Disruption Of TGF-β Signaling Research Articles

Heterogeneous Cardiac-Derived and Neural Crest-Derived Aortic Smooth Muscle Cells Exhibit Similar Transcriptional Changes After TGFβ Signaling Disruption.

Smooth muscle cells (SMCs) of cardiac and neural crest origin contribute to the developing proximal aorta and are linked to disease propensity in adults. We analyzed single-cell transcriptomes of aortic SMCs from adult mice to determine basal states and changes after disrupting TGFβ (transforming growth factor-β) signaling necessary for aortic homeostasis. A minority of Myh11 lineage-marked SMCs differentially expressed genes suggestive of embryological origin. Additional analyses in Nkx2-5 and Wnt1 lineage-marked SMCs derived from cardiac and neural crest progenitors, respectively, showed both lineages contributed to a major common cluster and each lineage to a minor distinct cluster. Common cluster SMCs extended from root to arch, cardiac subset cluster SMCs from root to ascending, and neural crest subset cluster SMCs were restricted to the arch. The neural crest subset cluster had greater expression of a subgroup of TGFβ-dependent genes. Nonetheless, conditional deletion of TGFβ receptors resulted in similar transcriptional changes among all SMC clusters. Several disease-associated transcriptional responses were comparable among SMC clusters in a mouse model of Marfan syndrome aortopathy, while many embryological markers of murine aortic SMCs were not detected in adult human aortas. There are multiple subtypes of cardiac-derived and neural crest-derived SMCs with shared or distinctive transcriptional profiles; neural crest subset cluster SMCs with increased expression of certain TGFβ-inducible genes are not spatially linked to the aortic root predisposed to aneurysms from aberrant TGFβ signaling; and loss of TGFβ responses after receptor deletion is uniform among SMC clusters.

The Role of SMAD7 in the Epigenetic Regulation of TGF-β Targets in the Metastasis of Ovarian Cancer.

The role of TGF-β signaling in the epigenetic modifications involved in ovarian cancer is not fully understood. This study investigated the relationship between TGF-β signaling, epigenetic modifications, and cellular behaviors in ovarian cancer. We found that E-cadherin, a key cell adhesion molecule, underwent epigenetic silencing via promoter DNA hypermethylation in ovarian cancer cell lines and that this was accompanied by the upregulation of vimentin, which is indicative of a mesenchymal and invasive phenotype. DNA-demethylating agents restored E-cadherin expression, which suggests that TGF-β signaling mediates this epigenetic silencing. Overexpression of SMAD7, an inhibitory component of TGF-β signaling, reversed E-cadherin silencing, which suggests a role of SMAD7 in modulating the epigenetic status. Functionally, SMAD7 overexpression inhibited the migration and invasion in ovarian cancer cells, which suggests its therapeutic potential for suppressing metastasis. Clinically, ovarian cancer patients with high SMAD7 expression had significantly longer disease-free survival. Mechanistically, SMAD7 overexpression decreased the acetylation of H3K9 and the binding of the transcriptional repressor TWIST1 at the E-cadherin promoter, which promoted its demethylation and reactivation. Disruption of TGF-β signaling upregulated SMAD4 target genes, which are silenced by epigenetic mechanisms, a finding that suggests broader therapeutic implications. Overall, our results provide insights into the role of TGF-β-mediated epigenetic regulation in ovarian cancer metastasis and underscore the therapeutic potential of targeting TGF-β signaling and its downstream effectors. Further research is needed to elucidate the underlying mechanisms and validate these therapeutic strategies.

Multiscale computational model of aortic remodeling following postnatal disruption of TGFβ signaling

The healthy adult aorta is a remarkably resilient structure, able to resist relentless cardiac-induced and hemodynamic loads under normal conditions. Fundamental to such mechanical homeostasis is the mechano-sensitive cell signaling that controls gene products and thus the structural integrity of the wall. Mouse models have shown that smooth muscle cell-specific disruption of transforming growth factor-beta (TGFβ) signaling during postnatal development compromises this resiliency, rendering the aortic wall susceptible to aneurysm and dissection under normal mechanical loading. By contrast, disruption of such signaling in the adult aorta appears to introduce a vulnerability that remains hidden under normal loading, but manifests under increased loading as experienced during hypertension. We present a multiscale (transcript to tissue) computational model to examine possible reasons for compromised mechanical homeostasis in the adult aorta following reduced TGFβ signaling in smooth muscle cells.

Arterial dissections: Common features and new perspectives.

