Inhibition Of TGF-β Signaling Research Articles

Ultrasmall Polyphenol-NAD+ Nanoparticle-Mediated Renal Delivery for Mitochondrial Repair and Anti-Inflammatory Treatment of AKI-to-CKD Progression.

As a central metabolic molecule, nicotinamide adenine dinucleotide (NAD+) can potentially treat acute kidney injury (AKI) and chronic kidney disease (CKD); however, its bioavailability is poor due to short half-life, instability, the deficiency of targeting, and difficulties in transmembrane transport. Here we designed a physiologically adaptive gallic acid-NAD+ nanoparticle, that had ultrasmall size and pH-responsiveness, passed through the glomerular filtration membrane to reach injured renal tubules, and efficiently delivered NAD+ into the kidneys. With an effective accumulation in the kidneys, it restored renal function, immune microenvironment homeostasis, and mitochondrial homeostasis of AKI mice via the NAD+-Sirt1 axis, and exerted strong antifibrotic effects on the AKI-to-CKD transition by inhibiting TGF-β signaling. It also exhibited excellent stability, biodegradable, and biocompatible properties, ensuring its long-term safety, practicality, and clinical translational feasibility. The present study shows a potential modality of mitochondrial repair and immunomodulation through nanoagents for the efficient and safe treatment of AKI and CKD. This article is protected by copyright. All rights reserved.

LP340, a novel histone deacetylase inhibitor, decreases liver injury and fibrosis in mice: role of oxidative stress and microRNA-23a

Effective therapy for liver fibrosis is lacking. Here, we examined whether LP340, the lead candidate of a new-generation of hydrazide-based HDAC1,2,3 inhibitors (HDACi), decreases liver fibrosis. Liver fibrosis was induced by CCl4 treatment and bile duct ligation (BDL) in mice. At 6 weeks after CCl4, serum alanine aminotransferase increased, and necrotic cell death and leukocyte infiltration occurred in the liver. Tumor necrosis factor-α and myeloperoxidase markedly increased, indicating inflammation. After 6 weeks, α-smooth muscle actin (αSMA) and collagen-1 expression increased by 80% and 575%, respectively, indicating hepatic stellate cell (HSC) activation and fibrogenesis. Fibrosis detected by trichrome and Sirius-red staining occurred primarily in pericentral regions with some bridging fibrosis in liver sections. 4-Hydroxynonenal adducts (indicator of oxidative stress), profibrotic cytokine transforming growth factor-β (TGFβ), and TGFβ downstream signaling molecules phospho-Smad2/3 also markedly increased. LP340 attenuated indices of liver injury, inflammation, and fibrosis markedly. Moreover, Ski-related novel protein-N (SnoN), an endogenous inhibitor of TGFβ signaling, decreased, whereas SnoN expression suppressor microRNA-23a (miR23a) increased markedly. LP340 (0.05 mg/kg, ig., daily during the last 2 weeks of CCl4 treatment) decreased 4-hydroxynonenal adducts and miR23a production, blunted SnoN decreases, and inhibited the TGFβ/Smad signaling. By contrast, LP340 had no effect on matrix metalloproteinase-9 expression. LP340 increased histone-3 acetylation but not tubulin acetylation, indicating that LP340 inhibited Class-I but not Class-II HDAC in vivo. After BDL, focal necrosis, inflammation, ductular reactions, and portal and bridging fibrosis occurred at 2 weeks, and αSMA and collagen-1 expression increased by 256% and 560%, respectively. LP340 attenuated liver injury, ductular reactions, inflammation, and liver fibrosis. LP340 also decreased 4-hydroxynonenal adducts and miR23a production, prevented SnoN decreases, and inhibited the TGFβ/Smad signaling after BDL. In vitro, LP340 inhibited immortal human hepatic stellate cells (hTERT-HSC) activation in culture (αSMA and collagen-1 expression) as well as miR23a production, demonstrating its direct inhibitory effects on HSC. In conclusions, LP340 is a promising therapy for both portal and pericentral liver fibrosis, and it works by inhibiting oxidative stress and decreasing miR23a.

