Growth Factor-beta Receptor Type Research Articles

An open-label, phase 1, multicenter study to evaluate the safety and preliminary anti-tumor activity of NT-112 in human leukocyte antigen-C*08:02–positive adult patients with unresectable, advanced, and/or metastatic solid tumors that are positive for the KRAS G12D mutation.

TPS2677 Background: The landscape of cancer treatment is evolving with the emergence of innovative immunotherapies. Among these, T cell receptor-engineered T cell (TCR-T) therapy has shown promise for patients with solid tumor malignancies. Targeting driver mutations, such as KRAS, may offer novel therapeutic options for patients with limited treatment options. Oncogenic driver mutations in KRAS typically involve single-base mutations at genomic hotspot locations. The KRAS isoform is mutated in 84% of RAS-driven cancers and in nearly 100% of pancreatic ductal adenocarcinoma (PDAC). The KRAS G12D mutation is the most frequent mutation in PDAC (28%), followed by colorectal cancers (12%). Despite its well-established target role in tumorigenesis, there is no FDA-approved therapy targeting the G12D variant. NT-112 is an autologous TCR-T cell therapy targeting the KRAS G12D mutation presented by HLA-C*08:02, armored by disruption of the gene encoding the transforming growth factor beta receptor type 2 (TGFBR2) in order to reduce the immunosuppressive effect of TGF-β in the tumor microenvironment. Methods: This first-in-human, open label, multicenter, Phase 1, dose escalation study aims to assess the safety, identify the MTD and evaluate potential anti-tumor activities of NT-112 in subjects with solid tumors. The study will enroll up to 24 adult subjects harboring the KRAS G12D mutation that are HLA-C*08:02 positive, who exhibit progressive or recurrent disease after at least one line of standard of care treatment. Enrolled patients will undergo leukapheresis, from which, CD4 and CD8 T cells will be enriched and activated ex vivo. The genes encoding the TGFBR2 and the endogenous TCR α and β chains are knocked out from the activated T cells utilizing clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). The HLA-C*08:02-restricted TCR specific to the KRAS G12D mutation is knocked-in in the TCR α locus. Subjects will undergo standard lymphodepletion followed by a single infusion of NT-112 and concurrent, daily, subcutaneous, recombinant IL-2 for up to 8 days. Dose escalation will occur across 3 ascending dose levels following the BOIN design. Subjects will be monitored for dose-limiting toxicities for 28 days following NT-112 infusion. Disease response will be assessed according to the RECIST v1.1 criteria. Comprehensive translational analysis will be conducted to gain insights into the mechanism of action, biomarkers associated with treatment response, and potential mechanisms of resistance. All subjects will be followed for up to 15 years. Clinical trial information: NCT06218914 .

Open-label, phase 1, multicenter study to evaluate the safety and preliminary anti-tumor activity of NT-175 in human leukocyte antigen-a*02:01-positive adult subjects with unresectable, advanced and/or metastatic solid tumors that are positive for the TP53 R175H mutation.

e14514 Background: The TP53 tumor suppressor gene is the most commonly mutated gene across all cancer types, and TP53 R175H is the most common recurrent mutation across indications, identified in 6.7% of patients with breast cancer, 6% of patients withs colorectal cancer, and 4.2% of patients with pancreatic cancer. No therapy is approved for treatment directly targeting TP53 R175H. NT-175 is an armored autologous T-cell product expressing an HLA-A*02:01-restricted TCR recognizing TP53 R175H with disruption of the endogenous TCR and the transforming growth factor beta receptor type 2 (TGFBR2) genes. Methods: This first-in-human phase I, open label study will assess the safety and preliminary anti-tumor activity, identify the maximum tolerated dose (MTD), and evaluate the persistence, and functional capacity of NT-175 in subjects with advanced, recurrent unresectable/metastatic solid tumors refractory to treatment options. The study will enroll approximately 24 subjects harboring TP53 R175H and HLA-A*02:01. Enrolled patients will undergo leukapheresis from which CD4 and CD8 T cells will be enriched and activated ex vivo. The endogenous TCR α and β chain gene are knocked out utilizing clustered Regularly Interspaced Short Palindromic repeats (CRISPR-Cas9) and the TCR encoding an HLA-A*02:01-restricted TCR specific to the TP53 R175H mutation is inserted in the TCR α locus. Additionally, the T cells are edited to disrupt the gene encoding the TGFBR2 to reduce the immunosuppressive effect of TGF-β in the tumor microenvironment. Subjects will undergo lymphodepletion chemotherapy with fludarabine and cyclophosphamide followed by a single infusion of NT-175 and subcutaneous recombinant IL-2 administration for up to 8 days. Dose escalation will occur across 3 ascending dose levels following the BOIN design. Subjects will be monitored for dose-limiting toxicities for 28 days after NT-175 infusion. Tumor response will be assessed by RECISTv1.1 criteria. All subjects will be followed for up to 15 years. Primary endpoints include safety, dose limited toxicities, MTD determination, and recommended Phase II dose. Secondary endpoints include preliminary efficacy measures. Translational analysis will evaluate potential biomarkers for insights into mechanism of action, response prediction, or resistance mechanisms. The study was activated in March 2023 and is open to accrual; 5 patients have been enrolled as of 4 Jan 2024.

