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The oncoprotein c-Ski has been implicated in the negative regulation of transforming growth factor-β (TGF-β) signaling owing to its ability to repress Smad transcriptional activity via recruitment of a transcriptional corepressor complex containing histone deacetylases. However, c-Ski has also been shown to localize to the cytoplasm, raising the interesting possibility that it might disable TGF-β signaling through alternative mechanisms. Here, we provide evidence that c-Ski can restrict TGF-β signaling by interacting directly with the activated TGF-β type I receptor (TβRI). We explored the physiologic relevance of the c-Ski/TβRI interaction and found that it can culminate in a constitutive association of TβRI with a nonfunctional R-Smad/Smad4 complex. Based on these findings, we hypothesize that the interaction between c-Ski and TβRI might interfere with nuclear translocation of the R-Smad/Smad4 complex, thereby attenuating TGF-β signaling. Such a mechanism may play a crucial role in tumor progression, because many tumors that express high levels of c-Ski also display impaired nuclear accumulation of Smads. Cancer Res; 70(21); 8457–66. ©2010 AACR.

Supplementary Figures 1-2 from The Oncoprotein c-Ski Functions as a Direct Antagonist of the Transforming Growth Factor-β Type I Receptor

Pancreatic ductal adenocarcinoma formation culminates in hyperactivation of transforming growth factor β signaling, which in turn induces selective depletion of β-cell mass and thereby leading to new-onset diabetes., Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that remains incurable because of late diagnosis, which renders any therapeutic intervention challenging. Most PDAC patients develop de novo diabetes, which exacerbates their morbidity and mortality. How PDAC triggers diabetes is still unfolding. Using a mouse model of KrasG12D-driven PDAC, which faithfully recapitulates the progression of the human disease, we observed a massive and selective depletion of β-cells, occurring very early at the stages of preneoplastic lesions. Mechanistically, we found that increased TGF beta (TGF-β) signaling during PDAC progression caused erosion of β-cell mass through apoptosis. Suppressing TGF-β signaling, either pharmacologically through TGF-β immunoneutralization or genetically through deletion of Smad4 or TGF-β type II receptor (TβRII), afforded substantial protection against PDAC-driven β-cell depletion. From a translational perspective, both activation of TGF-β signaling and depletion of β-cells frequently occur in human PDAC, providing a mechanistic explanation for the pathogenesis of diabetes in PDAC patients, and further implicating new-onset diabetes as a potential early prognostic marker for PDAC.

A prominent function of TGIF1 is suppression of transforming growth factor beta (TGF‐β) signaling, whose inactivation is deemed instrumental to the progression of pancreatic ductal adenocarcinoma (PDAC), as exemplified by the frequent loss of the tumor suppressor gene SMAD4 in this malignancy. Surprisingly, we found that genetic inactivation of Tgif1 in the context of oncogenic Kras, Kras(G12D), culminated in the development of highly aggressive and metastatic PDAC despite de‐repressing TGF‐β signaling. Mechanistic experiments show that TGIF1 associates with Twist1 and inhibits Twist1 expression and activity, and this function is suppressed in the vast majority of human PDACs by Kras(G12D)/MAPK‐mediated TGIF1 phosphorylation. Ablating Twist1 in Kras (G12D) ;Tgif1 (KO) mice completely blunted PDAC formation, providing the proof‐of‐principle that TGIF1 restrains Kras(G12D)‐driven PDAC through its ability to antagonize Twist1. Collectively, these findings pinpoint TGIF1 as a potential tumor suppressor in PDAC and further suggest that sustained activation of TGF‐β signaling might act to accelerate PDAC progression rather than to suppress its initiation.

RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-β (TGF-β) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-β signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-β signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-β. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-β pathway activation., Graphical abstract, Highlights • Identification of the endogenous RNF111 ubiquitylated substrates. • A new powerful method to profile protein ubiquitylation using pan UB nanobody. • Lysine K343 as a target for RNF111 ubiquitylation. • SKI and SKIL are the only identified RNF111 degradative targets in TGF-β signaling., In Brief In this study, we aimed to identify exhaustively the substrates of the E3 ubiquitin ligase RNF111 that activates TGF-β signaling. We performed quantitative ubiquitylome comparison of parental U2OS cells with CRISPR modified clones that express a truncated RNF111 devoid of RING domain using two approaches based on the enrichment of ubiquitylated proteins. Integrative proteomics comparison of ubiquitylome and proteome identifies SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 upon TGF-β stimulation.

Although E3 ubiquitin ligases are deemed to play key roles in normal cell function and homeostasis, whether their alterations contribute to cancer pathogenesis remains unclear. In this study, we sought to investigate potential mechanisms that govern WWP1/Tiul1 (WWP1) ubiquitin ligase activity, focusing on its ability to trigger degradation of TGFβ type I receptor (TβRI) in conjunction with Smad7. Our data reveal that the WWP1 protein is very stable at steady states because its autopolyubiquitination activity is silenced due to an intra-interaction between the C2 and/or WW and Hect domains that favors WWP1 monoubiquitination at the expense of its polyubiquitination or polyubiquitination of TβRI. Upon binding of WWP1 to Smad7, this functional interplay is disabled, switching its monoubiquitination activity toward a polyubiquitination activity, thereby driving its own degradation and that of TβRI as well. Intriguingly, a WWP1 point mutation found in human prostate cancer disrupts this regulatory mechanism by relieving the inhibitory effects of C2 and WW on Hect and thereby causing WWP1 hyperactivation. That cancer-driven alteration of WWP1 culminates in excessive TβRI degradation and attenuated TGFβ cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1.

