© 1999 Elsevier Science Ltd. All rights reserved. For article fee, see p. IV.TCB, Vol. 9, N¼ 12 (0962-8924) TIBS, Vol. 24, N¼ 12 (0968-0004) TIG, Vol. 15, N¼ 12 (0168-9525)PII: S0962-8924(99)01668-2 PII: S0968-0004(99)01480-2 PII: S0168-9525(99)01892-2

Millennium issue

M53

Frank McCormickmccormick@cc.ucsf.edu

UCSF CancerCenter, CancerResearch Institute,2340 Sutter St, San Francisco,CA 94115, USA.

T umours arise through stepwise mutations in proto-oncogenes and tumour-suppressor genes. The initial identi-fication of these genes and their functions suggested that

they affect discrete pathways, each making distinct contributions tothe full malignant phenotype: the Ras pathway drives uncontrolledproliferation, the retinoblastoma (Rb) pathway alters cell-cyclecontrol, the p53 pathway affects apoptosis, and so on. Morerecently, as we learn more about the complexities of these path-ways and their relevance in biological systems that are moresophisticated, it has become increasingly difficult to ascribe dis-tinct biological functions with precision. Furthermore, the path-ways intersect at multiple points, so that they can no longer beconsidered in isolation. Indeed, their interdependence has impor-tant implications for the stepwise model itself, as most singlemutational steps require others to reveal their selective advantage.Here, I review some of these issues in the context of two majorpathways that are misregulated in human cancers at high fre-quencies: the Ras pathway and the adenomatous polyposis coli(APC) pathway.

Ras: concerted pathways leading to cancerRas proteins activate multiple signalling pathways (summarizedin Fig. 1). Ras binds to at least three types of effector protein:kinases of the Raf family, phosphoinositide (PI) 3-kinases andRalGDS proteins. Several other candidate effector proteins havebeen identified, such as Rin1 and AF6, but their biological func-tions are not yet known1. The molecular mechanisms that lead toactivation of Ras effectors are complicated and unclear, and thedownstream consequences of activation are more complicatedstill. Raf and PI 3-kinase effector pathways influence cell prolifer-ation and apoptosis, and each effector might activate complextranscription programmes that could contribute in any numberof ways to tumour progression and survival. For example, acti-vation of the mitogen-activated protein (MAP) kinase cascadeinduces the transcription of proteases, such as urokinase-typeplasminogen activator (uPA)2, which could affect cell motilityand invasion, and transcription of proteins that induce angio-genesis, such as vascular endothelial growth factor (VEGF)3. It islikely that the RalGDS pathway and other effector pathways willbe equally complex.

Which of these pathways is the major driver of Ras-dependenttransformation? The answer is probably all of them. Ras effectorpathways interact synergistically, at least in cell-culture models oftransformation, and the powerful biological effects of Ras are the result of the concerted action of multiple activities4. Forexample, activated Raf is a weak oncogene in traditional 3T3 cell

focus-forming assays. Activated Rac barely scores in this assay,but the combination of Raf and Rac causes dramatic transformingactivity5. Synergistic interactions have been noted for Rho andRaf (Ref. 6), PI 3-kinase and Raf (Ref. 4), RalGDS and Erk (Ref. 7),and so on. Consistent with this, Ras transformation can bestrongly inhibited by blocking any one of these pathways, whichis encouraging from the perspective of therapeutic intervention.

Another encouraging aspect of this unexpected complexity isthe possibility that Ras transformation might be qualitatively different from the Ras signalling that occurs during normal acti-vation by growth factors: during this process, Ras effector pathwaysshow precise temporal fluctuations. Oncogenic Ras proteins, how-ever, are persistently switched on, and the synergistic interactionsthat appear to be the hallmark of Ras transformation might notoccur during normal signal transduction, when effector pathwaysare separated in time. For example, it is likely that transcriptionpatterns induced by mutant Ras will be the result of convergentsignals from distinct Ras effector pathways, leading to expressionof genes that are not expressed in normal cells, even when theyare proliferating rapidly.

Oncogenic Ras signalling could differ from normal Ras sig-nalling in another way: high levels of constitutively active Ras mightengage effector proteins that are not activated by normal Ras. Therole of Ras in PI 3-kinase activation could be an important exampleof this: Ras plays a minor role in normal signalling to PI 3-kinasefrom growth factor receptors, such as the platelet-derived growthfactor (PDGF) receptor and epidermal growth factor (EGF)receptor8, but oncogenic Ras is a potent activator of PI 3-kinase.Furthermore, the function of Ras might change during tumourdevelopment as levels of Ras proteins and their effectors arealtered. The stepwise increases in Ras levels that occur duringmouse models of carcinogenesis might reflect the engagement ofdifferent effectors, with different selective advantages to tumourgrowth at each step9. The idea that Ras could contribute totumour development through multiple pathways that change astumours develop is a far cry from our perception of ten years ago,when the hunt was on for a single, elusive Ras effector thataccounted for Ras transformation.