Suppression of the TGF-β signaling exacerbates degeneration of auditory neurons in kanamycin-induced ototoxicity in mice

Transforming growth factor-β (TGF-β) signaling plays a significant role in multiple biological processes, including inflammation, immunity, and cell death. However, its specific impact on the cochlea remains unclear. In this study, we aimed to investigate the effects of TGF-β signaling suppression on auditory function and cochlear pathology in mice with kanamycin-induced ototoxicity. Kanamycin and furosemide (KM-FS) were systemically administered to 8-week-old C57/BL6 mice, followed by immediate topical application of a TGF-β receptor inhibitor (TGF-βRI) onto the round window membrane. Results showed significant TGF-β receptor upregulation in spiral ganglion neurons (SGNs) after KM-FA ototoxicity, whereas expression levels in the TGF-βRI treated group remained unchanged. Interestingly, despite no significant change in cochlear TGF-β expression after KM-FS ototoxicity, TGF-βRI treatment resulted in a significant decrease in TGF-β signaling. Regarding auditory function, TGF-βRI treatment offered no therapeutic effects on hearing thresholds and hair cell survival following KM-FS ototoxicity. However, SGN loss and macrophage infiltration were significantly increased with TGF-βRI treatment. These results imply that inhibition of TGF-β signaling after KM-FS ototoxicity promotes cochlear inflammation and SGN degeneration.

MAD1 deficiency accelerates hepatocellular proliferation via suppressing TGF-β signaling

Numerous researches have reported on the regulatory network of liver regeneration induced by partial hepatectomy (PH). However, information on key molecules and/or signaling pathways regulating the termination stage of liver regeneration remains limited. In this study, we identify hepatic mitotic arrest deficient 1 (MAD1) as a crucial regulator of transforming growth factor β (TGF-β) in the hepatocyte to repress liver regeneration. MAD1 has a low expression level at the rapid proliferation phase but significantly increases at the termination phase of liver regeneration. We show that MAD1 deficiency accelerates hepatocyte proliferation and enhances mitochondrial biogenesis and respiratory. Mechanistically, MAD1 deficiency in hepatocytes enhances mitochondrial function and promotes hepatocyte proliferation by suppressing TGF-β signaling. Our study reveals MAD1 as a novel suppressor of hepatocyte proliferation, which may provide a new therapeutic target for the recovery of liver function after liver transplant and partial hepatectomy.

Inhibition of MERTK reduces organ fibrosis in mouse models of fibrotic disease.

Transforming growth factor-β (TGFβ) drives fibrosis and disease progression in a number of chronic disorders, but targeting this ubiquitously expressed cytokine may not yield a viable and safe antifibrotic therapy. Here, we sought to identify alternative ways to inhibit TGFβ signaling using human hepatic stellate cells and macrophages from humans and mice in vitro, as well as mouse models of liver, kidney, and lung fibrosis. We identified Mer tyrosine kinase (MERTK) as a TGFβ-inducible effector of fibrosis that was up-regulated during fibrosis in multiple organs in three mouse models. We confirmed these findings in liver biopsy samples from patients with metabolic dysfunction-associated fatty liver disease (MAFLD). MERTK also induced TGFβ expression and drove TGFβ signaling resulting in a positive feedback loop that promoted fibrosis in cultured cells. MERTK regulated both canonical and noncanonical TGFβ signaling in both mouse and human cells in vitro. MERTK increased transcription of genes regulating fibrosis by modulating chromatin accessibility and RNA polymerase II activity. In each of the three mouse models, disrupting the fibrosis-promoting signaling loop by reducing MERTK expression reduced organ fibrosis. Pharmacological inhibition of MERTK reduced fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis was established. Together, these data suggest that MERTK plays a role in modulating organ fibrosis and may be a potential target for treating fibrotic diseases.

BRCC3 Regulation of ALK2 in Vascular Smooth Muscle Cells: Implication in Pulmonary Hypertension.

