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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 TGFBR1 CA ; 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.

Idiopathic pulmonary fibrosis (IPF) is a chronic human disease with persistent destruction of lung parenchyma. Transforming growth factor-β1 (TGF-β1) signaling plays a pivotal role in the initiation and pathogenesis of IPF. As shown herein, TGF-β1 signaling down-regulated not only peroxisome biogenesis but also the metabolism of these organelles in human IPF fibroblasts. In vitro cell culture observations in human fibroblasts and human lung tissue indicated that peroxisomal biogenesis and metabolic proteins were significantly down-regulated in the lung of 1-month-old transgenic mice expressing a constitutively active TGF-β type I receptor kinase (ALK5). The peroxisome biogenesis protein peroxisomal membrane protein Pex13p (PEX13p) as well as the peroxisomal lipid metabolic enzyme peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and antioxidative enzyme catalase were highly up-regulated in TGF-β type II receptor and Smad3 knockout mice. This study reports a novel mechanism of peroxisome biogenesis and metabolic regulation via TGF-β1-Smad signaling: interaction of the Smad3 transcription factor with the PEX13 gene in chromatin immunoprecipitation-on-chip assay as well as in a bleomycin-induced pulmonary fibrosis model applied to TGF-β type II receptor knockout mice. Taken together, data from this study suggest that TGF-β1 participates in regulation of peroxisomal biogenesis and metabolism via Smad-dependent signaling, opening up novel strategies for the development of therapeutic approaches to inhibit progression of pulmonary fibrosis patients with IPF., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

TGF-β signaling is involved in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis, representing one of the four major pathways genetically altered in 100% of PDAC cases. TGF-β exerts complex and pleiotropic effects in cancers, notably via the activation of SMAD pathways, predominantly SMAD2/3/4. Though SMAD2 and 3 are rarely mutated in cancers, SMAD4 is lost in about 50% of PDAC, and the role of SMAD2/3 in a SMAD4-null context remains understudied. We herein provide evidence of a SMAD2/3 oncogenic effect in response to TGF-β1 in SMAD4-null human PDAC cancer cells. We report that inactivation of SMAD2/3 in SMAD4-negative PDAC cells compromises TGF-β-driven collective migration mediated by FAK and Rho/Rac signaling. Moreover, RNA-sequencing analyses highlight a TGF-β gene signature related to aggressiveness mediated by SMAD2/3 in the absence of SMAD4. Using a PDAC patient cohort, we reveal that SMAD4-negative tumors with high levels of phospho-SMAD2 are more aggressive and have a poorer prognosis. Thus, loss of SMAD4 tumor suppressive activity in PDAC leads to an oncogenic gain-of-function of SMAD2/3, and to the onset of associated deleterious effects., (© 2022. The Author(s).)

