Non-canonical TGF-β Pathway Research Articles

Clathrin switches transforming growth factor-β role to pro-tumorigenic in liver cancer

Upon ligand binding, tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), are recruited into clathrin-coated pits for internalization by endocytosis, which is relevant for signalling and/or receptor degradation. In liver cells, transforming growth factor-β (TGF-β) induces both pro- and anti-apoptotic signals; the latter are mediated by the EGFR pathway. Since EGFR mainly traffics via clathrin-coated vesicles, we aimed to analyse the potential role of clathrin in TGF-β-induced signalling in liver cells and its relevance in liver cancer. Real-Time PCR and immunohistochemistry were used to analyse clathrin heavy-chain expression in human (CLTC) and mice (Cltc) liver tumours. Transient knockdown (siRNA) or overexpression of CLTC were used to analyse its role on TGF-β and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGFB1 expression on prognosis and overall survival in patients with hepatocellular carcinoma (HCC). Clathrin expression increased during liver tumorigenesis in humans and mice. CLTC knockdown cells responded to TGF-β phosphorylating SMADs (canonical signalling) but showed impairment in the anti-apoptotic signals (EGFR transactivation). Experiments of loss or gain of function in HCC cells reveal an essential role for clathrin in inhibiting TGF-β-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-β signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. This work describes a novel role for clathrin in liver tumorigenesis, favouring non-canonical pro-tumorigenic TGF-β pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-β-targeted therapy. Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Cltc), altering the cellular response to TGF-β in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CLTC expression in HCC human samples could help select patients that would benefit from therapies targeting TGF-β.

Noncanonical TGFβ Pathway Relieves the Blockade of IL1β/TGFβ-Mediated Crosstalk between Tumor and Stroma: TGFBR1 and TAK1 Inhibition in Colorectal Cancer.

The aim of the study is blocking the recruitment of a protective stroma by altering the crosstalk between normal stromal cells and tumor cells for stripping tumors of the protection conferred by the microenvironment. A transcriptomic analysis of cocultured normal colonic fibroblasts and colorectal tumor cells was performed. We focused on the study of molecules that mediate the communication between both compartments and that entail fibroblasts' activation and the alteration of the sensitivity to chemotherapy. We identified targets for the blocking of the tumor-stroma interaction. Finally, we tested, in vivo, the blockade of the tumor-stroma interaction in orthotopic models derived from patients and in models of acquired resistance to oxaliplatin. IL1β/TGFβ1 are the triggers for fibroblasts' recruitment and conversion into carcinoma-associated fibroblasts (CAF) in colorectal cancer. CAFs then secrete proinflammatory factors that alter sensitivity in tumor cells, activating JAK/STAT and PI3KCA/AKT pathways. Blocking such crosstalk with a neutralizing IL1β antibody and a TGFBR1 inhibitor is relieved by the TAK1-mediated activation of the noncanonical TGFβ pathway, which induces a change in the cytokine/chemokine repertoire that maintains a sustained activation of AKT in tumor cells. TAK1 plus TGFBR1 inhibition blocks IL1β/TGFβ1-mediated fibroblast activation, decreasing the secretion of proinflammatory cytokines. In turn, tumor cells became more sensitive to chemotherapy. In vivo, the combination of a TAK1 inhibitor plus TGFBR1 inhibitor reduced the metastatic capacity of tumor cells and the recruitment of fibroblasts. Our findings provide a translational rationale for the inhibition of TAK1 and TGFBR1 to remove the chemoprotection conferred by CAFs.

