Invasion and Metastases tumor cell

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Invasion and MetastasesWilliam G. Stetler-StevensonMetastasis formation is the spread of cancer cells from a primary tumor to vital organs anddistant sites in the cancer patient's body. This process is the end result of a complex series ofgenetic alterations epigenetic events and host responses. The tendency of a primary tumor to

form a metastasis is the hallmar! of malignant cancer. Metastasis formation associated "ithmalignant transformation has significant diagnostic prognostic and therapeutic implications.#fter a diagnosis of cancer the primary tumor is resected and histologic examination of thistissue is performed for evidence of metastatic potential $local invasion%. The patient alsoundergoes clinical staging for evidence of metastatic involvement of other organs. Ma&or factorsdictating the course of cancer treatment are the malignant potential of the primary tumor $i.e. presence of local invasion and regional lymph node involvement% and the presence of distantmetastases. ne of the ma&or obstacles to effective cancer diagnosis is the detection of clinicallyoccult metastatic disease. nce the primary tumor is resected cancer therapy is directed at theelimination of metastases. The variation in si(e age dispersed anatomic location andheterogeneous composition of metastases ma!es complete eradication of metastatic disease by

currently available therapeutic strategies extremely difficult. )atients "ith metastases succumb toorgan failure secondary to anatomic compromise of organ function by nonfunctional tumor tissueor to complications associated "ith systemic therapy directed against the metastatic disease.The principal ob&ectives of current research in cancer invasion and metastasis are improvementof diagnostic mar!ers for detection of malignant potential and clinically silent $occult% metastasisformation as "ell as the design of more effective therapies to treat metastatic disease. Thesegoals re*uire a better understanding of the molecular events cellular processes and hostresponses involved in metastasis formation. The focus of current research efforts is directed atunderstanding the origins of cancer metastasis definition of malignant potential $i.e. delineatingthe metastatic propensity of a primary tumor from any given patient% and identification of genesspecifically associated "ith metastasis formation as potential therapeutic targets.

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• T+M, ),G,SSI #/ GTI0 IST#1I2IT3• )art of =chapter < - Invasion and Metastases=• It is "idely accepted that tumor formation or tumorigenesis re*uires several genetic

alterations either somatic or inherited that confer a selective gro"th advantage to theneoplastic cell population.> This concept is supported by studies on the molecularchanges in oncogenes and tumor suppressor genes that are associated "ith distinct pathologic lesions for example epithelial dysplasia adenoma or carcinoma in situ before the development of invasive carcinoma.?@ /uring tumor development initialrandom genetic alterations result in a tumor cell population "ith a proliferativeadvantage. These tumor cells become the progenitors of a clonal population that

eventually dominates the tumor mass. Subse*uently a second random genetic eventoccurs that is again clonally selected on the basis of improved fitness. In this vie" tumor progression is analogous to /ar"inian selection "ith repeated mutations and subse*uentdominance of the daughter cell population via expression of a phenotypicadvantage89Athat is gro"th autonomy resistance to apoptosis and so forth.>< #s theserandom advantageous mutations are selected they become fixed in future generations ofthe tumor cell population as it is the daughter cell that eventually overta!es theremaining nonmutant tumor cells. 7o"ever this clonal selection is an ongoing processand the dominant

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tumor cell population in the primary tumor changes through continuous accrual of ne"genetic events $4ig. <->%.



4igure <->. 0urrent models of tumor progression and metastatic origins. #5 0lonal progressionmodel of tumorigenesis. 15 0lonal dominance theory of tumor progression. 05 Metastatic variantmodel of tumor progression. /5 0urrent "or!ing hypothesis of tumor progression.

• #nalysis of the age-dependent incidence of most types of cancer suggests that at leastfour to seven rate-limiting stochastic events are re*uisite for tumor formation.B Themutation of specific genes is a highly inefficient process and ac*uisition of the geneticchanges re*uired for tumorigenesis may re*uire ?C to <C years. This is in contrast to thelarge number of genetic alterations that have been documented in human tumors manymore than are li!ely to occur "ithin a human life span. The limited series of mutations

re*uired for tumor formation compared "ith the relatively large number of geneticchanges observed in many cancers suggests that most tumors ac*uire an inherent geneticinstability sometimes referred to as the mutator phenotype.D The ac*uisition of geneticinstability combined "ith an enhanced proliferative capacity "ould provide a mechanismthat accounts for the large number of accrued genetic changes seen in many tumors. It has been suggested that ac*uisition of this genetic instability evidenced by subtle se*uencechanges alterations in chromosome number chromosomal translocations or geneamplification is the engine of tumor progression and of tumor heterogeneity.D Initialstochastic mutations conferring enhanced proliferative capacity are complemented bydefects in /# repair mechanisms $loss of pB@ function% resulting in acceleration of the/ar"inian selection process that leads to clonal expansion. This role for genetic

instability also accounts for the observation that humans and animals "ith inherentgenetic instability $e.g. those "ith xeroderma pigmentosa adenomatous polyposis coli%secondary to defects in /# repair or chromosomal segregation or both are prone todevelopment of cancers. The relative contributions of genetic instability and somaticmutation mechanisms to the process of tumor progression and metastasis remaincontroversial.E



,IGIS 4 MT#ST#TI0 T+M, 022S)art of =chapter < - Invasion and Metastases=The central *uestion "ith regard to metastasis formation and "ith significant clinicalimplications for the diagnosis and treatment of cancer metastasis is ho" metastases arise fromthe primary tumor. T"o dominant hypotheses have been formulated regarding the evolution of

cancer metastases. The follo"ing section briefly revie"s the origins of these hypotheses.Metastasis formation is the final step in tumor progression. This suggests that li!etumorigenesis neoplastic cells "ith metastatic capacity arise through a process analogous to/ar"inian natural selection aided by genetic instability. Initial genetic changes mutations inspecific oncogenes and inactivation of tumor suppressor genes result in ac*uisition of a selectivegro"th advantage that produces a dominant descendent population "ithin the primary tumormass. Subse*uent genetic changes confer additional advantageous phenotypes that include self-sufficiency in gro"th signals $gro"th autonomy% insensitivity to antigro"th signals evasion ofapoptosis sustained angiogenesis limitless replicative potential and the capacity for tissueinvasion and metastasis.F These changes and subse*uent genetic or epigenetic alterations "oulddominate the primary tumor cell population through clonal expansion $see 4ig. <->%. This clonal

progression model is consistent "ith numerous studies demonstrating similar patterns of geneexpression in the primary tumor and metastases derived from the same patient. This model ofmetastatic progression suggests that evolution of metastatic competence is a generic predisposition that can be expressed at any time during the process of tumor development andthat this phenotype "ill predominate in the primary tumor through clonal expansion.In contrast a second model suggests that metastases arise from rare highly metastatic variants"ithin the primary tumor. In this model metastatic competence does not confer a selectivegro"th advantage "ithin the primary so that these rare tumor cells "ith metastatic capacityremain a minor cell population in the primary tumor $see 4ig. <->%. This model is principally based on classic animal model experiments of 4idler et al.>C sho"ing that multiple variants ofmetastatic potential can be isolated from the primary tumor population by subcloning in vitro or by in vivo selection. This is an important concept in that it suggests that metastatic variants89Hpreexist89 in the primary tumor that not all cells of the primary tumor population share the

same propensity for metastasis formation and that metastasis results from selection of anaggressive rare subpopulation of tumor cells from the primary tumor population. 4urthermorethis model of tumor progression suggests that the metastatic potential of the primary tumor"ould be determined by the si(e or behavior or both of this highly aggressive subpopulation.Therefore detection of such rare variant cells "ithin the primary tumor population "ould be ofsignificant).>>B

prognostic value. This "ould suggest that determination of the average metastatic potential of theentire primary tumor cell population by assessment of molecular mar!ers associated "ithmetastatic potential may not reflect the presence of these highly metastatic variants.#re the clonal expansion and rare variant models for the origin of metastatic tumor cellsmutually exclusive or are they t"o conceptual frame"or!s in a continuous spectrum ofmechanisms for metastasis formation To study this experimentally Jerbel>> exploited therandom integration of foreign /# into the tumor cell genome to tag metastatic cells isolatedfrom a primary tumor population. Tumor cell clones tagged "ith different integration sites "eremixed "ith a single tagged metastatic clone and inoculated subcutaneously into syngeneic



experimental animals. The clonal evolution of primary tumors and metastatic foci "ere thenfollo"ed over time by restriction fragment length polymorphism analysis of the foreign /#integration sites. The results of these experiments indicate that a single clone initially present inthe mixture in as lo" as >K to ?K of the tumor cell inoculum gro"s to dominate the primarytumor and that this clone is metastatically competent. This suggested that if a rare metastatic

variant preexists "ithin the tumor cell population it can over time overgro" the primary tumormass. This mechanism is referred to as the clonal dominance theory of cancer progression.>>The underlying implication of these studies is that metastatic competence and gro"th dominancein the primary tumor are someho" lin!ed. If instead of a single metastatic clone mixed "ithtagged primary tumor cells the experiment "as conducted by pooling a large number $>C< or>CB% of metastatically competent tumor cell clones the results "ere similar in that the tumor cell populations in primary and metastatic tumors "ere dominated by a fe" $less than >C% clones.These findings suggest that cells "ithin the primary tumor that obtain metastatic potential alsohave a selective gro"th advantage. It must be noted ho"ever that the seemingly89Hgenetically89 convergent dominant clones although homogeneous "ith respect to the tag

