First-in-human study of GFH018, a small molecule inhibitor of transforming growth factor-ß receptor I inhibitor, in patients with advanced solid tumors.

BACKGROUND: Transforming growth factor-ß (TGF-ß) is a cytokine with multiple functions, including cell growth regulation, extracellular matrix production, angiogenesis homeostasis adjustment and et al. TGF-ß pathway activation promotes tumor metastasis/progression and mediates epithelial-mesenchymal transmission suppressing immunosurveillance in advanced tumors. GFH018, a small molecule inhibitor blocking TGF-ß signal transduction, inhibits the progression and/or metastasis of advanced cancers. This first-in-human study evaluated the safety, tolerability, pharmacokinetics (PK), and efficacy of GFH018 monotherapy in patients with advanced solid tumors. METHODS: This phase I, open-label, multicenter study used a modified 3+3 dose escalation and expansion design. Adult patients with advanced solid tumors failing the standard of care were enrolled. Starting at 5 mg, eight dose levels up to 85 mg were evaluated. Patients received GFH018 BID (14d-on/14d-off) starting on the 4th day after a single dose on cycle 1, day 1. Subsequent cycles were defined as 28 days. The study also explored the safety of 85 mg BID 7d-on/7d-off. Adverse events were graded using NCI criteria for adverse events (NCI-CTCAE v5.0). PK was analyzed using a noncompartmental method. Efficacy was evaluated using RECIST 1.1. Blood samples were collected for biomarker analysis. RESULTS: Fifty patients were enrolled and received at least one dose of GFH018. No dose-limiting toxicity occurred, and the maximum tolerated dose was not reached. Forty-three patients (86.0%) had at least one treatment-related adverse event (TRAE), and three patients (6.0%) had ≥ G3 TRAEs. The most common TRAEs (any grade/grade ≥3) were AST increased (18%/0%), proteinuria (14%/2%), anemia (14%/2%), and ALT increased (12%/0%). No significant cardiotoxicity or bleeding was observed. GFH018 PK was linear and dose-independent, with a mean half-life of 2.25-8.60 h from 5 - 85 mg. Nine patients (18.0%) achieved stable disease, and one patient with thymic carcinoma achieved tumor shrinkage, with the maximum target lesion decreased by 18.4%. Serum TGF-ß1 levels were not associated with clinical responses. The comprehensive recommended dose for Phase II was defined as 85 mg BID 14d-on/14d-off. CONCLUSIONS: GFH018 monotherapy presented a favorable safety profile without cardiac toxicity or bleeding. Modest efficacy warrants further studies, including combination strategies. TRIAL REGISTRATION: ClinicalTrial. gov ( https://www. CLINICALTRIALS: gov/ ), NCT05051241. Registered on 2021-09-02.

Targeting the TGF-ß signaling pathway: an updated patent review (2021-present).

INTRODUCTION: The TGF-ß signaling pathway is a complex network that plays a crucial role in regulating essential biological functions and is implicated in the onset and progression of multiple diseases. This review highlights the recent advancements in developing inhibitors targeting the TGF-ß signaling pathway and their potential therapeutic applications in various diseases. AREA COVERED: The review discusses patents on active molecules related to the TGF-ß signaling pathway, focusing on three strategies: TGF-ß activity inhibition, blocking TGF-ß receptor binding, and disruption of the signaling pathway using small molecule inhibitors. Combination therapies and the development of fusion proteins targeting multiple pathways are also explored. The literature search was conducted using the Cortellis Drug Discovery Intelligence database, covering patents from 2021 onwards. EXPERT OPINION: The development of drugs targeting the TGF-ß signaling pathway has made significant progress in recent years. However, addressing challenges such as specificity, systemic toxicity, and patient selection is crucial for their successful clinical application. Targeting the TGF-ß signaling pathway holds promise as a promising approach for the treatment of various diseases.

Synthesis and anti-liver fibrosis activity of imidazole and thiazole compounds containing amino acids.

Four series of imidazoles (15a-g, 20c, and 20d) and thiazoles (18a-g, 22a, and 22b) possessing various amino acids were synthesized and evaluated for activin receptor-like kinase 5 (ALK5) inhibitory activities in an enzymatic assay. Among them, compounds 15g and 18c showed the highest inhibitory activity against ALK5, with IC50 values of 0.017 and 0.025 µM, respectively. Compounds 15g and 18c efficiently inhibited extracellular matrix (ECM) deposition in TGF-ß-induced hepatic stellate cells (HSCs), and eventually suppressed HSC activation. Moreover, compound 15g showed a good pharmacokinetic (PK) profile with a favorable half-life (t1/2 = 9.14 h). The results indicated that these compounds exhibited activity targeting ALK5 and may have potential in the treatment of liver fibrosis; thus they are worthy of further study.