Background: Cancer patients at advanced stages experience a severe depletion of skeletal muscle compartment together with a decrease in muscle function, known as cancer cachexia. Cachexia contributes to reducing quality of life, treatment efficiency, and lifespan of cancer patients. However, the systemic nature of the syndrome is poorly documented. Here, we hypothesize that glucocorticoids would be important systemic mediators of cancer cachexia., Methods: To explore the role of glucocorticoids during cancer cachexia, biomolecular analyses were performed on several tissues (adrenal glands, blood, hypothalamus, liver, and skeletal muscle) collected from Apc Min/+ male mice, a mouse model of intestine and colon cancer, aged of 13 and 23 weeks, and compared with wild type age-matched C57BL/6J littermates., Results: Twenty-three-week-old Apc mice recapitulated important features of cancer cachexia including body weight loss (-16%, P < 0.0001), muscle atrophy (gastrocnemius muscle: -53%, P < 0.0001), and weakness (-50% in tibialis anterior muscle force, P < 0.0001), increased expression of atrogens (7-fold increase in MuRF1 transcript level, P < 0.0001) and down-regulation of Akt-mTOR pathway (3.3-fold increase in 4EBP1 protein content, P < 0.0001), together with a marked transcriptional rewiring of hepatic metabolism toward an increased expression of gluconeogenic genes (Pcx: +90%, Pck1: +85%), and decreased expression of glycolytic (Slc2a2: -40%, Gk: -30%, Pklr: -60%), ketogenic (Hmgcs2: -55%, Bdh1: -80%), lipolytic/fatty oxidation (Lipe: -50%, Mgll: -60%, Cpt2: -60%, Hadh: -30%), and lipogenic (Acly: -30%, Acacb: -70%, Fasn: -45%) genes. The hypothalamic pituitary-adrenal axis was activated, as evidenced by the increase in the transcript levels of genes encoding corticotropin-releasing hormone in the hypothalamus (2-fold increase, P < 0.01), adrenocorticotropic hormone receptor (3.4-fold increase, P < 0.001), and steroid biosynthesis enzymes (Cyp21a1, P < 0.0001, and Cyp11b1, P < 0.01) in the adrenal glands, as well as by the increase in corticosterone level in the serum (+73%, P < 0.05), skeletal muscle (+17%, P < 0.001), and liver (+24%, P < 0.05) of cachectic 23-week-old Apc mice. A comparative transcriptional analysis with dexamethasone-treated C57BL/6J mice indicated that the activation of the hypothalamic-pituitary-adrenal axis in 23-week-old Apc Min/+ mice was significantly associated with the transcription of glucocorticoid-responsive genes in skeletal muscle (P < 0.05) and liver (P < 0.001). The transcriptional regulation of glucocorticoid-responsive genes was also observed in the gastrocnemius muscle of Lewis lung carcinoma tumour-bearing mice and in KPC mice (tibialis anterior muscle and liver)., Conclusions: These findings highlight the role of the hypothalamic-pituitary-adrenal-glucocorticoid pathway in the transcriptional regulation of skeletal muscle catabolism and hepatic metabolism during cancer cachexia. They also provide the paradigm for the design of new therapeutic strategies., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Ovarian granulosa cell tumors (GCTs) are rare sex cord-stromal tumors, accounting for ~5% ovarian tumors. The etiology of GCTs remains poorly defined. Genetically engineered mouse models are potentially valuable for understanding the pathogenesis of GCTs. Mice harboring constitutively active TGFβ signaling (TGFBR1-CA) develop ovarian GCTs that phenocopy several hormonal and molecular characteristics of human GCTs. To determine molecular alterations in the ovary upon TGFβ signaling activation, we performed transcriptomic profiling of gene expression associated with GCT development using ovaries from 1-month-old TGFBR1-CA mice and age-matched controls. RNA-sequencing and bioinformatics analysis coupled with the validation of select target genes revealed dysregulations of multiple cellular events and signaling molecules/pathways. The differentially expressed genes are enriched not only for known GCT-related pathways and tumorigenic events but also for signaling events potentially mediated by neuroactive ligand-receptor interaction, relaxin signaling, insulin signaling, and complements in TGFBR1-CA ovaries. Additionally, a comparative analysis of our data in mice with genes dysregulated in human GCTs or granulosa cells overexpressing a mutant FOXL2, the genetic hallmark of adult GCTs, identified some common genes altered in both conditions. In summary, this study has revealed the molecular signature of ovarian GCTs in a mouse model that harbors the constitutive activation of TGFBR1. The findings may be further exploited to understand the pathogenesis of a class of poorly defined ovarian tumors.

Tissue-resident memory T (T RM ) cells are non-recirculating cells that exist throughout the body. Although T RM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze T RM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate T RM cell function, durability and malleability. We find that unequal responsiveness to TGFβ is a major driver of this diversity. Notably, dampened TGFβ signaling results in CD103 - T RM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFβ-responsive CD103 + T RM counterparts. Furthermore, whereas CD103 - T RM cells readily modified their phenotype upon relocation, CD103 + T RM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for T RM cell development, tissue adaptation of these cells confers discrete functional properties such that T RM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Antigen-specific CD8 + T cells in chronic viral infections and tumors functionally deteriorate, a process known as exhaustion. Exhausted T cells are sustained by precursors of exhausted (Tpex) cells that self-renew while continuously generating exhausted effector (Tex) cells. However, it remains unknown how Tpex cells maintain their functionality. Here, we demonstrate that Tpex cells sustained mitochondrial fitness, including high spare respiratory capacity, while Tex cells deteriorated metabolically over time. Tpex cells showed early suppression of mTOR kinase signaling but retained the ability to activate this pathway in response to antigen receptor signals. Early transient mTOR inhibition improved long-term T cell responses and checkpoint inhibition. Transforming growth factor-β repressed mTOR signaling in exhausted T cells and was a critical determinant of Tpex cell metabolism and function. Overall, we demonstrate that the preservation of cellular metabolism allows Tpex cells to retain long-term functionality to sustain T cell responses during chronic infection., Competing Interests: Declaration of interests F.S.-F.-G. has a funded research collaborative agreement with Miltenyi Biotec Australia and is a consultant for Biotheus. N.D.H. is founder and shareholder of oNKo-Innate Pty Ltd. The authors declare no other competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Following antigen-driven expansion in lymph node, transforming growth factor-β (TGFβ) is required for differentiation of skin-recruited CD8 + T cell effectors into epidermal resident memory T (Trm) cells and their epidermal persistence. We found that the source of TGFβ -supporting Trm cells was autocrine. In addition, antigen-specific Trm cells that encountered cognate antigen in the skin, and bystander Trm cells that did not, both displayed long-term persistence in the epidermis under steady-state conditions. However, when the active-TGFβ was limited or when new T cell clones were recruited into the epidermis, antigen-specific Trm cells were more efficiently retained than bystander Trm cells. Genetically enforced TGFβR signaling allowed bystander Trm cells to persist in the epidermis as efficiently as antigen-specific Trm cells in both contexts. Thus, competition between T cells for active TGFβ represents an unappreciated selective pressure that promotes the accumulation and persistence of antigen-specific Trm cells in the epidermal niche., Competing Interests: Declaration of Interests D.W.G. is a consultant and equity holder of Indalo Therapeutics, which provided compound CWHM-12. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1-Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast-specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1-Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype., (© 2020 The Authors. Genesis published by Wiley Periodicals, Inc.)

