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Carcinogenesis Advance Access originally published online on May 12, 2006
Carcinogenesis 2006 27(11):2148-2156; doi:10.1093/carcin/bgl068
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Escaping from the TGFß anti-proliferative control

Joan Seoane

Institució Catalana de Recerca i Estudis Avançats (ICREA), Medical Oncology Program Vall d'Hebron University Hospital Research Institute, Barcelona, Spain

*To whom correspondence should be addressed at: Medical Oncology Program, Vall d'Hebron University Hospital Research Institute, Psg. Vall d'Hebron 119-129, 08035 Barcelona, Spain. Email: jseoane{at}ir.vhebron.net


   Abstract
Top
 Abstract
Introduction
 TGFß signal...
 Dual role of TGFß...
 Regulation of cell proliferation...
 Evading the TGFß anti...
 Conclusion
 References
 
Transforming growth factor-ß (TGFß) has a crucial role in tissue homeostasis and disruption of the TGFß pathway has been implicated in many human diseases including cancer. As a potent inhibitor of epithelial cell proliferation, TGFß is a tumor suppressor. Tumor cells evade the antitumoral effect of TGFß, either by acquiring somatic mutations that blunt TGFß signaling or by selectively preventing the cytostatic responses to TGFß. During tumor progression, TGFß not only loses the anti-proliferative response but can also become an oncogenic factor. Recent work has provided insights into the specific molecular mechanisms involved in the loss of the TGFß anti-proliferative response. This review is an overview of the mechanisms that lead to the impairment of the tumor-suppressive function of TGFß in cancer. The understanding of how the TGFß signal is disrupted in cancer might facilitate the design and development of rational and successful therapeutic strategies.

Abbreviations: BMP, bone morphogenetic protein; EMT, epithelial-to-mesenchymal transition; MH1/2, Mad-homology 1/2; RSmads, receptor phosphorylated Smads; SARA, Smad anchor for receptor activation; TGFß, Transforming growth factor-ß; TßRI/II, TGFß type I/II receptor


   Introduction
Top
 Abstract
 Introduction
TGFß signal...
 Dual role of TGFß...
 Regulation of cell proliferation...
 Evading the TGFß anti...
 Conclusion
 References
 
Transforming growth factor-ß (TGFß) is a member of a large family of cytokines, which includes bone morphogenetic proteins (BMPs), Nodals, Activins and others, involved in the regulation of embryonic development and tissue homeostasis. TGFß is secreted by several cell types and it regulates a diverse array of cellular processes including cell proliferation, morphogenesis, migration, extracellular matrix production, cytokine secretion and apoptosis. The TGFß pathway is modulated by many other signaling cascades and the TGFß response is determined by the integration of the signals that a given cell receives (1–3).

The role of TGFß in carcinogenesis has been subject of study during the last decades. TGFß is a potent inhibitor of proliferation in epithelial cells and acts as a tumor suppressor. Tumor cells tend to escape from the TGFß anti-proliferative effect by either acquiring mutations in the components of the TGFß signal transduction pathway or by selectively disrupting the TGFß anti-proliferative response. In advanced tumors, TGFß not only tends to lose its tumor-suppressive function, but it becomes an oncogenic factor inducing invasion, angiogenesis, epithelial-to-mesenchymal transition (EMT), proliferation and, in certain cases, metastasis (4,5).

Our understanding of the molecular mechanisms implicated in the TGFß signal transduction pathway has dramatically increased over the last years. One by one, many of the TGFß molecular pathways that regulate specific gene responses have been elucidated shedding light on the biological and physiological relevance of TGFß in normal and disease states. Recent studies have identified several molecular mechanisms that allow a tumor cell to evade from the tumor suppressive effect of TGFß and are involved in the oncogenic switch of the TGFß response. This review summarizes our present knowledge on the molecular mechanisms involved in the disruption of the anti-proliferative response to TGFß. The understanding of the exact mechanisms involved in the control of tissue homeostasis by TGFß and how those mechanisms are disrupted in cancer is crucial to gain knowledge about cancer biology and might be helpful in order to design new tools for our fight against cancer.


   TGFß signal transduction
Top
 Abstract
 Introduction
 TGFß signal...
Dual role of TGFß...
 Regulation of cell proliferation...
 Evading the TGFß anti...
 Conclusion
 References
 
The active form of the TGFß ligand is a 25 kDa dimer of two polypeptides stabilized by hydrophobic interactions strengthened by a bisulfide bond. TGFß is synthesized as the C-terminal domain of a precursor form that is cleaved before secretion from the cell. The N-terminal TGFß propeptide, called latency associated peptide (LAP), remains non-covalently bound to TGFß after secretion inhibiting the binding of the active TGFß form to its receptors. In most cell types, TGFß is secreted as a large latent complex composed of the active TGFß form bound to LAP that in turn is covalently bound to a member of a family of latent TGFß-binding proteins (LTBPs). The latent TGFß complex interacts with components of the extracellular matrix through the remaining LTBP stored and providing a source of readily accessible ligand. The process of activation of the TGFß ligand involves the proteolytic cleavage of the LTBP molecule by serine proteases and the release or conformational modification of LAP (6,7).

