TGFb Activity Research Articles

Molecular mechanisms regulating natural menopause in the female ovary: a study based on transcriptomic data.

Natural menopause is an inevitable biological process with significant implications for women's health. However, the molecular mechanisms underlying menopause are not well understood. This study aimed to investigate the molecular and cellular changes occurring in the ovary before and after perimenopause. Single-cell sequencing data from the GTEx V8 cohort (30-39: 14 individuals; 40-49: 37 individuals; 50-59: 61 individuals) and transcriptome sequencing data from ovarian tissue were analyzed. Seurat was used for single-cell sequencing data analysis, while harmony was employed for data integration. Cell differentiation trajectories were inferred using CytoTrace. CIBERSORTX assessed cell infiltration scores in ovarian tissue. WGCNA evaluated co-expression network characteristics in pre- and post-perimenopausal ovarian tissue. Functional enrichment analysis of co-expression modules was conducted using ClusterprofileR and Metascape. DESeq2 performed differential expression analysis. Master regulator analysis and signaling pathway activity analysis were carried out using MsViper and Progeny, respectively. Machine learning models were constructed using Orange3. We identified the differentiation trajectory of follicular cells in the ovary as ARID5B+ Granulosa -> JUN+ Granulosa -> KRT18+ Granulosa -> MT-CO2+ Granulosa -> GSTA1+ Granulosa -> HMGB1+ Granulosa. Genes driving Granulosa differentiation, including RBP1, TMSB10, SERPINE2, and TMSB4X, were enriched in ATP-dependent activity regulation pathways. Genes involved in maintaining the Granulosa state, such as DCN, ARID5B, EIF1, and HSP90AB1, were enriched in the response to unfolded protein and chaperone-mediated protein complex assembly pathways. Increased contents of terminally differentiated HMGB1+ Granulosa and GSTA1+ Granulosa were observed in the ovaries of individuals aged 50-69. Signaling pathway activity analysis indicated a gradual decrease in TGFb and MAPK pathway activity with menopause progression, while p53 pathway activity increased. Master regulator analysis revealed significant activation of transcription factors FOXR1, OTX2, MYBL2, HNF1A, and FOXN4 in the 30-39 age group, and GLI1, SMAD1, SMAD7, APP, and EGR1 in the 40-49 age group. Additionally, a diagnostic model based on 16 transcription factors (Logistic Regression L2) achieved reliable performance in determining ovarian status before and after perimenopause. This study provides insights into the molecular and cellular mechanisms underlying natural menopause in the ovary. The findings contribute to our understanding of perimenopausal changes and offer a foundation for health management strategies for women during this transition.

Molecular heterogeneity of antiviral resident memory CD8 T cell in the female reproductive tract

Abstract Resident memory CD8 T cells (TRM) in the female reproductive tract (FRT) represent a primary defense mechanism against viruses. Presence of CD8 TRM in the reproductive mucosa is associated with accelerated viral control and rapid establishment of an antiviral state. However, little is known about the factors that control and maintain TRM in this important barrier organ. Here, using single-cell RNA-sequencing (scRNA-seq) and genetic methods, we characterized development of distinct FRT CD8 TRM subsets. Our scRNA-seq studies revealed that most FRT CD8 TRM belong to two transcriptionally distinct subsets that are marked by mutually exclusive surface expression of CD103 and Ly6C. CD103 +TRM were dependent on the cytokine TGF-b for development, as CD8 T cells lacking TGF-b receptors were significantly impaired at giving rise to CD103 +TRM. Local activation of TGF-b by av integrins was important for the maintenance of these FRT TRM. In comparison to the CD103 +TRM which resembled mature TRM, the Ly6C +TRM exhibited features of effector cells. Ly6C +TRM were regulated by the transcription factor T-bet and developed under distinct priming conditions compared to CD103 +TRM. Upon in vivo antigenic recall, the CD103 +subset elaborated greater cytotoxicity than the Ly6C +subset. Taken together, our findings provide key insights into molecular and developmental heterogeneity of FRT CD8 TRM which has implications for improving vaccination and local immunotherapy approaches. Supported by grants from NIH (P20GM121298)

Abstract 706: CRB-601, an avβ8 blocking antibody, prevents activation of TGFb and exhibits anti-tumor activity associated with immune cell remodeling of the tumor microenvironment