The role of Raf and the MAP kinase pathway in Ras signallingand transformation has been studied thoroughly and is wellestablished. Drugs that inhibit this pathway are in preclinicaldevelopment as potential anticancer therapies. The PI 3-kinasepathway was discovered more recently and is of tremendous cur-rent interest. Carcinomas are the major form of human cancer,and are derived from epithelial cells. Epithelial cells normally dieby apoptosis when they detach from their surrounding substrates:

Signalling networks that cause cancerFrank McCormick

Cancer is caused by the stepwise accumulation of mutations that affect growth control, differentiation andsurvival. The view that mutations affect discrete signalling pathways, each contributing to a specific aspect ofthe full malignant phenotype, has proved to be too simplistic. We now know that oncogenes and tumoursuppressors depend on one another for their selective advantage, and that they affect multiple pathways thatintersect and overlap. The interactive nature of each genetic change has important implications for cancertherapy and for the stepwise model of carcinogenesis.

Millennium issue Frank McCormick ¥ Intracellular signalling and cancer

M54

therefore, the suppression of apoptosis can be a crucial factor incarcinoma development. Oncogenic Ras proteins suppress apop-tosis through the PI 3-kinase pathway (Fig. 1), partly through theactivation of protein kinase B (PKB)10. The precise role of PKBin suppressing apoptosis is complex: PKB phosphorylates BAD11,a member of the Bcl-2 family that promotes cell death, and caspase 912, a protease that degrades cellular substrates as part ofthe process of apoptotic death. But PKB also regulates transcriptionthrough the phosphorylation of Forkhead proteins13,14, whichmight themselves regulate other effectors of apoptosis, suggestinga much more complex and indirect route towards survival.

The importance of PKB and the PI 3-kinase pathway in cancerhas been underscored by the discovery that PTEN (phosphataseand tensin homologue deleted from chromosome 10), also knownas MMAC 1 (mutated in multiple advanced cancers 1) or TEP1(TGF-b-regulated and epithelial cell-enriched phosphatase 1), is a phosphatase that degrades phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3], a product of PI 3-kinase and akey regulator of PKB15. Therefore, the loss of PTEN activatesPKB indirectly by allowing the accumulation of PtdIns(3,4,5)P3(Ref. 16). Activation of PKB by Ras could be biologically similarto loss of PTEN, if indeed they are on the same pathway and ifPKB is a major Ras effector. If so, Ras mutations and loss ofPTEN might be mutually exclusive, a possibility that has not yetbeen tested formally.

APC: more of the same?Mutational inactivation of the gene encoding APC occurs in themajority of human colon cancers and leads to accumulation of b-catenin. Many of the remaining colon cancers, as well as othertypes of cancer, have mutations in b-catenin itself, at sites that makethe protein resistant to APC-directed destruction17. b-catenin

binds to and activates transcription factors of the T-cell factor(TCF)/lymphoid enhancer factor (LEF) family, a signalling eventthat occurs normally in response to Wnt ligands. These discoveriesled to the search for TCF-inducible genes that might be targets ofthe b-catenin pathway in colon cancers. These searches haverevealed the usual suspects: Myc (also known as c-MYC)18 andcyclin D119, as well as the matrix metalloprotease matrilysin20,uPA21 and Cox-222 and a number of candidates whose biologicalfunctions are unknown23.

We identified the gene encoding cyclin D1 as a b-catenin-regulated gene and considered it an attractive target because of itspossible role in allowing cell proliferation under conditions thatpromote differentiation and senescence in normal cells. We pro-pose that, during early development of the colonic epithelium, b-catenin, TCF and cyclin D1 are all present at relatively highlevels. As differentiation occurs, the levels of b-catenin drop, possibly through reduced Wnt signalling. In the absence of b-catenin, TCF represses cyclin D1 promoter activity, leading toterminal differentiation. In rare cells expressing high levels of b-catenin, owing to loss of APC or mutation in the gene encod-ing b-catenin, proliferation continues, leading to clonal growthof cells: this could be the first step towards malignancy, eventhough these cells are not overtly transformed. This model, inwhich b-catenin serves to suppress differentiation, is consistentwith observations that b-catenin itself does not function as anoncogene in standard transformation assays.