An imbalance of antiproliferative BMP (bone morphogenetic protein) signaling and proliferative TGF-β (transforming growth factor-β) signaling is implicated in the development of pulmonary arterial hypertension (PAH). The posttranslational modification (eg, phosphorylation and ubiquitination) of TGF-β family receptors, including BMPR2 (bone morphogenetic protein type 2 receptor)/ALK2 (activin receptor-like kinase-2) and TGF-βR2/R1, and receptor-regulated (R) Smads significantly affects their activity and thus regulates the target cell fate. BRCC3 modifies the activity and stability of its substrate proteins through K63-dependent deubiquitination. By modulating the posttranslational modifications of the BMP/TGF-β-PPARγ pathway, BRCC3 may play a role in pulmonary vascular remodeling, hence the pathogenesis of PAH. Bioinformatic analyses were used to explore the mechanism of BRCC3 deubiquitinates ALK2. Cultured pulmonary artery smooth muscle cells (PASMCs), mouse models, and specimens from patients with idiopathic PAH were used to investigate the rebalance between BMP and TGF-β signaling in regulating ALK2 phosphorylation and ubiquitination in the context of pulmonary hypertension. BRCC3 was significantly downregulated in PASMCs from patients with PAH and animals with experimental pulmonary hypertension. BRCC3, by de-ubiquitinating ALK2 at Lys-472 and Lys-475, activated receptor-regulated Smad1/5/9 (Smad1/5/9), which resulted in transcriptional activation of BMP-regulated PPARγ, p53, and Id1. Overexpression of BRCC3 also attenuated TGF-β signaling by downregulating TGF-β expression and inhibiting phosphorylation of Smad3. Experiments in vitro indicated that overexpression of BRCC3 or the de-ubiquitin-mimetic ALK2-K472/475R attenuated PASMC proliferation and migration and enhanced PASMC apoptosis. In SM22α-BRCC3-Tg mice, pulmonary hypertension was ameliorated because of activation of the ALK2-Smad1/5-PPARγ axis in PASMCs. In contrast, Brcc3-/- mice showed increased susceptibility of experimental pulmonary hypertension because of inhibition of the ALK2-Smad1/5 signaling. These results suggest a pivotal role of BRCC3 in sustaining pulmonary vascular homeostasis by maintaining the integrity of the BMP signaling (ie, the ALK2-Smad1/5-PPARγ axis) while suppressing TGF-β signaling in PASMCs. Such rebalance of BMP/TGF-β pathways is translationally important for PAH alleviation.

Cyclic strain alters the transcriptional and migratory response of scleral fibroblasts to TGFβ

In glaucoma, scleral fibroblasts are exposed to IOP-associated mechanical strain and elevated TGFβ levels. These stimuli, in turn, lead to scleral remodeling. Here, we examine the scleral fibroblast migratory and transcriptional response to these stimuli to better understand mechanisms of glaucomatous scleral remodeling. Human peripapillary scleral (PPS) fibroblasts were cultured on parallel grooves, treated with TGFβ (2 ng/ml) in the presence of vehicle or TGFβ signaling inhibitors, and exposed to uniaxial strain (1 Hz, 5%, 12-24 hours). Axis of cellular orientation was determined at baseline, immediately following strain, and 24 hours after strain cessation with 0° being completely aligned with grooves and 90° being perpendicular. Fibroblasts migration in-line and across grooves was assessed using a scratch assay. Transcriptional profiling of TGFβ-treated fibroblasts with or without strain was performed by RT-qPCR and pERK, pSMAD2, and pSMAD3 levels were measured by immunoblot. Pre-strain alignment of TGFβ-treated cells with grooves (6.2±1.5°) was reduced immediately after strain (21.7±5.3°, p<0.0001) and restored 24 hours after strain cessation (9.5±2.6°). ERK, FAK, and ALK5 inhibition prevented this reduction; however, ROCK, YAP, or SMAD3 inhibition did not. TGFβ-induced myofibroblast markers were reduced by strain (αSMA, POSTN, ASPN, MLCK1). While TGFβ-induced phosphorylation of ERK and SMAD2 was unaffected by cyclic strain, SMAD3 phosphorylation was reduced (p=0.0004). Wound healing across grooves was enhanced by ROCK and SMAD3 inhibition but not ERK or ALK5 inhibition. These results provide insight into the mechanisms by which mechanical strain alters the cellular response to TGFβ and the potential signaling pathways that underlie scleral remodeling.

Protein Disulfide Isomerase 4 Is an Essential Regulator of Endothelial Function and Survival.

Endothelial autophagy plays an important role in the regulation of endothelial function. The inhibition of endothelial autophagy is associated with the reduced expression of protein disulfide isomerase 4 (PDIA-4); however, its role in endothelial cells is not known. Here, we report that endothelial cell-specific loss of PDIA-4 leads to impaired autophagic flux accompanied by loss of endothelial function and apoptosis. Endothelial cell-specific loss of PDIA-4 also induced marked changes in endothelial cell architecture, accompanied by the loss of endothelial markers and the gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition (EndMT). The loss of PDIA-4 activated TGFβ-signaling, and inhibition of TGFβ-signaling suppressed EndMT in PDIA-4-silenced endothelial cells in vitro. Our findings help elucidate the role of PDIA-4 in endothelial autophagy and endothelial function and provide a potential target to modulate endothelial function and/or limit autophagy and EndMT in (patho-)physiological conditions.

