A Continuum Model for Tumour Suppression Nature Pandolfi

REVIEWdoi:10.1038/nature10275

A continuum model for tumoursuppressionAlice H. Berger1, Alfred G. Knudson2 & Pier Paolo Pandolfi1

This year, 2011, marks the forty-year anniversary of the statistical analysis of retinoblastoma that provided the firstevidence that tumorigenesis can be initiated by as few as two mutations. This work provided the foundation for thetwo-hit hypothesis that explained the role of recessive tumour suppressor genes (TSGs) in dominantly inherited cancersusceptibility syndromes. However, four decades later, it is now known that even partial inactivation of tumoursuppressors can critically contribute to tumorigenesis. Here we analyse this evidence and propose a continuummodel of TSG function to explain the full range of TSG mutations found in cancer.

A lthough hereditary predisposition to cancer was known before1900, it was only then, after the rediscovery of Gregor Mendel’sonce-ignored nineteenth century work, that hereditary pre-

disposition to cancer could be rationalized. By then it was also knownthat the pattern of chromosomes in cancer cells is abnormal. The nextcontribution to understanding cancer genetics was made by TheodorBoveri, who proposed that some chromosomes might stimulate celldivision and others might inhibit it, but his idea was long overlooked.Now we know that there are genes of both types.

In this review, we summarize the history of the study of the latter typeof genes, tumour suppressor genes (TSGs), and the evidence supportinga role for both complete and partial tumour suppressor inactivation inthe pathogenesis of cancer. We integrate the classical ‘two-hit’ hypo-thesis of tumour suppression with a continuum model that accounts forsubtle dosage effects of tumour suppressors, and we discuss other excep-tions to the two-hit hypothesis such as the emerging concept of ‘obligatehaploinsufficiency’ in which partial loss of a TSG is more tumorigenicthan complete loss. The continuum model highlights the importance ofsubtle regulation of TSG expression or activity such as regulation bymicroRNAs (miRNAs). Finally, we discuss the implications of thismodel for the diagnosis and therapy of cancer.

The two-hit hypothesisThe first evidence for a genetic abnormality as a cause of cancer came withthe discovery, in 1960, of the abnormal ‘Philadelphia’ chromosome inchronic myelogenous leukaemia cells1. Later, in 1973, it was discoveredthat this chromosome was a translocation between chromosomes 9 and 22(ref. 2) and in 1977, a translocation of chromosomes 15 and 17 was iden-tified in acute promyelocytic leukaemia3. Eventually, the genes at the break-points of these translocations were cloned: BCR/ABL1 in 1983 (ref. 4) andPML/RARA in 1991 (ref. 5). Meanwhile, seminal work demonstrated thatthe cancer-causing gene within the avian sarcoma virus genome, v-src, wasactually a co-opted and mutated version of a normal cellular gene termed aproto-oncogene, now called c-src6,7. These observations demonstrated thatnormal cellular genes, when mutated or altered, are able to cause cancer,and were followed by the identification of numerous other cellular onco-genes that are activated by mutation, chromosome translocation or amp-lification. One of Boveri’s predictions was proven to be correct.

In the 1980s, scientists verified Boveri’s other prediction with the iden-tification of a second class of genes involved in cancer, tumour suppressor

genes, that inhibit cancer development and oppose oncogene function. Ina normal cell, a physiological balance between tumour suppressors andoncogenes maintains homeostasis and allows carefully regulated cell pro-liferation without unrestrained malignant tumour growth. Somatic cellfusion experiments pointed to the existence of such TSGs, because fusionof a normal cell with a malignant cell could revert the malignant cell to anormal phenotype8. One paradox that puzzled cancer researchers was thatcancer susceptibility syndromes usually display a dominant mode ofinheritance, whereas the proposed tumour suppressor genes seemed tofunction in a recessive manner in the in vitro cell fusion experiments.

The resolution of this problem was provided by the analysis of atumour of children, retinoblastoma, which is sometimes present evenat birth9. Statistical modelling indicated that hereditary cases probablydeveloped after only one somatic mutational event. It was proposed thatone mutant allele is inherited and the other is generated somaticallyduring growth of the developing eye. Because of the high chance ofthe second ‘hit’ occurring, almost all individuals with the inherited firsthit (the mutant allele) will develop retinoblastoma, and therefore thecancer-susceptibility phenotype is inherited in a dominant manner(Fig. 1). In contrast, tumour initiation requires both hits, and thereforetumorigenesis is recessive. A similar mechanism could be extended tosporadic retinoblastoma, the only difference being that in the sporadiccases both the first and second hits occur in somatic tissue. At the time ofthe original analysis, it was not known whether these two hits occurredin the same or distinct genes, but it was clear that the search for thesecondary mutation would start with the disease gene itself.