Abstract Background: Transforming growth factor-beta (TGFβ) is a promising immunotherapeutic target in cancer given the association of increased TGFβ signaling in the tumor microenvironment (TME) with immune cell exclusion, and poor clinical outcomes. TGFβ is expressed as a latent form (L-TGFβ) and presented on cell surfaces by L-TGFβ binding proteins (e.g. GARP and LRRC33) as part of the large latent complex, whereupon it is activated by binding to integrins, including integrin αvβ8. Corbus Pharmaceuticals is developing a humanized monoclonal antibody, CRB-601, that binds with high specificity and affinity to αvβ8 and blocks the critical interaction with L-TGFβ that promotes an immune excluded phenotype. Methods: Tumor growth was evaluated in mice bearing orthotopically implanted murine breast cancer EMT6 or colon cancer MC38 and treated with CRB-601, anti-PD1 antibody, or the combination. αvβ8 receptor occupancy (RO) was measured ex vivo in dissociated tumors by flow cytometry (FC). Tumor-infiltrating immune cell populations were characterized by FC, and pSMAD2/3 was measured by Western blot. Results: CRB-601 exhibited dose dependent tumor growth inhibition (TGI) in the EMT6 tumor model. TGI activity at 0.3mg/kg biw dose was 22.0 ± 3.8 %, and reached maximum antitumor activity at a dose between 3 mg/kg biw (41.3 ± 8.4%) and 10 mg/kg biw (48.4 ± 8.4 %). TGI was significantly augmented in combination with anti-PD1. These effects were associated with changes in TME immune cell populations, as characterized by marked increases in infiltrating T cells, NK cells and M1 polarized macrophages, thereby converting immune excluded EMT6 tumors to immune cell-inflamed tumors. Efficacy correlated with cell surface αvβ8 occupancy by CRB-601 as measured by FC in CD45- EMT6 tumor cells, increasing with CRB-601 dose from 5.5 ± 0.4% at 0.3 mg/kg to a maximum of 75.6 ± 23.8% at 10 mg/kg. CRB-601 treatment downregulated phosphorylation of SMAD proteins pSMAD2 and pSMAD3, consistent with blockade of the canonical TGFβ signaling pathway. When combined with anti-PD1, CRB-601 induced a T-cell memory response. In the MC38 model, mice cured by the combination treatment survived long term (>40 mo). Naïve mice adoptively transferred with splenocytes from these mice gained tumor-specific antitumor activity and rejected MC38 tumor cell inoculation. These splenocytes contained an immune cell population of IFN-γ producing cytotoxic T cells with a repertoire consistent with MC38 antigen recognition. Conclusions: CRB-601 is a potent and selective integrin αvβ8 blocking monoclonal antibody that can overcome tumor immune exclusion and enhance the activity of immune checkpoint inhibitors. CRB-601 antitumor activity correlated to increased αvβ8 RO and downregulation of TGFβ signaling. Investigational New Drug (IND) enabling studies are in progress. Citation Format: Daqing Wang, Vaishali Shinde, Maneesh Singh, Rachael Brake, Andrew Kolodziej. CRB-601, an avβ8 blocking antibody, prevents activation of TGFb and exhibits anti-tumor activity associated with immune cell remodeling of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 706.

P107 Decreased epithelial keratinization contributes to defective wound healing in Crohn's disease fistula

Abstract Background Up to 30% of Crohn's Disease (CD) patients suffer from perianal fistula creating a high disease burden. Treating CD fistula is challenging compared to cryptoglandular fistula, which have a significantly better wound healing response. Previous studies suggested epithelial to mesenchymal transition as an inducer of fistula formation, as markers were observed in fistula tracts. However, most studies compared CD fistula to normal mucosa, thus disregarding whether any observed difference was in fact a sign of healing (as CD fistula will have more active healing than uninjured mucosa) or a sign of pathology (as healing is not occurring effectively). Therefore, in this study we evaluated CD fistula, as well as both healthy mucosa (no active healing) and cryptoglandular fistula (effective healing) in order to determine the role of differences found. Methods Biopsies from the internal opening (n=20) of perianal fistula of CD patients were collected and compared to two publicly available datasets obtained from un-inflamed rectum (RISK cohort and GSE83245). In addition, curettage material of CD (n=54) and cryptoglandular fistula (n=15) was obtained. Expression profiling was performed using RNASeq. Results Comparing the mucosa at the internal opening of CD fistula to publicly available datasets of healthy rectum, 770 genes were differentially expressed. Pathway analysis revealed upregulation of proliferative and inflammatory processes, consistent with expected immune and wound healing responses. Interestingly, strongest upregulation was seen for processes of cornification and keratinization, including expression of IVL (involucrin) and FLGR2 (fillagrin2), involved in keratinocyte differentiation. To evaluate whether this was contributing to active wound healing or rather to persistent disease, we analyzed the actual tract in CD and cryptoglandular fistula. Strikingly, cornification and keratinization processes were more pronounced in cryptoglandular fistula, suggesting a role in effective healing rather than the persistent pathology seen in CD. Similar data was observed for TGFb, previously detected in CD fistula and thus considered a pathological contributor. While we confirm increased TGFb activity in CD fistula compared to normal mucosa, activity in cryptoglandular fistula tracts was higher than in CD fistula tracts. Again, this would suggest TGFb signaling as part of the wound healing response rather than part of the pathogenic mechanism. Conclusion Perianal fistulas show upregulation of keratinization and cornification pathways compared to healthy mucosa. Comparing CD and cryptoglandular fistula, we saw more cornification in the better healing cryptoglandular fistula, suggesting involvement in effective healing rather than pathology.