Abstract 2374: Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression

Abstract Introduction: While the genomic and proteomic characterization of pancreatic ductal adenocarcinoma (PDAC) is extraordinarily complex, the transforming growth factor-beta (TGF-β) pathway is critically important. TGF-β has a context dependent role- it is tumor suppressive in early PDAC and tumor promotive in metastatic PDAC. Our hypothesis is that non-canonical TGF-β signaling induces metastatic PDAC. Non-canonical signaling may help explain the paradoxical nature of TGF-β in PDAC. Methods: Two cell lines were utilized- Panc-1, a cell line derived from primary PDAC, was compared to Capan-1, a cell line derived from metastatic PDAC- in order to model an early PDAC (Panc-1) and a more aggressive PDAC (Capan-1). We investigated the gene and protein expression of markers of epithelial to mesenchymal transition (Snail and Twist), Akt, β-catenin, and PD-L1 upon treatment with TGF-β and the Akt inhibitor MK-2206. Results: While TGF-β increased Snail gene and protein expression in Panc-1 cells, it had nil effect on Capan-1 cells where TWIST1 protein expression increased with TGF-β. TGF-β increased pAkt expression in both cell lines. TGF-β increased active and total β-catenin expression in Capan-1 but not Panc-1 cell lines. We then found that PD-L1 expression was generally diminished by Akt inhibition in both cell lines but this was reversed with TGF-β receptor inhibition and gemcitabine treatment in the Capan-1 cells. Conclusions: Our data suggests that modulation of the non-canonical TGF-β pathway may lead to increased epithelial to mesenchymal transition through multiple mechanisms. PD-L1 expression is a response to chemotherapy in metastatic PDAC when multiple proteins in that pathway are inhibited. Our data suggests that (1) personalized therapy based on the degree of non-canonical TGF-β pathway activation will be critical, (2) patients with uncontrolled non-canonical TGF-β pathway activation may have lower survival, and (3) there is an opportunity to utilize anti-PD-L1 based therapy in combination with other therapy in selected patients based on their response to chemotherapy. Citation Format: S. Mazher Hussain, Gustavo Miranda-Carboni, Marcus A. Alvarez, Abul Elahi, Leah E. Hedrick, David Shibata, Evan S. Glazer. Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2374.

Abstract LB-120: Acetylated Kruppel-like factor 5 and transforming growth factor-β mediated drug resistance in prostate cancer

Abstract Purpose: In this study, we explored the role of Kruppel-like factor 5 (KLF5) and its acetylated form on lysine 369 in transforming growth factor-β (TGFβ) induced drug resistance to promote antitumor synergy and overcome drug resistance in advanced stage prostate cancer. Procedures: Two aggressive prostate cancer cell lines DU 145 and PC-3 were treated with docetaxel to measure cytotoxicity. To assess docetaxel resistance, we used in-vitro cell survival assay, colony survival assay and xenograft mouse model. In addition, TGFβ1 and SB505124 (TGFβ receptor I inhibitor) were used to explore TGFβ induced docetaxel resistance. To measure function of KLF5 and its different acetylation form on lysine 369, stable cell lines were constructed with different KLF5 acetylation status mimics after knocking out endogenous KLF5, utilizing the CRISPR-Cas9 system. Results: We found that KLF5 alone induced docetaxel resistance in vivo, but not in vitro. Moreover, endogenous knocking out of KLF5 inhibited TGFβ induced docetaxel resistance. In addition to previous findings that TGFβ recruits P300 acetyl-transferase to acetylate KLF5 on lysine 369, we further found that TGFβ induced docetaxel resistance depends on acetylation of KLF5. In addition, Mass Spectrometry analysis revealed that JUNB, a downstream target of non-canonical TGFβ pathway, is a direct binding target of acetylated KLF5 but not de-acetylated KLF5. Knocking down of JUNB in acetylated KLF5 mimic cell line inhibited TGFβ induced docetaxel resistance. Conclusions: These suggested that acetylated KLF5 is indispensable in TGFβ induced drug resistance through interaction with JUNB. Our study introduced a novel mechanism of acetylated KLF5 and JUNB mediated docetaxel resistance in prostate cancer and contributed to an advanced therapy combination for advanced prostate cancer patients. Citation Format: Yixiang Li, Baotong Zhang, Jin-Tang Dong. Acetylated Kruppel-like factor 5 and transforming growth factor-β mediated drug resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-120.

The motor protein Myo1c regulates transforming growth factor-β–signaling and fibrosis in podocytes

Transforming growth factor-β (TGF-β) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-β signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-β-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-β-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-β signaling through downregulation of canonical and non-canonical TGF-β pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-β signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-β responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-β-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.