The success of the two-hit model came with the identification ofdeletion of a locus on chromosome 13 in retinoblastoma tumours andthe subsequent cloning of the RB1 gene as mutated in familial retino-blastoma10–17. This discovery was made possible by the development ofrestriction fragment length polymorphism (RFLP) mapping technology.Using this approach, it was shown that many families with familialretinoblastoma did indeed harbour mutations or deletions of RB1 inthe germ line (the first hit), and tumours from retinoblastoma patientsnearly always contained mutation or loss of the other RB1 allele10–17 (thesecond hit). This latter event most often results from somatic mitoticrecombination, whereas in a minority of cases, the normal allele acquiresa distinct mutation. The role of recessive TSGs in dominantly inheritedcancer susceptibility syndromes could then be understood, and Boveri’ssecond prediction was substantiated.

1Cancer Genetics Program, Beth Israel Deaconess Cancer Center, Departments of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115,USA. 2Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

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These findings and others not only established the two-hit hypothesisas a model that explained hereditary cancer susceptibility, but alsoprovided a proof-of-principle that TSGs could be identified by the studyof chromosomal deletions and genetic linkage in hereditary cases of acancer. Such a strategy was critical to the ultimate identification of TP53(also called p53) as a TSG18 and was used successfully in the identifica-tion of other TSGs such as APC19, BRCA1 (ref. 20) and BRCA2 (ref. 21).Now the advent of high-throughput sequencing and genome-wide copynumber profiling is allowing further systematic identification of regionsof mutation and deletion in tumour genomes22,23.

It should be noted that it was initially proposed that two hits would besufficient for the initiation of some tumours such as paediatric cancers, butadditional mutations would be necessary for others. It is now thought thatnon-hereditary cancers require approximately four or more distinctmutation events that result in perturbation of critical cellular signallingpathways such as phosphoinositide-3-kinase (PI(3)K), p53 or RB24.Malignancy is thought to result from an iterative process of somaticmutation followed by clonal expansion25. In the context of hereditarycancer syndromes, full inactivation of the involved disease gene may bethe rate-limiting step for tumour initiation, but other events may occur,particularly to promote or enhance tumour progression. In this respect,the two-hit hypothesis should be applied directly to the TSG, with two hitsrepresenting the number of events necessary for inactivation of the TSG,but not necessarily for tumorigenesis. Haploinsufficiency, as we discussbelow, must also be interpreted in this context; in many cases, a gene mightbe haploinsufficient for a specific cellular function, but TSG haploinsuffi-ciency may promote tumour formation only in the context of othergenetic or environmental insults.

Haploinsufficiency and quasi-sufficiencyThe two-hit hypothesis clearly explained the differences in tumour numberand age of onset of retinoblastoma in hereditary versus sporadic cases ofretinoblastoma. Moreover, it led to the eventual cloning of RB1 as the firstbona fide human TSG. The hypothesis has been useful in cloning genes forhereditary cancers and for their non-hereditary counterparts. However,this success has led to a questionable dogma in the field that all importanttumour suppressors should behave according to the two-hit model.

Investigators sought to apply the principles learned from the two-hitmodel to sporadic cancers in order to identify novel TSGs. Many recur-ring regions of chromosomal deletion have been identified in sporadiccancers, indicative of the presence of a TSG. Unfortunately, it hasbecome apparent that not all regions of consistent loss are accompaniedby obvious aberrations on the other allele, raising the question of

whether these deletions may represent ‘passenger’ events not relevantto carcinogenesis. Indeed, very few of the identified regions of chromo-somal losses have yielded clear TSGs in those loci that conform to thetwo-hit hypothesis. This finding has led many to conclude that therelevant tumour suppressors in those regions have not been identifiedor do not exist, when in fact many genes in those loci are known to havetumour suppressive properties in vitro or in vivo.

Alternatively, these regions could harbour bona fide TSGs but mayrepresent regions/genes where single-copy mutation or loss has a role intumorigenesis. These single-copy events may even be selected for duringtumorigenesis instead of biallelic TSG loss. One possibility is that theinitial lesion, when reduced to homozygosity, may lead to cell death orsenescence. The lethality could be due to homozygosity of the diseasegene itself or of other distinct genes included in the initial hit, becauserecombination is a principal second hit in tumorigenesis26. Such a scenariowould result in an obligate haploinsufficiency in which selection pressureduring tumorigenesis favours partial, but not complete, loss of the TSG.