used to identify them are still heterogeneous in other characteristics due to the ongoing process

of genetic instability. These other characteristics may be secondary unrelated to gro"th ormetastatic potential $referred to as carrier mutations% or primary in that they enhance themetastatic phenotype. 4inally the clonal dominance model of Jerbel suggests that the clonal progression and metastatic variant models for the evolution of metastatic tumor cells maycontribute to the development of metastatic tumor cells and that these models are not mutuallyexclusive.The consideration of these models of tumor progression and origin of metastatic tumor cells isincreasingly important for the design of experiments to isolate and identify genes involved intumor metastasis. With the advent of complementary /# $c/#% microarray technologiesresearchers can simultaneously screen for the differential expression of thousands of genes in asingle experiment. 7o"ever to obtain useful information from these experiments "e mustunderstand the potential relationships bet"een primary tumors and metastasis. 4or examplec/# microarray experiments comparing the differential gene expression bet"een a primarytumor and metastatic lesion from the same patient yield different information than an experimentthat compares a series of nonmetastatic and metastatic tumors from different patients.

MI0,#,,#3 ##23SIS 4 T+M, ),G,SSI #/ MT#ST#SIS)art of =chapter < - Invasion and Metastases=The introduction of c/# microarray technology affords investigators the opportunity toexamine changes in expression of thousands of genes in a single experiment. Such experimentshave been used to explore the process of tumor progression and identify genes that may beinvolved in metastasis formation. )atterns of gene expression have been analy(ed for a variety of

primary tumors and their metastases. These studies include microarray profiling of primarytumor cells and metastatic lesions in animal models of tumor progression as "ell as analysis ofhuman tumor samples. The results of these experiments clearly indicate that specific patterns ofgene expression can be associated "ith specific tumor subsets and clinical outcomes. 7o"everthese experiments also present ne" insights into the origins of metastatic tumor cells and therelationship bet"een primary tumors and their metastases.c/# microarray $transcriptome% analysis of primary breast tumors and comparison of the geneexpression profiles of nonmetastatic and metastatic primary tumors result in identification of a



gene expression signature that predicts the probability of metastases.>? In these experiments thegene expression profiles across approximately ?BCCC genes "ere conducted using primary breast cancer tissue from t"o groups of patients5 those in "hom distant metastasis developed"ithin B years and those "ho remained disease free after a period of at least B years. #ll of these patients "ith sporadic breast cancer "ere lymph node negative and under age BB at the time of

diagnosis. The authors found that some BCCC genes "ere significantly regulated across this groupof patient samples and that simple hierarchic cluster analysis revealed t"o distinct groups of patients. In one group only @<K of patients had metastasis "ithin B years in comparison "iththe second group in "hich ECK of patients had metastatic progression. 4rom this study theauthors identified a set of EC genes to establish a prognosis profile that "ould predict clinicaloutcome in node-negative primary breast cancer. #pplication of this gene expression profiledemonstrated that it "as a more po"erful predictor of disease outcome than standard prognosticsystems based on clinical and histologic criteria.>@,esearchers have also compared the gene expression profiles of adenocarcinomas of multipletumor types to unmatched primary adenocarcinomas.>< In this study the investigators examinedthe expression profiles of metastatic nodules and compared these "ith primary adenocarcinomas

of similar tissue origin. These authors found a gene expression signature that distinguished primary from metastatic adenocarcinomas and that "as associated "ith metastasis and poorclinical outcome. 4urther refinement of the initial gene set identified >E uni*ue genes thatrecapitulated the distinction of primary tumors and metastasis across the entire set of tumortypes. #pplication of this subset of >E genes to specific tumor types for example breast cancerlung cancer prostate adenocarcinoma and medulloblastomas $brain tumors% revealed thegeneral utility of this subset in identifying primary tumors that "ere more li!ely to developdistant metastases demonstrating the prognostic value of this approach.>< This study also founda subset of primary tumors in "hich the gene expression signatures "ere identical to themetastatic tumors an observation consistent "ith the clonal evolution model of cancermetastasis.)rimary breast carcinomas and metastases from the same patient may also sho" very similargene expression profiles.>B).>>D

xamination of premalignant preinvasive and malignant mammary lesions obtained by lasercapture microdissection revealed extensive similarities in the gene expression patterns$transcriptome% bet"een these distinct stages of breast cancer progression.>D 0ollectively thesefindings suggest that the molecular program of a primary tumor may generally be retained in itsmetastasis "hich is again consistent "ith the clonal evolution model for the origin of metastatictumor cells. 7o"ever these methods cannot detect rare highly metastatic variants "ithin the primary tumor population and so do not exclude the presence of these variants or theircontribution to metastasis formation.Transcriptome analysis $c/# microarrays% has also been used to examine the changes in geneexpression that are associated "ith in vivo selection of highly metastatic tumor cell variants.>EThese experiments used techni*ues for the isolation of highly metastatic variants for example invivo selection identical to those originally used by 4idler et al.>C )oorly metastatic melanomacell lines either the murine 1>D4C or human #@EB) "ere in&ected intravenously into the tailvein of host mice. )ulmonary metastases "ere isolated and rein&ected via tail vein either t"o$#@EB% or three $1>D% times for selection of highly metastatic tumor cell lines. The gene



expression profiles of pulmonary metastasis "ere compared "ith the parental #@EB) or 1>D4Ccell lines gro"n as subcutaneous tumors. The results define a pattern of gene expression thatinvolved >B genes independent of tumor site that correlate "ith progression to a metastatic phenotype independent of the tumor microenvironment. 7o"ever many of the differentiallyexpressed genes that "ere identified in this study encode extracellular matrix $0M% proteins

suggesting that enhanced expression of specific 0M proteins may promote tumor cell survivalor angiogenesis or both.>E The pattern of transcriptome alterations observed in this animalmodel of progression are distinct and more restricted than those observed in studies of humanclinical material. #lthough this is due in part to the use of tumor cell lines as a starting point italso suggests that many of the cells "ithin the heterogeneous tumorigenic populations of the parental tumor cell lines $#@EB) or 1>B4C% are genetically primed for ac*uisition of metastaticability. This "as demonstrated by additional experiments in "hich introduction of a single gene$,ho0% identified in the original screen bac! into the parental cell population "as sufficient toconfer a high level of metastatic capacity.These findings are supported by a study on the formation of osteolytic bone metastases in breastcancer. Jang et al.>F intravenously in&ected the human breast cancer cell line M/#-M1-?@>

into athymic nude mice "hich resulted in formation of metastasis in bone and the adrenalmedulla. The authors isolated the cells from osteolytic bone metastasis and rein&ected them toobtain cells "ith a stable elevated capacity to form bone metastasis. Transcriptome analysis$c/# microarray% revealed that the parental M/#-M1-?@> cell line derived from a patient"ith metastatic breast cancer demonstrated the poor prognosis gene expression signature previously defined using human clinical samples.>? o enhanced expression of this poor prognosis pattern occurred in the osteolytic metastasis but this comparison did yield a bonemetastasis signature composed of a set of four genes. These four genes included the chemo!inereceptor 0;0,< interleu!in->> connective tissue gro"th factor and matrix metalloproteinase >$MM)->%. Tumor cell populations expressing only one of these four genes "ere not moreaggressive than the parental M/#-M1-?@> cells but expression of any three of the four genesresulted in metastatic activity that "as intermediate bet"een the parental cell and fully metastatic population $expressing all four genes%. These findings suggest that the parental M/#-M1-?@>cell population contains evidence of clonal evolution for the entire primary tumor cell populationto a 89Hpoor prognosis89 pattern of gene expression. 7o"ever superimposed on this poor

prognosis signature are variant cells of high metastatic potential "ith gene expression profiles for metastasis at a specific tissue or organ site. These findings are consistent "ith our hypothesis thatthe 89Hclonal evolution89 and 89Hmetastatic variant89 models of metastatic progression

represent nonexclusive mechanisms for the development of site-specific metastatic tumor cellsand that these models should not be considered mutually exclusive but rather opposite ends inthe spectrum for the origin of tumor metastases.0ollectively c/# microarray data support the concept that clonal evolution and metastaticvariants occur "ithin the same tumor $see 4ig. <->%. The clonal evolution of this primary tumorresults in a poor prognosis profile in tumors "ith enhanced metastatic potential. Superimpositionof additional genetic changes $metastatic variants% on the poor prognosis profile of geneexpression increases the potential for metastasis to a specific site or organ. These findingssuggest that tumor formation and metastasis may be under independent genetic control.