Optimizing Tenogenic Differentiation of Equine Adipose-Derived Mesenchymal Stem Cells (eq-ASC) Using TGFB3 Along with BMP Antagonists.

Objective Tendon repair strategies usually are accompanied by pathological mineralization and scar tissue formation that increases the risk of re-injuries. This study aimed to establish an efficient tendon regeneration method simultaneously with a reduced risk of ectopic bone formation. Materials and MethodsIn this experimental study, tenogenic differentiation was induced through transforming growth factor- β3 (TGFB3) treatment in combination with the inhibiting concentrations of bone morphogenetic proteins (BMP) antagonists, gremlin-2 (GREM2), and a Wnt inhibitor, namely sclerostin (SOST). The procedure’s efficacy was evaluated using real-time polymerase chain reaction (qPCR) for expression analysis of tenogenic markers and osteo- chondrogenic marker genes. The expression level of two tenogenic markers, SCX and MKX, was also evaluated by immunocytochemistry. Sirius Red staining was performed to examine the amounts of collagen fibers. Moreover, to investigate the impact of the substrate on tenogenic differentiation, the nanofibrous scaffolds that highly resemble tendon extracellular matrix was employed.Results Aggregated features formed in spontaneous normal culture conditions followed by up-regulation of tenogenic and osteogenic marker genes, including SCX, MKX, COL1A1, RUNX2, and CTNNB1. TGFB3 treatment exaggerated morphological changes and markedly amplified tenogenic differentiation in a shorter period of time. Along with TGFB3 treatment, inhibition of BMPs by GREM2 and SOST delayed migratory events to some extent and dramatically reduced osteo-chondrogenic markers synergistically. Nanofibrous scaffolds increased tenogenic markers while declining the expression of osteo-chondrogenic genes. ConclusionThese findings revealed an appropriate in vitro potential of spontaneous tenogenic differentiation of eq- ASCs that can be improved by simultaneous activation of TGFB and inhibition of osteoinductive signaling pathways.

Tacrolimus induces ligand independent TGFβ receptor signaling to promote renal fibrosis

BackgroundAlthough calcineurin inhibitors (CNIs) such as tacrolimus and cyclosporin have dramatically improved the quality of patient care, long‐term therapy causes irreversible damage to the kidneys in the form of renal fibrosis. These morphologic changes ultimately lead to a decline in renal function and can progress to end‐stage renal failure. These detrimental outcomes present a critical need to identify the driving mechanisms by which CNIs cause renal damage.ObjectiveIt is well established that TGFb is a major contributor to CNI‐induced renal fibrosis. However, the underlying mechanisms remain unknown. The objectives of this study are to 1) investigate whether TGFb secretion is required to stimulate TGFb receptor signaling in a model of CNI‐induced renal fibrosis and 2) investigate whether calcineurin plays a critical role in regulating TGFb receptor activity.Experimental DesignTo examine the role of calcineurin inhibition in altered TGFb receptor signaling, wild type mice were treated with either vehicle (0.01% ethanol) or 10 mg/kg tacrolimus for 7 days. To confirm in vivo findings, wild‐type mouse renal cortical fibroblasts were treated with either vehicle (0.01% ethanol) or 1nM tacrolimus for 24 hours in the presence and absence of anti‐TGFb neutralizing antibodies. TGFb receptor expression and activation, TGFb receptor downstream signaling mediators, profibrotic markers and calcineurin activity were analyzed.ResultsFindings demonstrate that tacrolimus‐induced loss of calcineurin activity is accompanied with enhanced TGFb receptor activation and signaling. Notably, increasing concentrations of anti‐TGFb neutralizing antibodies failed to abolish aberrant TGFb signaling and increased expression of profibrotic markers.ConclusionsTogether, these results demonstrate that 1) CNIs promote ligand‐independent TGFb signaling and 2) calcineurin plays a functional role in regulating TGFb receptor activity.SignificanceThis study elucidates a direct pharmacological mechanism by which CNIs promote renal fibrosis and provides a physiological role of calcineurin in regulating TGFb receptor activity. Further insight into the role of calcineurin in regulating TGFb receptor activity could improve the long‐term outcomes of patients on chronic CNI therapy.