Electric field induced the changes in structure and function of human transforming growth factor beta receptor type I: from molecular dynamics to docking

The transforming growth factor beta (TGF-β) signaling pathway is believed to play essential roles in several physiological activities, including cancer. TGF-β receptor type I (TBR-I) is a key membrane receptor protein in the TGF-β signaling pathway, which relates to many intracellular biological effects. In recent years, cold atmospheric plasma (CAP) has been found to have promising prospects in selective anticancer therapy and has confirmed its essential role in the TGF-β signaling pathway. However, the ambiguous effect of CAP-induced electric field (EF) on TBR-I still limits the application of CAP in clinical therapy. Molecular dynamics is applied to assess the effect of EF on the structure of the extracellular domain of TBR-I using a series of indicators and methods, and then we discuss the ligand binding ability of TBR-I. Results show that moderate EF intensities’ structural restraints may contribute to the structural stability and ligand-binding ability of TBR-I, but an EF higher than 0.1 V/nm will be harmful. What’s more, EF induces a change in the docking interface of TBR-I, showing the conformation and position of special sequences of residues decide the ligand binding surface. The relevant results suggest that CAP-induced EF plays a crucial role in receptor-receptor interaction and provides significant guidelines for EF-related anticancer therapy. Communicated by Ramaswamy H. Sarma

SPARC Is Highly Expressed in Young Skin and Promotes Extracellular Matrix Integrity in Fibroblasts via the TGF-β Signaling Pathway.

The matricellular secreted protein acidic and rich in cysteine (SPARC; also known as osteonectin), is involved in the regulation of extracellular matrix (ECM) synthesis, cell-ECM interactions, and bone mineralization. We found decreased SPARC expression in aged skin. Incubating foreskin fibroblasts with recombinant human SPARC led to increased type I collagen production and decreased matrix metalloproteinase-1 (MMP-1) secretion at the protein and mRNA levels. In a three-dimensional culture of foreskin fibroblasts mimicking the dermis, SPARC significantly increased the synthesis of type I collagen and decreased its degradation. In addition, SPARC also induced receptor-regulated SMAD (R-SMAD) phosphorylation. An inhibitor of transforming growth factor-beta (TGF-β) receptor type 1 reversed the SPARC-induced increase in type I collagen and decrease in MMP-1, and decreased SPARC-induced R-SMAD phosphorylation. Transcriptome analysis revealed that SPARC modulated expression of genes involved in ECM synthesis and regulation in fibroblasts. RT-qPCR confirmed that a subset of differentially expressed genes is induced by SPARC. These results indicated that SPARC enhanced ECM integrity by activating the TGF-β signaling pathway in fibroblasts. We inferred that the decline in SPARC expression in aged skin contributes to process of skin aging by negatively affecting ECM integrity in fibroblasts.

Abstract 5009: Inhibition of TGFBR2 signaling re-sensitizes TMZ-resistant glioblastoma cells to therapy

Abstract Glioblastoma (GBM), the most aggressive and lethal form of brain tumors, recurs in 90% of cases and the standard protocol of care—temozolomide (TMZ)—is an ineffective method to combat chemo-resistant GBM. It is therefore imperative that we identify, develop, and design novel therapeutics to effectively combat recurrent GBM (rGBM). Multi-potent stem-like cell subpopulations within GBM (also referred to as glioma stem cells or GSCs) play important roles in tumor propagation, therapeutic resistance, and recurrence following treatment. Understanding the molecular mechanisms that drive resistance in GSCs can lead to novel therapeutic approaches for rGBM. Bioinformatics analyses of transcriptome data sets from rGBM clinical specimens identified transforming growth factor-beta receptor type 2 (TGFBR2) as a putative driver of resistance and recurrence in GBM. We show that TMZ-resistant cells become more stem-like concurrent with higher expression of TGFBR2 protein and mRNA. Importantly, shRNA-mediated inhibition of TGFBR2 decreases proliferation of TMZ-resistant cells and enhances the effects of TMZ. We hypothesize that by developing targeted therapeutics to reduce TGFBR2 expression we will inhibit the growth capacity and/or re-sensitize rGBM cells to ionizing radiation (IR) or TMZ treatment by simultaneously targeting multiple putative oncogenes implicated in the induction of stem-like resistant GBM cell subsets. Future directions include inhibiting TGFBR2 signaling in clinical specimens derived from rGBM patients using two translatable approaches: (1) ITD1, a small molecule TGFBR2 inhibitor predicted to cross the blood-brain-barrier and (2) microRNAs to target multiple TGFBR2 target genes up-regulated in rGBM. Citation Format: Maya K. Johnson, Jack Korleski, Amanda Johnson, Sophie Sall, John Laterra, Hernando Lopez-Bertoni. Inhibition of TGFBR2 signaling re-sensitizes TMZ-resistant glioblastoma cells to therapy. [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 5009.