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)art of =chapter < - Invasion and Metastases=It has been demonstrated that transfection of oncogenic se*uences into the correct recipient cellcan result in ac*uisition of an invasive phenotype and metastatic competence. This "as firstdemonstrated by transfection of ,as se*uences into fetal mouse fibroblasts and has beenconfirmed for fibroblast and epithelial cells. Similar findings have been observed "ith

transfection of Mos ,af Src 4es and 4ms. #t first these results might suggest that themetastatic phenotype might arise from genetic alterations associated "ith tumor developmentand progression $tumorigenicity%. 7o"ever not all tumor cells ac*uire metastatic competenceafter oncogene transfection. 4urthermore metastatic competence could be dissociated fromtumorigenicity in ras-transfected rat embryo fibroblasts by adenovirus ?># gene expression.These findings can be explained by the fact that metastasis re*uires activation of additional89Hmetastasis effector genes89 or inactivation of 89Hmetastasis suppressor genes89 over and

above genetic alterations re*uired for tumorigenesis. This concept is also consistent "ith thefindings of Jang et al.>F "hich suggest that primary tumors may present a poor prognosis profile of gene expression but formation of site-specific metastasis re*uires additional changesin gene expression.

Inherent in this concept is the idea that &ust as there are tumor suppressor genes for exampleretinoblastoma gene or von 7ippel-2indau gene there may also be a metastasis suppressor gene.Several metastasis suppressor genes have been identified through their reduced expression inhighly metastatic tumor cells compared "ith tumorigenic but poorly or nonmetastatic tumorcells.> 1y definition reexpression of the metastasis suppressor gene in a tumor cell line resultsin loss of).>>E

metastatic competence in vivo "ithout a significant reduction in tumorigenicity. #t least eighttumor suppressor genes have been identified to date through a variety of methods includingsubtractive hybridi(ation differential display serial analysis of gene expression and mircroarrayanalysis.> These tumor suppressor genes affect many aspects of signal transduction includinggro"th factor receptor signaling cell-cell communication mitogen-activated protein !inase$M#)J% path"ay and transcription. The identity and functions of tumor suppressor genes have been revie"ed in detail else"here.>

MT#ST#TI0 0#S0#/)art of =chapter < - Invasion and Metastases=Investigators refer to the process of metastasis formation as the metastatic cascade $4ig. <-?%.This conceptual frame"or! divides the process into a series of discrete steps that can then beinvestigated for identification of cellular and molecular events re*uisite for metastasis to occur. Itis "ell established from clinical observations and from mechanistic studies that metastasis

formation is an inefficient process. What is the source of this inefficiency



4igure <-?. The pathogenesis of cancer metastasis. To produce metastases tumor cells mustdetach from the primary tumor invade the extracellular basement membrane and enter the

circulation survive in the circulation to arrest in the capillary bed adhere to subendothelial basement membrane gain entrance into the organ parenchyma respond to paracrine gro"thfactors proliferate and induce angiogenesis and evade host defenses. The pathogenesis ofmetastasis is therefore complex and consists of multiple se*uential selective and interdependentsteps "hose outcome depends on the interaction of tumor cells "ith homeostatic factors.2arge numbers of tumor cells and tumor cell clumps are shed into the vascular drainage of a primary tumor.?C It has been demonstrated experimentally that after intravenous in&ection ofhighly metastatic tumor cells only approximately C.C>K of these cells "ill form tumor foci. Thenumber of circulating tumor cells and tumor emboli correlates "ith the si(e and age of the primary tumorL that is larger tumors shed more tumor cells and emboli. 7o"ever the number ofcirculating tumor cells does not correlate "ith the clinical outcome of metastases.The inefficiency of tumor cells in completing the metastatic cascade is in part the result of thefact that successful formation of metastatic foci consists of several highly complex andinterdependent steps. ach step is rate limiting in that failure to complete any of these eventscompletely disrupts metastasis formation. Thus the steps involved in metastasis formation alsorepresent a /ar"inian selection process. nly those tumor cells that have ac*uired sufficientgenetic changes and accompanying alterations in gene expression can successfully complete there*uisite events to allo" metastasis formation.



#nother source of inefficiency in the metastatic cascade is revealed by studies on tumor cellsshedding into the circulation.?> In these experiments highly metastatic tumor cells "ere gro"nsubcutaneously in athymic nude mice and perfused in vivo to collect shed tumor cells. The shedtumor cells "ere collected and analy(ed for clonogenicity in soft agar resistance to apoptosisand ability to form tumors in vivo after in vitro expansion and rein&ection subcutaneously. These

properties of the tumor cells shed into the circulation "ere compared "ith those of tumor cellsisolated directly from the primary tumors by excision and dissociation into single-cellsuspensions. Some"hat surprisingly the authors found that shed tumor cells have a lo"metastatic potential compared to cells isolated directly from the primary tumor. Specifically cellsin shed tumor cell populations sho"ed an increase in apoptosis $<FK% compared "ith an averageapoptotic fraction of ?CK in the native tumors. This suggests that an additional source ofinefficiency in the metastatic cascade is due to the fact that most of the tumor cells shed into thecirculation are in the process of dying as they exit the tumor.?> Thus not all circulating tumorcells represent metastatically competent tumor cells capable of coloni(ing distant tissue sites.#s might be expected from the highly complex nature of metastasis formation no single gene product is exclusively

).>>Fresponsible for metastasis formation. Successful completion of many of the steps of themetastatic cascade is the result of ac*uisition of both positive effectors as "ell as the loss ofnegative regulators. +nrestrained gro"th is not sufficient to result in tumor metastasis. Tumormetastasis is not a passive process secondary to tumor gro"th and re*uires additional geneticchanges other than those associated "ith the tumorigenicity. Tumorigenicity and metastaticcompetence have some overlapping features but are clearly under separate genetic control.ngoing research into the steps of the metastatic cascade has identified gene products that canfacilitate completion of each of the steps outlined above. These are the molecular effectors oftumor metastasis. In many cases research has also identified gene products that function to bloc! successful completion of each of the steps in the metastatic cascade89Athat is metastasissuppressor genes. The idea that there is loss of negative effectors as "ell as positive phenotypicchanges associated "ith malignant progression and metastasis formation is no" "ellestablished.T+M, MI0,6I,MT5 /T,MI#T 4 MT#ST#TI0 )TTI#2 #/SIT 4 MT#ST#SIS)art of =chapter < - Invasion and Metastases=/uring investigation of the molecular events associated "ith specific steps in the metastaticcascade investigators fre*uently use animal models of metastasis. In these assays tumor cellsare either in&ected into experimental animals to form a primary tumor site that subse*uentlymetastasi(es or directly into the circulation to model the later phases of metastasis formation.#nimal models in "hich there is formation of a primary tumor and subse*uent metastasisformation are !no"n as spontaneous metastasis models. Intravenous in&ection of tumor cells bypassing the molecular and cellular events associated "ith tumor invasion focuses oncoloni(ation of the metastatic site and assays that use this techni*ue are referred to as tumorcoloni(ation models. The end point in both types of assay systems is the formation of visiblemetastases at a secondary site and both have led to the identification of specific molecularchanges in tumor cells that contribute to metastatic competence. 7o"ever these end point assays



are unable to examine the role of specific molecules at each individual step in the metastaticcascade.Subcutaneous xenografts of human metastatic tumor cells in nude or severe combinedimmunodeficiency disease mice often fail to recapitulate the behavior of the parent tumor ordemonstrate a spontaneously metastatic phenotype. 7o"ever "hen in&ected intravenously many

of these same human tumor cells are capable of forming metastatic colonies in the lung$coloni(ation assays%. In pioneering experiments 4idler and colleagues demonstrated that the primary tumor site can directly influence metastatic potential through tumor-host interactions.??+sing orthotopic $defined as in the normal or usual position breast tumors in breast tissue%implantation of tumor xenografts these investigators have demonstrated that the hostmicroenvironment has a profound influence on a number of tumor cell parameters.?? Theseinclude tumor gro"th invasive behavior response to chemotherapeutic agents and gro"thfactor and cyto!ine production as "ell as protease profiles for uro!inase and metalloproteinases.#n important host contribution to tumor progression is the fre*uent association of MM) production by stimulated stromal fibroblasts ad&acent to invading tumor cells.?@ In more recentstudies 4idler?< has extended these findings to demonstrate that tumor-host interactions also

influence gene expression patterns in metastatic foci and may contribute to tumor progression atthese sites. These findings are supported by transcriptome analysis of metastatic human tumorsin murine models "hich sho" that successful formation of metastatic foci is associated "ithalterations in gene expression of host tissues.>FThese findings suggest that favorable tumor-host interactions may facilitate the outgro"th ofmetastatic tumor cells "hereas unfavorable interactions "ould suppress metastasis formation.This concept is embodied in the 89Hseed-soil89 hypothesis of metastasis originally put forth by

