TGF-β signaling in tumor suppression and cancer progression

Transcription

1 TGF-β signaling in tumor suppression and cancer progression Rik Derynck 1, Rosemary J. Akhurst 2 & Allan Balmain 2,3 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 and tumorigenesis. The consequent changes in cell behavior and responsiveness result not only from genetic alterations such as activation of oncogenes or inactivation of tumor suppressor genes, but also from altered production of, or responsiveness to, stimulatory or inhibitory growth and differentiation factors. Among these, transforming growth factor β (TGF-β) and its signaling effectors act as key determinants of carcinoma cell behavior. The autocrine and paracrine effects of TGF-β on tumor cells and the 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 pathway and a promoter of tumor progression and invasion. Here we evaluate the role of TGF-β in tumor development and attempt to reconcile the positive and negative effects of TGF-β in carcinogenesis. In mammals, there are three different TGF-βs, β1, β2 and β3, which are encoded by different genes and which all function through the same receptor signaling systems 1,2. Of these, TGF-β1 is most frequently upregulated in tumor cells 3,4 and is the focus of 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 two prosegments, to which one of several latent TGF-β binding proteins is often linked 5 7. This latent complex represents an important safeguard against inadvertent activation, and may stabilize and target latent TGF-β to the extracellular matrix, where it is sequestered 7,8. The matrix thus acts as a reservoir from which TGFβ can readily be recruited without the need for new synthesis. The secretion of TGF-β as a latent complex necessitates the existence of a regulated activation process, which is most probably mediated through the activities of proteases that preferentially degrade the TGF-β prosegments and thereby release the highly stable, active TGF-β dimer. Because plasmin activates latent TGF-β 9,10 and plasminogen is converted into plasmin at sites of cell migration and invasion 11, we assume that endothelial and tumor cells are exposed to active TGF-β. Latent TGF-β can also be activated by the metalloproteases MMP-9 and MMP-2 (ref. 12), which are frequently expressed by malignant cells, especially at sites of tumor cell invasion 13,14. Other activation mechanisms might not depend on proteases. For example, the extracellular matrix protein thrombospondin 15,16 and the αvβ6 integrin, which is expressed at the surface of epithelial cells in response to inflammation 17, may mediate TGF-β activation through a conformational change in the TGF-β complex. Thus, different mechanisms may regulate TGF-β activation in different physiological contexts, and tumor cells are well equipped to activate TGF-β locally. Mechanisms of TGF-β signaling The TGF-β receptors. As the mechanisms of TGF-β receptor signaling have been extensively reviewed recently 18 23, we will summarize only the salient points (Fig. 1) before focusing on the role of TGF-β in cancer cells. TGF-β signals through a heteromeric cell-surface complex of two types of transmembrane serine/threonine kinases, named type I and type II receptors. Although there is only one type II TGF-β receptor (TβRII), there are three type I receptors (ALK1/TSR-1, ALK2/Tsk7L, ALK5/TβRI). Most gene expression responses are presumably mediated by the TβRI receptor. TβRII and TβRI show a widespread pattern of expression and are expressed by tumor cells. After TGF-β binds to the receptor complex, the TβRII kinase phosphorylates TβRI in the GS sequence, which is located upstream from the kinase domain. This phosphorylation activates the TβRI kinase that mediates TβRI autophosphorylation and phosphorylation of downstream target proteins. Several intracellular proteins have been shown to interact with TGF-β receptor complexes on the cell surface. Among these, FKBP12 interacts constitutively with the juxtamembrane domain of type I receptors and regulates the conformation of this domain and the signaling sensitivity Three proteins containing WD repeats associate with and are phosphorylated by the receptor complex after ligand-induced activation and regulate TGF-β receptor signaling. Whereas TRIP-1 interacts with TβRII (refs 28,29), the Bα subunit of the protein phosphatase 2A interacts with type I receptors 30, and STRAP interacts with both TβRII and TβRI to decrease TGF-β signaling 31,32. The regulatory roles of these proteins are well documented, but there is no convincing evidence to show that these WD-repeat proteins act as effectors of TGF-β responses, with the exception of the Bα subunit of protein phosphatase 2A, which may be involved in growth arrest through inactivation of the S6 kinase pathway Departments of Growth and Development, and Anatomy, Programs in Cell Biology and Developmental Biology, 2 Mount Zion Cancer Research Institute, 3 Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California, USA. Correspondence should be addressed to R.D. ( derynck@itsa.ucsf.edu). nature genetics volume 29 october

2 TGF-β EGF IFN-γ TNF-α stress signals Wnt RII RI Smad2/3 MAPK JAK1 NF-kB JNK p38 Ras Erk /MAPK Rho JNK RhoA p160rock TAK1 p38 MAPK PP2A S6K Smad4 Fig. 1 TGF-β-induced signaling through Smads, and several non-smad signaling mechanisms. After ligand-induced activation of the receptor, Smad2 and/or Smad3 interact transiently with the TβRI receptor (RI), and this interaction is stabilized by the FYVE protein SARA. Smad2 and Smad3 are phosphorylated on their C terminals by TβRI, and then dissociate from the receptor to form a heterotrimeric complex comprising two receptor-activated Smads and Smad4. This complex then translocates into the nucleus, where it interacts at the promoter with transcription factors with sequence-specific DNA binding to regulate gene expression. The heteromeric Smad complex also interacts with the CBP/p300 transcriptional coactivator, which connects the Smad complex with the general transcription factors (GTF). Smad7 inhibits activation 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 to EGF, 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 expression, 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 proteins. 