Riboflavin protects against pancreatic cancer metastasis by targeting TGF-ß receptor 1.

The inhibition of transforming growth factor-ß1 (TGF-ß1) signaling by targeting TGF-ß receptor 1 (TßR1) has been considered as an ideal approach for the prevention of pancreatic cancer metastasis. Utilizing a pharmacophore model for TßR1 inhibitors, candidate compounds with the potential TßR1 binding ability were screened from the U.S. Food and Drug Administration (FDA) database, and riboflavin (RF) with a highest fit value was chosen to investigate its binding ability to TßR1 and effect on TGF-ß1 signaling in pancreatic cancer cells. Molecular docking and cellular thermal shift assay (CETSA) proved that RF at pharmacological concentrations could directly bind to TßR1. Further studies showed that pharmacological concentrations of RF in vitro could block TGF-ß1 signaling, suppress the migration and invasion, and prevent epithelial-mesenchymal transition (EMT) process of pancreatic cancer cells in the absence or presence of TGF-ß1 stimulation, indicating that RF presented anti-metastatic effect in pancreatic cancer cells. Knockdown of TßR1 could significantly attenuate the effects of RF on the migration and EMT process in pancreatic cancer cells, further confirming that the anti-metastatic effect of RF was achieved by blocking TGF-ß1 signaling after binding to TßR1. Moreover, in a mouse model of pancreatic cancer metastasis, it was certified that RF administration could block lung and liver metastases, TGF-ß1 signaling and EMT process of pancreatic cancer in vivo. In summary, our findings showed that RF could block TGF-ß1 signaling by directly binding to TßR1, thereby suppressing the metastasis of pancreatic cancer cells by inhibiting EMT process both in vitro and in vivo.

Radiation-induced effects on TGF-ß and PDGF receptor signaling in cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) consist of heterogeneous connective tissue cells and are often constituting the most abundant cell type in the tumor stroma. Radiation effects on tumor stromal components like CAFs in the context of radiation treatment is not well-described. AIM: This study explores potential changes induced by ionizing radiation (IR) on platelet-derived growth factor (PDGF)/PDGFRs and transforming growth factor-beta (TGF-ß)/TGFßRs signaling systems in CAFs. METHODS AND RESULTS: Experiments were carried out by employing primary cultures of human CAFs isolated from freshly resected non-small cell lung carcinoma tumor tissues. CAF cultures from nine donors were treated with one high (1 × 18 Gy) or three fractionated (3 × 6 Gy) radiation doses. Alterations in expression levels of TGFßRII and PDGFRα/ß induced by IR were analyzed by western blots and flow cytometry. In the presence or absence of cognate ligands, receptor activation was studied in nonirradiated and irradiated CAFs. Radiation exposure did not exert changes in expression of PDGF or TGF-ß receptors in CAFs. Additionally, IR alone was unable to trigger activation of either receptor. The radiation regimens tested did not affect PDGFRß signaling in the presence of PDGF-BB. In contrast, signaling via pSmad2/3 and pSmad1/5/8 appeared to be down-regulated in irradiated CAFs after stimulation with TGF-ß, as compared with controls. CONCLUSION: Our data demonstrate that IR by itself is insufficient to induce measurable changes in PDGF or TGF-ß receptor expression levels or to induce receptor activation in CAFs. However, in the presence of their respective ligands, exposure to radiation at certain doses appear to interfere with TGF-ß receptor signaling.

Glycosaminoglycan modifications of betaglycan regulate ectodomain shedding to fine-tune TGF-ß signaling responses in ovarian cancer.