Syringin as TGF-βR1, HER2, EGFR, FGFR4 Kinase, and MMP-2 Inhibitor and Potential Cytotoxic Agent against ER+ Breast Cancer Cells

Background: Breast cancer is currently the most diagnosed cancer worldwide. Neoplastic cells and components of the tumor microenvironment trigger enzymes and receptors to facilitate cancer advancement. Syringin, a natural phenylpropanoid glycoside, has been reported to possess anti-cancer activity and affinity with numerous druggable targets of breast carcinoma. Objectives: This work aims to evaluate the effects of syringin on the growth of breast cancer cells (MCF-7) and normal dermal fibroblast cells (HDFn) and its ability to inhibit the protein targets of breast cancer. Methods: Syringin was investigated on cell lines in vitro via MTT assay. Using non-cell-based activity assay kits, its influence on the activity of transforming growth factor-beta receptor type 1 (TGF-βR1), human epidermal growth factor receptor (HER2), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor 4 (FGFR4), and matrix metalloproteinase-2 (MMP-2) was evaluated. Results: Syringin exhibited significant cytotoxicity against MCF-7 cells (IC50: 32.11 μM for 24 hours and 21.35 μM for 48 hours) and was non-toxic on healthy HDFn cells (IC50: >100 μM for 24 and 48 hours). It significantly suppressed the activity of cancer and angiogenesis regulating enzymes in vitro with commendable IC50 values on TGF-βR1 kinase (IC50: 6.48 μM), HER2 kinase (IC50: 7.18 μM), EGFR kinase (IC50: 12.38 μM), FGFR4 kinase (IC50: 16.03 μM), and MMP-2 (IC50: 16.07 μM). Conclusion: Findings showed the selective toxicity of syringin on breast cancer cells and its potential against pro-angiogenic enzymes. These discoveries strongly indicate the significance and therapeutic potential of syringin in targeted cancer therapy.

IMMU-27. SIMULTANEOUS TARGETING OF THE PD-L1 AND THE TGF-Β PATHWAYS WITH BINTRAFUSP ALFA IN EXPERIMENTAL GLIOMAS

Abstract Currently, immunotherapy is one of the most promising research areas in oncology, but immunotherapeutic strategies do not have proven clinical activity against glioblastoma so far. The immunosuppressive microenvironment of gliomas limits an appropriate anti-tumor immune response and represents a major challenge in the context of immunotherapy. Therefore, novel therapeutic strategies that reactivate the immune system and allow for potent anti-glioma immune responses are needed. Bintrafusp alfa is a bifunctional fusion protein that is composed of the extracellular domain of human transforming growth factor-beta receptor type II (TGF-βRII) and a human IgG1 monoclonal antibody blocking programmed death-ligand 1 (PD-L1). It is designed to target the TGF-β and the PDL-1 pathways simultaneously by specifically localizing to the tumor via the anti-PD-L1 moiety and sequestering TGF-β within the tumor microenvironment. We analyzed interrelations between the immunosuppressive factors TGF-β and PD-L1 in glioblastoma samples from patients and observed a significant correlation that points towards a co-regulation of both targets as part of a gene expression network. Next, we assessed the activity of bintrafusp alfa in human and murine glioma cell lines in vitro and observed interference with TGF-β-induced Smad2 phosphorylation as a bona fide response marker of TGF-β pathway activation and an increase in immune cell-mediated glioma cell killing. In vivo, bintrafusp alfa prolonged the survival of glioma-bearing immunocompetent mice, whereas the survival benefit was absent in immunodeficient Rag1-/- mice lacking T and B cell populations. Characterization of the tumor microenvironment by immunohistochemistry and high-dimensional flow cytometry confirmed the immune-activating effects of bintrafusp alfa. In summary, these encouraging results demonstrate that novel treatment options such as bintrafusp alfa may have the potential to overcome the lack of immunogenicity and reinvigorate anti-tumor immune responses to efficiently combat glioblastoma.