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 kinase pathways and the downregulation of S6 kinase activity. The mechanisms of activation of these non-smad signaling events and how they connect to the heteromeric TGF-β receptor complex remain to be characterized. P P GTF P P P STAT1 SMAD7 target genes c-jun ATF2 β-catenin Signaling through Smads. The only well-characterized signaling effector pathway that is initiated by activated TGF-β receptors is provided by the Smads, a small family of structurally related proteins (Fig. 1) 19 23,34,35. Smad2 and Smad3 are activated via carboxy-terminal phosphorylation by type I TGF-β receptor kinases and form heterotrimeric complexes with Smad4. These complexes then translocate into the nucleus and act as TGF-βinduced transcriptional activators of target genes. By contrast, Smad6 and Smad7 interfere with the activation of the effector Smads and act as inhibitory Smads. Both Smad6 and Smad7 can interact with type I receptors, thus competitively preventing the receptor-activated Smads from being phosphorylated; Smad6 also interferes with the formation of the heteromeric Smad complex. TGF-β signaling induces expression of Smad7, which provides a TGF-β induced negative feedback loop. The TGF-β protein activates or represses the transcription of defined target genes, and many of these responses are immediate responses to TGF-β receptor activation. So far, Smads have been shown primarily to activate transcription 19 23,34,35, but they are also involved in TGF-β mediated repression of transcription Smads also interact with many DNA-binding transcription factors; this interaction, together with the DNA-binding activity of Smads, specifies the promoter binding and transcriptional activation 21,34,35. For example, Smad3 can interact with bzip transcription factors such as c-jun (refs ), 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 and CBFA1 (refs. 37,44,47,48); nuclear receptors such as the vitamin D3 (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 transcriptional machinery through a direct physical interaction with the transcriptional coactivator CBP/p300, which is essential for the transcriptional activation function of Smads The high-affinity binding of the Smad transcription complex to a defined promoter sequence is mediated primarily by the interacting transcription factor, although Smad3 and Smad4, but not Smad2, have an intrinsic DNA-binding capability as well 39, A Smad-binding DNA sequence thus provides a favorable sequence context for Smad binding close to the highaffinity binding sequence for the Smad-interacting transcription factor 34,56,58. This DNA context dependent binding of a Smad to both the interacting transcription factor and the promoter DNA might explain why TGF-β activates only a subpopulation of the promoters that bind the transcription factor with which a Smad can interact. Crosstalk with Smad signaling. The TGF-β dependent recruitment of Smad complexes to the transcription machinery also allows the recruitment of additional coactivators or corepressors, which regulate the amplitude of transcriptional activation. Besides the interaction of the Smad complex with 118 nature genetics volume 29 october 2001

3 progress Fig. 2 TGF-β acts on both the tumor cell and its environment. Both tumor and stromal cells secrete elevated levels of TGF-β that are selfperpetuating via a cycle of enhanced protease activation of latent TGF-β, auto-induction of TGF-β1 gene activity and recruitment of TGFβ secreting cells such as macrophages and neutrophils. Most TGF-β activities, apart from the growth inhibition of early tumor cells, promote tumor progression. The TGF-β signaling pathway is thus a target for therapeutic intervention in invasive tumors. TGF-β activity can be targeted by using endogenous proteins such as soluble betaglycan or decorin, or by using artificial agents such as antisense oligonucleotides, antibodies or small-drug molecules selective for specific components of the signaling pathway. ECM, extracellular matrix; Fas-L, Fas ligand. CBP/p300, Smad4 can also engage another coactivator, named MSG1, into the transcription complex to enhance the Smad response 60,61. By contrast, recruitment of a corepressor decreases the Smad and TGF-β responsiveness. The corepressors Evi-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/or Smad2 and inhibit TGF-β responses. Thus, the expression of Smad-interacting corepressors by tumor cells might block some TGF-β responses, including its inhibitory effect on growth, and contribute to tumor progression. tumor cell The transcriptional activation by Smads, through their physical interactions and functional cooperativity with transcription factors, allows crosstalk with other signaling pathways 19,21,69,70. For example, the activation of mitogen-activated protein (MAP) kinase pathways, which is commonly observed in tumor cells, and of Jun kinase or p38 MAP kinase is likely to regulate TGF-β-induced transcription from promoters with TGF-β-responsive AP-1 (c-jun/c-fos) or CREB/ATF-binding sites and Smad-binding sites 39,42,43, Most notably, the expression of extracellular matrix proteins and proteases in response to TGF-β often requires an intact AP-1 binding promoter sequence 75,76, suggesting that there is transcriptional cooperation of Smads with the AP-1 complex and a dependence of these TGF-β responses on Ras/MAP kinase and/or phosphatidylinositol-3-oh kinase (PI-3-K) signaling 39,72,77. Ras/MAP kinase signaling also induces expression of TGF-β1 (refs. 73,78,79), which can be enhanced further by TGF-β signaling 73,80 and thus may explain the often-observed increase in expression of TGF-β1 by tumor cells. Because Ras signaling also induces the expression of urokinase and the consequent generation of plasmin 81, and activates expression of the cell-surface protease MMP-9 (refs. 12,82 84), Ras/MAP kinase signaling might enhance the cellautonomous effects of TGF-β1 and the effects of TGF-β activation on the tumor micro-environment. Ras signaling has also been proposed to inhibit TGF-β signaling. Specifically, extracellular signal regulated kinase (Erk) MAP kinase has been shown to phosphorylate Smad2 and Smad3, and this phosphorylation inhibits nuclear translocation of these TGFβ-activated Smads 85. This mechanism might explain why in some cells with hyperactive Ras signaling the response to TGF-β is inhibited 85,86. But other studies have not reported impaired nuclear translocation of Smads in Ras-transformed cells or in cells proteases thrombospondin integrins effects on tumor cell 1. growth inhibition (early) 2. invasion (late) latent TGF-β decorin betaglycan active TGF-β stromal cell effects on tumor environment 1. stroma proteases ECM production 2. endothelial cells angiogenisis 3. immune cells Fas-L activity NK cells T cells B cells with activated MAP kinase signaling 74,87, and impaired Smad signaling in Ras-transformed cells cannot be easily reconciled with the cooperativity between Ras/MAP kinase signaling and TGF-β signaling in tumor cell differentiation and behavior 87,88. Finally, Jun N-terminal kinase (JNK) mediated phosphorylation of Smad3 enhances its activation and nuclear translocation 74. Clearly, the crosstalk between these two pathways may depend on the physiological cell context, a general theme in TGF-β signaling. Several additional mechanisms of crosstalk with the Smad pathway may be relevant for the role of TGF-β signaling in cancer cells. For example, signaling by Wnt differentiation factors, which direct tissue specification and growth control, can intersect with TGF-β/Smad signaling because Smad3 can interact directly with β-catenin and LEF/TCF transcription regulators of Wnt signaling 89,90. Virally encoded nuclear proteins may also regulate Smad-mediated gene expression through interactions with the Smads at responsive promoters. For example, the human T cell leukemia virus type I (HTLV-1) encoded Tax protein is a potent repressor of Smad-mediated transcription 91, whereas the hepatitis B virus encoded nuclear px protein enhances Smad-mediated transcription in response to TGF-β 92. Perhaps conceptually similar, menin, a putative tumor suppressor associated with multiple endocrine neoplasia type 1 (MEN1), interacts with Smad3 in the nucleus, and its inactivation suppresses Smad3-mediated transcription and TGF-βinduced responses 93. The activation of some signaling pathways, including epidermal growth factor (EGF) receptor activation 94, interferon-γ signaling through STATs 95 and tumor-necrosis factor-α (TNF-α) induced activation of NF-κB 96, induces the expression of Smad7, which in turn inhibits TGF-β signaling through Smads (Fig. 1). nature genetics volume 29 october

4 normal cell BOB CRIMI TGF-β rapid growth loss of TGF-β signaling, e.g. TGFBR2 mutation benign lesion perturbation of TGF-β signaling migration/invasion Ras further genetic changes carcinoma Crosstalk with Smad signaling may also result from the ability of TGF-β to activate signaling pathways independently of Smads (Fig. 1). TGF-β can activate Erk MAP kinase 97 99, p38 MAP kinase 42,43 and JNK 71,74, although the extent and kinetics of activation differ among different cell lines and types. The MAP kinase kinase kinase TAK1, which is rapidly activated by TGFβ 100 but is also involved in other signaling pathways , may initiate these signaling cascades. The activation of p38 MAP kinase and JNK can enhance Smad signaling through either Smad phosphorylation 74 or the phosphorylation of c-jun and ATF-2 (refs. 42,43,71), which are transcription factors that cooperate with Smad3, resulting in functional crosstalk with Smadmediated transcription at defined promoters. In addition, TGF-β can activate or stabilize the small GTPases RhoA 105 and RhoB 106 ; these may in turn have roles in several responses to TGF-β, for example through a requirement of RhoB for activation of JNK 74. Finally, TGF-β induces an interaction of protein phosphatase 2A with S6 kinase, which regulates protein translation and growth control, decreasing its activity 33. Although the mechanisms of activation by TGF-β and the roles of these non-smad signaling cascades remain to be better characterized, these observations indicate that inactivation of the Smad pathways may not leave the cell unresponsive to TGF-β. TGF-β mediated growth arrest and apoptosis Most pertinent to our understanding of the role of TGF-β in carcinoma development is the fact that TGF-β is a potent inducer of growth inhibition in several cell types, including epithelial cells One key event that leads to TGF-β induced growth arrest is the induction of expression of the CDK inhibitors p15 INK4B (refs. 110,111) and/or p21 CIP1 (ref. 112), depending on the cell type. The inhibitor p21 CIP1 interacts with complexes of CDK2 and cyclin A or cyclin E and thereby inhibits CDK2 activity, preventing progression of the cell cycle 22,111. By contrast, p15 INK4B interacts with and inactivates CDK4 and CDK6, or associates with cyclin D complexes of CDK4 or CDK6. The latter interaction not only inactivates the catalytic activity of these metastasis Smad carcinoma metastasis Fig. 3 Alternative roles for TGF-β signaling in cancer progression. Alterations in the TGF-β signaling pathway contribute to increased tumor progression, invasion and metastasis by two different routes. First, normal epithelial cells are inhibited in growth by TGF-β. Early genetic loss of signaling components, such as TβRII, leads to increased synthesis of DNA, more rapid tumor growth and clonal expansion, which results in an increased probability of accumulating further mutations and cytogenetic changes. This ultimately drives tumor progression. Second, and more frequently, the TGF-β signaling pathway remains intact but is perturbed by epigenetic mechanisms, such as activation of the Ras pathway, which leads to a diminished growth inhibitory response but accentuation of activities that enhance tumor progression (see Fig. 2). Synergism between an elevated TGF-β signaling pathway and an enhanced Ras pathway leads to a direct increase in tumor cell plasticity, invasion and metastasis. CDKs but also displaces p21 CIP1 or the related p27 KIP1 from these complexes, allowing them to bind to and inactivate the CDK2 complexes with cyclin A and E 22,111,113. Induction of p15 INK4B or p21 CIP1 expression in response to TGF-β is brought about by Smad-mediated transcriptional activation 114,115. In contrast to many TGF-β responses that are mediated by Smad3 and Smad4, a heteromeric complex of Smad2, Smad3 and Smad4 induces transcription by interacting with Sp1 at the p15 INK4B (ref. 114) or the p21 CIP1 (ref. 115) promoter. Consequently, the Smad complex recruits the coactivator CBP/p300 into the complex and strongly potentiates the transcriptional activity of Sp1, which activates transcription of the p15 INK4B or p21 CIP1 genes 114,115. Additional mechanisms also contribute to TGF-β mediated growth arrest, again depending on the cell type 22. For example, TGF-β inhibits the