FAT1 inhibits AML autophagy and proliferation via downregulating ATG4B expression.

BACKGROUND: Emerging studies have shown that FAT atypical cadherin 1 (FAT1) and autophagy separately inhibits and promotes acute myeloid leukemia (AML) proliferation. However, it is unknown whether FAT1 were associated with autophagy in regulating AML proliferation. METHODS: AML cell lines, 6-week-old male nude mice and AML patient samples were used in this study. qPCR/Western blot and cell viability/3H-TdR incorporation assays were separately used to detect mRNA/protein levels and cell activity/proliferation. Luciferase reporter assay was used to examine gene promoter activity. Co-IP analysis was used to detect the binding of proteins. RESULTS: In this study, we for the first time demonstrated that FAT1 inhibited AML proliferation by decreasing AML autophagy level. Moreover, FAT1 weakened AML autophagy level via decreasing autophagy related 4B (ATG4B) expression. Mechanistically, we found that FAT1 reduced the phosphorylated and intranuclear SMAD family member 2/3 (smad2/3) protein levels, thus decreasing the activity of ATG4B gene promoter. Furthermore, we found that FAT1 competitively bound to TGF-ßR II which decreased the binding of TGF-ßR II to TGF-ßR I and the subsequent phosphorylation of TGF-ßR I, thus reducing the phosphorylation and intranuclear smad2/3. The experiments in nude mice showed that knockdown of FAT1 promoted AML autophagy and proliferation in vivo. CONCLUSIONS: Collectively, these results revealed that FAT1 downregulates ATG4B expression via inhibiting TGFß-smad2/3 signaling activity, thus decreasing the autophagy level and proliferation activity of AML cells. GENERAL SIGNIFICANCE: Our study suggested that the "FAT1-TGFß-smad2/3-ATG4B-autophagy" pathway may be a novel target for developing new targeted drugs to AML treatment.

Recombinant Human HAPLN1 Mitigates Pulmonary Emphysema by Increasing TGF-ß Receptor I and Sirtuins Levels in Human Alveolar Epithelial Cells.

Chronic obstructive pulmonary disease (COPD) will be the third leading cause of death worldwide by 2030. One of its components, emphysema, has been defined as a lung disease that irreversibly damages the lungs' alveoli. Treatment is currently unavailable for emphysema symptoms and complete cure of the disease. Hyaluronan (HA) and proteoglycan link protein 1 (HAPLN1), an HA-binding protein linking HA in the extracellular matrix to stabilize the proteoglycan structure, forms a bulky hydrogel-like aggregate. Studies on the biological role of the full-length HAPLN1, a simple structure-stabilizing protein, are limited. Here, we demonstrated for the first time that treating human alveolar epithelial type 2 cells with recombinant human HAPLN1 (rhHAPLN1) increased TGF-ß receptor 1 (TGF-ß RI) protein levels, but not TGF-ß RII, in a CD44-dependent manner with concurrent enhancement of the phosphorylated Smad3 (p-Smad3), but not p-Smad2, upon TGF-ß1 stimulation. Furthermore, rhHAPLN1 significantly increased sirtuins levels (i.e., SIRT1/2/6) without TGF-ß1 and inhibited acetylated p300 levels that were increased by TGF-ß1. rhHAPLN1 is crucial in regulating cellular senescence, including p53, p21, and p16, and inflammation markers such as p-NF-κB and Nrf2. Both senile emphysema mouse model induced via intraperitoneal rhHAPLN1 injections and porcine pancreatic elastase (PPE)-induced COPD mouse model generated via rhHAPLN1-containing aerosols inhalations showed a significantly potent efficacy in reducing alveolar spaces enlargement. Preclinical trials are underway to investigate the effects of inhaled rhHAPLN1-containing aerosols on several COPD animal models.

