perspectivesin cancerresearch oncogenes, antioncogenes ... · oncogenes, antioncogenes, and the...

(CANCER RESEARCH 49, 3713-3721, July IS, 1989]

Perspectivesin CancerResearch

Oncogenes, Antioncogenes, and the Molecular Bases of Multistep Carcinogenesis1

Robert A. Weinberg2

Whitehead Institute for BiomÃ©dicalResearch and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142

Tumorigenesis in humans and laboratory animals is a complex, mu 11Â¡stepprocess (1, 2). In humans, in whom the processhas been studied only indirectly, measurements of age-dependent tumor incidence indicate kinetics dependent on the fifthor sixth power of elapsed time (3). This suggests a successionof five or six independent steps, each of which is rate limitingon the process. In experimental models, such as mouse skintumorigenesis, the process has been broken down into at leastthree distinct steps: initiation, promotion, and progression (4,5). From the perspective of the organism, the multistep natureof tumorigenesis is easily rationalized; each step in the processrepresents a physiological barrier that must be breached inorder for a cell to progress further toward the end point ofmalignancy. Such multiple barriers conspire to ensure thatsuccessful completion of the tumorigenic process is a rarelyachieved event.

An unanswered question concerns the natures of these barriers to tumor inception and growth. A portion of the defensesmay well derive from systemic defenses against tumors; yetothers, confronted in this essay, reflect underlying mechanismsgoverning the behavior of individual cells. Our cells and likelythose of all metazoa would seem to be constructed so as topresent multiple impediments to full malignant transformation.Only recently has it been possible to search for the molecularand cellular mechanisms that govern multistep tumorigenesis.What are the rules that govern cell growth? How is the growth-regulatory circuitry laid out within the cell? And how canmultiple physiological controls be overridden to produce thederegulation of neoplasia?

Collaborating Oncogenes

The oncogene paradigm developed over the past decade hasproved to be particularly powerful in generating an explanationof cancer at the molecular level. Because cellular oncogenes aremutated forms of normal cellular genes, they provide clearindication of the genetic targets that suffer alteration at thehands of mutagenic carcinogens. Accordingly, a simple modelwould identify protooncogenes, the normal antecedents of cellular oncogenes, with the genetic targets the alteration of whichdefines each of the distinct steps in multistep cellular transformation. In this view, the evolutionary history of a tumor cellclone is demarcated by a series of oncogene activations, each ofwhich confers on the tumor cells some of the phenotypes thatin aggregate constitute fully malignant behavior.

An initial connection between oncogenes and the multistepnature of tumorigenesis was made 7 years ago through studiesof two viral oncogenes, the middle T (A/7") and large T (LT)

genes of polyomavirus (6). Neither was found to be able totransform rat embryo fibroblasts on its own. However, the two,working in collaboration, elicited a fully tumorigenic pheno-type. This suggested that each oncogene was specialized to

Received 2/2/89; accepted 4/19/89.1Part of the work described herein was supported by National Cancer Institute

Grant OIG 5 R35 CA39826.2American Cancer Society Research Professor.

induce part of the phenotypes required for full transformation.Was this a peculiarity of these viral oncogenes or were theprinciples transferable as well to the behavior of oncogenesderived from the cell genome?

The model was indeed extended to a number of oncogenes ofcellular origin. Thus, neither a ras nor a myc oncogene wasfound able to induce full transformation while the two, coin-troduced into rat embryo fibroblasts, achieved this end result(7). Analogously, a ras oncogene was found to collaborate withthe adenovirus EIA oncogene in the full transformation of babyrat kidney cells (8).

These rather simple experiments had a number of conceptualramifications that warrant mention. Like the polyomavirusgenes, the observed ability of ras and myc oncogenes to collaborate with one another in the transformation process showedthat each acts in a distinct, complementary way on cell pheno-type. Detailed studies in rodent cells showed that rus oncopro-teins can induce refractility, anchorage independence, andgrowth factor secretion even when expressed in low amounts;such expression did not favor immortalization in culture. Conversely, the myc oncoproteins appeared more adept at immortalization and less able to induce anchorage independence andgrowth factor secretion (9, 10).

Such distinctions in function have been seen in a variety ofcell backgrounds. They suggest that the cell is organized so asto respond in only limited ways to the transforming influencesof a single activated oncogene. Stated differently, they suggestthat an activated oncogene is able to control only a limitedsubset of the growth-regulatory circuits of the cell. These circumscribed actions of single oncogenes presumably reflect thecorrespondingly limited powers of antecedent protooncogenes,each of which has apparently been evolved to transduce onlypart of the complex information regulating cell growth andquiescence.

Yet other oncogenes could be placed into two functionalcategories based on their abilities to complement either a rasor a myc oncogene in transformation assays of rat embryo cells(10). Such a list has been extended in recent years (Table 1).Another line of work showed that oncogenes involved in thetransformation of avian hematopoietic cells also showed collaborative effects (11).