expression of c-myc (ref. 107), CDK4 (refs. 116,117) and CDC25A, a tyrosine phosphatase of CDK4 and CDK6 (refs. 118,119). High levels of c-myc render the cells resistant to the growth inhibitory activity of TGF-β (refs. 120,121), and downregulation of c-myc is required for the induction of p15 INK4B (ref. 121) and p21 CIP1 (ref. 122) expression. The interaction of c-myc in a complex at the p15 INK4B promoter correlates with transcriptional repression 123,124 ; TGF-β induced downregulation of c-myc thus allows derepression 124 and TGF-β induced transcription through Smads 114. Decreased expression of c-myc may also be involved in the downregulation of CDC25A expression in response to TGF-β (refs. 125,126). The TGF-β protein has also been shown to induce apoptosis in several cell types The initial events that follow receptor activation and result in cell death are not understood, although the Daxx adaptor protein has been proposed to mediate TGFβ induced apoptosis through its ability to interact with the TβRII receptor 134. Increased levels of Smad3 or Smad4 can induce apoptosis , and dominant-negative interference with Smad3 function protects against apoptosis 138. In addition, Smad7 can act as an effector of TGF-β induced cell death 131,139, but can also protect against cell death 133,140, depending on the context. 120 nature genetics volume 29 october 2001

5 progress These findings implicate Smad signaling in TGF-β mediated apoptosis, although the pro-apoptotic gene targets of Smad signaling remain to be identified. Downregulation of Bcl-X L (refs. 141,142) and activation of caspase 3 and 8 (refs. 141,143,144) have also been implicated in this process, and a mitochondrial septin, ARTS, has a role in the sensitivity of cells to TGF-β mediated apoptosis 132. The activation of tyrosine kinase receptor signaling 145, through stimulation of the PI-3-K/Akt or the Raf 87 signaling pathway, protects against TGF-β-induced apoptosis and is likely to contribute to carcinogenesis. TGF-β signaling as a tumor suppressor pathway Alterations in TGF-β receptor signaling. The role of TGF-β signaling as a tumor suppressor pathway is best illustrated by the presence of inactivating mutations in genes encoding TGF-β receptors and Smads in human carcinomas, and by studies of tumor development in mouse models. Somatic mutations in TGFBR2, the gene that encodes the TβRII receptor, occur most frequently in tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC). A repeat stretch of adenines in the TβRII coding sequence is prone to mutation in these patients, owing to germline defects in their capacity for DNA mismatch repair. Resulting nucleotide additions or deletions give rise to a truncated TβRII, which is incapable of signaling Mutations in TGFBR2 are frequently found in colon cancers , gastric cancers 150, and gliomas 155 with microsatellite instability, but are less common in microsatelliteinstability tumors from the endometrium, pancreas, liver and breast 150, Missense and inactivating mutations in the kinase domain of TβRII have also been reported to occur in colon cancers, which do not display microsatellite instability 160, and in various other carcinomas 22,161. Overall, inactivating TGFBR2 mutations may be present in 20 25% of all colon cancers. Albeit less common, inactivating mutations in TβRI, encoded by TGFBR1 have also been observed, most notably in ovarian cancers, metastatic breast cancers, pancreatic carcinomas and T-cell lymphomas Mutations in TGFBR1 have not, however, been found to be accompanied by TGFBR2 mutations 166. Altogether, these mutations suggest that TβRII and TβRI might function as tumor suppressors in the development of carcinomas. Various observations suggest that TGF-β receptor expression is often downregulated or that TGF-β receptor availability at the cell surface is impaired in tumor cells, and that these defects allow cells to escape the growth inhibitory activities of TGF-β. TGF-β receptor expression may also be reduced in tumor cells through altered levels or activities of transcription factors, such as the Ets transcription factors that are required for expression of TβRII (ref. 170). Transcriptional silencing may also result from hypermethylation of CpG islands in the TβRI or TβRII gene promoters, or from mutations in the TβRII promoter that interfere with transcription factor binding 170. Whereas decreased TβRII function confers resistance against the growth inhibitory effect of TGF-β, other TGF-β responses may not be similarly affected because they require different thresholds of signaling The tumor suppressor role of TβRII has been demonstrated by expressing wildtype TβRII in colon or breast carcinoma cells, which lack a functional TGFBR2 allele 177,178, and by overexpressing it in thyroid carcinomas 179. Expression of TβRII conferred growth inhibition and suppressed anchorage independence, and strongly reduced tumor formation in nude mice when compared with the parental cells. Overexpression of TGF-β1 or TβRII in the skin of transgenic mice also provided evidence for a tumor suppressor activity 180,181, and expression of dominant-negative forms of TβRII in the skin or mammary gland 182,183 increased tumor formation. In addition, mice with one inactivated allele of the gene encoding TGF-β1 (TGFB1) show increased propensity for developing carcinoma, either spontaneously 184 or after carcinogen exposure 185. In the latter case, haploinsufficiency of TGFB1 increased tumor susceptibility, as the tumors retained one wildtype allele 185. Consistent with these results, decreased expression of TβRII correlates with high tumor grade in human cancers 170 and experimental systems Although these observations suggest that loss of TGF-β responsiveness provides a distinct advantage for developing tumors, most tumors do not have inactivated TGF-β receptors; individuals with HNPCC, who frequently have TGFBR2 mutations in their tumors 149, have a better prognosis than individuals with sporadic colon cancer 190. Abrogation of TGF-β signaling, while leading to a loss of growth inhibition mediated by TGF-β and early tumor onset, paradoxically protects against tumor progression. TGF-β receptors can thus act as tumor suppressors at early stages of carcinoma development, but at later stages TGF-β responsiveness may provide distinct advantages for cancer progression (see below and Fig. 3). Smads as tumor suppressors. Mutations of the Smad2- and