)aget.?B )aget noted the propensity for some types of cancer to produce metastasis in specificorgans and that the metastatic site "as not simply a matter of chance. This idea "as laterchallenged by the proposal of ames "ing?D "ho suggested that the pattern of bloodcirculation leaving the site of the primary tumor "as a principal determinant for the site ofmetastases.7o"ever these theories are not mutually exclusive and evidence from more recent studiessupports contributions from both mechanisms. 4or example the direct drainage of the portalcirculation through the liver can account for this organ as a principal site of metastaticinvolvement in patients "ith advanced colorectal carcinomas. 7o"ever in a study in "hichcolorectal cancer cells of differing metastatic ability "ere implanted into the liver it "as foundthat gro"th regulation in the liver microenvironment influenced tumor cell gro"th although themolecular basis for these differences remains to be elucidated. #s described above the organmicroenvironment can influence gene expression and response to chemotherapy. Investigatorshave identified soluble cyto!ines and cell-adhesion molecules in the tissue microenvironmentthat modulate tumor cell responses to chemotherapy and therapy-induced cell death. #notherexample is the influence of chemo!ine receptors on organ-specific metastasis formation.0hemo!ines are "ell characteri(ed in their ability to modulate the 89Hhoming89 of

hematopoietic cells $lymphocytes% to specific organs. Studies have demonstrated that in sometumor types the malignant cells have a cyto!ine receptor pattern that allo"s homing to specificend organs. This occurs by 89Hmatching89 the cyto!ine receptor expression on the tumor cells

to the pattern of cyto!ines expressed in specific tissues. In breast cancer for example metastatictumor cells express high levels of the chemo!ine receptors 0;0,< and 0;0,E. These receptorshave specific ligands 0;02>? and 0;02?> "hich are expressed at high levels in bone



marro" liver and lymph nodes fre*uent sites of breast cancer metastases.?E eutrali(ing the0;0,< receptor in vivo inhibited metastasis formation. 0yto!ine receptor expression has also been observed in c/# microarray analysis of metastatic versus nonmetastatic tumors.>FThe mechanisms of tumor cell intravasation have not been as systematically investigated as other events in the metastatic cascade. This is due in part to the lac! of suitable model systems. # clear

role for protease activity in tumor cell intravasation has been sho"n using the chic!chorioallantoic membrane system.?F In this model human tumor cells are placed directly onto achorioallantoic membrane in "hich the epithelium and basement membrane have been disruptedallo"ing tumor cells direct access to the underlying connective tissue that is highly vasculari(ed.Tumor cell intravasation is then *uantified by using polymerase chain reaction amplification ofhuman-specific #lu).>>

genomic /# se*uences of tumor cells present in the chorioallantoic membrane on the otherside of the chic! embryo from the initial tumor cell inoculation. These experiments demonstratethat MM)s as "ell as uro!inase-type plasminogen activator and the uro!inase-type plasminogen

activator-receptor are involved in the escape of cells from the primary tumor.0hambers et al.? have used intravital videomicroscopy to study the events and mechanismsinvolved in tumor cell exit from the circulation $extravasation%. The results of these studies have profoundly changed current thin!ing about the metastatic process. It appears that circulatingtumor cells may remain viable in the circulation and extravasate up to @ days after theirintroduction into the circulation. Surprisingly metastatic and nonmetastatic cells extravasate andthis process is not protease dependent. 7o"ever only a small subset $> in <C% of cells gro" andexpand to form micrometastases and even fe"er $> in >CC% continue to gro" formingmacroscopic tumors. #lmost <CK of in&ected tumor cells remained as dormant solitary cancercells. These findings suggest that the control of postextravasation gro"th of individual cancercells is a dominant effect in metastatic inefficiency. 0ollectively these data as "ell as the studieson orthotropic effects demonstrate that the local environment of the target organ and primarytumor may profoundly influence the gro"th potential and metastatic competence of both the primary tumor and its metastases.4or the remainder of this chapter the molecular events associated "ith tumor cell invasion andmigration are examined "ith the aim of identifying the molecular mechanisms as "ell aseffector and suppressor genes that may become targets for ne" and effective cancer therapies.Immune modulation of cancer is discussed else"here as is the process of tumor-associatedangiogenesis.

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0ell motility is a critical component of the invasive phenotype. +nderstanding the molecularmechanisms that confer tumor cell motility should allo" identification of novel targets fordisrupting this process and preventing tumor dissemination. Tumor cell motility can becorrelated "ith metastatic behavior. When parameters such as pseudopod extension membraneruffling or vectorial translation are measured there is a *uantitative increase in metastatic tumorcells "hen compared "ith their nonmetastatic counterparts. # variety of stimuli have been sho"nto stimulate tumor cell motility in vitro including host-derived factors gro"th factors andtumor-secreted factors that function in an autocrine fashion to stimulate tumor cell motility.



#utocrine motility factor $#M4% is a DC-!/ glycoprotein produced by human melanoma cellsthat stimulates tumor cell migration. #M4 has been identified as neuroleu!inNphosphohexoseisomerase. #utotaxin $#T;% another autocrine motility agent is a >?B-!/ glycoprotein thatelicits chemotactic and chemo!inetic responses at picomolar to nanomolar concentrations inhuman melanoma cells. #T; possesses B89O-nucleotide )/ $0 @.>.<.>% activity@C binds

adenosine triphosphate $#T)% and is phosphorylated only on threonine $Thr?>C% "hich isre*uired for motility-stimulating activities. #T; possesses no detectable protein !inase activityto"ard histone myelin basic protein or casein. These results have led to the proposal that #T; iscapable of at least t"o alternative reaction mechanisms threonine $T-type% #T)ase and B89O-nucleotide )/N#T) pyrophosphatase "ith a common site $Thr?>C% for the formation ofcovalently bound reaction intermediates threonine phosphate and threonine adenylaterespectively.@C The identification of #M4 and #T; suggests that stimulation of tumor cellmovement occurs in response to autocrine mechanisms that are uni*ue to metastatic tumor cells.

T+M, I6#SI 4 T7 1#SMT MM1,#)art of =chapter < - Invasion and Metastases=

/uring the transition from benign to invasive carcinoma extensive changes occur in the *uantityorgani(ation and distribution of the subepithelial basement membrane. # primaryhistopathologic feature of malignant tumors is the disruption of the epithelial basementmembrane and the presence of cancer cells in the stromal compartment.@> 1enign proliferativedisorders such as fibrocystic disease sclerosing adenosis intraductal hyperplasia intraductal papilloma and fibroadenoma are all characteri(ed by disorgani(ation of the normal epithelialarchitecture. o matter ho" extensive this disorgani(ation may become ho"ever these benignlesions are al"ays characteri(ed by a continuous basement membrane that separates theneoplastic epithelium from the stroma.@> In contrast malignant tumors are characteri(ed by aloss of basement membrane around the invasive tumor cells in the stromal compartment. ncethe basement membrane barrier is compromised it is impossible to determine the *uantity or

location of tumor cells that may have escaped from the primary tumor. Thus local invasion is paramount to metastatic competence "hich is the hallmar! of malignant conversion.The ability to invade across basement membrane barriers is not uni*ue to malignant tumor cellsho"ever. /uring an inflammatory response nonneoplastic immune cells regularly cross thesubendothelial basement membranes as do endothelial cells during the angiogenic response.Trophoblasts invade the endometrial stroma and blood vessels to establish contact "ith thematernal circulation during development of the hemochorial placenta. onneoplastic invasivecells such as trophoblasts endothelial cells and inflammatory cells all use mechanisms forinvasion that are functionally similar to those of tumor cells. The difference bet"een thesenormal invasive processes and the pathologic nature of tumor metastasis is therefore one ofregulation. #n understanding of the factors that control cellular processes essential to cell

invasion should allo" identification of novel targets for therapeutic intervention to prevent andtreat angiogenesis and inflammatory diseases as "ell as tumor metastases.

IITI#TI 4 022 MIG,#TI)art of =chapter < - Invasion and Metastases=The initial events in cellular migration are changes in cell adhesion. These changes consist ofalterations in cell-cell adhesion as "ell as interactions of cells "ith the 0M. # variety of cell



surface receptors that mediate these interactions have been characteri(ed. These include thecadherins integrins immunoglobulin $Ig% superfamily members and 0/<<. Tumor cells mustdecrease cell- and matrix-adhesive interactions to escape from the primary tumor. 7o"ever atlater stages in the metastatic cascade tumor cells may need to increase adhesive interactions "ithcells or 0M or both such as during arrest

).>?Cand extravasation at a distant site. The apparent contribution of each class of cell-adhesionmolecule to invasive behavior "ill in some "ay be dependent on the tumor cell population andmodel system used to study these interactions. This chapter revie"s the contribution of changesin cell-cell adhesion to tumor progression before considering alterations in tumor cell adhesion tothe 0M.

02289:022 #/7SI5 MT#ST#SIS S+)),SS, )art of =chapter < - Invasion and Metastases=The ma&ority of human cancers arise in epithelial cells. Several types of &unctional structures

such as desmosomes tight &unctions and adherens-type &unctions tightly interconnect normalepithelial cells. The formation and maintenance of these contacts re*uire 0a?P-dependenthomophilic interactions mediated by the cell-adhesion molecules !no"n as cadherins $4ig. <-@%.0adherins are a superfamily of single-pass transmembrane glycoproteins that mediate 0a?P-dependent cell89:cell adhesion. The cadherin superfamily no" consists of five subfamilies.These are the classic type I and type II cadherins desmosomal cadherins protocadherins andcadherin-related proteins.@? The classic cadherin epithelial cadherin $-cadherin% mediateshomotypic cell adhesion in epithelial cells. -cadherin is a transmembrane glycoprotein that hasfive extracellular homologous domains $ectodomains% a single membrane-spanning region anda cytosolic domain. -cadherin is physically anchored to the actin cytos!eleton by cytoplasmic proteins termed catenins. QO-0atenin is also a ma&or component of the "nt signaling path"ay.



