TGF-β Signaling Molecules Research Articles

Serial Changes of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Treated with Atezolizumab Plus Bevacizumab.

Immune checkpoint inhibitors have promising outcomes in patients with hepatocellular carcinoma (HCC); however, there is no reliable biomarker for predicting disease progression. Circulating tumor cells (CTCs) derived from peripheral blood have attracted attention in monitoring therapeutic efficacy. In this study, CTCs were serially collected from HCC patients undergoing atezolizumab plus bevacizumab (Atezo+Bev), and changes in molecular expression and CTC numbers were analyzed to identify effective biomarkers. Changes in CTC numbers during Atezo+Bev reflected the tumor volume. Targeted RNA sequencing with next-generation sequencing (NGS) revealed that patients with elevated transforming growth factor (TGF)-β signaling molecules had a poorer response, whereas those with elevated apoptosis signaling molecules had a favorable response. In addition, compared with changes in CTC counts, changes in TGF-β signaling molecule expression in CTCs accurately and promptly predicted treatment response. Overall, NGS analysis of CTC-derived RNA showed that changes in TGF-β signaling molecules predict treatment response earlier than changes in CTC counts. These findings suggest that changes in the expression of TGF-β molecules in CTCs could serve as novel biomarkers for the early prediction of therapeutic response in patients with unresectable HCC undergoing Atezo+Bev.

Early changes in asporin levels in osteoarthritis of the temporomandibular joint

ObjectivesThe present study aimed to elucidate the pathogenesis of temporomandibular joint (TMJ) osteoarthritis (TMJ-OA) in a mouse model. We investigated morphological and histological changes in the head of mandible cartilage and early immunohistochemical (IHC) changes in transforming growth factor (TGF)-β, phosphorylated Smad-2/3 (p-Smad2/3), a TGF-β signaling molecule, and asporin. MethodsTMJ-OA was induced in a mouse model through unilateral partial discectomy. Micro-computed tomography (micro-CT) and safranin-O staining were performed to morphologically and histologically evaluate the degeneration of the head of mandible caused by TMJ-OA. IHC staining for TGF-β, p-Smad2/3, and asporin was performed to evaluate the changes in protein expression. ResultsIn the experimental group, three-dimensional (3D) morphometry revealed an enlarged head of mandible and safranin-O staining showed degeneration of cartilage tissue in the early stages of TMJ-OA compared to the control group. IHC staining revealed that TGF-β, p-Smad2/3, and asporin expression increased in the head of mandible cartilage before the degeneration of cartilage tissue, and subsequently decreased for a short period. ConclusionThe findings suggested a negative feedback relationship between the expression of asporin and the TGF-β/Smad transduction pathway, which may be involved in the degeneration of the head of mandible in the early stages of TMJ-OA. Asporin is a potential biomarker of the early stages of TMJ-OA, which ultimately leads to the irreversible degeneration of TMJ tissues.

Organosulfur Compounds in Aged Garlic Extract Ameliorate Glucose Induced Diabetic Cardiomyopathy by Attenuating Oxidative Stress, Cardiac Fibrosis, and Cardiac Apoptosis.

Diabetic cardiomyopathy has emerged as a major cause of cardiac fibrosis, hypertrophy, diastolic dysfunction, and heart failure due to uncontrolled glucose metabolism in patients with diabetes mellitus. However, there is still no consensus on the optimal treatment to prevent or treat the cardiac burden associated with diabetes, which urges the development of dual antidiabetic and cardioprotective cardiac therapy based on natural products. This study investigates the cardiotoxic profile of glucose and the efficacy of AGE against glucose-induced cardiotoxicity in H9c2 cardiomyocytes. The cellular metabolic activity of H9c2 cardiomyocytes under increasing glucose concentration and the therapeutic efficacy of AGE were investigated using the MTT cell cytotoxicity assay. The in vitro model was established in six groups known as 1. control, 2. cells treated with 25 µM glucose, 3. 100 µM glucose, 4. 25 µM glucose +35 µM AGE, 5. 100 µM glucose + 35 µM AGE, and 6. 35 µM AGE. Morphological and nuclear analyses were performed using Giemsa, HE, DAPI, and PI, respectively, whereas cell death was simultaneously assessed using the trypan blue assay. The antioxidant potential of AGE was evaluated by DCFH-DA assay, NO, and H202 scavenging assay. The activities of the antioxidant enzymes catalase and superoxide dismutase were also investigated. The antiglycative potential of AGE was examined by antiglycation assays, amylase zymography, and SDS PAGE. These results were then validated by in silico molecular docking and qRTPCR. Hyperglycemia significantly reduced cellular metabolic activity of H9c2 cardiomyocytes, and AGE was found to preserve cell viability approximately 2-fold by attenuating oxidative, fibrosis, and apoptotic signaling molecules. In silico and qRTPCR studies confirmed that organosulfur compounds target TNF-α, MAPK, TGF-β, MMP-7, and caspase-9 signaling molecules to ameliorate glucose-induced cardiotoxicity. AGE was found to be an antidiabetic and cardioprotective natural product with exceptional therapeutic potential for use as a novel herb-drug therapy in the treatment of diabetic cardiomyopathy in future therapies.