Arterial dissections, which involve an abrupt tear in the wall of a major artery resulting in the intramural accumulation of blood, are a family of catastrophic disorders causing major, potentially fatal sequelae. Involving diverse vascular beds, including the aorta or coronary, cervical, pulmonary, and visceral arteries, each type of dissection is devastating in its own way. Traditionally they have been studied in isolation, rather than collectively, owing largely to the distinct clinical consequences of dissections in different anatomical locations - such as stroke, myocardial infarction, and renal failure. Here, we review the shared and unique features of these arteriopathies to provide a better understanding of this family of disorders. Arterial dissections occur commonly in the young to middle-aged, and often in conjunction with hypertension and/or migraine; the latter suggesting they are part of a generalized vasculopathy. Genetic studies as well as cellular and molecular investigations of arterial dissections reveal striking similarities between dissection types, particularly their pathophysiology, which includes the presence or absence of an intimal tear and vasa vasorum dysfunction as a cause of intramural hemorrhage. Pathway perturbations common to all types of dissections include disruption of TGF-β signaling, the extracellular matrix, the cytoskeleton or metabolism, as evidenced by the finding of mutations in critical genes regulating these processes, including LRP1, collagen genes, fibrillin and TGF-β receptors, or their coupled pathways. Perturbances in these connected signaling pathways contribute to phenotype switching in endothelial and vascular smooth muscle cells of the affected artery, in which their physiological quiescent state is lost and replaced by a proliferative activated phenotype. Of interest, dissections in various anatomical locations are associated with distinct sex and age predilections, suggesting involvement of gene and environment interactions in disease pathogenesis. Importantly, these cellular mechanisms are potentially therapeutically targetable. Consideration of arterial dissections as a collective pathology allows insight from the better characterized dissection types, such as that involving the thoracic aorta, to be leveraged to inform the less common forms of dissections, including the potential to apply known therapeutic interventions already clinically available for the former.

TGF-β-dependent lymphoid tissue residency of stem-like T cells limits response to tumor vaccine

TGF-β signaling is necessary for CD8+ T cell differentiation into tissue resident memory T cells (TRM). Although higher frequency of CD8+ TRM cells in the tumor microenvironment is associated with better prognosis, TGF-β−blockade typically improves rather than worsens outcomes. Here we show that in a mouse melanoma model, in the tumor-draining lymph nodes (TDLN) rather than in the tumors themselves, stem-like CD8+ T cells differentiate into TRMs in a TGF-β and tumor antigen dependent manner. Following vaccination against a melanoma-specific epitope, most tumour-specific CD8+ T cells are maintained in a stem-like state, but a proportion of cells lost TRM status and differentiate into CX3CR1+ effector CD8+ T cells in the TDLN, which are subsequently migrating into the tumours. Disruption of TGF-β signaling changes the dynamics of these developmental processes, with the net result of improving effector CD8+ T cell migration into the tumours. In summary, TDLN stem-like T cells transiently switch from a TGF-β-dependent TRM differentiation program to an anti-tumor migratory effector development upon vaccination, which transition can be facilitated by targeted TGF-β blockade.

Deletion of Tgfβ signal in activated microglia prolongs hypoxia-induced retinal neovascularization enhancing Igf1 expression and retinal leukostasis.

Retinal neovascularization (NV) is the major cause of severe visual impairment in patients with ischemic eye diseases. While it is known that retinal microglia contribute to both physiological and pathological angiogenesis, the molecular mechanisms by which these glia regulate pathological NV have not been fully elucidated. In this study, we utilized a retinal microglia‐specific Transforming Growth Factor‐β (Tgfβ) receptor knock out mouse model and human iPSC‐derived microglia to examine the role of Tgfβ signaling in activated microglia during retinal NV. Using a tamoxifen‐inducible, microglia‐specific Tgfβ receptor type 2 (Tgfβr2) knockout mouse [Tgfβr2 KO (ΔMG)] we show that Tgfβ signaling in microglia actively represses leukostasis in retinal vessels. Furthermore, we show that Tgfβ signaling represses expression of the pro‐angiogenic factor, Insulin‐like growth factor 1 (Igf1), independent of Vegf regulation. Using the mouse model of oxygen‐induced retinopathy (OIR) we show that Tgfβ signaling in activated microglia plays a role in hypoxia‐induced NV where a loss in Tgfβ signaling microglia exacerbates and prolongs retinal NV in OIR. Using human iPSC‐derived microglia cells in an in vitro assay, we validate the role of Transforming Growth Factor‐β1 (Tgfβ1) in regulating Igf1 expression in hypoxic conditions. Finally, we show that Tgfβ signaling in microglia is essential for microglial homeostasis and that the disruption of Tgfβ signaling in microglia exacerbates retinal NV in OIR by promoting leukostasis and Igf1 expression.

Endothelial Cell TGF-β (Transforming Growth Factor-Beta) Signaling Regulates Venous Adaptive Remodeling to Improve Arteriovenous Fistula Patency.