4igure <-@. /isruption of cell-cell adhesion concomitant "ith tumor progression. pithelialcadherin $-cadherin% is a homotypic cell-adhesion molecule containing five homologousextracellular domains $ectodomains% that bind divalent calcium ions. 0alcium binding promoteshomophilic cell89:cell -cadherin complexes found in such structures as desmosomes tight &unctions and adherens-type &unctions. The cytoplasmic tail of -cadherin involved incell89:cell adhesion interacts "ith QO-catenin QR-catenin and p>?C0#S $p>?C%. 2oss of -cadherin

function by germline mutation promoter hypermethylation or destruction of the ectodomains by matrix metalloproteinase $MM)% activity results in an increase in free cytosolic QO-cateninlevels. Increased cytoplasmic QO-catenin can be directed to the proteosome complex by glycogensynthase !inase-@QO $GSJ@QO% phosphorylation and subse*uent interaction "ith the adenomatous polyposis coli $#)0% gene product. The fri((led $4,%-disheveled $/S7% path"ay for WTsignaling can do"n-regulate the activity of GSJ@QO. #ctivation of the WT signaling path"ay or loss of #)0 function facilitates the increase in cytosolic QO-catenin levels that are associated "ithloss of -cadherin function or mutations of the QO-catenin gene that result in reduced association"ith -cadherin cytoplasmic domain. Translocation of QO-catenin to the nucleus results inassociation "ith members of the T04N24-> transcription factor family. This is associated "ithgene expression associated "ith cell transformation and tumor gro"th $i.e. c-Myc cyclin />%. Itis note"orthy that this cascade of cellular transformation can be initiated by expression of anextracellular protease that culminates in enhanced chromosomal instability. G4 epidermalgro"th factorL ) phosphorylated amino acid residuesL T04 tissue coding factor. $4rom ref. @D"ith permission.%#ny disruption of the intracellular -cadherin89:catenin complex results in loss of cell adhesion.This includes changes in -cadherin expression or function as "ell as genes other than -cadherin re*uired for &unctional complex formation and function. #bundant evidence has beensho"n that -cadherin function is fre*uently lost during progression of many human cancers



including those arising in the breast prostate esophagus stomach colon s!in !idney lung andliver.@@ This loss of -cadherin function arises via several different mechanisms. In familialgastric carcinomas germline mutations in the -cadherin gene predispose an individual to thedevelopment of malignant cancer. Mutations in QO-catenin are found in many primary tumorsincluding prostatic cancer and melanoma as "ell as gastric and colon cancer. #nother

mechanism disrupting -cadherin).>?>

function during tumor progression is hypermethylation of the -cadherin promoter resulting indecreased gene expression. This has been found to be a ma&or mechanism in papillary thyroidcancer in that F@K of cases demonstrated hypermethylation of the -cadherin promoter.@< 3etanother mechanism to alter -cadherin function is proteolytic modification. 2ochter et al.@Breported that -cadherin function can be disrupted by degradation of -cadherin extracellulardomains by stromelysin-> a member of the MM) family that has been closely lin!ed "ith tumor progression. 0onstitutive expression of active stromelysin in mammary epithelial cells results incleavage of -cadherin and progressive phenotypic changes in vitro including loss of catenins

from cell-cell contacts do"n-regulation of cyto!eratins up-regulation of vimentin and MM)-.These changes result in a stable epithelial-to-mesenchymal transition of cellular phenotype. Invivo stromelysin expression promotes mammary carcinogenesis that includes genomic changesthat are distinct from those seen in other mouse breast cancer models.@D It has been reported thatloss of 7-cadherin expression occurs during the progression of breast cancer but little is !no"nabout the function of other cadherin family members during tumor progression. In summary adecrease in cell89:cell adhesion is associated "ith malignant conversion. 4orced expression of-cadherin in tumor cell lines results in reversion from an invasive to a benign tumor cell phenotype@@ implicating -cadherin as a metastasis suppressor.In normal cells QO-catenin is se*uestered in the intracellular adhesion complex "ith thecytoplasmic domain of -cadherin QR-catenin Q-catenin and p>?C0#S. 2oss of cell89:celladhesion results in disruption of the adhesion complex and an increase in free cytosolic QO-catenin. This free QO-catenin is bound by the adenomatous polyposis coli $#)0% gene product andis rapidly phosphorylated by glycogen synthase !inase-@QO. )hosphorylated QO-catenin issubse*uently degraded in the ubi*uitin-proteosome path"ay. In many colon cancer cells thetumor suppressor gene #)0 is nonfunctional. This can lead to accumulation of high levels ofcytoplasmic QO-catenin that are subse*uently translocated to the nucleus. The "nt-> protooncogene-initiated signaling path"ay "hich includes the fri((led and disheveled gene products bloc!s the activity of the glycogen synthase !inase-QO and results in accumulation of QO-catenin. In the nucleus free nonphosphorylated QO-catenin can bind to members of the T04N24-> family of transcription factors. It has been demonstrated that after inactivation of #)0function the increase in available cytosolic QO-catenin results in translocation to the nucleus"here it complexes "ith transcription factor Tcf-< and up-regulates c-Myc expression.@E It hasalso been sho"n that QO-catenin activates transcription from the cyclin /> promoter andcontributes to neoplastic transformation by causing accumulation of cyclin />.@F These findingslin! changes in cell89:cell adhesion "ith intracellular signaling oncogene expression and tumor cell gro"th. Thus loss of cadherin-mediated cell89:cell adhesion is an important event that hasmany far-reaching conse*uences for ac*uisition of the invasive phenotype and tumor progression.



ther types of cell89:cell adhesive interactions can actually facilitate metastasis formation. Theymay be particularly important during tumor cell arrest and extravasation. These moleculesinclude members of the Ig superfamily such as 0#M and 60#M->. This superfamily has a"ide variety of members involved in cellular immunity and signal transduction as "ell as celladhesion. Members of the Ig superfamily share the Ig homology unit that consists of EC- to >>C-

amino acid residues organi(ed into seven to nine QO-sheet structures. The diversity of superfamilymembers precludes generali(ation about their role in tumor cell invasion and metastasis.7o"ever the role of one family member seems straightfor"ard. 60#M-> is an endothelial cellcyto!ine-inducible counter-receptor for 62#-< integrin also !no"n as QR<QO>-integrin receptor.The role of integrin receptors is discussed separately in ,ole of Integrins in Tumor )rogressionlater in this chapter. ormally 62#-< is expressed on leu!ocytes and functions in mediatingleu!ocyte attachment to endothelial cells. 62#-< is also found on tumor cells in malignantmelanoma and metastatic sarcoma but not in adenocarcinomas. It is thought that expression of62#-< may facilitate interaction of circulating tumor cells "ith endothelium before tumor cellextravasation. This "as demonstrated by intravenous in&ection of human melanoma cells intonude mice pretreated "ith 62#-<89:inducing cyto!ines "hich results in an enhanced number of

lung metastases compared "ith no cyto!ine pretreatment of the mice.@ 0ell-cell adhesiveinteractions can either suppress or facilitate metastasis formation. ither role is dependent on thespecific context and molecular mechanisms of cell89:cell interaction.

022 M#T,I; IT,#0TIS #/ T+M, 022 MIG,#TI)art of =chapter < - Invasion and Metastases=#s stated previously in Initiation of 0ell Migration the interaction of the tumor cell "ith the0M in particular the basement membrane defines the invasive phenotype and tumor invasionis paramount to metastasis. It is no" recogni(ed that the 0M exerts a profound influence on the behavior of nonneoplastic cells and that cells can direct the assemblyNdisassembly of the matrix.This concept is !no"n as dynamic reciprocity and also applies to the interaction of malignant

tumor cells "ith the 0M. /uring the process of metastasis formation malignant tumor cellsmust interact "ith a variety of different types of 0M. These include the subepithelial basementmembrane of the tissue of origin stromal elements of the tissue of origin subendothelial basement membranes during extravasation and the stromal matrix and basement membranes ofthe organ$s% at the site of metastasis gro"th. #ttachment of nonneoplastic cells to the 0M is prere*uisite for cell survival. # fundamental difference for neoplastic cells is the loss ofanchorage re*uirement for cell survival and gro"th. The anchorage-independent gro"th of tumor cells may result from an uncoupling of cell survival signals transduced from the 0M via 0Mreceptors together "ith autonomous gro"th mechanisms associated "ith neoplastictransformation. Tumor cell interactions "ith the 0M have profound implications for cell-cycleregulation and for migration.