Abstract 728: Oral TGF-beta receptor1 inhibitor vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity

Abstract Osteosarcoma (OS) is an aggressive malignant bone cancer, with the lung as the most frequent site of metastasis. Unresectable pulmonary metastasis remains a significant challenge with a survival rate of less than 20%. Identification of novel therapeutic strategies are desperately needed. Transforming growth factor-β1 (TGF-β) is a potent immune suppressive cytokine in OS tumor microenvironment (TME). TGF-β1 expression is increased in the sera and tumor tissues of OS patients and this increase is associated with high-grade OS and lung metastases. Therefore, blocking TGF-β1 signaling may be a novel therapy for OS treatment. In this study, we show that blocking TGF-β1 signaling using the orally bioavailable small molecule TGF-βR1 inhibitor, Vactosertib, significantly inhibited OS proliferation in vitro and in vivo. Notably, Vactosertib inhibits c-Myc expression in the OS cells and oral administration of Vactosertib significantly reduces OS growth in vivo. Vactosertib increased immune effectors (e.g., IFNγ+CD8+ cells and NK cells) and inhibited immune suppressors (e.g., M2-like TAM, MDSC) in the OS TME. Our results suggest that inhibition of TGF-β1 signaling is an effective therapeutic strategy against OS through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response. Citation Format: Sung Hee Choi, Jay Myers, Suzanne Tomchuck, Melissa Bonner, Saada Eid, Daniel Kingsley, Kristen VanHeyst, Seong-Jin Kim, Byung-Gyu Kim, Alex Y. Huang. Oral TGF-beta receptor1 inhibitor vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 728.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6956.

Abstract 6272: Loss of SIRT6 diverts TGF-β signaling to promote liver cancer

Abstract Background: In the US, ~35% of individuals on the waitlist for a liver transplant who had hepatocellular cancer (HCC) also had metabolic dysfunction-associated steatohepatitis (MASH). Yet, key signaling mechanisms controlling cellular transformation in this obesity-driven HCC remain only partially understood. SIRT6 is NAD+-dependent protein deacetylases that limits the activity of the lipogenic transcription factors SREBP1/2. Reduced SIRT6 is associated with human fatty liver and cancer. However, SIRT6 also limits liver fibrosis by inhibiting TGF-β signaling through the deacetylation of SMAD2 and SMAD3. We found that Smad3 driven TGF-β signaling is modulated by βII-Spectrin (encoded by SPTBN1), and that a hot mutation in HCC- D1089Y encompasses the SPTBN1-SMAD3 binding site (Gastro 2018; 154:195-210). In stressed hepatocytes, βII-Spectrin promotes lipogenesis through interactions between caspase-cleaved forms of both SREBP1 and βII-Spectrin (Sci Transl Med 2021;13:624). Liver-specific knockout of βII-Spectrin (LSKO) blocks fatty liver and HCC. Here we explored the extent of βII-spectrin-D1089Y regulation of SIRT6 expression through SMAD3, and the combined roles of SIRT6 and SPTBN1 in human HCC. Methods: We generated a SPTBN1-D1089Y mutant mouse and examined SIRT6 in the SPTBN1-D1089Y mutant mouse liver tissues and MEFs. TGF-β/Smad3 pathway activity was analyzed in SPTBN1-D1089Y mutant MEFs via western blot and RNA-seq. SIRT6 expression was examined in mouse MAFLD/MASH (GSE156059). Structure-based molecular docking simulations using three-dimensional structural coordinates of human SIRT6 (ID: Q8N6T7) and β2SP (ID: Q01082) from the AlphaFold database, followed by interactions between SIRT6 and SPTBN1. Results: SIRT6 levels are decreased in SPTBN1-D1089Y mutant liver cancer cell line and mouse liver tissues. TGF-β/SMAD3 pathway activity was altered in SPTBN1-D1089Y mutant MEF cells with decreased phospho-SMAD3 levels. The structural model predicted specific interactions between human SPTBN1 and SIRT6. Overexpression of Flag-SIRT6 reduced βII-Spectrin acetylation in HepG2 cells. TGF-β induced SMAD3, βII-Spectrin coimmunoprecipitation with SIRT6. SIRT6 knockdown increases nSREBP1, SCD1 (encoded by a nSREBP1 target gene), and βII-Spectrin in HepG2 cells. SIRT6 RNA expression is significantly decreased in liver macrophages from mice with NAFLD/NASH. Conclusions: Our data indicated a SMAD dependent as well as a SMAD3 independent role of SPTBN1 in regulating SIRT6. βII-spectrin-D1089Y together with SIRT6 deficiency may increase the susceptibility to MASH/HCC via regulating the crosstalk between macrophage and hepatocytes or regulating MASH and HCC. Citation Format: Kazufumi Ohshiro, Xiyan Xiang, Xiaochun Yang, Krishanu Bhowmick, Mohammad I. Hassan, Taj Mohammad, Lopa Mishra. Loss of SIRT6 diverts TGF-β signaling to promote liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6272.