Transforming growth factor (TGFβ) is a secreted factor, which accumulates in tissues during many physio- and pathological processes such as embryonic development, wound healing, fibrosis and cancer. In order to analyze the effects of increased microenvironmental TGFβ concentration in vivo, we developed a conditional transgenic mouse model (Flpo/Frt system) expressing bioactive TGFβ in fibroblasts, a cell population present in the microenvironment of almost all tissues. To achieve this, we created the genetically-engineered [Fsp1-Flpo; FSF TGFβ CA ] mouse model. The Fsp1-Flpo allele consists in the Flpo recombinase under the control of the Fsp1 (fibroblast-specific promoter 1) promoter. The FSF TGFβ CA allele consists in a transgene encoding a constitutively active mutant form of TGFβ (TGFβ CA ) under the control of a Frt-STOP-Frt (FSF) cassette. The FSF TGFβ CA allele was created to generate this model, and functionally validated by in vitro, ex vivo and in vivo techniques. [Fsp1-Flpo; FSF TGFβ CA ] animals do not present any obvious phenotype despite the correct expression of TGFβ CA transgene in fibroblasts. This [Fsp1-Flpo; FSF TGFβ CA ] model is highly pertinent for future studies on the effect of increased microenvironmental bioactive TGFβ concentrations in mice bearing Cre-dependent genetic alterations in other compartments (epithelial or immune compartments for instance). These dual recombinase system (DRS) approaches will enable scientists to study uncoupled spatiotemporal regulation of different genetic alterations within the same mouse, thus better replicating the complexity of human diseases.

Pancreatic ductal adenocarcinoma (PDAC) is one of the solid tumors with the poorest prognosis. The stroma of this tumor is abundant and composed of extracellular matrix and stromal cells (including cancer-associated fibroblasts and immune cells). Nerve fibers invading this stroma represent a hallmark of PDAC, involved in neural remodeling, which participates in neuropathic pain, cancer cell dissemination and tumor relapse after surgery. Pancreatic cancer-associated neural remodeling is regulated through functional interplays mediated by physical and molecular interactions between cancer cells, nerve cells and surrounding Schwann cells, and other stromal cells. In the present study, we show that Schwann cells (glial cells supporting peripheral neurons) can enhance aggressiveness (migration, invasion, tumorigenicity) of pancreatic cancer cells in a transforming growth factor beta (TGFβ)-dependent manner. Indeed, we reveal that conditioned medium from Schwann cells contains high amounts of TGFβ able to activate the TGFβ-SMAD signaling pathway in cancer cells. We also observed in human PDAC samples that high levels of TGFβ signaling activation were positively correlated with perineural invasion. Secretome analyses by mass spectrometry of Schwann cells and pancreatic cancer cells cultured alone or in combination highlighted the central role of TGFβ in neuro-epithelial interactions, as illustrated by proteomic signatures related to cell adhesion and motility. Altogether, these results demonstrate that Schwann cells are a meaningful source of TGFβ in PDAC, which plays a crucial role in the acquisition of aggressive properties by pancreatic cancer cells.