The classification of oncogenes based on their collaborativepowers in transformation assays was paralleled in a strikingway by a totally distinct system of categorizing these genes:classifying them by the intracci hilar localization of their respective gene products. Those oncogenes that function like ras (bycollaborating with myc) encode cytoplasmic proteins whilethose that function like myc (collaborating with ras) specifynuclear proteins. This suggests that each group of oncoproteinsconverges on a common target or pathway, one in the cytoplasm, the other in the nucleus.

Not all oncogenes are seen to fit neatly into this scheme (12-19). Thus, yet other pathways or targets may exist that are notaddressed by this ras/myc paradigm. Nonetheless, because ofthis body of work, a terminology has evolved in which these

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ONCOGENES, ANTIONCOGENES, AND CARC1NOGENESIS

ke oncogenes encoding nuclear proteins are called ''nuclear oncogenes" while these ros-like oncogenes specifying cy-toplasmic proteins are called "cytoplasmic oncogenes."

The discovery of oncogene collaboration lent more substanceto the simple model of how multistep tumorigenesis works.Restated, it would propose that each step in the tumorigenicprocess reflects a mutation leading to the activation of one oranother cellular oncogene; the resulting activated oncogenesthen work together to induce the full neoplastic phenotypes ofthe cell. Yet it must be said that fundamental aspects of thismodel remain unproved. Is it true that multiple oncogenes areinvariably required to transform cells? And equally important,how instructive is this model in understanding multistep carci-nogenesis as it occurs in humans and in well-defined animalmodels of tumorigenesis?

Apparent Violations of Multistep Carcinogenesis: Full Transformation by Single Oncogenes

A number of experimental strategies would appear to violatethe rule that at least two oncogenes are required to transformnormal cells into fully malignant ones. To understand these, itis necessary to go back and review how the ras/myc (or poly-omavirus MT/LT or ros/ElA) oncogene collaborations werefirst observed. Monolayer cultures of embryo cells (fibroblastsor kidney cells) were transfected with individual oncogene-bearing plasmici DNAs or cotransfected with several plasmidscarrying distinct oncogenes. When single oncogenes (e.g., rasalone or myc alone) were transfected, the resulting monolayersyielded few if any foci upon reaching confluence. However,when two complementary oncogenes were cotransfected, fociappeared, the cells of which proved tumorigenic upon inoculation into appropriate hosts (6-8). These were the results underlying the conclusion that multiple oncogenes are required forfull transformation to tumorigenicity.

The simplest and most frequently observed deviation fromthis scheme derived from transfection of ros-like oncogenes intoestablished cell types like Rat-1 cells or NIH3T3 cells (2, 20).In this instance, single oncogenes indeed suffice to induce fullconversion to tumorigenicity in one step. Here the discrepancywith the multistep model is easily rationalized. Such 3T3 cellsdeviate from primary embryo cells in their established, immortalized phenotype. This immortalization can be rationalized as

Table 1 Functional classification of cooperating oncogene in rat embryofibroblasts

Classification of oncogenes on the basis of their ability to collaborate intransformation. For example, a ras oncogene can collaborate with mir, pS3,protein or adenovirus EIA to transform rat embryo fibroblasts.

Cytoplasmiconcogenes Nuclear oncogenes

Ha-rojKi-rasN-rassrc (90)Polyoma MT (6)

myc (7, 8)

L-myc (93)p53 (94, 95)AdenoElA(8)Polyoma LT (6, 7)SV40LT (7)Papillomavirus E7 (96)

erbBsrcfpsmilHn-rasrosyessea

In avian bone marrow cells

myc (11, 97)myb

a premalignant phenotype similar to that induced by a myconcogene and known to make a primary cell responsive totransformation by a ras oncogene acting alone. Viewed in thisway, established cells have already undergone changes reminiscent of those induced by certain nuclear oncogenes. Thesechanges make these cells unsuitable for studying the full andnatural process of tumorigenesis that begins with a fully normalcell.

Results like this refocus attention on primary cells that haveostensibly undergone few if any changes in their growth controlmechanisms following their explantation from an embryo. Theyshould represent good models of the cells that suffer transformation within a living tissue. Manipulations of such fullynormal cells make it clear that there are indeed conditionsunder which single oncogenes, acting on their own, can inducewhat appears to be total transformation. For example, when aras oncogene is cointroduced with a neomycin resistance markerinto embryo cells growing in monolayer, subsequent applicationof neomycin results in killing off of the great majority of cellsin the culture and in the outgrowth of a small minority ofoncogene-bearing, neomycin-resistant transfectants. Their descendants growing to confluence form a monolayer of refractilecells that are tumorigenic (9, 22).

A similar result is seen when embryo fibroblast monolayersare infected with a retrovirus such as Harvey sarcoma virus,which transduces a ras oncogene. If the virus is allowed tospread through the monolayer, thereby infecting the great majority of cells, then once again monolayers of fully transformed,refractile cells soon appear which are also tumorigenic. In boththese instances, the cooperation of a second oncogene seems tobe gratuitous (23).