SummaryMany types of human cancers having hyperactivated Wnt signaling display no causative alterations in known effectors of this pathway. Here, we report a function of TGIF in Wnt signaling. TGIF associates with and diverts Axin1 and Axin2 from the β-catenin destruction complex, therefore allowing β-catenin accrual. Intriguingly, activation of Wnt signaling induces the expression of TGIF, which unveils a feed-forward loop that ensures effective integration of Wnt signaling. In triple-negative breast cancers (TNBC), elevated levels of TGIF correlate with high Wnt signaling and poor survival of patients. Moreover, genetic experiments revealed that Tgif1 ablation impeded mammary tumor development in MMTV-Wnt1 mice, further underscoring a requirement of TGIF for oncogenic Wnt signaling.

SummaryPHRF1 functions as an essential component of the TGF-β tumor suppressor pathway by triggering degradation of the homeodomain repressor factor TGIF. This leads to redistribution of cPML into the cytoplasm, where it coordinates phosphorylation and activation of Smad2 by the TGF-β receptor. In acute promyelocytic leukemia (APL), acquisition of PML-RARα is known to impede critical aspects of TGF-β signaling, including myeloid differentiation. Although these defects are thought to rely on suppression of cPML activity, the mechanisms underlying this phenomenon remain enigmatic. Here, we find that an abnormal function of PML-RARα is to interfere with TGIF breakdown, presumably by competing with PHRF1 for binding to TGIF, culminating in cPML sequestration and inactivation. Enforcing PHRF1 activity is sufficient to restore TGF-β cytostatic signaling in human blasts and suppress APL formation in a mouse model of APL, providing proof-of-concept data that suppression of PHRF1 activity by PML-RARα represents a critical determinant in APL pathogenesis.

Cystic fibrosis (CF) lung disease severity is highly variable and dependent on several factors including genetic modifiers. Family with sequence similarity 13 member A (FAM13A) has been previously associated with lung function in the general population as well as in several chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), we examined whether FAM13A is a modifier gene of CF lung phenotype. We also studied how FAM13A may contribute to the physiopathological mechanisms associated with CF.We investigated the association of FAM13A with lung function in CF French patients (n=1222) by SNP-wise analysis and Versatile Gene Based Association Study. We also analyzed the consequences of FAM13A knockdown in A549 cells and primary bronchial epithelial cells from CF patients.We found that FAM13A is associated with lung function in CF patients. Utilizing lung epithelial A549 cells and primary human bronchial epithelial cells from CF patients we observed that IL-1β and TGFβ reduced FAM13A expression. Knockdown of FAM13A was associated with increased RhoA activity, induction of F-actin stress fibers and regulation of epithelial-mesenchymal transition markers such as E-cadherin, α-smooth muscle actin and vimentin.Our data show that FAM13A is a modifier gene of CF lung phenotype regulating RhoA activity, actin cytoskeleton dynamics and epithelial-mesenchymal transition.

Summary The homeodomain protein TGIF (TG-interacting factor) restricts TGF-β/Smad cytostatic signaling by interfering with the nucleocytoplasmic transit of the tumor suppressor cPML. Here, we identify PHRF1 as a ubiquitin ligase that enforces TGIF decay by driving its ubiquitination at lysine 130. In so doing, PHRF1 ensures redistribution of cPML into the cytoplasm, where it associates with SARA and coordinates activation of Smad2 by the TGF-β receptor. The PHRF1 gene resides within the tumor suppressor locus 11p15.5, which displays frequent loss in a wide variety of malignancies, including breast cancer. Remarkably, we found that the PHRF1 gene is deleted or silenced in a high proportion of human breast cancer samples and cancer cell lines. Reconstitution of PHRF1 into deficient cells impeded their propensity to form tumors in vivo, most likely because of the reemergence of TGF-β responsiveness. These findings unveil a paradigm behind inactivation of the cPML tumor suppressor network in human malignancies.

Fatty pancreas or pancreatic steatosis has been associated with a variety of debilitating diseases, such as obesity, diabetes, pancreatitis, and pancreatic ductal adenocarcinoma (PDAC). Currently, neither the mechanisms underlying the pathogenesis of fatty pancreas nor the physiopathologic impacts of this disorder are well established. Serendipitously, we found that conditional overexpression of Twist1 in pancreatic progenitor cells in mice resulted in fatty pancreas development, leading to pancreatic insufficiency, severe hyperglycemia, and growth retardation. Molecular and genetic studies demonstrated that Twist1 drives fatty pancreas formation by selectively reprogramming acinar cells towards the adipocyte lineage albeit without affecting islet or ductal cell fate. Moreover, loss-of-function genetic experiments demonstrated that Twist1 plays a causative role in glucose tolerance, pancreatitis genesis, and PDAC progression. Finally, high levels of Twist1 expression correlate with fatty pancreas formation in patients with pancreatitis or PDAC, implicating Twist1 as a prognostic marker and possible target for attenuating several lethal diseases associated with fatty pancreas development. Citation Format: Thien Ly Nguyen, Purba Singh, Parash Parajuli, Lianna Li, Celine Prunier, Santosh Kumar, Sailaja Eragmerdi, Seval Ozkan, Hao Me, Jussara do Carmo, John Hall, Azeddine Atfi. Twist1-driven fatty pancreas formation facilitates pancreatitis and pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A38.

