Shah, Vijay H.

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.

Chronic passive hepatic congestion (congestive hepatopathy) leads to hepatic fibrosis; however, the mechanisms involved in this process are not well understood. We developed a murine experimental model of congestive hepatopathy through partial ligation of the inferior vena cava (pIVCL). C57BL/6 and transgenic mice overexpressing tissue factor pathway inhibitor (SM22α‐TFPI) were subjected to pIVCL or sham. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, real‐time polymerase chain reaction, and western blot assays. Hepatic fibrosis and portal pressure were significantly increased after pIVCL concurrent with hepatic stellate cell (HSC) activation. Liver stiffness, as assessed by magnetic resonance elastography, correlated with portal pressure and preceded fibrosis in our model. Hepatic sinusoidal thrombosis as evidenced by fibrin deposition was demonstrated both in mice after pIVCL as well as in humans with congestive hepatopathy. Warfarin treatment and TFPI overexpression both had a protective effect on fibrosis development and HSC activation after pIVCL. In vitro studies show that congestion stimulates HSC fibronectin (FN) fibril assembly through direct effects of thrombi as well as by virtue of mechanical strain. Pretreatment with either Mab13 or Cytochalasin‐D, to inhibit β‐integrin or actin polymerization, respectively, significantly reduced fibrin and stretch‐induced FN fibril assembly. Conclusion : Chronic hepatic congestion leads to sinusoidal thrombosis and strain, which in turn promote hepatic fibrosis. These studies mechanistically link congestive hepatopathy to hepatic fibrosis. (H epatology 2015;61:648‐659)

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.

Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl−/−; or Yes, Src, and Fyn knockout mice (YSF−/−)] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl−/− MEF showed impaired matrix endocytosis, YSF−/− MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9.

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)

Significance Tumor cells adapt to hypoxia by modulating energy production and utilization. Endocytosis is an energy-consuming process that is suppressed during hypoxia. Our studies show that Dynamin 2 (DNM2), a key component of endocytic machinery, is transcriptionally suppressed by HIF-1. Genetic and pharmacological inactivation of DNM2 stabilized HIF-1α and HIF-2α. Furthermore, miR-199a, which is encoded from the opposite strand of DNM2 , exerts reciprocal negative regulation upon HIF-1α and HIF-2α. Overexpression of miR-199a decreased HIF-1α and HIF-2α, cell migration, and metastasis. These findings establish a regulatory loop between endocytic pathway and hypoxic response in tumor cells.

Hepatic stellate cells (HSC) and liver endothelial cells (LEC) migrate to sites of injury and perpetuate alcohol-induced liver injury. High-mobility group box 1 (HMGB1) is a protein released from the nucleus of injured cells that has been implicated as a proinflammatory mediator. We hypothesized that HMGB1 may be released from ethanol-stimulated liver parenchymal cells and contribute to HSC and LEC recruitment. Ethanol stimulation of rat hepatocytes and HepG2 cells resulted in translocation of HMGB1 from the nucleus as assessed by Western blot. HMGB1 protein levels were increased in the supernatant of ethanol-treated hepatocytes compared with vehicle-treated cells. Migration of both HSC and LEC was increased in response to conditioned medium for ethanol-stimulated hepatocytes (CMEtOH) compared with vehicle-stimulated hepatocytes (CMVEH) ( P < 0.05). However, the effect of CMEtOHon migration was almost entirely reversed by treatment with HMGB1-neutralizing antibody or when HepG2 cells were pretransfected with HMGB1-siRNA compared with control siRNA-transfected HepG2 cells ( P < 0.05). Recombinant HMGB1 (100 ng/ml) also stimulated migration of HSC and LEC compared with vehicle stimulation ( P < 0.05 for both HSC and LEC). HMGB1 stimulation of HSC increased the phosphorylation of Src and Erk and HMGB1-induced HSC migration was blocked by the Src inhibitor PP2 and the Erk inhibitor U0126. Hepatocytes release HMGB1 in response to ethanol with subsequent recruitment of HSC and LEC. This pathway has implications for HSC and LEC recruitment to sites of ethanol-induced liver injury.