A second possibility is that the single-copy mutation of a TSG func-tions as a dominant-negative mutation towards the wild-type gene/protein. After mutation of one allele, the mutant protein product inter-feres with the normal wild-type protein produced from the remainingwild-type allele. Because the complete normal function of the TSG isalready impaired with only one hit, there is no selection pressure by thetumour for loss or mutation of the wild-type allele.

A third possibility is that a gene or genes in the region of consistentdeletion shows haploinsufficiency for tumour suppressor function andsingle-copy loss of the TSG is sufficient for aberrant TSG function andpromotion of cancer (Fig. 1). In contrast with classical TSGs that areinsensitive to large reductions in expression or activity, the function ofhaploinsufficient TSGs is impaired by a 50% partial reduction inexpression or activity, or sometimes by subtler changes, as in a quasi-insufficiency of TSG function that we discuss below.

The role of haploinsufficiency in cancer has been met with scepticism,despite the well-established role of haploinsufficiency in numerousdevelopmental disorders such as, for instance, aniridia (caused by hap-loinsufficiency for PAX6) and Grieg’s syndrome (caused by haploinsuf-ficiency of GLI3)27. Just as increased dosage of genes can result indevelopmental syndromes, such as Down’s syndrome, increased dosageor activity of oncogenes is an established genetic mechanism of malig-nancy28, exemplified by the role of MYC amplification or RAS proteinhyperactivity in cancer. However, the notion that subtle decreases ingene dosage or protein activity can be relevant to cancer has gained onlylimited acceptance in the scientific community.

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Figure 1 | Paradigms of tumour suppression. A black gradient represents acontinuum of expression related to the number of alleles present (greynumbering). Left, the two-hit paradigm as exemplified by the tumoursuppressor, RB. Loss of one allele induces cancer susceptibility; loss of twoalleles induces cancer. Middle, classical haploinsufficiency. Loss of one allele issufficient for induction of cancer. Right, quasi-sufficiency and obligate

haploinsufficiency. Quasi-sufficiency refers to the phenomenon wherebytumour suppression is impaired after subtle expression downregulationwithout loss of even one allele. Obligate haploinsufficiency occurs when TSGhaploinsufficiency is more tumorigenic than complete loss of the TSG, usuallydue to the activation of fail-safe mechanisms following complete loss of TSGexpression.

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One reason for this scepticism stems from the difficulty in definitivelyproving that a haploinsufficient TSG is involved in tumorigenesis.Whereas the rare TSGs that fully conform to the two-hit hypothesiscan be identified by their homozygous deletion or mutation in cancer,haploinsufficient TSGs cannot be identified in such a manner.Moreover, large regions of the genome, encompassing many genes,can be targeted by allelic loss and likewise, many genes may acquiresomatic mutations during the course of tumour development. It isassumed that only a portion of these genes is responsible for the cancer,while the rest are passenger mutations. What then could be the goldstandard by which to determine which gene or genes in the region arecausative and which are non-involved bystanders? Unlike the analysis oftraditional TSGs, no one assay or approach offers definitive proof that ahaploinsufficient TSG is causally involved in tumorigenesis. Instead, anintegrated approach involving human genetic analysis, in vitro and exvivo functional studies, and murine cancer genetic modelling is criticalto ascertain whether a gene may function as a haploinsufficient gene incancer. Although there are caveats of each analysis on its own, a body ofevidence from each of these analyses can strongly implicate a gene indosage-sensitive tumour suppression.

Throughout this review, we use PTEN as a model dosage-sensitiveTSG, but numerous other TSGs exhibit haploinsufficiency and dosage-sensitivity. Notably, p53 shows haploinsufficiency. Mice with heterozyg-ous p53 mutation (p531/2) show an intermediate survival to that of p53homozygous mutants and wild-type mice, and tumours that develop inthe p531/2 animals do not always display loss of the remaining wild-type allele29. Similarly, tumours in patients with Li–Fraumeni syndrome,a cancer susceptibility syndrome caused by germ line mutation of p53,do not always exhibit loss of the wild-type p53 allele, suggesting thathaploinsufficiency of p53 may be sufficient for tumour initiation inhumans as well30. A caveat with these analyses is that the effect of p53haploinsufficiency on cancer initiation in mouse models and Li–Fraumeni patients may be due to complex non-cell autonomous effectsstemming from the partial loss of p53 throughout the body. However, exvivo analysis of murine p53 heterozygous thymocytes showed that these

cells have an impaired apoptotic response to ionizing radiation or etopo-side31. Similarly, a p53-deleted HCT116 isogenic cell line expressed only25% the normal level of p53 messenger RNA and showed impairment ininduction of p53-responsive genes and apoptosis after exposure to ultra-violet radiation32. These studies demonstrate that reductions in p53dosage and function can have an impact on a cell’s ability to respondto oncogenic stimuli and strengthen the notion that p53 haploinsuffi-ciency may drive tumorigenesis.