Arteriovenous fistulae (AVF) are the gold standard for vascular access for hemodialysis. Although the vein must thicken and dilate for successful hemodialysis, excessive wall thickness leads to stenosis causing AVF failure. Since TGF-β (transforming growth factor-beta) regulates ECM (extracellular matrix) deposition and smooth muscle cell (SMC) proliferation-critical components of wall thickness-we hypothesized that disruption of TGF-β signaling prevents excessive wall thickening during venous remodeling. A mouse aortocaval fistula model was used. SB431542-an inhibitor of TGF-β receptor I-was encapsulated in nanoparticles and applied to the AVF adventitia in C57BL/6J mice. Alternatively, AVFs were created in mice with conditional disruption of TGF-β receptors in either SMCs or endothelial cells. Doppler ultrasound was performed serially to confirm patency and to measure vessel diameters. AVFs were harvested at predetermined time points for histological and immunofluorescence analyses. Inhibition of TGF-β signaling with SB431542-containing nanoparticles significantly reduced p-Smad2-positive cells in the AVF wall during the early maturation phase (days 7-21) and was associated with decreased AVF wall thickness that showed both decreased collagen density and decreased SMC proliferation. SMC-specific TGF-β signaling disruption decreased collagen density but not SMC proliferation or wall thickness. Endothelial cell-specific TGF-β signaling disruption decreased both collagen density and SMC proliferation in the AVF wall and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. Endothelial cell-targeted TGF-β inhibition may be a translational strategy to improve AVF patency.

Intercellular coupling between peripheral circadian oscillators by TGF-β signaling.

Coupling between cell-autonomous circadian oscillators is crucial to prevent desynchronization of cellular networks and disruption of circadian tissue functions. While neuronal oscillators within the mammalian central clock, the suprachiasmatic nucleus, couple intercellularly, coupling among peripheral oscillators is controversial and the molecular mechanisms are unknown. Using two- and three-dimensional mammalian culture models in vitro (mainly human U-2 OS cells) and ex vivo, we show that peripheral oscillators couple via paracrine pathways. We identify transforming growth factor-β (TGF-β) as peripheral coupling factor that mediates paracrine phase adjustment of molecular clocks through transcriptional regulation of core-clock genes. Disruption of TGF-β signaling causes desynchronization of oscillator networks resulting in reduced amplitude and increased sensitivity toward external zeitgebers. Our findings reveal an unknown mechanism for peripheral clock synchrony with implications for rhythmic organ functions and circadian health.

Abstract 1405: Colorectal carcinogenesis in Smad4/SPTBN1 mutants alter the intestinal microbiome and resistance to 5FU

Abstract Background/Aims: Emerging data shows a rise in colorectal cancer (CRC) incidence in young men and women that is often chemoresistant, with potential new risk factors including alterations in the microbiome that remain understudied. We have recently observed altered microbiome with modulation of the gut immune response through crosstalk between sensors, microbes, and the TGF-β signaling pathway. Interestingly, we observed that human CRC cell lines with disruption of TGF-β had increased sensitivity to cisplatin and other DNA cross-linking agents. Yet at the same time, human CRC with loss of Smad4 portends poor prognosis and resistance to chemotherapy. Therefore, it was unclear whether the epithelial loss of Smad4 or stromal loss of Smad4 expression could be responsible for chemoresistance. Here, we investigated the role of chemotherapy with disruption of TGF-β signaling and an altered intestinal microbiome in colorectal carcinogenesis. Methods: CRCs induced by azoxymethane (AOM)/dextran sodium sulfate (DSS) in wild type (WT) and TGF-β signaling deficient mice (SKO: Smad4+/- and Smad4+/-/Sptbn1+/-) were treated with 5-Fluoro-Uracil (5FU). Shotgun metagenomic sequencing was performed in fecal samples from WT and SKO mice before and after treatment. Results: Our analyses revealed that SKO mice are more susceptible to AOM/DSS induced CRC as demonstrated by increased multiplicity (SKO vs WT: 10.25±1.5 vs 5.5±1.4, p=0.01) and tumor size (SKO vs WT: 3.63±0.7mm vs 2.17±0.3mm, p=0.02). CRC that develops in mice with disrupted TGF-β signaling is chemoresistance to 5FU and progress to liver metastasis confirmed by histological and immunohistochemical analysis. Interestingly, SKO mice display a unique gut microbiome signature compared to the WT mice. SKO mice had significantly reduced abundances of beneficial species of B. vulgutus (0.056±0.0078 vs 0.018±0.0052) and P. distasonis (0.007±0.001 vs 0.003±0.001). In addition to E. Boltae, and Mordavella sp., the relative abundance of Bacteroides was significantly reduced in AOM/DSS induced tumors and recovered to basal levels after 5FU treatment in WT mice but not in SKO mice with deficient TGF-β signaling (e.g. Bacteroides dorei: WT basal vs WT-CRC vs WT-CRC-5FU: 0.28±0.08 vs 0.05 ±0.01 vs 0.45±0.17; SKO basal vs SKO CRC vs SKO-CRC-5FU: 0.13±0.02 vs 0.06 ±0.007 vs 0.07±0.01). Conclusions: Our study identified unique gut microbiome species corresponding to 5FU resistance through interactions with key immune-related pathways such as TGF-β members that are implicated in CRC development and drug resistance. The in vivo studies suggest a cell non-autonomous role of the TGF-β pathway in CRC chemoresistance. Collectively, the altered microbiome composition from impaired TGF-β signaling could promote colorectal carcinogenesis and drug resistance. Manipulating these specific species associated with 5FU could potentially increase drug response. Citation Format: Shuyun Rao, Zhuanhuai Wang, Xiaochun Yang, Lindsay M. Hopson, Stephanie S. Singleton, Wilma Jogunoori, Raja Mazumder, Vincent Obias, Paul Lin, Bao-Ngoc Nguyen, Michael Yao, Larry Miller, Jon White, Lopa Mishra. Colorectal carcinogenesis in Smad4/SPTBN1 mutants alter the intestinal microbiome and resistance to 5FU [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1405.