Role of platelet GARP in TGFB activation

Abstract Introduction Transforming growth factor (TGF)β is known to be a central player in the control of cardiac fibroblast properties and fibrosis. However, cellular and molecular mechanisms that trigger its activation remain poorly understood. Platelets are considered as a major source of TGFβ and recent evidence suggest that they are involved in TGFβ activation via Glycoprotein A Repetitions Predominant (GARP) present on their surface. Purpose The present study sought to evaluate the role of platelet GARP in TGFβ activation using platelet specific GARP knockout mice. Methods We generated a new Cre transgenic mouse strain that allowed Megakaryocyte/platelet specific invalidation of GARP (GpIba-Cre x GARPfl/fl). The impact of GARP deficiency on platelet function was measured in vitro by flow cytometry using thrombin and CRP. Serum production of total and active TGFβ was assessed by ELISA. Results Platelet count and other hematological parameters were normal in platelet specific GARP knockout mice, except platelet volume, which was increased by 10.3%, as compared to wild-type platelets. Stimulation by thrombin and CRP increased GARP exposure at platelet surface. However, platelets without GARP displayed normal agonist induced activation, as reflected by CD62P and αIIbβ3 exposure. Interestingly, the generation of active TGFβ was drastically impaired in the serum of platelet specific GARP knockout mice, while the amount of total TGFβ was not affected. Conclusion We provided evidence that platelet GARP is a crucial contributor to the systemic activation of TGFβ. Future work will aim to determine its role in cardiac fibroblast myodifferentiation and fibrosis. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): FRIA

Equine pregnancy-specific glycoprotein CEACAM49 secreted by endometrial cup cells activates TGFB.

In early equine pregnancy, a highly invasive trophoblast cell subpopulation, the chorionic girdle cells, invade the endometrium and form endometrial cups (EC). These cells express classical MHC molecules, thereby stimulating a humoral and cellular immune response, resulting in a massive accumulation of maternal CD4+ and CD8+ T cells around the EC. Nevertheless, no immediate destruction of endometrial cups by maternal lymphoid cells occurs, presumably due to immune tolerance. Although the environment of EC is rich in TGFB and in FOXP3+, CD4+ T cells, the mechanisms leading to tolerance have not been elucidated. Recently, we discovered that equine trophoblast cells secrete pregnancy-specific glycoproteins (PSGs). Since human and murine PSGs activate latent TGFB, we hypothesized that equine PSGs may have a similar activity. We performed plasmon surface resonance experiments to show that equine PSG CEACAM49 can directly bind to the latency-associated peptide (LAP) of both TGFB1 and TGFB2. We then found that the binding of CEACAM49 leads to the activation of TGFB1 as determined by both ELISA and cell-based assays. Furthermore, the activation of TGFB is a unique function of PSGs within the human CEA family, because CEACAM1, 3, 5, 6, 8 do not activate this cytokine. This finding further strengthens the classification of CEACAM49 as an equine PSG. Based on our results, we hypothesize that activation of latent TGFB in the EC environment by equine PSGs secreted by invasive trophoblast cells, could contribute to the generation of regulatory T cells (Tregs) to maintain immune tolerance.

ECM1 Prevents Activation of Transforming Growth Factor β, Hepatic Stellate Cells, and Fibrogenesis in Mice

Activation of TGFB (transforming growth factor β) promotes liver fibrosis by activating hepatic stellate cells (HSCs), but the mechanisms of TGFB activation are not clear. We investigated the role of ECM1 (extracellular matrix protein 1), which interacts with extracellular and structural proteins, in TGFB activation in mouse livers. We performed studies with C57BL/6J mice (controls), ECM1-knockout (ECM1-KO) mice, and mice with hepatocyte-specific knockout of EMC1 (ECM1Δhep). ECM1 or soluble TGFBR2 (TGFB receptor 2) were expressed in livers of mice after injection of an adeno-associated virus vector. Liver fibrosis was induced by carbon tetrachloride (CCl4) administration. Livers were collected from mice and analyzed by histology, immunohistochemistry, in situ hybridization, and immunofluorescence analyses. Hepatocytes and HSCs were isolated from livers of mice and incubated with ECM1; production of cytokines and activation of reporter genes were quantified. Liver tissues from patients with viral or alcohol-induced hepatitis (with different stages of fibrosis) and individuals with healthy livers were analyzed by immunohistochemistry and in situ hybridization. ECM1-KO mice spontaneously developed liver fibrosis and died by 2 months of age without significant hepatocyte damage or inflammation. In liver tissues of mice, we found that ECM1 stabilized extracellular matrix-deposited TGFB in its inactive form by interacting with αv integrins to prevent activation of HSCs. In liver tissues from patients and in mice with CCl4-induced liver fibrosis, we found an inverse correlation between level of ECM1 and severity of fibrosis. CCl4-induced liver fibrosis was accelerated in ECM1Δhep mice compared with control mice. Hepatocytes produced the highest levels of ECM1 in livers of mice. Ectopic expression of ECM1 or soluble TGFBR2 in liver prevented fibrogenesis in ECM1-KO mice and prolonged their survival. Ectopic expression of ECM1 in liver also reduced the severity of CCl4-induced fibrosis in mice. ECM1, produced by hepatocytes, inhibits activation of TGFB and its activation of HSCs to prevent fibrogenesis in mouse liver. Strategies to increase levels of ECM1 in liver might be developed for treatment of fibrosis.

