Active TGF-beta Research Articles

PO-431 A novel strategy to treat advanced, late-stage tumours with real-time tumour vaccination

IntroductionNovel anti-cancer immune therapeutic strategies, like various new antibody formats and/or tumour vaccines, show promising results in patients.However, due to the ‘immune-escape phenomenon’ driven by tumor-secreted Transforming Growth Factor-beta (TGF-beta), the host immune system of cancer bearing patients frequently fails to control tumour re-growth.Real-Time Tumour Vaccincation (RealTVac) approach aims at avoiding this „immune-escape phenomenon‘ by intratumoral inhibition of active TGF-beta isotypes while simultanously and synergistically inducing an efficient immune response by a highly potent combination of immune stimulating factors.Material and methodsThe synergistic effects of the proposed combination of a TGF-beta inhibitor with immunostimulating cytokines are demonstrated in in-vitro and in-vivo experiments:Effects of RealTVac components (ALK5-Inhibitor SD208 combined with T-cell stimulating interleukin-2 (IL-2) and Dendritic Cell (DC)-stimulatingGranulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) in a pharmaceutical composition upon human peripheral blood mononuclear cells (PBMCs) proliferation and allogeneous cytotoxicity against human malignant glioma cells were studied in-vitro (Cell Proliferation Assay, CARE-LASS Assay).Effects of intratumoral RealTVac components (see above) upon in-vivo growth of malignant melanoma were studied in a syngeneic B16-Mouse Model.Results and discussionsBoth, immune cell proliferation and tumour cell cytotoxicity were significantly enhanced using components of RealTVac therapy.Initial experiments in a syngenic B16 melanoma xenograft model in immunocompetent mice indicate that Real-Time Tumour Vaccination of established subcutaneous B16 tumour xenografts reduced local tumour growth compared to untreated controls.ConclusionReal-time tumour vaccination (RealTVac) activates human immune cells in vitro and inhibits growth of established B16 subcutaneous melanoma tumours in vivo.We hypothesised that local, intratumoral generation of tertiary lymphoid structures by combined activation of T-cells and DC by immunostimulating/-modulating cytokines in TGF-beta-free context induces the host anti-tumour immune responses to personalisedTumor-Associated Antigens (TAAs) in real-time mode.Thus, intratumoral immunisation as applied for RealTVac may create a new paradigm for cancer therapy in the near future (Marabelle et al., Clin. Cancer Res., 2014).

A novel mouse model of testicular granulosa cell tumors.

What is the role of dysregulated transforming growth factor beta (TGFB) signaling in the development of sex cord-stromal tumors in the testis? Overactivation of TGFB signaling results in the development of testicular tumors resembling granulosa cell tumors (GrCTs). In an earlier study, we demonstrated that constitutively active TGFB receptor 1 (TGFBR1) in ovarian somatic cells promotes the development of ovarian GrCTs. However, the consequence of dysregulation of TGFB signaling in the pathobiology of the testis, remains poorly defined. To identify the impact of dysregulation of TGFB signaling on the testis, we generated mice with constitutive activation of TGFBR1 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase. The effect of constitutively active TGFBR1 on testis development and the timeline of testicular tumor formation were examined. We further investigated the molecular features of testicular tumors and determined the expression of beta-catenin (CTNNB1) known to be involved in testicular GrCT development. Male mice with constitutive activation of TGFBR1 were examined at various developmental stages (i.e. from 1 week up to 6 months) along with controls. Testis samples were collected and processed for histological and molecular analyses, including haematoxylin and eosin (H and E) staining, real-time PCR, immunohistochemistry, immunofluorescence and western blotting. Immunostaining/immunoblotting and real-time PCR experiments were performed using at least three animals per genotype. Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tail t-test and reported when P value is <0.05. Mice harboring constitutively active TGFBR1 in the testes developed tumors resembling testicular GrCTs, a rare type of tumors in the testis. The formation of testicular tumors led to altered cell proliferation, loss of germ cells and defective spermatogenesis. Immunohistochemically, these tumors were positive for inhibin alpha (INHA), forkhead box O1 (FOXO1), and more importantly, forkhead box L2 (FOXL2), a protein specifically expressed in the ovary and required for normal granulosa cell differentiation and function. Consistent with the immunohistochemical findings, FOXL2 proteins were only detectable in testes of TGFBR1-CAAcre mice but not those of controls by western blotting, suggesting potential alteration of Sertoli cell fate. To explore mechanisms underlying the tumor-promoting effect of TGFBR1 overactivation, we examined the expression of CTNNB1. The results revealed increased expression of CTNNB1 in testicular tumors in TGFBR1-CAAcre mice. Collectively, this study uncovered tumorigenic function of enhanced TGFB signaling in the testis. N/A. This study was performed using mice, and the direct relevance of the experimental paradigm and findings to human testicular GrCTs awaits further investigation. Of note, constitutive activation of TGFBR1 was employed to enhance TGFB/SMAD signaling activity and may not be interpreted as the genetic cause of the disease. This mouse model may prove to be a useful addition to the mouse genetics toolkit for GrCT research. Our finding that dysregulation of TGFB signaling results in the development of testicular GrCTs supports a common origin between Sertoli cells and granulosa cells, and highlights the paramount importance of balanced TGFB signaling in reproduction and development. This research was supported by the National Institutes of Health grant R03HD082416 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development and the New Faculty Start-up Funds from Texas A&M University awarded to Q.L. The authors declare no competing interest.