Epithelial to mesenchymal transition is mediated by both TGF-β canonical and non-canonical signaling during axolotl limb regeneration

Axolotls have the amazing ability to regenerate. When compared to humans, axolotls display a very fast wound closure, no scarring and are capable to replace lost appendages perfectly. Understanding the signaling mechanism leading to this perfect healing is a key step to help develop regenerative treatments for humans. In this paper, we studied cellular pathways leading to axolotl limb regeneration. We focus on the wound closure phase where keratinocytes migrate to close the lesion site and how epithelial to mesenchymal transitions are involved in this process. We observe a correlation between wound closure and EMT marker expression. Functional analyses using pharmacological inhibitors showed that the TGF-β/SMAD (canonical) and the TGF-β/p38/JNK (non-canonical) pathways play a role in the rate to which the keratinocytes can migrate. When we treat the animals with a combination of inhibitors blocking both canonical and non-canonical TGF-β pathways, it greatly reduced the rate of wound closure and had significant effects on certain known EMT genes.

Crosstalk between the TGF-β and WNT signalling pathways during cardiac fibrogenesis.

Cardiac fibrosis is referred to as an excessive accumulation of stromal cells and extracellular matrix proteins in the myocardium. Progressive fibrosis causes stiffening of the cardiac tissue and affects conduction of electrical impulses, leading to heart failures in a broad range of cardiac conditions. At the cellular level, activation of the cardiac stromal cells and myofibroblast formation are considered as hallmarks of fibrogenesis. At the molecular level, transforming growth factor β (TGF-β) is traditionally considered as a master regulator of the profibrotic processes. More recently, the WNT signalling pathway has also been found to be implicated in the development of myocardial fibrosis. In this review, we summarize current knowledge on the involvement of TGF-β and WNT downstream molecular pathways to cardiac fibrogenesis and describe a crosstalk between these two profibrotic pathways. TGF-β and WNT ligands bind to different receptors and trigger various outputs. However, a growing body of evidence points to cross-regulation between these two pathways. It has been recognized that in cardiac pathologies TGF-β activates WNT/β-catenin signalling, which in turn stabilizes the TGF-β/Smad response. Furthermore both, the non-canonical TGF-β and non-canonical WNT signalling pathways, activate the same mitogen-activated protein kinases (MAPKs): the extracellular signal-regulated kinase (Erk), the c-Jun N-terminal kinases (JNKs) and p38. The crosstalk between TGF-β and WNT pathways seems to play an essential role in switching on the genetic machinery initiating profibrotic changes in the heart. Better understanding of these mechanisms will open new opportunities for development of targeted therapeutic approaches against cardiac fibrosis in the future.

TGF-β Determines the Pro-migratory Potential of bFGF Signaling in Medulloblastoma

The microenvironment shapes cell behavior and determines metastatic outcomes of tumors. We addressed how microenvironmental cues control tumor cell invasion in pediatric medulloblastoma (MB). We show that bFGF promotes MB tumor cell invasion through FGF receptor (FGFR) invitro and that blockade of FGFR represses brain tissue infiltration invivo. TGF-β regulates pro-migratory bFGF function in a context-dependent manner. Under low bFGF, the non-canonical TGF-β pathway causes ROCK activation and cortical translocation of ERK1/2, which antagonizes FGFR signaling by inactivating FGFR substrate 2 (FRS2), and promotes a contractile, non-motile phenotype. Under high bFGF, negative-feedback regulation of FRS2 by bFGF-induced ERK1/2 causes repression of the FGFR pathway. Under these conditions, TGF-β counters inactivation of FRS2 and restores pro-migratory signaling. These findings pinpoint coincidence detection of bFGF and TGF-β signaling by FRS2 as a mechanism that controls tumor cell invasion. Thus, targeting FRS2 represents an emerging strategy to abrogate aberrant FGFR signaling.

Complement Activation During Ischemia/Reperfusion Injury Induces Pericyte-to-Myofibroblast Transdifferentiation Regulating Peritubular Capillary Lumen Reduction Through pERK Signaling.