Abstract 6964: Regulation of TGF-β signaling via a novel TβRIII sheddase

Abstract In a normal cell, TGF-β signaling serves as a tumor suppressor by reducing proliferation and promoting apoptosis. Paradoxically, the function of TGF-β signaling reverses in a cancer cell and begins to promote oncogenic pathways such as proliferation, survival, evasion of the immune system, and epithelial to mesenchymal transition (EMT). This has made TGF-β signaling a very attractive therapeutic target for a wide variety of human carcinomas. As a result, many small molecule inhibitors of TGF-β signaling have been developed, but none have achieved FDA approval for use in human cancers due to the deleterious effects of inhibiting TGF-β responsiveness in normal cells. In effort to overcome this obstacle, we aim to restore the normal biological mechanisms that regulate TGF-β signaling. Specifically, the membrane bound co-receptor TβRIII can be proteolytically cleaved and shed from the membrane, resulting in an extracellular protein capable of binding and sequestering TGF-β ligand. This decoupling of ligand binding from the receptor allows TβRIII to suppress signaling. TβRIII shedding decreases TGF-β mediated oncogenic phenotypes such as migration and metastasis in multiple cancer types. However, this shedding is often lost in cancer. Therefore, restoration of TβRIII shedding could be a viable therapeutic approach for multiple cancer types. One potential mechanism for the loss of shedding is the negative regulation of the protease responsible for shedding (sheddase). However, the identity of the sheddase still remains unknown. Here, our goal is to identify the sheddase and uncover regulatory mechanisms that could be leveraged to restore TβRIII shedding in cancer. To achieve this goal, a library of protease CRISPR knockout viruses was screened to determine which proteases have an effect on shedding. The quantity of TβRIII shed into the cell culture media was measured via ELISA for each of these CRISPR knockout cell lines, and the candidates with the largest reduction of shedding were retained for further investigation. The list of candidates was further screened in silico via gene set enrichment analysis of human patient data against a TGF-β signaling gene set signature. Only the candidates demonstrating a negative correlation between sheddase candidate expression and TGF-β signaling activity were studied further. To further validate these candidate sheddases, knockdown and overexpression cell lines were created and characterized. Overexpression of the most promising candidate reduced TGF-β mediated phenotypes such as migration and invasion, while knockdown of this candidate increased the same phenotypes. Knockdown also increased expression of TGF-β and EMT markers. Importantly, these effects of sheddase knockdown were neutralized in the presence of the TGF-β inhibitor galunisertib or when the cells were rescued with exposure to purified soluble TβRIII, indicating the effects are mediated through TβRIII shedding. Citation Format: Benjamin M. Greulich, John Pawlak, John Post, Elena Karas, Gerard Blobe. Regulation of TGF-β signaling via a novel TβRIII sheddase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6964.

Abstract 2495: CCL3 predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining a basal-like ecotype enriched in LIF-producing macrophages