Similarly, other classical cancer susceptibility genes such as BRCA1and BRCA2 may show haploinsufficiency and/or dosage effects. Primarybreast and ovarian cells from patients with heterozygous germ linemutation of BRCA1 or BRCA2 have altered mRNA profiles comparedto BRCA wild-type cells, indicating that single hits in these genes canconfer phenotypic differences that could, in principle, have an effect onbreast and/or ovarian tumorigenesis33. Indeed, ex vivo studies of humanBRCA1 mutant breast cells have found that these cells show enhancedcolony formation potential34 and show impaired lineage commitment indifferentiation assays35. Although tumour formation in BRCA1 andBRCA2 carriers does appear to require the second hit, these one-hitpremalignant changes could have an impact on tumorigenesis by eitherpromoting loss of the second TSG allele or of other TSGs, or by alteringthe cellular phenotype to predispose to certain tumour subtypes.

Another haploinsufficient TSG is the transcription factor PAX5. PAX5is found mutated, deleted or translocated in approximately 30% of acutelymphoblastic leukaemia cases, but the aberrant mutations/deletions arealmost invariably mono-allelic and seem to function as hypomorphs, notdominant negative alleles36. Similarly, the E3 ligase gene FBW7 (alsoknown as FBXW7) is mutated in approximately 10% of human colorectalcancers37. In 70% of these cases, the mutations are mono-allelic, indi-cative of haploinsufficiency. Conditional mono-allelic deletion of Fbw7in the murine intestine cooperated with the tumorigenic APCmin allele inintestinal tumorigenesis, albeit to a lesser degree than biallelic deletion ofFbw7, indicating that Fbw7 is dosage-sensitive37.

Like complete loss of TSGs, the effect of TSG haploinsufficiency canbe highly tissue-specific and context-dependent (Fig. 2). Thus, thecellular and molecular context in which TSG function is altered willdetermine the outcome of such impairment of TSG function. The dif-fering outcomes of TSG expression level in differing situations mayreflect distinct thresholds of protein expression or activity needed forcertain processes or in differing cell contexts. For example, in one celltype there may be other compensatory proteins that mask the potentialphenotype caused by TSG haploinsufficiency, whereas in other cellsthese proteins are not expressed and so the haploinsufficiency of theTSG manifests as tissue-specific cancer susceptibility.

Two hits for tumour suppressionThe genetic background of the individual or tumour will also have animpact on the phenotypic outcome of TSG haploinsufficiency. In somecases, combinatorial effects could arise whereby haploinsufficiency ormutation of other genetic loci is required for a phenotype caused byTSG haploinsufficiency. A powerful example of this context dependencyis provided by the interaction between Pten deficiency or haploinsuffi-ciency and the presence or absence of mutated p53 in mouse models(ref. 38; Fig. 2). In the context of wild-type p53, Pten haploinsufficiencyis actually more tumorigenic in the prostate than complete loss of Pten,because the latter triggers a p53-dependent fail-safe senescence mechanismcalled Pten-loss-induced cellular senescence (PICS). Haploinsufficiency ofPten enhances proliferation without activating PICS, making partial loss ofPten more detrimental in this context than complete loss of Pten (Fig. 2).Similarly, complete loss of Pten in the murine haematopoietic compart-ment induces haematopoietic stem cell exhaustion or bone marrow failure,unless loss of Pten is accompanied by other genetic events39. After accu-mulation of aneuploidy or other mutations, such as loss of p53, completeloss of Pten in the blood induces fatal leukaemias39,40 (Fig. 2).

This phenomenon defines a novel paradigm of obligate haploinsuffi-ciency in which selection pressure during tumour growth may favour

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Figure 2 | Tissue specificity and context dependency of tumoursuppression. The phenotypic outcome of a reduction in PTEN expression isdifferentially manifested depending on tissue type and genetic background.BM, bone marrow; HSC, haematopoietic stem cell; LA, lymphadenopathy; PD,poorly differentiated; SM, splenomegaly; WD, well-differentiated. The effect ofcomplete loss of PTEN is highly context-dependent due to the obligatehaploinsufficiency caused by PTEN loss-induced cellular senescence (PICS).Data summarized here come from multiple groups and studies of geneticallyengineered mice with differing PTEN alleles and expression38–40,52,65–68,70,71.