Abstract 1984: Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer

Abstract Background: Cancer stem cells that escape suppression by key pathways such as TGF-β represent one of the mechanisms for poor prognosis in hepatocellular cancer (HCC). Our mouse models with disruption of TGF-β signaling (β2SP+/-/Smad3+/-) develop HCC and phenocopy an epigenetically human cancer stem cell disorder, providing clues towards the role of TGF-β in the switch in stem cell transformation to HCC. An examination of HCC genomics revealed a surprising and strong correlation between TGF-β and Sirtuins (SIRTs) expression. SIRTs are also implicated in cancer stem cell and HCC suppression. Here we hypothesized that the TGF-β pathway harnesses Sirtuin members to maintain stem cell homeostasis, which may mediate the progression of HCC. Methods: Overall Survival (OS) analysis was carried out by using early-stage HCC (TCGA, Firehose Legacy, stage N1, n=186) from cBioportal. ChIP assay was performed in HepG2 cells using anti-Smad3 and normal rabbit IgG. Biotinylated SIRT6 promoter with/without SMAD binding sites was used as probes to pull down the DNA-interacting proteins in HepG2 cells treated with/without TGF-β for 2h. Results: Analysis of TCGA HCC dataset (cBioportal, stage N1, n=186) indicated reduced OS for HCC patients with decreased SIRT6 together with loss of SPTBN1 in the early stage (Median survival months: 40.37 vs 83.18, p=0.0417). • Chip-qPCR and the DNA-protein pulldown assay revealed that Smad3 positively upregulates the SIRT6 mRNA expression (Smad3 vs IgG, SBE1: 3.27±0.53 vs 0.99±0.01, p<0.05; SBE2: 3.32±0.74 vs 1.12±0.3, p<0.05) through direct binding to the SIRT6 promoter in HepG2 cells after TGF-β treatment. • Co-IP analysis revealed directly binding of SIRT6 with β2sp and Smad3. • Overexpression of SIRT6 represses Smad3 and β2sp expression (p<0.01) and results in inhibition of the TGF-β target genes (p<0.05) such as MMP9 (68.2% inhibited), HMGA2 (53.4% inhibited), and PAI-1 (35.4% inhibited); whereas knockdown of SIRT6 increases β2sp expression in HepG2 cells (3 fold, p<0.05). • Similar to mice with disruption of SIRT6 (SIRT6-/-) that develop liver inflammation, steatosis with progression to tumorigenesis, mice with TGF-β signaling defect (Smad3+/-Sptbn1+/-) also develop fatty liver with progression to tumorigenesis under HFD. Interestingly, we found that SIRT6 was significantly downregulated in MEF cells from mice with loss of TGF-β signaling (Smad3+/-Sptbn1+/-). Conclusions: Our findings suggest that the TGF-β signaling participates in positively regulating the expression of SIRT6. In turn, SIRT6 acts as a negative regulator of TGF-β signaling through de-acetylating Smad3 and β2sp. SIRT6 and TGF-β signaling exert integrative yet complex roles in inhibiting hepatic steatosis, inflammation, and HCC that when disrupted lead to poor prognosis in HCC. The data provide new combined driving roles for the two pathways in suppressing NASH and HCC. Citation Format: Xiyan Xiang, Sobia Zaidi, Shuyun Rao, Kazufumi Ohshiro, Zhanhuai Wang, Wilma Jogunoori, Chuxia Deng, Patricia Latham, Lopa Mishra. Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1984.