862 Absence of collagen VII binding to thrombospondin 1 promotes activation of TGF-beta in recessive dystrophic epidermolysis bullosa

862 Absence of collagen VII binding to thrombospondin 1 promotes activation of TGF-beta in recessive dystrophic epidermolysis bullosa

Effect of collagen length distribution and timing for repair on the active TGF-β concentration in tendon

ABSTRACTThe composition of extracellular matrix (ECM) in tendon depends on the secretion profile of resident cells known as tenocytes. For tissues with a mechanical role like tendon, mechanical strain is known to play an important role in determining the secretion profile of resident cells. Previously we explored the idea of estimating average concentrations of ECM molecules as a function of tendon strain magnitude and number of loading cycles. Specifically, we developed a model of the mechanical fatigue damage of tendon collagen fibers and introduced elementary cell responses (ECRs) by which local cellular-level responses to the strain environment, combined with the fatigue damage model, were scaled up to predict tissue-level responses. Using this approach, we demonstrated that the proposed model is capable of estimating average concentrations of ECM molecules that qualitatively accord with experimental observations. In this study, we increase model realism by extending this approach to consider the implications of a non-uniform collagen fiber distribution, and the influence of time delay on repair of damaged collagen fibers. Using this approach, we focus the study on the average tenocyte secretion profile for active transforming growth factor beta (TGF-β), and discover that increasing fiber length dispersion and/or increasing repair delay leads to increasing active TGF-β concentrations, and reduced sensitivity of average concentration profile of TGF-β to tendon strain.

A novel transforming growth factor beta-induced long noncoding RNA promotes an inflammatory microenvironment in human intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a deadly liver primary cancer associated with poor prognosis and limited therapeutic opportunities. Active transforming growth factor beta (TGFβ) signaling is a hallmark of the iCCA microenvironment. However, the impact of TGFβ on the transcriptome of iCCA tumor cells has been poorly investigated. Here, we have identified a specific TGFβ signature of genes commonly deregulated in iCCA cell lines, namely HuCCT1 and Huh28. Novel coding and noncoding TGFβ targets were identified, including a TGFβ‐induced long noncoding RNA (TLINC), formerly known as cancer susceptibility candidate 15 (CASC15). TLINC is a general target induced by TGFβ in hepatic and nonhepatic cell types. In iCCA cell lines, the expression of a long and short TLINC isoform was associated with an epithelial or mesenchymal phenotype, respectively. Both isoforms were detected in the nucleus and cytoplasm. The long isoform of TLINC was associated with a migratory phenotype in iCCA cell lines and with the induction of proinflammatory cytokines, including interleukin 8, both in vitro and in resected human iCCA. TLINC was also identified as a tumor marker expressed in both epithelial and stroma cells. In nontumor livers, TLINC was only expressed in specific portal areas with signs of ductular reaction and inflammation. Finally, we provide experimental evidence of circular isoforms of TLINC, both in iCCA cells treated with TGFβ and in resected human iCCA. Conclusion: We identify a novel TGFβ‐induced long noncoding RNA up‐regulated in human iCCA and associated with an inflammatory microenvironment. (Hepatology Communications 2018;2:254‐269)