Once activated, TGFß signals through two classes of receptors, the TGFß type I receptor (TßRI) and the TGFß type II receptor (TßRII). In addition, endoglin and betaglican, also called accessory receptors, bind TGFß with low affinity and present it to the TßRI and TßRII. Type I and II receptors are serine/threonine kinase receptors that form a heterodimeric complex upon TGFß binding. TGFß interacts with the ectodomain of the TßRII and allows the subsequent incorporation of the TßRI generating a ligand-receptor complex formed by a ligand dimer, two TßRI and two TßRII. The TßRII appears to be a constitutively active kinase that, when the ligand–receptor complex is formed, phosphorylates a characteristic SGSGSG sequence, called the GS domain, present in the type I receptor. Phosphorylation of the TßRI GS domain leads to the activation of its kinase and turns the GS region into a Smad binding site. Once activated by the TßRII, the TßRI phosphorylates Smads through its catalytic domain (8).

Smads are transcription factors that in the basal state are mostly localized in the cytoplasm. Smad2 and 3 are phosphorylated by TßRI and are commonly referred to as receptor phosphorylated Smads (RSmads). Smad4, also called co-Smad, is a partner of the RSmads and it is not phosphorylated by the TGFß receptors. Moreover, two Smad proteins serve as inhibitors of the TGFß signal, Smad6 and 7. They are not phosphorylated by the TGFß receptors and act as decoys interfering with Smad–receptor or Smad–Smad interaction as well as facilitating the proteasome-mediated degradation of the TGFß receptors (9).

Smad proteins consist of two globular domains coupled by a linker region. The N-terminal domain, Mad-homology 1 (MH1) domain, contains the DNA binding module formed by a ß-hairpin structure conserved in all Smads that recognizes the 5'-AGAC-3' DNA sequence. The linker region is a flexible segment that contains binding sites for ubiquitin ligases, phosphorylation sites and, in the case of Smad4, a nuclear export signal (NES). The C-terminal domain, Mad-homology 2 (MH2) domain, is highly conserved and mediates the interaction of Smads with a large plethora of proteins. It contains the Smad transactivation domain and in the case of RSmads, the MH2 domain has a C-terminal motif, Ser–X–Ser, which is phosphorylated by the activated TßRI (8,9).

Phosphorylation of the two serine residues in the C-terminal motif generates an acidic tail that binds to a basic pocket in the Smad4 MH2 domain. RSmad bound to Smad4 forms oligomers that interact with DNA-binding partners to form transcriptional complexes in the nucleus. Depending on the gene target or the DNA-binding cofactor present in the complex, RSmad–Smad4 complexes can be heterodimers (RSmad–Smad4) or heterotrimers (two RSmads and one Smad4) (8–10).

In the basal state, Smads are constantly shuttling in and out of the nucleus. Once phosphorylated by the TßRI, RSmads accumulate in the nucleus. Smad intra-cellular localization depends on its interaction with retention factors. The affinity of phosphorylated Smads for cytosolic retention factors is lower than the one of unphosphorylated Smads and, concomitantly, phospho-Smads have higher affinity for nuclear retention factors. This results in an accumulation of phosphorylated Smads in the nucleus (3). The Smad anchor for receptor activation (SARA) is the best characterized of the cytosolic retention factors for RSmads. SARA facilitates phoshorylation of Smad by making Smads accessible to the activated receptor. Phosphorylation of Smads induces a decrease in their affinity for SARA and promotes the release of Smads from their cytosolic retention. Other proteins have been described to play a similar adaptor role besides SARA (11). Once in the nucleus, phosphorylated Smads are sequestered by nuclear retention factors such as the Smad DNA-binding partner FoxH1 (3,8).

Smad2, 3 and 4 undergo nuclear import by means of direct interactions with nucleoporins in an importin-independent mechanism. Smad3 and 4 can also interact with importin-ß that facilitates their nucleocytoplasmic dynamics (8,12). In the nucleus, the Smad complex lands on gene promoters to regulate transcription. Smads in isolation have low affinity for DNA and they associate with DNA-binding partners in order to bind gene promoters. Many Smad-binding cofactors have been described, listed in (2,11,13). The DNA-binding partner of Smad is usually a transcription factor that interacts on one side with the Smad complex and on the other with their cognate sequences on DNA. Thus, the DNA-binding cofactor facilitates the binding of the Smad complex to a particular promoter region containing suitable DNA binding elements for Smads and the Smad partner. The presence of a particular cofactor in the Smad complex dictates which gene promoters are targeted. Moreover, many Smad cofactors facilitate the binding of either co-activators or co-repressors, which determine the transcriptional activity of the Smad complex (9).

DNA-binding cofactors are critical for TGFß signal transduction and they are responsible for the pleiotropic and versatile responses found in such an apparently linear pathway. In many cases, the activity or the expression of the DNA-binding partner is regulated by other signaling pathways. In this sense, the TGFß–Smad pathway is modulated and integrated in the signaling context of the cell (1,9).

