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nature genetics • volume 29 • october 2001 117

TGF-β signaling in tumor suppression andcancer progression

Rik Derynck1, Rosemary J. Akhurst2 & Allan Balmain2,3

Epithelial and hematopoietic cells have a high turnover and their progenitor cells divide continuously,making them prime targets for genetic and epigenetic changes that lead to cell transformation andtumorigenesis. The consequent changes in cell behavior and responsiveness result not only from genet-ic alterations such as activation of oncogenes or inactivation of tumor suppressor genes, but also fromaltered production of, or responsiveness to, stimulatory or inhibitory growth and differentiation fac-tors. Among these, transforming growth factor β (TGF-β) and its signaling effectors act as key deter-minants of carcinoma cell behavior. The autocrine and paracrine effects of TGF-β on tumor cells andthe tumor micro-environment exert both positive and negative influences on cancer development.Accordingly, the TGF-β signaling pathway has been considered as both a tumor suppressor pathwayand a promoter of tumor progression and invasion. Here we evaluate the role of TGF-β in tumor devel-opment and attempt to reconcile the positive and negative effects of TGF-β in carcinogenesis.

1Departments of Growth and Development, and Anatomy, Programs in Cell Biology and Developmental Biology, 2Mount Zion Cancer Research Institute,3Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California, USA. Correspondence should be addressedto R.D. (e-mail: [email protected]).

In mammals, there are three different TGF-βs, β1, β2 and β3,which are encoded by different genes and which all functionthrough the same receptor signaling systems1,2. Of these, TGF-β1is most frequently upregulated in tumor cells3,4 and is the focusof most studies on the role of TGF-β in tumorigenesis.

The TGF-β protein is released as an inactive ‘latent’ complex,comprising a TGF-β dimer in a non-covalent complex with twoprosegments, to which one of several ‘latent TGF-β–binding pro-teins’ is often linked5–7. This latent complex represents an impor-tant safeguard against ‘inadvertent’ activation, and may stabilizeand target latent TGF-β to the extracellular matrix, where it issequestered7,8. The matrix thus acts as a reservoir from which TGF-β can readily be recruited without the need for new synthesis.

The secretion of TGF-β as a latent complex necessitates theexistence of a regulated activation process, which is mostprobably mediated through the activities of proteases thatpreferentially degrade the TGF-β prosegments and therebyrelease the highly stable, active TGF-β dimer. Because plasminactivates latent TGF-β9,10 and plasminogen is converted intoplasmin at sites of cell migration and invasion11, we assumethat endothelial and tumor cells are exposed to active TGF-β.

Latent TGF-β can also be activated by the metalloproteasesMMP-9 and MMP-2 (ref. 12), which are frequently expressedby malignant cells, especially at sites of tumor cell inva-sion13,14. Other activation mechanisms might not depend onproteases. For example, the extracellular matrix proteinthrombospondin15,16 and the αvβ6 integrin, which isexpressed at the surface of epithelial cells in response toinflammation17, may mediate TGF-β activation through aconformational change in the TGF-β complex. Thus, differentmechanisms may regulate TGF-β activation in different physi-ological contexts, and tumor cells are well equipped to activateTGF-β locally.

Mechanisms of TGF-β signalingThe TGF-β receptors. As the mechanisms of TGF-β receptor sig-naling have been extensively reviewed recently18–23, we will sum-marize only the salient points (Fig. 1) before focusing on the roleof TGF-β in cancer cells. TGF-β signals through a heteromericcell-surface complex of two types of transmembrane serine/thre-onine kinases, named ‘type I’ and ‘type II’ receptors. Althoughthere is only one type II TGF-β receptor (TβRII), there are threetype I receptors (ALK1/TSR-1, ALK2/Tsk7L, ALK5/TβRI). Mostgene expression responses are presumably mediated by the TβRIreceptor. TβRII and TβRI show a widespread pattern of expres-sion and are expressed by tumor cells. After TGF-β binds to thereceptor complex, the TβRII kinase phosphorylates TβRI in the‘GS sequence’, which is located upstream from the kinasedomain. This phosphorylation activates the TβRI kinase thatmediates TβRI autophosphorylation and phosphorylation ofdownstream target proteins.

Several intracellular proteins have been shown to interact withTGF-β receptor complexes on the cell surface. Among these,FKBP12 interacts constitutively with the juxtamembrane domainof type I receptors and regulates the conformation of this domainand the signaling sensitivity24–27. Three proteins containing WDrepeats associate with and are phosphorylated by the receptorcomplex after ligand-induced activation and regulate TGF-βreceptor signaling. Whereas TRIP-1 interacts with TβRII (refs28,29), the Bα subunit of the protein phosphatase 2A interactswith type I receptors30, and STRAP interacts with both TβRIIand TβRI to decrease TGF-β signaling31,32. The regulatory rolesof these proteins are well documented, but there is no convincingevidence to show that these WD-repeat proteins act as effectorsof TGF-β responses, with the exception of the Bα subunit of pro-tein phosphatase 2A, which may be involved in growth arrestthrough inactivation of the S6 kinase pathway33.

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118 nature genetics • volume 29 • october 2001

Signaling through Smads. The only well-characterized signal-ing effector pathway that is initiated by activated TGF-β receptorsis provided by the Smads, a small family of structurally relatedproteins (Fig. 1) 19–23,34,35. Smad2 and Smad3 are activated viacarboxy-terminal phosphorylation by type I TGF-β receptorkinases and form heterotrimeric complexes with Smad4. Thesecomplexes then translocate into the nucleus and act as TGF-β-induced transcriptional activators of target genes. By contrast,Smad6 and Smad7 interfere with the activation of the effectorSmads and act as ‘inhibitory’ Smads. Both Smad6 and Smad7 caninteract with type I receptors, thus competitively preventing the‘receptor-activated’ Smads from being phosphorylated; Smad6also interferes with the formation of the heteromeric Smad com-plex. TGF-β signaling induces expression of Smad7, which pro-vides a TGF-β–induced negative feedback loop.