Pericytes are one of the principal sources of scar-forming myofibroblasts in chronic kidneys disease. However, the modulation of pericyte-to-myofibroblast transdifferentiation (PMT) in the early phases of acute kidney injury is poorly understood. Here, we investigated the role of complement in inducing PMT after transplantation. Using a swine model of renal ischemia/reperfusion (I/R) injury, we found the occurrence of PMT after 24 h of I/R injury as demonstrated by reduction of PDGFRβ+/NG2+ cells with increase in myofibroblasts marker αSMA. In addition, PMT was associated with significant reduction in peritubular capillary luminal diameter. Treatment by C1-inhibitor (C1-INH) significantly preserved the phenotype of pericytes maintaining microvascular density and capillary lumen area at tubulointerstitial level. In vitro, C5a transdifferentiated human pericytes in myofibroblasts, with increased αSMA expression in stress fibers, collagen I production, and decreased antifibrotic protein Id2. The C5a-induced PMT was driven by extracellular signal-regulated kinases phosphorylation leading to increase in collagen I release that required both non-canonical and canonical TGFβ pathways. These results showed that pericytes are a pivotal target of complement activation leading to a profibrotic maladaptive cellular response. Our studies suggest that C1-INH may be a potential therapeutic strategy to counteract the development of PMT and capillary lumen reduction in I/R injury.

Ubiquitin System

Ever since the discovery of ubiquitin in 1975[...].

Long non-coding RNAs AC026904.1 and UCA1: a "one-two punch" for TGF-β-induced SNAI2 activation and epithelial-mesenchymal transition in breast cancer.

Transforming growth factor-β (TGF-β) has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) during cancer progression, mainly by activating a set of pleiotropic transcription factors including SNAI2/Slug. However, the involvement of long non-coding RNAs (lncRNAs) in TGF-β-induced Slug activation and EMT remains largely unknown.Methods: In this study, we used microarray analysis to compare lncRNA expression profiles between TGF-β treated and untreated breast cancer cells. Then, the clinical significance of lncRNAs in breast cancer was investigated by qPCR and Kaplan-Meier survival analysis. The molecular mechanisms and EMT-promoting effects in vitro were analyzed by confocal laser microscopy, Western blotting, chromosome conformation capture (3C), chromatin isolation by RNA purification (ChIRP), ChIP, luciferase reporter assay and transwell migration assay. Lastly, the pro-metastatic effects in vivo were evaluated by bioluminescent imaging and hematoxylin and eosin (H&E) staining.Results: We observed that TGF-β induced genome-wide changes in lncRNA levels in breast cancer cells, among which AC026904.1 and UCA1 were highly expressed in metastatic breast cancer and closely associated with poor prognosis. Mechanistic study revealed that AC026904.1 and UCA1 were upregulated by non-canonical and canonical TGF-β pathways, respectively. Further analysis showed that AC026904.1 functions as an enhancer RNA in the nucleus, whereas UCA1 exerts a competitive endogenous RNA (ceRNA) activity in the cytoplasm. In addition, the biological functions of these two lncRNAs converged on the activation and maintenance of Slug, constituting a one-two punch in promoting EMT and tumor metastasis.Conclusion: These findings uncover for the first time that AC026904.1 and UCA1 could cooperatively upregulate Slug expression at both transcriptional and post-transcriptional levels, exerting critical roles in TGF-β-induced EMT. The present work provides new evidence that lncRNAs function as key regulators of EMT and hold great promise to be used as novel biomarkers and therapeutic targets for metastatic breast cancer.

Suppression of TAK1 pathway by shear stress counteracts the inflammatory endothelial cell phenotype induced by oxidative stress and TGF-\u03b21

Endothelial dysfunction is characterised by aberrant redox signalling and an inflammatory phenotype. Shear stress antagonises endothelial dysfunction by increasing nitric oxide formation, activating anti-inflammatory pathways and suppressing inflammatory pathways. The TAK1 (MAP3K7) is a key mediator of inflammation and non-canonical TGF-β signalling. While the individual roles of TAK1, ERK5 (MAPK7) and TGF-β pathways in endothelial cell regulation are well characterised, an integrative understanding of the orchestration of these pathways and their crosstalk with the redox system under shear stress is lacking. We hypothesised that shear stress counteracts the inflammatory effects of oxidative stress and TGF-β1 on endothelial cells by restoring redox balance and repressing the TAK1 pathway. Using human umbilical vein endothelial cells, we here show that TGF-β1 aggravates oxidative stress-mediated inflammatory activation and that shear stress activates ERK5 signalling while attenuating TGF-β signalling. ERK5 activation restores redox balance, but fails to repress the inflammatory effect of TGF-β1 which is suppressed upon TAK1 inhibition. In conclusion, shear stress counteracts endothelial dysfunction by suppressing the pro-inflammatory non-canonical TGF-β pathway and by activating the ERK5 pathway which restores redox signalling. We propose that a pharmacological compound that abates TGF-β signalling and enhances ERK5 signalling may be useful to counteract endothelial dysfunction.