The TGFß–Smad pathway is terminated mainly through two mechanisms. Experiments using TGFß receptor inhibitors have suggested that once the signal stops, RSmads get rapidly dephosphorylated and, hence, inactivated and exported out of the nucleus (14). Very recently, pyruvate dehydrogenase phosphatase has been identified in a functional screen in Drosophila S2 cells as the phosphatase of Smad1 activated by the TGFß family member BMP (15). However, pyruvate dehydrogenase phosphatase does not dephosphorylate TGFß-activated Smad2 and 3. Thus, the phosphatase involved in the dephosphorylation of Smad2 and 3 is still unknown. Besides dephosphorylation, the RSmad transcriptional activity is terminated via proteasome-mediated degradation. Receptor phosphorylated RSmads are ubiquitinated in their linker region by members of the Smurf family of HECT-domain E3 ubiquitin ligases and targeted to the 26S proteasome for degradation. Ubiquitination of RSmad controls both the extent and the length of the TGFß response in a given cell (3,9) (Figure 1).


Figure 1
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  		Fig. 1 The TGFß–Smad pathway. TGFß is synthesized as a dimeric proprotein (pro-TGFß). In the Golgi apparatus, the dimeric propeptides are cleaved from the mature TGFß dimer but still remain attached to the ligand generating a latent TGFß form. Once TGFß is liberated from the latent complex, it can bind a TGFß receptor complex formed by the type I and the type II TGFß receptors. Upon TGFß binding, the type II receptor phosphorylates the type I receptor inducing the activity of its serine/threonine kinase. Anchor proteins like SARA capture RSmads for presentation to the activated type I receptor. This facilitates the phosphorylation of RSmads by the type I receptor. RSmads and the co-Smad, Smad4, are continuously shuttling in and out of the nucleus. Receptor-mediated phosphorylation of RSmad induces their accumulation in the nucleus and their association with the co-Smad, Smad4. In the nucleus, the Smad complex interacts with different DNA-binding cofactors that facilitate the binding of the complex to specific gene promoters. Recruitment of transcriptional co-activators or co-repressors allows the Smad complex to regulate gene transcription. Termination of the TGFß signal is mediated by RSmad dephosphorylation or proteasome-mediated degradation. In addition and depending on the cellular context, TGFß can activate Smad-independent pathways such as the Ras–Erk, PI3K–Akt, TAK1/MEKK1, PP2A, Rho and Par6 pathways.

 
Over the last decade, several groups have described the TGFß-mediated activation of Smad-independent pathways (11,16,17). Ras and several members of the mitogen-activated protein kinase (MAPK) pathways, extracellular signal regulated kinase (ERK)-1, ERK-2, p38 and cJun N-terminal kinase (JNK) have been shown to be activated in response to TGFß in some cell types, in certain cases very rapidly, minutes after ligand addition (11,16,17). Ras was observed to be rapidly activated by TGFß in intestinal and lung epithelial cells (17). ERK activation by TGFß may be mediated by TGFß-induced Ras. TAK1 and MEKK1, both MAPK kinase kinases (MEKK), have been implicated in the process of the activation of JNK and p38. Despite these evidences, the exact molecular mechanisms of the activation of Ras and the MAPK pathways are still unclear (11,16).

In certain cell types, TGFß is able to activate the phosphoinositide-3 kinase (PI3K)–Akt pathway. An association of both TßRII and TßRI with the regulatory subunit of PI3K, p85, has been observed (18). Moreover, constitutively active TßRI upregulates the PI3K catalytic activity, and transgenic mice expressing an active mutant of TßRI in the mammary epithelium exhibit an increased PI3K activity (19). On the other hand, TGFß can activate or stabilize Rho-like GTPases (RhoA, Rac and Cdc42) in some cell lines (20–22). The mechanisms of the activation of Rho-like GTPases are not completely discerned although Ras activation in response to TGFß may lead to such activation. The protein phosphatase PP2A has been involved in the TGFß pathway. PP2A associates with the activated type I receptor recruiting the p70 ribosomal protein S6 kinase that is then dephosphorylated and inactivated. The resulting decrease in p70 S6 kinase activity contributes to the anti-proliferative response mediated by TGFß (23).

In mesenchymal cells and not in epithelial cells, TGFß induces an Smad-independent activation of Pak2 via the activation of PI3K or the Rho-like small GTPases, Rac1 or Cdc42 (24,25). Pak2 in turn induces the activation of c-Abl (25). Recently, it has been shown that the activated TGFß receptor complex can also signal through Par6. In polarized epithelial cells, the TGFß receptor complex is recruited to tight junctions where it binds Par6. Upon TGFß binding to the TGFß receptor complex, the TßRII phosphorylates TßRI and Par6. Phosphorylated Par6 recruits the ubiquitin ligase Smurf1 leading to the ubiquitylation and degradation of RhoA (26). Par6 is the first known substrate of TßRII that is not a TGFß-receptor protein. Interestingly, TGFß-mediated signaling through Par6 leads to the downregulation of RhoA in contrast to what has been observed in other systems where TGFß induces activity of Rho-like GTPases. This exemplifies how the TGFß response depends on the cellular context (Figure 1).