The TGF-β protein activates or represses the transcription ofdefined target genes, and many of these responses are immediateresponses to TGF-β receptor activation. So far, Smads have beenshown primarily to activate transcription19–23,34,35, but they arealso involved in TGF-β–mediated repression of transcrip-tion36–38. Smads also interact with many DNA-binding tran-scription factors; this interaction, together with theDNA-binding activity of Smads, specifies the promoter bindingand transcriptional activation21,34,35. For example, Smad3 caninteract with bZIP transcription factors such as c-Jun (refs.39–41), ATF-2 (refs. 42,43) or CREB (ref. 44); basic

helix–loop–helix transcription factors such as TFE3 (refs. 45,46);runt domain transcription factors such as AML-1, AML-2 andCBFA1 (refs. 37,44,47,48); nuclear receptors such as the vitaminD3 (ref. 49), glucocorticoid (ref. 50) and androgen receptors(refs. 38,51), and STAT-3 (ref. 52).

The receptor-activated Smads are linked to the general tran-scriptional machinery through a direct physical interactionwith the transcriptional coactivator CBP/p300, which is essen-tial for the transcriptional activation function of Smads53–55.The high-affinity binding of the Smad transcription complex toa defined promoter sequence is mediated primarily by the inter-acting transcription factor, although Smad3 and Smad4, butnot Smad2, have an intrinsic DNA-binding capability aswell39,56–59. A Smad-binding DNA sequence thus provides afavorable sequence context for Smad binding close to the high-affinity binding sequence for the Smad-interacting transcrip-tion factor34,56,58. This DNA context–dependent binding of aSmad to both the interacting transcription factor and the pro-moter DNA might explain why TGF-β activates only a subpop-ulation of the promoters that bind the transcription factor withwhich a Smad can interact.

Crosstalk with Smad signaling. The TGF-β–dependentrecruitment of Smad complexes to the transcription machin-ery also allows the recruitment of additional coactivators orcorepressors, which regulate the amplitude of transcriptionalactivation. Besides the interaction of the Smad complex with

Fig. 1 TGF-β-induced signaling through Smads, and several non-Smad signaling mechanisms. After ligand-induced activation of the receptor, Smad2 and/or Smad3interact transiently with the TβRI receptor (RI), and this interaction is stabilized by the FYVE protein SARA. Smad2 and Smad3 are phosphorylated on their C terminalsby TβRI, and then dissociate from the receptor to form a heterotrimeric complex comprising two receptor-activated Smads and Smad4. This complex then translocatesinto the nucleus, where it interacts at the promoter with transcription factors with sequence-specific DNA binding to regulate gene expression. The heteromeric Smadcomplex also interacts with the CBP/p300 transcriptional coactivator, which connects the Smad complex with the general transcription factors (GTF). Smad7 inhibitsactivation of Smad2 and/or Smad3 by the receptors, and Smad7 expression is induced on stimulation of one of several signaling pathways—for example, in response toEGF, interferon-γ (IFN-γ) or tumor necrosis factor-α (TNF-α). Several other signaling pathways also regulate both signaling by Smads and Smad-mediated gene expres-sion, as exemplified here by the activation of JNK and p38 MAP kinase signaling in response to various stress signals, and β-catenin signaling in response to Wnt pro-teins. TGF-β also induces activation of Ras, RhoB and RhoA, as well as of TAK1 and protein phosphatase 2A, which leads to the activation of several MAP kinasepathways and the downregulation of S6 kinase activity. The mechanisms of activation of these non-Smad signaling events and how they connect to the het-eromeric TGF-β receptor complex remain to be characterized.

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Fig. 2 TGF-β acts on both the tumor cell and itsenvironment. Both tumor and stromal cellssecrete elevated levels of TGF-β that are self-perpetuating via a cycle of enhanced proteaseactivation of latent TGF-β, auto-induction ofTGF-β1 gene activity and recruitment of TGF-β–secreting cells such as macrophages andneutrophils. Most TGF-β activities, apart fromthe growth inhibition of early tumor cells,promote tumor progression. The TGF-β signal-ing pathway is thus a target for therapeuticintervention in invasive tumors. TGF-β activitycan be targeted by using endogenous pro-teins such as soluble betaglycan or decorin, orby using artificial agents such as antisenseoligonucleotides, antibodies or small-drugmolecules selective for specific components ofthe signaling pathway. ECM, extracellularmatrix; Fas-L, Fas ligand.

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CBP/p300, Smad4 can also engageanother coactivator, named MSG1,into the transcription complex toenhance the Smad response60,61. Bycontrast, recruitment of a corepres-sor decreases the Smad and TGF-βresponsiveness. The corepressorsEvi-1 (ref. 62) and c-Ski (ref. 63,64),as well as SnoN (ref. 65), TGIF (ref.66), SNIP1 (ref. 67) and SIP1 (ref.68), can interact with Smad3 and/orSmad2 and inhibit TGF-β responses.Thus, the expression of Smad-inter-acting corepressors by tumor cellsmight block some TGF-β responses,including its inhibitory effect ongrowth, and contribute to tumorprogression.

The transcriptional activation by Smads, through their physicalinteractions and functional cooperativity with transcription factors,allows crosstalk with other signaling pathways19,21,69,70. For example,the activation of mitogen-activated protein (MAP) kinase pathways,which is commonly observed in tumor cells, and of Jun kinase or p38MAP kinase is likely to regulate TGF-β-induced transcription frompromoters with TGF-β-responsive AP-1 (c-Jun/c-Fos)– orCREB/ATF-binding sites and Smad-binding sites39,42,43,71–74.

Most notably, the expression of extracellular matrix proteins andproteases in response to TGF-β often requires an intact AP-1–bind-ing promoter sequence75,76, suggesting that there is transcriptionalcooperation of Smads with the AP-1 complex and a dependence ofthese TGF-β responses on Ras/MAP kinase and/or phosphatidyli-nositol-3-OH kinase (PI-3-K) signaling39,72,77. Ras/MAP kinasesignaling also induces expression of TGF-β1 (refs. 73,78,79), whichcan be enhanced further by TGF-β signaling73,80 and thus mayexplain the often-observed increase in expression of TGF-β1 bytumor cells. Because Ras signaling also induces the expression ofurokinase and the consequent generation of plasmin81, and acti-vates expression of the cell-surface protease MMP-9 (refs.12,82–84), Ras/MAP kinase signaling might enhance the cell-autonomous effects of TGF-β1 and the effects of TGF-β activationon the tumor micro-environment.