TGF-β signaling molecules in Hydra: role of BMP and BMP inhibitors during pattern formation.

Understanding the evolution of body plans has been one of the major areas of investigation in developmental and evolutionary biology. Cnidaria, the sister group to bilaterians, provides an opportunity to elucidate the origin and evolution of body axes. Hydra, a freshwater cnidarian, is a useful model to study signaling pathways governing pattern formation, which are conserved up to vertebrates including humans. The transforming growth factor β (TGF-β) signaling pathway is one of the fundamental pathways that regulate axis formation and organogenesis during embryonic development. In this article, we discuss the TGF-β pathway members identified in Hydra along with other cnidarians with an emphasis on bone morphogenetic proteins (BMPs) and their inhibitors. TGF-β members, especially those involved in BMP signaling pathway, are mainly involved in maintaining the Organizer region and patterning the body axis in Hydra. Identification of other members of this pathway in Hydra and fellow cnidarians would provide insights into the evolution of body axes and pattern formation in more complex metazoans.

Moxibustion enables protective effects on rheumatoid arthritis-induced myocardial injury transforming growth factor beta1 signaling and metabolic reprogramming.

To examine the effects of moxibustion on myocardial injury and myocardial metabolomics in rats with rheumatoid arthritis (RA) based on the transforming growth factor beta1 (TGF-β1)/Smads signaling pathway. One hundred rats were treated with saline [normal control (NC) group] or complete Freund's adjuvant (CFA) by right plantar injection for the RA model group, and the latter were randomly divided into 4 groups. Tripterygium wilfordii polyglycoside tablets (, TPT) have anti-inflammatory and are widely used in the clinical treatment of RA, therefore serving as a positive control group. Three days post injection rats were given TPT tablet (TPT group), acupuncture therapy (APT group), and moxibustion treatment (MOX group) for 15 consecutive days, while NC group and model group were equally grasped and fixed and received normal saline. Rat joint swelling scores and arthritis index (AI) were evaluated in each group before the CFA challenge, therapy and after receiving therapy. Myocardial ultrastructure was observed by electron microscope. Enzyme-linked immunosorbent assay was used to detect cardiac troponin I (cTnI) levels in rat myocardial tissue. Quantitative reverse transcription polymerase chain reaction and Western blotting analysis were used to measure the mRNA and protein levels of TGF-β signaling molecules including TGF-β1, Smad2, Smad3, Smad4, and Smad7. Myocardial metabolomics was analyzed using gas chromatography-mass spectrometer. Compared with model group, RA model rats receiving TPT, acupuncture, or moxibustion therapy all showed reduced joint swelling scores and AI (all P < 0.01) and improved myocardial damage, whereas rats treated with moxibustion were found to be more marked. Consistently, the expressions of cTnI, TGF-β1, Smad2, Smad3, and Smad4 were found to be elevated in model rat group in contrast to NC rats and were significantly downregulated in TPT, APT and MOX group when compared with model group, while the levels of Smad7 showed the opposite result (all P < 0.01). Moreover, the dissection of metabolomics suggested a novel metabolite biomarker panel including D-Xylulose 5-phosphate, dihydroxyacetone phosphate, arachidonic acid, etc was defined and implicated in amino acid, glucose, and fatty acid metabolic processes as revealed by principal component analysis and partial least squares discriminant analysis. Moxibustion prevents RA-induced inflammatory response and offers potent therapeutic effects on myocardial dysfunctions. The protective effects might be associated with its role in TGF-β1 inactivation and metabolic reprogramming.

Abstract P321: &lt;RKER-012, A Novel Activin Receptor Type Ilb (actrllb) Ligand Trap, Inhibited Mediators Of Dysregulated Vascular Remodeling In Pulmonary Arterial Endothelial And Smooth Muscle Cells&gt;