664 Thrombospondin 1 is a major activator of TGF-beta in Recessive Dystrophic Epidermolysis Bullosa

664 Thrombospondin 1 is a major activator of TGF-beta in Recessive Dystrophic Epidermolysis Bullosa

Abstract 407: Divergent Functions of Thrombospondin Genes in Mammals

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 various aspects of cell- matrix interactions. Thbs1 and 2 can also alter angiogenesis and modulate MMP- as well as TGF beta activity. More recently, we have shown that they can also serve an intracellular chaperone function along the secretory pathway in response to ER stress (Lynch et. al. Cell 2012). 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 gain and loss of function mouse models for Thbs1 as a representative of group A and Thbs3 for group B. Our overall conclusion was that Thbs4 serves a protective function in the heart while Thbs1 and Thbs3 are either overtly maladaptive or they predispose to worsening heart disease with injury stimulus. Taken together, this is the first study comparing Thbs subfamilies, unraveling previously unrecognized functions of Thbs1 and 3 in the mammalian heart, which appears to oppose the protective function of Thbs4.

Effect of intravenous immunoglobulin on the function of Treg cells derived from immunosuppressed mice with Pseudomonas aeruginosa pneumonia.

AimThe present study aimed to investigate the effect of intravenous immunoglobulin (IVIG) on regulatory T (Treg) cells derived from immunosuppressed mice with Pseudomonas aeruginosa (PA) pneumonia.MethodsA total of 108 BALB/c mice were randomly divided into the following groups: control group (Control), immunosuppressed group (IS), PA pneumonia group (PA), PA pneumonia in immunosuppressed group (IS + PA), PA pneumonia with IVIG treatment in immunocompetent group (PA + IVIG) and PA pneumonia with IVIG treatment in immunosuppressed group (IS + PA + IVIG). Each group comprised 18 mice. The combined PA pneumonia in immunosuppressed model and the treatment models were established. The mice in each group were sacrificed at 4, 8, and 24 h time points. The general condition and pathological changes in the lung tissues of the mice were monitored. Reverse transcription-polymerase chain reaction was used to detect the forkhead box P3 (FOXP3) mRNA relative expression level in the lung tissues. The enzyme-linked immunosorbent assay was used to detect the serum concentration of active transforming growth factor beta (TGF-β).ResultsNo inflammatory response were exhibited in the lung tissues of the mice in Control group and IS group, while varying degrees of acute lung injury were revealed in the mice in PA group, IS + PA group, PA + IVIG group and IS + PA + IVIG group. Lung tissue injury was most apparent at the 8 h time point, and it indicated the greatest effect in IS + PA group. Whereas tissue damages were alleviated in PA + IVIG group and IS + PA + IVIG group compared with IS + PA group. In addition, tissue damage lessened in PA + IVIG group compared with PA group and IS + PA + IVIG group. FOXP3 mRNA expression levels in the lung tissues and the serum concentration of TGF-β were lower in IS group, PA group, IS + PA group and IS + PA + IVIG group at the 4, 8 and 24 h time points, respectively compared with Control group. FOXP3 mRNA expression levels decreased in PA + IVIG group at the 4h time point and TGF-β serum concentrations decreased at the 4 and 8h time points compared with Control group, and subsequently increased.ConclusionsIn the immunosuppred model with PA pneumonia, the immune system was greatly compromised. IVIG partially restored the immunosuppressed functions of Treg cells, suppressed the overactivated immune system and ameliorated the development of the disease.