These results conflict with the model that requires at leasttwo oncogenes for transformation. The tension between thetwo-gene model and these experimental results can be addressedby introducing another element into the logic: the environmentof the oncogene-bearing cell. When monolayers of embryo cellsare transfected with a single oncogene (e.g., ras), only a smallnumber of cells initially acquire an oncogene, and each of thesecells is surrounded by normal untransfected neighbors. Underthese conditions, the occasional ros-transformants remain in-apparent; they are unable to expand clonally to form visiblefoci. In contrast, use of neomycin selection, as described above,results in killing the normal neighbors, leaving only pure populations of ras transformants, the proliferation of which is nowunfettered. An analogous situation pertains upon high multiplicity infection of monolayer cells by an oncogene-transducingvirus; the normal neighbors of an oncogene-bearing cell are nolonger present, having been recruited into the cohort of transformants through viral spread. The resulting pure populationsof transformants also grow aggressively.

All this suggests that neighboring normal cells exert a normalizing or inhibitory influence on the growth of ras transformants. When their presence and influence are absent, then purepopulations of ras transformants can proliferate to producelarge progeny clones. This makes the simple and importantpoint that the growth properties of a cell depend not only onits own genotype (e.g., its complement of oncogenes) but on itsenvironment as well, a conclusion drawn by others from anumber of analogous experiments (24-30).

This point is made quite dramatically by recent /// vivoexperiments involving use of ros-transformed mouse skin ker-atinocytes. When a culture of these is mass transformed by highmultiplicity infection with Harvey sarcoma virus, then thesecells form rapidly growing squamous carcinomas upon implan-

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ONCOGENES, ANTIONCOGENES, AND CARCINOGENESIS

tation onto the back of a mouse. In contrast, when these samecells are rÃ©implantÃ©e)together with a 4-fold excess of normaldermal fibroblasts, then only small nonprogressing nodules areobserved (31). These dermal fibroblasts represent a cell typewith which keratinocytes normally coexist within the skin. Onceagain the environment of oncogene-bearing cells constitutes a

strong determinant of their future growth properties. In theseseveral examples, normal, closely apposed cells strongly deterthe proliferation of ras transformants. An analogous result hasalso been observed with wye-transformed cells (32, 33).

In Vivo Tumorigenesis with Single Viral Oncogenes

The above data suggest that a single oncogene, acting on itsown, is able to induce tumorigenesis under special conditionsin which the oncogene-bearing cell is isolated from the influences of normal neighbors. Strong testimonial to this is alsoprovided by two decades of work on in vivo c;irebiogenesis usinga variety of rapidly oncogenic retroviruses, each of which transduces a single cellular oncogene in its genome. Included in thislist are the aforementioned Harvey sarcoma virus (ras), Abelsonleukemia virus (Â«/>/),feline sarcoma virus (fes), and avianmyelocytomatosis viruses (myc) (34). In each case, the presenceof a coinfecting, replication-competent helper virus ensures thatthese rapidly tumorigenic viruses are not only able to infect aninitially encountered target cell but also to spread centrifugali yto all neighboring cells, recruiting them into the population oftransformants. This mimics the in vitro monolayer model inwhich mass infection eliminates normal neighbors by recruitingthem into the cohort of transformants.

Here too, single oncogenes acting on their own appear sufficient for tumorigenesis. But appearances are misleading. Thelarge population of virus-infected transformants may well proliferate only for a limited time before a few of its progenysustain secondary changes that enable them to grow as trulyautonomous, malignant cells. This possibility can be addressedby analysis of the clonality of tumor cell populations emergingfrom such virus-inoculated tissues. Discovery of polyclonalpopulations would indicate that many if not most of the initiallytransformed cells can be tumorigenic without requiring raresecondary changes; mono- or oligoclonality would indicate thatonly a few of the initially large number of transformants acquirethe secondary changes necessary to propel them to a tumorigenic state. A recent study indicates that in one case suchtumors are indeed mono- or oligoclonal and that the initialburst of proliferation seen after mass infection by a transforming retrovirus may not suffice to create a stably growing tumormass (35). The nature of such secondary changes is presentlyobscure; their implied existence suggests that even upon masstransformation of a cell population by a single oncogene, subsequent events must intervene before the cells are genuinelymalignant.

Mouse Models of Tumorigenesis

Oncogene-mediated transformation studied in vitro acquiresvalidity only if it reflects aspects of tumorigenesis as it occursin vivo. In vitro transformation can indeed mimic certain earlysteps in tumor formation by creating cells of a similar genotype.For example, strong evidence has been reported which showsthat the initiating event in mouse skin carcinogenesis is thecreation, through chemically induced mutation, of an activatedras oncogene. This genotype can be mimicked readily throughuse of transducing retroviruses which serve to convey an exog

enous ras oncogene into an apparently small proportion ofkeratinocytes infected in situ in the skin. The small minority ofinfected cells function as if they has been altered by an initiatingcarcinogen, yielding papillomas upon treatment by a tumorpromoter (36).