PS-091-Targeted disruption of TGF-B activation by an AVB1 integrin inhibitor significantly reduces liver fibrosis in CCl4 mice and Human NASH liver slices

PS-091-Targeted disruption of TGF-B activation by an AVB1 integrin inhibitor significantly reduces liver fibrosis in CCl4 mice and Human NASH liver slices

The relationship between substrate topography and stem cell differentiation in the musculoskeletal system.

It is well known that biomaterial topography can exert a profound influence on various cellular functions such as migration, polarization, and adhesion. With the development and refinement of manufacturing technology, much research has recently been focused on substrate topography-induced cell differentiation, particularly in the field of tissue engineering. Even without biological and chemical stimuli, the differentiation of stem cells can also be initiated by various biomaterials with different topographic features. However, the underlying mechanisms of this biological phenomenon remain elusive. During the past few decades, many researchers have demonstrated that cells can sense the topography of materials through the assembly and polymerization of membrane proteins. Following the activation of RHO, TGF-b or FAK signaling pathways, cells can be induced into various differentiation states. But these signaling pathways often coincide with canonical mechanical transduction pathways, and no firm conclusion has been reached among researchers in this field on topography-specific signaling pathways. On the other hand, some substrate topographies are reported to have the ability to inhibit differentiation and maintain the 'stemness' of stem cells. In this review, we will summarize the role of topography in musculoskeletal system regeneration and explore possible topography-related signaling pathways involved in cell differentiation.

The Beneficial Effects of Sitagliptin, a Dipeptedyl Peptidase-4 (DPP-4) Inhibitor on Experimentally Induced Non-Alcoholic Fatty Liver Disease in Rats

AbstractBackground: Non alcoholic fatty liver disease (NAFLD) is an increasing health problem which affects about 1/3 of the population worldwide, it is strongly associated with metabolic syndrome with oxidative stress and insulin resistance are the mechanisms mostly involved in its pathogenesis. Dipeptidyl peptidase-4 inhibitors are antidiabetic agents expected to have beneficial effects beyond their glycemic control.Aim of the Study: The present work was designed to study the beneficial effects of sitagliptin (a DPP-4I) on a dietary model of NAFLD.Material and Methods: 30 male albino rats were allocated in 3 groups. The effects of sitagliptin, was assessed in groups III. Body weight and blood pressure were recorded and blood samples were tested for total cholesterol, triglycerides and liver transaminases, glucose and insulin levels. Livers for GSH, MDA, TGF-B and DPP-4 activity and for histopatholg-ical examination. Aortae were removed for in vitro vascular reactivity studies.Results: Induction of NAFLD model by HFD for 20 weeks resulted in significant increase in body weight, blood pressure, liver index and epididymal weight. Significant reduction of these parameters was observed with 4 week treatment with sitagliptin. There was significant increase in all biochemical parametres in HFD fed rats compared to that of normal with significant improvement in sitagliptin treated group. HFD model groups showed notable steatosis, lobular inflammation and fibrosis which was improved with sitagliptin use. Also NAFLD rats developed endothelial dysfunction which was significantly ameliorated in sitagliptin treated group.Conclusion: The results of the present work showed that the DPP4Is have the potential to favorably influence the course of NAFLD process and its complications. DPP4I (sitagliptin) has many beneficial effects including scavenging of free radicals, and enhancing the activity of antioxidant effects. Large clinical trials are needed to determine the effectiveness of DPP-4 inhibitors for the prevention and treatment of NAFLD

Syzygium Samarangense Leaves Ointment Enhances Wound Healing Process Of Skin Burn Based On Collagen