Smad4-encoding gene sequences, but not those of Smad3 or the inhibitory Smad6 or Smad7, have been detected in several carcinomas 22,191, but are uncommon overall. These observations suggest that some Smads act as tumor suppressors. Inactivation of the genes encoding Smad2 (MADH2) or Smad4 (MADH4) occurs by loss of entire chromosome segments, small deletions, frameshift, nonsense or missense mutations 22,191. MADH4 mutations occur primarily in pancreatic carcinomas, in which the MADH4 gene was first identified as DPC4 (deleted in pancreatic carcinomas) 192, and in colon carcinomas, and less frequently in other types of carcinomas 22,191. Whereas biallelic inactivation of MADH4 often occurs in pancreatic and colon carcinomas, haploinsufficiency of the MADH4 locus may also contribute to progression of cancer 193,194. The occurrence of MADH4 mutations in the germline of a subset of juvenile polyposis families 195 further supports the notion that Smad4 acts as a tumor suppressor. In contrast to MADH4, inactivating mutations of MADH2 are rare and occur primarily in colorectal and lung carcinomas Finally, enhanced Smad7 levels, as observed in pancreatic carcinomas 200 (possibly as a result of TGF-β signaling) may also decrease Smad responsiveness. Tumor-associated mutations in the Smad4 and Smad2 proteins occur most frequently in the MH2 domain, which mediates heteromeric complex formation and transcriptional activation 22,191. C-terminal deletions or mutations often inactivate the Smad, but also provide dominant-negative interference with wildtype Smad function Other mutations confer decreased stability through increased ubiquitin-mediated degradation 204. Mutations in the MH1 domain of Smad4 interfere with its DNA-binding ability 205,206. Most if not all mutations impair Smad function; however, some mutations might alter TGF-β signaling to the advantage of the tumor. Indeed, the Smad2 mutations D450E and P445H, which are found in colorectal carcinomas, may enhance the invasive behavior of the tumor cells 207, as do some mutations in the type I TGF-β receptor 162. Studies that use mice carrying an inactivated allele of MADH4 support a role for this gene in tumor suppression. Whereas homozygous inactivation of MADH4 leads to early embryonic lethality, heterozygous mice are viable but develop intestinal polyps that can progress to carcinomas 193,208,209. When combined with an inactivated allele of the adenomatous polyposis coli (Apc) gene, simultaneous loss of the wildtype alleles at both loci results in the development of multiple polyps and in progression to heterogeneous invasive adenocarcinomas 208. nature genetics volume 29 october

6 Mice with homozygous inactivation of the gene encoding Smad3 (MADH3) have also been shown to develop colon carcinomas 210, although this phenotype was not seen in two similar studies 211,212. This discrepancy could be due to differences in genetic background or in environmental factors associated with animal husbandry. For example, the absence of Smad3 expression might impair TGF-β mediated immunosuppression and contribute to immune or inflammatory responses that predispose to cancer formation. Although a role of MADH3 as tumor suppressor gene is conceivable, no inactivating MADH3 mutations have been observed in human tumors. The occurrence of MADH2, but not MADH3, mutations can be rationalized by the crucial role of Smad2 in the TGFβ induced expression of p21 CIP1 or p15 INK4B CDK inhibitors 114,115. Inactivation of Smad2 might therefore inactivate the growth arrest response without affecting the Smad3- mediated gene expression that provides an advantage for tumor development, that is, the induction of some extracellular matrix proteins by TGF-β. The loss of Smad4 function may not abolish TGF-β responsiveness, even though Smad4 is generally perceived as essential for TGF-β responses. Indeed, mouse fibroblasts derived from Madh4 / embryos 213, as well as some Smad4-deficient tumor cell lines 214,215, retain at least some TGF-β responses. This is consistent with the observation that TGF-β induced synthesis of fibronectin can occur in the absence of Smad4 (ref. 71). Thus, the high frequency of MADH4 deletions in tumors might represent a selective perturbation of TGF-β signaling, rather than its complete abrogation. In addition, loss of Smad4 expression may enhance Ras signaling and progression to undifferentiated carcinomas 216, further emphasizing the crosstalk between TGF-β and Ras signaling. Moreover, different TGF-β responses may have differential sensitivity to Smad signaling. Accordingly, overexpression of Smad7 in a carcinoma cell line suppressed TGF-β induced growth arrest, without affecting TGF-β induced expression of plasminogen activator inhibitor 1, and enhanced anchorage-independent growth and tumorigenicity 200. Finally, inactivating MADH4 mutations have been found in conjunction with mutations in TGFBR2 or TGFBR1 (ref. 160), which strongly suggests that Smad4 has tumor suppressive activities that are unrelated to TGF-β signaling. Other mechanisms of impaired TGF-β responsiveness. Upregulated expression of proto-oncogenes or expression of their oncogenic counterparts may also provide a mechanism by which tumor cells downregulate TGF-β responsiveness and escape its tumor suppressor effects. For example, elevated expression of the proto-oncogene c-ski in melanoma 217 has been correlated with decreased TGF-β responsiveness, presumably owing to repression of Smad-mediated transcription 63,64. Expression of c-myc may also be involved in the growth inhibitory response to TGF-β. The downregulation of c-myc expression by TGF-β, as observed in epithelial cells, is lost in various cancer cell lines, concomitant with the loss of the growth inhibitory response to TGF-β 36. The EWS-FLI1 oncogene, which incorporates a partial coding sequence for the Ets family transcription factor Fli-1 and was first identified in Ewing sarcoma, represses expression of TβRII and may account for decreased TGF-β responsiveness 218. In addition, the Tax protein, which is encoded by HTLV-1 and is thought to contribute to leukemogenesis in adult T-cell leukemia, is a potent repressor of Smad-mediated transcription 91. Clearly, tumor cells have developed several strategies to escape the growth inhibitory response of TGF-β, and most carcinoma cells are resistant to growth inhibition by TGF-β. Finally, the mannose-6-phosphate receptor may also have a role