Hyperactive Non-Canonical TGF-β Pathway Signaling in Fanconi Anemia Bone Marrow Stromal Cells Contributes to Growth Suppression

Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life due to attrition of hematopoietic stem cells (HSCs). FA is caused by autosomal recessive or X-linked mutations in one of nineteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to genotoxic DNA cross-linking agents. Although its mechanism is unknown, bone marrow failure in FA may be the result, directly or indirectly, of hyperactivation of cell-autonomous or microenvironmental growth-suppressive pathways induced, in part, due to genotoxic stress. We have recently identified canonical transforming growth factor-β (TGF-β) pathway-mediated growth suppression of HSCs as a cause of bone marrow failure in FA (Zhang H et al, Cell Stem Cell, 2016). We have shown that TGF-β pathway inhibition rescues genotoxic stress, proliferation defects and engraftment defects of FA-deficient HSCs, and ameliorates bone marrow failure in FA mice. Previous studies have suggested that bone marrow stromal fibroblasts from human FA patients and FA pathway-deficient mouse models, like HSCs, are hypersensitive to genotoxic stress and have impaired growth. Here, we therefore investigated the possible suppressive function of the TGF-β pathway in bone marrow stromal cells derived from FA mice and patients with FA.Methods: We established primary stromal cell lines from bone marrow of FA-deficient mice (Fancd2-/- mice) or wild-type sibling control mice. Primary bone marrow stromal cultures were also established from FA patients or normal healthy donors. The stromal cells were characterized and evaluated for growth kinetics, mitomycin C (MMC) sensitivity, chromosome breakage, inflammatory signals and response to the TGF-β inhibitors. CRISPR/Cas-9 technology was used to knockdown specific genes in stromal cells.Results: As expected,the primary bone marrow stromal cells from Fancd2-/- mice exhibited classical FA phenotypes, including hypersensitivity to a DNA cross-linking agent, MMC, and increased MMC-induced chromosomal radials. Fancd2-/- stromal cells also demonstrated a growth defect characterized by an enrichment of cells in G1 and elevated p21 expression. Interestingly, the FA stromal cells derived from FA patients or from Fancd2-/- mice expressed constitutively elevated levels of phosphorylated (activated) ERK1/2 (pERK) compared to control cells. In order to determine whether the factor responsible for inducing ERK1/2 phosphorylation in the murine FA stromal cells was cell-intrinsic or cell-extrinsic, we examined the conditioned media from the stromal cells. Indeed the FA stromal cells secreted a high level of TGF-β cytokine responsible for increased pERK levels, and expressed a high level of secreted TGF-β mRNA. The high level of pERK indicated that the TGF-β non-canonical pathway was hyperactive in the FA stromal cells. Interestingly, CRISPR/Cas9-mediated knockdown of Tgfbr1 or inhibition of the TGF-β pathway by a treatment with a small molecule inhibitor of TGFβR1 or a neutralizing antibody against TGF-β in these cells reduced pERK levels, promoted DNA repair and rescued MMC sensitivity. In addition, a MEK inhibitor also significantly improved the clonogenic growth of Fancd2-/- stromal cells. However, CRISPR/Cas9-mediated knockdown of Smad3, a downstream target of the canonical TGF-β pathway, did not rescue the growth inhibition of FA stromal cells in MMC, further indicating that hyperactivation of the canonical pathway is less relevant to their growth defect. Collectively, these results demonstrated that the hyperactive TGF-β pathway increases phosphorylation of ERK1/2 in FA stromal cells through the non-canonical signaling pathway and impairs their growth after genotoxic stress.Conclusions: The primary FA bone marrow stromal cells exhibit hyperactive non-canonical TGF-β pathway signaling and blocking this pathway improves their growth under genotoxic stress. The TGF-β signaling pathway-mediated growth suppression in bone marrow stromal cells may account, at least in part, for defective microenvironment, impaired HSC function and bone marrow failure in FA. This work suggests that the TGF-β signaling pathway may be a potential therapeutic target for the treatment of bone marrow failure in FA. DisclosuresShimamura:TransCellular Therapeutics: Other: Husband is founder. No revenue to date.; Novartis: Other: In discussion regarding possible clinical trial for aplastic anemia; Glaxo Smith Kline: Honoraria.