Cancer cachexia is characterized by extreme skeletal muscle loss that results in high morbidity and mortality. The incidence of cachexia varies among tumor types, being lowest in sarcomas, whereas 90% of pancreatic ductal adenocarcinoma (PDAC) patients experience severe weight loss. How these tumors trigger muscle depletion is still unfolding. Serendipitously, we found that overexpression of Twist1 in mouse muscle progenitor cells, either constitutively during development or inducibly in adult animals, caused severe muscle atrophy with features reminiscent of cachexia. Using several genetic mouse models of PDAC, we detected a massive increase in Twist1 expression in muscle undergoing cachexia. In cancer patients, elevated levels of Twist1 are associated with greater degrees of muscle wasting. Both genetic and pharmacologic inactivation of Twist1 in muscle progenitor cells afforded substantial protection against cancer-mediated cachexia, which translated into meaningful survival benefits, implicating Twist1 as a possible target for attenuating muscle cachexia in cancer patients. Citation Format: Parash Parajuli, Santosh Kumar, Audrey Loumaye, Purba Singh, Sailaja Eragamreddy, Thien Ly Nguyen, Seval Ozkan, Mohammed S. Razzaqe, Céline Prunier, Jean-Paul Thissen, Azeddine Atfi. Twist1 activation in muscle progenitor cells during development or adulthood causes severe muscle loss reminiscent of human cancer cachexia [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A40.

Pancreatic ductal adenocarcinoma (PDAC) is a disease that remains incurable due to diagnosis at advanced stages, which renders any therapeutic intervention impracticable. The vast majority of PDAC patients develop new-onset diabetes, which often culminates in dysfunction of vital organs, leading to severe morbidity and high mortality. How PDAC development leads to diabetes is still unfolding. More than 90% of PDAC cases have an oncogenic mutation that affects the proto-oncogene Kras, mostly G12D. Using several mouse models of KrasG12D-driven PDAC, which faithfully recapitulate the human disease, we observed a massive depletion of beta cell mass, occurring even at early stages of pancreatic intraepithelial neoplasia (PanIN). This phenomenon was selective for beta cells, as PanIN or PDAC formation did not affect any other type of islet cells, including alpha and delta cells, which produce Glucagon and Somatostatin, respectively. Intriguingly, both alpha and delta cells retained their localization at the periphery of islets that are devoid of beta cells, giving rise to PanIN-like structures. Such pattern was observed when KrasG12D expression was combined with genetic inactivation of other tumor suppressor genes frequently inactivated in human PDAC, including TP53 and p16Ink4a. Lastly, we also detected depletion of beta cells and presence of remnant islets resembling PanIN in human PDAC, attesting to the clinical relevance of our findings, and further rise the attractive question of whether restoration of beta cell mass could be utilized to curb PDAC driven by oncogenic Kras. Citation Format: Thienly Nguyen, Parash Parajuli, Asrar Ahmad, Purba Singh, Sailaja Eragameddy, Gopalakrishnan Ramakrishnan, Laurence Levy, Oliver Ferrigno, Yin-Yuan Mo, Radhika Pochampally, Mohammed S Razzaque, Celine Prunier, Azeddine Atfi. Restoration of beta cell mass as a synthetic lethal strategy to overcome oncogenic Kras-driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B32.

The homeodomain protein TGIF function as a potent inhibitor of transforming growth factor beta (TGF-β) cytostatic signaling, whose inactivation is deemed instrumental to the pathogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) driven by oncogenic Kras. Surprisingly, we found that deletion of Tgif in the context of mutant Kras (KrasG12D) expression culminated in the development of highly metastatic PDAC, despite leading to hyperactivation of TGF-(β/Smad) signaling. Mechanically, we found that TGIF associated with and antagonized Twist1 by a process involving Smad4, a tumor suppressor that is frequently inactivated in human PDAC. Along these lines, Twist1 overexpression synergized with KrasG12D to induce PDAC progression, whereas its ablation completely blocked the malignant phenotype driven by the combined KrasG12D expression and TGIF ablation. Thus, by demonstrating that TGIF functions as a tumor suppressor in PDAC owing to its ability to antagonize the pro-malignant transcription factor Twist1, our studies provide key insights into the role of Twist1 that could be exploited to design synthetic lethal therapeutics against oncogenic Kras, which has proven so far to be intractable. Citation Format: Parash Parajuli, Purba Singh, Zhe Wang, Santosh Kumar, Lena Li, Sailaja Eragamreddi, Thien Ly Nguyen, Subhi Talal Younes, Xu Zhang, Keli Xu, Mohammed Razzaque, Céline Prunier, Azeddine Atfi. Tgif inactivation defines a synthetic lethal interaction among oncogenic Kras and Twist1 in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B33.

The TGIF homoeodomain protein functions as an important negative regulator in the TGF-beta signalling pathway. The inhibitory function of TGIF is executed in part through its ability to sequester the tumour suppressor cytoplasmic promyelocytic leukaemia (cPML) in the nucleus, thereby preventing the phosphorylation of Smad2 by the activated TGF-beta type I receptor. Here, we report on the identification of PCTA (PML competitor for TGIF association), a TGIF antagonist that promotes TGF-beta-induced transcriptional and cytostatic responses. We provide evidence that PCTA functions in TGF-beta signalling by relieving the suppression of Smad2 phosphorylation by TGIF. Furthermore, we demonstrate that PCTA selectively competes with cPML for TGIF association, resulting in the accumulation of cPML in the cytoplasm, where it associates with SARA and coordinates the access of Smad2 for phosphorylation by the activated TGF-beta type I receptor. Thus, our findings on the mode of action of PCTA provide new and important insights into the molecular mechanism underlying the antagonistic interplay between TGIF and cPML in the TGF-beta signalling network.