,2 4 0/<< I T+M, I6#SI #/ MT#ST#SIS)art of =chapter < - Invasion and Metastases=0/<< is a transmembrane glycoprotein "ith a large ectodomain and single cytoplasmic domain.0/<< is involved in cell adhesion to hyaluronan $7#%. The gene encoding 0/<< is on the shortarm of human chromosome >> and contains constant and variable exons.<C #s a result of thisgene structure a number of



).>??

differentially spliced isoforms of 0/<< can be generated. The isoform containing no variantexon se*uences is referred to as standard 0/<< $0/<<s%. # total of nine variant regions canencode protein se*uences v? to v>C. #lternatively spliced messenger ,# variants of 0/<<

$0/<<v% can contain one or more variant coding regions. More than @C different splice variantshave been detected by polymerase chain reaction analysis. In addition to these variants there arealso cell type89:specific differences in glycosylation of the core protein. The pattern ofglycosylation and presence of variant exons influence the ability of 0/<< to function in 7# binding.Several lines of evidence suggest that 0/<< expression plays a role in metastasis formation.0linical studies demonstrate that a variety of different types of cancer express high cell surfacelevels of 0/<< "hich correlate "ith a poorer clinical outcome compared "ith tumors that havelo" 0/<< surface expression.<><? 4orced expression of 0/<< v< to vE confers metastaticability to a nonmetastasi(ing rat pancreatic carcinoma cell line and metastasis formation could be bloc!ed using anti-0/<< variant89:specific antibodies. 7o"ever the exact role of 0/<<v in

metastasis formation remains elusive.In some tumors 0/<<-associated increases in tumor gro"th and metastatic potential correlate"ith 0/<<-mediated cell attachment to 7#.<@ 0/<< also functions in 7# upta!e anddegradation correlated "ith invasive tumor cell behavior.<< These studies demonstrate that0/<< aggregation on the cell surface creates a binding site for the active MM)-. It is postulatedthat bound MM)- may liberate 0M-bound 7# and facilitates tumor cell 7# upta!e anddegradation. These findings lin! cell adhesion and 0M turnover. In addition they suggest that0/<< may function at different stages of tumor cell invasion and metastasis and that the specific0/<< role may depend on the specific stage of metastasis formation that is examined.

,2 4 ITG,IS I T+M, ),G,SSI

)art of =chapter < - Invasion and Metastases=Integrins are heterodimeric transmembrane proteins that are formed by the noncovalentassociation of QR and QO subunits.<B<D 0onsiderable redundancy "ithin cell-0M interactionmediated by integrins exists as most integrins bind to several individual matrix proteins and0M components such as laminin fibronectin vitronectin and collagens can bind to severaldifferent integrin receptors. This suggests that integrins are capable of providing the cell "ithdetailed information about the surrounding 0M environment "hich is then integrated at thecellular level to generate a cellular response $4ig. <-<%. It is no" "ell established that integrinscan signal across the cell membrane in both directions.<E 1inding of 0M ligands to integrins is!no"n to initiate signal transduction path"ays that can result in cell proliferation differentiationmigration or cell death $apoptosis anoi!is%. This is referred to as outside-in signaling. It is also

!no"n that intracellular events can modulate the binding activity of integrins for their ligands inthe 0ML this is referred to as inside-out signaling. Integrin clustering and ligand occupancy arecrucial for the initiation of intracellular integrin-mediated signal path"ays.



4igure <-<. ,ole of integrins in tumor cell invasion and metastasis5 integration of !inase and phosphatase activities. 1inding of extracellular matrix $0M% components to integrin receptorsinitiates an intracellular signaling cascade that results in formation of a focal adhesion complexthat consists of cytos!eletal and signal transduction molecules. 2igand binding to the integrinreceptor results in integrin clustering and association of signal transduction molecules. Integrin

receptor clustering induces autophosphorylation of focal adhesion !inase $4#J% on tyrosine @E.Subse*uently an Src homology ? $S7?%-containing $Shc% adapter protein of the Src !inasefamily that binds to specific phosphotyrosine residues $3@E% on 4#J is recruited to theintegrin-4#J complex. ,ecruitment of additional proteins such as QR-actinin talin and paxillinto this complex connects the focal adhesion complex to the filamentous actin cytos!eleton.Interaction of Shc "ith 4#J results in additional sites of phosphorylation on the 4#J moleculeand subse*uent recruitment of additional S7? adapter proteins such as Grb? and the nucleotideexchange factor Sos. These interactions lead to activation of the mitogen-activated protein$M#)% path"ay that stimulates tumor cell gro"th adhesion and migration. Similarly receptorsfor gro"th factors can transiently associate "ith the focal adhesion complex to synergisticallyactivate the M#) !inase path"ay. 4#J activation also acts upstream of the #!tNprotein !inase 1

$)J1% signaling path"ay that promotes cell survival. #ssociation of the pFB subunit of phosphatidylinositol @ !inase $)I@J% "ith tyrosine @E in 4#J mediates this effect. The rapidelevation of phosphatidylinositol$@<B%triphosphate $)I)@% lipid product of )I@J activitystimulates the #!tN)J1 path"ay leading to enhanced cell survival. 0r!-associated substrate$p>@C0#S% is another S7?- and S7@-containing signal transduction that associates "ith 4#J onintegrin binding to the 0M. Interaction of p>@C0#S "ith 4#J is mediated by a proline-richregion on 4#J $residues E>?->EF% that interacts "ith the S7@ domain of p>@C0#S. #ctivation of p>@C0#S promotes cell migration and invasion "hich are associated "ith enhanced metastatic



behavior. The tumor suppressor gene )T inhibits cell adhesion migration and invasion. Thisinhibition is mediated by direct )T dephosphorylation of 4#J and Shc. This leads to negativeregulation of the p>@C0#S path"ay that affects cell attachment migration and invasion. /o"n-regulation of the M#)J path"ay by )T dephosphorylation of 4#J and Shc negativelyaffects cell gro"th in addition to attachment and migration. )T is also !no"n to directly

dephosphorylate )I)@ and negatively regulate the do"nstream #!tN)J1 cell survival path"ay.)T may also disrupt this path"ay indirectly by dephosphorylation of 4#J "hich alters )I@Jactivation. Thus integrin-mediated regulation of cell gro"th adhesion migration and invasionis a complex net"or! of signal transduction cascades that have positive $!inase% and negative$phosphatase% regulatory elements. G4 epidermal gro"th factorL r! extracellularsignal89:regulated !inaseL MJ M#) !inase or ,J !inase.The roles of specific integrins in tumor progression and metastasis formation are dichotomous.The decreased expression of some integrins is associated "ith cellular transformation andtumorigenesis. These include QRBQO> and QR?QO> integrins. 7T-? colon carcinoma cells lac!ingQRBQO> expression "ere either significantly less tumorigenic or completely nontumorigenic "henforced to express QRB.<F 2oss of QR?QO> expression in breast epithelial cells correlates "ith the

transformed phenotype and reexpression of this integrin abrogates the malignant phenotype.<n the other hand expression of some integrins directly correlates "ith tumorigenicity andtumor progression. 4or example the QRvQO@ integrin is expressed in metastatic melanoma but notin benign melanocytic lesions.BC #ntibodies against QRv integrins bloc!ed the gro"th of humanmelanoma xenografts in nude mice.B> Integrin QRD expression is increased in oropharyngeal and bladder cancers as "ell as lung tumors.@ Tumor progression is associated "ith expression ofQR@QO> in F?K of tumors. The molecular events associated "ith enhanced expression of thisintegrin and tumor progression are not "ell defined. The role of integrins in tumor invasion andmetastasis may only be secondarily related to gro"th control. Integrins are also directly involvedin cell migration.Integrin-mediated signal transduction involves direct activation of signaling path"ays andcollaborative signaling $also referred to as cooperative signaling% in "hich integrins modulatesignaling events initiated through receptor tyrosine !inases.B? The cis association of integrins"ith other receptors on the same cell surface results in formation of multireceptor complexes.2ittle evidence has been sho"n that these complexes signal exclusively through integrin-specific path"aysL instead they cooperate "ith the other receptors to influence a variety of signaling path"ays. /irect integrin signaling starts after the engagement of these receptors "ith theircognate 0M ligands "hich results in lateral clustering of the integrin receptors. 2ateralaggregation leads to interaction of the integrin cytoplasmic domains to form complexes that lin!to the cytos!eleton. This results in organi(ation of cell structures !no"n as focal adhesions.#utophosphorylation of the focal adhesion !inase $4#J% "as among the first integrin-mediatedsignaling events to be identified. This autophosphorylation event on tyrosine @E results inrecruitment of Src family protein !inases that in turn leads to phosphorylation of additionaltyrosine residues on 4#J. )hosphorylation of 4#J can result in activation of the extracellularsignal89:regulated !inaseNM#)J path"ay. #ctivation of the M#)J path"ay has been lin!ed toinduction of cell migration. The activation of M#)J can be mediated by Grb-? recruitment to4#J "hich then binds Sos a guanine nucleotide exchange factor for ,as.B@ #lternativelyactivation of the M#)J path"ay can result from 4#J phosphorylation of paxillin and the 0r!-associated substrate $p>@C0as% that leads to binding of lin! proteins such as c!. In addition



phosphorylation of 4#J at tyrosine @E creates a binding site for the regulatory subunit of phosphoinositol @ !inase and triggers activation of this signaling path"ay.The !no"ledge that 4#J is tyrosine phosphorylated on integrin activation suggests that focaladhesion-associated protein tyrosine phosphatases $)T)% could modulate 4a! function. Several)T)s that interact "ith components of the focal adhesion complex have been identified. These