in the tumor suppressor activity of TGF-β. This receptor binds latent TGF-β and is required for activation of TGF-β by plasminogen conversion into plasmin 219. Loss of heterozygosity at the receptor locus and inactivating receptor mutations occur with high incidence in hepatocellular and squamous lung carcinomas 220,221, and are early events during carcinogenesis 222. As the levels of mannose-6-phosphate receptor and loss of heterozygosity of this locus correlate with TGF-β1 activation, it is conceivable that part of its tumor suppressor role is linked to activation of TGF-β1 (ref. 223). Cell-autonomous stimulation of tumor development In spite of the tumor suppressor activity of TGF-β1, tumor cells often show increased production of this growth factor 3,4, and considerable evidence documents its tumor-promoting role through its effects on tumor cell invasion and changes in the tumor microenvironment. Tumor metastasis depends on various factors, including the ability of tumor cells to migrate and invade the stroma, and to migrate in and out of blood and lymphatic vessels. The epithelial to mesenchymal differentiation of tumor cells has an important role in this invasive phenotype 224. Accordingly, fibroblastoid or spindle-cell tumors of epithelial origin have been characterized as highly malignant and invasive 88, This type of transdifferentiation and invasion of epithelial cells into the underlying mesenchyme is not restricted to cancer progression; it also occurs during developmental processes such as placentation 224. During cardiac 230,231, palatal 232,233 and hair-follicle development 234, TGF-βs are involved in epithelial to mesenchymal transdifferentiation, and related family members serve the same function in mesoderm induction 235. This morphological transition is characterized by extensive changes in the expression of cell-adhesion molecules and by a switch from a cytoskeleton of mainly cytokeratin intermediate filaments to one comprising predominantly vimentin 88,228,229,236. The ability of epithelial or carcinoma cells to undergo epithelial to mesenchymal transition in culture correlates with cell changes that facilitate invasion and metastasis in vivo 88,176,229. The TGF-β protein can induce an epithelial to mesenchymal transition in cultures of normal and transformed breast epithelial cells 88,229, , squamous carcinoma cells 176,239, ovarian adenosarcoma cells 240 and melanoma cells 241. The phenotypic changes have been best characterized in NMuMG cells, in which TGF-β induces changes in cell shape, downregulates the expression of E-cadherin, ZO-1, vinculin and keratin, and induces the expression of vimentin and N-cadherin, which has been shown to increase cell motility and scattering 105,236,238. The mechanism of the TGF-β-induced epithelial to mesenchymal transition is complex, and Ras/PI-3-K signaling and TGF-βinduced activation of Smad and RhoA signaling seem to have distinct roles in the process 105,216,238,242. Whereas TGF-β induces this phenotypic transition in some cell lines, growth factors that act through tyrosine kinase receptors and activation of Ras signaling induce similar changes in other cell systems 224,243. In some cases, both signaling cascades need to be enhanced to achieve epithelial to mesenchymal transition 88. These findings suggest that these two pathways synergize in the transition of squamous to spindle carcinoma formation. This synergy explains why, for example, the invasive growth of Ras- or Raf-transformed epithelial cells in vitro depends on intact autocrine TGF-β signaling 87,229. Accordingly, spindle-cell carcinoma tissue has an increased mutant Hras gene dosage when compared with squamous cells originating from the same skin tumors 227. Together, these observations suggest that activation of TGF-β signaling and an involvement of Ras/MAP 122 nature genetics volume 29 october 2001

7 progress kinase signaling may be essential for carcinoma cell invasion and metastasis, whether through epigenetic alterations of autocrine growth factor signaling or genetic alterations. This interpretation is also consistent with the cooperation of both pathways in mesoderm induction 235, and in epithelial to mesenchymal differentiation and invasion during hair-follicle formation 234 and cardiogenesis 244. The cell-autonomous role of TGF-β in the spindle-cell phenotype and invasive behavior of carcinomas has been well documented for skin carcinomas in mice 176,180. Transgenic expression of activated TGF-β1 in mouse epidermis in vivo increased the malignant conversion to carcinomas and the incidence of spindle-cell carcinomas, even though the hyperplastic response to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate and the number of papillomas after treatment with 7,12-dimethylbenz- [a]-anthracene were reduced 180. In another study, expression of a dominant-negative TβRII prevented squamous carcinoma cells from undergoing an epithelial to mesenchymal transition in response to TGF-β in vivo 176. Similar observations on the role of TGF-β signaling have been made using a Ras-transformed mammary epithelial cell line and a fibroblastoid colon carcinoma cell line 229. Expression of a dominant-negative TβRII prevented TGF-β-induced epithelial to mesenchymal changes and reverted the colon carcinoma cells to an epithelial phenotype. In addition, blocking TβRII function suppressed the in vitro invasion and the highly metastatic phenotype of this colon carcinoma cell line 229. Finally, restoration of TβRII signaling in HNPCC cells with an endogenous TβRII mutation rendered the cells invasive in contrast to their normally non-invasive phenotype 229. The increased incidence of aggressive skin neoplasia associated with the use of cyclosporin (for example, in kidney transplant patients) may also result from a cell-autonomous effect mediated by TGF-β. Cyclosporin induces invasive behavior in adenocarcinoma cells in vitro and enhances tumor growth in immunodeficient SCID-beige mice, whereas anti-tgf-β antibodies prevent the cyclosporin-induced increase in metastasis 245. TGF-β has also been shown to be an autocrine effector of breast tumor metastasis 246. By using a breast cancer cell line that metastasizes to bone in nude mice, it was shown that dominant-negative interference with TβRII function decreases tumor formation and metastasis, and the resulting destruction of bone 246. In this model, TGF-β1 induced the expression and