Rationale: Imbalance in TGF-β signaling molecules SMAD2/3 and SMAD1/5/9 is a key driver of vascular dysfunction and pulmonary arterial hypertension (PAH) pathophysiology. KER-012, a novel ActRIIB ligand trap, designed to bind activin (Act) A and ActB to inhibit SMAD2/3: thereby permitting bone morphogenic protein (BMP) ligand signaling via SMAD1/5/9 to potentially rebalance TGF-β signaling and restore vascular homeostasis in PAH and associated disorders. Methods: Using hypoxia (1% O2) to mimic the pulmonary cell environment of PAH, we evaluated the effect of KER-012 or RKER -012 (a research form of KER-012) on TGF-β signaling in human pulmonary arterial endothelial cells (HPAECs) and smooth muscle cells (HPASMCs), two key cell types involved in the vascular remodeling in PAH. Gene expression of hypoxia/normoxia cultured cells was assessed by qPCR, phosphorylation of SMAD2 (pSMAD2) by alphaLISA, and ActA protein levels by ELISA. Results: HPAECs and HPASMCs both responded to TGF-β ligands implicated in PAH. ActA and Growth Differentiation Factor (GDF)11 increased SMAD2 phosphorylation and BMP9 increased SMAD1 phosphorylation (pSMAD1), confirming activation of the respective signaling pathways. KER-012 inhibited ActA and GDF11 induced pSMAD2, but not BMP9 induced pSMAD1. In hypoxic HPAECs, ActA gene expression increased &gt;3-fold and BMPR2 expression decreased &gt;2-fold relative to normoxia. ActA protein levels from hypoxic HPAEC supernatant also increased 10-fold, which was associated with an observed increase in pSMAD2 activation. While RKER-012 did not affect ActA gene expression, it did reduce ActA protein levels in supernatant to normoxic levels. This reduction in ActA may represent the fraction not bound to RKER-012, and potentially demonstrates a reduction in ActA availability for SMAD2/3 activation. Conclusions: ActA inhibition by RKER-012 restored homeostatic SMAD2 signaling in HPAECs and HPASMCs in an in vitr o PAH hypoxia model. RKER012 has also shown not to effect BMP signaling in these cells. These studies suggest that RKER-012 can potentially correct imbalanced SMAD signaling in PAH, partially via binding and inhibition of ActA protein.

Oral delivery of nerolidol alleviates cyclophosphamide-induced renal inflammation, apoptosis, and fibrosis via modulation of NF-κB/cleaved caspase-3/TGF-β signaling molecules

Cyclophosphamide (CP) is one of the most extensively used antineoplastic drug, but the nephrotoxicity caused by this drug is a major limiting factor for its use. Nerolidol (NERO) is a natural bioactive compound with diverse pharmacological actions. In Vitro and in vivo study was performed using HK-2 renal cells and Swiss Albino mice. Cell lines and animals were treated with NERO 25 and 50 µM + 30 µM CP (in vitro), 200 and 400 mg/kg, p.o. NERO from day 1 to day 15 + 200 mg/kg, i.p. CP on day 17 as single intraperitoneal injection (in vivo). The makers of oxidative stress, renal-specific injury markers, inflammation, apoptosis, fibrosis, and histopathological changes were studied. The study’s outcome showed a significant reduction in the level of malonaldehyde and interleukin-6 (p < 0.01), tumor necrosis factor-α, IL-1β (p < 0.001), and an increase in the superoxide dismutase, catalase, glutathione and interleukin-10 level (p < 0.01), in the in vivo study when treated with NERO 400 and compared with CP 200. In Vitro study showed reduced expression of nuclear factor kappa light chain enhancer of activated B cells, cleaved caspase-3, kidney injury molecule-1 and transforming growth factor-β-1 (p < 0.001), when treated with NERO 50 µM whereas NERO 25 µM only reduced the level of cleaved caspase-3 (p < 0.05) when compared with 30 µM. NERO 400 also reduced uric acid (p < 0.05), urea (p < 0.01), blood urea nitrogen, and serum creatinine levels (p < 0.001) and increased the level of blood-urea-nitrogen/creatinine ratio (p < 0.001). Additionally, the level of fibrosis-specific markers such as transforming growth factor-β1, hyaluronic acid (p < 0.01), 4-hydroxyproline, a collagen-rich area in Masson’s’ trichome stain, and Smad3 expression was also significantly reduced (p < 0.001). Furthermore, the outcome of multiple renal staining showed structural reversal aberrations, reduction of the thick basement membrane, and glycogen level toward normal when treated with NERO 400. Thus, the study showed a novel mechanistic modality of NERO against cyclophosphamide-induced renal toxicity. The outcome of this study can be considered a step closer to the development of an adjuvant to mitigate cyclophosphamide-induced renal toxicity among patients treated with cyclophosphamide.

Ploidy Status of Ovarian Cancer Cell Lines and Their Association with Gene Expression Profiles