In pathologies including cancer, aberrant Transforming Growth Factor-ß (TGF-ß) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-ß responses. Betaglycan/type III TGF-ß receptor (TßRIII), is an established co-receptor for the TGF-ß superfamily known to bind directly to TGF-ßs 1-3 and inhibin A/B. Betaglycan can be membrane-bound and also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. Its extracellular domain undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. We report the unexpected discovery that the heparan sulfate glycosaminoglycan chains on betaglycan are critical for the ectodomain shedding. In the absence of such glycosaminoglycan chains betaglycan is not shed, a feature indispensable for the ability of betaglycan to suppress TGF-ß signaling and the cells' responses to exogenous TGF-ß ligands. Using unbiased transcriptomics, we identified TIMP3 as a key inhibitor of betaglycan shedding thereby influencing TGF-ß signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-ß signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan chains of betaglycan for shedding and influence on TGF-ß signaling responses. Dysregulated shedding of TGF-ß receptors plays a vital role in determining the response and availability of TGF-ßs', which is crucial for prognostic predictions and understanding of TGF-ß signaling dynamics.

Prodigiosin Inhibits Transforming Growth Factor ß Signaling by Interfering Receptor Recycling and Subcellular Translocation in Epithelial Cells.

Prodigiosin (PG) is a naturally occurring polypyrrole red pigment produced by numerous microorganisms including some Serratia and Streptomyces strains. PG has exhibited promising anticancer activity; however, the molecular mechanisms of action of PG on malignant cells remain ambiguous. Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that governs a wide array of cellular processes in development and tissue homeostasis. Malfunctions of TGF-ß signaling are associated with numerous human cancers. Emerging evidence underscores the significance of internalized TGF-ß receptors and their intracellular trafficking in initiating signaling cascades. In this study, we identified PG as a potent inhibitor of the TGF-ß pathway. PG blocked TGF-ß signaling by targeting multiple sites of this pathway, including facilitating the sequestering of TGF-ß receptors in the cytoplasm by impeding the recycling of type II TGF-ß receptors to the cell surface. Additionally, PG prompts a reduction in the abundance of receptors on the cell surface through the disruption of the receptor glycosylation. In human Caucasian lung carcinoma cells and human hepatocellular cancer cell line cells, nanomolar concentrations of PG substantially diminish TGF-ß-triggered phosphorylation of Smad2 protein. This attenuation is further reflected in the suppression of downstream target gene expression, including those encoding fibronectin, plasminogen activator inhibitor-1, and N-cadherin. SIGNIFICANCE STATEMENT: Prodigiosin (PG) emerges from this study as a potent TGF-ß pathway inhibitor, disrupting receptor trafficking and glycosylation and reducing TGF-ß signaling and downstream gene expression. These findings not only shed light on PG's potential therapeutic role but also present a captivating avenue towards future anti-TGF-ß strategies.

Cholesterol modulates type I/II TGF-ß receptor complexes and alters the balance between Smad and Akt signaling in hepatocytes.

Cholesterol mediates membrane compartmentalization, affecting signaling via differential distribution of receptors and signaling mediators. While excessive cholesterol and aberrant transforming growth factor-ß (TGF-ß) signaling characterize multiple liver diseases, their linkage to canonical vs. non-canonical TGF-ß signaling remained unclear. Here, we subjected murine hepatocytes to cholesterol depletion (CD) or enrichment (CE), followed by biophysical studies on TGF-ß receptor heterocomplex formation, and output to Smad2/3 vs. Akt pathways. Prior to ligand addition, raft-dependent preformed heteromeric receptor complexes were observed. Smad2/3 phosphorylation persisted following CD or CE. CD enhanced phospho-Akt (pAkt) formation by TGF-ß or epidermal growth factor (EGF) at 5 min, while reducing it at later time points. Conversely, pAkt formation by TGF-ß or EGF was inhibited by CE, suggesting a direct effect on the Akt pathway. The modulation of the balance between TGF-ß signaling to Smad2/3 vs. pAkt (by TGF-ß or EGF) has potential implications for hepatic diseases and malignancies.

68Ga-FAPI PET imaging monitors response to combined TGF-ßR inhibition and immunotherapy in metastatic colorectal cancer.

