Constitutively active transforming growth factor β receptor 1 in the mouse ovary promotes tumorigenesis

Despite the well-established tumor suppressive role of TGFβ proteins, depletion of key TGFβ signaling components in the mouse ovary does not induce a growth advantage. To define the role of TGFβ signaling in ovarian tumorigenesis, we created a mouse model expressing a constitutively active TGFβ receptor 1 (TGFBR1) in ovarian somatic cells using conditional gain-of-function approach. Remarkably, these mice developed ovarian sex cord-stromal tumors with complete penetrance, leading to reproductive failure and mortality. The tumors expressed multiple granulosa cell markers and caused elevated serum inhibin and estradiol levels, reminiscent of granulosa cell tumors. Consistent with the tumorigenic effect, overactivation of TGFBR1 altered tumor microenvironment by promoting angiogenesis and enhanced ovarian cell proliferation, accompanied by impaired cell differentiation and dysregulated expression of critical genes in ovarian function. By further exploiting complementary genetic models, we substantiated our finding that constitutively active TGFBR1 is a potent oncogenic switch in mouse granulosa cells. In summary, overactivation of TGFBR1 drives gonadal tumor development. The TGFBR1 constitutively active mouse model phenocopies a number of morphological, hormonal, and molecular features of human granulosa cell tumors and are potentially valuable for preclinical testing of targeted therapies to treat granulosa cell tumors, a class of poorly defined ovarian malignancies

Premature Senescence and Increased TGFβ Signaling in the Absence of Tgif1

Transforming growth factor β (TGFβ) signaling regulates cell cycle progression in several cell types, primarily by inducing a G1 cell cycle arrest. Tgif1 is a transcriptional corepressor that limits TGFβ responsive gene expression. Here we demonstrate that primary mouse embryo fibroblasts (MEFs) lacking Tgif1 proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. We also provide evidence that the effects of loss of Tgif1 on proliferation and senescence are not limited to primary cells. The increased DNA damage in Tgif1 null MEFs can be partially reversed by culturing cells at physiological oxygen levels, and growth in normoxic conditions also partially rescues the proliferation defect, suggesting that in the absence of Tgif1 primary MEFs are less able to cope with elevated levels of oxidative stress. Additionally, we show that Tgif1 null MEFs are more sensitive to TGFβ-mediated growth inhibition, and that treatment with a TGFβ receptor kinase inhibitor increases proliferation of Tgif1 null MEFs. Conversely, persistent treatment of wild type cells with low levels of TGFβ slows proliferation and induces senescence, suggesting that TGFβ signaling also contributes to cellular senescence. We suggest that in the absence of Tgif1, a persistent increase in TGFβ responsive transcription and a reduced ability to deal with hyperoxic stress result in premature senescence in primary MEFs

iNKT cell development is orchestrated by different branches of TGF-β signaling

Invariant natural killer T (iNKT) cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although various steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling carefully and specifically orchestrates several steps of iNKT cell development. In vivo, this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1γ branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1γ/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors, allowing TGF-β to fine tune the iNKT cell differentiation program

Inactivation of TIF1γ Cooperates with KrasG12D to Induce Cystic Tumors of the Pancreas

Inactivation of the Transforming Growth Factor Beta (TGFβ) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1γ) has recently been proposed to be involved in TGFβ signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1γ in pancreatic carcinogenesis. Using conditional Tif1γ knockout mice (Tif1γlox/lox), we selectively abrogated Tif1γ expression in the pancreas of Pdx1-Cre;Tif1γlox/lox mice. We also generated Pdx1-Cre;LSL-KrasG12D;Tif1γlox/lox mice to address the effect of Tif1γ loss-of-function in precancerous lesions induced by oncogenic KrasG12D. Finally, we analyzed TIF1γ expression in human pancreatic tumors. In our mouse model, we showed that Tif1γ was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-KrasG12D;Smad4lox/lox mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1γ don't have strictly redundant functions. Finally, we report that TIF1γ expression is markedly down-regulated in human pancreatic tumors by quantitative RT–PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1γ is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1γ in the multifaceted functions of TGFβ in carcinogenesis and development

Epithelial TGFβ engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features

The pro-tumourigenic role of epithelial TGFβ signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFβ signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFβ signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFβ signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFβ signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC’s with born to be bad traits

Identification of NF-kappaB Responsive Elements in Follistatin Related Gene (FLRG) Promoter

International audienceFollistatin related gene (FLRG) has been previously identified from a chromosomal translocation observed in a B-cell chronic lymphocytic leukemia (B-CLL). FLRG (alternative names: follistatin-related protein, FSRP/follistatin-like-3, FSTL3) is a secreted glycoprotein highly similar to follistatin. Like follistatin, FLRG is involved in the regulation of various biological effects through its binding to members of the transforming growth factor beta (TGFbeta) superfamily such as activin A and myostatin. We have previously shown that TGFbeta and activin A are potent inducers of FLRG transcriptional activation through the Smad proteins. Using a biochemical approach, we investigated whether tumor necrosis factor alpha (TNFalpha) could regulate FLRG expression since TNFalpha plays a critical role in hematopoietic malignancies. We demonstrate that TNFalpha activates FLRG expression at the transcriptional level. This activation depends on a promoter region containing four 107-108 bp DNA repeats, which are evolutionary conserved in primates. These repeats carry a strong phylogenetic signal, which is not common among non-coding sequences. Each DNA repeat contains one TNFalpha responsive element (5'-GGGAGAG/TTCC-3') able to bind nuclear factor kappaB (NF-kappaB) transcription factors. We also show that TGFbeta, through the Smad proteins, potentates the effect of TNFalpha on FLRG expression. This cooperation is unexpected since TGFbeta and TNFalpha usually have opposite biological effects. In all, this work brings new insights in the understanding of FLRG regulation by cytokines and growth factors. It opens attractive perspectives of research that should allow us to better understand the role of FLRG during tumorigenesis

