Transforming Growth Factor-beta Receptor Type Research Articles

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.

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.

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.

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.

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.

A Novel Strategy for the Preparation of Codon-Optimized Truncated Ulp1 and its Simplified Application to Cleavage the SUMO Fusion Protein.

Ubiquitin-like protease 1 (Ulp1) of Saccharomyces cerevisiae emerges as a fundamental tool to obtain the natural N-terminal target protein by cleavage of the small ubiquitin-related modifier (SUMO) fusion protein. However, the costly commercial Ulp1 and its complicated procedures limit its application in the preparation of the target protein with natural N-terminal sequence. Here, we describe the preparation of bioactive codon-optimized recombinant truncated Ulp1 (Leu403-Lys621) (rtUlp1) of S. cerevisiae in Escherichia coli using only one-step with Ni–NTA affinity chromatograph, and the application of rtUlp1 to cleave the SUMO fusion protein by simply mixing the purified rtUlp1, SUMO fusion protein and DL-Dithiothreitol in Tris–HCl buffer. The optimal expression level of non-fusion protein rtUlp1 accounts for approximately 50 % of the total cellular protein and 36 % of the soluble form by addition of isopropyl β-D-l-thiogalactopyranoside at a final concentration of 0.4 mM at 18 °C for 20 h. The purification of target protein rtUlp1 was conducted by Ni–NTA affinity chromatography. The final yield of rtUlp1 was 45 mg/l in flask fermentation with a purity up to 95 %. Furthermore, the high purity of rtUlp1 could effectively cleave the SUMO-tTβRII fusion protein (SUMO gene fused to truncated transforming growth factor-beta receptor type II gene) with the above simplified approach, and the specific activity of the rtUlp1 reached up to 2.8 × 104 U/mg, which is comparable to the commercial Ulp1. The preparation and application strategy of the rtUlp1 with commonly available laboratory resources in this study will be convenient to the cleavage of the SUMO fusion protein to obtain the natural N-terminal target protein, which can be implemented in difficult-to-express protein functional analysis.

A Cis-Membrane FRET-Based Method for Protein-Specific Imaging of Cell-Surface Glycans

Metabolic labeling of glycans with chemical reproters (e.g., alkyne or azide) in conjunction with bioorthogonal chemistry is a powerful tool for imaging glycome; however, this method lacks protein-specificity and therefore is not applicable to imaging glycosylation of a specific protein of interest (POI). Here we report the development of a cis-membrane FRET-based methodology that allows protein-specific imaging of glycans on live cells. We exploit metabolic glycan labeling in conjunction with site-specific protein labeling to simultaneously install a FRET acceptor and a donor onto the glycans and the extracellular terminal of the protein of interest, respectively. The intramolecular donor-acceptor distance for the POI falls within the range for effective FRET, whereas the intermolecular FRET is disfavored since the excess acceptors on other proteins are distant from the donor. We demonstrated the capability of this cis-membrane FRET imaging method by visualizing the sialylation of several important cell surface receptors including integrin αXβ2, epidermal growth factor receptor, and transforming growth factor-beta receptor type I. Furthermore, our imaging experiments revealed that the sialylation might be important for β2 integrin activation. Our methodology should enable the live-cell studies on how glycosylation regulates the functions and dynamics of various cell-surface proteins.

TGF-βRI kinase activity mediates Emdogain-stimulated in vitro osteoclastogenesis

Emdogain, containing an extract of fetal porcine enamel matrix proteins, is a potent stimulator of in vitro osteoclastogenesis. The underlying molecular mechanisms are, however, unclear. Here, we have addressed the role of transforming growth factor-beta receptor type 1 (TGF-βRI) kinase activity on osteoclastogenesis in murine bone marrow cultures. Inhibition of TGF-βRI kinase activity with SB431542 abolished the effect of Emdogain on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand or tumor necrosis factor-alpha. SB431542 also suppressed the Emdogain-mediated increase of OSCAR, a co-stimulatory protein, and dendritic cell-specific transmembrane protein and Atp6v0d2, the latter two being involved in cell fusion. Similar to transforming growth factor-beta1 (TGF-β), Emdogain could not compensate for the inhibition of IL-4 and IFNγ on osteoclast formation. When using the murine macrophage cell line RAW246.7, SB431542 and the smad-3 inhibitor SIS3 blocked Emdogain-stimulated expression of the transcription factor NFATc1. Taken together, the data suggest that TGF-βRI kinase activity is necessary to mediate in vitro effects of Emdogain on osteoclastogenesis. Based on these in vitro data, we can speculate that at least part of the clinical effects of Emdogain on osteoclastogenesis is mediated via TGF-β signaling.