Abstract 95: The Role of TGFβ Signaling in a Fibrotic cMyBP-C HCM/HF Model

Purpose: Hypertrophic cardiomyopathy (HCM) is considered one of the most common genetic heart disorders with a prevalence of about 1 in 500 people, with 35% of those affected being attributed to mutations within the gene encoding cardiac myosin-binding protein C (cMyBP-C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40kDa fragment in mouse cardiomyocytes ( Mybp3 40kDa ) leads to HCM, fibrosis and heart failure, mimicking human disease progression. The transforming growth factor-β (TGFβ) signaling pathway has been implicated in a variety of fibrotic processes. The goal of this study is to define the role of TGFβ signaling in distinct cell populations, the cardiomyocyte and fibroblast, in the cMyBP-C HCM/HF model. Methods and results: Masson’s Trichrome staining, PCR arrays, immunohistochemistry and western blots were performed to characterize the fibrotic progression in Mybp3 40kDa transgenic mice. Cardiac fibrosis was initially detected 4 weeks after transgene expression. Extensive interstitial fibrosis and severe atrial fibrosis were detected at 16 weeks. Both canonical and non-canonical TGFβ pathways were active during fibrotic progression. To specifically block TGFβ signaling in Mybp3 40kDa transgenic mice, compound mutant mice were generated, in which the tgfbr1 or tgfbr2 alleles were ablated, either in cardiomyocytes or in activated fibroblasts (myofibroblasts) by αMHC-Cre or Periostin-MerCreMer-Cre respectively. Blockage of TGFβ signaling in either cardiomyocytes or myofibroblasts alleviated cardiac fibrosis. Furthermore, treatment with the non-canonical TGFβ signaling inhibitor MMI-0100 also alleviated cardiac fibrosis and increased the life span of the Mybp3 40kDa transgenic mice. Conclusions: TGFβ signaling is activated in the Mybp3 40kD HCM/HF model. Genetic or pharmaceutical inhibition of TGFβ signaling inhibited fibrosis and increased the life span in this model.

Puerarin Attenuates Cardiac Hypertrophy Partly Through Increasing Mir-15b/195 Expression and Suppressing Non-Canonical Transforming Growth Factor Beta (Tgfβ) Signal Pathway.

BackgroundPrevious studies demonstrated that puerarin has therapeutic effects on cardiac hypertrophy. This study aimed to explore whether the effect of puerarin on attenuating cardiac hypertrophy is related to regulation of microRNAs (miRNAs) and the transforming growth factor beta (TGFβ) signal pathway.Material/MethodsThe therapeutic effect of puerarin was assessed using an angiotensin (Ang) II-induced heart hypertrophy model in mice. The primary cardiomyocytes were used as an in vitro model. MiR-15 family expression was quantified using qRT-PCR analysis. The expression of the genes involved in canonical and non-canonical TGFβ signal pathways was measured using qRT-PCR and Western blot analysis. In vitro cardiac hypertrophic features were assessed by quantifying cardiac hypertrophic genes and measurement of cell surface, protein synthesis, and total protein content.ResultsPuerarin attenuated cardiac hypertrophy and increased miR-15b and miR-195 expression in the mouse cardiac hypertrophy model and in primary cardiomyocytes. It suppressed both canonical and non-canonical TGFβ signal pathways, partially through miR-15b and miR-195. Puerarin reduced mRNA expression of cardiac hypertrophic genes, reduced cell surface area, and lowered the rate of protein synthesis and the total protein content induced by Ang II. Knockdown of endogenous miR-15b and miR-195 partly abrogated these effects. Knockdown of endogenous p38, but not Smad2/3/4, presented similar effects as miR-15b.ConclusionsPuerarin administration enhances miR-15b and miR-195 expression in an Ang II-induced cardiac hypertrophy model, through which it suppresses both canonical and non-canonical TGFβ signal pathways at the same time. However, the effect of puerarin on attenuating cardiac hypertrophy is mainly through the non-canonical TGFβ pathway.