The proportion of cells infected by such a transformingretrovirus in a target tissue would seem to be a critical parameterin virus-induced tumor initiation. Nonviral tumors start out assmall nests of partially transformed cells surrounded by a largeamount of normal tissue, a state which would appear to bemimicked by infecting scattered skin keratinocytes with Harveysarcoma virus. A contrasting situation results upon efficientmass infection and transformation of all the cells in a tissue.This must drastically alter an important element in the dynamics of tumorigenesis by depriving initiated, oncogene-bearingcells of close contact with normal neighbors. Such a large massof initiated cells may quickly undertake clonal expansion sincethey are no longer confronted with the inhibitory influences ofnormal tissue.

This notion has important bearing on another frequentlyused model of tumorigenesis, that involving transgenic mice.As many as 20 different mouse lines have been derived over thepast 5 years in which activated oncogenes are inserted into themouse germ line (37). Invariably, the expression of these oncogenes is driven by a tissue-specific transcriptional promoterthat may also be regulated in a stage-specific manner. The resultis usually the appearance of a tumor at a site that is predictedby the nature of the promoter chosen to regulate oncogeneexpression. Thus, the mouse mammary tumor virus transcriptional promoter engenders largely mammary tumors while theinsulin gene promoter favors pancreatic tumors.

By creating cohorts of mice with well-timed onsets of predictable tumors, these transgenic models would seem to provideideal experimental models of spontaneous tumorigenesis. Butthey fall short in one important aspect. Instead of creatingsmall, isolated nests of initiated, oncogene-bearing cells, thesetransgenes create tissues in which virtually all the cells areexpressing an activated oncogene. In so doing, the transgenicmodel fails to address one of the most important aspects oftumorigenesis, i.e., the interactions of transformants with theirnormal neighbors during the early stages of this process.

Early Steps: Transcending a Hostile Environment

Repeated mention has been made here of a critical early stepin tumorigenesis, the process by which a small, early preneo-plastic cell clone expands in spite of the inhibitory influencesof normal neighbors. Contrary to earlier discussion in whichras and myc oncogenes were given equal weight, the attentionin this discussion has been focused on the role of ras oncogenesin affecting the behavior of early tumor cell clones. The bias isintentional in that activated ras oncogenes have been found ina number of preneoplastic murine and human tumor modelsincluding those of the skin, colon, and hematopoietic system(38-40). This suggests that ras activation is often a relativelyearly event in tumor formation.

How are isolated premalignant ras transformants normallyable to expand to the large clonal sizes that then permit the lowprobability secondary changes necessary for truly autonomousneoplastic growth? Clearly in the vast majority of cases, rastransformants fail to do so, remaining as isolated single cellsor small pockets of cells that are hemmed in by their environment. This point is made most dramatically by calculating howmany cells in the human body have through accidents of DNA

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replication acquired activating oncogenic point mutations intheir ras genes. The number, variously calculated at 105-106,

vastly exceeds the number of observable premalignant lesionsany one of us will experience in a lifetime.

An escape from environmental inhibition can be forced experimentally through use of tumor promoters. To cite thepreviously mentioned case, small nests of initiated rax transformants can be driven into macroscopic papillomas by TPA.3

By the present logic, this tumor promoter contributes to skintumorigenesis through its ability to allow ros-bearing keratin-ocytes to transcend the growth-inhibitory influences of theirnormal neighbors, including perhaps those of the dermal libroblasts to which they are closely apposed within the skin. Analogously, in the rat embryo fibroblast monolayer model, TPAapplication enables the focal outgrowth of ras transformants,the presence of which would otherwise be inapparent (41). Thispromoter acts as an agent that confers special growth advantageon ros-bearing cells; conversely, the initiated, ros-bearing cellsbehave as if they were especially responsive to the growth-stimulatory influences of the promoter. A similar suppressionof growth by normal neighbors and TPA-induced reversal ofthis suppression has been seen with UV-irradiated C3H lOT'/z

mouse fibroblasts (30).It is unclear how TPA acts to propel forward the growth of

the raj-bearing cells described above. One obvious answercomes from the observations that gap junction communicationbetween normal and tumor cells correlates with the susceptibilities of tumor cells to inhibition by their normal neighbors, andthe fact that TPA dramatically reduces the gap junction-mediated communication that normally couples the metabolismof neighboring cells (42-45). In so doing, the TPA may interdictthe flow of growth-inhibitory signals from normal cells to aninitiated neighbor, thus giving the latter a free hand to proliferate (32, 33, 42-46). While this mechanism may explain someof the effects of TPA, I suspect that the biological reality ismore complex. The kinase C enzyme activated by TPA sitsastride a central mitogenic pathway within the cell. Thus, TPAwould seem to provide a strong growth impetus to a ras-bearingcell in addition to liberating it from the inhibitory influences ofits environment.