Abstract 309: Blockade of Transforming Growth Factor Beta Activity in Elastase-Induced Aortic Injury in Mice Induces a Human-Like Abdominal Aneurysm

Background: Abdominal aortic aneurysm (AAA) carries important morbidity and mortality and is resistant to medical therapy. Current experimental models do not accurately reproduce the major features of the human disease. There are 2 major categories of mouse models of AAA: those that induce medial dissection, which is not a major characteristic of human AAA, and those that induce aortic dilatation but are self-contained and do not progress to rupture. Methods: We hypothesized that blockade of TGFβ activity, a guardian of vascular integrity and immune homeostasis, and a major causal factor in genetically triggered thoracic aortic aneurysms in humans, would impair vascular healing in models of ‘non-dissecting’ abdominal aortic dilatation, and would lead to continuous aneurysmal growth until rupture. We tested this hypothesis in the elastase-induced abdominal aortic dilatation model in mice. We analyzed AAA development and progression using ultrasound in vivo, advanced synchrotron-based ultrahigh resolution imaging ex-vivo, and a combination of biological, histological and flow cytometry-based cellular and molecular approaches in vitro. Results: We show that systemic blockade of TGFβ activity using a neutralizing mouse monoclonal anti-TGFβ antibody induces a transition from a model of self-contained aortic dilatation to a model of sustained aneurysmal growth culminating in rupture. TGFβ blockade enhances leukocyte infiltration and extracellular matrix degradation, and leads to sustained aneurysmal aortic dilatation, associated with the formation of an intra-luminal thrombus infiltrated with neutrophils, as seen in the human disease. Persistent AAA growth throughout the duration of the experiment is associated with wall disruption without medial dissection, and culminates in fatal aortic wall rupture. Monocyte/macrophage depletion substantially limits AAA severity. Conclusions: Endogenous TGFβ activity is required for the resolution of elastase-induced aortic injury. We expect that this new model will improve our understanding of the pathophysiology of AAA, and will be useful to identify new therapeutic targets.

Endoplasmic Reticulum Oxidative Stress Triggers Tgf-Beta-Dependent Muscle Dysfunction by Accelerating Ascorbic Acid Turnover

Endoplasmic reticulum (ER) and oxidative stress are two related phenomena that have important metabolic consequences. As many skeletal muscle diseases are triggered by oxidative stress, we explored the chain of events linking a hyperoxidized ER (which causes ER and oxidative stress) with skeletal muscle dysfunction. An unbiased exon expression array showed that the combined genetic modulation of the two master ER redox proteins, selenoprotein N (SEPN1) and endoplasmic oxidoreductin 1 (ERO1), led to an SEPN1-related myopathic phenotype due to excessive signalling of transforming growth factor (TGF)-beta. The increased TGF-beta activity in the genetic mutants was caused by accelerated turnover of the ER localized (anti-oxidant) ascorbic acid that affected collagen deposition in the extracellular matrix. In a mouse mutant of SEPN1, which is dependent on exogenous ascorbic acid, a limited intake of ascorbic acid revealed a myopathic phenotype as a consequence of an altered TGF-beta signalling. Indeed, systemic antagonism of TGF-beta re-established skeletal muscle function in SEPN1 mutant mice. In conclusion, this study sheds new light on the molecular mechanism of SEPN1-related myopathies and indicates that the TGF-beta/ERO1/ascorbic acid axis offers potential for their treatment.

TGF-beta and glutathione promote tissue repair in cigarette smoke induced injury.