Ras signaling has also been proposed to inhibit TGF-β signal-ing. Specifically, extracellular signal–regulated kinase (Erk) MAPkinase has been shown to phosphorylate Smad2 and Smad3, andthis phosphorylation inhibits nuclear translocation of these TGF-β-activated Smads85. This mechanism might explain why in somecells with hyperactive Ras signaling the response to TGF-β isinhibited85,86. But other studies have not reported impairednuclear translocation of Smads in Ras-transformed cells or in cells

with activated MAP kinase signaling74,87, and impaired Smad sig-naling in Ras-transformed cells cannot be easily reconciled withthe cooperativity between Ras/MAP kinase signaling and TGF-βsignaling in tumor cell differentiation and behavior87,88. Finally,Jun N-terminal kinase (JNK)–mediated phosphorylation ofSmad3 enhances its activation and nuclear translocation74.Clearly, the crosstalk between these two pathways may depend onthe physiological cell context, a general theme in TGF-β signaling.

Several additional mechanisms of crosstalk with the Smadpathway may be relevant for the role of TGF-β signaling in can-cer cells. For example, signaling by Wnt differentiation factors,which direct tissue specification and growth control, can inter-sect with TGF-β/Smad signaling because Smad3 can interactdirectly with β-catenin and LEF/TCF—transcription regulatorsof Wnt signaling89,90. Virally encoded nuclear proteins may alsoregulate Smad-mediated gene expression through interactionswith the Smads at responsive promoters. For example, thehuman T cell leukemia virus type I (HTLV-1)–encoded Tax pro-tein is a potent repressor of Smad-mediated transcription91,whereas the hepatitis B virus–encoded nuclear pX proteinenhances Smad-mediated transcription in response to TGF-β92.Perhaps conceptually similar, menin, a putative tumor suppres-sor associated with multiple endocrine neoplasia type 1(MEN1), interacts with Smad3 in the nucleus, and its inactiva-tion suppresses Smad3-mediated transcription and TGF-β-induced responses93. The activation of some signaling pathways,including epidermal growth factor (EGF) receptor activation94,interferon-γ signaling through STATs95 and tumor-necrosis fac-tor-α (TNF-α)–induced activation of NF-κB96, induces theexpression of Smad7, which in turn inhibits TGF-β signalingthrough Smads (Fig. 1).

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Crosstalk with Smad signaling may also result from the abilityof TGF-β to activate signaling pathways independently of Smads(Fig. 1). TGF-β can activate Erk MAP kinase97–99, p38 MAPkinase42,43 and JNK71,74, although the extent and kinetics of acti-vation differ among different cell lines and types. The MAPkinase kinase kinase TAK1, which is rapidly activated by TGF-β100 but is also involved in other signaling pathways101–104, mayinitiate these signaling cascades. The activation of p38 MAPkinase and JNK can enhance Smad signaling through eitherSmad phosphorylation74 or the phosphorylation of c-Jun andATF-2 (refs. 42,43,71), which are transcription factors that coop-erate with Smad3, resulting in functional crosstalk with Smad-mediated transcription at defined promoters.

In addition, TGF-β can activate or stabilize the small GTPasesRhoA105 and RhoB106; these may in turn have roles in severalresponses to TGF-β, for example through a requirement of RhoBfor activation of JNK74. Finally, TGF-β induces an interaction ofprotein phosphatase 2A with S6 kinase, which regulates proteintranslation and growth control, decreasing its activity33.Although the mechanisms of activation by TGF-β and the rolesof these non-Smad signaling cascades remain to be better charac-terized, these observations indicate that inactivation of the Smadpathways may not leave the cell unresponsive to TGF-β.

TGF-β–mediated growth arrest and apoptosisMost pertinent to our understanding of the role of TGF-β in car-cinoma development is the fact that TGF-β is a potent inducer ofgrowth inhibition in several cell types, including epithelialcells107–109. One key event that leads to TGF-β–induced growtharrest is the induction of expression of the CDK inhibitorsp15INK4B (refs. 110,111) and/or p21CIP1 (ref. 112), depending onthe cell type. The inhibitor p21CIP1 interacts with complexes ofCDK2 and cyclin A or cyclin E and thereby inhibits CDK2 activ-ity, preventing progression of the cell cycle22,111. By contrast,p15INK4B interacts with and inactivates CDK4 and CDK6, orassociates with cyclin D complexes of CDK4 or CDK6. The latterinteraction not only inactivates the catalytic activity of these

CDKs but also displaces p21CIP1 or the related p27KIP1 fromthese complexes, allowing them to bind to and inactivate theCDK2 complexes with cyclin A and E22,111,113.

Induction of p15INK4B or p21CIP1 expression in response toTGF-β is brought about by Smad-mediated transcriptional acti-vation114,115. In contrast to many TGF-β responses that are medi-ated by Smad3 and Smad4, a heteromeric complex of Smad2,Smad3 and Smad4 induces transcription by interacting with Sp1at the p15INK4B (ref. 114) or the p21CIP1 (ref. 115) promoter.Consequently, the Smad complex recruits the coactivatorCBP/p300 into the complex and strongly potentiates the tran-scriptional activity of Sp1, which activates transcription of thep15INK4B or p21CIP1 genes114,115.

Additional mechanisms also contribute to TGF-β–mediatedgrowth arrest, again depending on the cell type22. For example,TGF-β inhibits the expression of c-Myc (ref. 107), CDK4 (refs.116,117) and CDC25A, a tyrosine phosphatase of CDK4 andCDK6 (refs. 118,119). High levels of c-Myc render the cells resis-tant to the growth inhibitory activity of TGF-β (refs. 120,121),and downregulation of c-Myc is required for the induction ofp15INK4B (ref. 121) and p21CIP1 (ref. 122) expression. The inter-action of c-Myc in a complex at the p15INK4B promoter correlateswith transcriptional repression123,124; TGF-β–induced downreg-ulation of c-Myc thus allows derepression124 and TGF-β–inducedtranscription through Smads114. Decreased expression of c-Mycmay also be involved in the downregulation of CDC25A expres-sion in response to TGF-β (refs. 125,126).