Background: The World Health Organization (WHO) estimates the number of death in 2014 caused by burns is 265,000. Burns can caused skin damage as well as other complication problems such as dehydration, infection, and other multiple organ failures. Syzygium samarangense leaves contain flavonoids and saponins that can increase the activation of macrophages and TGF-B which is important to accelerate the process of collagen formation and wound healing process. Objective: Knowing the effect of Syzygium samarangense leaf extract on the healing process of burn based on collagen. Method: This in vivo study use true experimental design. We made burns by placing a coin that already heated in oven at 70ºC for 10 seconds. Rattus Wistar as experimental animals dividedinto 6 groups (n = 4) with details of Group A (normal), B (positive), C (negative). Groups D, E, and F were the groups that given ointment extract topically in doses of 15%, 30%, and 45% each day’s. Termination is done on day 7. Test statistics by using Anova and Kruskal Wallis. Results and discussion: From this research, Syzygium samarangense leaf extract can reduce the wound area (p <0,05) and increase the amount of collagen (p <0,05). Conclusion: Syzygium samarangense leaf extract ointment can accelerate the healing process of burns.
 Keywords : Burns, Syzygium samarangense Leaves ointment, Saponin, Flavonoid, Collagen

Abstract P1-08-10: Introduction of H1047R oncogenic mutation of PI3K p110alpha subunit in HER2-overexpressing mammary epithelial cells confers a "stem-like" phenotype and acute sensitivity to HSP90 inhibition

Abstract The Human Epidermal Growth Factor Receptor 2 (HER2) oncogene is amplified in one-fifth of breast cancers (BC). However, development of resistance against standard anti-HER2 therapies poses a major clinical challenge. Anti-tumor efficacy of HER2-targeting agents depends on inhibition of the downstream phosphatidylinositol-3 kinase (PI3K) signaling cascade. Gain-of-function somatic mutations in the gene encoding the PI3K catalytic subunit p110alpha (PIK3CA), co-expressed in about 40% of HER2+ BC, have been implicated in conferring resistance to HER2 monoclonal antibody herceptin. The single amino acid alteration H1047R within the kinase domain of PIK3CA is one of three hot spot mutations prevalent in BC. Previously, we demonstrated that introduction of H1047R mutation in HER2-overexpressing MCF10A mammary epithelial cells enhances cellular transformation and decreases herceptin sensitivity by inducing secretion of endogenous ErbB ligand heregulin. However, genetic ablation of HER3, the major co-receptor for HER2 and the solitary receptor for heregulin, was insufficient for complete inhibition of cell growth, indicating the existence of additional mechanism/s responsible for the heightened aggressiveness and decreased drug sensitivity of HER2/H1047RPI3K cells. In the current study, we looked further into the molecular changes within these cells that might be responsible for these phenomena. When compared with the HER2/WTPI3K cells, the HER2/H1047RPI3K cells revealed a significant increase in CD44high/CD24low/negative populations, common markers of BC stem cells, as well as molecular and phenotypic changes associated with epithelial-to-mesenchymal transition. These observations are in agreement with previously published report on mouse model of HER2/H1047RPI3K BC. Further analyses demonstrated additional stem cell-associated characteristics in HER2/H1047RPI3K cells, such as expression of angiogenic and inflammatory cytokines, ability to induce chemotaxis and invasion, activation of TGFb and NFKb signaling pathways. Connectivity map (CMap) analysis of the gene expression signatures from HER2/H1047RPI3K cells revealed a negative association with those from BC cells treated with 17AAG, an inhibitor of the heat shock protein 90 (HSP90). In line with this, HER2/H1047RPI3K-expressing cells are found to be more sensitive to HSP90 inhibition compared to the pan-ErbB inhibitor lapatinib. Cancer stem cells are implicated in drug resistance and tumor recurrence. Enrichment of cell population expressing high levels of stem cell markers and stem cell-related features could be one of major mechanisms by which BC cells co-expressing HER2 and H1047RPI3K adapt to anti-HER2 therapeutic agents. Acute dependence on molecular chaperone HSP90 provides a unique, yet practical opportunity to effectively inhibit tumors harboring both molecular alterations, since HSP90 inhibitors have already shown encouraging clinical activity in herceptin-resistant setting. Citation Format: Surendran S, Bhola N, Arteaga CL, Chakraborty K, Chakrabarty A. Introduction of H1047R oncogenic mutation of PI3K p110alpha subunit in HER2-overexpressing mammary epithelial cells confers a "stem-like" phenotype and acute sensitivity to HSP90 inhibition [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-10.

Inhibitors of Arg-Gly-Asp-Binding Integrins Reduce Development of Pancreatic Fibrosis in Mice.