are )T) QR and )T)-)ST $)T) rich in proline glutamic acid serine and threonine% "hichnegatively regulate Src and paxillin respectively.B< It has been sho"n that )T)-)ST89:deficient cells have a defect in cell motility that correlates "ith an increase in the si(e).>?@

and number of focal adhesions.BB This defect appears to be due to in part to the constitutiveincrease in tyrosine phosphorylation of paxillin as "ell as p>@C0#S and 4#J.# )T) that interacts directly "ith 4#J is )T. )T "as identified as a tumor suppressorgene on human chromosome >C*?@ "hich is fre*uently mutated or deleted in a "ide variety ofhuman cancers including gliomas prostate breast lung bladder endometrial !idney andoropharyngeal cancers.B@BD This gene encodes a phosphatase domain and also has extensive

se*uence homology to the cytos!eletal protein tensin. )T functions as a dual-specificity phosphatase in that not only is it a )T) but it also dephosphorylates the lipid phosphatidylinositol$@<B%triphosphate $)I)@%. The current vie" is that phosphatase activitiesfunction as tumor suppressor activities. The lipid phosphatase activity regulates levels of )I)@that in turn regulate activation of the protein !inase 1 $)J1N#!t% path"ay "hich is protectiveagainst programmed cell death $apoptosis anoi!is%. #s a )T) )T can also regulate the phosphorylation status of 4#J and Shc "hich in turn regulate cell adhesion migrationcytos!eletal organi(ation and M#)J activation.To summari(e loss of )T function results in alterations in integrin-mediated signaling via the4#J and )I)@ path"ays "hich results in a migratory and invasive phenotype.B@BD The protein!inase activities function to promote cell invasion. The).>?<

activities of these !inases are countered by the )T)s "hich act as tumor suppressors.

022 MIG,#TI)art of =chapter < - Invasion and Metastases=0ell migration re*uires transmission of propulsive force from the 0M to the cytos!eleton of themigrating tumor or endothelial cell $4ig. <-B%. ,epetitive assembly of cytos!eletal elements toform membrane ruffles lamellipodia filopodia and pseudopodia accomplishes cellmovement.BEBF 2amellipodia are broad flat sheet-li!e structures compared to filopodia "hichare thin cylindric pro&ections. 0ell movement begins "ith protrusion of a filopod or lamellipod.

These are formed by polymeri(ation of actin to form elongated central filaments in the filopodand a broader cross-"eave mesh in the lamellipod.B #t the leading edge of the protrudingstructures integrins concentrate in specific regions and after ligation "ith 0M ligands formfocal adhesions. These focal adhesions are anchored to the actin filaments. Microin&ectionstudies have sho"n that integrin association "ith the ,ho family of guanosine triphosphatases iscritical for organi(ation and assembly of the actin cytos!eleton. It has been demonstrated thatthere is a hierarchic cascade among these guanosine triphosphatases that controls formation of



specific cytos!eletal structures. 0dc<? and ,ac control formation of filopodia and lamellipodiarespectively "hereas ,ho controls stress fiber formation and focal adhesions.DC

4igure <-B. 0ell migration re*uires transmission of force from the extracellular matrix $0M% tothe cytos!eleton. This is accomplished in distinct steps several of "hich are illustrated. 4irstthere is protrusion of a cellular pro&ection $filopodia or lamellipodia% that is mediated by actin polymeri(ation. The second step involves organi(ation of the focal adhesion complex andconnection "ith the actin cytos!eleton. This is accomplished via mechanisms described in 4igure<-<. vidence suggests that if the integrin component of these complexes is not activated they

continually recycle across the cell surface "ithout cell movement. Step @ entails engagement ofthe integrin "ith the 0M that is mediated by a change in integrin affinity. Traction force isgenerated and transmitted by the contraction and reinforcement of the actin cytos!eleton. Thefinal step in cell locomotion is the release or disruption of the focal adhesion complex at thetrailing edge of the migrating cell. Step B is accomplished by release of components of theintegrin complex or via proteolytic disruption of focal adhesion !inase $4#J% connections "iththe filamentous action cytos!eleton.The binding of integrins to their 0M ligands provides adhesive traction. The subse*uentcontraction of the actin filaments results in for"ard propulsion of the cell body. #s the cellmoves ne" pro&ections occur at the leading edge and are anchored "ith ne" focal adhesion. #sthe cell moves for"ard the focal adhesions appear to move in a retrograde fashion on the cell

surface. This apparent movement of the focal adhesions has been observed using fluorescent-tagged beads coated "ith integrin substrates or antiintegrin antibodies.D> These coated beads canalso be used to measure the integrin-cytos!eletal traction forces generated. This has led to thedemonstration that the strength of the integrin-cytos!eletal interaction can be modulated by therigidity of the extracellular substrate. When cells are on a rigid substrate there is morerestraining force that prevents movement of the focal adhesion. 0ells can detect this change inthe substrate and respond by increasing the force generated by cytos!eletal lin!age to the focaladhesion so that the cell pulls harder. This is referred to as reinforcement of the integrin-



cytos!eletal attachments. /ata suggest that Src !inases may be selectively involved in regulatingthis reinforcement.D> ,esearchers have sho"n that beads binding to the fibronectin receptor infibroblast containing either "ild-type or Src-deficient cells sho" similar reinforcement of thefibronectin-receptor89:actin cytos!eletal lin!age. In contrast "hen they use vitronectin there islittle reinforcement of the vitronectin receptor in "ild-type Src-expressing cells but a strong

reinforcement in the Src-deficient fibroblasts. These authors also sho" that !inase-defective Srcselectively associates "ith the QRv ).>?B

integrin subunit of the vitronectin receptor but not "ith the fibronectin receptor. Thisobservation suggests that either Src is normally a selective inhibitor of reinforcement duringforce generation through the vitronectin receptor or that Src promotes the turnover of lin!s bet"een cytos!eletal and integrin components of the focal adhesion complex.Investigators have used a QO> integrin89:G4) chimera to follo" focal adhesion cycling over thecell surface.D? These experiments demonstrate that in stationary cells focal adhesions "erehighly motile and moved in a linear fashion to the cell center. In motile cells the focal adhesions

remained stationary and only moved at the trailing edge of the cell. These authors postulate acellular 89Hclutch-li!e mechanism89 in "hich there is alteration in integrin affinity in response

to migratory stimulus. ,egulation of integrin-ligand interactions by inside-out signaling helpsdetermine the nature of cellular responses to the 0M that is "hether a cell becomes migratoryor remains stationary.The last step in integrin-mediated cell migration is the release of the 0M-integrin-cytos!eletalattachments at the trailing edge of the cell.BE T"o mechanisms have been identified in thisdetachment. The first involves the release of the integrins from the cell surface. Integrin releasefrom the cell membrane has been observed in fibroblast migration and by tumor cell lines invitro.D@ # second mechanism that mediates release of the trailing edge of the cell isdestabili(ation of cytos!eletal lin!ages intracellularly by either proteolytic activity or phosphatase activity. 0alpain is a 0a?P-dependent protease that locali(es to focal adhesions andregulates retraction in 07 cells migrating on fibronectin by destabili(ing cytos!eletallin!ages.D<

),T#SS I T+M, 022 I6#SI)art of =chapter < - Invasion and Metastases=)roteolytic remodeling of the 0M has been recogni(ed as essential for tumor cell invasion.Tumor cells must be able to move through connective tissue barriers such as the basementmembrane and interstitial matrix to spread from their site of origin. #lthough a variety of proteases have been implicated in this process the family of proteases that has received the mostattention has been the MM)s. MM)s and their specific inhibitors the tissue inhibitors of

metalloproteinases $TIM)s% play important roles in physiologic remodeling of the 0M.#pproximately ?D MM)s and four TIM)s have been characteri(ed in humans and otheranimals.?@DB The MM)s share a common domain structure $4ig. <-D% although not all domainsare represented in all family members. #ll of the en(ymes have a signal peptide se*uenceL a propeptide domain $prodomain%L a catalytic domain "hich includes a highly conserved bindingsite for the catalytic (inc ionL and a hemopexin-li!e domain. T"o family members MM)-? andMM)- have a gelatin-binding domain containing three fibronectin type II repeats inserted intothe catalytic domain &ust on the amino side of the active site se*uence. 4ive family members



have a carboxy-terminal transmembrane domain after the hemopexin domain. This subgroup isalso !no"n as the membrane-type MM)s or MT-MM)s.).>?D

These MT-MM)s reside on the cell surface in contrast to the other family members "hich are

all secreted as proen(ymes into the extracellular milieu.

4igure <-D. Matrix metalloproteinase $MM)% family members. #5 /omain structure of MM)s.Simplified schematic domain structure of the MM)s. Most MM)s have the same basic domainstructure consisting of signal pro catalytic hinge and hemopexin-li!e domains. MM)-E lac!sthe hemopexin-li!e domain and the gelatinases and membrane-type MM)s have additionaldomains. The arro" indicates the location of the furin ,;J, cleavage site of MM)->> and themembrane-type MM)s. The length of each segment is roughly proportional to the number ofamino acids that comprise the domain. 15 merging roles of MM)s in tumor progression.