Vactosertib, TGF-ß receptor I inhibitor, augments the sensitization of the anti-cancer activity of gemcitabine in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) exhibits a pronounced extracellular matrix (ECM)-rich response, which is produced by an excessive amount of transforming growth factor ß (TGF-ß), resulting in tumor progression and metastasis. In addition, TGF-ß signaling contributes to rapidly acquired resistance and incomplete response to gemcitabine. Recently, selective inhibitors of the TGF-ß signaling pathway have shown promise in PDAC treatment, particularly as an option for augmenting responses to chemotherapy. Here, we investigated the synergistic anticancer effects of a small-molecule TGF-ß receptor I kinase inhibitor (vactosertib/EW-7197) in the presence of gemcitabine, and its mechanism of action in pancreatic cancer. Vactosertib sensitized pancreatic cancer cells to gemcitabine by synergistically inhibiting their viability. Importantly, the combination of vactosertib and gemcitabine significantly attenuated the expression of major ECM components, including collagens, fibronectin, and α-SMA, in pancreatic cancer compared with gemcitabine alone. This resulted in potent induction of mitochondrial-mediated apoptosis, gemcitabine-mediated cytotoxicity, and inhibition of tumor ECM by vactosertib. Additionally, the combination decreased metastasis through inhibition of migration and invasion, and exhibited synergistic anti-cancer activity by inhibiting the TGF-ß/Smad2 pathway in pancreatic cancer cells. Furthermore, co-treatment significantly suppressed tumor growth in orthotopic models. Therefore, our findings demonstrate that vactosertib synergistically increased the antitumor activity of gemcitabine via inhibition of ECM component production by inhibiting the TGF-ß/Smad2 signaling pathway. This suggests that the combination of vactosertib and gemcitabine may be a potential treatment option for patients with pancreatic cancer.

Loss of BTK ameliorates the pathological cardiac fibrosis and dysfunction.

Cardiac fibrosis is a common irreversible pathological feature of diverse heart disorders. Uncontrolled cardiac fibrosis contributes to maladaptive cardiac remodeling and eventually heart failure. However, the molecular determinants of ischemic and non-ischemic pathological cardiac fibrosis remain largely unknown. Here, we investigated the role of Bruton's tyrosine kinase (BTK) in cardiac fibrosis and remodeling of mice under various pathological conditions. BTK expression was increased in myocardium of mice after pressure overload or myocardial infarction (MI). BTK was mainly located in cardiac fibroblasts of myocardium, and its expression in isolated cardiac fibroblasts was also upregulated following TGF-ß treatment. The deficiency or pharmacological inhibition of BTK with the second-generation inhibitor Acalabrutinib attenuated cardiac fibrosis, preserved cardiac function and prevented adverse cardiac remodeling, which protected against heart failure in mice following pressure overload or MI. BTK deficiency or inhibitor treatment significantly decreased the expression of pro-fibrotic molecules in isolated cardiac fibroblasts and inhibited the transition of fibroblasts to myofibroblasts in response to diverse pathological stresses. BTK directly bound and phosphorylated TGF-ß receptor Ⅰ (TßRⅠ) at tyrosine 182, and then promoted the activation of downstream SMAD-dependent or -independent TGF-ß signaling, leading to the enhanced transition of fibroblasts to pro-fibrotic myofibroblasts and the excessive extracellular matrix gene expression. Our finding uncovers a driving role of BTK in cardiac fibrosis and dysfunction following pressure overload and MI stress, and highlights novel pathogenic mechanisms in ischemic and non-ischemic maladaptive cardiac remodeling, which presents as a promising target for the development of anti-fibrotic therapy.

Ochratoxin a induces hepatic fibrosis through TGF-ß receptor I/Smad2/3 signaling pathway.

Ochratoxin A (OTA) is a mycotoxin generated by Penicillium and Aspergillus species. It is often found in cereals. We hypothesized that OTA exposure induces epithelial-mesenchymal transition (EMT), leading to liver fibrosis. In this research, we explored whether the TGF-ß receptor I (TGF-ß RI)/Smad2/3 signaling pathway is related to EMT-induced hepatic fibrosis. In vitro and in vivo experiments, mRNA and protein expression of liver fibrosis-related markers such as fibronectin, α-smooth muscle actin (α-SMA) and E-cadherin were assessed. The levels of alkaline phosphatase, alanine transaminase, aspartate aminotransferase, and total bilirubin, which are used to assess damage, increased. We also confirmed the increase in mRNA and protein expression of TGF-ß RI, Smad2, and Smad3. The expression of liver fibrosis-related markers was decreased by siRNA-mediated silencing of Smad2/3, as well as TGF-RI suppression. Liver cells exposed to OTA showed enhanced TGF-ß RI expression on the cell membrane. These results demonstrated that OTA induces hepatic fibrosis through TGF-ß RI and Smad2/3 pathways in vitro and in vivo.