We studied the effect of cigarette smoke extract (CSE) on a three-dimensional (3-D) co-culture model with epithelial cells and mesenchymal cells to clarify how epithelial cells protect lung tissue from cigarette smoke in-vivo. Two types of gels were prepared. The one was the co-culture of human fetal lung fibroblasts (HFL-I) embedded in type-I collagen gel, with alveolar epithelial cells (A549) cultured covering the top of the gel. The other was HFL-I cells alone. After 48 hours from CSE exposure, gel contraction, levels of fibronectin, transforming growth factor (TGF)-beta and GSH were assessed. CSE inhibited fibroblast-mediated gel contraction and this inhibition was lessened in co-culture associated with higher GSH concentration and TGF-beta1 level as compared to the level in HFL-I cells alone. CSE lowered fibronectin level to a lesser extent in co-culture as compared to the level in HFL-I cells alone. Exogenous TGF-beta1 restored the inhibition of gel contraction by CSE independent of GSH level. Cigarette smoke may interfere with 3-D co-culture gel contraction by diminishing GSH, fibronectin and TGF-beta1 action in the epithelial-mesenchymal interaction.

Abstract P107: Hypertension Enhances the Differentiation of Cardiac Fibroblasts Into Myofibroblasts After TGF-beta1 Treatment

Objectives: In cardiac fibrosis associated with hypertension, TGF-beta1 plays a key role by acting on differentiation of cardiac fibroblasts (CF) into alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts. In this study, we tested the effect of TGF-beta1 during the myofibroblast differentiation process of CF from normotensive and hypertensive rats. Methods: CF were obtained by enzymatic digestion of hearts isolated from Spontaneously Hypertensive (hCF) and normotensive Wistar Kyoto (nCF) rats (n=5 rat/group). Gene and protein expression in CF was evaluated by Western blot and qRT-PCR analyses, respectively. Immunohistochemistry analysis for integrin alpha-v beta-5 was performed on rat cardiac tissue (n=5 rat/group). Results: Cultured hCF showed an enhanced SMAD2/3 activation and alpha-SMA protein expression after treatment with TGF-beta1 (5 ng/ml) in comparison with nCF. Alpha-SMA up-regulation was further confirmed by qRT-PCR analysis that showed a significant increase in alpha-SMA gene expression in hCF after TGF-beta1 treatment (2.78±0.25 vs 2.01±0.21 fold increase, p &lt;0.05). Moreover, immunostaining on cardiac tissues revealed a higher expression of integrin alpha-v beta-5 in hypertensive vs normotensive rat hearts (345.3±170.0 vs 48.2±22.3 mm 2 of integrin-positive area, p &lt;0.05). This result was also confirmed in vitro ; indeed, integrin alpha-v beta-5 gene expression in hCF increased 2.8-fold in basal condition and 5.12-fold after TGF-beta1 treatment when compared to untreated nCF. Conclusions: Taken together, these results suggest that hCF are more prone to upregulate integrin alpha-v beta-5 and consequently differentiate into myofibroblasts in vitro under TGF-beta1 treatment. Thus, targeting alpha-v beta-5 might open a novel prospective for the treatment of fibrosis in hypertensive hearts likely reducing integrin-mediated TGF-beta1 activation.

Abstract 3741: Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma

Abstract Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both ‘seed and soil’ theory and efficacy of combination regimen (immunomodulation) for better clinical outcome. Citation Format: Katja Wosikowski, Kathy Hasenbach, Jutta Petschenka, Diana Barea Roldan, Sebastian Kreiter, Ugur Sahin, Guillaume Serin, Julie-Orlane Redon, Marc Hillairet de Boisferon, Francis Bichat, Hanna Kohonen, Michel Janicot. Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3741.

Arthrofibrotic remodeling after knee replacement surgery, a stress-related disease? - Clinic, biochemical model, therapy, research -