As a cancer type potentially dominated by copy number variations, ovarian cancer shows hyperploid karyotypes and large-scale chromosome alterations, which might be promising biomarkers correlated with tumor metastasis and chemoresistance. Experimental studies have provided more information about the roles of aneuploids and polyploids in ovarian cancer. However, ploidy evaluation of ovarian cancer cell lines is still limited, even in some ploidy-related research. Herein, the ploidy landscape of 51 ovarian cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were analyzed, and the ploidy statuses of 13 human ovarian cancer cell lines and 2 murine cell lines were evaluated using G-banding and flow cytometry. Most human ovarian cancer cell lines were aneuploid, with modal numbers of 52-86 and numerical complexity ranging from 5 to 12. A2780, COV434 and TOV21G were screened as diploid cell lines, with a modal number of 46, a low aneuploid score and a near-diploid ploidy value. Two murine cell lines, both OV2944-HM1 and ID-8, were near-tetraploid. Integrated information on karyotypes, aneuploid score and ploidy value supplied references for a nondiploid model construction and a parallel analysis of diploid versus aneuploid. Moreover, the gene expression profiles were compared between diploid and aneuploid cell lines. The functions of differentially expressed genes were mainly enriched in terms of protein function regulation, TGF-β signaling and cell adhesion molecules. Genes downregulated in the aneuploid group were mainly related to metabolism and protein function regulation, and genes upregulated in the aneuploid group were mainly involved in immune regulation. Differentially expressed genes were randomly distributed on all chromosomes, while chromosome 1 alteration might contribute to immune-related alterations in aneuploid cell lines. Chromosome 19 alteration might be potentially significant for aneuploid ovarian cancer cell lines and patients, which needs further verification in ploidy research.

Natural plant extracts mediated expression regulation of TGF-β receptors and SMAD genes in human cancer cell lines.

Transforming growth factor beta (TGF-β) superfamily has key role in cell proliferation which leads to tumor promoting activities at metastatic stage of cancer. Inhibition of transforming growth factor beta receptor (TGFβR) signaling pathway can provide better therapeutic strategy to control cancer. Natural products are best known for their safety, less toxic nature, antioxidant characteristics making them a promising candidate to inhibit TGFβR signaling pathway. Crude methanolic extracts (CMEs) of 16 selected plants were prepared by using maceration method and subjected to phytochemical assays for identification of major phytometabolites particularly cancer chemopreventive antioxidant constituents. Total flavonoid content of all plants CME was > 0.6mg/ml exhibiting the Cichorium intybus contains comparatively highest amount of total flavonoid content (0.53mg/ml). Scanvenging activity of all plants was determined having IC50 ranges between 2 and 88 (µg/ml) while Moringa oleifera revealed the maximum scavenging activity (IC50 2.03µg/ml). Comparative cytotoxicity of plant extracts was evaluated in HUH and MCF-7 cell lines using 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) colorimetric assay. The nine active plant extracts i.e. Fagonia cretica, Argemone Mexicana, Rubus fruticosus, M. oleifera, Punica granatum, Cichorium intybus, Xanthium strumarium, Carissa opaca, Cyperus rotundus were identified based on their high antiproliferative activity > 50% against cancer cell lines and subjected to relative expression studies. Modulation of TGFβ signaling molecules (i.e.TGFβR1, 2 & 3, SMAD3, SMAD5) and ubiquitous proteins i.e. SMURF1 and SMURF2 genetic expression by potent extracts was determined by RT-PCR using GAPDH (housekeeping gene) as gene of reference. This present study revealed that CME of Fagonia cretica and Argemone mexicana significantly inhibit TGF beta mediated signaling cascade by downregulating the gene expression fold change > 1 of TGFβR 1, 2 & 3 and receptor associated complex protein SMAD3 as compared to control.

Aberrant Activation Mechanism of TGF-β Signaling in Epithelial-mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is an important program in epithelial cancer cells to acquire the motility and invasion, which promotes cancer metastasis to remote organs. EMT is induced by various secreted factors, such as transforming growth factor-β (TGF-β) and epidermal growth factor (EGF). TGF-β ligand activates Smad-dependent and -independent pathways by binding to TGF-β receptors. In Smad-dependent pathway, the activated TGF-β receptor phosphorylates Smad2/3 and accelerates its association with Smad4, leading to their nuclear translocation. Smad2/3-4 complex promotes the expression of EMT-inducing transcription factors, such as Snail and Slug. In Smad-independent pathway, mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways are activated and required for TGF-β-induced EMT. Smad-independent pathway is similar to downstream of receptor tyrosine kinases, and therefore EGFR signaling is known to induce EMT synergize with TGF-β signaling. We explored a new mechanism of EGFR-mediated activation of TGF-β signaling and found that c-Abl kinase activates TGF-β signaling. Based on our proteomic analysis, we identified several TGF-β signaling molecules as nuclear c-Abl substrates, including transcriptional intermediary factor 1-γ (TIF1γ/TRIM33/Ectodermin), a suppressor of TGF-β signaling. c-Abl-mediated phosphorylation of TIF1γ inhibits its binding to Smad3, thereby increasing Smad3's transcriptional activity and promoting EMT. TIF1γ phosphorylation is also involved in the EGFR-caused aberrant activation of TGF-β signaling, suggesting that EGFR/c-Abl pathway activates TGF-β signaling through phosphorylation of nuclear substrates and promotes EMT. Our findings provide new insights into the activation machinery of TGF-β signaling, and further studies are required to clarify the clinical significance of the EGFR/c-Abl pathway in cancer metastasis.