International audience; Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological processes through two types of Ser/Thr transmembrane receptors: the TGF-beta type I receptor and the TGF-beta type II receptor (TbetaRII). Upon ligand binding, TGF-beta type I receptor activated by TbetaRII propagates signals to Smad proteins, which mediate the activation of TGF-beta target genes. In this study, we identify ADAM12 (a disintegrin and metalloproteinase 12) as a component of the TGF-beta signaling pathway that acts through association with TbetaRII. We found that ADAM12 functions by a mechanism independent of its protease activity to facilitate the activation of TGF-beta signaling, including the phosphorylation of Smad2, association of Smad2 with Smad4, and transcriptional activation. Furthermore, ADAM12 induces the accumulation of TbetaRII in early endosomal vesicles and stabilizes the TbetaRII protein presumably by suppressing the association of TbetaRII with Smad7. These results define ADAM12 as a new partner of TbetaRII that facilitates its trafficking to early endosomes in which activation of the Smad pathway is initiated.

The homeodomain protein TGIF functions as a negative modulator for multiple classes of transcription factors. Loss of function mutations in a single copy of TGIF result in holoprosencephaly, a developmental anomaly leading to severe forebrain and craniofacial malformations. However, the mechanisms by which these mutations disrupt the functions of TGIF remain to be elucidated. Here we show that a holoprosencephaly mutation (P63R) interferes with the ability of TGIF to act as a corepressor for c-Jun and Smad2, suggesting that this holoprosencephaly mutation may lead to a general defect in the TGIF protein. In fact, we observed that the P63R mutation affects folding of the TGIF protein, resulting in the disruption of the diffuse nuclear staining pattern characteristic of wild-type (WT) TGIF and the accumulation of TGIF in nuclear aggregates. We also show that the mutant TGIF.P63R is degraded more rapidly when compared with WT TGIF and that this degradation occurs through the ubiquitin-proteasome pathway. Furthermore, we observed that TGIF.P63R homodimerizes with WT TGIF to sequester it into nuclear aggregates and to enhance its ubiquitin-dependent degradation. These results reveal an important mechanism for the degradation of TGIF through the ubiquitin-proteasome pathway, whose deregulation might contribute to the development of human holoprosencephaly.

Summary The homeodomain protein TGIF has been implicated in the negative regulation of TGF-β signaling. In this study, we report an unexpected role of TGIF in the inhibition of Smad2 phosphorylation, which occurs by a mechanism independent of its association with Smad2. This inhibitory function of TGIF is executed in concert with c-Jun, which facilitates the interaction of TGIF with cPML, resulting in the nuclear sequestration of cPML and the disruption of the cPML-SARA complex. Notably, knockdown of TGIF by siRNA caused increased association of cPML with SARA and cytoplasmic accumulation of cPML. Furthermore, c-Jun −/− fibroblasts exhibit enhanced association of cPML with SARA. c-Jun −/− fibroblasts also lose their sensitivity to TGIF-mediated disruption of the cPML-SARA complex and of nuclear sequestration of cPML. We suggest that the interaction of TGIF with cPML through c-Jun may negatively regulate TGF-β signaling through controlling the localization of cPML and, consequently, the assembly of the cPML-SARA complex.

Transforming growth factor beta (TGFbeta) induces an epithelial to mesenchymal transition (EMT) during both physiological and pathological processes; however, the mechanism underlying this transition is not fully elucidated. Here, we have demonstrated that TGFbeta induces the expression of the adaptor molecule disabled-2 (Dab2) concomitant with the promotion of EMT. We show that TGFbeta induces a transient accumulation of Dab2 to the membrane and increases Dab2 binding to beta1 integrin. Furthermore, small interfering RNA (siRNA)-mediated silencing of Dab2 expression in mouse mammary gland epithelial cells results in inhibition of integrin activation, shown by a decrease of both TGFbeta-induced focal adhesion kinase phosphorylation and cellular adherence, leading to apoptosis and inhibition of EMT. Forced re-expression of human Dab2, not targeted by the mouse siRNA sequence, rescues cells from apoptosis and restores TGFbeta-mediated integrin activation and EMT. These results are confirmed in the F9 teratocarcinoma cell line, a model for retinoic acid-induced visceral endoderm differentiation in which we demonstrate that ablation of retinoic acid-induced Dab2 expression levels, by stable siRNA silencing of Dab2, blocks visceral endoderm differentiation. Our findings indicate that Dab2 plays an important regulatory role during cellular differentiation and that induction of differentiation in the absence of Dab2 expression commits the cell to apoptosis.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell proliferation and the loss of responsiveness to TGF-beta may contribute to the development of human cancers. In hepatocellular carcinomas, the potential role of TGF-beta signaling as a tumor suppressor pathway can be illustrated by the presence of mutations in genes encoding TGF-beta receptors or downstream components of this signaling such as Smad2. Although Smad2 is mutated in hepatocellular carcinomas, the alteration of TGF-beta signaling with respect to tumor progression remains to be established. Using the HepG2 hepatoma cells, we showed here that expression of Smad2.Q407R, a missense mutation found in human hepatocellular carcinoma, was less effective than expression of wild-type Smad2 in enhancing the ability of TGF-beta to induce transcription from the Mix.2 promoter. This effect was specifically associated with a decrease in the steady-state level of Smad2.Q407R, presumably because of an enhancement of its ubiquitination and degradation through the proteasome machinery. More importantly, we found that the unstability of Smad2.Q407R was reversed when this mutant undergoes homo-oligomerization with wild-type Smad2 or hetero-oligomerization with Smad3 within the cells. Therefore, our findings allowed us to propose a novel mechanism for suppression of the deleterious effect of a tumor-derived mutation of Smad2, which loss may lead to dysregulated cell proliferation during tumorigenesis.