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haploinsufficiency of a TSG over complete loss in certain scenarios(Fig. 1). However, in advanced cancers with p53 mutation or loss,PICS cannot be induced and complete loss of Pten enhances prolifera-tion and tumorigenesis to a greater degree than Pten haploinsufficiency.This remarkable finding is likely to explain why complete loss of PTEN istypically restricted to advanced cancers and also emphasizes the import-ance of single-hit haploinsufficiency of TSGs in cancer initiation.Understanding the combinatorial and contextual dependencies of geneticevents should not only enhance our ability to effectively treat tumourswith differing combinations of genetic hits, but will also inform chemo-preventative strategies for eradicating cancer by attacking mechanismsnecessary for its development.

Like PTEN, many other TSGs have obligate haploinsufficiency. Oneexample is the microRNA-processing enzyme DICER1. Mono-allelicdeletion or mRNA downregulation of DICER1 is observed in diversecancers41,42. In murine models, mono-allelic conditional deletion ofDicer1 enhances lung tumorigenesis driven by oncogenic Kras41, sarcomadevelopment induced by KrasG12D and loss of p53 (ref. 41), and retino-blastoma formation induced by deletion of Rb1 and p107 (ref. 42). In thelung model, complete inactivation of Dicer1 improved survival comparedto animals with mono-allelic Dicer1 deletion41. Tumours that did developin the Dicer1flox/flox animals retained one wild-type allele and partialDicer1 expression, strongly suggesting that partial loss of Dicer1 pro-motes tumorigenesis.

A recent in vivo short hairpin RNA (shRNA)-based screeningapproach in mice identified lymphoma tumour suppressors, includingone, the cell cycle regulator Rad17, which seemed to function as a TSGwith obligate haploinsufficiency43. The use of multiple hairpins targetingRad17 led to the discovery that only hairpins that partially suppressedRad17 promoted lymphomagenesis, whereas hairpins that completelyrepressed Rad17 expression were selected against in the lymphomas.This study highlights the utility of shRNA knockdown and screeningtechnologies for the identification of haploinsufficient TSGs.

NPM1, found mutated and translocated in haematopoietic malignancies,also shows obligate haploinsufficiency. Whereas single-allelic deletion ofNpm1 induces tumour formation in mice44, biallelic loss induces embryoniclethality and is also incompatible with cell growth after conditional deletionex vivo45. Although more data are needed the tumour-suppressive isoformof Trp63, TAp63, may also show obligate haploinsufficiency in mice;heterozygosity for TAp63 promoted tumorigenesis to a greater degreethan complete loss of TAp63 in genetically engineered mice, and tumoursthat developed in the heterozygous mice did not display loss of the wild-type allele46. Similarly, PinX1, recently identified as a haploinsufficienttumour suppressor47, may also show obligate haploinsufficiency; hetero-zygous loss of PinX1 in a mouse model promoted cancer development inmultiple organs, and tumours did not lose the wild-type allele or expres-sion of PinX1 (ref. 47).

A genome-wide screening study in yeast indicated that about 3% ofthe genes in the yeast genome showed haploinsufficiency when assayedunder specific growth conditions48. These haploinsufficient genes wereenriched for ribosomal genes and genes that are highly expressed.Currently, mammalian tumour suppressors that show haploinsuffi-ciency do not seem to conform to one specific functional class.Besides p53 and PTEN, dozens of other TSGs show haploinsufficiencyfor tumour suppression49,50, many of which we have discussed above.These TSGs come from many functional classes, including transcriptionfactors (for example, PAX5 and TAp63), cell cycle checkpoint genes(RAD17), E3 ligases (FBW7), ribosomal or associated proteins(NPM1), and other diverse genes such as DICER1 and PINX1.

A continuum model for tumour suppressionRather than TSG function and activity following discrete, step-wisechanges, evidence is emerging that supports the dosage-dependencyof TSG function. Data from an allelic series of targeted Pten alleles thatresults in varying levels of Pten in vivo in genetically engineered mice51,52

demonstrate the tight correlation between Pten expression level and

function. Taking advantage of a null Pten allele in combination with aPten ‘hy’ allele, which is expressed at a lower level than the wild-type allele,an allelic series was created in which Pten expression follows a decreasingpattern in the order Pten1/1 . Ptenhy/1 . Pten1/2 . Ptenhy/2.Surprisingly, Ptenhy/1 animals, with cells expressing 80% of the normallevel of Pten, did display many of the phenotypes observed in Pten1/2

animals, such as lymphadenopathy, splenomegaly and mammary glandtumours52. Moreover, survival of Ptenhy/1 animals is compromised com-pared to Pten1/1 controls, but not to the same degree as the survivalimpairment in Pten1/2 animals52. Thus, the tumour incidence and sur-vival of the animals correlate with the Pten expression level52. Critically,mammary tumours from Ptenhy/1 animals do not show loss of Ptenexpression or loss of the wild-type Pten allele and exhibit a Pten dose-dependent increase in activation of Akt, further demonstrating that asubtle reduction in Pten dose can result in deregulated signalling andcancer52. Similarly, in the murine prostate, a progressive lowering ofPten levels correlates with Akt activation, prostate enlargement, prolifera-tion of prostate cells and tumorigenesis51. These in vivo studies demon-strate that even a subtle 20% reduction of protein level could constitute animportant hit involved in the development of cancer. We termed this state:TSG ‘quasi-insufficiency’52 (Fig. 1).