Mechanics of ascending aortas from TGFβ-1, -2, -3 haploinsufficient mice and elastase-induced aortopathy

Transforming growth factor-beta (TGFβ-1, -2, -3) ligands act through a common receptor complex yet each is expressed in a unique and overlapping fashion throughout development. TGFβ plays a role in extra-cellular matrix composition with mutations to genes encoding TGFβ and TGFβ signaling molecules contributing to diverse and deadly thoracic aortopathies common in Loeys-Dietz syndrome (LDS). In this investigation, we studied the TGFβ ligand-specific mechanical phenotype of ascending thoracic aortas (ATA) taken from 4-to-6 months-old Tgfb1+/−, Tgfb2+/−, and Tgfb3+/− mice, their wild-type (WT) controls, and an elastase infusion model representative of severe elastolysis. Heterozygous mice were studied at an age without dilation to elucidate potential pre-aortopathic mechanical cues. Our findings indicate that ATAs from Tgfb2+/− mice demonstrated significant wall thickening, a corresponding decrease in biaxial stress, decreased biaxial stiffness, and a decrease in stored energy. These results were unlike the pathological elastase model where decreases in biaxial stretch were found along with increases in diameter, biaxial stress, and biaxial stiffness. ATAs from Tgfb1+/− and Tgfb3+/−, on the other hand, had few mechanical differences when compared to wild-type controls. Although aortopathy generally occurs later in development, our findings reveal that in 4-to-6 month-old animals, only Tgfb2+/− mice demonstrate a significant phenotype that fails to model ubiquitous elastolysis.

C-peptide ameliorates high glucose-induced podocyte dysfunction through the regulation of the Notch and TGF-β signaling pathways

The podocyte is one of the main components of the glomerular filtration barrier in the kidney, and its injury may contribute to proteinuria, glomerulosclerosis and eventually kidney failure. C-peptide, a cleavage product of proinsulin, shows therapeutic potential for treating diabetic nephropathy (DN). The aim of this study was to investigate the effect of C-peptide on high glucose-induced podocyte dysfunction. In the present study, we found that the protective effects of islet transplantation were superior to simple insulin therapy for the treatment of DN in streptozotocin (STZ)-treated rats. And such superiority may due to the function of C-peptide secreted at the implanted site. Based on this background, we determined that the application of C-peptide significantly prevented high glucose-induced podocyte injury by increasing the expression of nephrin and synaptopodin. Meanwhile, C-peptide suppressed high glucose-induced epithelial-mesenchymal transition (EMT) and renal fibrosis via decreasing the expression of snail, vimentin, α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF). Moreover, the Notch and transforming growth factor-β (TGF-β) signaling pathways were activated by high glucose, and treatment with C-peptide down-regulated the expression of the Notch signaling molecules Notch 1 and Jagged 1 and the TGF-β signaling molecule TGF-β1. These findings suggested that C-peptide might serve as a novel treatment method for DN and podocyte dysfunction.

Discoidin domain receptor 2: An emerging pharmacological drug target for prospective therapy against osteoarthritis.

Discoidin domain receptor2 (DDR2), a cell membrane tyrosine kinase on chondrocytes surface plays main role in cell-ECM interaction during the progressive degeneration of articular cartilage in osteoarthritis. The degraded component of ECM, type II collagen upon DDR2 binding provokes synthesis of matrix metalloproteinases (MMPs), responsible for severe destruction of joint tissues. DDR2 knockout has been investigated to decline the expression of MMP-1 and 13. Previously, various molecules were effective in preclinical level against different targets in OA, but found to be collapsed in clinical trial due to insufficient target specificity and clinical toxicity. Review emphasizes the role of DDR2 in the degeneration of cartilage in osteoarthritis (OA) and its blocking by DDR2 antagonist attenuates the disease severity. DDR2 in chondrocytes contributes paramount role in degradation of cartilage at early stage of osteoarthritis via collagen 2 binding through the felicitation of TGF-β signaling molecule and other triggering factors. DDR2 involvement in regulation of matrix metalloproteinase (MMP), cross talking interaction in maintenance of ECM-chondrocytes, bone developments, interference RNA and designing the DDR2 antagonists have been critically investigated. The exploration may conclude that the DDR2 could be the novel pharmacological target to prevent the progression of osteoarthritis at early stage because of over expression of DDR2 and MMP which further promotes severe cartilage degeneration. Owing to pharmacological specificity of DDR2 in OA as drug target, it is to be hypothesized that development of safe molecules as DDR2 antagonist could be the good option in the treatment of OA with promising landmark.