The Smad proteins are key intracellular effectors of transforming growth factor-beta (TGF-beta) cytokines. The ability of Smads to modulate transcription results from a functional cooperativity with the coactivators p300/cAMP-response element-binding protein-binding protein (CBP), or the corepressors TGIF and Ski. The c-Jun N-terminal kinase (JNK) pathway, another downstream target activated by TGF-beta receptors, has also been suggested to inhibit TGF-beta signaling through interaction of c-Jun with Smad2 and Smad3. Here we show that c-Jun directly interacts with Ski to enhance the association of Ski with Smad2 in the basal state. Interestingly, TGF-beta signaling induces dissociation of c-Jun from Ski, thereby relieving active repression by c-Jun. Moreover, activation of JNK pathway suppressed the ability of TGF-beta to induce dissociation of c-Jun from ski. Thus, the formation of a c-Jun/Ski complex maintains the repressed state of Smad2-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-beta signaling by the JNK cascade.

The Sma and Mad related (Smad) family proteins are critical mediators of the transforming growth factor-β (TGF-β) superfamily signaling. After TGF-β-mediated phosphorylation and association with Smad4, Smad2 moves to the nucleus and activates expression of specific genes through cooperative interactions with DNA-binding proteins, including members of the winged-helix family of transcription factors, forkhead activin signal transducer (FAST)-1 and FAST2. TGF-β has also been described to activate other signaling pathways, such as the c-Jun N-terminal Kinase (JNK) pathway. Here, we show that activation of JNK cascade blocked the ability of Smad2 to mediate TGF-β-dependent activation of the FAST proteins. This inhibitory activity is mediated through the transcriptional factor c-Jun, which enhances the association of Smad2 with the nuclear transcriptional corepressor TG-interacting factor (TGIF), thereby interfering with the assembly of Smad2 and the coactivator p300 in response to TGF-β signaling. Interestingly, c-Jun directly binds to the nuclear transcriptional corepressor TGIF and is required for TGIF-mediated repression of Smad2 transcriptional activity. These studies thus reveal a mechanism for suppression of Smad2 signaling pathway by JNK cascade through transcriptional repression.

Transforming growth factor beta (TGF-beta) is a potent natural antiproliferative agent that plays an important role in suppressing tumorigenicity. In numerous tumors, loss of TGF-beta responsiveness is associated with inactivating mutations that can occur in components of this signaling pathway, such as the tumor suppressor Smad2. Although a general framework for how Smads transduce TGF-beta signals has been proposed, the physiological relevance of alterations of Smad2 functions in promoting tumorigenesis is still unknown. Here, we show that expression of Smad2.P445H, a tumor-derived mutation of Smad2 found in human cancer, suppresses the ability of the Smads to mediate TGF-beta-induced growth arrest and transcriptional responses. Smad2.P445H is phosphorylated by the activated TGF-beta receptor at the carboxy-terminal serine residues and associates with Smad3 and Smad4 but is unable to dissociate from the receptor. Upon ligand-induced phosphorylation, Smad2.P445H interacts stably with wild-type Smad2, thereby blocking TGF-beta-induced nuclear accumulation of wild-type Smad2 and Smad2-dependent transcription. The ability of the Smad2.P445H to block the nuclear accumulation of wild-type Smad2 protein reveals a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

In this study, we examined the effect of the stable expression of Smad7 in two different cell lines on apoptosis induced by various stimuli including TGF-beta, serum withdrawal, loss of cell adhesion (anoikis) and TNF-alpha. Smad7 increased TGF-beta-mediated apoptosis in Mv1Lu cells as well as anoikis and/or serum withdrawal-induced apoptosis in Mv1Lu and MDCK cells. Smad7 markedly decreased the activity of the survival NF-kappaB transcription factor in MDCK cells. Interestingly, the stable expression of oncogenic Ras in MDCK cells which suppressed Smad7 inhibition of NF-kappaB also suppressed Smad7 potentiation of serum withdrawal-induced apoptosis and anoikis. In addition, Smad7 inhibited TNF-alpha stimulation of NF-kappaB and increased TNF-alpha-mediated apoptosis in MDCK cells. Our results provide the first evidence that Smad7 induces sensitization of cells to different forms of cell death. They moreover demonstrate that Smad7 inhibits the survival NF-kappaB factor, providing a potential mechanism whereby Smad7 potentiates cell death.

The transforming growth factor beta (TGF-beta) plays an important role in constraining cellular proliferation, but it is also a potent inducer of programmed cell death or apoptosis. Here, we demonstrate that TGF-beta can have an opposite effect, acting as a survival factor to prevent c-Myc-induced cell death in Rat-1 fibroblasts. However, in marked contrast to TGF-beta, Smad2, which is a critical intracellular mediator of the TGF-beta signaling pathway, functions as an antagonist to induce increased cell death. The protective activity of TGF-beta was associated with the activation of c-Jun N-terminal Kinase (JNK) and was not linked to the ability of TGF-beta to promote cell cycle progression. Expression of dominant-interfering forms of various components of the JNK signaling pathway, including Rac1, Cdc42, mitogen-activated protein kinase kinase 4 (MKK4), and c-Jun, abolished TGF-beta-mediated cell survival. Furthermore, overexpression of the constitutively activated mutant RacL61F37A, which selectively stimulates JNK cascade but not G1 cell cycle progression or actin polymerization, was sufficient to prevent apoptosis induced by c-Myc. These findings describe a differential effect of two separated signaling pathways of TGF-beta and indicate for the first time that Smad2 can act as antagonist to suppress TGF-beta-dependent cell survival. Oncogene (2000) 19, 1277 - 1287.