Abstract The TGFβ receptor inhibitor galunisertib (GAL) exhibited promising efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the randomized phase 2 H9H-MC-JBAJ study. However, the identification of predictive biomarkers remains of unique importance. We used a 279 multi-analyte panel and confirmed CCL3 as the most significant plasma protein for chemoresistance in patients receiving gemcitabine (GEM) [overall survival (mOS) (95%CI), high vs low, 3.6 (2.5-4.0) vs 10.1 (7.2-17.0) months, HR (95%CI)= 3.92 (2.14-7.20); p&amp;lt;0.001]. GAL dramatically reverted this resistance and CCL3 was the most significant predictive marker for the combination of this agent plus GEM [mOS (95%CI), GAL + GEM vs. placebo + GEM, 9.0 (5.5-12.1) vs 3.6 (2.5-4.0) months, HR (95%CI)= 0.39 (0.23-0.68); p&amp;lt;0.001]. We delved into the mechanisms behind this exceptional response in PDAC with this prognostically negative ecotype sustained by CCL3 in 6 different murine PDAC models. Mice bearing high-Ccl3 tumors had a significantly shorter mOS, a higher infiltration of M2-macrophages (TAMs), and a more mesenchymal/basal-like phenotype than did models with undetectable Ccl3.Ccl3 was stably expressed in DT4313 and PAN610, and silenced in RC416 cells. In co-culture models, DT4313Ccl3 and PAN610Ccl3 had a more mesenchymal phenotype and induced a M2 polarization of RAW264.7 murine TAMs than did controls. Mice bearing DT4313Ccl3 or PAN610Ccl3 tumors had a significantly shorter mOS, an higher infiltration of M2-polarized TAMs and a more mesenchymal/basal-like phenotype than controls. The expression of Tgfβ1 ligand and the phosphorylation of Smad2 were triggered only in TAMs if co-cultured with Ccl3-high RC416scr, DT4313Ccl3 or PAN610Ccl3 cells. This activation of Tgfβ pathway was blocked by maraviroc, a selective inhibitor of Ccl3-receptor Ccr5. In co-culture, GAL reverted the mesenchymal phenotype of PDAC cells and the M2 polarization of TAMs. By using an 111 different cytokine immunoassay, Leukemia Inhibitory Factor (Lif) was the most significantly factor overexpressed by TAMs if stimulated by Ccl3. GAL suppressed this Lif expression. In mice bearing DT4313Ccl3 tumors GEM was ineffective, whereas it prolonged mOS in those with DT4313NTC tumors. Conversely, GAL plus GEM doubled mOS duration vs. GEM, modulated M2- TAMs infiltration, the mesenchymal/basal-like phenotype and plasma Lif only in mice bearing DT4313Ccl3 tumors, but not in those with DT4313NTC controls. In summary, we demonstrated that tumor-derived Ccl3 activates an autocrine Tgfβ signaling in TAMs, triggering their M2-phenotype and secretion of Lif. This Tgfβ-Lif axis sustains paracrinally a mesenchymal/basal-like PDAC ecotype resistant to GEM. Inhibition of Tgfβ signaling modulates this poor prognosis ecotype, and explains the exceptional response to GAL in patients with high-CCL3 PDAC. Citation Format: Silvia Pietrobono, Monica Bertolini, Veronica De Vita, Enza Scarlato, Federica Fazzini, Fabio Sabbadini, Domenico Mangiameli, Alberto Quinzii, Simona Casalino, Davide Melisi. CCL3 predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining a basal-like ecotype enriched in LIF-producing macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2495.

Prenatal dexamethasone exposure impairs rat blood-testis barrier function and sperm quality in adult offspring via GR/KDM1B/FSTL3/TGFβ signaling.

Both epidemiological and animal studies suggest that adverse environment during pregnancy can change the offspring development programming, but it is difficult to achieve prenatal early warning. In this study we investigated the impact of prenatal dexamethasone exposure (PDE) on sperm quality and function of blood-testis barrier (BTB) in adult offspring and the underlying mechanisms. Pregnant rats were injected with dexamethasone (0.1, 0.2 and 0.4 mg·kg-1·d-1, s.c.) from GD9 to GD20. After weaning (PW4), the pups were fed with lab chow. At PW12 and PW28, the male offspring were euthanized to collect blood and testes samples. We showed that PDE significantly decreased sperm quality (including quantity and motility) in male offspring, which was associated with impaired BTB and decreased CX43/E-cadherin expression in the testis. We demonstrated that PDE induced morphological abnormalities of fetal testicle and Sertoli cell development originated from intrauterine. By tracing to fetal testicular Sertoli cells, we found that PDE dose-dependently increased expression of histone lysine demethylases (KDM1B), decreasing histone 3 lysine 9 dimethylation (H3K9me2) levels of follistatin-like-3 (FSTL3) promoter region and increased FSTL3 expression, and inhibited TGFβ signaling and CX43/E-cadherin expression in offspring before and after birth. These results were validated in TM4 Sertoli cells following dexamethasone treatment. Meanwhile, the H3K9me2 levels of FSTL3 promoter in maternal peripheral blood mononuclear cell (PBMC) and placenta were decreased and its expression increased, which was positively correlated with the changes in offspring testis. Based on analysis of human samples, we found that the H3K9me2 levels of FSTL3 promoter in maternal blood PBMC and placenta were positively correlated with fetal blood testosterone levels after prenatal dexamethasone exposure. We conclude that PDE can reduce sperm quality in adult offspring rats, which is related to the damage of testis BTB via epigenetic modification and change of FSTL3 expression in Sertoli cells. The H3K9me2 levels of the FSTL3 promoter and its expression in the maternal blood PBMC can be used as a prenatal warning marker for fetal testicular dysplasia.