Abstract 5459: Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis

Abstract Development of hepatocellular carcinoma (HCC), which remains lethal, is associated with alterations in multiple factors including the transforming growth factor beta (TGF-β) signaling pathway. Previously we have uncovered a unique role for TGF-β signaling molecules, Smad3 and its adaptor β2SP, in suppressing stem cell transformation into cancer. Yet, while TGF-β plays a pleiotropic role including regulating stem cell differentiation, proliferation, and inflammation, mechanistic insight into the dichotomy of TGF-β, and its role in stem cell transformation remains poorly understood for these cancers. Here, we took an integrated approach to identify and validate effects of changes in this pathway in HCC and identify potential therapeutic targets such as IGF2. We extended our mechanistic studies associated with the regulation of insulin-like growth factor 2 (IGF2) in the context of the TGF-β-pathway, by utilizing both human liver cancer cell lines and TGF-β signaling-deficient mice (β2SP+/- and β2SP+/-/Smad3+/-) that develop liver cancers. We found the following: 1) IGF2 promotes stemness, overexpression leading to increased expression of stem cell genes that include SOX2 and ALDH1A1, and sphere formation in liver cancer cells; 2) A marked increase in IGF2 mRNA and protein in HepG2 cells and Huh7 cells with β2SP stable knockdown; 3) TGF-β signaling through β2SP/Smad3 significantly inhibits IGF2 transcription; ChIP assays validated binding of Smad3 at the human IGF2 promoter region between -859bp to -688bp upstream of the IGF2 transcriptional start site; 4) TCGA based integrated analyses identify specific HCC patient groups with altered TGF-β members and high expression of IGF2 that could be potentially targetable for IGF2; 5) TCGA analyses of 33 cancers also revealed that potentially “switching off” TGF-β-pathway (through decreased expression levels) leads to increased stemness validating findings of our TGF-β deficient mouse models that phenocopy a human cancer stem cell syndrome. In summary, TGF-β negatively regulates IGF2 transcription: loss of TGF-β signaling through β2SP/Smad3 leads to IGF2 activation, contributing to tumor progression. TGF-β-β2SP/Smad3 are important for IGF2 repression and maintaining stem cell homeostasis, which may result from TGF-β/β2SP/Smad3 and/or CTCF-dependent regulation of IGF2. Our studies provide a framework for new animal models of liver and gastrointestinal cancers and future new therapeutics. Citation Format: Shuyun Rao, Sobia Zaidi, Kazufumi Ohshiro, Jian Chen, Shoujun Gu, Wilma Jogunoori, Jon White, Bibhuti Mishra, Shulin Li, Rehan Akbani, Lopa Mishra. Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5459.

Grainyhead-like 2 (GRHL2) knockout abolishes oral cancer development through reciprocal regulation of the MAP kinase and TGF-\u03b2 signaling pathways

Grainyhead-Like 2 (GRHL2) is an epithelial-specific transcription factor that regulates epithelial morphogenesis and differentiation. Prior studies suggested inverse regulation between GRHL2 and TGF-β in epithelial plasticity and potential carcinogenesis. Here, we report the role of GRHL2 in oral carcinogenesis in vivo using a novel Grhl2 knockout (KO) mouse model and the underlying mechanism involving its functional interaction with TGF-β signaling. We developed epithelial-specific Grhl2 conditional KO mice by crossing Grhl2 floxed mice with those expressing CreER driven by the K14 promoter. After induction of Grhl2 KO, we confirmed the loss of GRHL2 and its target proteins, while Grhl2 KO strongly induced TGF-β signaling molecules. When exposed to 4-nitroquinoline 1-oxide (4-NQO), a strong chemical carcinogen, Grhl2 wild-type (WT) mice developed rampant oral tongue tumors, while Grhl2 KO mice completely abolished tumor development. In cultured oral squamous cell carcinoma (OSCC) cell lines, TGF-β signaling was notably induced by GRHL2 knockdown while being suppressed by GRHL2 overexpression. GRHL2 knockdown or KO in vitro and in vivo, respectively, led to loss of active p-Erk1/2 and p-JNK MAP kinase levels; moreover, ectopic overexpression of GRHL2 strongly induced the MAP kinase activation. Furthermore, the suppressive effect of GRHL2 on TGF-β signaling was diminished in cells exposed to Erk and JNK inhibitors. These data indicate that GRHL2 activates the Erk and JNK MAP kinases, which in turn suppresses the TGF -β signaling. This novel signaling represents an alternative pathway by which GRHL2 regulates carcinogenesis, and is distinct from the direct transcriptional regulation by GRHL2 binding at its target gene promoters, e.g., E-cadherin, hTERT, p63, and miR-200 family genes. Taken together, the current study provides the first genetic evidence to support the role of GRHL2 in carcinogenesis and the underlying novel mechanism that involves the functional interaction between GRHL2 and TGF-β signaling through the MAPK pathways.