Upregulated expression of transforming growth factor-ß receptor I/II in an endemic Osteoarthropathy in China.

BACKGROUND: Kashin-Beck disease (KBD) is a chronic, deforming, endemic osteochondropathy that begins in patients as young as 2-3 years of age. The pathogenesis of KBD remains unclear, although selenium (Se) deficiency and T-2 toxin food contamination are both linked to the disease. In the present study, we evaluated transforming growth factor-ß receptor (TGF-ßR I and II) levels in clinical samples of KBD and in pre-clinical disease models. METHODS: Human specimens were obtained from the hand phalanges of eight donors with KBD and eight control donors. Animal models of the disease were established using Sprague-Dawley rats, which were fed an Se-deficient diet for 4 weeks and later administered the T-2 toxin. Cartilage cellularity and morphology were examined by hematoxylin and eosin staining. Expression and localization of TGF-ßRI and II were evaluated using immunohistochemical staining and western blotting. RESULTS: In the KBD samples, chondral necrosis was detected based on cartilage cell disappearance and alkalinity loss in the matrix ground substance. In the necrotic areas, TGF-ßRI and II staining were strong. Positive percentages of TGF-ßRI and II staining were higher in the cartilage samples of KBD donors than in those of control donors. TGF-ßRI and II staining was also increased in cartilage samples from rats administered T-2 toxin or fed on Se-deficient plus T-2 toxin diets. CONCLUSION: TGF-ßRI and II may be involved in the pathophysiology of KBD. This study provides new insights into the pathways that contribute to KBD development.

Clinical efficacies, underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management.

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN.

Hedgehog signaling in gastrointestinal carcinogenesis and the gastrointestinal tumor microenvironment.

The Hedgehog (HH) signaling pathway plays important roles in gastrointestinal carcinogenesis and the gastrointestinal tumor microenvironment (TME). Aberrant HH signaling activation may accelerate the growth of gastrointestinal tumors and lead to tumor immune tolerance and drug resistance. The interaction between HH signaling and the TME is intimately involved in these processes, for example, tumor growth, tumor immune tolerance, inflammation, and drug resistance. Evidence indicates that inflammatory factors in the TME, such as interleukin 6 (IL-6) and interferon-γ (IFN-γ), macrophages, and T cell-dependent immune responses, play a vital role in tumor growth by affecting the HH signaling pathway. Moreover, inhibition of proliferating cancer-associated fibroblasts (CAFs) and inflammatory factors can normalize the TME by suppressing HH signaling. Furthermore, aberrant HH signaling activation is favorable to both the proliferation of cancer stem cells (CSCs) and the drug resistance of gastrointestinal tumors. This review discusses the current understanding of the role and mechanism of aberrant HH signaling activation in gastrointestinal carcinogenesis, the gastrointestinal TME, tumor immune tolerance and drug resistance and highlights the underlying therapeutic opportunities.

EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells.

BACKGROUND: The Ecotropic viral integration site 5 (EVI5), an important protein in regulating cell cycle, cytokinesis and cellular membrane traffic, functions as a stabilizing factor maintaining anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 in S/G2 phase. However, the mechanism by which EVI5 promotes malignant transformation of non-small cell lung cancer (NSCLC) remains unknown. In the present study, we addressed the role of EVI5 in NSCLC by regulating tumor growth, migration and invasion. METHODS: The expression levels of EVI5 and miR-486-5p in NSCLC tissues and cells were measured by real-time PCR. Meanwhile, EVI5 and its associated protein expression were analyzed by western blot and co-immunoprecipitation assay. Flow cytometry was performed to determine cell proliferation and apoptosis. CCK-8 and clonogenic assays were used to analyze cell viability. Wound healing, transwell migration and matrigel invasion assays were utilized to assess the motility of tumor cells. To investigate the role of EVI5 in vivo, lung carcinoma xenograft mouse model was applied.. RESULTS: EVI5 was upregulated in NSCLC tissues and cell lines when compared with that in normal tissues and cell line. Knockdown of EVI5 in vitro inhibited tumor cell proliferation, migration and invasion in NSCLC cells. Further, inoculation of EVI5-deficient tumor cells into nude mice suppressed tumor proliferation and metastasis compared to control mice inoculated with unmanipulated tumor cells. These data indicated that EVI5 promote the proliferation of NSCLC cells which was consistent with our previous results. Additionally, we showed that EVI5 was directly regulated by miR-486-5p, and miR-486-5p-EVI5 axis affected the NSCLC migration and invasion through TGF-ß/Smad signaling pathway by interacting with TGF-ß receptor II and TGF-ß receptor I. CONCLUSIONS: Based on these results, we demonstrated a new post-transcriptional mechanism of EVI5 regulation via miR-486-5p and the protumoral function of EVI5 in NSCLC by interacting with Emi1 and/or TGF-ß receptors, which provides a new insight into the targeted therapy of NSCLC.