We recently showed that the antiapoptotic function of insulin requires nuclear factor kappaB (NF-kappaB) activation (Bertrand, F., Atfi, A., Cadoret, A., L'Allemain, G., Robin, H., Lascols, O., Capeau, J., and Cherqui, G. (1998) J. Biol. Chem. 273, 2931-2938). Here we sought to identify the NF-kappaB-dependent survival genes that are activated by insulin to mediate this function. Insulin increased the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2) mRNA and protein in Chinese hamster ovary cells overexpressing insulin receptors (IRs). This effect required (i) IR activation since it was abrogated by IR mutation at tyrosines 1162 and 1163 and (ii) NF-kappaB activation since it was abolished by overexpression of dominant-negative IkappaB-alpha(A32/36) and mimicked by overexpression of the NF-kappaB c-Rel subunit. TRAF2 contributed to insulin protection against serum withdrawal-induced apoptosis since TRAF2 overexpression mimicked insulin protection, whereas overexpression of dominant-negative TRAF2-(87-501) reduced this process. Along with its protective effect, overexpressed TRAF2 increased basal and insulin-stimulated NF-kappaB activities. All effects were inhibited by IkappaB-alpha(A32/36), suggesting that an amplification loop involving TRAF2 activation of NF-kappaB is implicated in insulin antiapoptotic signaling. We also show that insulin increased manganese-superoxide dismutase (Mn-SOD) mRNA expression through NF-kappaB activation and that Mn-SOD contributed to insulin antiapoptotic signaling since expression of antisense Mn-SOD RNA decreased this process. This study provides the first evidence that insulin activates the NF-kappaB-dependent survival genes encoding TRAF2 and Mn-SOD and thereby clarifies the role of NF-kappaB in the antiapoptotic function of insulin.

CCN5 is a member of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family and was identified as an estrogen-inducible gene in estrogen receptor-positive cell lines. However, the role of CCN5 in breast carcinogenesis remains unclear. We report here that the CCN5 protein is localized mostly in the cytoplasm and in part in the nucleus of human tumor breast tissue. Using a heterologous transcription assay, we demonstrate that CCN5 can act as a transcriptional repressor presumably through association with histone deacetylase 1 (HDAC1). Microarray gene expression analysis showed that CCN5 represses expression of genes associated with epithelial-mesenchymal transition (EMT) as well as expression of key components of the transforming growth factor β (TGF-β) signaling pathway, prominent among them TGF-βRII receptor. We show that CCN5 is recruited to the TGF-βRII promoter, thereby providing a mechanism by which CCN5 restricts transcription of the TGF-βRII gene. Consistent with this finding, CCN5, we found, functions to suppress TGF-β-induced transcriptional responses and invasion that is concomitant with EMT. Thus, our data uncovered CCN5 as a novel transcriptional repressor that plays an important role in regulating tumor progression functioning, at least in part, by inhibiting the expression of genes involved in the TGF-β signaling cascade that is known to promote EMT.

The oncoprotein c-Ski has been implicated in the negative regulation of transforming growth factor-β (TGF-β) signaling owing to its ability to repress Smad transcriptional activity via recruitment of a transcriptional corepressor complex containing histone deacetylases. However, c-Ski has also been shown to localize to the cytoplasm, raising the interesting possibility that it might disable TGF-β signaling through alternative mechanisms. Here, we provide evidence that c-Ski can restrict TGF-β signaling by interacting directly with the activated TGF-β type I receptor (TβRI). We explored the physiologic relevance of the c-Ski/TβRI interaction and found that it can culminate in a constitutive association of TβRI with a nonfunctional R-Smad/Smad4 complex. Based on these findings, we hypothesize that the interaction between c-Ski and TβRI might interfere with nuclear translocation of the R-Smad/Smad4 complex, thereby attenuating TGF-β signaling. Such a mechanism may play a crucial role in tumor progression, because many tumors that express high levels of c-Ski also display impaired nuclear accumulation of Smads. Cancer Res; 70(21); 8457–66. ©2010 AACR.

The multifunctional cytokine transforming growth factor beta (TGFbeta) exerts many of its effects through its regulation of extracellular matrix components, including fibronectin (FN). Although expression of both TGFbeta and FN are essential for embryonic development and wound healing in the adult, overexpression leads to excessive deposition of extracellular matrix observed in many fibroproliferative disorders. We previously have demonstrated that TGFbeta-stimulated FN induction requires activation of the c-Jun N-terminal kinase (JNK) pathway; however, the signaling molecules that link the TGFbeta receptors to the JNK pathway remain unknown. We show here that the cytosolic adaptor protein disabled-2 (Dab2) directly stimulates JNK activity, whereas stable small interfering RNA-mediated ablation of Dab2 in NIH3T3 mouse fibroblasts and A10 rat aortic smooth muscle cells demonstrates that its expression is required for TGFbeta-mediated FN induction. We demonstrate that TGFbeta treatment stimulates the association of Dab2 with the mitogen-activated protein kinase kinase kinase, TAK1. Attenuation of cellular TAK1 levels by transient double-stranded RNA oligonucleotide transfection as well as overexpression of kinase-deficient TAK1 leads to abrogation of TGFbeta-stimulated FN induction. Furthermore, cell migration, another JNK-dependent response, is attenuated in NIH3T3-siDab2-expressing clones. We, therefore, delineate a signaling pathway proceeding from the TGFbeta receptors to Dab2 and TAK1, leading to TGFbeta-stimulated JNK activation, FN expression, and cell migration.