Another well-characterized potential target of the b-cateninpathway is the MYC proto-oncogene18. Like cyclin D1, Myc is anattractive biological target because of its obvious role in cell pro-liferation, and, like cyclin D1, it is highly expressed in manycolon cancer cell lines and primary tumours. Other putative tar-gets of the pathway, such as the UPA receptor (uPAR), matrilysin

FIGURE 1. Ras activates multiple effector pathways. Ras proteins in their active, GTP-bound state, bind directly to RalGDS, phosphoinositide(PI) 3-kinase or Raf. Each of these events triggers a cascade of downstream events that lead to proteins affecting many aspects of cellgrowth and survival. Ultimately, each pathway leads to proteins that are involved in the transcriptional regulation of gene expression.Abbreviations: GSK3b, glycogen synthase kinase 3b; HbEGF, heparin-binding epithelial growth factor; PKB/Akt, protein kinase B.

TCB•TIBS•TIG

Ras

PI 3-kinaseRalGDS Raf

PKB/Akt

PDK

? RacRal

Mek

Forkhead

GSK3β BadE1F2

? Caspase-9

Rho ? Ets

Erk?

?

?

? Fas ligand FosHbEGFCyclin D1p21?

Millennium issueFrank McCormick ¥ Intracellular signalling and cancer

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and Cox-2, can be justified equally easily. Which of these targetsis the more important? By analogy with the Ras pathway, wemight anticipate that the APC–b-catenin pathway leads to mul-tiple, interactive effects. Like Ras, the function of b-cateninmight change during tumour development.

When pathways collideExpression of cyclin D1 is regulated by activated Ras, forging apotential link from growth-factor signalling to cell-cycle progressionthrough G1 to S phase. Ras has multiple effects on cyclin D1: theRaf–MAPK branch of the Ras pathway regulates transcription ofthe gene encoding cyclin D124 and the PI 3-kinase branch affectsstability of the cyclin D1 protein, through the action of PKB andof glycogen synthase kinase 3b25. Perhaps these distinct effectsprovide the basis of synergy between these two Ras effector path-ways26. These observations, together with its central role in growthregulation, appear to make cyclin D1 a prime target of Ras acti-vation. However, inhibition of signalling from b-catenin to TCFin colon cancer cells inhibits cyclin D1 expression, even in thepresence of activated Ras19. This suggests that, in these cells, lossof APC and accumulation of b-catenin is the primary cause ofincreased cyclin D1 expression. Another argument against the ideaof cyclin D1 as a major Ras target comes from the observationthat Ras mutations occur in tumours in which the gene encodingRb is mutated and the Rb checkpoint is defective. In thesetumours, expression of cyclin D1 should be irrelevant as the Rbprotein is thought to be the major substrate for cyclin D1–cyclin-dependent kinase (CDK) complexes. Perhaps cyclin D1 is indeeda Ras target in some tumours at certain stages of tumour develop-ment, but its potential role needs to be considered in the contextof other intersecting pathways that affect its function.

Glycogen synthase kinase 3 (GSK3), like cyclin D1, is part ofthe Ras and b-catenin pathways, raising difficult questions aboutits precise mode of regulation. It is a substrate for PKB, as dis-cussed above, but is also a key component in the pathway thatleads from Wnt ligands to b-catenin (Fig. 2). In both pathways,GSK3 activity is decreased by upstream signalling, but the down-stream consequences are quite different: signals from PKB lead todecreased phosphorylation of glycogen synthase, signals from the

Wnt pathway lead to decreased phosphorylation of b-catenin andincreased b-catenin stability. The mechanism by which decreasedGSK3 could lead to two distinct downstream signals is fascinat-ing but, as yet, unresolved.

MDM2 is another protein that might be regulated by mul-tiple pathways. Most of its biological properties have been attrib-uted to its effects on p53 degradation, and it is a transcriptionaltarget of p53 itself. This led to an elegant model in which p53induces a feedback loop that results in its own destruction27. Butmore-recent analysis reveals that MDM2 is an immediate-earlygene that is turned on by growth-factor signalling28, suggesting adirect pathway from mitogenic signals to suppression of p53activity, and also explaining why MDM2 is expressed in manytumour cells that lack functional p53. Recently, a role forMDM2 that is distinct from p53 degradation has been proposed,possibly through Rb and E2F29: this might be another way inwhich MDM2 affects cell growth, and another example of inter-secting signalling pathways.