Endothelial Microparticle-Mediated Transfer of microRNA-19b Inhibits the Function and Distribution of Lymphatic Vessels in Atherosclerotic Mice

In recent years, the function of the lymphatic system in atherosclerosis has attracted attention due to its role in immune cell trafficking, cholesterol removal from the periphery, and regulation of the inflammatory response. However, knowledge of the mechanisms regulating lymphangiogenesis and lymphatic function in the pathogenesis of atherosclerosis is limited. Endothelial microparticles carrying circulating microRNA (miRNA)s are known to mediate cell–cell communication, and our previous research showed that miRNA-19b in EMPs (EMPmiR-19b) was significantly increased in circulation and atherosclerotic vessels, and this increase in EMPmiR-19b promoted atherosclerosis. The present study investigated whether atherogenic EMPmiR-19b influences pathological changes of the lymphatic system in atherosclerosis. We first verified increased miR-19b levels and loss of lymphatic system function in atherosclerotic mice. Atherogenic western diet-fed ApoE-/- mice were injected with phosphate-buffered saline, EMPs carrying control miRNA (EMPcontrol), or EMPmiR-19b intravenously. The function and distribution of the lymphatic system was assessed via confocal microscopy, Evans blue staining, and pathological analysis. The results showed that lymphatic system dysfunction existed in the early stage of atherosclerosis, and the observed pathological changes persisted at the later stage, companied by an increased microRNA-19b level. In ApoE-/- mice systemically treated with EMPmiR-19b, the distribution, transport function, and permeability of the lymphatic system were significantly inhibited. In vitro experiments showed that miRNA-19b may damage the lymphatic system by inhibiting lymphatic endothelial cell migration and tube formation, and a possible mechanism is the inhibition of transforming growth factor beta receptor type II (TGF-βRII) expression in lymphatic endothelial cells by miRNA-19b. Together, our findings demonstrate that atherogenic EMPmiR-19b may destroy lymphatic system function in atherosclerotic mice by downregulating TGF-βRII expression.

Variable Expression Levels of the Hematopoietic Stem Cell Surface Antigen ESAM Depict Heterogeneity and Fluctuations in Leukemic Stem Cells

Traditionally, hematopoietic stem cells (HSCs) have been regarded as a homogeneous cell population. However, many studies have shown that populations of HSCs are essentially heterogenous. Moreover, several recent studies have proposed that HSCs exhibit fluctuating biological characteristics. Although the physiological significance of this heterogeneity and fluctuation is unclear, we presume that these qualities enable HSCs to flexibly demonstrate their self-renewal or differentiation abilities dependent on physiological demands. It is, therefore, unsurprising that such heterogeneity and fluctuation is also seen in leukemic stem cells (LSCs), and that they are involved in the development of resistance to treatment.We have previously demonstrated that the endothelial-cell selective adhesion molecule (ESAM) is an important marker of HSCs in humans and mice (Blood 2009, Exp Hematol 2013), and that the expression level of this molecule indicates the activation status of HSCs (J Immunol 2012). Furthermore, we found that a subset of leukemic cells exhibit elevated levels of ESAM expression on their surfaces (Exp Hematol 2013). Approximately two-thirds of acute myelogenous leukemia cases in humans were found to be positive for ESAM expression. Since ESAM was found to be useful in monitoring the fluctuating status of HSCs after 5-FU treatment, we posited that the marker could be used to characterize LSCs.In this study, we used ESAM expression levels as an index to analyze the heterogeneity and fluctuation in LSC populations. Flow cytometric analysis of the human leukemia cell lines KG1a and CMK revealed wide-ranging heterogeneity in ESAM expression levels, with up to a 1000-fold difference in variation, even among individual cells derived from the same clone. When these cell populations were roughly fractionated into ESAM-high or ESAM-low expression groups, the ESAM-high expression group showed a higher proliferative capacity in culture. Interestingly, however, the expression levels of ESAM were not maintained indefinitely at the same level in these groups. In four weeks, both ESAM-high and ESAM-low cell fractions developed heterogeneous ESAM expression distributions similar to that in the parent cell lines. Furthermore, when single cells were sorted and cultured, each cell showed a self-renewal ability and the heterogenous nature of ESAM expression levels was reconstructed in each population derived from these single cells. These results suggest that populations of LSCs develop heterogeneity and have a fluctuating nature.When the transcriptomes of ESAM-low and ESAM-high KG1a cells were compared using RT-PCR, the expression of the transforming growth factor beta receptor type 2 (TGFBR2) gene was found to be higher in the ESAM-low cells. Reportedly, the transforming growth factor beta (TGFβ) signaling pathway is involved in various biological processes such as cell proliferation, differentiation, and migration. In tumor cells, this pathway is involved in reducing proliferation and accelerating invasion. Based on this information, it was considered likely that the TGFβ signaling pathway is involved in the heterogeneity of ESAM expression. When TGFβ1 (5 ng/ml) was added to a culture of KG1a cells (grown in RPMI 1640 medium with 10% fetal bovine serum (FBS)), the heterogeneity of ESAM expression in the population became more pronounced, mainly due to a decrease in ESAM expression in some cells.A transcriptomic analysis of cells exhibiting decreased ESAM expression after TGFβ1 stimulation showed that these cells had a higher expression of TGFBR2 than cells not exhibiting a decrease in ESAM expression. Since the presence of 10% FBS in the culture medium provides natural TGFβ1 at a concentration of ~1 ng/ml, we conducted culture experiments without FBS. When KG1a cells were cultured in serum-free medium, the ESAM-low fraction disappeared and the heterogeneity of ESAM expression could not be replicated. However, the heterogeneity of ESAM expression in KG1a cultures could be replicated in serum-free medium by the addition of 1 ng/ml TGFβ1. These results suggest that the TGFβ signaling pathway is involved in inducing heterogeneity in ESAM expression in cell populations. The fluctuation of ESAM expression levels could be caused by differences in TGF sensitivity, which in turn, may confer certain benefits to LSCs, such as drug resistance and promoting invasiveness. DisclosuresYokota:Celgene: Research Funding; MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:MOCHIDA PHARMACEUTICAL: Speakers Bureau; Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Kyowa Hakko Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Fujimotoseiyaku: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Nippon Shinyaku: Research Funding; Toyama Chemical: Research Funding; Shionogi: Research Funding.