Later Steps: Collaborating Secondary Genetic Changes

The scenario of tumor evolution drawn here echoes schemeswidely accepted in the field of carcinogenesis. Initiating carcinogens create a critical genetic change (in this case involving aras gene); any resulting initiated cells expand clonali y under theinfluences of a tumor promoter (e.g., TPA) until they form alarge enough clone of descendants to permit the occurrence oflow probability, secondary genetic changes; and these secondarychanges create alÃelesthat collaborate with the initially inducedras oncogene to produce a fully tumorigenic cell that is nolonger dependent on the promoter for its continued growth(47-49).

Missing from such a scheme is a clear understanding of thenature of the genes that are activated late in tumorigenesisduring progression and serve as collaborators with the onco-genes created initially. Obvious candidates for these collaborators are the myc gene and analogously acting "nuclear oncogenes" like N-myc, L-myc, and p53 (Table 1; Ref. 50). These

all act synergistically in the in vitro transformation assay withras-like oncogenes. One or the other of these, activated during

3The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate;pl05-Rb, M, I05.000 Rb gene-encoded protein.

tumor progression, could well serve as the second gene requiredfor autonomous neoplastic growth.

Regrettably, faith in such a model is not reinforced by muchdata collected over the past 5 years. Very few tumors have beenfound that carry both activated ras oncogenes and aberrationsof nuclear oncogenes like myc. A first reaction to such failureis that the scheme of ras-myc collaboration is incorrect anddoes not address the biological realities of tumor cell biology.However, I suggest that a more useful recourse is to restate thelesson of ras/myc collaboration in a more general form: thatthe conversion of a normal cell into one that is fully tumorigenicinvolves at least two types of change in cell physiology, oneoccurring in the cytoplasm and one in the nucleus. The cyto-plasmic changes may be induced by ras or other analogouslyacting oncogenes; those in the nucleus may be achieved by mycor other genetic changes that create phenocopies of the myc-induced state. These required nuclear changes may depend ongenes that operate on totally different principles than those ofthe well-characterized nuclear oncogenes.

A similar dilemma is posed by the existence of spontaneouslyimmortalized cells (e.g., NIH3T3, Balb/c3T3, C3H lOT'/z)

which ras can readily transform to a tumorigenic state. Thesecells appear to lack wye-like oncogenes that might mediatetheir immortalization and ras responsiveness. Perhaps theirmyc-like phenotypes may also be achieved through mechanismsthat do not depend upon the mutation of nuclear oncogeneslike myc.

The Search for myc Surrogates

What clues do we have in the search for the elusive geneticchanges that can mimic physiologically the activation of nuclearoncogenes? This question can be approached by examining thephysiological effects of myc on the cell, myc is an immortalizinggene, but unexpectedly this trait may not be central to its rolein tumorigenesis. The relationship of immortalization in tissueculture to in vivo growth properties of tumor cells is at bestobscure. Many biopsied tumor cells are not immortal in culture.Moreover, it has been shown that immortalization of cells inculture does not necessarily confer responsiveness to the transforming effects of oncogenes like ras (51). By this logic, theabilities of myc (or El A) to confer immortalization and responsiveness to ras are physiologically separable qualities and it isthe latter quality (ras responsiveness and associated oncogenecollaboration) that is more central to tumorigenesis.

What then is the essence of the collaborating powers of myc(or myc-like oncogenes)? Here, once again, the embryo fibroblast monolayer assay proves instructive. If ray-transformedcells are unable to induce foci in the presence of normal neighbors while ras+myc cells growth strongly, then one mechanismof action of myc is clear: myc enables ras transformants toignore or override the inhibitory influences of normal neighboring cells.

How myc oncogenes can do this mechanistically is not at allapparent. One clue may derive from the peculiar behavior ofmyc and the group of other nuclear oncogenes that it represents.Expression of the normal (protooncogene) versions of thesegenes is highly regulated and almost always positively correlatedwith growth, myc itself is expressed at low basal levels inquiescent fibroblasts and its expression is substantially increased and maintained upon entrance into the active cell cycle.myc and other nuclear oncogenes are turned on rapidly inresponse to a number of cell mitogens.