Abstract P1-01-22: TGFβ-Smad3-TMEPAI axis drives the tumor progression of triple negative breast cancer

Abstract Background: In the absence of mutations and/or deletions in the TGF-β signaling molecules, how triple negative breast cancer (TNBC) cells become resistant to growth inhibitory signaling of TGF-β was addressed by screening of several TNBC cell lines for Smad proteins involved in TGF-β signaling. Unexpectedly, altered Smad2 and Smad3 protein levels were detected in breast cancer cells relative to normal mammary epithelial cells, suggesting a novel mechanism to escape from TGF-β mediated growth inhibition. In order to reflect the true functional status of the endogenous molecules, Smad2 or Smad3 were individually knocked down and tested its effect on pro-oncogenic behavior of TGF-β. Materials and Methods: All cell lines were cultured according to the recommended standard procedures. TMEPAI knockdown was achieved by using lentiviral vectors and knockdown of Smad2 and Smad3 was achieved by using retroviral vectors. DNA transfections and luciferase assays were performed according to vendor instructions. Cell proliferation was measured by quantitation of total cellular DNA. Immunoblotting, invasion and immunohistochemical assays were performed using standard methods. Bioinformatics: Triple negative breast cancer patient dataset (GSE58812) was used to compare Smad2, Smad3, Smad4 and TMEPAI/PMEPA1 expressions correlated with overall survival using the PROGgene tool by using median gene expression value as a dividing point. Results: While Smad2 deficiency has no effect on breast cancer cell behavior, Smad3 deficiency reduced growth and invasion capacity of breast cancer cells. Interestingly, Smad3 deficiency was associated with reduced gene expressions of transmembrane prostate androgen induced (TMEPAI or PMEPA1) gene and EMT inducing transcription factors and increased expression of cell cycle inhibitors. In contrast, Smad2 deficiency had opposite effect on these regulators. Importantly, the decreased growth and invasion and their associated gene expressions in Smad3 knockdown cells were largely reversed by overexpression of TMEPAI. Meta-analysis of Jezequel dataset of triple negative breast cancer patients suggested higher TMEPAI/PMEPA1 and lower Smad2 expressions are significantly associated with decreased survival. Our results support the idea that development of several triple negative breast cancers may involve expansion of cell populations with altered Smad2 and Smad3 levels resulting in Smad3 dependent expression of TMEPAI, which provides a competitive advantage for cancer cells to grow and metastasize in presence of TGF-β. Moreover, following meta-analysis of published microarray datasets in lung and gastric cancers also revealed that increased TMEPAI and Smad3 expression and decreased Smad2 expressions were significantly associated with poorer patient prognosis in non-small cell lung adenocarcinomas and gastric cancers suggesting universality of this phenomenon. Conclusion: We identified TGF-β-Smad3-TMEPAI signaling axis as a driver of tumor progression in triple negative breast cancer. Our results suggest that novel therapeutics targeting TMEPAI will selectively inhibit oncogenic activity of TGF-β and promote its tumor suppressive activity in treating TNBC. Citation Format: Saikumar P, Singha PK, Pandeswara S, Manjeri VA. TGFβ-Smad3-TMEPAI axis drives the tumor progression of triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-22.

Connexin 43 Hemichannels Regulate the Expression of Wound Healing-Associated Genes in Human Gingival Fibroblasts

Connexin 43 (Cx43) is the most ubiquitous connexin in various cells, and presents as hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We have recently shown that Cx43 abundance was strongly reduced in fibroblasts of human gingival wounds, and blocking Cx43 function in cultured human gingival fibroblasts (GFBLs) strongly regulated the expression of wound healing-related genes. However, it is not known whether these responses involved Cx43 HCs or GJs. Here we show that Cx43 assembled into distinct GJ and HC plaques in GFBLs both in vivo and in vitro. Specific blockage of Cx43 HC function by TAT-Gap19, a Cx43 mimetic peptide, significantly upregulated the expression of several MMPs, TGF-β signaling molecules, Tenascin-C, and VEGF-A, while pro-fibrotic molecules, including several extracellular matrix proteins and myofibroblast and cell contractility-related molecules, were significantly downregulated. These changes were linked with TAT-Gap19-induced suppression of ATP signaling and activation of the ERK1/2 signaling pathway. Collectively, our data suggest that reduced Cx43 HC function could promote fast and scarless gingival wound healing. Thus, selective suppression of Cx43 HCs may provide a novel target to modulate wound healing.

Biomarker modulation in patients treated with TRC105 in combination with anti-VEGF therapy.