Aims and Objectives:The predicted incidence of arthrofibrosis after knee replacement surgery is 5 - 10%. Arthrofibrosis is defined as painful impairment of joint flexibility occurring postoperatively. To counteract symptoms and to accomplish quality standards (E/F 0-0-90) several therapeutic strategies as for instance physiotherapy or mobilization under anaesthesia are applied. Although it is known that mechanical and emotional stress induce myofibroblast differentiation, extracellular matrix (ECM) synthesis and xylosyltransferase (XT) activity, biochemical processes of fibrotic diseases receive little attention in orthopaedics. Until today, neither an adequate disease model nor a diagnostic marker for arthrofibrosis has been described.Materials and Methods:Surgical procedures (e.g. endoprosthesis, cruciate ligament replacement) initiate physiological wound healing and the release of inflammatory mediators from platelets, damaged tissue and other cells of the immune system. Secreted cytokines such as transforming growth factor (TGF-beta 1) or platelet derived growth factor (PDGF) promote myofibroblast proliferation and differentiation. Mechanical strain (stretching exercises with pain inhibition) leads to activation of latent TGF-beta 1, which triggers scarring via ECM/XT synthesis and prevents myofibroblast apoptosis. Our recent study carried out a decreased platelet count in arthrofibrosis patients compared to healthy controls. Thus, an elevated platelet consumption might take place. Analogous to TGF-beta 1, PDGF also inhibits myofibroblast apoptosis and expression of matrix metalloproteinase expression, which degrade ECM. Emotional stress might not only matter in other fibrotic diseases e.g. tako-tsubo cardiomyopathy (TTC) but also in arthrofibrosis. Sympathicotonic destabilization (due to pain, frustration or social burden) results in increased catecholamine synthesis and sympathetic activity. These effects are recognizable by vegetative symptoms (e.g. insomnia, sweating, muscle tension). Catecholamines directly activate fibroblasts and increase ECM synthesis by binding to Beta 2-adrenergic receptors. In TTC, a decrease in catecholamines and profibrotic cytokines restores organ functionality.Results:Causal therapy Several therapeutic approaches might exert a positive influence on degradation of fibrotic tissue: less of postisometric stretching, avoidance of emotional workload, positive expectations due to medical consultation about reversibility of fibrotic mechanisms, appropriate medication as well as osteopathy.Conclusion:Research Operating surgeons are able to detect an unfavorable development of arthrofibrosis relatively early. Nevertheless, they are afraid to name the possible complication. Therefore, quantification of a reliable biomarker would bring great advantages for diagnostics of pre- and after-treatment. Our current studies examine whether increased XT activity might display a biomarker in synovial fluid.

Surface proteome analysis identifies platelet derived growth factor receptor-alpha as a critical mediator of transforming growth factor-beta-induced collagen secretion

Fibroblasts are extracellular matrix-producing cells in the lung. Fibroblast activation by transforming growth factor-beta leads to myofibroblast-differentiation and increased extracellular matrix deposition, a hallmark of pulmonary fibrosis. While fibroblast function with respect to migration, invasion, and extracellular matrix deposition has been well-explored, little is known about the surface proteome of lung fibroblasts in general and its specific response to fibrogenic growth factors, in particular transforming growth factor-beta.We thus performed a cell-surface proteome analysis of primary human lung fibroblasts in presence/absence of transforming growth factor-beta, followed by characterization of our findings using FACS analysis, Western blot, and siRNA-mediated knockdown experiments.We identified 213 surface proteins significantly regulated by transforming growth factor-beta, platelet derived growth factor receptor-alpha being one of the top down-regulated proteins. Transforming growth factor beta-induced downregulation of platelet derived growth factor receptor-alpha induced upregulation of platelet derived growth factor receptor-beta expression and phosphorylation of Akt, a downstream target of platelet derived growth factor signaling. Importantly, collagen type V expression and secretion was strongly increased after forced knockdown of platelet derived growth factor receptor-alpha, an effect that was potentiated by transforming growth factor-beta. We therefore show previously underappreciated cross-talk of transforming growth factor-beta and platelet derived growth factor signaling in human lung fibroblasts, resulting in increased extracellular matrix deposition in a platelet derived growth factor receptor-alpha dependent manner. These findings are of particular importance for the treatment of lung fibrosis patients with high pulmonary transforming growth factor-beta activity.

FRI-028 - Role of Alphav Integrin-Mediated TGF-Beta Activation in Hepatocarcinogenesis

FRI-028 - Role of Alphav Integrin-Mediated TGF-Beta Activation in Hepatocarcinogenesis

Thrombospondin-1 Affects Bovine Luteal Function via Transforming Growth Factor-Beta1-Dependent and Independent Actions.