Thus, protein function can be a continuum that is related to the levelof expression or activity of the TSG rather than to discrete step-by-stepchanges in gene copy number. We therefore propose a shift from theclassical discrete model of tumour suppression to a continuum model(Fig. 3). This model takes into account the fact that subtle regulation ofTSGs can have profound consequences on cancer susceptibility and/orprogression, and in turn has important implications for cancer diagnosisand therapy. Note that whereas the function of PTEN seems to bestrongly coupled to its expression level, for other TSGs it will be theactivity of the protein and not the expression level that may be critical.This situation is readily observed in tumours with p53 mutation wherethe mutant allele is highly expressed but functionally inactive or actingas a dominant negative.

Like the effect on TSGs, dosage can be extremely important for regu-lating the cellular effects of oncogene expression. Markedly different

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Figure 3 | The continuum model of tumour suppression. The classicaldiscrete, step-wise model of tumour suppression (left) is contrasted with acontinuum model of tumour suppression and oncogenesis (centre and right,respectively). In the discrete model, tumorigenesis is induced by eithercomplete loss of a TSG (two-hit paradigm, dark blue) or after single-copy loss ofa TSG (haploinsufficiency, light blue). In contrast, we propose a continuummodel (centre and right), in which tumour suppression is related to acontinuum of TSG expression, rather than to discrete changes in DNA copynumber. A continuum of increasing TSG expression will generally be negativelycorrelated with malignancy (centre, light green), whereas increasing oncogeneexpression will generally be positively correlated with malignancy (right, red).A linear relationship is depicted for schematic purposes, but the dose–responserelationship need not be linear. In some cases, fail-safe mechanisms are inducedby complete loss of TSG expression or by massive oncogene overexpression. Inthese cases, complete loss of TSG expression (centre, dark green) or massiveoverexpression of an oncogene (right, orange) will be negatively correlated withmalignancy, as shown.

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consequences for the cell and for cancer development can occur depend-ing on the level of oncogene expression. For example, high expression ofoncogenic, mutation of RAS-family genes drives senescence, not prolif-eration53. Similarly, robust expression of MYC induces apoptosis, notproliferation54. These examples mirror the paradigm of PICS and obligatehaploinsufficiency and suggest that tumours may select for optimalexpression of oncogenes. Data to support this notion exist already inthe rampant amplification of mutated oncogenes, such as the amplifica-tion of the mutated EGFR gene in human lung cancer55,56. Such dataindicate that it is not only the mutation of an oncogene that is importantfor cancer, but also its expression level. In this respect, the continuummodel could also apply to oncogenes (Fig. 3).

It seems now that some TSGs are exquisitely sensitive to dose (forexample, PTEN), some are intermediately sensitive (for example, p53)and others are more resistant to expression level and/or activity changes(for example, RB1). Those that are most resistant to changes in express-ion or activity, such as RB1, will be most likely to conform to the two-hithypothesis, whereas those that are exquisitely sensitive to dose mayexhibit frequent monoallelic deletion/mutation or be misregulated incancer via other means, such as miRNA overexpression. Moreover,tumour suppressors that show obligate haploinsufficiency due to thelethality of homozygous inactivation of the TSG will never show com-plete inactivation in cancer unless there are other context- or genotype-dependent events that favour their complete inactivation (as in the caseof Pten/p53 discussed above).

The dogma that every TSG must behave according to the two-hitparadigm could significantly slow the pace of cancer research if poten-tially critical genes are dismissed as unimportant. The pace of cancerdrug development will in turn be delayed by a lack of appreciation of theinvolvement of these potentially druggable pathways in tumourdevelopment and maintenance. A very real challenge for cancer biolo-gists in the next decade will be first to define the TSGs affected byhaploinsufficiency and then to model these subtle dosage effects in away that can yield quantitative models of cancer risk or outcome.Because human genetics analysis alone is often not enough to identifycritical haploinsufficient genes unequivocally, mouse models will proveessential to define which genes show haploinsufficiency for TSG func-tion and in which specific cellular and genetic contexts.