Signaling by the Wnt family of secreted glycoproteins has been demonstrated to be essential both in normal embryonic development and in the pathogenesis of a variety of diseases, including cancer. This signaling pathway is exquisitely regulated by a large and complex array of proteins, which act as agonists or antagonists of signal transduction, modulating the Wnt signal extracellularly, in the cytoplasm and in the nucleus. Here, we will briefly review the canonical Wnt signaling pathway and consider molecular defects in Wnt signaling components known to promote uncontrolled cell growth following induction of Wnt signaling. We will also focus on two recently identified factors in this pathway, that seem to act as Wnt signaling antagonists, one functioning in the cytoplasm called Disabled-2 and the other in the nucleus named Chibby.

The phosphorylation of Smad2 and Smad3 by the transforming growth factor (TGF)-beta-activated receptor kinases and their subsequent heterodimerization with Smad4 and translocation to the nucleus form the basis for a model how Smad proteins work to transmit TGF-beta signals. The transcriptional activity of Smad2-Smad4 or Smad3-Smad4 complexes can be limited by the corepressor Ski, which is believed to interact with Smad complexes on TGF-beta-responsive promoters and represses their ability to activate TGF-beta target genes by assembling on DNA a repressor complex containing histone deacetylase. Here we show that Ski can block TGF-beta signaling by interfering with the phosphorylation of Smad2 and Smad3 by the activated TGF-beta type I receptor. Furthermore, we demonstrate that overexpression of Ski induces the assembly of Smad2-Smad4 and Smad3-Smad4 complexes independent of TGF-beta signaling. The ability of Ski to engage Smad proteins in nonproductive complexes provides new insights into the molecular mechanism used by Ski for disabling TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) and insulin-like growth factors (IGFs) play critical roles in the control of myogenesis. Insulin-like growth factor-binding protein-5 (IGFBP-5), by regulating the bioavailability of IGFs, is involved in controlling IGF-dependent differentiation. We investigated the effects of TGF-beta on the IGFBP-5 production induced by IGFs in mouse myoblasts. TGF-beta leads to a decrease in IGFBP-5 synthesis at both transcript and protein levels, and blocked muscle differentiation. The Smad proteins and the c-Jun N-terminal kinase (JNK) have been shown to be involved in TGF-beta signaling pathways. We provide evidence that the JNK pathway, rather than Smad proteins, is involved in the response of muscle cells to TGF-beta. This factor failed to stimulate the GAL4-Smad 2/3 transcriptional activities of the constructs used to transfect myoblasts. Moreover, stable expression of the antagonistic Smad7 did not abolish the inhibitory effect of TGF-beta on IGFBP-5 production whereas expression of a dominant-negative version of MKK4, an upstream activator of JNK, did. We also showed, using a specific inhibitor, that the p38 mitogen-activated protein kinase (p38 MAPK) was not involved in the inhibition of IGFBP-5 production. Thus, TGF-beta-mediated IGFBP-5 inhibition is independent of Smads and requires activation of the JNK signaling pathway.

Smad proteins are central mediators of the transcriptional effects of transforming growth factor beta (TGF-beta) superfamily that regulate a wide variety of biological processes. Smad7, an inhibitory Smad protein that prevents TGF-beta signaling by interacting with the activated type I TGF-beta receptor, was recently shown to induce sensitization of cells to different forms of cell death. Here we examined the effect of Smad7 on the c-Jun N-terminal kinase (JNK) cascade and investigated the role of this cascade in both the inhibitory and apoptotic functions of Smad7. The transient and stable expression of Smad7 caused a strong and sustained activation of JNK. Expression of a dominant-interfering mutant of mitogen-activated protein kinase kinase 4, which completely abolished Smad7-induced activation of JNK, had no effect on Smad7-mediated inhibition of TGF-beta signaling, indicating that the inhibitory function of Smad7 is independent of the JNK cascade. In contrast, expression of the dominant-interfering mutant of mitogen-activated protein kinase kinase 4 impaired the ability of Smad7 to promote cell death. These experiments reveal a novel link between Smad7 and the JNK cascade, which is essential for potentiation of cell death by this inhibitory Smad.

The TEL/PDGFR beta (T/P) fusion protein isolated from patients bearing a t(5;12) translocation is transforming when expressed in haematopoietic cells. To examine the signal transduction events activated by this protein, we measured the effect of T/P on activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) in mouse bone marrow-derived Ba/F3 cells. Significant increase in the activity of JNK/SAPK1 was observed in transient transfection as well as in Ba/F3 cells stably expressing T/P. This activation was abrogated when the T/P-expressing cells were treated with a specific inhibitor of the PDGFR beta tyrosine kinase, indicating that the activity of the PDGFR beta part of the fusion protein was involved in JNK/SAPK activation. Expression of a dominant negative mutant of mitogen-activated protein kinase kinase 4 (MKK4), a direct activator of JNK/SAPK, prevented T/P-induced JNK/SAPK activation. In addition, inhibition of phosphoinositide-3 OH kinase (PI-3 kinase), a promoting survival factor, potentiated the effect of T/P on JNK/SAPK activation. Interestingly, expression of T/P was shown to initiate an apoptotic response that was enhanced by treatment of cells with the PI-3 kinase inhibitor LY294002, suggesting that T/P mediated cell death through activation of JNK/SAPK signalling pathway. Consistent with this hypothesis, expression of the dominant negative mutant of MKK4 decreased T/P-mediated apoptosis, while a dominant-negative mutant of PI-3 kinase enhances cell death. These findings indicate that activation of JNK/SAPK by T/P is related to apoptosis rather than cell proliferation and transformation.