The TGF-β protein has also been shown to induce apoptosis inseveral cell types127–133. The initial events that follow receptor acti-vation and result in cell death are not understood, although theDaxx adaptor protein has been proposed to mediate TGF-β–induced apoptosis through its ability to interact with the TβRIIreceptor134. Increased levels of Smad3 or Smad4 can induce apop-tosis135–137, and dominant-negative interference with Smad3 func-tion protects against apoptosis138. In addition, Smad7 can act as aneffector of TGF-β–induced cell death131,139, but can also protectagainst cell death133,140, depending on the context.

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Fig. 3 Alternative roles for TGF-β sig-naling in cancer progression. Alter-ations in the TGF-β signaling pathwaycontribute to increased tumor pro-gression, invasion and metastasis bytwo different routes. First, normalepithelial cells are inhibited in growthby TGF-β. Early genetic loss of signal-ing components, such as TβRII, leadsto increased synthesis of DNA, morerapid tumor growth and clonalexpansion, which results in anincreased probability of accumulatingfurther mutations and cytogeneticchanges. This ultimately drives tumorprogression. Second, and more fre-quently, the TGF-β signaling pathwayremains intact but is perturbed byepigenetic mechanisms, such as acti-vation of the Ras pathway, whichleads to a diminished growthinhibitory response but accentuationof activities that enhance tumor pro-gression (see Fig. 2). Synergismbetween an elevated TGF-β signalingpathway and an enhanced Ras path-way leads to a direct increase intumor cell plasticity, invasion andmetastasis.

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These findings implicate Smad signaling in TGF-β–mediatedapoptosis, although the pro-apoptotic gene targets of Smad signal-ing remain to be identified. Downregulation of Bcl-XL (refs.141,142) and activation of caspase 3 and 8 (refs. 141,143,144) havealso been implicated in this process, and a mitochondrial septin,ARTS, has a role in the sensitivity of cells to TGF-β–mediated apop-tosis132. The activation of tyrosine kinase–receptor signaling145,through stimulation of the PI-3-K/Akt146–148 or the Raf87 signalingpathway, protects against TGF-β-induced apoptosis and is likely tocontribute to carcinogenesis.

TGF-β signaling as a tumor suppressor pathwayAlterations in TGF-βreceptor signaling. The role of TGF-β signal-ing as a tumor suppressor pathway is best illustrated by the presenceof inactivating mutations in genes encoding TGF-β receptors andSmads in human carcinomas, and by studies of tumor developmentin mouse models. Somatic mutations in TGFBR2, the gene thatencodes the TβRII receptor, occur most frequently in tumors frompatients with hereditary non-polyposis colorectal cancer(HNPCC). A repeat stretch of adenines in the TβRII codingsequence is prone to mutation in these patients, owing to germlinedefects in their capacity for DNA mismatch repair. Resultingnucleotide additions or deletions give rise to a truncated TβRII,which is incapable of signaling149–151.

Mutations in TGFBR2 are frequently found in colon can-cers149–151, gastric cancers150,152–154 and gliomas155 withmicrosatellite instability, but are less common in microsatellite-instability tumors from the endometrium, pancreas, liver andbreast150,156–159. Missense and inactivating mutations in thekinase domain of TβRII have also been reported to occur in coloncancers, which do not display microsatellite instability160, and invarious other carcinomas22,161. Overall, inactivating TGFBR2mutations may be present in 20–25% of all colon cancers. Albeitless common, inactivating mutations in TβRI, encoded byTGFBR1 have also been observed, most notably in ovarian can-cers, metastatic breast cancers, pancreatic carcinomas and T-celllymphomas162–166. Mutations in TGFBR1 have not, however,been found to be accompanied by TGFBR2 mutations166. Alto-gether, these mutations suggest that TβRII and TβRI might func-tion as tumor suppressors in the development of carcinomas.

Various observations suggest that TGF-β receptor expression isoften downregulated or that TGF-β receptor availability at the cellsurface is impaired167–170 in tumor cells, and that these defectsallow cells to escape the growth inhibitory activities of TGF-β.TGF-β receptor expression may also be reduced in tumor cellsthrough altered levels or activities of transcription factors, such asthe Ets transcription factors that are required for expression ofTβRII (ref. 170). Transcriptional silencing may also result fromhypermethylation of CpG islands in the TβRI or TβRII gene pro-moters, or from mutations in the TβRII promoter that interferewith transcription factor binding170. Whereas decreased TβRIIfunction confers resistance against the growth inhibitory effect ofTGF-β, other TGF-β responses may not be similarly affectedbecause they require different thresholds of signaling171–176.

The tumor suppressor role of TβRII has been demonstrated byexpressing wildtype TβRII in colon or breast carcinoma cells,which lack a functional TGFBR2 allele177,178, and by overexpress-ing it in thyroid carcinomas179. Expression of TβRII conferredgrowth inhibition and suppressed anchorage independence, andstrongly reduced tumor formation in nude mice when comparedwith the parental cells. Overexpression of TGF-β1 or TβRII in theskin of transgenic mice also provided evidence for a tumor sup-pressor activity180,181, and expression of dominant-negativeforms of TβRII in the skin or mammary gland182,183 increasedtumor formation. In addition, mice with one inactivated allele of

the gene encoding TGF-β1 (TGFB1) show increased propensityfor developing carcinoma, either spontaneously184 or after car-cinogen exposure185. In the latter case, haploinsufficiency ofTGFB1 increased tumor susceptibility, as the tumors retainedone wildtype allele185. Consistent with these results, decreasedexpression of TβRII correlates with high tumor grade in humancancers170 and experimental systems186–189.