Regulated activation of the cytokine TGF-β by integrins α v β 6 and α v β 8 expressed on keratinocytes is required for residence of epidermal-resident memory T cells, but whether skin-derived signals also affect recirculating memory cells in the skin remains unclear. Here, we show that after resolution of skin vaccinia virus (VV) infection, antigen-specific circulating memory CD8 + T cells migrated into skin. In mice lacking α v β 6 and α v β 8 integrins (Itgb6 -/- Itgb8 fl/fl -K14-cre), the absence of epidermal-activated TGF-β resulted in a gradual loss of E- or P-selectin-binding central and peripheral memory populations, which were rescued when skin entry was inhibited. Skin recirculating memory cells were required for optimal host defense against skin VV infection. These data demonstrate that skin migration can persist after resolution of local skin infection and that the cytokine environment within this nonlymphoid tissue shapes the differentiation state and persistence of the central and peripheral memory-T-cell pool., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: The demographics of oncology residents has changed since 2010 with the increase in the size of promotions. The evolution of the residents' aspirations towards research and their future exercise in parallel with these demographic changes has not been assessed., Methods: A questionnaire was developed by a working group from GERCOR (cooperative group in oncology), involving clinicians, researchers, GERCOR members, and residents. It consisted of 62 questions divided into 7 sections: demographics, medical thesis, post-residency, mobility, publication activity, basic research, and clinical/translational research. The national survey was published online by the Association d'enseignement et de recherche des internes en oncologie (AERIO)., Results: In total, 143 residents participated, of which 116 (81.1%) completed the questionnaire entirely. The population was representative of the current demographics, with a majority of women (65.0%), a median age of 28 years, and 39.7% of residents from Paris region. The unsupervised analysis revealed four profiles of residents, including one group strongly committed to research (16.8%), one group with moderate involvement (41.3%) and one group that did not seem interested in research (14.7%). Uncertainty about future position and lack of time and interaction with researchers appeared to be the main barriers to involvement of residents in research., Discussion: This national survey provided useful information about the residents' perspective to academic research. It may serve as a basis for proposing measures adapted to their expectations., (Copyright © 2019 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Uterine gland development, also known as adenogenesis, is a key uterine morphogenic process indispensable for normal uterine function and fertility. Our earlier studies have reported that overactivation of TGFB receptor 1 (TGFBR1) in the mouse uterus using progesterone receptor (Pgr)-Cre recombinase causes female infertility, defective decidualization, and reduced uterine gland formation, a developmental milestone of postnatal uterus. To understand mechanisms that underpin the disrupted uterine gland formation in mice with sustained activation of TGFBR1, we raised the question of whether early postnatal adenogenesis was compromised in these mice. Experiments were designed using mice with constitutive activation of TGFBR1 driven by Pgr-Cre to determine the timing of adenogenic defects and potential mechanisms associated with dysregulation of adenogenic genes, luminal epithelial cell proliferation and endometrial fibrotic changes. Uterine tissues from mice with constitutive activation of TGFBR1 were collected during the critical time window of adenogenesis and analyzed together with age-matched controls. Multiple approaches including immunohistochemistry, immunofluorescence, Trichrome staining, quantitative real-time PCR, western blot, conditional knockout and human endometrial cell culture were utilized. TGFBR1 activation in the mouse uterus suppressed adenogenesis during postnatal uterine development, concomitant with the aberrant differentiation of uterine stromal cells. Analysis of transcript expression of WNT pathway components revealed dysregulation of adenogenesis-associated genes. Notably, the adenogenic defects occurred in spite of the increased proliferation of uterine luminal epithelial cells, accompanied by increased expression of genes associated with fibrotic changes. Moreover, the adenogenic defects were alleviated in mice where TGFBR1 was activated in presumably half of the complement of uterine cells. Our results suggest that altered differentiation of endometrial stromal cells and formation of stromal compartment promote adenogenic defects., Competing Interests: The authors have declared that no competing interests exist.