Abstract 2910: A TGF-β-ALDH2 axis controls liver- brain-gut microbiome driven obesity, metabolic syndrome and cancer

Abstract Background/Aims: ALDH2 (Aldehyde dehydrogenase 2) is associated with multiple human diseases including cancers, Asian flush syndrome (deficiencies affect 35%-40% of East Asians), and alcoholic liver disease. Yet, oncogenic mechanisms and pathways that ALDH2 interacts with remain unclear. Previously we have demonstrated that TGF-β-deficient mutants derived from the loss of Smad3 and its adaptor Sptbn1 are exquisitely sensitive to alcohol, with impaired DNA damage repair. ALDH2 levels are altered in the liver tissues of the mouse mutants, and the Sptbn1-/- phenotype is similar to ALDH2-FancD2 mutants. We, therefore, hypothesized that disruption of TGF-β signaling combined with ALDH2 deficiency would increase the susceptibility of liver diseases and cancer. Methods: Aldh2-/- mice were intercrossed with Sptbn1+/-, Smad3+/- mice. Control mice and intercrosses were fed with high-fat diet (HFD) or chow diet or alcohol diet, or hepatic vagotomy followed by phenotypic and mechanistic analyses through RNA-seq, lipidomics, metabolomics, western blot analyses, RTPCR, structure modeling, cell fractionation, and immunohistochemistry. Fecal samples from these mice underwent shotgun metagenomic sequencing. Results: Strikingly, compared to WT, Aldh2-/-Sptbn1+/- (ASKO) mice on a normal diet develop metabolic syndromes with truncal obesity, insulin resistance, with increased blood glucose (272.3±28.6mg/dl vs 189.9 ±7.0mg/dl, p<0.05), serum triglyceride (185.2±40.0 mg/dl vs 83.7 ±7.8 mg/dl, p<0.05). Nonalcoholic steatohepatitis (NASH), and cancer, with raised ALT and AST levels, also develop in the mutant mice. HFD exacerbated obesity and NASH in Aldh2-/-Sptbn1+/- on HFD with substantial additional visceral fat accumulation and hyperglycemia with Zone 3 hepatic macro-steatosis and inflammation, which correlated with increased fatty acid metabolism and gluconeogenesis. ASKO mice had significantly altered neurotransmitter receptors in the liver including cholinergic receptors (e.g., Chrnb1, and Chrna2) and altered gut microbiome composition with increased abundance of S. pseudoporcinus (Aldh2-/-Smad3+/- vs WT: 85.6±29 vs 2.71 ±1.44, p<0.05) and decreased A. propionicum (ASKO vs WT: 65±21 vs 168±31, p<0.05). Conclusions: Aldh2-/-Sptbn1+/- mice develop metabolic syndrome with alterations in the cholinergic pathway and microbiome species, suggesting a disruption in afferent vagal activity. ALDH2/SPTBN1 is therefore potentially a major liver-brain-gut vagal regulator of obesity. Aldh2 and TGF-β signaling are important in maintaining normal gut microbiome composition. These studies highlight the potential role of the gut-liver axis in regulating obesity and liver disease. With > 35% Asian population harboring ALDH2 alterations, our studies potentially have a high impact on these patient populations with a high risk of metabolic syndrome. Citation Format: Shuyun Rao, Xiaochun Yang, Zhanhuai Wang, Kazufumi Ohshiro, Sobia Zaidi, Wilma Jogunoori, Bryan Nguyen, Keith A. Crandall, Patricia S. Latham, Kirti Shetty, Lopa Mishra. A TGF-β-ALDH2 axis controls liver- brain-gut microbiome driven obesity, metabolic syndrome and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2910.

Adventitial remodeling protects against aortic rupture following late smooth muscle-specific disruption of TGFβ signaling

Altered signaling through transforming growth factor-beta (TGFβ) increases the risk of aortic dissection in patients, which has been confirmed in mouse models. It is well known that altered TGFβ signaling affects matrix turnover, but there has not been a careful examination of associated changes in structure-function relations. In this paper, we present new findings on the rupture potential of the aortic wall following late postnatal smooth muscle cell (SMC)-specific disruption of type I and II TGFβ receptors in a mouse model with demonstrated dissection susceptibility. Using a combination of custom computer-controlled biaxial tests and quantitative histology and immunohistochemistry, we found that loss of TGFβ signaling in SMCs compromises medial properties but induces compensatory changes in the adventitia that preserve wall strength above that which is needed to resist in vivo values of wall stress. These findings emphasize the different structural defects that lead to aortic dissection and rupture – compromised medial integrity and insufficient adventitial strength, respectively. Relative differences in these two defects, in an individual subject at a particular time, likely reflects the considerable phenotypic diversity that is common in clinical presentations of thoracic aortic dissection and rupture. There is, therefore, a need to move beyond examinations of bulk biological assays and wall properties to cell- and layer-specific studies that delineate pathologic and compensatory changes in wall biology and composition, and thus the structural integrity of the aortic wall that can dictate differences between life and death.

The Roles of TGF-β Signaling in Cerebrovascular Diseases.

