understood only in the context of osteotropic metastases, especially the osteolytic metastases initiated by breast cancer cells.

• In some cases, the metastatic tropisms of cancer cells can be explained by the organization of the circulation between the primary tumor site and the target site of metastasis. However, in many other cases, the reasons why cancer cells metastasize from primary tumors to certain target organs are poorly under­stood.

• The acquisition of invasive and metastatic powers does not appear to involve major changes in the genotype of cancer cells within the primary tumor.

Thought questions

1. What arguments can be m ustered for or against the notion cell metastases? In what way do these supports affect the that invasion and metastasis are likely to be orchestrated by ultimate success of the colonization procedure? specific mutant alleles that are acquired by cancer cells late 6. How might primary tumors exhibit metastatic powers as in tumor progression? soon as they form?

2. What explanations can be offered for the inefficiency of col­ 7. Would the ability to prevent metastasis have demonstrable onization by the cells within micrometastases? effects on the clinical course of some human tumors but

3. What arguments suggest that the ability to metastasize is not others? expressed either by the bulk of cancer cells in a primary 8. What evidence supports the involvement of the EMT in tumor or only by a minoriry of cells that are specialized to human tumor pathogenesis, and what evidence argues do so? against it?

4. What evidence suggests that genetic and phenotypic evolu­ 9. How might the ability to accurately determine the progno­tion of cancer cells can occur in sites within the body that sis of a diagnosed prostate or mammary tumor lead to dra­are far removed from the primary tumor? matic changes in the practice of clinical oncology?

5. What specific types of physiologic support may be supplied by tissues that are frequently the sites of successful cancer

Additional reading

Akhurst RJ & Balmain A (1999) Genetic events and the role of TGF­ Braun S, Pantel K, Muller Pet al. (2000) Cytokeratin-positive cells in ~ in epithelial tumor progression . I Pathol. 187, 82-90. the bone marrow and survival of patients with Stage I. II or III

breast cancer. N. Engl. I Med. 342, 525-533. Barrallo-Gimeno A & Nieto MA (2005) The Snail genes as inducers of ce ll movement and survival: implications in development and Brinkerhoff CD & Matrisian LM (2002) Matrix metalloproteinases: a cancer. Development 1 32.31 51-3161 . . tail of a frog that became a prince. Nat. ReIC Mol. CeJl BioI. 3,

207-214.Birchmeier C. Birchmeier W, Gherardi E& Vande Woude GF (2003) Met. metastasis, motility and more. Nat. ReIC Mol. Cell BioI. 4, Bray D (2002) Cell Movements. New York Garland Science . 915-925 Burridge K & Wennerberg K (2004) Rho and Rac take center stage.

Blasi F & Carmeliet P (2002) uPAR: a versatile signalling orchestrator. Cell 116, 167-179. Nat. ReIC Mol. Cell BioI. 3, 932-943. Cavallaro U & Christofori G (2001) Cell adhesion in tumor invasion

Brabletz B, lung A. Spaderna S et ai, (2005) Migrating cancer stem and metastasis: loss of the glue is not enough. Biochim. Biophys. cells-an integrated concept of malignant tumour progression. Acta/Revs. Cancer 1552. 39-45. Nat. ReIC Cancer 5.744-749 653

Chapter 14: Moving Out: Invasion and Metastasis

Cavallaro U & Christofori G (2004) Cell adhesion and signaling by cadherins and Ig-CAMs in cancer. Nat. Rev. Cancer 4, 118-132.

Chambers AF, Groom AC & MacDonald IC (2002) Dissemination and grow th of cancer cells in metastatic sites. Nat. Rev. Cancer 2, 563-572.

Comoglio PM & Trusolino L (2002) Invasive growth: from development to metastasis. J. Clin. Invest. 109, 857-862 .

Condeelis JS (2001) Lamellipodia in invasion. Semin. Cancer BioI. 11,119-128.

Conway EM & Carmeliet P (2003) Cardiovascular biology: signalling silenced. Nature 425,139-141.

Coussens LM, Fingleton B & Matrisian LM (2002) Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 295, 2387-2393 .

Coussens L & Werb Z (2002) Inflammation and cancer. Nature 420, 860-867.

deCaestecker MP, Piek E & Roberts AB (2000) Role of transforming growth factor-beta signaling in cancer. J. Nat!. Cancer Inst. 92, 1388-1402.

Derynck R, Akhurst RJ & Balmain A (2001 ) TGF-beta signaling in tumor suppression and cancer progression. Nat. Genet. 29, 117-129.

Egeblad M & Werb Z (2002) New functions for the matrix metalloproteinase in cancer progression. Nat. Rev. Cancer 2, 161-174.

Feldner J & Brandt BH (2002) Cancer cell motility-on the road from c-erbB-2 receptor steered signaling to actin reorganization. Exp. Cell Res. 272, 93-108.

Fidler IJ (2001) in Cancer Principles and Practice of Oncology (VT. DeVita, jr., S. Hellman, SA Rosenberg eds), pp 135-152 . Philadelphia: Lippincott-Raven.

Fidler IJ (2001) Seed and soil revisited: contribution of the organ microenvironment to cancer metastasis. Cancer Metastasis: Bioi. Clin. Aspects 10, 257-269.

Fidler IJ (2003) The pathogenesis of cancer metastasis the "seed and soil" hypothesis revisited. Nat. Rev. Cancer 3, 453-458.

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