Thrombospondin-1 (THBS1) and transforming growth factor-beta1 (TGFB1) are specifically up-regulated by prostaglandin F2alpha in mature corpus luteum (CL). This study examined the relationship between the expression of THBS1 and TGFB1 and the underlying mechanisms of their actions in luteal endothelial cells (ECs). TGFB1 stimulated SMAD2 phosphorylation and SERPINE1 levels in dose- and time-dependent manners in luteal EC. THBS1 also elevated SERPINE1; this effect was abolished by TGFB1 receptor-1 kinase inhibitor (SB431542). The findings here further imply that THBS1 activates TGFB1 in luteal ECs: THBS1 increased the effects of latent TGFB1 on phosphorylated SMAD (phospho-SMAD) 2 and SERPINE1. THBS1 silencing significantly decreased SERPINE1 and levels of phospho-SMAD2. Lastly, THBS1 actions on SERPINE1 were inhibited by LSKL peptide (TGFB1 activation inhibitor); LSKL also counteracted latent TGFB1-induced phospho-SMAD2. We found that TGFB1 up-regulated its own mRNA levels and those of THBS1. Both compounds generated apoptosis, but THBS1 was significantly more effective (2.5-fold). Notably, this effect of THBS1 was not mediated by TGFB1. THBS1 and TGFB1 also differed in their activation of p38 mitogen-activated protein kinase. Whereas TGFB1 rapidly induced phospho-p38, THBS1 had a delayed effect. Inhibition of p38 pathway by SB203580 did not modulate TGFB1 effect on cell viability, but it amplified THBS1 actions. THBS1-stimulated caspase-3 activation coincided with p38 phosphorylation, suggesting that caspase-induced DNA damage initiated p38 phosphorylation. The in vitro data suggest that a feed-forward loop exists between THBS1, TGFB1, and SERPINE1. Indeed all these three genes were similarly induced in the regressing CL. Their gene products can promote vascular instability, apoptosis, and matrix remodeling during luteolysis.

Transcriptional activity of TGFβ1 and its receptors genes in thyroid gland

Determination of gene-candidates' profile expression responsible for fibrosis, immunosuppression, angiogenesis, and neoplasia processes in the pathogenesis of thyroid gland disease. Sixty-three patients underwent thyroidectomy: 27 with non-toxic nodular goitre (NG), 22 with toxic nodular goitre (TNG), six with papillary cancer (PTC), and eight with Graves' disease (GD). In thyroid tissues, transcriptional activity of TGFbeta1 and its receptors TGFbetaRI, TGFbetaRII, and TGFbetaRIII genes were assessed using RT-qPCR (Reverse Transcriptase Quantitative Polymerase Chain Reaction). Molecular analysis was performed in tissues derived from GD and from the tumour centre (PTC, NG, TNG) and from peripheral parts of the removed lobe without histopathological lesions (tissue control). Control tissue for analysis performed in GD was an unchanged tissue derived from peripheral parts of the removed lobe of patients surgically treated for a single benign tumour. Strict regulation observed among transcriptional activity of TGFb1 and their receptor TGFbetaRI-III genes in control tissues is disturbed in all pathological tissues - it is completely disturbed in PTC and GD, and partially in NG and TNG. Additionally, higher transcriptional activity of TGFb1 gene in PTC in comparison with benign tissues (NG, GD) and lower expression of mRNA TGFbRII (than in TNG, GD) and mRNA TGFbetaRIII than in all studied benign tissues (NG, TNG, GD) suggests a pathogenetic importance of this cytokine and its receptors in PTC development. In GD tissue, higher transcriptional activity of TGFbetaRII and TGFbetaRIII genes as compared to other pathological tissues was observed, indicating a participation of the receptors in the pathomechanism of autoimmune thyroid disease (AITD). TGFbeta1 blood concentrations do not reflect pathological processes taking place in thyroid gland. (Endokrynol Pol 2016; 67 (4): 375-382).