11546 Background: TRC105, an endoglin-targeting monoclonal antibody with anti-angiogenic and anti-tumor activity, is being evaluated in multiple diseases. Here we report on pharmacodynamic and prognostic biomarkers in patients (pts) treated with both TRC105 and anti-VEGF agents. Methods: Plasma samples were collected from pts on three phase 2 trials combining TRC105 with an anti-VEGF agent: axitinib in metastatic renal cell carcinoma (mRCC), pazopanib in advanced soft tissue sarcoma, and bevacizumab in glioblastoma (GBM). Baseline and on-treatment levels of 22 soluble protein biomarkers were assessed. Results: Soluble endoglin markedly increased after TRC105 treatment in all pts ( p&lt; 0.001) as previously reported. BMP9 (a ligand for endoglin) and TGFβR3 (a type III TGFβ receptor) decreased in sarcoma pts at Cycle 2 Day 1 (C2D1) and generally remained below baseline throughout the course of treatment (BMP9, p= 0.004; TGFβR3, p= 0.003). Although TGFβR3 was decreased at C2D1 in mRCC (p = 0.030), no clear patterns were observed over time. Overall BMP9 levels did not change in response to therapy in either mRCC or GBM. Osteopontin (OPN) levels, a downstream effector of TGFβ signaling, were increased in sarcoma pts [ p= 0.002 at C2D15, p&lt; 0.001 at C4D1 and end of study (EOS)]; however, in mRCC ( p= 0.010) and GBM ( p= 0.003), OPN was only elevated at EOS. Increases in PlGF and VEGFD, and decreases in VEGFR2 were observed across all studies, as previously noted for VEGF inhibitors. In the mRCC trial, 5 of 18 pts exhibited a ≥30% tumor reduction. Lower OPN ( p= 0.026) and higher TGFβR3 ( p= 0.003) levels at baseline correlated with radiographic response to treatment. In the sarcoma trial, 6 of 19 pts responded (CHOI criteria) in which lower baseline levels of ICAM1 ( p= 0.018) and TSP2 ( p= 0.042) correlated with stable disease. Conclusions: In these trials, increases of soluble endoglin in response to TRC105 were observed, independent of the presence of any specific VEGF inhibitor. Differential regulation of BMP9, TGFβR3, and OPN suggests potential disease-specific modulation of key TGFβ signaling molecules in response to dual therapy. Baseline levels of OPN and TGFβR3 showed potential prognostic value in mRCC. Confirmation in larger trials is needed.

Age and sex differences in human skeletal muscle fibrosis markers and transforming growth factor-β signaling.

The aim of the study was to determine whether higher fibrosis markers in skeletal muscle of older adults are accompanied by increased expression of components of the canonical TGF-β signal transduction pathway. Fourteen healthy young (21-35years; 9 males and 5 females) and seventeen older (55-75years; 9 males and 8 females) participants underwent vastus lateralis biopsies to determine intramuscular mRNA and protein expression of fibrogenic markers and TGF-β signaling molecules related to TGF-β1 and myostatin. Expression of mRNA encoding the pro-fibrotic factors; axin 2, collagen III, β-catenin and fibronectin, were all significantly higher (all p<0.05) in the older participants (350, 170, 298, and 641%, respectively). Furthermore, axin 2 and β-catenin mRNA were significantly higher in older females than older males (p<0.05). Gene expression of ActRIIB, myostatin, and TGF-β1 were higher in older adults compared to younger adults (all p<0.05). There was, however, no difference in the total protein content of myostatin, myoD or myogenin (all p>0.05), whereas Smad3 protein phosphorylation was 48% lower (p<0.05) in muscle from older adults. Increased abundance of mRNA of fibrotic markers was observed in muscle from older adults and was partly accompanied by altered abundance of pro-fibrotic ligands in a sex specific manner.

Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling.

Cancer upregulated gene 2 (CUG2) enhances cell migration and invasion, but the underlying mechanism has not been revealed. Herein, CUG2 decreased the expression of E-cadherin and increased the expression of N-cadherin and vimentin, characteristics of the epithelial-mesenchymal transition (EMT). A CUG2 deletion mutant, lacking interaction with nucleophosmin 1 (NPM1), or suppression of NPM1 reduced wound healing and cell invasion, indicating that CUG2-mediated EMT requires NPM1. CUG2 enhanced activation of Smad2/3 and expression of Snail and Twist, while the CUG2 silence decreased these TGF-β signaling pathways, leading to suppression of EMT. NPM silence also inhibited the CUG2-induced TGF-β signaling. These results suggest that TGF-β signaling is involved in CUG2-induced EMT. Treatment with EW-7197, a novel inhibitor of TGF-β signaling, diminished CUG2-mediated EMT and inhibition of Akt, ERK, JNK, and p38 MAPK, non-canonical TGF-β signaling molecules, also decreased expression of Smad2/3, Snail and Twist, leading to inhibition of EMT. The results confirm that TGF-β signaling is essential for CUG2-mediated EMT. Interestingly, TGF-β enhanced CUG2 expression. We further found that both CUG2-induced TGF-β production and TGF-β-induced CUG2 up-regulation required a physical interaction between Sp1 and Smad2/3 in the CUG2 and TGF-β promoter, as demonstrated by a promoter reporter assay, immunoprecipitation, and ChIP assay. These results indicated close crosstalk between CUG2 and TGF-β. Conversely, suppression of CUG2 or NPM1 did not completely inhibit TGF-β-induced EMT, indicating that the effect of TGF-β on EMT is dominant over the effect of CUG2 on EMT. Collectively, our findings suggest that CUG2 induces the EMT via TGF-β signaling.