Treatment of a patient with vertebral and subclavian aneurysms in the setting of a TGFBR2 mutation

We present the case of a patient diagnosed with hypermobile Ehlers-Danlos syndrome with aneurysms of the subclavian and vertebral arteries. Molecular testing demonstrated transforming growth factor-beta receptor type 2 mutation. She was not a candidate for an open repair; therefore, a hybrid approach involving right vertebral ligation and bypass from the right common carotid to the vertebral C1-2 interface, endovascular exclusion of the left vertebral artery, and stent grafting of the left subclavian/axillary artery was used. The left vertebral embolization proved ineffective, requiring a right-to-left vertebral catheterization with glue occlusion. Based on her proper molecular diagnosis, she underwent prophylactic root and ascending aortic repair.

Abstract A69: The novel strategy for treatment of pancreactic ductal adenocarcinoma targeting tumor microenvironment.

Abstract Background and Aim: Previously, we have reported a genetically engineered mouse pancreatic ductal adenocarcinoma (PDAC) progression model which has pancreatic-specific transforming growth factor-beta receptor type II knockout in the context of Kras activation. This model shows PDAC with 100 % penetrance and recapitulates the signature of human PDAC well. Using this model, we explored novel treatment for PDAC targeting tumor microenvironment. Materials and Methods: To investigate whether the mice model is suitable for the drug screening, the mice were treated with gemcitabine or S-1, which are the standard drug for human PDAC, To the next, for single agent treatment, mice were treated with axitinib (A) or sunitinib (S), which are multi-kinase inhibitors, or candesartan (C) or telmisartan (T), which are anigiotensin II receptor blockers (ARBs), respectively. For combined agent experiment, mice were treated with A, C, or T combined with gemcitabine or S-1. Treatment continued until 8 weeks of age. Moreover, for the survival analysis, the drug treatment was continued until the mice became distressed. In vivo anti-tumor effect and survival time were assessed. Immunostaining of tumor tissue for caspase 3, Ki67, CD31, F4/80, α-SMA, and VEGF was performed. Azan staining also performed for the assessment of fibrosis in the tumor. Results: Gemcitabine and S-1 showed anti-proliferative effect and prolonged overall survival of these mice compared to control, as well as human cases. Median survival time of single use of A and S group was significantly longer and that of C and T group was tended to be longer than that of control group. The entire drug-treatment group showed significantly stronger anti-tumor effect in vivo compared to control. Combined treatment led to statistically significant longer survival and more anti-tumor effect than that of single agent-treated group. A and S group showed significantly higher caspase 3 staining and lower Ki67 staining than that of control, however, C and T group showed no change of these staining, compared to control. Microvessel density, F4/80, and α-SMA staining, VEGF expression, and azan staining of the entire drug-treated group was significantly lower than that of control. Conclusion: Targeting tumor microenvironment, such as angiogenesis, immune cell infiltration and fibrosis, using multi-kinase inhibitor or ARB in addition to cytotoxic agents, such as gemcitabine or S-1, may be a promising therapeutics for PDAC. Citation Format: Motohisa Tada, Hideaki Ijichi, Koji Miyabayashi, Yoshinari Asaoka, Dai Mohri, Tsuneo Ikenoue, Rintarou Mikata, Takeshi Ishihara, Fumihiko Kanai, Masao Omata, Harold L. Moses, Osamu Yokosuka. The novel strategy for treatment of pancreactic ductal adenocarcinoma targeting tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A69.

Abstract A39: The exploration of novel strategy for treatment of pancreactic ductal adenocarcinoma targeting tumor microenvironment with multi-kinase inhibitors