Abstract 301: RealTVac, a novel strategy to treat advanced, late-stage tumors with real-time tumor vaccination

Abstract Novel anti-cancer immune therapeutic strategies, like various new antibody formats and/or tumor vaccines, show promising results in patients. However, due to the “immune-escape phenomenon” driven by tumor-secreted Transforming Growth Factor-beta (TGF-beta), the host immune system of cancer bearing patients frequently fails to control tumor re-growth. The RealTVac® approach aims at avoiding this “immune-escape phenomenon” by intratumoral inhibition of active TGF-beta isotypes while simultanously and synergistically inducing an efficient immune response by a highly potent combination of immune stimulating factors. The synergistic effects of the proposed combination of a TGF-beta inhibitor with immunostimulating cytokines upon human immune cell activation are demonstrated in in-vitro experiments. Both, immune cell proliferation and tumor cell cytotoxicity were significantly enhanced. Initial experiments in a syngenic B16 melanoma xenograft model in immunocompetent mice indicate that the RealTVac® therapy of established subcutaneous B16 tumor xenografts reduced local tumor growth compared to untreated controls. To summarize, the goal of the local application of RealTVac® is to allow the host immune system to scan in real-time all Tumor Associated Antigens (TAAs) being currently expressed by those tumor cells that are exposed to sufficient TGF-beta inhibition. The repetitive administration of the RealTVac® approach is furthermore expected to provide a continuous update of the immune response, reflecting the changing pattern of TAAs related to the dynamic intratumoral heterogeneity in malignant tumors. Citation Format: Piotr Jachimczak, Andreas Mitsch, Achim Aigner. RealTVac, a novel strategy to treat advanced, late-stage tumors with real-time tumor vaccination. [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 301. doi:10.1158/1538-7445.AM2015-301

Abstract 2215: Transcriptional co-regulation of Nox4 by p53 and SMAD3

Abstract Recent studies suggest p53 plays an important role in TGF-beta-mediated cell signaling and migration. Previously, we showed that wild-type (WT) and mutant forms of p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (Nox4). We found that WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration, whereas tumor-associated mutant p53 proteins enhanced Nox4 mRNA and protein expression and cell migration by both TGF-beta-dependent and independent mechanisms (Boudreau et al. Br J Cancer. 2014 May 13;110(10):2569-82). In this study, we examined the basis of human Nox4 promoter regulation by p53 and SMAD3. By constructing a deletion series of Nox4 promoter-reporter constructs, we discovered two regions (-4.76kb to -3.9kb and -1.8kb to - 0.2kb) upstream of the transcription start site that induce promoter activity upon activation of the TGF-beta/SMAD3 pathway. Interestingly, TGF-beta/SMAD3-mediated Nox4 promoter activity was abolished by heterologous expression of p53-WT in p53-null cell lines Hep3B (hepatocytes) and H1299 (lung epithelial). This activity was repressed by wild-type p53 in a dose-dependent manner. In contrast, expression of common tumor-associated mutant-p53 proteins (R175H, R248H, R249Q, R273H) did not repress but enhanced Nox4 promoter activity. We observed this enhancement of Nox4 promoter activity by mutant p53 (R273H) in H1299 cells expressing T204D, a constitutively active TGF-beta receptor. When individual p53 response elements were mutated, however, both untreated and R273H-expressing cells showed 50% reduction in promoter activity. These results indicate that the Nox4 promoter region between -3.97kb and -4.76kb, containing candidate p53 response elements, plays a role in modulating Nox4 expression. Preliminary chromatin immunoprecipitation results suggest that endogenous mutant p53 R249S in PLC/PRF/5 hepatoma cells complexes with the Nox4 promoter region between -3.97kb and -4.76kb. Furthermore, constructs lacking this region show significantly reduced Nox4 promoter activity in response to TGF-beta. Our results provide a basis for Nox4 co-regulation by SMAD3 and mutant p53 that may be involved in tumor progression and metastasis. Citation Format: Howard E. Boudreau, Jonathan J. Park, Thomas L. Leto. Transcriptional co-regulation of Nox4 by p53 and SMAD3. [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 2215. doi:10.1158/1538-7445.AM2015-2215