Staurosporine induces chondrogenesis of chick embryo wing bud mesenchyme in monolayer cultures through canonical and non-canonical TGF-β pathways.

Staurosporine has been known to induce chondrogenesis in monolayer cultures of mesenchymal cells by dissolving actin stress fibers. The aim of this study was to further elucidate how the alteration of actin filaments by staurosporine induces chondrogenesis. Specifically, we examined whether the transforming growth factor (TGF)-β pathway is implicated. SB505124 strongly suppressed staurosporine-induced chondrogenesis without affecting the drug's action on the actin cytoskeleton. Staurosporine increased the phosphorylation of TGF-β receptor I (TβRI) but had no significant effect on the expression levels of TGF-β1, TGF-β2, TGF-β3, TβRI, TβRII, and TβRIII. Phosphorylation of Smad2 and Smad3 was not increased by staurosporine. However, SB505124 almost completely suppressed the phosphorylation of Smad2 and Smad3. In addition, inhibition of Smad3 blocked staurosporine-induced chondrogenesis. Inhibition of Akt, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) suppressed chondrogenesis induced by staurosporine. Phosphorylation of Akt, p38 MAPK, and JNK was increased by staurosporine. SB505124 reduced the phosphorylation of Akt and p38 MAPK, while it had no effect on the phosphorylation of JNK. The phosphorylation level of extracellular signal-regulated kinase (ERK) was not significantly affected by staurosporine. In addition, inhibition of ERK with PD98059 alone did not induce chondrogenesis. Taken together, these results suggest that staurosporine induces chondrogenesis through TGF-β pathways including canonical Smads and non-canonical Akt and p38 MAPK signaling.

Dysregulation of the Transforming Growth Factor β Pathway in Induced Pluripotent Stem Cells Generated from Patients with Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome with clinical features of red cell aplasia and variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations in ribosomal protein genes but the pathogenic mechanism is still unknown. We generated induced pluripotent stem cells from DBA patients carrying RPS19 or RPL5 mutations. Transcriptome analysis revealed the striking dysregulation of the transforming growth factor β (TGFβ) signaling pathway in DBA lines. Expression of TGFβ target genes, such as TGFBI, BAMBI, COL3A1 and SERPINE1 was significantly increased in the DBA iPSCs. We quantified intermediates in canonical and non-canonical TGFβ pathways and observed a significant increase in the levels of the non-canonical pathway mediator p-JNK in the DBA iPSCs. Moreover, when the mutant cells were corrected by ectopic expression of WT RPS19 or RPL5, levels of p-JNK returned to normal. Surprisingly, nuclear levels of SMAD4, a mediator of canonical TGFβ signaling, were decreased in DBA cells due to increased proteolytic turnover. We also observed the up-regulation of TGFβ1R, TGFβ2, CDKN1A and SERPINE1 mRNA, and the significant decrease of GATA1 mRNA in the primitive multilineage progenitors. In summary our observations identify for the first time a dysregulation of the TGFβ pathway in the pathobiology of DBA.

Abstract 2396: Hampering the crosstalk between fibroblasts and tumor cells reveals the need for blocking both canonical and non-canonical TGFβ pathways