Although these observations suggest that loss of TGF-βresponsiveness provides a distinct advantage for developingtumors, most tumors do not have inactivated TGF-β receptors;individuals with HNPCC, who frequently have TGFBR2 muta-tions in their tumors149, have a better prognosis than individualswith sporadic colon cancer190. Abrogation of TGF-β signaling,while leading to a loss of growth inhibition mediated by TGF-βand early tumor onset, paradoxically protects against tumor pro-gression. TGF-β receptors can thus act as tumor suppressors atearly stages of carcinoma development, but at later stages TGF-βresponsiveness may provide distinct advantages for cancer pro-gression (see below and Fig. 3).

Smads as tumor suppressors. Mutations of the Smad2- andSmad4-encoding gene sequences, but not those of Smad3 or theinhibitory Smad6 or Smad7, have been detected in several carci-nomas22,191, but are uncommon overall. These observations sug-gest that some Smads act as tumor suppressors. Inactivation ofthe genes encoding Smad2 (MADH2) or Smad4 (MADH4)occurs by loss of entire chromosome segments, small deletions,frameshift, nonsense or missense mutations22,191. MADH4mutations occur primarily in pancreatic carcinomas, in whichthe MADH4 gene was first identified as DPC4 (deleted in pancre-atic carcinomas)192, and in colon carcinomas, and less frequentlyin other types of carcinomas22,191.

Whereas biallelic inactivation of MADH4 often occurs inpancreatic and colon carcinomas, haploinsufficiency of theMADH4 locus may also contribute to progression of can-cer193,194. The occurrence of MADH4 mutations in thegermline of a subset of juvenile polyposis families195 furthersupports the notion that Smad4 acts as a tumor suppressor. Incontrast to MADH4, inactivating mutations of MADH2 are rareand occur primarily in colorectal and lung carcinomas196–199.Finally, enhanced Smad7 levels, as observed in pancreatic carci-nomas200 (possibly as a result of TGF-β signaling) may alsodecrease Smad responsiveness.

Tumor-associated mutations in the Smad4 and Smad2 proteinsoccur most frequently in the MH2 domain, which mediates het-eromeric complex formation and transcriptional activation22,191.C-terminal deletions or mutations often inactivate the Smad, butalso provide dominant-negative interference with wildtype Smadfunction201–203. Other mutations confer decreased stabilitythrough increased ubiquitin-mediated degradation204. Mutationsin the MH1 domain of Smad4 interfere with its DNA-bindingability205,206. Most if not all mutations impair Smad function;however, some mutations might alter TGF-β signaling to theadvantage of the tumor. Indeed, the Smad2 mutations D450E andP445H, which are found in colorectal carcinomas, may enhancethe invasive behavior of the tumor cells207, as do some mutationsin the type I TGF-β receptor162.

Studies that use mice carrying an inactivated allele of MADH4support a role for this gene in tumor suppression. Whereashomozygous inactivation of MADH4 leads to early embryoniclethality, heterozygous mice are viable but develop intestinalpolyps that can progress to carcinomas193,208,209. When com-bined with an inactivated allele of the adenomatous polyposiscoli (Apc) gene, simultaneous loss of the wildtype alleles at bothloci results in the development of multiple polyps and in progres-sion to heterogeneous invasive adenocarcinomas208.

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Mice with homozygous inactivation of the gene encodingSmad3 (MADH3) have also been shown to develop colon carci-nomas210, although this phenotype was not seen in two similarstudies211,212. This discrepancy could be due to differences ingenetic background or in environmental factors associatedwith animal husbandry. For example, the absence of Smad3expression might impair TGF-β–mediated immunosuppres-sion and contribute to immune or inflammatory responsesthat predispose to cancer formation. Although a role ofMADH3 as tumor suppressor gene is conceivable, no inactivat-ing MADH3 mutations have been observed in human tumors.The occurrence of MADH2, but not MADH3, mutations canbe rationalized by the crucial role of Smad2 in the TGF-β–induced expression of p21CIP1 or p15INK4B CDKinhibitors114,115. Inactivation of Smad2 might therefore inacti-vate the growth arrest response without affecting the Smad3-mediated gene expression that provides an advantage fortumor development, that is, the induction of some extracellu-lar matrix proteins by TGF-β.

The loss of Smad4 function may not abolish TGF-β respon-siveness, even though Smad4 is generally perceived as essentialfor TGF-β responses. Indeed, mouse fibroblasts derived fromMadh4–/– embryos213, as well as some Smad4-deficient tumorcell lines214,215, retain at least some TGF-β responses. This is con-sistent with the observation that TGF-β–induced synthesis offibronectin can occur in the absence of Smad4 (ref. 71). Thus, thehigh frequency of MADH4 deletions in tumors might representa selective perturbation of TGF-β signaling, rather than its com-plete abrogation.

In addition, loss of Smad4 expression may enhance Ras signal-ing and progression to undifferentiated carcinomas216, furtheremphasizing the crosstalk between TGF-β and Ras signaling.Moreover, different TGF-β responses may have differential sensi-tivity to Smad signaling. Accordingly, overexpression of Smad7in a carcinoma cell line suppressed TGF-β–induced growtharrest, without affecting TGF-β–induced expression of plas-minogen activator inhibitor 1, and enhanced anchorage-inde-pendent growth and tumorigenicity200. Finally, inactivatingMADH4 mutations have been found in conjunction with muta-tions in TGFBR2 or TGFBR1 (ref. 160), which strongly suggeststhat Smad4 has tumor suppressive activities that are unrelated toTGF-β signaling.

Other mechanisms of impaired TGF-β responsiveness.Upregulated expression of proto-oncogenes or expression oftheir oncogenic counterparts may also provide a mechanism bywhich tumor cells downregulate TGF-β responsiveness andescape its tumor suppressor effects. For example, elevated expres-sion of the proto-oncogene c-Ski in melanoma217 has been corre-lated with decreased TGF-β responsiveness, presumably owing torepression of Smad-mediated transcription63,64.