Identification of NF-kappaB responsive elements in follistatin related gene: NF-kappaB activates FLRG transcription

Follistatin related gene (FLRG) has been previously identified from a chromosomal translocation observed in a B-cell chronic lymphocytic leukemia (B-CLL). FLRG (alternative names: follistatin-related protein, FSRP / follistatin-like-3, FSTL3) is a secreted glycoprotein highly similar to follistatin. Like follistatin, FLRG is involved in the regulation of various biological effects through its binding to members of the transforming growth factor beta (TGFΒ) superfamily such as activin A and myostatin. We have previously shown that TGFβ and activin A are potent inducers of FLRG transcriptional activation through the Smad proteins. Using a biochemical approach, we investigated whether tumor necrosis factor alpha (TNFΑ) could regulate FLRG expression since TNFΑ plays a critical role in hematopoietic malignancies. We demonstrate that TNFΑ activates FLRG expression at the transcriptional level. This activation depends on a promoter region containing four 107-108 bp DNA repeats evolutionary conserved in primates. These repeats carry a strong phylogenetic signal, which is not very common among non-coding sequences. Each DNA repeat contains one TNFΑ responsive element (5'-GGGAGAG/TTCC-3') able to bind nuclear factor kappaB (NF-ΚB) transcription factors. We also show that TGFΒ, through the Smad proteins, potentates the effect of TNFΑ on FLRG expression. This cooperation is unexpected since TGFΒ and TNFΑ usually have opposite biological effects. In all, this work brings new insights in the understanding of FLRG regulation by cytokines and growth factors. It opens attractive perspectives of research that should allow us to better understand the role of FLRG during tumorigenesis

Drosophila TGIF Proteins Are Transcriptional Activators

The information carried by transforming growth factor β (TGF-β) signaling molecules induces profound responses in target cells. To restrict this information to appropriate cells, TGF-β signaling pathways are tightly regulated by dynamic interactions with transcriptional activators and repressors. Numerous cross-species experiments have shown that TGF-β family members and their signal transduction machinery (receptors and Smad signal transducers) are functionally conserved between vertebrates and invertebrates. TG-interacting factor (TGIF) is a homeodomain-containing transcriptional corepressor of TGF-β-dependent gene expression in mammals that is associated with holoprosencephaly in humans. Here we report a biochemical analysis of TGIF from zebra fish and Drosophila. Our study reveals an unprecedented role reversal between vertebrate and invertebrate TGIF proteins. Zebra fish TGIF, like its mammalian relative, interacts with general corepressors and represses TGF-β-responsive gene expression. We identified a tandem duplication of TGIF genes in Drosophila. In contrast to vertebrate TGIFs, both Drosophila TGIFs strongly activate transcription. We also demonstrate that Drosophila TGIF proteins physically interact with both Mad and dSmad2, suggesting a role in Dpp and activin signaling. Thus, dTGIF may be the first transcription factor in the Drosophila activin pathway. Overall, our study suggests that assumptions about the functional equivalence of conserved proteins must be validated experimentally

Sertoli Cell-Specific Activation of Transforming Growth Factor Beta Receptor 1 Leads to Testicular Granulosa Cell Tumor Formation

The transforming growth factor β (TGFβ) superfamily, consisting of protein ligands, receptors, and intracellular SMAD transducers, regulates fundamental biological processes and cancer development. Our previous study has shown that sustained activation of TGFβ receptor 1 (TGFBR1) driven by anti-Mullerian hormone receptor type 2 (Amhr2)-Cre in the mouse testis induces the formation of testicular granulosa cell tumors (TGCTs). As Amhr2-Cre is expressed in both Sertoli cells and Leydig cells, it remains unclear whether the activation of TGFBR1 in Sertoli cells alone is sufficient to induce TGCT formation. Therefore, the objective of this study was to determine whether Sertoli cell-activation of TGFBR1 drives oncogenesis in the testis. Our hypothesis was that overactivation of TGFBR1 in Sertoli cells would promote their transdifferentiation into granulosa-like cells and the formation of TGCTs. To test this hypothesis, we generated mice harboring constitutive activation of TGFBR1 in Sertoli cells using anti-Mullerian hormone (Amh)-Cre. Disorganized seminiferous tubules and tumor nodules were found in TGFBR1CA; Amh-Cre mice. A histological analysis showed that Sertoli cell-specific activation of TGFBR1 led to the development of neoplasms resembling granulosa cell tumors, which derailed spermatogenesis. Moreover, TGCTs expressed granulosa cell markers including FOXL2, FOXO1, and INHA. Using a dual fluorescence reporter line, the membrane-targeted tdTomato (mT)/membrane-targeted EGFP (mG) mouse, we provided evidence that Sertoli cells transdifferentiated toward a granulosa cell fate during tumorigenesis. Thus, our findings indicate that Sertoli cell-specific activation of TGFBR1 leads to the formation of TGCTs, supporting a key contribution of Sertoli cell reprogramming to the development of this testicular malignancy in our model