Abstract Background: Since fibroblasts are the predominant stromal cell type in colorectal carcinomas (CRC), it is reasonable to assume that fibroblast-tumor cell crosstalk play an important role in the production and maintenance of the malignant phenotype. Thus, it is very relevant to abort the supply of such factors responsible for this crosstalk in order to hamper tumor growth. In this work, we studied the crosstalk between Normal Colonic Fibroblasts (NCF) and tumor cells, its relation with de novo chemoresistance and fibroblast activation, providing hints for the treatment of CRC. Methods: After 5 days of Transwell coculture between fibroblasts and tumor cells we obtained mRNA of each cell type to obtain differentially expressed genes compared to monocultures (Affymetrix GeneChip Human Gene 1.0 ST Array). From genes with fold change>2, we selected those codifying for secreted or membrane proteins in both tumor cells and fibroblast. Protein-protein interaction (PPI) analysis was carried out by means of BIANA software searching for fibroblast secreted proteins with receptors in tumor cells plasma membrane and viceversa. We also studied the main pathways involved in such crosstalk in control conditions or pharmacologically disrupted. In addition we performed functional assays (proliferation, migration and citotoxicity against Oxaliplatin and 5FU). We also performed a cytokine array (Raybiotech) to characterise products mediating such crosstalk. Results: After integrating our transcriptomic/PPI data with information obtained by means of GSEA we stated our working hypothesis as: cytokines released by NCF stimulate tumor cells to secrete IL1β and TGFβ1. These two soluble factors are responsible for the NCF activation. Activated fibroblasts in turn began to produce large amounts of IL1β/TGFβ1 targets, mainly chemokines, cytokines, EGFR-ligands and PAI-1, signalling basically through IL6R, CCR2 (JAK-STAT pathway), CCR6, EGFR and CXCR3 (PI3K-AKT pathway), pathways involved in chemoresistance in CRC. Hampering the crosstalk by means of a blocking IL1β antibody and TGFBR1 inhibitor sensitizes tumor cells to Oxaliplatin and 5FU and induces a change in the cytokines mediating the crosstalk. Cell signalling reveals an activation of non-canonical TGFβ1 evidenced by TAK1 phosphorilation and NFkβ activation. Conclusions: Disrupting the crosstalk between fibroblasts and tumor cells with TGFBR1 inhibitor and a blocking IL1β antibody induces a change in the cytokine repertoire of both cells that activates NFkβ pathway in fibroblasts and a sustained activation of AKT in tumor cells. We can avoid such activation inhibiting non-canonical TGFβ pathway with a TAK1 inhibitor. The combined use of canonical and non-canonical TGFβ inhibitors could an interesting approach to overcome microenvironment-mediated drug resistance. Citation Format: NATALIA GUILLEN, REBECA SANZ-PAMPLONA, SAMUEL GONÇALVES, RAMON SALAZAR, GABRIEL CAPELLA, DAVID G. MOLLEVI. Hampering the crosstalk between fibroblasts and tumor cells reveals the need for blocking both canonical and non-canonical TGFβ pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2396. doi:10.1158/1538-7445.AM2015-2396

TGFβ‐induced TAZ upregulation and the underlying mechanisms

TGFβ is a prime inducer of myofibroblast (MF) transition from various precursors. TAZ, a Hippo pathway‐regulated transcriptional coactivator has been shown to crosstalk with TGFβ signaling, primarily as a Smad3 nuclear retention factor. During our studies aimed at assessing the role of TAZ in fibrogenic phenotype shifts, we observed that TGFβ caused a substantial (»5‐fold) and fast (6 h) rise in TAZ protein level in two MF precursors, pericyte‐like (10T1/2) cells and renal fibroblasts. TGFβ increased TAZ in tubular epithelial cells as well, an effect that was strongly potentiated by cell contact uncoupling, a prerequisite for epithelial‐MF transition. Interrogating the underlying signaling in 10T1/2 cells, we found that the Smad3 inhibitor SIS3 failed to abolish TGFβ‐induced TAZ upregulation, whereas inhibition of Akt or p38 abrogated this effect. These findings imply that non‐canonical TGFβ pathways are central to the control of TAZ. While interference with TAZ‐degrading pathways (Casein Kinase δ/ε, GSK3β) increased TAZ expression with similar magnitude and kinetics as TGFβ, cycloheximide‐provoked TAZ degradation was not prevented by TGFβ. Instead, TGFβ increased TAZ mRNA. Since myocardin‐related transcription factor (MRTF) is a TGFβ‐regulated key inducer of MF transition, we treated cells with its inhibitor, CCG‐1423. The drug strongly suppressed TGFβ‐triggered TAZ expression and the concomitant induction of a TAZ‐dependent luciferase reporter. Thus, TGFβ enhances TAZ transcription in an Akt‐, p38‐ and MRTF‐dependent manner, signifying a hitherto under‐recognized TAZ regulatory mechanism with potentially key roles in the pathobiology of organ fibrosis.

GW25-e0832 TNF receptor-associated factor 6 (TRAF6) mediates the angiotensin-induced non-canonical TGFβ pathway activation and differentiation of c-kit+ cardiac stem cells

GW25-e0832 TNF receptor-associated factor 6 (TRAF6) mediates the angiotensin-induced non-canonical TGFβ pathway activation and differentiation of c-kit+ cardiac stem cells