Background & AimsPancreatic stellate cells (PSCs) regulate the development of chronic pancreatitis (CP) and are activated by the cytokine transforming growth factor β (TGFB). Integrins of the αv family promote TGFB signaling in mice, probably by interacting with the Arg-Gly-Asp (RGD) sequence of the TGFB latency-associated peptide, which frees TGFB to bind its cellular receptors. However, little is known about the role of integrins in the development of CP. We investigated the effects of small-molecule integrin inhibitors in a mouse model of CP.MethodsWe induced CP in C57BL/6 female mice by repeated cerulein administration. An active RGD peptidomimetic compound (Center for World Health and Medicine [CWHM]-12) was delivered by continuous infusion, starting 3 days before or 5 days after cerulein administration began. Pancreata were collected and parenchymal atrophy, fibrosis, and activation of PSCs were assessed by histologic, gene, and protein expression analyses. We measured CWHM-12 effects on activation of TGFB in co-culture assays in which rat PSC cells (large T immortalized cells [LTC-14]) activate expression of a TGFB-sensitive promoter in reporter cells.ResultsPancreatic tissues of mice expressed messenger RNAs encoding subunits of RGD-binding integrins. Cerulein administration increased expression of these integrins, altered pancreatic cell morphology, and induced fibrosis. The integrin inhibitor CWHM-12 decreased acinar cell atrophy and loss, and substantially reduced fibrosis, activation of PSCs, and expression of genes regulated by TGFB. CWHM-12 also reduced established fibrosis in mice and blocked activation of TGFB in cultured cells.ConclusionsBased on studies of a mouse model of CP and cultured PSCs, integrins that bind RGD sequences activate PSCs and promote the development of pancreatic fibrogenesis in mice. Small-molecule antagonists of this interaction might be developed for treatment of pancreatic fibrotic diseases.

Abstract 1932: RNA-seq differential transcription analysis of TCGA colorectal cancer (CRC) transcriptomes reveals subtype-specific isoform usage

Abstract Background: Differential transcription analysis may reveal transcriptome aberrations associated with cancer progression and help identify important drivers in molecularly distinct subtypes of a disease. Methods: We developed a data-driven bioinformatics pipeline (DiffSplice) that performs differential transcription analysis at the alternative splicing (AS) level and allows accurate transcriptome reconstruction through joint analysis of all RNA-seq samples. In this study, we applied DiffSplice to RNA-seq data from TCGA CRC (n = 686) and evaluated the expression patterns of the detected AS events across the four consensus molecular subtypes (CMS) of the disease (n = 511): CMS1 - MSI Immune (hypermutated, n = 76); CMS2 - Canonical (epithelial, WNT/MYC activation, n = 220); CMS3 - Metabolic (epithelial, enriched for KRAS mutations, n = 72); and CMS4 - Mesenchymal (stromal invasion, TGFB activation, n = 143). Results: A total of 15,613 AS events were detected, including 5,128 events that involved novel splice junctions not cataloged in UCSC GAF2.0 transcriptome annotation. Exon skipping was the most common pattern with 6,482 occurrences, followed by 4,962 alternative splice sites, 2,689 alternative 5′/3′ transcription sites and 1,262 retained introns. In the subset of samples with CMS classification, a cross-subtype comparative analysis revealed 1,835 differentially transcribed loci from 1,634 genes, including 759 novel junctions (FDR<0.01). Of particular interest, we found that KRAS 4A expression levels were higher in CMS3 samples (p = 0.0070), in line with the known effect of this isoform as a negative regulator of mutant KRAS alleles. In addition, expression of the CD44v isoform, a marker of epithelial cancer stem cells, was higher in CMS2 and CMS3 samples (p = 0.015). Alternatively, CD44s isoform expression levels were higher in CMS4 samples (p<2.2e-16), consistent with previous literature showing that cancer stem cells of tumors that have undergone mesenchymal transition predominantly express the standard CD44 isoform that contains no variant exons. Finally, variance of the transcription profiles suggested potential intra-subtype heterogeneity and an opportunity to further refine these global patterns. Discussion: Here we present an in-depth transcriptome analysis of TCGA CRC data across the molecular subtypes of the disease. We were able to identify differential transcription patterns in an unbiased way by using a scalable and efficient computational approach that models both annotated and novel splice variants simultaneously. Our pipeline revealed a number of promising candidates that may be acting as key modulators in specific disease subsets and deserve further investigation. Citation Format: Yin Hu, Rodrigo Dienstmann, Justin Guinney. RNA-seq differential transcription analysis of TCGA colorectal cancer (CRC) transcriptomes reveals subtype-specific isoform usage. [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 1932. doi:10.1158/1538-7445.AM2015-1932

Abstract 360: Differential Analysis of Thrombospondin Proteins in Cardiac Disease