Pancreatic ductal adenocarcinoma (PDAC) is the digestive cancer with the poorest prognosis, with a 5-year overall survival rate of 7%. Complete surgical resection followed by adjuvant chemotherapy is the only treatment with curative intent. However, many patients with an apparently localized disease who may undergo primary tumor resection already have micro-metastatic disease and will promptly develop metastases. Considering the significant rate of morbidity and mortality upon pancreatic surgery, the pre-operative identification of patients with an aggressive disease is therefore a major clinical issue. Although tumor size, differentiation, margins, and lymph node invasion are the main "classical" prognostic factors, they are not sufficient to fully predict early disease recurrence. In the last decade, multi-omics high-throughput analyses have provided a new insight into PDAC biology and have led to the description of multiple molecular subtypes, with a significant prognostic value for most of them, but that have not yet been transposed to routine clinical practice, mainly due to poor availability of tumor tissue material prior to surgical resection. In this review, we provide an overview of the current status of clinico-pathological and molecular biomarkers (tumor and blood) to predict early recurrence, and their implications for clinical practice and future research development., (Copyright © 2018 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy. However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction of paracrine senescence was observed within 24 hours and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence, we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended on macrophage-derived transforming growth factor-β1 (TGFβ1) ligand. In acetaminophen poisoning, inhibition of TGFβ receptor 1 (TGFβR1) improved mouse survival. TGFβR1 inhibition reduced senescence and enhanced liver regeneration even when delivered beyond the therapeutic window for treating acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Study Question: What is the role of dysregulated transforming growth factor beta (TGFB) signaling in the development of sex cord-stromal tumors in the testis?, Summary Answer: Overactivation of TGFB signaling results in the development of testicular tumors resembling granulosa cell tumors (GrCTs)., What Is Known Already: In an earlier study, we demonstrated that constitutively active TGFB receptor 1 (TGFBR1) in ovarian somatic cells promotes the development of ovarian GrCTs. However, the consequence of dysregulation of TGFB signaling in the pathobiology of the testis, remains poorly defined., Study Design, Size, Duration: To identify the impact of dysregulation of TGFB signaling on the testis, we generated mice with constitutive activation of TGFBR1 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase. The effect of constitutively active TGFBR1 on testis development and the timeline of testicular tumor formation were examined. We further investigated the molecular features of testicular tumors and determined the expression of beta-catenin (CTNNB1) known to be involved in testicular GrCT development., Participants/materials, Setting, Methods: Male mice with constitutive activation of TGFBR1 were examined at various developmental stages (i.e. from 1 week up to 6 months) along with controls. Testis samples were collected and processed for histological and molecular analyses, including haematoxylin and eosin (H and E) staining, real-time PCR, immunohistochemistry, immunofluorescence and western blotting. Immunostaining/immunoblotting and real-time PCR experiments were performed using at least three animals per genotype. Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tail t-test and reported when P value is <0.05., Main Results and the Role of Chance: Mice harboring constitutively active TGFBR1 in the testes developed tumors resembling testicular GrCTs, a rare type of tumors in the testis. The formation of testicular tumors led to altered cell proliferation, loss of germ cells and defective spermatogenesis. Immunohistochemically, these tumors were positive for inhibin alpha (INHA), forkhead box O1 (FOXO1), and more importantly, forkhead box L2 (FOXL2), a protein specifically expressed in the ovary and required for normal granulosa cell differentiation and function. Consistent with the immunohistochemical findings, FOXL2 proteins were only detectable in testes of TGFBR1-CAAcre mice but not those of controls by western blotting, suggesting potential alteration of Sertoli cell fate. To explore mechanisms underlying the tumor-promoting effect of TGFBR1 overactivation, we examined the expression of CTNNB1. The results revealed increased expression of CTNNB1 in testicular tumors in TGFBR1-CAAcre mice. Collectively, this study uncovered tumorigenic function of enhanced TGFB signaling in the testis., Large-Scale Data: N/A., Limitations, Reasons for Caution: This study was performed using mice, and the direct relevance of the experimental paradigm and findings to human testicular GrCTs awaits further investigation. Of note, constitutive activation of TGFBR1 was employed to enhance TGFB/SMAD signaling activity and may not be interpreted as the genetic cause of the disease., Wider Implications of the Findings: This mouse model may prove to be a useful addition to the mouse genetics toolkit for GrCT research. Our finding that dysregulation of TGFB signaling results in the development of testicular GrCTs supports a common origin between Sertoli cells and granulosa cells, and highlights the paramount importance of balanced TGFB signaling in reproduction and development., Study Funding/competing Interest(s): This research was supported by the National Institutes of Health grant R03HD082416 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development and the New Faculty Start-up Funds from Texas A&M University awarded to Q.L. The authors declare no competing interest.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, mostly due to its resistance to treatment. Of these, checkpoint inhibitors (CPI) are inefficient when used as monotherapy, except in the case of a rare subset of tumors harboring microsatellite instability (< 2%). This inefficacy mainly resides in the low immunogenicity and non-inflamed phenotype of PDAC. The abundant stroma generates a hypoxic microenvironment and drives the recruitment of immunosuppressive cells through cancer-associated-fibroblast activation and transforming growth factor β secretion. Several strategies have recently been developed to overcome this immunosuppressive microenvironment. Combination therapies involving CPI aim at increasing tumor immunogenicity and promoting the recruitment and activation of effector T cells. Ongoing studies are therefore exploring the association of CPI with vaccines, oncolytic viruses, MEK inhibitors, cytokine inhibitors, and hypoxia- and stroma-targeting agents. Adoptive T-cell transfer is also under investigation. Moreover, translational studies on tumor tissue and blood, prior to and during treatment may lead to the identification of biomarkers with predictive value for both clinical outcome and response to immunotherapy., Competing Interests: Conflict-of-interest statement: Neuzillet C reports non-financial support from OSE Immunotherapeutics; Hilmi M and Bartholin L have no potential conflicts of interest relevant to this article were reported.

Background: Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes., Methods: A mouse model containing constitutively active TGFBR1 (TGFBR1 CA ) using Gdf9-iCre (termed TGFBR1-CA G9Cre ) was generated. Hematoxylin and eosin (H & E) staining, follicle counting, and immunohistochemistry and immunofluorescence analyses using antibodies directed to Ki67, forkhead box L2 (FOXL2), forkhead box O1 (FOXO1), inhibin alpha (INHA), and SRY (sex determining region Y)-box 9 were performed to determine the characteristics of the TGFBR1-CA G9Cre ovary. Terminal deoxynucleotidyl transferase (TdT) labeling of 3'-OH ends of DNA fragments, real-time PCR, and western blotting were used to examine apoptosis, select gene expression, and TGFBR1 activation. RNAscope in situ hybridization was used to localize the expression of GLI-Kruppel family member GLI1 (Gli1) in ovarian tumor tissues., Results: TGFBR1-CA G9Cre females were sterile. Sustained activation of TGFBR1 led to altered granulosa cell proliferation evidenced by high expression of Ki67. At an early age, these mice demonstrated follicular defects and development of ovarian granulosa cell tumors, which were immunoreactive for granulosa cell markers including FOXL2, FOXO1, and INHA. Further histochemical and molecular analyses provided evidence of overactivation of TGFBR1 in the granulosa cell compartment during ovarian pathogenesis in TGFBR1-CA G9Cre mice, along with upregulation of Gli1 and Gli2 and downregulation of Tgfbr3 in ovarian tumor tissues., Conclusions: These results reinforce the role of constitutively active TGFBR1 in promoting ovarian tumorigenesis in mice. The mouse model created in this study may be further exploited to define the cellular and molecular mechanisms of TGFB/activin downstream signaling in granulosa cell tumor development. Future studies are needed to test whether activation of TGFB/activin signaling contributes to the development of human granulosa cell tumors.