Abstract Background and Aim: Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease in humans. Previously, we have reported a genetically engineered mouse PDAC progression model which has pancreatic-specific transforming growth factor-beta receptor type II knockout in the context of Kras activation (Ijichi H, et al 2006). This model shows PDAC with 100% penetrance and recapitulates the signature of human PDAC well. Using this model, we explored novel treatment for PDAC. Materials and Methods: At first, to investigate whether the mice model is suitable for the drug screening, the mice were treated with gemcitabine (12.5 mg/kg) by intraperitoneal (i.p.) injection or S-1 (8.4 mg/kg) per oral, which are the standard drug for human PDAC. To the next, for single agent treatment, mice were treated orally 6 times a week with vehicle (0.5 % carboxymethyl cellulose, CMC), sunitinib (40 mg/kg), and axitinib (30mg/kg), both of which are multikinase inhibitors targeting vascular endothelial growth factor receptor (VEGFR) and platelet derived growth factor receptor (PDGFR) from the 3 weeks of age. For combined agent experiment, mice were treated orally with vehicle (0.5 % CMC) or axitinib from the 3 weeks of age and also treated with saline or gemcitabine, i.p. twice a week from the 4 weeks of age. Treatment continued until 8 weeks of age. Moreover, for the survival analysis, the drug treatment was continued until the mice became distressed according to the same schedule stated above. In vivo anti-tumor effect and survival time were assessed. Immunostaining of tumor tissue for caspase 3, Ki67, CD31, F4/80 and VEGF was performed. Azan staining also performed for the assessment of fibrosis in the tumor. Results: Gemcitabine and S-1 showed antiproliferative effect and prolonged overall survival of these mice compared to control, as well as human cases. Median survival time of single use of axtinib and sunitinib group was significantly longer (p <0.01) than that of control group. Axitinib and sunitinib group showed significantly stronger anti-tumor effect in vivo (p <0.01). In the combined treatment experiment, gemcitabine plus axitinib-treated group showed statistically significant longer survival and more anti-tumor effect than that of gemcitabine or axtinib alone-treated group (p <0.01). Axitinib and sunitinib group showed significantly higher caspase 3 stainng and lower Ki67 staining than that of control (p <0.01). Microvessel density (CD 31 staining) of axitinib and sunitinib group was significantly lower than that of control (p <0.01). F4/80 staining was significantly lower in axitinib and sunitinib-treated group than that of control (p <0.05). VEGF expression of axitinib and sunitinib group was significantly lower than that of control a (p <0.001). Azan staining showed significantly lower fibrosis in axitinib and sunitinib-treated group compared to control (p <0.01). Conclusion: Targeting not only cancer cells but also tumor microenvironment, such as angiogenesis, infiltration of immune cells, and fibrosis, with the use of multikinase inhibitors in addition to gemcitabine, may be a promising therapeutics for PDAC.

Concordant Promoter Methylation of Transforming Growth Factor-Beta Receptor Types I and II Occurs Early in Esophageal Squamous Cell Carcinoma

IntroductionTransforming growth factor-β (TGF-β) is a pleiotropic growth factor with multiple functions through its type I (TGFBR1) and type II (TGFBR2) receptors. A reduction or loss of expression of TGFBRs enables cancer cells to escape the growth inhibitory effect of TGF-β and to gain a growth advantage. ObjectiveThe promoter methylation status and expression of TGFBR1, TGFBR2 and Smad4 gene were investigated in esophageal squamous cell carcinoma (ESCC). DesignMethylation-specific polymerase chain reaction approach was used to detect the methylation status. Immunohistochemistry and reverse transcription-polymerase chain reaction method were used to examine the protein and messenger RNA expression, respectively. ResultsBoth the ESCC and the high-grade dysplastic tissues showed hypermethylation of TGFBR1 and TGFBR2, and the hyper-methylation of TGFBR1 and TGFBR2 in ESCC tissues was significantly associated with decreased messenger RNA and protein expression (P < 0.05). When stratified for tumor lymph node metastasis stages, TGFBR1 and TGFBR2 gene methylation was more frequent in stage III and stage IV tumor tissues than that in stage I and stage II tumor tissues (P < 0.05). Simultaneous methylation of the 2 receptors was progressively increased along with the increasing of tumor lymph node metastasis stage and decreasing of histological differentiation. Smad4 hypermethylation was only detected in 7 ESCC tumor tissues and associated with the loss of Smad4 expression. The decreased protein expression of TGFBR1, TGFBR2 and Smad4 was correlated with increased expression of TGF-β1 in ESCC. ConclusionsThese data suggest that promoter methylation of TGFBR1 and TGFBR2 may exist in the early stage of ESCC and play important roles in TGFBR1 and TGFBR2 gene silencing.

Abstract 4383: Targeting tumor microenvironment with gemcitabine is useful for the treatment of pancreatic ductal adenocarcinoma