secretion of parathyroid hormone-releasing peptide (PTHrP), resulting in the recruitment of bone-resorbing osteoclasts. In complementary experiments, tumor cells expressing a partially activated TβRI accelerated bone destruction and reduced survival 246. This finding is likely to be relevant for the clinical correlation that women with PTHrP-positive breast cancer are more likely to develop metastatic disease than those with PTHrP-negative tumors 246. In summary, in vitro and in vivo studies link autocrine TGF-β responsiveness to epithelial to mesenchymal transdifferentiation, the acquisition of an invasive phenotype, and metastatic behavior. The TGF-β-dependent, reversible nature of this phenotype may explain why many metastatic tumors show a well-differentiated epithelial phenotype, presumably depending on the local tissue micro-environment and levels of active TGF-β. Effects of TGF-β on the tumor micro-environment The increased expression and activation of TGF-β1 by tumor cells profoundly affects the micro-environment. Increased protease expression and plasmin generation by tumor cells 11 enhances activation of TGF-β and degradation of the extracellular matrix, with a consequent release of stored TGF-β. But a concomitant increase in production of TGF-β1 stimulates synthesis of extracellular matrix proteins and chemoattraction of fibroblasts. Together, all these changes result in a micro-environment that promotes tumor growth and invasion and angiogenesis 247. Tumor angiogenesis is crucial for tumor growth and invasion, as blood vessels deliver nutrients and oxygen to the tumor cells and allow them to intravasate the blood system, which leads to metastasis. TGF-β1 acts as a potent inducer of angiogenesis in several assays , and mouse models defective in TGF-β signaling components indicate the importance of TGF-β1 in normal vascular development. Targeted inactivation of the gene encoding TGF-β1 or TβRII results in embryonic lethality owing to defective vasculogenesis and angiogenesis 253,254, whereas angiogenesis-defective phenotypes are also seen in mice with null mutations of the genes encoding TβRI (ref. 255), ALK-1, a TGFβ type I receptor that is expressed in endothelial cells and signals through Smad1 and Smad5 (ref. 256) endoglin, an endothelially restricted TGF-β type III receptor (refs. 257,258) and Smad5 (refs. 259,260). Several models indicate the role of tumor cell secreted TGF-β1 in tumor angiogenesis. Increased expression of TGF-β1 in transfected prostate carcinoma or Chinese hamster ovary cells enhanced tumor angiogenesis in immunodeficient mice 261,262, and local administration of neutralizing antibodies to TGF-β1 strongly reduced tumor angiogenesis 261. Intraperitoneal injection of TGF-β antibodies reduced angiogenesis and tumorigenicity of a renal carcinoma cell line that lacks TβRII, and is therefore not responsive to TGF-β, in natural killer (NK)-, T- and B-cell deficient mice 263. This cell line also lacks the von Hippel Lindau (VHL) tumor suppressor gene, and reintroduction of this gene decreased TGF-β1 synthesis, suggesting that VHL regulates TGFβ1 levels and has tumor suppressor effects through an anti-angiogenic mechanism involving TGF-β1 (ref. 263). Finally, re-expression of Smad4 in Smad4-deficient pancreas cancer cells suppresses tumor development primarily by repressing tumor angiogenesis 264. In human breast tumors, high levels of TGF-β1 messenger RNA are associated with increased microvessel density, and both parameters correlate with poor patient prognosis 265. Diagnostic studies on other carcinoma types suggest that high tumor burden and circulating plasma levels of TGF-β1 are associated with enhanced tumor angiogenesis and poor patient prognosis In one study, TGF-β1 levels were also correlated with expression levels of the angiogenic growth factor vascular endothelial growth factor (VEGF) 270. The mechanisms of angiogenesis stimulation by TGF-β1 presumably combine direct and indirect effects. TGF-β induces expression of VEGF, which directly acts on endothelial cells to stimulate cell proliferation and migration 271. TGF-β also induces capillary formation of endothelial cells, which are cultured on collagen matrix 249,252. Indirect stimulation of angiogenesis by TGF-β1 may occur through the potent chemoattractant activity of TGF-β for monocytes, which release angiogenic cytokines 248,250,272,273. TGF-β1 induced changes in the microenvironment provide favorable conditions for endothelial cell migration and capillary formation. Moreover, TGF-β induced expression of the metalloproteases MMP-2 and MMP-9, and downregulation of the protease inhibitor TIMP in tumor and endothelial cells , are expected to enhance the migratory and invasive properties of endothelial cells required for angiogenesis 280,281. Thus, both the direct effects of TGF-β and its indirect effects on the tumor micro-environment stimulate tumor angiogenesis. Secretion and activation of TGF-β1 by tumor cells also stimulate tumor development and progression through escape from immunosurveillance. TGF-β inhibits the proliferation and functional differentiation of T lymphocytes, lymphokineactivated killer cells, NK cells, neutrophils, macrophages and nature genetics volume 29 october

8 B cells 273,282,283. Several observations show that TGF-β expression by tumor cells promotes tumorigenicity by locally repressing immune functions. For example, the increased expression of TGF-β1 by tumor cells enhanced their tumorigenicity and prevented activation of cytotoxic T-lymphocyte function when compared with parental cells 284. This repression was presumably elaborated by the ability of TGF-β to inhibit the expression and function of interleukin 2 (IL-2) and IL-2 receptors 285,286. Accordingly, the inhibition of the cytotoxic T-lymphocyte response by EMT6 mammary tumor cells, which produce high levels of TGF-β1, was overcome by ectopic expression of IL-2 in these cells 287. Thus, TGFβ mediated suppression of the cytotoxic T-cell response promotes tumor development 288. TGF-β1 expression by tumor cells may also inhibit other immune functions. Increased expression of TGF-β1 by tumor cells decreased NK cell activity 289 and promoted tumor formation in nude mice that lack T cells 289,290. In addition, anti TGF-β antibodies suppressed tumor formation and metastasis of a breast carcinoma cell line in nude mice, while enhancing NK cell function 291,292. This suppression was