This provides a clue and a speculation; perhaps equally3716




important negative regulatory mechanisms also govern nuclearprotooncogene expression. Perhaps antimitogenic signals impinging on the cell actively suppress the expression of myc-likegenes. Such negative environmental influences may encompasssignals involved in maintaining contact inhibition, signalsthrough which growth-inhibitory hormones like interferon andtransforming growth factor ÃŸshut down cell growth, indeedeven the signals used by normal cells to shut down the growthof ras transformants. While poorly documented, these inhibitory mechanisms may all operate through their ability to suppress expression of /wye-like genes including myc itself (52-56).The genetic changes that create the constitutively expressedmyc oncogene may serve to uncouple the regulation of this genefrom the environmental influences that usually act to shut downthe normal myc gene. From the viewpoint of cell physiology,such uncoupling of nuclear genes from extracellular signals,achieved through their genetic alteration, may create the sameend results as are seen upon uncoupling of cell-to-cell contact

by TPA.One often depicts myc oncogenes as constitutively expressed

and in this way no longer dependent on the mitogenic stimulation normally required to elicit and maintain their expression(57). Perhaps we should think of myc oncogenes in an entirelydifferent light: as genes that can remain on in spite of thepresence of antimitogenic influences that normally operate toshut down expression. This translates further into possibleinsight as to how myc oncogenes enable ras transformants toignore their normal neighbors; the myc oncogene, unlike itsnormal protooncogene, can remain on in spite of extracellularinfluences that would normally force its shutdown.

Creating Constitutively Activated myc-like Genes

We know rather little about how nuclear protooncogenes areregulated either positively or negatively. A number of researchgroups have focused on the DNA sequences that are linked totheir transcriptional promoters and serve to increase use ofthese promoters. Virtually nothing is known of the m-actingsequences that allow the cell to repress expression of theseprotooncogenes. Clearly by deleting or inactivating negativelyacting regulatory sequences of these genes, one may achievemany of the conditions required for their constitutive expression, thereby making these genes into oncogenes (58, 59).

An alternative molecular mechanism may achieve the sameend result. Rather than deleting the as-acting regulators ofexpression, other types of damage may serve to delete from thecell the diffusible trans-acting factors that normally act tomediate shutdown of genes like myc. This idea deserves generalization: a phenocopy of the myc oncogene-induced state maybe achieved by knocking out the signal transducing pathwaysthat normally shut down cell growth in response to environmental growth-inhibitory influences. The lost elements of thesesignaling pathways may be either regulators of nuclear protooncogene expression or even the targets of nuclear oncoproteinaction.

Such an idea forces the restructuring of the concept of oncogene collaboration. The events that often serve to collaboratewith ras activation may not be the activations of nuclear oncogenes; instead, the inactivation of negative regulatory pathwaysand the genes that encode them may be the most frequent wayof achieving the same end result. After all, it is usually far easierto knock out gene function than to create the hyperactive alÃelesthat we recognize as oncogenes.

Tumor cell genomes may often contain activated oncogenes

coexisting with inactivated versions of negative regulatorygenes, the two sets of changes collaborating to confer iumori-genicity. In the case of many raj-bearing tumors observed todate, complementary wye-like changes may be sustainedthrough gene inactivation. Moreover, immortalization/establishment in vitro and the acquisition of ras responsiveness mayalso be associated with the loss of negative regulatory genes(60).

Tumor Suppressor Genes: Dominance and Recessiveness

The existence of such negative regulatory genes is suggestedby an extensive literature describing the consequences of fusingnormal cells with tumorigenic cells (61-65). A frequent outcome is the loss of tumorigenicity by the hybrid cell. Thissupports the notion that the creation of the tumorigenic stateoften involves loss of growth-regulatory genes that are restoredto the tumor cell upon fusion with a normal partner. These lostnegative regulators may be termed tumor suppressor genes inthe sense that they serve to regulate normal cell growth; theirloss removes a critical constraint on proliferation that in turncan contribute to tumorigenicity.

Some have viewed the nontumorigenic phenotype of thesehybrids as evidence that the genes responsible for the tumorphenotype act recessively, being unable to elicit tumorigenicityin the presence of the wild-type intact alÃelesthat act domi-nantly. But this interpretation is unwarranted. If tumorigenesisis dependent on mutations in a number of critical genes, someof the mutations may create dominant, deregulated alÃeles,while other essential mutations in the same cells may createrecessive, inactivated alÃeles.If only one of the essential, contributory steps in tumorigenesis involves the creation of inactive, recessive alÃeles,then the phenotype of tumorigenicity asa whole will behave as if it too were recessive. As a consequence,it has little meaning to speak of tumorigenicity as a dominantor recessive phenotype. One can only use these terms meaningfully to describe the behavior of specific gene alÃelesacting inthe presence of their wild-type homologues.

To cite an example, in certain cases a ras oncogene-containingcell has been reverted to nontumorigenicity following fusionwith a normal partner (65, 66). Here the interpretation hasbeen that the ras oncogene acts recessively, i.e., that the normalcell contains gene products that override and suppress theactivity of ras (63, 66). But if the malignant state of the ras-bearing cell depends both on the activation of a ras oncogeneand on the inactivation of a negative regulatory gene, then suchinterpretation are untenable. The dominance or recessivenessof a ras oncogene can be known only by gauging its activitiesrelative to those of its wild-type alÃele.And the reversion ofthese cells to a nonmalignant state may only signal the essentialinvolvement of other genes, fully unrelated to ras, the inactivation of which acts collaboratively with ras activation to createthe end point of tumorigenicity. The isolation of one suchcandidate gene, which partially suppresses the malignant phenotype of a ras-transformed cell, has recently been reported(67).

