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The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload-induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-β receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload-induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-β mutant transgene. Interestingly, cardiac fibroblast-specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-β-Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.

In pancreatic ductal adenocarcinoma (PDAC), features of epithelial–mesenchymal transition (EMT) are often seen in tumor tissue, and such features correlate with poor prognosis. Solitary infiltration of tumor cells represents a morphological phenotype of EMT, and we previously reported that a high degree of solitary cell infiltration correlates with EMT-like features, including reduced E-cadherin and elevated vimentin levels. Using solitary cell infiltration to evaluate the degree of EMT, gene-expression profiling of 12 PDAC xenografts was performed, and SMAD3 was identified as an EMT-related gene. Immunohistochemistry using clinical specimens (n=113) showed that SMAD3 accumulated in the nuclei of tumor cells, but was not detected in most epithelial cells in the pancreatic duct. Moreover, SMAD3 upregulation correlated with malignant characteristics, such as higher tumor grade and lymph node metastasis, as well as with EMT-like features. SMAD4, which plays a key role in transforming growth factor-β (TGF-β) signaling, is inactivated in approximately half of PDAC cases. In this study, the nuclear accumulation of SMAD3 was immunohistochemically detected even in SMAD4-negative cases. SMAD3 knockdown resulted in upregulated E-cadherin, downregulated vimentin, and reduced cell motility in pancreatic cancer cells regardless of SMAD4 status. In addition, TGF-β-treatment resulted in EMT induction in cells carrying wild-type SMAD4, and EMT was suppressed by SMAD3 knockdown. Patients with upregulated SMAD3 and a high degree of solitary cell infiltration had shorter times to recurrence and shorter survival times after surgery, and multivariate analysis showed that both factors were independent prognostic factors linked to unfavorable outcomes. These findings suggest that SMAD3 in PDAC is involved in the promotion of malignant potential through EMT induction in tumor cells regardless of SMAD4 status and serves as a potential biomarker of poor prognosis.

Background Dysregulation or abnormality of the programmed cell death (PCD) pathway is closely related to the occurrence and development of many tumors, including acute myeloid leukemia (AML). Studying the abnormal characteristics of PCD pathway-related molecular markers can provide a basis for prognosis prediction and targeted drug design in AML patients. Methods A total of 1394 genes representing 13 different PCD pathways were examined in AML patients and healthy donors. The upregulated genes were analyzed for their ability to predict overall survival (OS) individually, and these prognostic genes were subsequently combined to construct a PCD-related prognostic signature via an integrated approach consisting of 101 models based on ten machine learning algorithms. RNA transcriptome and clinical data from multiple AML cohorts (TCGA-AML, GSE106291, GSE146173 and Beat AML) were obtained to develop and validate the AML prognostic model. Results A total of 214 upregulated PCD-related genes were identified in AML patients, 39 of which were proven to be prognostic genes in the training cohort. On the basis of the average C-index and number of model genes identified from the machine learning combinations, a PCD index was developed and validated for predicting AML OS. A prognostic nomogram was then generated and validated on the basis of the PCD index, age and ELN risk stratification in the Beat AML cohort and the GSE146173 cohort, revealing satisfactory predictive power (AUC values ≥ 0.7). With different mutation patterns, a higher PCD index was associated with a worse OS. The PCD index was significantly related to higher scores for immunosuppressive cells and mature leukemia cell subtypes. As the gene most closely related to the PCD index, the expression of SMAD3 was further validated in vitro. AML cells harboring KMT2A rearrangements were more sensitive to the SMAD3 inhibitor SIS3, and the expression of the autophagy-related molecular marker LC3 was increased in KMT2A -rearranged cell lines after SIS3 monotherapy and combined treatment. Conclusion The PCD index and SMAD3 gene expression levels have potential prognostic value and can be used in targeted therapy for AML, and these findings can lead to the development of effective strategies for the combined treatment of high-risk AML patients.

Transforming growth factor‐beta (TGF‐β) functions as a potent proliferation inhibitor and apoptosis inducer in the early stages of breast cancer, yet promotes cancer aggressiveness in the advanced stages. The dual effect of TGF‐β on cancer development is known as TGF‐β paradox, and the remarkable functional conversion of TGF‐β is a pivotal and controversial phenomenon that has been widely investigated for decades. This phenomenon may be attributed to the cross talk between TGF‐β signaling and other pathways, including EGF receptor (EGFR) signaling during cancer progression. However, the underlying mechanism by which TGF‐β shifts its role from a tumor suppressor to a cancer promoter remains elusive. In this study, TGF‐β is positively correlated with EGFR expression in breast cancer tissues, and a functional linkage is observed between TGF‐β signaling and EGFR transactivation in breast cancer cell lines. TGF‐β promotes the migration and invasion abilities of breast cancer cells, along with the increase in EGFR expression. EGFR is also essential for TGF‐β‐induced enhancement of these abilities of breast cancer cells. Canonical Smad3 signaling and ERK/Sp1 signaling pathways mediate TGF‐β‐induced EGFR upregulation. Hence, our study provided insights into a novel mechanism by which TGF‐β supports breast cancer progression. Transforming growth factor (TGF)‐β promotes the migration and invasion abilities of breast cancer cells through the upregulation of EGFR expression. Canonical Smad3 signaling and ERK/Sp1 signaling pathways are required for the TGF‐β‐induced upregulation of EGFR and the enhancement of migration and invasion abilities of breast cancer cells. Hence, our findings provided insights into a novel mechanism by which TGF‐β favors breast cancer progression.