Thrombospondin (Thbs) proteins are multidomain, matricellular proteins comprised of 5 genes that each share similar domains and have been largely ascribed the same functional characteristics The Thbs protein family is divided in 2 subgroups based on multimerization as trimers, (group A: Thbs1 and 2), or as pentamers (group B: Thbs3, 4 and 5). Thbs proteins modulate aspects of cell- matrix interactions, and more recently we have shown that they can also serve an intracellular chaperone function along the secretory pathway in response to ER stress. Thbs1 and 2 can also alter angiogenesis and modulate MMP activity as well as TGFb activity. The overall aim of this study is to functionally compare the two Thbs subgroups and elucidate their involvement in cardiac homeostasis and disease. Hence we analyzed Thbs1 as a representative of group A and Thbs3 for group B. Cardiomyocyte specific overexpression of either Thbs1 or Thbs3 results in robust activation of an ATF6- dependent ER stress response, similar to our previous working published with Thbs4. However, Thbs1 overexpressing mice suffer from cardiomyopathy and die within 12 weeks. In contrast, Thbs3 overexpressing mice are completely normal at baseline and Thbs3 overexpression even increased survival rates after MI similar to Thbs4 from our previous work. However, Thbs3 hearts also showed increased chamber dilation after MI and lower heart function suggesting that Thbs3 also has maladaptive effects. In contrast to loss of Thbs4, which die within the first week of TAC mediated pressure overload, Thbs3 KO mice are protected from TAC induced hypertrophy. Moreover, the combination of Thbs3/4 dKO more closely phenocopies the effect of Thbs3 deletion, as these mice show a significant reduction in hypertrophy and lung congestion after long term TAC. This is the first study comparing the function of different Thbs isoforms in the heart, which suggests a novel role of Thbs3.

ENHANCED BIOACTIVE TGF- LEVELS IN THE MYOCARDIUM SUGGESTS EARLY PATHOLOGICAL TRANSITION IN ASYMPTOMATIC SEVERE MITRAL REGURGITATION

The optimal timing of surgery for asymptomatic severe mitral regurgitation (MR) remains debatable. Excessive TGF-b activation is known to lead to pathological remodeling of the ventricular myocardium. Myocardial tissue and blood samples were collected from patients with severe MR undergoing mitral

ER stress inhibition reduces cardiac damage and improves endothelial function in hypertensive mice

Vascular complications and cardiac damage are major cause of mortality in hypertension. In this study we explored the beneficial effect of ER stress inhibition on vascular and cardiac damage in hypertensionMice were infused with Ang‐II with or without ER stress inhibitors (Tudca and PBA) for 2 weeks Mice infused with Ang‐II displayed an increase in SBP, cardiac hypertrophy and fibrosis associated with enhanced collagenI content, TGFb activity and ER stress markers which were blunted by ER stress inhibitors. AngII induced hypertension increased ER stress in aorta and mesenteric resistance arteries (MRA) enhanced TGFb activity in aorta but not in RA, reduced eNOS phosphorylation and endothelium‐dependent relaxation (EDR) in aorta and MRA. ER stress inhibition reduced TGFb fnactivity, enhanced eNOS phosphorylation and EDR. TGFb pathway inhibition improved EDR in aorta while the reduction in ROS levels ameliorated EDR in MRA onlyER stress inhibition reduces cardiac damage and improves vascular function in Ang II‐induced hypertension. ER stress could be a potential target for cardiovascular therapy

Engineered tissue based detection of PAR1 inhibition in thrombin‐induced profibrotic phenotypes of cardiac fibroblasts

Study objective To determine the effects of PAR1 (Protease-activated receptor) activation on profibrotic response in cardiac fibroblast (CF) using isolated cells and cardiac connective tissue equivalents (CCTEs). Methods Rat CFs were mixed with collagen to generate CCTEs, and they were treated for 24 hrs with thrombin and/or a PAR1 antagonist. Their tissue stiffness and cellular contractility were measured using a high-throughput mechanical measurement system. Treating with thrombin and/or PAR1 antagonists, the protein expression/activity of procollagen (type I), TGFb, MMP2/9, ERK1/2 were analyzed using ELISA, immunoblotting and zymography. Results Thrombin treatment stiffened and contracted CCTE by 38% and 25% respectively over controls (n=15). In isolated CFs, thrombin activated ERK1/2 as well as increased MMP2/9 activities and procollagen and TGFb secretion over the control. PAR1 and ERK1/2 inhibitors attenuated these effects. Conclusions Thrombin stimulated CCTEs to increase tissue stiffness and cell contractility. This profibrotic CCTE remodeling was correlated with ERK1/2 activation, increased TGFb and procollagen secretion and activation of MMP2/9. The anti-fibrotic effects of PAR1 inhibition strongly suggests that PAR1 signaling plays a critical role in ERK1/2 mediated thrombin-induced cardiac fibrosis.