Circular RNA Plek promotes fibrogenic activation by regulating the miR-135b-5p/TGF-βR1 axis after spinal cord injury.

Objectives: The spinal cord rarely repairs itself when damaged; however, methods for encouraging nerves to regrow are on the horizon. Although circular RNAs (circRNAs) contribute to various biological processes, including neuronal processes, their functions in the subacute phase of spinal cord injury (SCI) have not been elucidated. In this study, we identified a novel circRNA, named CircPlek, with increased expression in spinal tissues after SCI.Materials and Methods: We predicted a regulatory relationship between CircPlek and miR-135b-5p, which showed the most obvious decrease in post-SCI expression. We established the CircPlek/miR-135b-5p/transforming growth factor-beta receptor type I (TGF-βR1) axis using a bioinformatics approach and further evaluated the potential function of the interaction network in vitro.Results: We confirmed that in TGF-β1-induced fibroblasts, the overexpression of miR-135b-5p or/and inhibition of CircPlek inhibited fibrosis activation via the Smad pathway. Inhibitors of miR-135b-5p had antagonistic effects on CircPlek.Conclusions: the CircPlek/miR-135b-5p/TGF-βR1 axis may exert important functions in SCI and is a potential therapeutic target.

In Ovo and In Silico Evaluation of the Anti-Angiogenic Potential of Syringin.

IntroductionCancer is considered as one of the deadliest human diseases today. Angiogenesis, the propagation of new blood vessels from pre-existing vasculature, is a critical step in the progression of cancer as it is essential in the growth and metastasis of tumors. Hence, suppression of angiogenesis is a promising approach in cancer therapy. Syringin, a phenylpropanoid glycoside with a molecular formula of C17H24O9, has been found to exhibit chemopreventive effects. However, its anti-angiogenic activity and the underlying mechanism of action are still unknown.MethodsIn this work, in ovo chorioallantoic membrane (CAM) assay has been conducted to evaluate the effect of syringin on neovascularization. Additionally, reverse molecular docking studies have been performed in order to identify the probable enzyme targets in the angiogenesis pathway.ResultsTreatment with syringin showed significant dose-dependent inhibition of blood vessel length and junctions in the CAM of duck eggs; the anti-angiogenic activity of syringin at 100 µM and 200 µM is comparable with 200 µM of the positive control celecoxib. The results of reverse docking studies indicate that syringin binds the strongest to dihydrofolate reductase (DHFR) and, to some extent, with transforming growth factor-beta receptor type 1 (TGF-βR1), vascular endothelial growth factor receptor 2 (VEGFR2), and matrix metalloproteinase-2 (MMP-2). Furthermore, ADMET models revealed that syringin potentially possesses excellent pharmacokinetic and toxicity profiles.ConclusionThis study demonstrates the potential of syringin as an anti-angiogenic agent and elicits further investigations to establish its application in cancer suppression.