   Dual role of TGFß in cancer
Top
 Abstract
 Introduction
 TGFß signal...
 Dual role of TGFß...
Regulation of cell proliferation...
 Evading the TGFß anti...
 Conclusion
 References
 
TGFß is a potent anti-proliferative factor in epithelial and hematopoietic cells (1). Moreover, TGFß can act as an inducer of apoptosis in some cell types. The exact mechanisms of the TGFß-mediated induction of apoptosis are still not completely uncovered. Several gene responses have been implicated in the apoptotic effects of TGFß. The TGFß–Smad pathway induces the TGFß-inducible-early-response gene (TIEG-1), a transcriptional factor that induces apoptosis and inhibits proliferation in several cell types (27). TGFß-induced apoptosis in hepatoma cells involves the induction of the death-associated protein kinase (DAPK) (28). SHIP (SH2-domain-containing inositol-5-phosphatase) is upregulated by TGFß in hematopoietic cells and inhibits the pro-survival Akt signaling (29). Moreover, in gastric carcinoma cells TGFß induces the Fas receptor leading to activation of caspases (30). ARTS (apoptotic-related protein in the TGFß signaling pathway) has been identified as a protein required for TGFß-mediated apoptosis in rat prostate carcinoma cells (31), and the adaptor protein Daxx has been described to be a mediator of TGFß-induced apoptosis through JNK activation (32). Finally, two reports have shown that Akt modulates the TGFß pathway in hepatoma cells through the physical interaction of Smad3 and Akt. Akt when bound to Smad3 inhibits Smad3 transcriptional activity and Smad3-induced apoptosis in hepatoma cells (33,34).

Owing to its anti-proliferative and apoptotic functions, TGFß is a tumor suppressor factor. During tumor progression, tumor cells tend to lose the tumor-suppressive responses to TGFß. In some cases, tumor cells acquire somatic mutations in components of the TGFß–Smad signal transduction pathway (Smads and TGFß receptors) in order to evade the TGFß anti-proliferative function. In many other tumors, the TGFß signal transduction pathway is not affected but cells become specifically resistant to the anti-proliferative response to TGFß. In those tumors, TGFß can become an oncogenic factor, inducing proliferation, angiogenesis, invasion and metastasis (4,5,35). In addition, TGFß induces a blockade on the immune cellular proliferation and differentiation with a particular effect on T cells. Thus, in those tumors that are resistant to the TGFß anti-proliferative response, TGFß facilitates tumor progression by blocking the antitumoral immune response (36,37). Usually, tumors where TGFß acts as an oncogenic factor express high levels of TGFß, in many instances owing to the fact that TGFß is able to induce its own expression generating a malignant autocrine loop.


   Regulation of cell proliferation by TGFß
Top
 Abstract
 Introduction
 TGFß signal...
 Dual role of TGFß...
 Regulation of cell proliferation...
Evading the TGFß anti...
 Conclusion
 References
 
In order to study the mechanisms implicated in the loss of the TGFß anti-proliferative function, it is important to understand the TGFß cytostatic response in epithelial cells. In normal epithelial cells, TGFß regulates the expression of several genes promoting cell cycle arrest. In these cells, TGFß rapidly induces two cyclin-dependent kinase inhibitors, p21Cip1 and p15Ink4b, and downregulates Myc, Id1 and Id2, all three transcription factors involved in proliferation and inhibition of differentiation. These five genes are considered part of the TGFß cytostatic program shared at least by skin, lung and mammary epithelial cells (1). Some of the mechanisms involved in the transcriptional regulation of the TGFß cytostatic gene responses have been recently elucidated. In all cases, except for Id2, an activated Smad complex bound to a DNA-binding cofactor lands on the gene promoter and regulates transcription. In response to TGFß, phosphorylated Smad3, Smad4 and the transcription factor FoxO interact to form a nuclear complex that binds to a specific region of the p21Cip1 promoter and activates transcription (38). In addition, activated Smads can interact and synergize with Sp1 trancription factors bound to the proximal region of the p21Cip1 promoter (39). Transcription of the p15Ink4b gene is also induced by a Smad complex that binds to a discrete region of the upstream promoter. Some results indicate that a DNA-binding cofactor facilitates the binding of the Smad complex to the p15Ink4b promoter but its identity is still unknown (40). Sp1 transcription factors are also involved in TGFß-mediated induction of p15Ink4b (41). Myc downregulation by TGFß is mediated by an E2F4/5-Smad3–Smad4 complex. This complex inhibits Myc expression by recruiting p107, a pocket protein that acts as a transcriptional repressor. This is a very rapid and cell cycle independent process (42). Id proteins are negative regulators of basic helix–loop–helix transcription factors that are implicated in differentiation. Moreover, Id factors interact with the pocket protein Rb inducing cell proliferation (43). Therefore, by downregulating Id proteins, TGFß promotes differentiation and represses proliferation. An ATF3–Smad complex is responsible for Id1 downregulation. ATF3 is a transcriptional repressor that is previously induced by TGFß via a Smad-containing complex. Thus, a two-step mechanism is needed for Id1 repression that becomes a so-called ‘self-enabled’ gene response where the Id1 gene repression is mediated by a Smad complex that depends on a prior Smad-mediated response (44). Id2 is the only gene of the cytostatic program that is not directly regulated by Smads. Myc, in cooperation with Max, transcriptionally activates Id2 through an E-box found in the Id2 promoter (45). Hence, downregulation of Myc by TGFß prevents Id2 transcriptional activation. Furthermore, TGFß in certain cell types is able to induce Mad expression. Mad is a competitor of Myc for Max forming a Mad–Max complex that binds to the E-box of the Id2 promoter and represses Id2 expression (46) (Figure 2).