BACKGROUNDImproving and predicting tumor response to immunotherapy remains challenging. Combination therapy with a transforming growth factor-ß receptor (TGF-ßR) inhibitor that targets cancer-associated fibroblasts (CAFs) is promising for the enhancement of efficacy of immunotherapies. However, the effect of this approach in clinical trials is limited, requiring in vivo methods to better assess tumor responses to combination therapy.METHODSWe measured CAFs in vivo using the 68Ga-labeled fibroblast activation protein inhibitor-04 (68Ga-FAPI-04) for PET/CT imaging to guide the combination of TGF-ß inhibition and immunotherapy. One hundred thirty-one patients with metastatic colorectal cancer (CRC) underwent 68Ga-FAPI and 18F-fluorodeoxyglucose (18F-FDG) PET/CT imaging. The relationship between uptake of 68Ga-FAPI and tumor immunity was analyzed in patients. Mouse cohorts of metastatic CRC were treated with the TGF-ßR inhibitor combined with KN046, which blocks programmed death ligand 1 (PD-L1) and CTLA-4, followed by 68Ga-FAPI and 18F-FDG micro-PET/CT imaging to assess tumor responses.RESULTSPatients with metastatic CRC demonstrated high uptake rates of 68Ga-FAPI, along with suppressive tumor immunity and poor prognosis. The TGF-ßR inhibitor enhanced tumor-infiltrating T cells and significantly sensitized metastatic CRC to KN046. 68Ga-FAPI PET/CT imaging accurately monitored the dynamic changes of CAFs and tumor response to combined the TGF-ßR inhibitor with immunotherapy.CONCLUSION68Ga-FAPI PET/CT imaging is powerful in assessing tumor immunity and the response to immunotherapy in metastatic CRC. This study supports future clinical application of 68Ga-FAPI PET/CT to guide precise TGF-ß inhibition plus immunotherapy in CRC patients, recommending 68Ga-FAPI and 18F-FDG dual PET/CT for CRC management.TRIAL REGISTRATIONCFFSTS Trial, ChiCTR2100053984, Chinese Clinical Trial Registry.FUNDINGNational Natural Science Foundation of China (82072695, 32270767, 82272035, 81972260).

Advanced biosensors for nanomaterial-based detection of transforming growth factor alpha and beta, a class of major polypeptide regulators.

Transforming growth factors (TGFs) regulate several cellular processes including, differentiation, growth, migration, extracellular matrix production, and apoptosis. TGF alpha (TGF-α) is a heterogeneous molecule containing 160 amino acid residues. It is a potent angiogenesis promoter that is activated by JAK-STAT signaling. Whereas TGF beta (TGF-ß) consists of 390-412 amino acids. Smad and non-Smad signaling both occur in TGF beta. It is linked to immune cell activation, differentiation, and proliferation. It also triggers pre-apoptotic responses and inhibits cell proliferation. Both growth factors have a promising role in the development and homeostasis of tissues. Defects such as autoimmune diseases and cancer develop mechanisms to modulate checkpoints of the immune system resulting in altered growth factors profile. An accurate amount of these growth factors is essential for normal functioning, but an exceed or fall behind the normal level is alarming as it is linked to several disorders. This demands techniques for TGF-α and TGF-ß profiling to effectively diagnose diseases, monitor their progression, and assess the efficacy of immunotherapeutic drugs. Quantitative detection techniques including the emergence of biosensing technology seem to accomplish the purpose. Until the present time, few biosensors have been designed in the context of TGF-α and TGF-ß for disease detection, analyzing receptor binding, and interaction with carriers. In this paper, we have reviewed the physiology of transforming growth factor alpha and beta, including the types, structure, function, latent/active forms, signaling, and defects caused. It involves the description of biosensors on TGF-α and TGF-ß, advances in technology, and future perspectives.

Inhibition of transforming growth factor-ß signals suppresses tumor formation by regulation of tumor microenvironment networks.

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-ß (TGF-ß) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-ß receptor containing both TGF-ß type I (TßRI) and type II (TßRII) receptors (TßRI-TßRII-Fc), which trapped all TGF-ß isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TßRI-TßRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TßRI-TßRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TßRI-TßRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1ß (IL-1ß) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1ß and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-ß signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1ß/EREG pathways and that TßRI-TßRII-Fc protein is a promising tool for targeting the TME networks.

The usefulness of the genetic panel in the classification and refinement of diagnostic accuracy of Mexican patients with Marfan syndrome and other connective tissue disorders.