Avoiding destruction by immune cells is a hallmark of cancer, yet how tumors ultimately evade control by natural killer (NK) cells remains incompletely defined. Using global transcriptomic and flow-cytometry analyses and genetically engineered mouse models, we identified the cytokine-TGF-β-signaling-dependent conversion of NK cells (CD49a - CD49b + Eomes + ) into intermediate type 1 innate lymphoid cell (intILC1) (CD49a + CD49b + Eomes + ) populations and ILC1 (CD49a + CD49b - Eomes int ) populations in the tumor microenvironment. Strikingly, intILC1s and ILC1s were unable to control local tumor growth and metastasis, whereas NK cells favored tumor immunosurveillance. Experiments with an antibody that neutralizes the cytokine TNF suggested that escape from the innate immune system was partially mediated by TNF-producing ILC1s. Our findings provide new insight into the plasticity of group 1 ILCs in the tumor microenvironment and suggest that the TGF-β-driven conversion of NK cells into ILC1s is a previously unknown mechanism by which tumors escape surveillance by the innate immune system.

Background & Aims: Transforming growth factor beta (TGFβ) acts either as a tumor suppressor or as an oncogene, depending on the cellular context and time of activation. TGFβ activates the canonical SMAD pathway through its interaction with the serine/threonine kinase type I and II heterotetrameric receptors. Previous studies investigating TGFβ-mediated signaling in the pancreas relied either on loss-of-function approaches or on ligand overexpression, and its effects on acinar cells have so far remained elusive., Methods: We developed a transgenic mouse model allowing tamoxifen-inducible and Cre-mediated conditional activation of a constitutively active type I TGFβ receptor (TβRI CA ) in the pancreatic acinar compartment., Results: We observed that TβRI CA expression induced acinar-to-ductal metaplasia (ADM) reprogramming, eventually facilitating the onset of KRAS G12D -induced pre-cancerous pancreatic intraepithelial neoplasia. This phenotype was characterized by the cellular activation of apoptosis and dedifferentiation, two hallmarks of ADM, whereas at the molecular level, we evidenced a modulation in the expression of transcription factors such as Hnf1β , Sox9, and Hes1 ., Conclusions: We demonstrate that TGFβ pathway activation plays a crucial role in pancreatic tumor initiation through its capacity to induce ADM, providing a favorable environment for KRAS G12D -dependent carcinogenesis. Such findings are highly relevant for the development of early detection markers and of potentially novel treatments for pancreatic cancer patients.

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., Competing Interests: The authors declare no conflict of interest.

Solid tumors often display chemotherapy resistance. Pancreatic ductal adenocarcinoma (PDAC) is the archetype of resistant tumors as current chemotherapies are inefficient. The tumor stroma and extracellular matrix (ECM) are key contributors to PDAC aggressiveness and to limiting the efficacy of chemotherapy. Lysyl oxidase (LOX) family members mediate collagen cross-linking and thus promote ECM stiffening. Our data demonstrate increased LOX, LOXL1, and LOXL2 expression in PDAC, and that the level of fibrillar collagen, which is directly dependent of LOX family activity, is an independent predictive biomarker of adjuvant "Gemcitabine-based chemotherapy" benefit. Experimentally in mice, increased LOX family activity through LOXL2 promotes chemoresistance. This effect of LOX family activity seems to be due to decreased gemcitabine intra-tumoral diffusion. This observation might be explained by increased fibrillar collagen and decreased vessel size observed in tumors with increased LOX family activity. In conclusion, our data support that LOX family activity is both a novel target to improve chemotherapy as well as a novel biomarker to predict gemcitabine benefit in PDAC. Beyond the PDAC, it is possible that targeting LOX family activity might improve efficacy of chemotherapies against different kinds of solid tumors., Competing Interests: The authors declare no conflicts of interest.

Group 3 innate lymphoid cells (ILC3s) are composed of subsets that are either positive or negative for the natural cytotoxicity receptor (NCR) NKp46 (encoded by Ncr1). ILC3s are located at mucosal sites, such as in the intestine and lung, where they are exposed to billions of commensal microbes and potentially harmful pathogens. Together with T cells, the various ILC3 subsets maintain the balance between homeostasis and immune activation. Through genetic mapping, we identified a previously uncharacterized subset of NCR(-) ILC3s in mice that transiently express Ncr1, demonstrating previously undescribed heterogeneity within the ILC3 population. In addition, we showed that sustained Notch signaling was required for the maintenance of the NCR(+) phenotype and that the cytokine transforming growth factor-β (TGF-β) impaired the development of NCR(+) ILC3s. Thus, the plasticity of ILC3s is regulated by the balance between the opposing effects of Notch and TGF-β signaling, maintaining homeostasis in the face of continual challenges., (Copyright © 2016, American Association for the Advancement of Science.)

Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8(+) tissue-resident memory T cells (TRM cells) require active transforming growth factor-β1 (TGF-β) for epidermal residence. Here we found that integrins αvβ6 and αvβ8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-β. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvβ6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-β, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-β by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication., Competing Interests: The authors declare no competing financial interests.