Figure 2
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  		Fig. 2 The TGFß cytostatic program in epithelial cells. TGFß regulates the expression of several cytostatic genes in order to induce an arrest in the G1 phase of the cell cycle. TGFß induces two Cdk inhibitors, p15Ink4b and p21Cip1, and downregulates the expression of Myc, Id1 and Id2. These five gene responses are considered part of the epithelial TGFß cytostatic program. TGFß induces p21Cip1 via a Smad–FoxO complex that interacts with a specific region of the p21Cip1 promoter. p15Ink4b is induced by a Smad complex that contains a still unknown DNA-binding cofactor. Transcription of Myc and Id1 is repressed by a Smad–E2F4/5-p107 complex and a Smad–ATF3 complex, respectively. Id2 is not regulated directly by Smads. Myc is an inducer of Id2 transcription and TGFß-mediated downregulation of Myc protein levels prevents Id2 transactivation by Myc.

 
Besides the Smad-dependent gene responses involved in the cytostatic program, it has been described that TGFß can promote cell cycle arrest through a Smad-independent pathway. The binding of the TGFß receptor complex to the regulatory subunit of PP2A facilitates the dephosphorylation and inhibition of p70 S6 kinase and contributes to the TGFß anti-proliferative response (23), as mentioned above.


   Evading the TGFß anti-proliferative effect
Top
 Abstract
 Introduction
 TGFß signal...
 Dual role of TGFß...
 Regulation of cell proliferation...
 Evading the TGFß anti...
Conclusion
 References
 
The TGFß cytostatic program is often lost in cancer. In certain cases, this is due to the disruption of TGFß signaling caused by somatic mutations in components of the TGFß pathway. The TßRII receptor is inactivated by mutations in gastrointestinal cancers with microsatellite instability. Many sporadic cancers acquire microsatellite instability as a result of defects in DNA mismatch repair leading to nucleotide additions or deletions in simple repeated sequences called microsatellites. The TßRII contains a 10 bp polyadenine repeat in the extracellular domain, and in most sporadic colon and gastric cancers with microsatellite instability the polyadenine repeat acquire base additions or deletions generating frameshift mutations that yield a truncated and inactive TßRII product. Individuals with hereditary non-polyposis colon cancer (HNPCC), a familial syndrome characterized by a high incidence of colon, endometrial and gastric cancers, also present mutations in the 10 bp polyadenine stretch. In most cases, both TßRII alleles have mutations in the polyadenine repeat although, in some cases, the second allele is inactivated by a different mutation in the kinase domain. This indicates that TßRII shares the two-hit inactivation mechanism found in other tumor suppressor genes. Interestingly, mutations in the kinase domain of TßRII have also been found in microsatellite stable colon cancers. Furthermore, other inactivating mutations elsewhere in TßRII have been described in T-cell lymphoma, and head and neck carcinomas. With a lower incidence, mutations in the TßRI sequence have been described in ovarian, breast and pancreatic cancers as well as some T-cell lymphomas (1,7,47).

Inactivating mutations in Smad2 and 4 are frequently found in some cancers. Missense, nonsense, and frameshift mutations, as well as small or large deletions are found in both Smads. Most of the missense mutations are found in the Smad MH2 domain and preclude the formation of the Smad2/3–Smad4 heterocomplex (7,48). Tumor derived missense mutations have been also found in the MH1 domain of Smad2 and 4. These mutations yield an increase in the affinity of the MH1 domain towards the MH2 domain locking the molecule in an inactive conformation (49). Smad2 mutations are found in colorectal and lung cancers although with very low frequency. Interestingly, Smad3 has not been found mutated in human cancer to date; however, lack of Smad3 protein expression has been described in acute lymphocytic leukemia (1,7,50).