Background Lung adenocarcinoma (LUAD) is the leading cause of death worldwide. However, the roles of long noncoding RNAs (lncRNAs) hijacked by super-enhancers (SEs), vital regulatory elements of the epigenome, remain elusive in the progression of LUAD metastasis. Methods SE-associated lncRNA microarrays were used to identify the dysregulated lncRNAs in LUAD. ChIP-seq, Hi-C data analysis, and luciferase reporter assays were utilized to confirm the hijacking of LINC01977 by SE. The functions and mechanisms of LINC01977 in LUAD were explored by a series of in vitro and in vivo assays. Results We found that LINC01977 , a cancer-testis lncRNA, was hijacked by SE, which promoted proliferation and invasion both in vitro and in vivo. LINC01977 interacted with SMAD3 to induce its nuclear transport, which facilitated the interaction between SMAD3 and CBP/P300, thereby regulating the downstream target gene ZEB1. Additionally, SMAD3 up-regulated LINC09177 transcription by simultaneously binding the promoter and SE, which was induced by the infiltration of M2-like tumor-associated macrophages (TAM2), subsequently activating the TGF-β/SMAD3 pathway. Moreover, LINC01977 expression was positively correlated with TAM2 infiltration and SMAD3 expression, especially in early-stage LUAD. Higher chromatin accessibility in the SE region of LINC01977 was observed with high expression of TGF-β. Early-stage LUAD patients with high LIN01977 expression had a shorter disease-free survival. Conclusions TAM2 infiltration induced a rich TGF-β microenvironment, activating SMAD3 to bind the promoter and the SE of LINC01977 , which up-regulated LINC01977 expression. LINC01977 also promoted malignancy via the canonical TGF-β/SMAD3 pathway. LINC01977 hijacked by SE could be a valuable therapeutic target, especially for the treatment of early-stage LUAD.

Background Increasing evidences indicate that circular RNAs exert critical function in regulating bladder cancer progression. However, the expressive patterns and roles of circular RNAs in bladder cancer remain less investigated. Methods circRIP2 was identified and evaluated by RNA-sequencing and qPCR; in vitro effects of circRIP2 were determined by CCK8, clone forming, wound healing and trans-well assays; while mice subcutaneous tumor model was designed for in vivo analysis. Western blot, RNA pulldown assay, miRNA capture and dual luciferase assessment were applied for mechanistic studies. Results circRIP2 was identified as a conserved and dramatically repressed circular RNA in bladder cancer. Patients that displayed higher circRIP2 expression negatively associate with the grade, stage, metastasis as well as outcome of bladder cancer. In vitro and in vivo studies suggest that circRIP2 enables to promote bladder cancer progression via inducing EMT. Regarding the mechanism, we performed RNA-sequencing analysis, RNA pulldown with biotin-labeled circRIP2-specific probe, dual luciferase reporter assay. It was found that circRIP2 enables to sponge miR-1305 to elevate Tgf-β2 in bladder cancer, and inducing EMT via Tgf-β2/smad3 pathway. Blocking Tgf-β2 in bladder cancer deprives circRIP2 induced cancer progression and EMT. Conclusions Taken together, our study provides the first evidence that circRIP2 expresses differentially in bladder cancer and negatively along with the cancer progression; effective circRIP2 activity accelerates bladder cancer progression via inducing EMT by activating miR-1305/Tgf-β2/smad3 pathway. The research implies that circRIP2 might be a potential biomarker and therapeutic target for bladder cancer patients.

Transforming Growth Factor β (TGFβ) pathway has been recognized as one of the major processes involved in fibrotic diseases including the Atherosclerosis Cardiovascular Disease (ASCVD). Many drugs have been proposed and are under clinical trials for modulation of the TGFβ pathway by targeting TGFβ receptor. Recently, various proof of the concept studies have advocated that the inhibition of TGFβ-mediated Smad pathway could produce more focused effect with less off target toxicities in ASCVD. As these studies lack the mechanism and binding profile of Smad3 modulators, characterization of binding pattern for Smad3 inhibitors can provide a platform for the lead optimization against ASCVD. We utilized dimeric inhibitors from the PubChem dataset (PubChem ID: 630) of Smad3-FoxH1 binding inhibitors to generate binding hypothesis of Smad3 inhibitors. The selected compounds from the dataset were docked and ligand-protein complexes were simulated for 250 ns for further sampling of conformational space and to obtain stable binding hypothesis. Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF) and hydrogen bond analysis of ligand-protein complexes after simulations revealed that Asn320 in Smad3 provides an efficient inhibition site for the two most potent small inhibitors (hereby named SM1 and SM2 ) of Smad3-FoxH1 binding. Although the diverse nature of compounds produce variable interaction patterns with FoxH1 binding site in Smad3, Gln315, Gln364 and Arg367 were observed to be the most common hydrogen bond interaction points in this binding site. Additionally, two compounds (hereby named SM8 and SM19) detached from the FoxH1 binding site and formed a highly stable complex around Tyr323 via hydrophobic complementarity, suggesting a new binding site for modulation of Smad3 activity.