Targeting the TGFß pathway with galunisertib, a TGFßRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade.

BACKGROUND: TGFß signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFß's immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFß pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses. RESULTS: In vitro treatment with galunisertib reversed TGFß and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti-tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFß and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy. CONCLUSIONS: Together these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy.

ATP6V1H regulates the growth and differentiation of bone marrow stromal cells.

ATP6V1H encodes subunit H of vacuolar ATPase (V-ATPase) and may regulate osteoclastic function. The deficiency of ATP6V1H caused bone loss in human, mouse and zebrafish. In this report, we identified the mechanisms by which ATP6V1H regulates proliferation and differentiation of bone marrow stromal cells (BMSCs). We found that ATP6V1H was expressed in BMSCs, and Atp6v1h+/- BMSCs exhibited the lower proliferation rate, cell cycle arrest and reduced osteogenic differentiation capacity, as well as the increased adipogenic potentials. Histologic analysis confirmed less bone formation and more fatty degeneration in Atp6v1h+/- mice in the different age groups. Q-PCR analysis revealed that loss of ATP6V1H function downregulated the mRNA level of TGF-ß1 receptor, and its binding molecule, subunit ß of adaptor protein complex 2 (AP-2), suggesting ATP6V1H regulates the proliferation and differentiation of BMSCs by interacting with TGF-ß receptor I and AP-2 complex.

A polyethylenimine-modified carboxyl-poly(styrene/acrylamide) copolymer nanosphere for co-delivering of CpG and TGF-ß receptor I inhibitor with remarkable additive tumor regression effect against liver cancer in mice.

Cancer immunotherapy based on nanodelivery systems has shown potential for treatment of various malignancies, owing to the benefits of tumor targeting of nanoparticles. However, induction of a potent T-cell immune response against tumors still remains a challenge. In this study, polyethylenimine-modified carboxyl-styrene/acrylamide (PS) copolymer nano-spheres were developed as a delivery system of unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides and transforming growth factor-beta (TGF-ß) receptor I inhibitors for cancer immunotherapy. TGF-ß receptor I inhibitors (LY2157299, LY) were encapsulated to the PS via hydrophobic interaction, while CpG oligodeoxynucleotides were loaded onto the PS through electrostatic interaction. Compared to the control group, tumor inhibition in the PS-LY/CpG group was up to 99.7% without noticeable toxicity. The tumor regression may be attributed to T-cell activation and amplification in mouse models. The results highlight the additive effect of CpG and TGF-ß receptor I inhibitors co-delivered in cancer immunotherapy.

Focal adhesion molecule Kindlin-1 mediates activation of TGF-ß signaling by interacting with TGF-ßRI, SARA and Smad3 in colorectal cancer cells.