Transforming growth factor-β (TGF-β) is a major immunosuppressive cytokine that maintains immune homeostasis and prevents autoimmunity through its antiproliferative and anti-inflammatory properties in various immune cell types. We provide genetic, pharmacologic, and biochemical evidence that a critical target of TGF-β signaling in mouse and human natural killer (NK) cells is the serine and threonine kinase mTOR (mammalian target of rapamycin). Treatment of mouse or human NK cells with TGF-β in vitro blocked interleukin-15 (IL-15)-induced activation of mTOR. TGF-β and the mTOR inhibitor rapamycin both reduced the metabolic activity and proliferation of NK cells and reduced the abundances of various NK cell receptors and the cytotoxic activity of NK cells. In vivo, constitutive TGF-β signaling or depletion of mTOR arrested NK cell development, whereas deletion of the TGF-β receptor subunit TGF-βRII enhanced mTOR activity and the cytotoxic activity of the NK cells in response to IL-15. Suppression of TGF-β signaling in NK cells did not affect either NK cell development or homeostasis; however, it enhanced the ability of NK cells to limit metastases in two different tumor models in mice. Together, these results suggest that the kinase mTOR is a crucial signaling integrator of pro- and anti-inflammatory cytokines in NK cells. Moreover, we propose that boosting the metabolic activity of antitumor lymphocytes could be an effective strategy to promote immune-mediated tumor suppression., (Copyright © 2016, American Association for the Advancement of Science.)

In recent years, the importance of the cell biological process of epithelial-mesenchymal transition (EMT) has been established via an exponentially growing number of reports. EMT has been documented during embryonic development, tissue fibrosis, and cancer progression in vitro, in animal models in vivo and in human specimens. EMT relates to many molecular and cellular alterations that occur when epithelial cells undergo a switch in differentiation that generates mesenchymal-like cells with newly acquired migratory and invasive properties. In addition, EMT relates to a nuclear reprogramming similar to the one occurring in the generation of induced pluripotent stem cells. Via such a process, EMT is gradually established to promote the generation and maintenance of adult tissue stem cells which under disease states such as cancer, are known as cancer stem cells. EMT is induced by developmental growth factors, oncogenes, radiation, and hypoxia. A prominent growth factor that causes EMT is transforming growth factor β (TGF-β).A series of molecular and cellular techniques can be applied to define and characterize the state of EMT in diverse biological samples. These methods range from DNA and RNA-based techniques that measure the expression of key EMT regulators and markers of epithelial or mesenchymal differentiation to functional assays of cell mobility, invasiveness and in vitro stemness. This chapter focuses on EMT induced by TGF-β and provides authoritative protocols and relevant reagents and citations of key publications aiming at assisting newcomers that enter this prolific area of biomedical sciences, and offering a useful reference tool to pioneers and aficionados of the field.

The transcription accessory factor TIF1γ/TRIM33/RFG7/PTC7/Ectodermin functions as a tumor suppressor that promotes development and cellular differentiation. However, its precise function in cancer has been elusive. In the present study, we report that TIF1γ inactivation causes cells to accumulate chromosomal defects, a hallmark of cancer, due to attenuations in the spindle assembly checkpoint and the post-mitotic checkpoint. TIF1γ deficiency also caused a loss of contact growth inhibition and increased anchorage-independent growth in vitro and in vivo. Clinically, reduced TIF1γ expression in human tumors correlated with an increased rate of genomic rearrangements. Overall, our work indicates that TIF1γ exerts its tumor-suppressive functions in part by promoting chromosomal stability., (©2015 American Association for Cancer Research.)

Despite increasing evidence pointing to the essential involvement of the transforming growth factor beta (TGFB) superfamily in reproduction, a definitive role of TGFB signaling in the uterus remains to be unveiled. In this study, we generated a gain-of-function mouse model harboring a constitutively active (CA) TGFB receptor 1 (TGFBR1), the expression of which was conditionally induced by the progesterone receptor (Pgr)-Cre recombinase. Overactivation of TGFB signaling was verified by enhanced phosphorylation of SMAD2 and increased expression of TGFB target genes in the uterus. TGFBR1 Pgr-Cre CA mice were sterile. Histological, cellular, and molecular analyses demonstrated that constitutive activation of TGFBR1 in the mouse uterus promoted formation of hypermuscled uteri. Accompanying this phenotype was the upregulation of a battery of smooth muscle genes in the uterus. Furthermore, TGFB ligands activated SMAD2/3 and stimulated the expression of a smooth muscle maker gene, alpha smooth muscle actin (ACTA2), in human uterine smooth muscle cells. Immunofluorescence microscopy identified a marked reduction of uterine glands in TGFBR1 Pgr-Cre CA mice within the endometrial compartment that contained myofibroblast-like cells. Thus, constitutive activation of TGFBR1 in the mouse uterus caused defects in uterine morphology and function, as evidenced by abnormal myometrial structure, dramatically reduced uterine glands, and impaired uterine decidualization. These results underscore the importance of a precisely controlled TGFB signaling system in establishing a uterine microenvironment conducive to normal development and function., (© 2015 by the Society for the Study of Reproduction, Inc.)