Expression of c-Myc may also be involved in the growthinhibitory response to TGF-β. The downregulation of c-Mycexpression by TGF-β, as observed in epithelial cells, is lost invarious cancer cell lines, concomitant with the loss of thegrowth inhibitory response to TGF-β36. The EWS-FLI1 onco-gene, which incorporates a partial coding sequence for the Etsfamily transcription factor Fli-1 and was first identified inEwing sarcoma, represses expression of TβRII and mayaccount for decreased TGF-β responsiveness218. In addition,the Tax protein, which is encoded by HTLV-1 and is thought tocontribute to leukemogenesis in adult T-cell leukemia, is apotent repressor of Smad-mediated transcription91. Clearly,tumor cells have developed several strategies to escape thegrowth inhibitory response of TGF-β, and most carcinomacells are resistant to growth inhibition by TGF-β.

Finally, the mannose-6-phosphate receptor may also have arole in the tumor suppressor activity of TGF-β. This receptorbinds latent TGF-β and is required for activation of TGF-β byplasminogen conversion into plasmin219. Loss of heterozygosityat the receptor locus and inactivating receptor mutations occurwith high incidence in hepatocellular and squamous lung carci-nomas220,221, and are early events during carcinogenesis222. Asthe levels of mannose-6-phosphate receptor and loss of heterozy-gosity of this locus correlate with TGF-β1 activation, it is con-ceivable that part of its tumor suppressor role is linked toactivation of TGF-β1 (ref. 223).

Cell-autonomous stimulation of tumor developmentIn spite of the tumor suppressor activity of TGF-β1, tumor cellsoften show increased production of this growth factor3,4, and con-siderable evidence documents its tumor-promoting role throughits effects on tumor cell invasion and changes in the tumor micro-environment. Tumor metastasis depends on various factors,including the ability of tumor cells to migrate and invade thestroma, and to migrate in and out of blood and lymphatic vessels.The epithelial to mesenchymal differentiation of tumor cells hasan important role in this invasive phenotype224. Accordingly,fibroblastoid or ‘spindle-cell’ tumors of epithelial origin havebeen characterized as highly malignant and invasive88,225–229.

This type of transdifferentiation and invasion of epithelial cellsinto the underlying mesenchyme is not restricted to cancer pro-gression; it also occurs during developmental processes such asplacentation224. During cardiac230,231, palatal232,233 and hair-fol-licle development234, TGF-βs are involved in epithelial to mes-enchymal transdifferentiation, and related family members servethe same function in mesoderm induction235. This morphologi-cal transition is characterized by extensive changes in the expres-sion of cell-adhesion molecules and by a switch from acytoskeleton of mainly cytokeratin intermediate filaments to onecomprising predominantly vimentin88,228,229,236. The ability ofepithelial or carcinoma cells to undergo epithelial to mesenchy-mal transition in culture correlates with cell changes that facili-tate invasion and metastasis in vivo88,176,229.

The TGF-β protein can induce an epithelial to mesenchymaltransition in cultures of normal and transformed breast epithelialcells88,229,236–238, squamous carcinoma cells176,239, ovarianadenosarcoma cells240 and melanoma cells241. The phenotypicchanges have been best characterized in NMuMG cells, in whichTGF-β induces changes in cell shape, downregulates the expres-sion of E-cadherin, ZO-1, vinculin and keratin, and induces theexpression of vimentin and N-cadherin, which has been shownto increase cell motility and scattering105,236,238.

The mechanism of the TGF-β-induced epithelial to mesenchy-mal transition is complex, and Ras/PI-3-K signaling and TGF-β-induced activation of Smad and RhoA signaling seem to havedistinct roles in the process105,216,238,242. Whereas TGF-β inducesthis phenotypic transition in some cell lines, growth factors thatact through tyrosine kinase receptors and activation of Ras sig-naling induce similar changes in other cell systems224,243. In somecases, both signaling cascades need to be enhanced to achieveepithelial to mesenchymal transition88. These findings suggestthat these two pathways synergize in the transition of squamousto spindle carcinoma formation.

This synergy explains why, for example, the invasive growthof Ras- or Raf-transformed epithelial cells in vitro depends onintact autocrine TGF-β signaling87,229. Accordingly, spindle-cellcarcinoma tissue has an increased mutant Hras gene dosagewhen compared with squamous cells originating from the sameskin tumors227. Together, these observations suggest that acti-vation of TGF-β signaling and an involvement of Ras/MAP

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kinase signaling may be essential for carcinoma cell invasionand metastasis, whether through epigenetic alterations ofautocrine growth factor signaling or genetic alterations. Thisinterpretation is also consistent with the cooperation of bothpathways in mesoderm induction235, and in epithelial to mes-enchymal differentiation and invasion during hair-follicle for-mation234 and cardiogenesis244.

The cell-autonomous role of TGF-β in the spindle-cell pheno-type and invasive behavior of carcinomas has been well docu-mented for skin carcinomas in mice176,180. Transgenic expressionof activated TGF-β1 in mouse epidermis in vivo increased themalignant conversion to carcinomas and the incidence of spin-dle-cell carcinomas, even though the hyperplastic response to thetumor promoter 12-O-tetradecanoylphorbol 13-acetate and thenumber of papillomas after treatment with 7,12-dimethylbenz-[a]-anthracene were reduced180. In another study, expression of adominant-negative TβRII prevented squamous carcinoma cellsfrom undergoing an epithelial to mesenchymal transition inresponse to TGF-β in vivo176. Similar observations on the role ofTGF-β signaling have been made using a Ras-transformed mam-mary epithelial cell line and a fibroblastoid colon carcinoma cellline229. Expression of a dominant-negative TβRII preventedTGF-β-induced epithelial to mesenchymal changes and revertedthe colon carcinoma cells to an epithelial phenotype. In addition,blocking TβRII function suppressed the in vitro invasion and thehighly metastatic phenotype of this colon carcinoma cell line229.Finally, restoration of TβRII signaling in HNPCC cells with anendogenous TβRII mutation rendered the cells invasive in con-trast to their normally non-invasive phenotype229.