Regulation of TSG activityThe continuum model of tumour suppression holds that precise regu-lation of TSG expression and activity is critical and thus, mechanisms ofregulation of expression and function may powerfully contribute to

tumour suppression. One of the most widespread networks of expressionregulation is the post-transcriptional regulatory network of miRNAs andcompeting endogenous RNAs (ceRNAs). In addition to these networks,TSG dosage/activity is regulated by transcriptional, translational andpost-translational mechanisms (Fig. 4). These networks open up therealm of genes that could be contextually important for tumorigenesis,especially when considering mechanisms that tune TSG activity and notonly those that function as on/off switches.

As an example, a powerful PTEN-targeting miRNA network wasrecently identified. miRNAs are small non-coding RNA molecules thatregulate protein expression through inhibition of protein translation orinduction of direct cleavage of their mRNA targets via RISC57. Many ofthe PTEN-targeting miRNAs are amplified or overexpressed in cancersuch as miR-26 in glioma58 and miR-22 in prostate cancer59. The regu-lation of coding RNAs by miRNAs is made additionally complexthrough the interaction of miRNAs with ceRNAs60,61. We coined theterm ‘‘competing endogenous RNA’’ (ceRNA) to refer to an RNA—coding or noncoding—that shares miRNA binding sites with anotherRNA with which it competes for miRNA binding60,61. Any two or moremRNAs with shared miRNA recognition elements (MRE) can act asceRNAs towards each other by competing for miRNA binding andthereby co-regulating each other’s expression. As the expression of theceRNA increases, the miRNA binding sites on the ceRNA compete withthe miRNA binding sites on the other transcript, and less miRNA isavailable for binding to the TSG transcript (Fig. 4). ceRNA expression(for example, expression of the PTEN pseudogene60) hence results insustained TSG expression by preventing miRNA binding to the TSG andsubsequent mRNA or protein downregulation. Conversely, loss ofceRNA expression could result in low TSG expression through diversionof additional miRNA molecules to the TSG transcript60,61. Even non-coding ceRNAs that are not expressed at the protein level could functionas TSGs by regulating expression of protein-coding TSGs, so mutationor deletion of ceRNA genes could be important in cancer60,61.

Implications for cancer susceptibility and therapyAn appreciation of the continuous nature of tumour suppressive functionmust in turn lead to a re-evaluation of the role of TSG dosage in cancersusceptibility, diagnosis and treatment. Subtle variation in TSG expres-sion or activity could underlie genetic variation in cancer susceptibility inthe population. Inherent, constitutive differences in gene expressioncould be caused by polymorphic variation in TSG promoter regions orin miRNA binding sites or other regulatory regions. Importantly,synonymous single nucleotide polymorphisms (SNPs) that do not

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Post-translationalmodification/degradation

ceRNA

Figure 4 | Mechanisms of regulation of TSG dosage. The interactionbetween coding and non-coding factors determines final TSG dosage. Classicmechanisms such as DNA copy number (1), transcriptional regulation (2) andepigenetic silencing (3) can affect expression of TSG mRNA. TSG mRNA levelor translation into protein is then regulated by miRNAs (5). The availability of

miRNAs for TSG downregulation is further regulated by ceRNA-mediatedsponging effects (4). Finally, additional translational regulation (6) or post-translational modifications contribute to the final protein expression, functionand effective dosage. The protein structure shown is PTEN (Protein Data Bankaccession code 1D5R).

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change the protein sequence of an expressed gene could still have apowerful impact by altering the level of TSG expression through altera-tion of miRNA binding. Moreover, SNPs in non-coding RNAs couldhave an impact on tumour development through ceRNA effects.Alternatively, on the basis of a continuum model, susceptibility to cancercould be induced by environmental factors through subtle transcriptionalup- or downregulation of relevant genes. Development of drugs thatprevent and counter these fluctuations could be used as chemopreventa-tive agents to reduce the impact of environmental carcinogens or tocorrect the effects of inherited predisposing factors.

Just as transcriptional or post-transcriptional regulation may deter-mine cancer susceptibility, it could also be harnessed for cancer preven-tion and therapy (Fig. 5). As an example, statins, which are already inclinical use, were recently found to upregulate PTEN through PPARcsignalling62. Statins or PTEN-enhancing drugs could therefore be usednot only to treat dyslipidaemic cases but also to elevate PTEN expressionin patients with tumours with lowered levels of PTEN (Fig. 5). Such anapproach would require a quantitative analysis of the expression levelsof TSGs and oncogenes and not solely a genetic assessment of theirpresence or absence.

TSG-targeting microRNAs add another dimension to genotype/phenotype considerations in targeted cancer prevention and therapy.Overexpression of one or more of TSG-targeting miRNAs could confercancer susceptibility, and tumours not showing genetic alterations in theTSG but with aberrant TSG levels due to miRNA misregulation couldbehave similarly to tumours with genetic deletion or mutation of thegene. Mapping the interactions between miRNAs and TSGs could beuseful for defining and predicting tumour response to therapy.