Background/Aims: Endothelial-to-mesenchymal transition (EndMT) plays significant roles under various pathological conditions including cardiovascular diseases, fibrosis, and cancer. EndMT of endothelial progenitor cells (EPCs) contributes to neointimal hyperplasia following cell therapy Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) that promotes metastasis and cancer. MicroRNA-145 (miR-145) is a tumor suppressor that has been reported to inhibit SMAD3-mediated epithelial-to-mesenchymal transition (EMT) of cancer cells. In the present study, we investigated the role of MALAT1 and miR-145 in EndMT of human circulating EPCs induced by transforming growth factor beta1 (TGF-β1). Methods: Human circulating EPCs were isolated and characterized by fluorescence-activated cell sorting (FACS). Expression levels of EndMT markers were assessed by qRT-PCR and western blotting. Alpha-smooth muscle actin (α-SMA) expression was measured by cell immunofluorescence staining. The regulatory relationship between MALAT1 and miR-145 and its target genes, TGFBR2 (TGFβ receptortype II) and SMAD3 (mothers against decapentaplegic homolog 3) was analyzed using the luciferase reporter assay. Results: We found that EndMT of EPCs induced by TGF-β1 is accompanied by increased MALAT1 expression and decreased miR-145 expression, and MALAT1 and miR-145 directly bind and reciprocally repress each other in these cells. Dual-Luciferase Reporter assay indicated that miR-145 inhibits TGF-β1-induced EndMT by directly targeting TGFBR2 and SMAD3. Conclusions: MALAT1 modulates TGF-β1-induced EndMT of EPCs through regulation of TGFBR2 and SMAD3 via miR-145. Thus, the MALAT1-miR-145-TGFBR2/SMAD3 signaling pathway plays a key role in TGF-β1-induced EndMT.

Trimethylamine N-oxide (TMAO), a gut microbe-derived metabolite of dietary choline and other trimethylamine-containing nutrients, has been linked to increased cardiovascular disease risk. It is unknown whether TMAO plays a role in the development of cardiac hypertrophy. Transverse aortic constriction (TAC) was performed to induce cardiac hypertrophy in Sprague-Dawley (SD) rats. We observed that TMAO levels were significantly elevated in SD rats after 6 weeks of TAC, suggesting the potential role of TMAO in regulating cardiac hypertrophy. In cultured cardiomyocytes, TMAO treatment stimulated cardiac hypertrophy, as indicated by increased cell area of cardiomyocytes and expression of hypertrophic markers including atrial natriuretic peptide (ANP) and beta-myosin heavy chain (β-MHC). Additionally, TMAO treatment induced cardiac hypertrophy and cardiac fibrosis in SD rats. Reducing TMAO synthesis by antibiotics (Abs) attenuated TAC-induced cardiac hypertrophy and fibrosis. Furthermore, pharmacological inhibition of Smad3 by SIS3 significantly reduced the expression of ANP and β-MHC, and cardiomyocyte cell size in TMAO-treated group. These data for the first time demonstrate that gut microbe-derived metabolite TMAO induces cardiac hypertrophy and fibrosis involving Smad3 signaling, suggesting that inhibition of gut microbes or generation of TMAO may become a potential target for the prevention and treatment of cardiac hypertrophy.

Phosphorylation of Smad3 is a critical mediator of TGF-β signaling, which plays an important role in regulating innate immune responses. However, whether Smad3 activation can be regulated in innate immune cells in TGF-β-independent contexts remains poorly understood. Here, we show that Smad3 is activated through the phosphorylation of its C-terminal residues (pSmad3C) in murine and human macrophages in response to bacterial and viral ligands, and this activation is mediated by activin A in a TGF-β-independent manner. Specifically, infectious ligands, such as LPS, induced secretion of activin A through the transcription factor STAT5 in macrophages, and activin A signaling in turn activated pSmad3C. This activin A/Smad3 axis controlled mitochondrial ATP production and ATP conversion into adenosine by CD73 in macrophages, enforcing an antiinflammatory mechanism. Consequently, mice with a deletion of activin A receptor 1b specifically in macrophages (Acvr1bfl/fl-Lyz2cre) succumbed more to sepsis as a result of uncontrolled inflammation and exhibited exacerbated skin disease in a mouse model of imiquimod-induced psoriasis. Thus, we have revealed a previously unrecognized natural brake to inflammation in macrophages that occurs through the activation of Smad3 in an activin A-dependent manner.