The increased incidence of aggressive skin neoplasia associatedwith the use of cyclosporin (for example, in kidney transplantpatients) may also result from a cell-autonomous effect mediatedby TGF-β. Cyclosporin induces invasive behavior in adenocarci-noma cells in vitro and enhances tumor growth in immunodefi-cient SCID-beige mice, whereas anti-TGF-β antibodies preventthe cyclosporin-induced increase in metastasis245. TGF-β hasalso been shown to be an autocrine effector of breast tumormetastasis246. By using a breast cancer cell line that metastasizesto bone in nude mice, it was shown that dominant-negativeinterference with TβRII function decreases tumor formation andmetastasis, and the resulting destruction of bone246. In thismodel, TGF-β1 induced the expression and secretion of parathy-roid hormone-releasing peptide (PTHrP), resulting in therecruitment of bone-resorbing osteoclasts. In complementaryexperiments, tumor cells expressing a partially activated TβRIaccelerated bone destruction and reduced survival246. This find-ing is likely to be relevant for the clinical correlation that womenwith PTHrP-positive breast cancer are more likely to developmetastatic disease than those with PTHrP-negative tumors246.

In summary, in vitro and in vivo studies link autocrine TGF-βresponsiveness to epithelial to mesenchymal transdifferentiation,the acquisition of an invasive phenotype, and metastatic behav-ior. The TGF-β-dependent, reversible nature of this phenotypemay explain why many metastatic tumors show a well-differenti-ated epithelial phenotype, presumably depending on the localtissue micro-environment and levels of active TGF-β.

Effects of TGF-β on the tumor micro-environmentThe increased expression and activation of TGF-β1 by tumor cellsprofoundly affects the micro-environment. Increased proteaseexpression and plasmin generation by tumor cells11 enhancesactivation of TGF-β and degradation of the extracellular matrix,with a consequent release of stored TGF-β. But a concomitantincrease in production of TGF-β1 stimulates synthesis of extracel-lular matrix proteins and chemoattraction of fibroblasts.

Together, all these changes result in a micro-environment thatpromotes tumor growth and invasion and angiogenesis247.

Tumor angiogenesis is crucial for tumor growth and invasion,as blood vessels deliver nutrients and oxygen to the tumor cellsand allow them to intravasate the blood system, which leads tometastasis. TGF-β1 acts as a potent inducer of angiogenesis inseveral assays248–252, and mouse models defective in TGF-β sig-naling components indicate the importance of TGF-β1 in nor-mal vascular development. Targeted inactivation of the geneencoding TGF-β1 or TβRII results in embryonic lethality owingto defective vasculogenesis and angiogenesis253,254, whereasangiogenesis-defective phenotypes are also seen in mice with nullmutations of the genes encoding TβRI (ref. 255), ALK-1, a TGF-β type I receptor that is expressed in endothelial cells and signalsthrough Smad1 and Smad5 (ref. 256) endoglin, an endotheliallyrestricted TGF-β type III receptor (refs. 257,258) and Smad5(refs. 259,260).

Several models indicate the role of tumor cell–secreted TGF-β1in tumor angiogenesis. Increased expression of TGF-β1 in trans-fected prostate carcinoma or Chinese hamster ovary cellsenhanced tumor angiogenesis in immunodeficient mice261,262,and local administration of neutralizing antibodies to TGF-β1strongly reduced tumor angiogenesis261. Intraperitoneal injectionof TGF-β antibodies reduced angiogenesis and tumorigenicity ofa renal carcinoma cell line that lacks TβRII, and is therefore notresponsive to TGF-β, in natural killer (NK)-, T- and B-cell–defi-cient mice263. This cell line also lacks the von Hippel–Lindau(VHL) tumor suppressor gene, and reintroduction of this genedecreased TGF-β1 synthesis, suggesting that VHL regulates TGF-β1 levels and has tumor suppressor effects through an anti-angio-genic mechanism involving TGF-β1 (ref. 263).

Finally, re-expression of Smad4 in Smad4-deficient pancreascancer cells suppresses tumor development primarily by repressingtumor angiogenesis264. In human breast tumors, high levels ofTGF-β1 messenger RNA are associated with increased microvesseldensity, and both parameters correlate with poor patient progno-sis265. Diagnostic studies on other carcinoma types suggest thathigh tumor burden and circulating plasma levels of TGF-β1 areassociated with enhanced tumor angiogenesis and poor patientprognosis266–269. In one study, TGF-β1 levels were also correlatedwith expression levels of the angiogenic growth factor vascularendothelial growth factor (VEGF)270.

The mechanisms of angiogenesis stimulation by TGF-β1 pre-sumably combine direct and indirect effects. TGF-β inducesexpression of VEGF, which directly acts on endothelial cells tostimulate cell proliferation and migration271. TGF-β also inducescapillary formation of endothelial cells, which are cultured oncollagen matrix249,252. Indirect stimulation of angiogenesis byTGF-β1 may occur through the potent chemoattractant activityof TGF-β for monocytes, which release angiogeniccytokines248,250,272,273.

TGF-β1–induced changes in the microenvironment providefavorable conditions for endothelial cell migration and capillaryformation. Moreover, TGF-β–induced expression of the metallo-proteases MMP-2 and MMP-9, and downregulation of the proteaseinhibitor TIMP in tumor and endothelial cells274–279, are expectedto enhance the migratory and invasive properties of endothelialcells required for angiogenesis280,281. Thus, both the direct effects ofTGF-β and its indirect effects on the tumor micro-environmentstimulate tumor angiogenesis.

Secretion and activation of TGF-β1 by tumor cells alsostimulate tumor development and progression through escapefrom immunosurveillance. TGF-β inhibits the proliferationand functional differentiation of T lymphocytes, lymphokine-activated killer cells, NK cells, neutrophils, macrophages and

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B cells273,282,283. Several observations show that TGF-β expres-sion by tumor cells promotes tumorigenicity by locallyrepressing immune functions. For example, the increasedexpression of TGF-β1 by tumor cells enhanced their tumori-genicity and prevented activation of cytotoxic T-lymphocytefunction when compared with parental cells284. This repres-sion was presumably elaborated by the ability of TGF-β toinhibit the expression and function of interleukin 2 (IL-2) andIL-2 receptors285,286. Accordingly, the inhibition of the cyto-toxic T-lymphocyte response by EMT6 mammary tumor cells,which produce high levels of TGF-β1, was overcome byectopic expression of IL-2 in these cells287. Thus, TGF-β–mediated suppression of the cytotoxic T-cell response pro-motes tumor development288.