Smad4, also called deleted in pancreatic carcinoma locus 4 (DPC4), was identified as a candidate tumor suppressor gene in chromosome 18q21 that is deleted or mutated in ~50% of pancreatic carcinomas. Besides pancreatic cancer, mutations in Smad4 have been identified in ~10% of all colon cancers (1,7). Interestingly, in pancreatic cancer there is a high incidence of Smad4 mutations whereas TßRII mutations are predominantly found in gastrointestinal tumors. This suggests that the selective advantage of a given mutation in a specific component of the TGFß pathway depends on the tumor type. The high preponderance of Smad4 mutations in pancreatic cancer suggests that in pancreatic tumor cells there is a selective advantage for specifically inactivating Smad4 and acquiring Smad4-independent responses. In pancreatic cancer cells, Smad2 and 3 can translocate into the nucleus and regulate transcription in response to TGFß even in the absence of Smad4 (51). In addition, knockdown of Smad4 in keratinocyte and pancreatic cell lines has shown that the TGFß cytostatic response might be dependent on Smad4 and that by inactivating Smad4, tumor cells evade the TGFß anti-proliferative response (52). On the other hand, Smad4-independent responses might facilitate tumor progression through the induction of EMT (52). These results indicate that mutations in Smad4 might switch the TGFß response into an oncogenic one. Nevertheless, two reports have shown that knockdown of Smad4 did not or just partially silenced the TGFß anti-proliferative effect (53,54). The difference between these results could be due to the different levels of Smad4 knockdown and/or the different cell lines used in the studies. Altogether, this suggests that the role of Smad4 in the TGFß-mediated cell cycle arrest might depend on the tumor type and most likely on the oncogenic insults acquired by the tumor cells.

The involvement of the TGFß–Smad pathway in tumor progression has been a subject of debate. Smad-independent pathways were implicated in TGFß oncogenic responses, whereas Smad-dependent pathways were considered to be tumor-suppressive pathways. The fact that the Smad-independent induction of PP2A leads to cell cycle arrest (23) and the following two reports argue against these claims. Two independent laboratories have shown that Smad4 is required for breast cancer bone metastasis (55,56). Smad4 knockdown in a breast cancer cell line that has already lost the TGFß anti-proliferative response inhibited the frequency of bone metastasis in nude mice indicating that, in breast cancer, Smad4-dependent responses are involved in TGFß-induced tumor progression and metastasis (55,56). Interestingly, Smad4 mutations are not found in breast cancer.

Many tumors with an intact, non-mutated TGFß pathway lack the TGFß anti-proliferative response. In those tumors, instead of inhibiting proliferation, TGFß can become an oncogenic factor. Work over recent years has begun to uncover several mechanisms involved in the inhibition of the TGFß anti-proliferative pathway in tumors with no mutations in the TGFß receptors or Smads.

High levels of Myc repress the induction of p15Ink4b and p21Cip1 by TGFß (57,58). Miz-1, a zinc finger protein with a POZ domain, binds specifically to a region close to the transcriptional initiator of the p15Ink4b and p21Cip1 genes and recruits Myc to both promoters (40,59–61). The Myc–Miz-1 complex acts as a transcriptional repressor complex and prevents the induction of p15Ink4b by TGFß (40), as well as the induction of p21Cip1 by TGFß (60) and by other inducers such as p53 (60–63). Recently, the mechanism of Myc–Miz-1 transcriptional repression has been elucidated. The Myc–Miz-1 complex recruits the DNA methyltransferase, Dnmt3a, to the p21Cip1 promoter facilitating the methylation and silencing of the gene (64). Together, these results indicate that, in order to have full induction of p21Cip1 and p15Ink4b, Myc has to be previously downregulated by TGFß. Hence, tumors with Myc amplification or overexpression might specifically lose the TGFß anti-proliferative response keeping the rest of the TGFß responses intact.

The induction of p21Cip1 by TGFß is mediated by a Smad complex that contains the transcription factor FoxO (38). The transcriptional activity of FoxO factors is regulated by the PI3K–Akt pathway. Akt phosphorylates FoxO in three serine/threonine residues promoting FoxO translocation into the cytosol (65). Thus, FoxO factors are excluded from the nucleus in tumor cells with high levels of Akt activity. In these cells, a nuclear Smad–FoxO complex cannot be formed and, thus, the induction of p21Cip1 by TGFß and, consequently, the TGFß anti-proliferative response are lost (38). In addition, it has been reported that Akt can interact directly with Smad3 in hepatoma cells preventing its phosphorylation and nuclear translocation and inhibiting TGFß-mediated apoptosis (33,34). A hyperactive Akt pathway might also block the tumor-suppressive effect of TGFß by directly inhibiting Smad3 function.

The Ras/MAPK pathway can impair TGFß signaling through the phosphorylation of the linker region of Smad2. Phosphorylation at several sites of the linker region results in nuclear exclusion of Smad2 inhibiting TGFß signaling and possibly conferring resistance to TGFß-mediated cell cycle arrest in cancer cells harboring a hyperactive Ras/MAPK pathway (66,67). In addition, in a canine kidney-derived epithelial cell line hyperactivation of the Raf/MAPK pathway results in an increased autocrine production of TGFß and a downregulation of Smad3 levels leading to transdifferentiation of the cells into a mesenchymal phenotype (68). In this case, the Raf/MAPK pathway promotes the downregulation of Smad3 and contributes to the loss of the TGFß anti-proliferative function observed in the EMT.