Cerebrovascular diseases are one of the leading causes of death worldwide, however, little progress has been made in preventing or treating these diseases to date. The transforming growth factor-β (TGF-β) signaling pathway plays crucial and highly complicated roles in cerebrovascular development and homeostasis, and dysregulated TGF-β signaling contributes to cerebrovascular diseases. In this review, we provide an updated overview of the functional role of TGF-β signaling in the cerebrovascular system under physiological and pathological conditions. We discuss the current understanding of TGF-β signaling in cerebral angiogenesis and the maintenance of brain vessel homeostasis. We also review the mechanisms by which disruption of TGF-β signaling triggers or promotes the progression of cerebrovascular diseases. Finally, we briefly discuss the potential of targeting TGF-β signaling to treat cerebrovascular diseases.

Transforming Growth Factor-β Signaling Pathway in Colorectal Cancer and Its Tumor Microenvironment.

Transforming growth factor-beta (TGF-β) signaling is one of the important cellular pathways that play key roles for tissue maintenance. In particular, it is important in the context of inflammation and tumorigenesis by modulating cell growth, differentiation, apoptosis, and homeostasis. TGF-β receptor type 2 (TGFBR2) mutations affected by a mismatch repair deficiency causes colorectal cancers (CRCs) with microsatellite instability, which is, however, associated with relatively better survival rates. On the other hand, loss of SMAD4, a transcription factor in the TGF-β superfamily signaling, promotes tumor progression. Loss of heterozygosity on chromosome 18 can case SMAD4-deficient CRC, which results in poorer patients’ survival. Such bidirectional phenomenon driven by TGF-β signaling insufficiency reflects the complexity of this signaling pathway in CRC. Moreover, recent understanding of CRC at the molecular level (consensus molecular subtype classification) provides deep insight into the important roles of TGF-β signaling in the tumor microenvironment. Here we focus on the TGF-β signaling in CRC and its interaction with the tumor microenvironment. We summarize the molecular mechanisms of CRC tumorigenesis and progression caused by disruption of TGF-β signaling by cancer epithelial cells and host stromal cells.

Identification and Profiling of Environmental Chemicals That Inhibit the TGFβ/SMAD Signaling Pathway.

The transforming growth factor beta (TGFβ) superfamily of secreted signaling molecules and their cognate receptors regulate cell fate and behaviors relevant to many developmental and disease processes. Disruption of TGFβ signaling during embryonic development can, for example, affect morphogenesis and differentiation through complex pathways that may be SMAD (Small Mothers Against Decapentaplegic) dependent or SMAD independent. In the present study, the SMAD Binding Element (SBE)-beta lactamase (bla) HEK 293T cell line, which responds to the activation of the SMAD2/3/4 complex, was used in a quantitative high-throughput screening (qHTS) assay to identify potential TGFβ disruptors in the Tox21 10K compound library. From the primary screening we identified several kinase inhibitors, organometallic compounds, and dithiocarbamates (DTCs) that inhibited TGFβ1-induced SMAD signaling of reporter gene activation independent of cytotoxicity. Counterscreen of SBE antagonists on human embryonic neural stem cells demonstrated cytotoxicity, providing additional evidence to support evaluation of these compounds for developmental toxicity. We profiled the inhibitory patterns of putative SBE antagonists toward other developmental signaling pathways, including wingless-related integration site (WNT), retinoic acid α receptor (RAR), and sonic hedgehog (SHH). The profiling results from SBE-bla assay identify chemicals that disrupt TGFβ/SMAD signaling as part of an integrated qHTS approach for prioritizing putative developmental toxicants.

Cyclophilin A contributes to aortopathy induced by postnatal loss of smooth muscle TGFBR1.

Recent recognition that TGF-β signaling disruption is involved in the development of aortic aneurysms has led to renewed investigations into the role of TGF-β biology in the aortic wall. We previously found that the type I receptor of TGF-β (TGFBR2) receptor contributes to formation of ascending aortic aneurysms and dissections (AADs) induced by smooth muscle cell (SMC)-specific, postnatal deletion of Tgfbr1 (Tgfbr1iko). Here, we aimed to decipher the mechanistic signaling pathway underlying the pathogenic effects of TGFBR2 in this context. Gene expression profiling demonstrated that Tgfbr1iko triggers an acute inflammatory response in developing AADs, and Tgfbr1iko SMCs express an inflammatory phenotype in culture. Comparative proteomics profiling and mass spectrometry revealed that Tgfbr1iko SMCs respond to TGF-β1 stimulation via robust up-regulation of cyclophilin A (CypA). This up-regulation is abrogated by inhibition of TGFBR2 kinase activity, small interfering RNA silencing of Tgfbr2 expression, or inhibition of SMAD3 activation. In mice, Tgfbr1iko rapidly promotes CypA production in SMCs of developing AADs, whereas treatment with a CypA inhibitor attenuates aortic dilation by 56% (P = 0.003) and ameliorates aneurysmal degeneration (P = 0.016). These protective effects are associated with reduced aneurysm-promoting inflammation. Collectively, these results suggest a novel mechanism, wherein loss of type I receptor of TGF-β triggers promiscuous, proinflammatory TGFBR2 signaling in SMCs, thereby promoting AAD formation.-Zhou, G., Liao, M., Wang, F., Qi, X., Yang, P., Berceli, S. A., Sharma, A. K., Upchurch, G. R., Jr., Jiang, Z. Cyclophilin A contributes to aortopathy induced by postnatal loss of smooth muscle TGFBR1.