Each of these key cell regulators, cyclin D1, GSK3 andMDM2 is controlled at multiple levels by several distinct path-ways: furthermore, these pathways are altered in cancer cells andplay direct causal roles in the development of cancer. Otherexamples are easy to find: recently, c-Fos, a well-established tran-scriptional target of the Ras pathway, was shown to be regulatedby p5330. Myc protein is stabilized by Ras31, and Myc mRNA isinduced by the b-catenin pathway18. uPAR has been identified asa target of the b-catenin pathway, as described above: it is also atarget of the Raf–MEK–ERK pathway2. The recent discoverythat high levels of b-catenin induce expression of p53 is anotherexample of this complexity32.

Functional steps into cancerIn the current view of multistep carcinogenesis, each new mu-tational step leads to a clonal expansion of cells bearing the newmutation: in this way, the probability that multiple mutationsoccur in the same cell is greatly increased. Without this clonalexpansion of mutant cells, the chances of accumulating severalvery rare events in the same cell becomes vanishingly small. The strong interdependence of oncogenic events discussed here

FIGURE 2. Common targets in the Ras and Wnt/adenomatous polyposis coli (APC) pathway. Signal-transduction pathways intersect andoverlap. In this example, Ras and Wnt pathways independently send signals that converge on glycogen synthase kinase 3 (GSK3) to inhibitits activity. GSK3 is involved in targeting cyclin D1 and b-catenin for degradation. Cyclin D1 is also a direct target of transcription ofanother Ras pathway and of b-catenin/T-cell factor (TCF).

TCB•TIBS•TIG

Ras Wnt

GSK3

Cyclin D1

Frizzled

APC-mediated degradation

Degradation

PI 3-kinase PKB

Raf

Ets

Dishevelled

β-catenin

TCF

Mek

Erk

Millennium issue Frank McCormick ¥ Intracellular signalling and cancer

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constrains this model and strongly suggests that mutationsshould occur in a preferred order.

For example, loss of the Rb checkpoint, whether directly throughmutation in Rb itself, or through mutations in p16, APC, b-catenin or other events that lead to upregulation of cyclin-D–CDK activity, allows cells to escape senescence and differentiation.This alone could be a first step towards cancer. However, loss offunctional Rb allows the ras oncogene to transform cells: in pri-mary cells, Ras promotes cell-cycle arrest and senescence, probablythrough induction of the CDK4/CDK6 inhibitor protein p16 bythe Raf–MAP kinase effector pathway33. For this reason, Ras acti-vation might lead only to clonal expansion of cells in which theRb checkpoint is already defective. Therefore, loss of the Rbcheckpoint creates a permissive cellular context for clonal expan-sion driven by oncogenic Ras. It also leads to increased E2F tran-scriptional activity, which drives cells towards the S phase of thecell cycle. However, high levels of E2F leads to apoptosis34. Thismight be suppressed through loss of functional p53 or by activationof other pathways promoting cell survival.

In this example, loss of Rb facilitates activation of Ras andnecessitates loss of p53 (or an equivalent event) for tumour pro-gression. Clearly, the biological consequences of mutating these threemajor pathways are co-dependent and contain considerable func-tional overlap. This leads to a condition described by Weinstein asgene addiction35, in which the full malignant phenotype dependson the continued interaction between pathways. This has obviousimplications for cancer therapy and encourages the view thattherapeutic intervention by inhibiting a single key target couldhave profound effects on the growth and survival of tumour cells.

Concluding remarksOur current view of the stepwise progression of cancer, most elegantly illustrated by the development of colorectal cancer (see article by Cahill et al. in this issue), contains unexpectedlydeterministic elements, in which mutations in pathways thatonce seemed distinct strongly influence subsequent mutations,either by providing a more permissive cellular environment or by creating the need for further genetic changes for cell survival.This view has been created by projecting our functional analysisof oncogene and tumour suppressors in vitro onto the geneticframework of tumour progression in vivo. The functionalapproach probably represents an overestimate of possible inter-actions: in any one cell, Ras might only perform a subset of thefunctions illustrated in Fig. 1, for example. On the other hand,genetic analysis probably underestimates the complexity oftumour progression. Indeed, whole-genome scans of gene-copy-number changes in tumours reveal a much greater level of complexity than previously imagined36. To determine whichpathways are crucial to the process of cancer development, wewill need new ways of manipulating these pathways in vivo andmeasuring their activities quantitatively. The availability of phar-macological inhibitors of these pathways, and new ways ofmanipulating gene function in vivo, could address some aspectsof the first problem. Reagents that detect activated states of signalling proteins, such as phospho-specific antibodies, are a first step towards the second. As pathways appear to becomemore complex and entangled we will need, more than ever, todistinguish between what is functionally possible and what is biologically important.

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