Citations

Abstract Introduction: Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under TGF-β stimulation, hepatic stellate cells (HSCs), which are liver specific pericytes, transdifferentiate into myofibroblasts that promote tumor implantation and growth in the liver. The regulation of this HSC activation process in the liver however remains poorly understood. In this study, we tested a hypothesis that actin binding protein vasodilator-stimulated phosphoprotein (VASP), previously implicated in focal adhesion and migration of HSCs, may regulate the TGF-β mediated HSC activation process by interacting with and regulating TGF-β receptors. Methods: VASP shRNA and siRNA were used to knockdown VASP in primary human HSCs and LX2, an immortalized HSC cell line. TGF-β activation of HSCs was quantitated by immunofluorescence staining (IF) for alpha-smooth muscle actin (α-SMA) and Western blot analysis (WB) for α-SMA, fibronectin and P-SMAD2. Total TGF-β receptor levels were determined by WB, and plasma membrane TβRII was quantitated by biotinylation of cell surface proteins approach. Protein subcellular localization was investigated by IF and protein interactions were determined by double IF and co-immunoprecipitation (IP). Regulation of stellate cell VASP by HSC/tumor interactions was investigated by IF on human colorectal liver metastases and the role of stellate cell VASP in tumor growth was studied in a tumor/HSC coimplantation mouse model. Results: TGF-β1 induced more than 60% of human primary HSCs into myofibroblasts as determined by α-SMA IF. Two different VASP shRNAs significantly reduced this effect of TGF-β1 on HSC activation (P<0.05). The effect of VASP knockdown on HSC activation was confirmed in LX2 cells as well as using a VASP siRNA approach. VASP knockdown increased total TGF receptor protein levels with no effects on SMAD2 and 3 protein levels. VASP interacted with TβRII as evidenced by double IF and co-IP. Biotin-labeling of cell surface proteins and IF revealed that VASP knockdown reduced TβRII protein level at the plasma membrane and that VASP overexpression increased TβRII at the plasma membrane. Concurrently, VASP knockdown reduced plasma membrane localization of Rab11a, a small GTPase that marks recycling endosomes and is essential for recycling of plasma membrane receptors. VASP/Rab11 interactions were confirmed by IF and co-IP. Furthermore, we found that VASP knockdown HSCs significantly reduced tumor growth in mice as compared to control HSCs in a HSC/tumor coimplantation model. In patients, metastasis of colorectal cancer cells in liver sinusoids induced upregulation of VASP and α-SMA in adjacent HSCs as revealed by IF on biopsies of human colorectal liver metastases. Conclusions: Our studies demonstrate a requirement of VASP in the TGF-β dependent HSC activation in the tumor microenvironment and ensuing tumor growth. VASP interacts with Rab11 and regulates Rab11 positive recycling endosomal function thereby promoting TβRII recycling back to the plasma membrane in HSCs. VASP in the tumor microenvironment thus presents a therapeutic target for reducing tumor implantation and metastatic growth in the liver. Citation Format: Kangsheng Tu, Jiachu Li, Chunsheng Liu, Vijay H. Shah, Ningling Kang. Vasodilator-stimulated phosphoprotein promotes TGF-beta mediated myofibroblastic activation by regulating recycling of TGF-beta receptor II to the plasma membrane. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A70. doi:10.1158/1538-7445.CHTME14-A70

Platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) signaling are required for hepatic stellate cell (HSC) activation under pathological conditions such as liver metastatic tumor growth. These two signaling pathways are functionally divergent; PDGF signaling promotes proliferation and migration of HSCs, and TGF-β induces transdifferentiation of quiescent HSCs into myofibroblasts. Although PDGF signaling is implicated in TGF-β-mediated epithelial mesenchymal transition of tumor cells, the role of PDGF receptors in TGF-β activation of HSCs has not been investigated. Here we report that PDGF receptor-α (PDGFR-α) is required for TGF-β signaling of cultured human HSCs although HSCs express both PDGF-α and -β receptors. PDGFR-α knockdown inhibits TGF-β-induced phosphorylation and nuclear accumulation of SMAD2 with no influence on AKT or ERK phosphorylation associated with noncanonical TGF-β signaling. PDGFR-α knockdown suppresses TGF-β receptor I (TβRI) but increases TβRII gene transcription. At the protein level, PDGFR-α is recruited to TβRI/TβRII complexes by TGF-β stimulation. PDGFR-α knockdown blocks TGF-β-mediated internalization of TβRII and induces accumulation of TβRII at the plasma membrane, thereby inhibiting TGF-β phosphorylation of SMAD2. Functionally, knockdown of PDGFR-α reduces paracrine effects of HSCs on colorectal cancer cell proliferation and migration in vitro. In mice and patients, colorectal cancer cell invasion of the liver induces upregulation of PDGFR-α of HSCs. In summary, our finding that PDGFR-α knockdown inhibits SMAD-dependent TGF-β signaling by repressing TβRI transcriptionally and blocking endocytosis of TGF-β receptors highlights a convergence of PDGF and TGF-β signaling for HSC activation and PDGFR-α as a therapeutic target for liver metastasis and other settings of HSC activation.

Abstract Introduction: Transforming growth factor beta (TGF-β) within the tumor microenvironment induces transdifferentiation of quiescent pericytes and related stromal cells into myofibroblasts that promote tumor growth. However mechanisms governing TGF-β mediated myofibroblastic activation remain poorly understood. IQ motif containing GTPase activating protein 1 (IQGAP1) exerts diverse cellular functions, but its role in the tumor microenvironment is unexplored. In this study, we tested a hypothesis that IQGAP1 may interact with TGF-β receptor II (TβRII) and regulate its signaling in mesenchymal-type cells that activate into tumor associated myofibroblasts such as hepatic stellate cells (HSCs), which are resident liver pericytes, and that IQGAP1 of HSCs regulates liver metastatic growth. Experimental procedures: Retroviral and lentiviral based vectors were used to deliver TβRII, IQGAP1 or IQGAP1 shRNA into human primary HSCs. IQGAP1/TβRII interactions were determined by immunofluorescence staining, immunoprecipitation, in vitro GST pull down and in vitro protein binding assays. TGF-β1 activation of HSCs into myofibroblasts was determined by α-SMA immunofluorescence and Western Blot analyses for α-SMA, fibronectin and P-Smad2. The role of stellate cell IQGAP1 in tumorigenesis was assessed by in vitro assays of conditioned media, a HSC/tumor cell co-implantation mouse model and experimental liver metastasis model performed on IQGAP1 knockout mice. Additionally, IQGAP1 protein levels in the myofibroblasts of 29 human colorectal liver metastases were investigated by immunofluorescence staining. Results: IQGAP1 is recruited to TβRII by TGF-β1 with the C-terminal a.a. 1503-1657 region of IQGAP1 mediating IQGAP1/TβRII binding. Through scaffolding the E3 ubiquitin ligase Smurf1 and TβRII, IQGAP1 promotes ubiquitination, lysosomal and proteasomal degradation of TβRII, thus suppressing TβRII and TGF-β1 activation of HSCs into myofibroblasts in vitro. In mice, co-implantation of IQGAP1 knockdown HSCs promotes myofibroblastic activation of HSCs, tumor implantation and growth. In an experimental liver metastasis model, IQGAP1 deficiency in the tumor microenvironment promotes myofibroblastic activation and liver metastatic growth. Indeed, IQGAP1 knockdown in HSCs upregulates paracrine signaling molecules such as stromal derived factor 1 (SDF-1/CXCL12) and hepatic growth factor (HGF) that confer a stimulatory effect on proliferation, migration and survival of tumor cells. Additionally, we found that 24 out of 29 colorectal cancer patients display varying degrees of reduction of IQGAP1 protein in the myofibroblasts of their liver metastases as compared to IQGAP1 expression levels observed in activated HSCs and portal myofibroblasts of the adjacent non-tumorous control liver. Conclusion: Our studies demonstrate that IQGAP1 in the tumor microenvironment suppresses TβRII and the TGFβ1 dependent myofibroblastic activation switch with these events constraining tumor growth and metastasis. Citation Format: Chunsheng Liu, Haitham Abdelhakim, Vijay H. Shah, Ningling Kang. IQGAP1 in the tumor microenvironment suppresses TGF-beta mediated myofibroblastic activation and ensuing tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B19.