21P Double immunofluorescence (dIF) in analysing protein coexpression in tumours

Multiplex immunofluorescence is widely used nowadays for assessment of multiple mutually exclusive markers in one slide. It is especially helpful to distinguish various cell subpopulations based on expression of different markers in the tumor microenvironment. However, it is also a good way of studying coexpression of molecular markers of different cellular processes and signaling pathways in the same cell, including the same subcellular compartment, in tumor tissues obtained directly from patients. We used dIF to assess features of epithelial-mesenchymal transition (EMT), cancer stem cells (CSC) and their possible regulators (androgen (AR) and estrogen (ER) receptors, transforming growth factor beta receptor type II (TGFbRII)) in a training cohort of prostate cancer (PC) and endometrial cancer (EC) tissues. dIF staining of tissue sections showed that EMT markers E-cadherin and N-cadherin were coexpressed on the membranes of tumor cells, and cells expressing both markers were most likely representing those in EMT as a transitional metastable process. Opposingly, CSC markers CD44 and CD133 direct coexpression was not found at least in the studied cases, though both markers demonstrated membranous staining with slightly different patterns. ER alpha and beta subtypes were also coexpressed in the nuclei of a minor percent of cells in EC, mostly in stroma. AR and TGFbRII were simultaneously expressed in most PC tumor cells, the former in nucleus and the latter in cytoplasm, so no direct coexpression in the same compartment was seen. The described staining patterns obtained using dIF give additional information to the data that can be obtained with single marker staining with immunohistochemistry, so that interrelations between cellular proteins can be additionally analyzed as a prognostic or predictive biomarker. dIF is a good way of studying coexpression of markers in the same subcellular compartment. Thorough selection of fluorophores and filters enables assessment of staining of separate markers with further study of coexpression using image analysis software. Further quantitative assessment of studied markers in a larger sample cohort is needed to get data on possible links between EMT and CSC and their regulators in PC.

Twist1-induced miR-199a-3p promotes liver fibrosis by suppressing caveolin-2 and activating TGF-\u03b2 pathway

The activation of hepatic stellate cells (HSCs) participates in liver fibrosis, and emerging evidences indicate that microRNAs (miRNAs) are abnormally expressed during HSC activation. However, the potential roles of miRNAs in liver fibrosis still remain elusive. Therefore, this study aimed to investigate the role of miR-199a-3p in liver fibrosis and its underlying mechanism. We found that miR-199a-3p expression was dramatically upregulated during HSC activation in vitro, and during liver fibrogenesis in CCl4-treated rats, and its liver expression was increased in the patients with cirrhosis. By the luciferase assay and RT-qPCR, we revealed that the expression of miR-199a-3p in HSCs was driven by the transcription factor Twist1 which could be further induced by TGF-β treatment. Functional studies showed that inhibition of miR-199a-3p in both human LX2 cells and rat HSCs significantly decreased the expression of fibrotic markers, such as fibronectin and connective tissue growth factor (CTGF), whereas the forced expression of miR-199a-3p exhibited opposite effects, demonstrating the role of miR-199a-3p in promoting HSC activation. Mechanistically, miR-199a-3p plays an important role in TGF-β signalling pathway activation through targeting CAV2 that negatively regulates the expression of transforming growth factor-beta receptor type I (TGFβRI). Importantly, administration of antagomiR-199a-3p in the CCl4-treated mice significantly ameliorated hepatic fibrosis. In conclusion, Twist1-induced miR-199a-3p mediates the activation of HSCs by suppressing CAV2 expression and subsequently increasing TGFβRI expression to promote TGF-β pathway. Our findings highlight the therapeutic potential of miR-199a-3p for hepatic fibrosis.

Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail.

Nutrients are utilized and re-constructed by endodermal epithelial cells (EECs) of yolk sac membrane (YSM) in avian species during embryonic development. Sterol O-acyltransferase 1 (SOAT1) is the key enzyme to convert cholesterol to cholesteryl ester for delivery to growing embryos. During embryonic development, yolk absorption is concomitant with significant changes of SOAT1 mRNA concentration and enzyme activity in YSM. Presence of microRNAs (miRNAs) are observed in the embryonic liver and muscle during avian embryogenesis. However, the expression of miRNAs in YSM during embryogenesis and the involvement of miRNAs in lipid utilization are not known. Using a miRNA sequencing technique, we found several miRNA candidates and confirmed their expression patterns individually by real time PCR. MiRNA candidates were selected based on the expression pattern and their possible roles in inhibiting transforming growth factor beta receptor type 1 (TGFBR1) that would regulate the function of SOAT1. Similar to SOAT1 mRNA, the gga-miR-181a-5p expression was gradually elevated during embryonic development. However, the expression of gga-miR-429-3p in YSM was gradually decreased during embryonic development. The inhibitory effects of gga-miR-181a-5p or gga-miR-429-3p on the potential targets (SOAT1 and TGFBR1) were demonstrated by transient miRNA transfections in EECs. We also found that mutated TGFBR1 3'UTR prevented the direct pairings of gga-miR-181a-5p and gga-miR-429-3p. Treatment of TGFBR1 inhibitor, LY364947, further decreased SOAT1 transcription. Similar results were also observed by the miRNA transfection studies. The results showed the vital participations of gga-miR-181a-5p and gga-miR-429-3p in regulating TGFβ pathway, and affecting downstream SOAT1 expression and function in the YSM. This is indicative of possible regulation of avian yolk lipid utilization by changing YSM miRNA expressions.