Marfan syndrome (MFS) is a multisystem genetic disorder with over 3000 mutations described in the fibrillin 1 (FBN1) gene. Like MFS, other connective tissue disorders also require a deeper understanding of the phenotype-genotype relationship due to the complexity of the clinical presentation, where diagnostic criteria often overlap. Our objective was to identify mutations in patients with connective tissue disorders using a genetic multipanel and to analyze the genotype-phenotype associations in a cohort of Mexican patients. We recruited 136 patients with MFS and related syndromes from the National Institute of Cardiology. Mutations were identified using next-generation sequencing (NGS). To examine the correlation between mutation severity and severe cardiovascular conditions, we focused on patients who had undergone Bentall-de Bono surgery or aortic valve repair. The genetic data obtained allowed us to reclassify the initial clinical diagnosis across various types of connective tissue disorders. The transforming growth factor beta receptor 2 (TGFBR2) rs79375991 mutation was found in 10 out of 16 (63%) Loeys-Dietz patients. We observed a high prevalence (65%) of more severe mutations, such as frameshift indels and stop codons, among patients requiring invasive treatments like aortic valve-sparing surgery, Bentall and de Bono procedures, or aortic valve replacement due to severe cardiovascular injury. Although our study did not achieve precise phenotype-genotype correlations, it underscores the importance of a multigenetic panel evaluation. This could pave the way for a more comprehensive diagnostic approach and inform medical and surgical treatment decision-making.

17ß-Estradiol mediates TGFBR3/Smad2/3 signaling to attenuate the fibrosis of TGF-ß1-induced bovine endometrial epithelial cells via GPER.

Abnormal function and fibrosis of endometrium caused by cows' endometritis pose difficult implantation of embryos and uterine cavity adhesions. 17ß-Estradiol (E2) serves as the most effective aromatized estrogen, and its synthetase and receptors have been detected in the endometrium. Studies have demonstrated the positive role of estrogen in combating pathological fibrosis in diverse diseases. However, it is still unknown whether E2 regulates endometrium fibrosis in bovine endometritis. Herein, we evaluated the expression patterns of transforming growth factor-ß1 (TGF-ß1), epithelial-mesenchymal transformation (EMT)-related proteins (α-SMA, vimentin N-cadherin and E-cadherin), cytochrome P450 19A1 (CYP19A1), and G protein-coupled estrogen receptor (GPER) in bovine healthy endometrium and Inflammatory endometrium. Our data showed that the inflamed endometrium presented low CYP19A1 and GPER expression, and significantly higher EMT process versus the normal tissue. Moreover, we established a TGF-ß1-induced fibrosis model in BEND cells, and found that E2 inhibited the EMT process of BEND cells in a dose-dependent manner. The anti-fibrotic effect of E2 was blocked by the GPER inhibitor G15, but not the estrogen nuclear receptors (ERs) inhibitor ICI182780. Moreover, the GPER agonist G1 inhibited fibrosis and Smad2/3 phosphorylation but increased the expression of TGFBR3 in BEND cells. Transfection with TGFBR3 small interfering RNA blocked the effect of G1 on fibrosis of BEND cells and upregulated the expression of P-Smad2/3. Our in vivo data also showed that E2 and G1 affected uterus fibrosis in mice endometritis model caused by LPS, which was associated with the inhibition of TGFBR3/Smad2/3 signaling. In conclusion, our data implied that E2 alleviates the fibrosis of TGF-ß1-induced BEND cells, which is associated with the GPER mediation of TGFBR3/Smad2/3 signaling.

Chimeric TßRII-SE/Fc overexpression by a lentiviral vector exerts strong antitumoral activity on colorectal cancer-derived cell lines in vitro and on xenografts.

The TGF signaling pathway is a key regulator of cancer progression. In this work, we report for the first time the antitumor activity of TßRII-SE/Fc, a novel peptibody whose targeting domain is comprised of the soluble endogenous isoform of the human TGF-ß type II receptor (TßRII-SE). Overexpression of TßRIISE/Fc reduces in vitro cell proliferation and migration while inducing cell cycle arrest and apoptosis in human colorectal cancer-derived cell lines. Moreover, TßRII-SE/Fc overexpression reduces tumorigenicity in BALB/c nude athymic mice. Our results revealed that TRII-SE/Fc-expressing tumors were significantly reduced in size or were even incapable of developing. We also demonstrated that the novel peptibody has the ability to inhibit the canonical TGF-ß and BMP signaling pathways while identifying SMAD-dependent and independent proteins involved in tumor progression that are modulated by TßRII-SE/Fc. These findings provide insights into the underlying mechanism responsible for the antitumor activity of TßRII-SE/Fc. Although more studies are required to demonstrate the effectiveness and safety of the novel peptibody as a new therapeutic for the treatment of cancer, our initial in vitro and in vivo results in human colorectal tumor-derived cell lines are highly encouraging. Our results may serve as the foundation for further research and development of a novel biopharmaceutical for oncology.