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF‐β) stimulation, hepatic stellate cells (HSCs), which are liver‐specific pericytes, transdifferentiate into tumor‐associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator‐stimulated phosphoprotein (VASP) of HSCs regulated the TGF‐β‐mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up‐regulation of VASP and alpha‐smooth muscle actin (α‐SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF‐β‐mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF‐β receptor II (TβRII) and Rab11, a Ras‐like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11‐dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF‐β1 stimulation. Conclusions: Our study demonstrates a requirement of VASP for TGF‐β‐mediated HSC activation in the tumor microenvironment by regulating Rab11‐dependent recycling of TβRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver. (Hepatology 2015;61:361–374)

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1.

Abstract Purpose: Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. Experimental Design: 3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and clonogenic assays were used to test cytoxicity of the combination treatment in vivo. To examine the mechanism of synergy, one-step viral growth curves, terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assays, and Western blot analyses were performed. In vivo assessment of synergistic antitumor activity was conducted in a U87 glioma model. Results: MTS and clonogenic assays showed a strong synergistic interaction between MV-CEA and RT in glioblastoma multiforme cells including both primary and established glioma lines. Furthermore, significant antitumor efficacy was observed in vivo in a subcuteneous U87 xenograph model. There was significant prolongation of survival (P = 0.001) in the combination treatment group as compared with single modality– or control-treated animals. One-step viral growth curves showed increased viral burst size by up to 2 log in MV/RT combination–treated cells, as compared with single agent MV-CEA–treated glioma cells. Changes in CEA levels and expression of viral N and H protein were also consistent with increased viral production. Furthermore, TUNEL assays and Western blot analysis showed increase in apoptosis in MV/RT combination–treated cells. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected glioma cells from MV-CEA/RT–induced cleavage of poly(ADP-ribose) polymerase (PARP), indicating that the apoptotic death in combination-treated cells is mostly mediated via the extrinsic caspase pathway. The Fas/Fas ligand interaction blocking antibody NOK-1 blocked MV/RT–induced PARP cleavage whereas the Fas agonistic antibody CH11 increased PARP cleavage in MV/RT combination–treated cells. Reverse transcription-PCR, fluorescence-activated cell sorting analysis and immunohistochemistry showed up-regulation of Fas in combination-treated tumor in vitro and in vivo cells. Conclusions: There is synergy between MV-CEA and RT in vitro and in vivo. The synergistic effect of the combination seems to be due to increase in viral burst size and increase in apoptotic cell death. This latter effect is mostly mediated via the extrinsic caspase-8 pathway, activated via increased signaling through the Fas death receptor pathway. These results could have translational implications in glioma therapy.

Recent experimental studies suggest C-reactive protein (CRP) may be a potential mediator of atherosclerosis and its complications. However, there is growing criticism of in vitro CRP studies that use commercial CRP preparations containing biologically active contaminants. The effects of commercial CRP, dialyzed commercial CRP (dCRP) to remove azide, and sodium azide (NaN 3 ) alone at equivalent concentrations to the undialyzed preparation were tested at varying concentrations on human umbilical vein endothelial cells (HUVEC), circulating endothelial outgrowth cells (EOC), and endothelial progenitor cells (EPC) in vitro. CRP and NaN 3 alone exhibited equivalent concentration-dependent, proapoptotic effects on HUVEC, EOC, and EPC ( P <0.01 versus control), whereas dCRP had no such effect. Similarly, CRP and NaN 3 alone caused equivalent concentration-dependent decreases in migration, proliferation, and matrigel tube formation ( P <0.01 versus control) in EOC and HUVEC, whereas dCRP had absolutely no effect on these biological functions at any of the concentrations used. We conclude that proapoptotic, antiproliferative, antimigratory, and antiangiogenic effects of this commercial CRP preparation on a number of endothelial cell phenotypes in culture may be explained by the presence of sodium azide in this preparation. This study has implications for interpretation of in vitro studies using CRP preparations containing azide at equivalent or higher concentrations.