SILAC-Based Quantification of TGFBR2-Regulated Protein Expression in Extracellular Vesicles of Microsatellite Unstable Colorectal Cancers

Microsatellite unstable (MSI) colorectal cancers (CRCs) are characterized by mutational inactivation of Transforming Growth Factor Beta Receptor Type 2 (TGFBR2). TGFBR2-deficient CRCs present altered target gene and protein expression. Such cellular alterations modulate the content of CRC-derived extracellular vesicles (EVs). EVs function as couriers of proteins, nucleic acids, and lipids in intercellular communication. At a qualitative level, we have previously shown that TGFBR2 deficiency causes overall alterations in the EV protein content. To deepen the basic understanding of altered protein dynamics, this work aimed to determine TGFBR2-dependent EV protein signatures in a quantitative manner. Using a stable isotope labeling with amino acids in cell culture (SILAC) approach for mass spectrometry-based quantification, 48 TGFBR2-regulated proteins were identified in MSI CRC-derived EVs. Overall, TGFBR2 deficiency caused upregulation of several EV proteins related to the extracellular matrix and nucleosome as well as downregulation of proteasome-associated proteins. The present study emphasizes the general overlap of proteins between EVs and their parental CRC cells but also highlights the impact of TGFBR2 deficiency on EV protein composition. From a clinical perspective, TGFBR2-regulated quantitative differences of protein expression in EVs might nominate novel biomarkers for liquid biopsy-based MSI typing in the future.

Twist1-Induced miR-199a-3p Promotes Liver Fibrosis by Suppressing Caveolin-2 and Activating TGF-β Pathway

Background: The activation of hepatic stellate cells (HSCs) participates in liver fibrosis, and emerging evidences indicate that microRNAs (miRNAs) are abnormally expressed during HSC activation. However, the potential roles of miRNAs in liver fibrosis still remain elusive. Methods: The differential expression of miRNAs between the quiescent and activated HSCs was measured by miRNA microarray analysis, which was further validated by RT-qPCR in animal models and patients. The effect of miR-199a-3p on CAV2 expression was examined by Western blotting using miRNA mimics or inhibitor. Induction of miR-199a-3p expression by TWIST1 was explored by the luciferase assay and RT-qPCR. The role of miR-199a-3p in liver fibrosis was systematically investigated in vitro and in vivo. Findings: miR-199a-3p expression was dramatically upregulated during HSC activation in vitro, and during liver fibrogenesis in CCl4-treated rats, and was increased in livers from high-fat-diet-induced mice or the patients with cirrhosis. The expression of miR-199a-3p was driven by Twist1 which could be further induced by TGF-β treatment. Functional studies showed that inhibition of miR-199a-3p in both human LX2 cells and rat HSCs significantly decreased the expression of fibrotic markers, whereas the forced expression of miR-199a-3p exhibited opposite effects, demonstrating the role of miR-199a-3p in promoting HSCs activation. Mechanistically, miR-199a-3p plays an important role in TGF-β signalling pathway activation through targeting CAV2 that negatively regulates the expression of transforming growth factor-beta receptor type I (TGFβRI). Importantly, administration of antagomiR-199a-3p in the CCl4 treated mice significantly ameliorated hepatic fibrosis. Interpretation: Twist1-induced miR-199a-3p mediates the activation of HSCs by suppressing CAV2 expression and subsequently increasing TGFβRI expression to promote TGF-β pathway. Our findings highlight the therapeutic potential of miR-199a-3p for hepatic fibrosis. Funding Statement: This study was supported by National Natural Science Foundation of China (81570554 and 81770568 to LY; 81572739 to YP), and by National Key R&D Program of China (2016YFA0502204 to YP). TS is supported by a joint PhD studentship by King’s College London, UK and China Scholarship Council, China. Declaration of Interests: None of the authors has any potential conflict of interests to disclose. Ethics Approval Statement: All animal studies were approved by the Medical Ethics Committee of the West China Hospital of Sichuan University. All specimens were collected upon obtaining informed consent from all patients, and the study was approved by the Human Ethics Committee of Sichuan University.

Aberrant signal transduction in Indian triple-negative breast cancer patients.