not seen in nude beige mice, which lack both T and NK cells, which implicates TGFβ1 induced suppression of NK cells in cancer progression 292,293. TGF-β mediated suppression of neutrophil function may also be involved in tumor progression. Indeed, Fas ligand expressing carcinoma cells underwent neutrophil-mediated rejection, but this rejection did not occur at a site with high levels of TGF-β or when TGF-β1 was injected at the tumor site 294. Finally, strong downregulation of the expression of major histocompatibility complex (MHC) class II antigens by TGF-β may make the tumor cell surface less immunogenic. This regulation contributes to the local immunosuppression and the escape of TGF-β1- expressing tumor cells from immune surveillance 298,299. Potential avenues toward a cancer therapy Considering the complex roles of TGF-β signaling in tumor suppression and progression, which are context- and stage-dependent, any strategy to exploit this knowledge for therapeutic purposes is fraught with difficulties. For example, stimulating TGF-β signaling might suppress tumor growth, but might also promote tumor invasion and metastasis, particularly of cells that are no longer sensitive to the growth inhibitory effect of TGF-β. To suppress cancer progression by exogenous administration of TGF-β, induction of TGF-β synthesis or enhanced TGF-β signaling would require that the tumors are sensitive to the growth inhibitory effects of TGF-β; unfortunately, however, resistance to the growth inhibitory effect of TGF-β is often acquired in early tumor development, often before diagnosis. It has been proposed, on the one hand, that the beneficial effects of tamoxifen in preventing or treating breast cancer may be due, in part, to its ability to induce expression of TGF-β 300. On the other hand, tumor resistance to tamoxifen correlates with increased expression of TGF-β1, which is possibly a reflection of insensitivity to the antiproliferative effects of TGF-β 301,302. These elevated levels of TGF-β1 and the consequent suppression of NK cell activity may be responsible for the failure of tamoxifen therapy. Accordingly, tamoxifen resistance of a breast tumor cell line has been reversed through the use of anti-tgf-β antibodies 293,303. As indicated by this example, inhibiting TGF-β1 expression or activation may represent a valid approach to treatment of carcinoma. Inhibiting TGF-β production should affect the cellautonomous response and the effects of TGF-β on its micro-environment, whereas interfering with the TGF-β responsiveness of the tumor cells would primarily inhibit its cellautonomous effects. But any approach that inhibits TGF-β signaling in tumor cells might induce the regression of advanced cancers while stimulating the growth of otherwise quiescent premalignant lesions. The most sensible approach may therefore be to activate TGFβ in a preventive setting, but to inhibit signaling in advanced metastatic cancer. Inhibiting the production and/or signaling of TGF-β through antisense RNA, anti TGF-β antibodies or soluble TGF-β receptors might inhibit cancer progression. For example, antisense RNA inhibition of TGF-β2 synthesis by breast cancer cells enhanced the cytotoxicity of NK cells 293, which is consistent with the ability of anti TGF-β therapy to decrease suppression of the NK cell response 289,292. In other studies, antisense RNA inhibition of TGF-β1 or TGF-β2 synthesis in breast cancer, mesothelioma cells or glioma cells restored the tumor immunogenicity and the cytotoxic T-lymphocyte response, and inhibited tumor development As discussed above, antibodies against TGF-β have also been shown to inhibit tumor formation through an increase in NK cell activity 292 or a decrease in microvessel density, without directly affecting cell proliferation 261,263 or the innate tumorigenicity of cancer cells 245. In addition, administration of an anti-endoglin immunotoxin targeted the endothelial cells and selectively inhibited tumor angiogenesis and carcinoma development 308. Sequestration of TGF-β through the use of soluble receptor ectodomains may provide another approach to inhibit cancer progression. Indeed, expression of a soluble TβRII (ref. 309) or the soluble proteoglycans betaglycan 310 or decorin 311,312, which bind TGF-β, inhibited tumor formation and/or metastasis and even induced tumor regression, depending on the tumor model. These approaches to facilitating immune rejection might be enhanced further by combinatorial therapy with IL-2. For example, the combination of anti-tgf-β antibodies with IL-2 reduced tumor formation in a highly metastatic melanoma cell line, which was unaffected by either treatment alone 313. In future studies, it would be desirable to retain the TGFβ mediated stimulation of apoptosis in tumor cells, but to inhibit the cell-autonomous (invasion and metastatic spread) and non cell-autonomous (angiogenesis and immunosuppression) activities of this multifaceted molecule. In view of the important role of the Ras pathway in these effects, combinatorial approaches involving inhibitors of the Ras pathway coupled with inhibitors of TGF-β might lead to a marked synergistic inhibition of tumor growth. Future insight into the TGF-β signaling mechanisms that lead to epithelial to mesenchymal transition and invasion, or to apoptosis, might lead to the design of more targeted and more effective therapeutic interventions. Acknowledgments It is impossible to mention every important contribution to our understanding of the mechanisms of TGF-β signaling and its role in cancer, and we apologize to those investigators whose contributions could not be acknowledged. Relevant research in the laboratories of the authors is funded by NIH grants to R.D.; NIH, March of Dimes and American Heart Association grants to R.J.A.; and an NIH grant to A.B. We thank J. Qing for the design and drawing of Fig. 1, and A. Roberts, B. Lechleider and C. Arteaga for their comments. Received 8 August; accepted 29 August Roberts, A.B. & Sporn, M.B. in Peptide Growth Factors and their Receptors Handbook of Experimental Pharmacology (eds Sporn, M.B. & Roberts, A.B.) (Springer, Heidelberg, 1990) Massagué, J. TGF-β signal transduction. Annu. Rev. Biochem. 67, (1998). Review 3. Derynck, R. et al. Synthesis of messenger RNAs for transforming growth factors α and β and the epidermal growth factor receptor by human tumors. Cancer Res. 47, (1987). 124 nature genetics volume 29 october 2001