International audience; The proper tissue-specific regulation of gene expression is essential for development and homeostasis in metazoans. However, the illegitimate expression of normally tissue-restricted genes-like testis- or placenta-specific genes-is frequently observed in tumors; this promotes transformation, but also allows immunotherapy. Two important questions are: how is the expression of these genes controlled in healthy cells? And how is this altered in cancer? To address these questions, we used an unbiased approach to test the ability of 350 distinct genetic or epigenetic perturbations to induce the illegitimate expression of over 40 tissue-restricted genes in primary human cells. We find that almost all of these genes are remarkably resistant to reactivation by a single alteration in signaling pathways or chromatin regulation. However, a few genes differ and are more readily activated; one is the placenta-expressed gene ADAM12, which promotes invasion. Using cellular systems, an animal model, and bioinformatics, we find that a non-canonical but druggable TGF-β/KAT2A/TAK1 axis controls ADAM12 induction in normal and cancer cells. More broadly, our data show that illegitimate gene expression in cancer is an heterogeneous phenomenon, with a few genes activatable by simple events, and most genes likely requiring a combination of events to become reactivated.

The TGF-β pathway plays a biphasic role during cancerogenesis. My laboratory identified a new protein, WWP1, as a negative regulator of TGF-β signaling. WWP1 is an E3 ubiquitin ligase that triggers polyubiquitination and degradation of TGF-β type I receptor. A genomic amplification of WWP1 is found in a large portion of mammary and prostatic tumors, suggesting a key role for WWP1 during carcinogenesis related to TGF-β. My thesis project was to determine the regulation of the catalytic activity of WWP1 and a new molecular mechanism of action of WWP1 whose deregulation can be implicated in cancerogenesis. My results indicate that at steady states, WWP1 is monoubiquitinated, its polyubiquitination activity being silenced due to the inhibitory effects of C2 or/and WW domains on its Hect domain. In presence of substrates, WWP1 is « opened » and induces polyubiquitination and degradation of its substrates. Moreover, a WWP1 mutation found in prostate cancer disrupts this regulatory mechanism. It possesses an increased ligase activity towards itself and its substrates, which leads to the attenuation of TGF-β cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1. We also identified STARD13 as a novel WWP1 interacting partner. STARD13 has a RhoGAP activity, and is considered as a tumor suppressor. We have shown that STARD13 mediates the association of WWP1 with the GTPase RhoA, ultimately leading to RhoA polyubiquitination and degradation. Interestingly, the WWP1/STARD13 complex is involved in the actin cytoskeleton rearrangement by preferentially targeting the active form of RhoA for degradation. These results reveal a previously unrecognized role for WWP1, which could play a key role in the migration of cancer cells during metastasis. Characterization of new regulation and action mechanisms for WWP1 should allow identifying whether WWP1 is a diagnosis biomarker in cancer and/or a new therapeutic target for the development of anticancer drugs.; La voie de signalisation TGF-β joue un rôle biphasique durant la cancérogenèse. Mon laboratoire a identifié une nouvelle protéine inhibitrice de la voie TGF-β, WWP1. WWP1 est une E3 ubiquitine ligase qui induit la polyubiquitination et la dégradation du récepteur de type I au TGF-β. De plus, le gène WWP1 est amplifié dans une large proportion de cancers mammaires et prostatiques, suggérant que WWP1 pourrait jouer un rôle clé dans les processus de cancérogenèse liés au TGF-β. Mon projet de thèse était donc de caractériser la régulation de l’activité catalytique de WWP1 ainsi que son mécanisme d’action dans la cellule. Mes résultats montrent qu’à l’état basal, WWP1 est monoubiquitinée, son activité de polyubiquitination étant réduite par l’effet inhibiteur qu’exercent les domaines C2 et/ou WW sur son domaine HECT. En présence de substrats, la protéine WWP1 « s’ouvre » et peut alors induire la polyubiquitination et la dégradation de ses substrats. De plus, nous avons observé qu’un mutant de WWP1, détecté dans un cancer de la prostate, est incapable de s’autoréguler selon ce modèle. Il présente une plus forte activité ligase envers lui-même et ses substrats, ce qui entraîne une atténuation de la réponse cytostatique du TGF-β pouvant conférer une activité oncogénique à WWP1. De plus, nous avons identifié STARD13 comme un nouveau partenaire de WWP1. STARD13 est une protéine à activité RhoGAP, considérée comme un suppresseur de tumeur. Nous avons montré que STARD13 permet l’association de WWP1 avec la GTPase RhoA, entraînant ainsi la polyubiquitination et la dégradation de RhoA. De façon intéressante, le complexe WWP1/STARD13 est impliqué dans le remodelage de l’architecture du cytosquelette en dégradant préférentiellement la forme activée de RhoA. Ces résultats ont permis d’identifier un nouveau rôle de WWP1 qui pourrait jouer un rôle essentiel durant la migration des cellules cancéreuses lors du processus métastatique. La caractérisation de nouveaux mécanismes de régulation et d’action de WWP1 devrait permettre à terme d’identifier si WWP1 est un marqueur diagnostique dans le cancer et/ou une nouvelle cible thérapeutique pour le développement de médicaments anticancéreux.