Abstract 4360: ALDH2 and the TGF-β/SMAD3 adaptor, Sptbn1 suppress hepatic steatosis and obesity

Abstract Background: Previously we have demonstrated that TGF-β-deficient mutants derived from the loss of Smad3 and the Smad adaptor β2SP (Sptbn1) phenocopy a human cancer stem cell syndrome, are exquisitely sensitive to alcohol, with dysregulation DNA damage repair and have raised levels of aldehyde dehydrogenase (ALDH2). Moreover, β2SP and β2SP/Smad3 mutant mice develop multiple liver abnormalities that include steatosis, chronic hepatitis, inflammation, and cancer. ALDH2 is a key intermediate enzyme for aldehyde and alcohol metabolism. We, therefore, hypothesized that disruption of TGF-β signaling combined with ALDH2 deficiency would increase the susceptibility of liver cancer. Methods: Aldh2 knockout mice were intercrossed with Sptbn1 heterozygous knockout mice to explore the role of Aldh2 and Sptbn1 in liver steatosis and liver cancer. Conditional knockout of Sptbn1 mice was generated by the intercross of Albumin-Cre mice with mice carrying Loxp sites flanked exons of Sptbn1. Sptbn1 conditional mice were fed with high-fat diet or chow diet. Further analysis included blood lipid panel analysis and tissue profiles including western and RNA analysis, lipidomics and metabolomics analysis of liver tissues from the mutant mice and wild-type controls. Results: Interestingly, we found that Aldh2-/-Sptbn1+/- mice on a normal diet demonstrated early signs of truncal obesity. Within 6 months of age, we observed a greater than 23% increase in body weight, with predominantly abdominal fat accumulation in Aldh2-/-Sptbn1+/- mice compared to Aldh2-/- mice or wild type (WT) mice (Aldh2-/-Sptbn1+/- vs Aldh2-/- or WT, p<0.05). A marked increase in zone 3 hepatic micro and macro-steatosis with some inflammation was observed in liver tissues of mutant Aldh2-/-Sptbn1+/- mice as well as the conditional Sptbn1 knockout (Alb-Cre+Sptbn1F/+) mice in a high-fat diet. In addition to significant increased blood glucose levels in Aldh2-/-Sptbn1+/- mice compared with Aldh2-/- mice (165±23 VS 135±13, p<0.05), further analysis revealed that the fatty liver phenotype accompanied with abnormal expression of genes related to glucose uptake, metabolisms and homeostasis as demonstrated by decrease of glucose transporter and increased expression of Ceramide which is known to promote insulin resistance. Moreover, we also observed disruption of lipid metabolism as demonstrated by significantly increased serum and liver triglyceride and cholesterol ester levels in Aldh2-/-Sptbn1+/- mice. Conclusions: Our data suggest that the TGF-β pathway plays an important role in maintaining normal glucose metabolism homeostasis and patients with SPTBN1 and ALDH2 defects may have increased susceptibility to obesity as well as fatty liver (NASH), and cancer. Citation Format: Shuyun Rao, Sobia Zaidi, Wilma Jogunoori, Shoujun Gu, Jon White, Bibhuti Mishra, Chu-Xia Deng, Patricia Latham, Lopa Mishra. ALDH2 and the TGF-β/SMAD3 adaptor, Sptbn1 suppress hepatic steatosis and obesity [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 4360.

Cigarette Smoke Induces the Risk of Metabolic Bone Diseases: Transforming Growth Factor Beta Signaling Impairment via Dysfunctional Primary Cilia Affects Migration, Proliferation, and Differentiation of Human Mesenchymal Stem Cells.