Provocatively, tumours with partial loss of TSGs like PTEN that showobligate haploinsufficiency might be treated with agents to fully suppressthe expression of the TSG, thereby suppressing tumour growth viasenescence or other fail-safe mechanisms (Fig. 5). Alternatively, when aTSG is completely lost, activation of alternative tumour-suppressive

pathways or synthetic lethality approaches could be considered63

(Fig. 5). The success of such strategies would obviously depend on theintegrity of the fail-safe response and the expression level of the TSG,once again requiring a careful annotation of the molecular characteristicsof the cancer lesion subjected to treatment. The need for the same carefulannotation is not restricted to targeted therapies but applies to any cancertherapy because variations of oncogene or TSG dosage may well be at thecore of failure of more conventional radio- or chemotherapies.

On this basis, the scientific challenge will be to develop quantitativeassays that can assess and score subtle differences in TSG gene expres-sion to allow accurate diagnostics and prediction of risk/prognosis. Suchdiagnostics will also be important for analysis and prediction of responseto therapeutics, because subtle variations could also determine responseto therapy. A tumour with a partial downregulation of a TSG couldrespond differently from a tumour that has either no alteration of theTSG or complete mutation/loss of the gene (Fig. 5). Critically, in caseswhere a tumour shows downregulation of a TSG via transcriptionalrepression, epigenetic downregulation, or aberrant miRNA andceRNA regulation, there is an opportunity to explore therapeutic res-toration of TSG expression.

The recent explosion of knowledge in the field of non-coding RNAbiology has opened a new frontier in understanding gene dosage regu-lation and its involvement in tumorigenesis. In the months and years tocome, careful experimentation is required to map out the networks ofgene regulation of TSGs and oncogenes to allow drug development andcreation of diagnostics from this information. Forty years after the two-hit hypothesis, we are refining and extending our understanding of TSGinactivation in cancer. Milestones made in these past forty years havecontributed to the decline in cancer deaths observed in the US since 1990(ref. 64). The next decades promise further relief from the burden ofcancer as we continue to tease out its molecular genetic basis.

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Approach Therapeutic intervention

Tumours with

partial loss

(of TSG

expression/

activity)

-Use drugs that upregulate TSG expression

- Inhibition of TSG-downregulating miRNAs

- Derepression of epigenetic silencing via DNAdemethylating agents or histone modifying drugs

DNA methyltransferase inhibitors,

histone deacetylase inhibitors

Restoration

of TSG

pathway

or function

1

Induction/

enhancement

of fail-safe

mechanisms

2

Applicable

tumour type

-Further suppress TSG expression

- Activate alternative TSG pathways

- Synthetic lethality: use compound genetic effects

VO-OHpic PTEN

Complete loss of PTEN Senescence

Skp2 targeting in PTEN+/– cells Senescence

Tumours with

partial or

complete loss

Tumours with

partial loss

Nutlin p53

Statins PPARγ PTEN

miRNA decoys miRNA binding TSG expression

Figure 5 | Opportunities for therapeutic intervention after partial orcomplete loss of TSG expression. Tumour genotype ultimately determineswhich therapies can be considered for intervention. Broadly, two generalapproaches can be taken: restoration of the TSG pathway or function (upperpanel) or induction of fail-safe mechanisms by complete downregulation of theTSG (lower panel). In tumours with downregulation of TSG expression in theabsence of complete mutation or deletion, expression of the TSG can beattempted using drugs that induce the TSG transcription, inhibit miRNA-induced TSG downregulation, or relieve epigenetic silencing. Alternatively, fail-safe mechanisms may be induced by further downregulation of the TSG, suchas inhibition of PTEN by the small molecule VO-OHpic69. In tumours withmutation or complete loss of the TSG, activation of other TSG pathways couldbe pursued. Alternatively, if a synthetic lethality relationship with the TSG isknown, then the synthetic lethal gene can be targeted to induce cell death in thecells with complete loss of the TSG.

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Acknowledgements We thank L. Salmena, L. Poliseno and all Pandolfi laboratorymembers for advice and critical discussions. This work was supported in part by NIHcore grantCA06927 and anappropriation from the Commonwealth of Pennsylvania tothe Fox Chase Cancer Center to A.G.K. and NIH grant R01CA142787 to A.H.B. andP.P.P.

Author Contributions A.H.B., A.G.K. andP.P.P. together contributed toall aspects of thiswork.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence should be addressed to P.P.P.([email protected]).

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