The TIM)s also share a high level of homology including >? conserved cysteine residues thatare all involved in intramolecular disulfide bonds. TIM)s are divided into t"o domains by thedisulfide-bonding pattern. #n amino-terminal domain contains the inhibitory site and a carboxy-terminal domain has other binding interactions. 1ecause most of these en(ymes are secreted intheir proen(yme forms activation is a !ey regulatory step. Many of the MM)s are activated by

an initial protease cleavage "ith the prodomain by another MM) or by a serine protease such as plasmin or uro!inase-type plasminogen activator. This destabili(es the bond bet"een a conserved prodomain cysteine sulfhydryl group and the catalytic (inc in the active site. The bond brea!sand the prodomains are released "hich frees the active site for catalysis. +nli!e the other familymembers "hich are typically activated outside the cell MM)->@ and the MT-MM) subgroupare activated intracellularly by a furin-dependent cleavage of a conserved ,;J, se*uence thatlies bet"een the prodomain and the catalytic domain. The activation mechanisms have been thesub&ect of a revie".DD#n early indication of the importance of MM)s in tumor biology "as the characteri(ation in>FC of an MM) secreted from a melanoma cell line that "as able to degrade basementmembrane collagen type I6 and "as initially referred to as type I6 collagenase $no" MM)-?%.DE

This "as follo"ed by numerous studies sho"ing that secretion of MM)s enhanced tumor cellinvasion in experimental model systems and that inhibition of protease activity by TIM)s or bysynthetic metalloproteinase inhibitors impeded invasion. 4or example the invasion of 7T->CFCfibrosarcoma cells through Matrigel $a reconstituted basement membrane% is enhanced by theaddition of activated MM)-? and inhibited by the addition of TIM)-? or by (inc chelators.DFSuch in vitro evidence has been supported by the results of in vivo experiments using transfectedcell lines. 4or example "hen M3+@2 bladder carcinoma cells are transfected "ith mmp-? theyhave enhanced metastatic potential "hereas transfecting the highly metastatic 2M0> cell line"ith timp-? reduces its metastatic potential. Mice that are genetically engineered to overexpressMM)-@ in breast epithelial cells develop spontaneous breast carcinomas possibly through theeffects of MM)-@ on the -cadherinNQO-catenin system.@D These experiments and many othersli!e them have demonstrated the !ey role of MM)-initiated degradation in tumor invasion andmetastasis. The ability of TIM)s to inhibit tumor gro"th and metastasis suggests that targetedinhibition of MM)s could be an effective therapeutic strategy for cancer therapy. 7o"everclinical trials of synthetic MM) inhibitors have been disappointing.?@The role of these en(ymes in this process is more complicated than a 89Hdegradation e*ualsinvasion89 paradigm "ould suggest. +ninhibited matrix degradation may lead to complete

dissolution of matrix proteins and "ould prevent tumor cells from being able to formattachments to each other or to matrix proteins "hich is a necessary part of the tumor invasionmechanism. Thus there is an implied balance bet"een active proteases and inhibitors that "ouldresult in an optimal invasive phenotype. #s a demonstration of this principle "hen #?CBFmelanoma cells are transfected "ith either sense or antisense timp-? the invasive potential isdecreased. Increasing TIM)-? expression in this cell line enhances cell attachment and decreasesmotility "hereas decreasing expression decreases cell attachment and motility. Thus although protease activity in tumor cell invasion is abnormal it cannot be totally unregulated.#nother possible explanation for the ineffectiveness of synthetic metalloproteinase inhibitors inclinical trials is that MM)s have been sho"n to have effects other than removal of structural barriers to invasion.DB 4or example some MM)s act on other proteins to reveal hidden biologicactivities. MM)s such as MM)-? and MT->-MM) specifically degrade the Q? chain of laminin-B a structural protein in the basement membrane to reveal a site on the QR@ chain that has



chemotactic properties.D #lthough the physiologic role of this fragment may be to act as a"ound-related chemoattractant in tumors this peptide may attract tumor cells to brea!s in the basement membrane. In contrast MM)s have also been sho"n to process monocytechemoattractant protein-@ $M0)-@%.EC 7o"ever in this case the MM) processing of M0)-@"hich results in removal of five amino acids from the -terminus results in loss of

chemoattractant function and conversion of M0)-@ into an agonist that bloc!s inflammatory cellrecruitment. 4urther complicating their role in tumor progression MM)s have been sho"n todegrade plasminogen into angiostatin the angiogenesis inhibitor.E> Studies have focused on theinteraction bet"een cell surface adhesion molecules and the MM) family. ne obviousinteraction is the simple degradation of cell surface adhesion molecules. 4or example MM)-@degrades -cadherin on mammary epithelial cells inducing an increased expression of vimentinand decreased expression of !eratin.@B The cell matrix adhesion molecule 0/<< is also cleaved by MM)s permitting detachment from the matrix.E? /ecreased cell-cell or cell matrix adhesioncan be a proinvasive phenotype. 7o"ever beyond mere degradation of adhesion molecules cellsmay control the scope of degradative activity by binding the soluble MM)s "ith cell surfaceadhesion molecules thus limiting degradation to a (one in the immediate vicinity of the cell. n

endothelial cells the integrin QRvQO@ binds MM)-? through the MM)'s hemopexin domain inresponse to angiogenic stimuli.E@ Similarly 0/<< has been sho"n to bind MM)- to the surfaceof breast carcinoma and melanoma cells.<< Interestingly stimulation of melanoma cells byantibodies to 0/<< increased expression of MM)-?E< suggesting that cell surface receptorsmight not only provide a mechanism for locali(ing protease activity but also may serve to initiatean autocrine-stimulating loop mechanism for increased expression of MM)s.

002+SI)art of =chapter < - Invasion and Metastases=In this chapter the current concepts of tumorigenesis have been described in relation to the originof metastatic tumor cells. 0urrent understanding suggests that the clonal expansion and rare

metastatic variant models for the origin of tumor metastasis are not mutually exclusive butrepresent t"o ends of a mechanistic spectrum. )rimary cancers evolve through a process thatcontains elements of both theories as "ell as a profound influence from tumor cell-host cellinteractions $microenvironment%. Studies using c/# microarray analysis demonstrate thatspecific patterns of overall gene expression can reveal the prognosis and eventual outcome insome tumor types. #dditional studies sho" that metastatic propensity for a specific site$osteolytic metastasis% can also be identified by a small subset of gene expression superimposed).>?E

on the poor prognosis profile. Interestingly the molecular mechanisms that mediate invasivegro"th and metastasis are also found in embryonic development and tissue repair processes. This

suggests that tumor progression and metastasis formation represent an exaggeration of thenormal developmental processes that has become dysregulated. 4inally it is increasinglyapparent that the stromal microenvironment profoundly influences the process of tumor progression including metastasis formation. 7o"ever the understanding of ho" the manycellular events are integrated and coordinated bet"een tumor cells and host stromal componentsremains incomplete.,4,0S>. o"ell )0. The clonal evolution of tumor cell populations. Science >EDL><5?@.



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D. 2oeb 2#. Mutator phenotype may be re*uired for multistep carcinogenesis. 0ancer ,es>>LB>5@[email protected]. ,a&agopalan 7 o"a! M# 6ogelstein 1 et al. The significance of unstable chromosomes incolorectal cancer. at ,ev 0ancer ?CC@[email protected]. 7anahan / Weinberg ,#. The hallmar!s of cancer. 0ell ?CCCL>CC5BE.. 4idler I Jrip!e M2. Metastasis results from pre-existing cells "ithin a malignant tumor.Science >EEL>E5F@.>C. 4idler I 7art I,. 1iological diversity in metastatic neoplasms5 origins and implications.Science >F?L?>E5F.>>. Jerbel ,S. Gro"th dominance of the metastatic cancer cell5 cellular and molecular aspects.#dv 0ancer ,es >CLBB5FE.

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phosphodiesteraseN#T) pyrophosphatase and #T)ase activities. 1iol 0hem >EL?E?5D.@>. 2iotta 2# Steeg )S Stetler-Stevenson WG. 0ancer metastasis and angiogenesis5 animbalance of positive and negative regulation. 0ell >>LD<5@?E.@?. Su(u!i ST. )rotocadherins and diversity of the cadherin superfamily. 0ell Sci>DL>C5?DC.@@. 1rac!e M0 van ,oy 4M Mareel MM. The -cadherinNcatenin complex in invasion andmetastasis. 0urr Top Microbiol Immunol >DL?>@5>?@.@<. Graff ,. /istinct patterns of -cadherin 0pG island methylation in papillary follicular7urthle's cell and poorly differentiated human thyroid carcinoma. 0ancer ,es >FLBF5?CD@.@B. 2ochter # Galosy S Muschler et al. Matrix metalloproteinase stromelysin-> triggers acascade of molecular alterations that lead to stable-epithelial-to-mesenchymal conversion and a

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Invasion and Metastases tumor cell

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