Molecular Nature of Tumor Suppressor Genes

The use of somatic cell hybridization has served well to pointout the existence of tumor suppressor genes but has proved tobe a cumbersome tool in learning more about them. In order tounderstand these genetic factors, we need to identify these geneswith discrete genetic loci, to isolate these as molecular clones,

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ONCOGENES. ANTIONCOGENES, AND CARCINOGENESIS

and to study the biochemical modes of action of their encodedproteins.

The Rb gene, the loss of which predisposes to retinoblastomasand osteosarcomas, has proved to be the most tractable of apossibly large class of such genes. A conceptual breakthroughin our understanding of retinoblastoma origins came from thehypothesis of Knudson (68, 69) that all retinoblastoma tumorsmust sustain mutations in two distinct genes prior to tumordevelopment. In 1979, Yunis and Ramsay (70) provided thefirst evidence that at least one of these mutations creates inactivealÃeles.This was suggested by their observation of the occasionaldeletion of genetic material on chromosome 13ql4 in retinoblastomas. This suggested further that this chromosomal regionharbored a discrete gene (i.e., Rb) that served repeatedly as thetarget of genetic inactivation events occurring during the formation of these tumors.

The identity of the second of Knudson's hypothesized target

genes was soon apparent; it was the surviving, hitherto intactcopy of the Rb gene on the homologous wild-type chromosome.Study of a closely linked chromosomal marker gene, esteraseD, showed that its heterozygosity, often observed in the normaltissues of a retinoblastoma patient, was reduced to homozygos-ity in the tumor cells (71, 72). By analogy, the implications forRb were clear; the second step in tumorigenesis was the loss ofthe surviving intact alÃeleof the Rb gene, it being replaced by acopy of the initially mutated one. This established the pointthat both copies of the Rb gene need to be lost or inactivatedin order for phenotype to be affected. In this sense the Rb geneshows the properties of a negative regulatory gene of the typedescribed earlier.

A similar phenomenology has been derived for the Wilm's

locus, in which interstitial deletions and reductions to homozygosity have been observed associated with a chromosome 11locus (73). Moreover, the paradigm has been generalizedthrough use of polymorphic restriction enzyme site markers inwhich a number of distinct tumor types have been associatedwith reductions to homozygosity of particular chromosomalloci (74). In most of these cases, direct evidence of gene inactivation is still lacking and the recessiveness of the involvedalÃelesis imputed from the observed homozygosities.

In the case of the Rb locus, a candidate gene has been isolated:it is a 190-kilobase stretch of chromosome 13ql4 that is expressed in many tissues and specifies the structure of a M,105,000 phosphoprotein (75-78). The importance of this protein, or rather its loss, to the genesis of retinoblastoma isunderscored by recent evidence which shows that this proteinis present in normal retinoblasts but absent from 16 of 16retinoblastomas.4 To date the Rb gene stands as the only

example of a potentially large class of genes that has yielded tomolecular cloning.

The isolation of the Rb gene leaves a number of unresolvedquestions and paradoxes. The most direct proof that the clonedgene is indeed the Rb gene must come from introduction of acloned intact copy into retinoblastoma cells with observedrestitution of normal growth control. A recent report of thishas appeared in the literature (79). The cloning of this genealso reveals apparent paradoxes. While the gene is expressed ina rather wide array of tissues, ostensibly participating in theirgrowth control, its inactivation seems to lead to only a narrowrange of tumor types, notably retinoblastomas, soft tissue andosteosarcomas, small cell carcinomas of the lung, and bladdercarcinomas (80-84). Equally perplexing is the species distri-

4J. Horowitz and R. A. Weinberg, manuscript in preparation.

bution of retinoblastoma incidence. This type of tumor has beenobserved to occur spontaneously only in our own species, yetthe gene is present and apparently active in all mammals.

The Function of the Rb Protein

The central issue surrounding the Rb gene is the function ofits encoded protein, pl05-Rb. How do its properties fit withthe model of gene inactivation and wye-like phenotypes drawnabove? While little is known about the biochemistry of the Rb-encoded protein, its involvement in the physiology of growthregulation has been highlighted by a dramatic discovery madein the spring of 1988. This discovery directly ties the Rb proteinto the multistep pathways of transformation discussed here.Specifically, it places the Rb protein in the middle of the arenaof nuclear, wye-like oncogenes.

The work originated in the laboratory of Ed Harlow of ColdSpring Harbor who together with others had shown that theoncoprotein encoded by the EIA oncogene of human adenovi-rus type 5 is found complexed with a variety of host cell proteinsin virus-transformed cells (85, 86). These proteins number morethan six. Their multiplicity may provide a molecular explanation for the multiple functions exerted by the EIA oncogene,which include cell immortalization, oncogene collaboration,and regulation of transcription of a number of viral and hostgenes. Each of the host cell target proteins to which EIAcomplexes may represent a regulator of a distinct cellularpathway, the activities of which may be modulated followingcomplex formation.