Tenascin-X is the largest member of the tenascin (TN) family of evolutionary conserved extracellular matrix glycoproteins, which also comprises TN-C, TN-R and TN-W. Among this family, TN-X is the only member described so far to exert a crucial architectural function as evidenced by a connective tissue disorder (a recessive form of Ehlers-Danlos syndrome) resulting from a loss-of-function of this glycoprotein in humans and mice. However, TN-X is more than an architectural protein, as it displays features of a matricellular protein by modulating cell adhesion. However, the cellular functions associated with the anti-adhesive properties of TN-X have not yet been revealed. Recent findings indicate that TN-X is also an extracellular regulator of signaling pathways. Indeed, TN-X has been shown to regulate the bioavailability of the Transforming Growth Factor (TGF)-β and to modulate epithelial cell plasticity. The next challenges will be to unravel whether the signaling functions of TN-X are functionally linked to its matricellular properties.

Background: Nuclear protein 1 (Nupr1) is a major factor in the cell stress response required for Kras(G12D)-driven formation of pancreatic intraepithelial neoplastic lesions (PanINs). We evaluated the relevance of Nupr1 in the development of pancreatic cancer., Methods: We investigated the role of Nupr1 in pancreatic ductal adenocarcinoma (PDAC) progression beyond PanINs in Pdx1-cre;LSL-Kras(G12D);Ink4a/Arf(fl/fl)(KIC) mice., Results: Even in the context of the second tumorigenic hit of Ink4a/Arf deletion, Nupr1 deficiency led to suppression of malignant transformation involving caspase 3 activation in premalignant cells of KIC pancreas. Only half of Nupr1-deficient;KIC mice achieved PDAC development, and incident cases survived longer than Nupr1(wt);KIC mice. This was associated with the development of well-differentiated PDACs in Nupr1-deficient;KIC mice, which displayed enrichment of genes characteristic of the recently identified human classical PDAC subtype. Nupr1-deficient;KIC PDACs also shared with human classical PDACs the overexpression of the Kras-activation gene signature. In contrast, Nupr1(wt);KIC mice developed invasive PDACs with enriched gene signature of human quasi-mesenchymal (QM) PDACs. Cells derived from Nupr1-deficient;KIC PDACs growth in an anchorage-independent manner in vitro had higher aldehyde dehydrogenase activity and overexpressed nanog, Oct-4 and Sox2 transcripts compared with Nupr1(wt);KIC cells. Moreover, Nupr1-deficient and Nurpr1(wt);KIC cells differed in their sensitivity to the nucleoside analogues Ly101-4b and WJQ63. Together, these findings show the pivotal role of Nupr1 in both the initiation and late stages of PDAC in vivo, with a potential impact on PDAC cell stemness., Conclusions: According to Nupr1 status, KIC mice develop tumours that phenocopy human classical or QM-PDAC, respectively, and present differential drug sensitivity, thus becoming attractive models for preclinical drug trials.

Transforming growth factor β (TGF-β) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-β entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-β complex is a crucial step in the regulation of TGF-β function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-β complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-β to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion-dependent mechanism. We identify α11β1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-β and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells.

Several mechanisms underlying tumor progression have remained elusive, particularly in relation to transforming growth factor beta (TGF-β). Although TGF-β initially inhibits epithelial growth, it appears to promote the progression of advanced tumors. Defects in normal TGF-β pathways partially explain this paradox, which can lead to a cascade of downstream events that drive multiple oncogenic pathways, manifesting as several key features of tumorigenesis (uncontrolled proliferation, loss of apoptosis, epithelial-to-mesenchymal transition, sustained angiogenesis, evasion of immune surveillance, and metastasis). Understanding the mechanisms of TGF-β dysregulation will likely reveal novel points of convergence between TGF-β and other pathways that can be specifically targeted for therapy.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 10(6) acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

Background/objectives: Pdx1-Cre; LSL-KRAS(G12D) mice develop premalignant pancreatic ductal lesions that can possibly progress spontaneously to pancreatic ductal adenocarcinoma (PDAC). Although Pdx1-Cre is expressed in the embryonic endoderm, which gives rise to all pancreatic lineages, the possible consequences of KRAS(G12D) expression in the endocrine compartment have never been finely explored., Methods: We examined by histology whether Pdx1-driven expression of KRAS(G12D) could induce islets of Langerhans defects., Results: We observed in Pdx1-Cre; LSL-KRAS(G12D) early disorganization of the endocrine compartment including i) hyperplasia affecting all the endocrine lineages, ii) ectopic onset of Ck19-positive (ductal-like) structures within the endocrine islets, and iii) the presence of islet cells co-expressing glucagon and insulin, all occurring before the onset of ducts lesions., Conclusions: This work indicates that expression of KRAS(G12D) in Pdx1-expressing cells during embryogenesis affects the endocrine pancreas, and highlights the need to deepen possible consequences on both glucose metabolism and PDAC initiation., (Copyright © 2013 IAP and EPC. Published by Elsevier B.V. All rights reserved.)