It is well established that smoking has detrimental effects on bone integrity and is a preventable risk factor for metabolic bone disorders. Following orthopedic surgeries, smokers frequently show delayed fracture healing associated with many complications, which results in prolonged hospital stays. One crucial factor responsible for fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) at early stages, a mechanism mediated by transforming growth factor β (TGF-β). Although it is known that smokers frequently have decreased TGF-β levels, little is known about the actual signaling occurring in these patients. We investigated the effect of cigarette smoke on TGF-β signaling in MSCs to evaluate which step in the pathway is affected by cigarette smoke extract (CSE). Single-cell-derived human mesenchymal stem cell line (SCP-1 cells) were treated with CSE concentrations associated with smoking up to 20 cigarettes a day. TGF-β signaling was analyzed using an adenovirus-based reporter assay system. Primary cilia structure and downstream TGF-β signaling modulators (Smad2, Smad3, and Smad4) were analyzed by Western blot and immunofluorescence staining. CSE exposure significantly reduced TGF-β signaling. Intriguingly, we observed that protein levels of phospho-Smad2/3 (active forms) as well as nuclear translocation of the phospho-Smad3/4 complex decreased after CSE exposure, phenomena that affected signal propagation. CSE exposure reduced the activation of TGF-β modulators under constitutive activation of TGF-β receptor type I (ALK5), evidencing that CSE affects signaling downstream of the ALK5 receptor but not the binding of the cytokine to the receptor itself. CSE-mediated TGF-β signaling impaired MSC migration, proliferation, and differentiation and ultimately affected endochondral ossification. Thus, we conclude that CSE-mediated disruption of TGF-β signaling in MSCs is partially responsible for delayed fracture healing in smokers.

The role of different TGFβ signatures in predicting outcome in high grade serous ovarian carcinoma.

e14262 Background: Ovarian cancer (OV) is the most lethal gynecologic cancer in the US. Despite initial responsiveness to chemotherapy, > 50% experience recurrence. Recurrent disease is characterized by low response to chemotherapy and poor prognosis, especially platinum-resistant disease. Thus, there is an urgent need to identify new targeted therapy to improve the outcome of this aggressive cancer. The Transforming Growth Factor (TGFβ) family controls different cellular responses in development and cell homeostasis. Disruption of TGFβ signaling has been implicated in many cancers, including OV, but with paradox effects. It is unclear if TGFβ family genes can serve as a predictive marker and be selected for future therapy target in OV. Methods: We used quantified RNAseq data from 374 OV samples in The Cancer Genomic Atlas (TCGA) to extract 4 different sets of TGFβ pathway genes including: 3-gene ( TGFB1, TGFB2, TGFB3), 6-gene ( TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3), 43-genes from cBioPortal, or 173-genes from Calon et al, 2012 to predict colorectal cancer relapse. Expression counts for these genes were normalized and log transformed to generate a TGFβ score based on the quartiles of the average. Samples were categorized in to low (lowest quartile), medium (middle 2 quartiles), and high (highest quartile) expression groups. Overall (OS) and progression-free (PFS) survivals were compared among groups using Kaplan-Meier method. Results: Interestingly, the minimum TGFβ 6-gene set can predict high grade serous ovarian cancer patient survival among all 4 gene panels using TCGA data, with average age of 60 years (range: 38 –81). Majority of the patients were stage IIIIV. OV patients with highest quartile, TGFβ signature expression have in general less somatic mutations and copy number alterations (average mutation count = 41, average CNA fraction = 0.5) compared to lowest quartile TGFβ expression group (average mutation count = 52, average CNA fraction = 0.6). Patients with TGFB 6-gene expression scored high showed worse OS (36.2 months vs 63.5 months, log-rank p = 0.001) and worse PFS (18.6 months vs 31.4 months, log-rank p = 0.09). Details of outcome by each specified gene signature will be outlined later. Conclusions: Our analysis suggested that the narrowed 6-gene TGFβ family expression profile could potential serve as a prognosis biomarker for OV. With proper validation and mechanistic investigation, existing TGFβ inhibitors may hold promise for targeted or combination therapies for OV. Targeting both TGFβ and PD1 might be of therapeutic value.

TGF-\u03b2 signaling alters H4K20me3 status via miR-29 and contributes to cellular senescence and cardiac aging

Cellular senescence is a well-orchestrated programmed process involved in age-related pathologies, tumor suppression and embryonic development. TGF-β/Smad is one of the predominant pathways that regulate damage-induced and developmentally programmed senescence. Here we show that canonical TGF-β signaling promotes senescence via miR-29-induced loss of H4K20me3. Mechanistically, oxidative stress triggers TGF-β signaling. Activated TGF-β signaling gives rise to acute accumulation of miR-29a and miR-29c, both of which directly suppress their novel target, Suv4-20h, thus reducing H4K20me3 abundance in a Smad-dependent manner, which compromises DNA damage repair and genome maintenance. Loss of H4K20me3 mediated by the senescent TGF-β/miR-29 pathway contributes to cardiac aging in vivo. Disruption of TGF-β signaling restores H4K20me3 and improves cardiac function in aged mice. Our study highlights the sequential mechanisms underlying the regulation of senescence, from senescence-inducing triggers to activation of responsive signaling followed by specific epigenetic alterations, shedding light on potential therapeutic interventions in cardiac aging.