Abstract (Background and Aim) Previously, we have reported a genetically engineered mouse pancreatic ductal adenocarcinoma (PDAC) progression model which has pancreatic-specific transforming growth factor-beta receptor type II knockout in the context of Kras activation. This model shows PDAC with 100 % penetrance and recapitulates the signature of human PDAC well. Using this model, we explored novel treatment for PDAC targeting tumor microenvironment. (Materials and Methods) To investigate whether the mice model is suitable for the drug screening, the mice were treated with gemcitabine, which is the standard drug for human PDAC, by i.p. injection. To the next, for single agent treatment, mice were treated with axitinib, which is a multi-kinase inhibitor, or candesartan, which is an anigiotensin II receptor blocker (ARB), respectively. For combined agent experiment, mice were treated with axitinib or candesartan and gemcitabine. Treatment continued until 8 weeks of age. Moreover, for the survival analysis, the drug treatment was continued until the mice became distressed according to the same schedule stated above. In vivo anti-tumor effect and survival time were assessed. Immunostaining of tumor tissue for caspase 3, Ki67, CD31, F4/80, α-SMA, and VEGF was performed. Azan staining also performed for the assessment of fibrosis in the tumor. (Results) Gemcitabine showed anti-proliferative effect and prolonged overall survival of these mice compared to control, as well as human cases. Median survival time of single use of axtinib group was significantly longer and that of candesartan group was tended to be longer than that of control group. Axitinib and candesartan group showed significantly stronger anti-tumor effect in vivo. In the combined treatment experiment, gemcitabine plus axitinib or candesartan group showed statistically significant longer survival and more anti-tumor effect than that of gemcitabine, axtinib or candesartan alone-treated group. Axitinib group showed significantly higher caspase 3 staining and lower Ki67 staining than that of control, however, candesartan group showed no change of these staining, compared to control. Microvessel density, F4/80, and α-SMA staining of axitinib and candesartan group was significantly lower than that of control. VEGF expression of candesartan group was significantly lower than that of control. Azan staining showed significantly lower fibrosis in axitinib and candesartan group compared to control. (Conclusion)Targeting tumor microenvironment, such as angiogenesis, immune cell infiltration and fibrosis, using multi-kinase inhibitor or ARB in addition to gemcitabine, may be a promising therapeutics for PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4383. doi:1538-7445.AM2012-4383

Annuloaortic ectasia in a 16 year-old boy with Loeys–Dietz syndrome

Loeys-Dietz syndrome (LDS) is a rare genetic disorder with an autosomal dominant inheritance due to mutations in the transforming growth factor beta-receptor type 1 or type 2. The disease is characterised by the triad of hypertelorism, bifid uvula or cleft palate, arterial tortuosity and aortic aneurysms. These phenotypic characteristics distinguish LDS from other connective tissue disorders related to transforming growth factor beta-receptor. Patients with LDS have a high risk of aortic dissection or rupture at a younger age and smaller aortic diameters. So, clinical suspicion of LDS followed by genotyping is important to prevent aortic dissection, leading cause of death, by surgical treatment.

Thymoma Associated With Loeys-dietz Syndrome Type I

Loeys-Dietz syndrome is a rare autosomal dominant disorder characterized by the involvement of cardiovascular, craniofacial and skeletal systems. The main etiology of the disease is the mutation in the transforming growth factor betareceptor type 1 and 2 genes. Today, Loeys-Dietz syndrome has been classified into two subtypes due to the presence of craniofacial involvement. These patients have progressive aortic enlargement which increases the risk of dissection and rupture. So, delay in the diagnosis may be associated with poor prognosis. We present a new case with thymoma diagnosed as Loeys-Dietz syndrome type 1. Also, this is the first report of a tumor in Loeys-Dietz syndrome according to the current literature review. (Marmara Medical Journal 2012;25:103-6)

A Case of Near Total Aortic Replacement in an Adolescent With Loeys-Dietz Syndrome

Loeys-Dietz syndrome is a recently described autosomal dominant disorder caused by mutations in the genes for transforming growth factor-beta receptor type 1 or 2 (TGF-ßR 1/2). The syndrome predisposes patients to aortic aneurysm and dissections, along with craniofacial and musculoskeletal abnormalities. Here we report the case of an adolescent who underwent serial near total aortic replacement, from the aortic valve to the descending aorta. Loeys-Dietz syndrome was confirmed in this case by the detection of a mutation in the TGF-ßR 2 gene.

Cloning and expression analysis of the transforming growth factor-beta receptors type 1 and 2 in the rainbow trout Oncorhynchus mykiss

Transforming growth factor-β (TGF-β) binding to the TGF-β type I (TGFBR1) and type II (TGFBR2) receptors delivers a plethora of cell-type specific effects. Moreover, the responses to TGF-β are tuned by regulatory mechanisms at the receptor level itself. To further elucidate TGF-β family signal transduction in teleosts, we therefore cloned the first complete set of a putative TGF-β receptor complex in salmonids. Rainbow trout TGFBR1 and TGFBR2 are transmembrane proteins with a serine/threonine kinase domain and are highly conserved within vertebrates. High expression levels in muscle and brain indicate regulation of the TGF-β system in muscular and nervous systems. Lipopolysaccharide (LPS) induced expression of both receptor chains in RTgill cells while bacterial and viral mimics modulated the two receptors inversely in head kidney (HK) macrophages. In addition, T cell mitogens lowered receptor levels in HK leukocytes. These data provide the first insights into TGF-β type I and II receptor modulation during immune responses in teleost fish.