Hepsin promotes breast tumor growth signaling via the TGFß-EGFR axis.

Hepsin, a type II transmembrane serine protease, is commonly overexpressed in prostate and breast cancer. The hepsin protein is stabilized by the Ras-MAPK pathway, and, downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, such as the hepatocyte growth factor (HGF) and transforming growth factor beta (TGFß) pathway. However, how exactly active hepsin promotes cell proliferation machinery to sustain tumor growth is not fully understood. Here, we show that genetic deletion of the gene encoding hepsin (Hpn) in a WAP-Myc model of aggressive MYC-driven breast cancer inhibits tumor growth in the primary syngrafted sites and the growth of disseminated tumors in the lungs. The suppression of tumor growth upon loss of hepsin was accompanied by downregulation of TGFß and EGFR signaling together with a reduction in epidermal growth factor receptor (EGFR) protein levels. We further demonstrate in 3D cultures of patient-derived breast cancer explants that both basal TGFß signaling and EGFR protein expression are inhibited by neutralizing antibodies or small-molecule inhibitors of hepsin. The study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGFß and EGFR pathways, warranting consideration of hepsin as a potential indirect upstream target for therapeutic inhibition of TGFß and EGFR pathways in cancer.

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

The transforming growth factor ß (TGFß) 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 TGFß 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.

Simultaneously targeting extracellular vesicle trafficking and TGF-ß receptor kinase activity blocks signaling hyperactivation and metastasis.

Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-ß) signaling drives metastasis and is strongly enhanced during cancer progression. Yet, the use of on-target TGF-ß signaling inhibitors in the treatment of cancer patients remains unsuccessful, highlighting a gap in the understanding of TGF-ß biology that limits the establishment of efficient anti-metastatic therapies. Here, we show that TGF-ß signaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle (sEV) secretion. Demonstrating sEV's unique role, TGF-ß signaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-ß. Further, genetic disruption of sEV secretion in highly-metastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-ß signaling to nearly-normal levels. Otherwise, TGF-ß signaling activity in non-invasive breast cancer cells is inherently low, but can be amplified by sEVs, enabling invasion and metastasis of poorly-metastatic breast cancer cells. Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers, treatment with dimethyl amiloride (DMA) decreases sEV secretion, TGF-ß signaling activity, and breast cancer progression in vivo. Targeting both the sEV trafficking and TGF-ß signaling by combining DMA and SB431542 at suboptimal doses potentiated this effect, normalizing the TGF-ß signaling in primary tumors to potently reduce circulating tumor cells, metastasis, and tumor self-seeding. Collectively, this study establishes sEVs as critical elements in TGF-ß biology, demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-ß signaling levels in breast cancer cells.

Anti-Müllerian Hormone promotes human osteoblast differentiation and calcification by modulating osteogenic gene expression.

OBJECTIVE: To investigate whether the Anti-Müllerian Hormone (AMH), an ovarian hormone belonging to the Transforming Growth Factor ß superfamily, may represent a possible candidate for use as a bone anabolic factor. METHODS: We performed in vitro studies on Human Osteoblasts (HOb) to evaluate the expression and the functionality of AMHRII, the AMH receptor type-2, and investigate the effects of exogenous AMH exposure on osteogenic gene expression and osteoblast functions. RESULTS: We reported the first evidence for the expression and functionality of AMHRII in HOb cells, thus suggesting that osteoblasts may represent a specific target for exogenous AMH treatment. Furthermore, the exposure to AMH exerted a stimulatory effect on HOb cells leading to the activation of osteogenic genes, including the upregulation of osteoblastic transcription factors such as RUNX and OSX, along with increased deposition of mineralized nodules. CONCLUSION: Our findings proved interesting clues on the stimulatory effects of AMH on mature osteoblasts expressing its specific receptor, AMHRII. This study may therefore have translation value in opening the perspective that AMH may be an effective candidate to counteract the bone loss in osteoporotic patients by selectively targeting osteoblast with minimal off-target effect.

Roles of DSCC1 and GINS1 in gastric cancer.