Podocan promoting skeletal muscle post-injury regeneration by inhibiting TGF-β signaling pathway.

Podocan, the fifth member of Small Leucine-Rich Proteoglycan (SLRP) family of extracellular matrix components, is poorly known in muscle development. Previous studies have shown that Podocan promotes C2C12 differentiation in mice. In this study, we elucidated the effect of Podocan on skeletal muscle post-injury regeneration and its underlying mechanism. Injection of Podocan protein promoted the process of mice skeletal muscle post-injury regeneration. This effect seemed to be from the acceleration of muscle satellite cell differentiation invivo. Meanwhile, Podocan promoted myogenic differentiation invitro by binding with TGF-β1 to inhibit the activity of the TGF-β signaling pathway. These results indicated that Podocan had the potential roles to enhance skeletal muscle post-injury regeneration. Its mechanism is likely the regulation of the expression of p-Smad2 and p-Smad4 related to the TGF-β signaling pathway by interacting with TGF-β1.

Ursolic acid inhibits glioblastoma through suppressing TGFβ-mediated epithelial-mesenchymal transition (EMT) and angiogenesis

Found in many fruits and plants, Ursolic acid (UA), a pentacyclic triterpene that occurs naturally, is recognized for its anti-cancer effects, especially in combating glioblastoma. However, the intricate molecular mechanisms underpinning its anti-tumor actions are still not fully understood, despite the recognition of these effects. By examining the functions of epithelial-mesenchymal transition (EMT) and angiogenesis, crucial for glioblastoma progression, and their regulation through Transforming Growth Factor Beta (TGFβ) – a key marker for glioblastoma, our research aims to fill this knowledge gap. This study explores how ursolic acid can block the progression of glioblastoma by precisely targeting TGFβ-triggered EMT and angiogenesis. The findings show that UA successfully blocks the spread, movement, and invasion of glioblastoma cells. Accompanying this, there is a significant reduction in the expression of TGFβ and crucial EMT indicators like snail and vimentin. Furthermore, UA shows a reduction in angiogenesis that depends on the dosage, highlighted by decreased vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Interestingly, increased TGFβ expression in U87 and U251 glioblastoma cell lines was found to weaken UA's anti-tumor properties, shedding more light on TGFβ′s critical function in glioblastoma's pathology. Supporting these laboratory results, UA also showed considerable inhibition of tumor growth in a glioblastoma xenograft mouse model. Overall, our research emphasizes Ursolic acid's promise as a new treatment for glioblastoma and clarifies its action mechanism, mainly by inhibiting TGFβ signaling and thereby EMT and angiogenesis.

Rare flavanone-diarylheptanoid hybrids from Typha angustifolia shows anti breast cancer activity via activating TGF-β1/Smad signaling pathway

Four new flavanone-diarylheptanoid hetero dimers, typhatifolins A−D (1−4), were separated from the pollen of a widely distributed medicinal plant Typha angustifolia. Structures of these rare hybrids were elucidated by detailed interpretation of spectroscopic data, and their absolute configurations were determined on the basis of Mosher's method and ECD analyses. All the four compounds showed moderate to significant cytotoxicities against a panel of tumor cell lines with IC50 values ranging from 0.67 to 12.48 μM. Further in vitro antitumor evaluation for typhatifolin B (TTB, 2) on two breast cancer cells (4T1 and MDA-MB231) revealed that it could remarkably induce cell apoptosis and G0/G1 cycle arrest, as well as block cell migration and invasion. Mechanistically, TTB could exert its antitumor effect via activating the TGF-β1 (transforming growth factor beta 1) signaling pathway as evidenced by RNA-seq analysis and immunoblotting experiments, which was further corroborated by treating cancer cells with a TGF-β signaling inhibitor. Lastly, the in vivo anti breast cancer activity was demonstrated by applying the mixture of typhatifolins A−D to a preclinical animal model.