Another way through which tumor cells with a hyperactive Ras/MAPK pathway selectively evade the TGFß anti-proliferative response is via the stabilization of TGIF. TGIF is a Smad co-repressor that is involved in the regulation of p15Ink4b induction by TGFß. Hyperactive Ras promotes the phosphorylation of TGIF preventing its ubiquitination and inducing TGIF protein stabilization. TGIF competes with p300, a transcriptional co-activator, for the binding to Smad. The Smad complex that binds to the p15Ink4b promoter in tumor cells with high Ras activity contains TGIF instead of p300 and acts as a repressor complex of p15Ink4b transcription (69).

Both high Akt and high Ras activity blunt the TGFß anti-proliferative response. Interestingly, TGFß is able to induce Akt and Ras in a Smad-independent manner in certain cellular contexts (11,16,17), as described above. Hence, it might be possible that the induction of Akt and Ras activity by TGFß could selectively block TGFß-induced cell cycle arrest and apoptosis facilitating the switch of the TGFß effect towards oncogenesis. Along those lines, it has been described that the balance between the induction of the Smad and Ras–MAPK pathways by TGFß defines the cellular response to TGFß and controls the induction of epithelial-to-mesenchymal transdifferentiation during cancer progression (11,16).

Recently, it has been reported that CDK2 and CDK4 phosphorylate the linker region of Smad3. This phosphorylation inhibits Smad3 transcriptional activity and, hence, its ability to induce p15Ink4b, downregulate c-Myc, and promote cell cycle arrest. Cancer cells with high levels of CDK activity may have an impaired response to TGFß and evade to the TGFß-mediated inhibition of proliferation (70).

In glioma, the expression of the forkhead transcription factor, FoxG1, selectively prevents the induction of p21Cip1 by TGFß. FoxG1 is the cellular homologue of the viral oncogene Qin, and a transcriptional repressor that binds the Smad–FoxO complex on the p21Cip1 promoter repressing gene transcription. Consequently, the expression of FoxG1 in glioma allows tumor cells to escape from the p21Cip1-mediated cell cycle arrest induced by TGFß (38).

Another proto-oncogene, Evi-1, is a zinc finger protein that was shown to bind Smad3 preventing its transcriptional activity and, hence, inhibiting Smad3-mediated cell cycle arrest. The transcriptional co-repressor CtBP was shown to interact with Evi-1 and be required for the loss of the anti-proliferative response to TGFß. Evi-1 is implicated in the leukemic transformation of hematopoietic cells and it is a component of the AML/Evi-1 fusion gene generated by 3;21 translocation. The fusion protein contains the entire Evi-1 protein raising the possibility that the overexpression of Evi-1 as part of the fusion inhibits the TGFß cytostatic program. Another gene translocation implicated in TGFß signaling is the EWS–Fli gene translocation found in Ewing Sarcoma. EWS–Fli represses expression of TßRII and may account for decreased TGFß responsiveness and prevent the TGFß tumor-suppressive function observed in Ewing sarcoma.

Smad7 has been implicated in the repression of the anti-proliferative function of TGFß. Smad7 is an inhibitory Smad that is induced by TGFß and represses TGFß signaling, generating a negative feedback loop (9). Aberrant expression of Smad7 and disruption of the feedback regulation results in an inactivation of the TGFß–Smad pathway and impairs TGFß-mediated inhibition of proliferation (71). Interestingly, a transgenic mouse model overexpressing Smad7 in the pancreas developed pre-malignant ductal lesions (72). Furthermore, Smad7 has been found to be upregulated in pancreatic, endometrial and colon cancers (73–75), suggesting that these tumors evade the TGFß antitumorigenic response through the acquisition of abnormally high levels of Smad7. The Stat and the NF{kappa}B pathways can induce Smad7 expression. Induction of Smad7 by the IFN-{gamma}–Stat pathway blocks the TGFß–Smad pathway (76) and recently it has been described that the hyperactivation of Stat3 in mice promotes gastric adenomas due in part to the inhibition of the TGFß anti-proliferative response (77). NF{kappa}B in turn is also able to activate Smad7 gene transcription (78). Hence, Smad7 is a pivotal element in the cross-talk between the TGFß pathways, and the Stat and NF{kappa}B pathways. This suggests that tumor cells with high activity of the Stat and NF{kappa}B pathways might have high expression of Smad7 repressing TGFß signaling.