TGF-β1 expression by tumor cells may also inhibit otherimmune functions. Increased expression of TGF-β1 by tumorcells decreased NK cell activity289 and promoted tumor forma-tion in nude mice that lack T cells289,290. In addition, anti–TGF-βantibodies suppressed tumor formation and metastasis of abreast carcinoma cell line in nude mice, while enhancing NK cellfunction291,292. This suppression was not seen in nude beigemice, which lack both T and NK cells, which implicates TGF-β1–induced suppression of NK cells in cancer progression292,293.

TGF-β–mediated suppression of neutrophil function may alsobe involved in tumor progression. Indeed, Fas ligand–expressingcarcinoma cells underwent neutrophil-mediated rejection, butthis rejection did not occur at a site with high levels of TGF-β orwhen TGF-β1 was injected at the tumor site294. Finally, strongdownregulation of the expression of major histocompatibilitycomplex (MHC) class II antigens by TGF-β295–297 may make thetumor cell surface less immunogenic. This regulation contributesto the local immunosuppression and the escape of TGF-β1-expressing tumor cells from immune surveillance298,299.

Potential avenues toward a cancer therapyConsidering the complex roles of TGF-β signaling in tumor sup-pression and progression, which are context- and stage-depen-dent, any strategy to exploit this knowledge for therapeuticpurposes is fraught with difficulties. For example, stimulatingTGF-β signaling might suppress tumor growth, but might alsopromote tumor invasion and metastasis, particularly of cells thatare no longer sensitive to the growth inhibitory effect of TGF-β.To suppress cancer progression by exogenous administration ofTGF-β, induction of TGF-β synthesis or enhanced TGF-β signal-ing would require that the tumors are sensitive to the growthinhibitory effects of TGF-β; unfortunately, however, resistance tothe growth inhibitory effect of TGF-β is often acquired in earlytumor development, often before diagnosis.

It has been proposed, on the one hand, that the beneficialeffects of tamoxifen in preventing or treating breast cancer maybe due, in part, to its ability to induce expression of TGF-β300.On the other hand, tumor resistance to tamoxifen correlateswith increased expression of TGF-β1, which is possibly areflection of insensitivity to the antiproliferative effects ofTGF-β301,302. These elevated levels of TGF-β1 and the conse-quent suppression of NK cell activity may be responsible forthe failure of tamoxifen therapy. Accordingly, tamoxifen resis-tance of a breast tumor cell line has been reversed through theuse of anti-TGF-β antibodies293,303.

As indicated by this example, inhibiting TGF-β1 expression oractivation may represent a valid approach to treatment of carci-noma. Inhibiting TGF-β production should affect the cell-autonomous response and the effects of TGF-β on itsmicro-environment, whereas interfering with the TGF-β respon-siveness of the tumor cells would primarily inhibit its cell-

autonomous effects. But any approach that inhibits TGF-β sig-naling in tumor cells might induce the regression of advancedcancers while stimulating the growth of otherwise quiescent pre-malignant lesions.

The most sensible approach may therefore be to activate TGF-β in a preventive setting, but to inhibit signaling in advancedmetastatic cancer. Inhibiting the production and/or signaling ofTGF-β through antisense RNA, anti–TGF-β antibodies or solu-ble TGF-β receptors might inhibit cancer progression. For exam-ple, antisense RNA inhibition of TGF-β2 synthesis by breastcancer cells enhanced the cytotoxicity of NK cells293, which isconsistent with the ability of anti–TGF-β therapy to decreasesuppression of the NK cell response289,292. In other studies, anti-sense RNA inhibition of TGF-β1 or TGF-β2 synthesis in breastcancer, mesothelioma cells or glioma cells restored the tumorimmunogenicity and the cytotoxic T-lymphocyte response, andinhibited tumor development304–307. As discussed above, anti-bodies against TGF-β have also been shown to inhibit tumor for-mation through an increase in NK cell activity292 or a decrease inmicrovessel density, without directly affecting cell prolifera-tion261,263 or the innate tumorigenicity of cancer cells245.

In addition, administration of an anti-endoglin immunotoxintargeted the endothelial cells and selectively inhibited tumorangiogenesis and carcinoma development308. Sequestration ofTGF-β through the use of soluble receptor ectodomains may pro-vide another approach to inhibit cancer progression. Indeed,expression of a soluble TβRII (ref. 309) or the soluble proteogly-cans betaglycan310 or decorin311,312, which bind TGF-β, inhibitedtumor formation and/or metastasis and even induced tumorregression, depending on the tumor model. These approaches tofacilitating immune rejection might be enhanced further bycombinatorial therapy with IL-2. For example, the combinationof anti-TGF-β antibodies with IL-2–reduced tumor formation ina highly metastatic melanoma cell line, which was unaffected byeither treatment alone313.

In future studies, it would be desirable to retain the TGF-β–mediated stimulation of apoptosis in tumor cells, but toinhibit the cell-autonomous (invasion and metastatic spread)and non–cell-autonomous (angiogenesis and immunosuppres-sion) activities of this multifaceted molecule. In view of theimportant role of the Ras pathway in these effects, combinatorialapproaches involving inhibitors of the Ras pathway coupled withinhibitors of TGF-β might lead to a marked synergistic inhibitionof tumor growth. Future insight into the TGF-β signaling mecha-nisms that lead to epithelial to mesenchymal transition and inva-sion, or to apoptosis, might lead to the design of more targetedand more effective therapeutic interventions.

AcknowledgmentsIt is impossible to mention every important contribution to ourunderstanding of the mechanisms of TGF-β signaling and its role in cancer,and we apologize to those investigators whose contributions could not beacknowledged. Relevant research in the laboratories of the authors is fundedby NIH grants to R.D.; NIH, March of Dimes and American HeartAssociation grants to R.J.A.; and an NIH grant to A.B. We thank J. Qing forthe design and drawing of Fig. 1, and A. Roberts, B. Lechleider and C.Arteaga for their comments.

Received 8 August; accepted 29 August 2001.

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