The aim of this study is to correlate the prominin-1 or CD133 association with functional pathway markers of cancer stemness in Indian triple-negative breast cancer (TNBC) patient samples. TNBC samples were confirmed for the absence of hormone receptors (estrogen receptor-ER/progesterone receptor) and human epidermal growth factor receptor-2 or proto-oncogene neu or erbB2 or CD340 by immunohistochemical analysis. Formalin-fixed paraffin-embedded samples of patients were used to collect the total RNA. Then, one-step reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the cancer stemness-related transcript levels in the different samples. The RT-PCR products were analyzed semi-quantitatively on agarose gels. The band intensities of respective samples for different transcripts were analyzed by densitometry. TNBC-confirmed samples had shown increased levels of CD133 transcript than control tissues. Further, elevated CD133 transcripts are correlated with higher transcript levels of NOTCH1/FZD7/transforming growth factor-beta receptor Type III R/patched-1 pathway mediators. This work has clearly indicated that there is a correlation between CD133 and functional pathways that control cancer stem cells in TNBC. These observations may indicate the possible association between cancer stemness and TNBC malignancy.

Proteomic Analysis Reveals Distinctive Protein Profiles Involved in CD8 T Cell-Mediated Murine Autoimmune Cholangitis

Abstract Autoimmune cholangitis arises from abnormal innate and adaptive immune responses in liver and T cells are critical drivers in this process, but little is known about the regulation of their functional behavior during disease development. We previously reported that mice with a T cell-restricted expression of a dominant negative form of transforming growth factor beta receptor type II (dnTGFbRII) spontaneously develop autoimmune cholangitis that resembles human primary biliary cholangitis (PBC). Adoptive transfer of CD8+ but not CD4+ T cells into Rag1−/− mice reproduced the disease, demonstrating a critical role for CD8 T cells in pathogenesis. Herein, we took advantage of SOMA scan technology to perform proteomic analysis of serum samples from dnTGFbRII and B6 control mice at different ages. In addition, we analyzed CD8 protein profiles after adoptive transfer of splenic CD8+ cells into Rag1−/− recipients. The use of the unique aptamer technology of SOMA scans reveals critical distinct profiles of CD8 cells, which are key to biliary mediation. 254 proteins were significantly increased, while 216 proteins significantly decreased in recipient hepatic CD8+ cells compared to donor splenic CD8+ cells. In contrast to donor splenic CD8+ cells, recipient hepatic CD8+ cells express distinct protein profiles involved in chemokine signaling, focal adhesion, T cell receptor and natural killer cell mediated cytotoxicity pathways.

Proteomic analysis reveals distinctive protein profiles involved in CD8+ T cell-mediated murine autoimmune cholangitis.

Autoimmune cholangitis arises from abnormal innate and adaptive immune responses in the liver, and T cells are critical drivers in this process. However, little is known about the regulation of their functional behavior during disease development. We previously reported that mice with T cell-restricted expression of a dominant negative form of transforming growth factor beta receptor type II (dnTGFβRII) spontaneously develop an autoimmune cholangitis that resembles human primary biliary cholangitis (PBC). Adoptive transfer of CD8+ but not CD4+ T cells into Rag1-/- mice reproduced the disease, demonstrating a critical role for CD8+ T cells in PBC pathogenesis. Herein, we used SOMAscan technology to perform proteomic analysis of serum samples from dnTGFβRII and B6 control mice at different ages. In addition, we analyzed CD8 protein profiles after adoptive transfer of splenic CD8+ cells into Rag1-/- recipients. The use of the unique SOMAscan aptamer technology revealed critical and distinct profiles of CD8 cells, which are key to biliary mediation. In total, 254 proteins were significantly increased while 216 proteins were significantly decreased in recipient hepatic CD8+ cells compared to donor splenic CD8+ cells. In contrast to donor splenic CD8+ cells, recipient hepatic CD8+ cells expressed distinct profiles for proteins involved in chemokine signaling, focal adhesion, T cell receptor and natural killer cell-mediated cytotoxicity pathways.

Tgfbr2 inactivation facilitates cellular plasticity and development of Pten-null prostate cancer.

Mutations in tumors can create a state of increased cellular plasticity that promotes resistance to treatment. Thus, there is an urgent need to develop novel strategies for identifying key factors that regulate cellular plasticity in order to combat resistance to chemotherapy and radiation treatment. Here we report that prostate epithelial cell reprogramming could be exploited to identify key factors required for promoting prostate cancer tumorigenesis and cellular plasticity. Deletion of phosphatase and tensin homolog (Pten) and transforming growth factor-beta receptor type 2 (Tgfbr2) may increase prostate epithelial cell reprogramming efficiency in vitro and cause rapid tumor development and early mortality in vivo. Tgfbr2 ablation abolished TGF-β signaling but increased the bone morphogenetic protein (BMP) signaling pathway through the negative regulator Tmeff1. Furthermore, increased BMP signaling promotes expression of the tumor marker genes ID1, Oct4, Nanog, and Sox2; ID1/STAT3/NANOG expression was inversely correlated with patient survival. Thus, our findings provide information about the molecular mechanisms by which BMP signaling pathways render stemness capacity to prostate tumor cells.