Ski and SnoN are two negative regulators of the TGFß signaling. They are SAND domain-containing transcriptional co-repressors that when overexpressed block TGFß-mediated cell cycle arrest. Ski is the cellular homologue of an avian viral oncogene and its transforming activity is dependent on the ability to inhibit the TGFß anti-proliferative response (79,80). Ski and SnoN can interact with Smad2, 3 and 4 and are recruited to the Smad-binding element of TGFß-responsive promoters (79). Moreover, Ski and SnoN are able to recruit the co-repressor complex N-Cor/Sin3/HDAC to the Smad complex and repress the ability of the Smads to activate their TGFß target genes (79). Binding to Ski or SnoN interferes with the interaction of Smad4 and the phosphorylated Smads and disrupts the active RSmad–Smad4 heteromeric complex (79). Ski is highly expressed in primary invasive melanoma and displays both nuclear and cytoplasmic localization. In the cytoplasm, Ski binds to Smad3 preventing its nuclear localization and transcriptional function. Among other TGFß responses, Ski prevents the induction of p21Cip1 by TGFß. Moreover, Ski can directly associate with retinoblastoma (Rb) and repress its activity (80,81). Thus, Ski might be preventing TGFß-mediated cell cycle arrest via two ways: repressing p21Cip1 induction and, on the other hand, directly inhibiting Rb function (80). Melanomas, breast cancers, and carcinomas of the esophagus as well as many human tumor cell lines express high levels of Ski and/or SnoN (82–84) indicating that some human tumors escape from the TGFß tumor-suppressive function due to Ski and SnoN overexpression.

Very recently, the catalytic subunit of Tert (telomerase reverse transcriptase) has been involved in the TGFß pathway. Tert overexpression confers a growth advantage to primary murine embryonic fibroblasts in part due to the abrogation of the anti-proliferative effect of TGFß through a still unknown mechanism (85). Moreover, Tert overexpression in p16 null mammary epithelial cells blocks the TGFß anti-proliferative response (86). Thus, high Tert activity might also contribute to the loss of the TGFß cytostatic program in tumors.


   Conclusion
Top
 Abstract
 Introduction
 TGFß signal...
 Dual role of TGFß...
 Regulation of cell proliferation...
 Evading the TGFß anti...
 Conclusion
References
 
Work over the last years has considerably increased our knowledge about the molecular mechanisms implicated in the TGFß signal transduction pathway. At a first glance, the TGFß pathway seems quite linear, but it is turning to be complex, finely regulated and integrated with other signaling pathways. Owing to its anti-proliferative and apoptotic effect on epithelial cells, TGFß is considered to be a tumor suppressor factor. During cancer progression, tumor cells evade TGFß action either by acquiring mutations in components of the TGFß pathway or by selectively inhibiting its anti-proliferative response. In the latter case, TGFß not only loses its anti-tumoral function but can also become an oncogenic factor inducing proliferation, angiogenesis, invasion, metastasis and immune suppression. To date, several mechanisms have been implicated in the disruption of the ability of TGFß to promote cell cycle arrest. Still many more have to be uncovered. Depending on the tumor type, cells might acquire some of these mechanisms in isolation or in combination to evade the tumor suppressive effect of TGFß (Figure 3). The mechanisms through which the cell escapes from the anti-proliferative effect of TGFß influence the switch of the TGFß function towards malignancy and determine the oncogenic outcome of the TGFß pathway.


Figure 3
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  		Fig. 3 Escaping from the TGFß anti-proliferative response. Tumors escape from the TGFß anti-proliferative response via two distinct mechanisms. Some tumor cells acquire inactivating mutations in several components of the TGFß pathway leading to the disruption of the TGFß signaling. Mutations in the TßRI, TßRII, Smad2 and Smad4 have been found in colon and pancreatic cancers as well as other tumor types. On the other hand, certain tumors evade the TGFß-mediated cell cycle arrest by selectively avoiding the pathway that leads to the induction of the TGFß cytostatic program. High levels of Myc, high activity of Akt or the Ras/MAPK pathway as well as stabilization of TGIF, expression of FoxG1, Ski/SnoN, Evi-1, high levels of Smad7 in some cases due to its induction by Stat and NFkB pathways, and high Tert expression through a still unknown mechanism prevent the ability of TGFß to inhibit proliferation.

 
Advances in the study of the molecular mechanisms that govern tumor progression are providing hopeful therapeutic benefits in cancer and have pushed rational molecular targeting to the cutting-edge of cancer therapy. Owing to its oncogenic role, the TGFß pathway is nowadays being evaluated as a potential therapeutic target (47,87,88). The dual and complex role of TGFß in oncogenesis presents a unique challenge that has to be addressed to be able to select the patient population that may benefit from an anti-TGFß therapy. The understanding of the exact mechanisms involved in the malignant transformation of TGFß will improve patient stratification and the development of successful therapeutic strategies as well as provide new therapeutic targets to restore the normal TGFß action.


   Acknowledgments
 
Work in the laboratory of J.S. is supported by the Spanish Ministry of Health grant FIS(PI040766), and the European Commission Marie Curie International Reintegration grant (MIRG-CT-2005-017121).

Conflict of Interest Statement: None declared.


   References
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 Dual role of TGFß...
 Regulation of cell proliferation...
 Evading the TGFß anti...
 Conclusion
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