One of these host cell target proteins was found to be p 105-Rb (87). This association is not merely adventitious, sincemutations that inactivate the transforming activities of EIAalso knock out its ability to complex with pl05-Rb. Sinceinactivation of the Rb gene and resulting loss of pl05-Rb iscritical to the formation of retinoblastomas, it is tempting tospeculate that a similar result is achieved epigenetically throughthe ability of the EIA oncoproteins to complex with and functionally inactivate the AA-encoded protein.

This strengthens the hand of those who would call Rb an"antioncogene." Here we see a direct physical confrontation

between an oncogene product and that of the Rb gene. Nevertheless, the term is misleading in that it implies that the role ofRb is to antagonize oncogene function. More palatable is theterm "tumor suppressor." Ultimately the term "growth suppressor" may be seen to mirror most closely the normal func

tions of the Rb and its analogues.Other complexes similar to Rb:ElA have been discovered

more recently. They are found between Rb and oncoproteins ofat least two other DN A tumor viruses, those of the SV40 largeT (88) and human papilloma type 16 E7 (89) oncogene.Through an apparent process of convergent evolution, threedifferent groups of DNA tumor viruses (adeno-, papilloma,SV40) have developed oncoproteins that specifically complexwith pl05-Rb. In the case of SV40 large T, a point mutationaffecting only 1 of the 708 amino acid residues of the proteininactivates its transforming powers and at the same time destroys its ability to form complexes with pl05-Rb (88). Thisargues that these associations are central to the ability of theseoncogenes to contribute to transformation.

All this highlights the unexpectedly central position thatpl05-Rb occupies in the growth-regulatory circuitry of the cell,being involved in a number of distinct mechanisms of transformation. Of equal interest is the biochemical basis that thisprovides to mechanisms of oncogene collaboration. All three of

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the oncogenes have been shown to function as myc-\ike genes,each able to immortalize and to collaborate with ras oncogenesin transformation of primary cells (Table 1). The oncogenecollaboration test, described earlier, has been a purely functionalmeans of classifying oncogenes. It is satisfying that these oncogenes, functionally allied through this test, can now be partially understood in terms of a shared molecular mechanism.The interactions of these three different DNA virus oncopro-teins with a common cellular target suggest that they act bymimicking an endogenous cellular protein. An attractive candidate would be the cellular myc protein itself, but the evidenceis still lacking.

These interactions provide support for the notion describedearlier that inactivation of a tumor suppressor gene yieldsphenotypes that resemble those resulting from myc-tike oncogenes. How physiological changes like immortalization andoncogene collaboration are achieved at the molecular levelremains unclear. It is tempting to speculate that the Rh geneproduct is a component of a growth-inhibitory signalling chain.When the Rb protein is lost through alteration of its gene, theresponsiveness of the cell to negative signals may be compromised or lost. Similarly, by complexing with the Rb protein,DNA virus oncoproteins may inactivate its function and in thisway deprive the cell of a vital link needed to transduce growth-

inhibitory signals.

Oncogenes and Antioncogenes in Multistep Carcinogenesis

These models and recent findings have implications for thegenesis of retinoblastoma tumors. It has been argued thatinactivation of both copies of the Rb gene suffices to createthese retinal tumors (68, 69). But ifRb inactivation creates only/wye-like changes in the cell, then it may be the case thatcomplementary changes in a cytoplasmic oncogene may berequired to create a truly autonomously growing tumor. Such anotion is not yet addressed by direct experimental studies ofretinoblastoma genomes.

Another type of tumor cell genome, that of colon carcinomacells, has already yielded evidence of activated ras oncogenesoccurring together with reductions to homozygosity of loci onchromosomes 17 and 18 (40). The inactive state of these homo-zygous alÃeleshas not yet been shown. Nonetheless, this appearsto provide an attractive tumor model in which oncogene activation and antioncogene inactivation collaborate to create thefull malignant phenotype.

Where will all this take us? It should be clear that oncogenespresent only part of the answer to the puzzle of multisteptumorigenesis. The vista of the negative genetic regulators ofcell growth, termed variously antioncogenes, tumor suppressorgenes, or growth suppressor genes, is just opening. These geneswill be hard to find and study since their existence is mostapparent when they have undergone inactivation. However, thestruggle to find them will be worth the effort. They will tell usas much about cancer as oncogenes, perhaps even more. Andthey will fill a large gap in our current picture of the growth-regulatory circuitry of the cell.

Acknowledgments

The author would like to thank Ehry Anderson for excellent help inpreparing this manuscript. He is an American Cancer Society ResearchProfessor.

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1989;49:3713-3721. Cancer Res Robert A. Weinberg Multistep CarcinogenesisOncogenes, Antioncogenes, and the Molecular Bases of

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