Gastric carcinoma is a common malignant tumor originating from gastric mucosal epithelium. However, role of DS-cell cycle-dependent protein 1 (DSCC1) and GINS1 in gastric carcinoma remains unclear. The gastric carcinoma datasets GSE79973 and GSE118916 were downloaded from gene expression omnibus. Multiple datasets were merged and batched. Differentially expressed genes (DEGs) were screened and weighted gene co-expression network analysis was performed. Functional enrichment analysis, gene set enrichment analysis and immune infiltration analysis were performed. Construction and analysis of protein-protein interaction Network. Survival analysis and comparative toxicogenomics database were performed. A heat map of gene expression was drawn. Target Scan screen miRNAs regulating DEGs. Two thousand forty-four DEGs were identified. According to gene ontology analysis, in biological process, they were mainly enriched in cell migration, transforming growth factor ß receptor signaling pathway, angiogenesis, and steroid metabolism process. In cellular component, they were mainly enriched in extracellular vesicles, basement membrane, endoplasmic reticulum lumen, and extracellular space. In molecular function, they focused on extracellular matrix structural components, protein binding, platelet-derived growth factor binding, and catalytic activity. In Kyoto encyclopedia of genes and genomes, they were mainly enriched in protein digestion and absorption, metabolic pathways, fatty acid degradation, Glycerophospholipid metabolism, ether lipid metabolism. Gene set enrichment analysis showed that DEGs were mainly enriched in transforming growth factor ß receptor signaling pathway, steroid metabolism process, basement membrane, endoplasmic reticulum lumen, structural components of extracellular matrix, platelet-derived growth factor binding, Glycerophospholipid metabolism, ether lipid metabolism. The results of immune infiltration analysis showed that expression of T cell CD4 memory resting was lower in the samples of gastric cancer. The core genes (TRIP13, CHEK1, DSCC1, GINS1) are protective factors, their expression shows a downward trend with increase of risk score. Comparative toxicogenomics database analysis showed that TRIP13, CHEK1, DSCC1, GINS1 were related to gastric tumors, gastric diseases, tumors, inflammation, and necrosis. DSCC1 and GINS1 are highly expressed in gastric cancer. Higher expression levels of DSCC1 and GINS1, worse the prognosis.

MicroRNA-499-5p inhibits transforming growth factor-ß1-induced Smad2 signaling pathway and suppresses fibroblast proliferation and collagen synthesis in rat by targeting TGFß-R1.

BACKGROUND: Artial fibrosis has been recognized as a typical pathological change in atrial fibrillation. Although present evidence suggests that microRNA-499-5p (miR-499-5p) plays an important role in the development of atrial fibrosis, the specific mechanism is not fully understood. Therefore, this study attempted to assess the influence of miR-499-5p on atrial fibroblasts and explore the potential molecular mechanism. METHODS: Atrial fibroblasts from sprague dawley rat were respectively transfected with miR-499-5p mimic, miR-499-5p negative control and miR-499-5p inhibitor, atrial fibroblasts without any treatment were also established. Cell counting kit-8 assay and transwell assay were used to detect the proliferation and migration of atrial fibroblasts in each group. Expressions of miR-499-5p, TGF-ß1, smad2, α-SMA, collagen-I and TGFß-R1 in mRNA and protein level were subsequently detected via quantitative real-time polymerase chain reaction and western blot. Furthermore, the prediction of the binding sites of miR-499-5p and TGFß-R1 was performed via the bioinformatics online software TargetScan and verified by dual luciferase reporter. RESULTS: By utilizing miR-499-5p-transfected atrial fibroblasts model, expression of miR-499-5p in the miR-499-5p mimic group was upregulated, while it was downregulated in the miR-499-5p inhibitors group. Upregulated miR-499-5p expression led to to a significant decrease in the proliferative and migratory ability of cultured atrial fibroblasts, while downregulated miR-499-5p expression led to a significant increase in the proliferative and migratory ability of cultured atrial fibroblasts. Additionally, upregulated miR-499-5p expression made a significant rise in TGF-ß1-induced mRNA and protein expression of TGF-ß1, TGFß-R1, smad2, α-SMA and collagen-I in atrial fibroblasts. Furthermore, results from the dual luciferase reporter conformed that miR-499-5p may repress TGFß-R1 by binding the 3'UTR of TGFß-R1 directly. CONCLUSIONS: miR-499-5p is able to inhibit the activation of transforming growth factor ß-induced Smad2 signaling and eventually suppressed the proliferation, migration and invasion of atrial fibroblasts and collagen synthesis by targeting TGFß-R1.