Kindlin-1, an integrin-interacting protein, has been implicated in TGF-ß/Smad3 signaling. However, the molecular mechanism underlying Kindlin-1 regulation of TGF-ß/Smad3 signaling remains elusive. Here, we reported that Kindlin-1 is an important mediator of TGF-ß/Smad3 signaling by showing that Kindlin-1 physically interacts with TGF-ß receptor I (TßRI), Smad anchor for receptor activation (SARA) and Smad3. Kindlin-1 is required for the interaction of Smad3 with TßRI, Smad3 phosphorylation, nuclear translocation, and finally the activation of TGF-ß/Smad3 signaling pathway. Functionally, Kindlin-1 promoted colorectal cancer (CRC) cell proliferation in vitro and tumor growth in vivo, and was also required for CRC cell migration and invasion via an epithelial to mesenchymal transition. Kindlin-1 was found to be increased with the CRC progression from stages I to IV. Importantly, raised expression level of Kindlin-1 correlates with poor outcome in CRC patients. Taken together, we demonstrated that Kindlin-1 promotes CRC progression by recruiting SARA and Smad3 to TßRI and thereby activates TGF-ß/Smad3 signaling. Thus, Kindlin-1 is a novel regulator of TGF-ß/Smad3 signaling and may also be a potential target for CRC therapeutics.

Oncogenic STRAP functions as a novel negative regulator of E-cadherin and p21(Cip1) by modulating the transcription factor Sp1.

We have previously reported the identification of a novel WD-domain protein, STRAP that plays a role in maintenance of mesenchymal morphology by regulating E-cadherin and that enhances tumorigenicity partly by downregulating CDK inhibitor p21(Cip1). However, the functional mechanism of regulation of E-cadherin and p21(Cip1) by STRAP is unknown. Here, we have employed STRAP knock out and knockdown cell models (mouse embryonic fibroblast, human cancer cell lines) to show how STRAP downregulates E-cadherin and p21(Cip1) by abrogating the binding of Sp1 to its consensus binding sites. Moreover, ChIP assays suggest that STRAP recruits HDAC1 to Sp1 binding sites in p21(Cip1) promoter. Interestingly, loss of STRAP can stabilize Sp1 by repressing its ubiquitination in G1 phase, resulting in an enhanced expression of p21(Cip1) by >4.5-fold and cell cycle arrest. Using Bioinformatics and Microarray analyses, we have observed that 87% mouse genes downregulated by STRAP have conserved Sp1 binding sites. In NSCLC, the expression levels of STRAP inversely correlated with that of Sp1 (60%). These results suggest a novel mechanism of regulation of E-cadherin and p21(Cip1) by STRAP by modulating Sp1-dependent transcription, and higher expression of STRAP in lung cancer may contribute to downregulation of E-cadherin and p21(Cip1) and to tumor progression.

Inhibition of intimal hyperplasia in murine aortic allografts by the oral administration of the transforming growth factor-beta receptor I kinase inhibitor SD-208.

BACKGROUND: Transforming growth factor-beta (TGF-ß) plays a significant role in the pathogenesis of the intimal hyperplasia of transplant arteriosclerosis (TA). The aim of this study was to evaluate the efficacy of an oral inhibitor of TGF-ß receptor I kinase (SD-208) on the development of TA. METHODS: BALB/c (H-2(d)) donor aortas were transplanted into C57BL/6 (H-2(b)) recipients, and the mice then received different doses (40 or 60 mg/kg) of SD-208 or control vehicle by daily gavage for 8 weeks. The grafts were analyzed by histology and morphometry at 1, 2, 4, 6 and 8 weeks after transplantation. The effects of TGF-ß and SD-208 on neointimal smooth muscle-like cell (SMLC) and vascular smooth muscle cell (VSMC) proliferation and migration were evaluated, and the expression levels of Smad3, P-Smad3, connective tissue growth factor (CTGF) and collagen I were determined by in vitro experiments. RESULTS: The intimal hyperplasia of the SD-208-treated group was significantly reduced compared with the vehicle-treated control group (32% and 48% reduction for 40 mg/kg and 60 mg/kg SD-208 compared with the controls, respectively [n = 5], p < 0.05). SD-208 reduced SMLC proliferation and the production of intimal collagen by 21% and 75%, respectively, in the grafts. SD-208 also abolished the promoting effect of TGF-ß on SMLC proliferation and migration but did not affect TGF-ß inhibition of VSMCs in vitro. CTGF, a protein downstream of TGF-ß, was downregulated with the inhibition of Smad3 phosphorylation by SD-208, both in vitro and in vivo. Moreover, we found that the endogenous Smad3 in SMLCs was upregulated from 2 weeks after transplantation and was 64% higher than in VSMCs at 8 weeks. CONCLUSION: These results demonstrate that SD-208 can effectively reduce the formation of intimal hyperplasia of TA in the murine aortic allograft model.