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

In pathologies including cancer, aberrant Transforming Growth Factor-β (TGF-β) 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-β responses. Betaglycan/type III TGF-β receptor (TβRIII), is an established co-receptor for the TGF-β superfamily known to bind directly to TGF-βs 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-β signaling and the cells' responses to exogenous TGF-β ligands. Using unbiased transcriptomics, we identified TIMP3 as a key inhibitor of betaglycan shedding thereby influencing TGF-β 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-β signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan chains of betaglycan for shedding and influence on TGF-β signaling responses. Dysregulated shedding of TGF-β receptors plays a vital role in determining the response and availability of TGF-βs’, which is crucial for prognostic predictions and understanding of TGF-β signaling dynamics.

Lingual Denervation Improves the Efficacy of Anti-PD-1 Immunotherapy in Oral Squamous Cell Carcinomas by Downregulating TGFβ Signaling.

Intratumoral nerve infiltration relates to tumor progression and poor survival in oral squamous cell carcinoma (OSCC). How neural involvement regulates antitumor immunity has not been well characterized. This study aims to investigate molecular mechanisms of regulating tumor aggressiveness and impairing antitumor immunity by nerve-derived factors. We performed the surgical lingual denervation in an immunocompetent mouse OSCC model to investigate its effect on tumor growth and the efficacy of anti-PD-1 immunotherapy. A trigeminal ganglion neuron and OSCC cell coculture system was established to investigate the proliferation, migration, and invasion of tumor cells and the PD-L1 expression. Both the neuron-tumor cell coculture in vitro model and the OSCC animal model were explored. Lingual denervation slowed down tumor growth and improved the efficacy of anti-PD-1 treatment in the OSCC model. Coculturing with neurons not only enhanced the proliferation, migration, and invasion but also upregulated TGFβ-SMAD2 signaling and PD-L1 expression of tumor cells. Treatment with the TGFβ signaling inhibitor galunisertib reversed nerve-derived tumor aggressiveness and downregulated PD-L1 on tumor cells. Similarly, lingual denervation in vivo decreased TGFβ and PD-L1 expression and increased CD8+ T-cell infiltration and the expression of IFNγ and TNFα within tumor. Neural involvement enhanced tumor aggressiveness through upregulating TGFβ signaling and PD-L1 expression in OSCC, while denervation of OSCC inhibited tumor growth, downregulated TGFβ signaling, enhanced activities of CD8+ T cells, and improved the efficacy of anti-PD-1 immunotherapy. This study will encourage further research focusing on denervation as a potential adjuvant therapeutic approach in OSCC. This study revealed the specific mechanisms for nerve-derived cancer progression and impaired antitumor immunity in OSCC, providing a novel insight into the cancer-neuron-immune network as well as pointing the way for new strategies targeting nerve-cancer cross-talk as a potential adjuvant therapeutic approach for OSCC.

CD109 Attenuates Bleomycin-induced Pulmonary Fibrosis by Inhibiting TGF-β Signaling.

Pulmonary fibrosis is a fatal condition characterized by fibroblast and myofibroblast proliferation and collagen deposition. TGF-β plays a pivotal role in the development of pulmonary fibrosis. Therefore, modulation of TGF-β signaling is a promising therapeutic strategy for treating pulmonary fibrosis. To date, however, interventions targeting TGF-β have not shown consistent efficacy. CD109 is a GPI-anchored glycoprotein that binds to TGF-β receptor I and negatively regulates TGF-β signaling. However, no studies have examined the role and therapeutic potential of CD109 in pulmonary fibrosis. The purpose of this study was to determine the role and therapeutic value of CD109 in bleomycin-induced pulmonary fibrosis. CD109-transgenic mice overexpressing CD109 exhibited significantly attenuated pulmonary fibrosis, preserved lung function, and reduced lung fibroblasts and myofibroblasts compared with wild-type (WT) mice. CD109-/- mice exhibited pulmonary fibrosis comparable to WT mice. CD109 expression was induced in variety types of cells, including lung fibroblasts and macrophages, upon bleomycin exposure. Recombinant CD109 protein inhibited TGF-β signaling and significantly decreased ACTA2 expression in human fetal lung fibroblast cells invitro. Administration of recombinant CD109 protein markedly reduced pulmonary fibrosis in bleomycin-treated WT mice invivo. Our results suggest that CD109 is not essential for the development of pulmonary fibrosis, but excess CD109 protein can inhibit pulmonary fibrosis development, possibly through suppression of TGF-β signaling. CD109 is a novel therapeutic candidate for treating pulmonary fibrosis.