Kang, Ningling

Background and Aims: Transforming growth factor-beta 1 (TGFβ1) induces HSC activation into metastasis-promoting cancer-associated fibroblasts (CAFs), but how the process is fueled remains incompletely understood. We studied metabolic reprogramming induced by TGFβ1 in HSCs. Approaches and Results: Activation of cultured primary human HSCs was assessed by the expression of myofibroblast markers. Glucose transporter 1 (Glut1) of murine HSC was disrupted by Cre recombinase/LoxP sequence derived from bacteriophage P1 recombination (Cre/LoxP). Plasma membrane (PM) Glut1 and glycolysis were studied by biotinylation assay and the Angilent Seahorse XFe96 Analyzer. S.c. HSC/tumor co-implantation and portal vein injection of MC38 colorectal cancer cells into HSC-specific Glut1 knockout mice were performed to determine in vivo relevance. Transcriptome was obtained by RNA sequencing of HSCs and spatialomics with MC38 liver metastases. TGFβ1-induced CAF activation of HSCs was accompanied by elevation of PM Glut1, glucose uptake, and glycolysis. Targeting Glut1 or Src by short hairpin RNA, pharmacologic inhibition, or a Src SH3 domain deletion mutant abrogated TGFβ1-stimulated PM accumulation of Glut1, glycolysis, and CAF activation. Mechanistically, binding of the Src SH3 domain to SH3 domain–binding protein 5 led to a Src/SH3 domain–binding protein 5/Rab11/Glut1 complex that activated Rab11-dependent Glut1 PM transport under TGFβ1 stimulation. Deleting the Src SH3 domain or targeting Glut1 of HSCs by short hairpin RNA or Cre recombinase/LoxP sequence derived from bacteriophage P1 recombination suppressed CAF activation in mice and MC38 colorectal liver metastasis. Multi-omics revealed that Glut1 deficiency in HSCs/CAFs suppressed HSC expression of tumor-promoting factors and altered MC38 transcriptome, contributing to reduced MC38 liver metastases. Conclusion: The Src SH3 domain–facilitated metabolic reprogramming induced by TGFβ1 represents a target to inhibit CAF activation and the pro-metastatic liver microenvironment.

Despite the importance of the vasculature during tumorigenesis and during antiangiogenesis treatment, there are few methods available to visualize conveniently the three‐dimensional microvascular structure of tumors. Micro‐computed tomography provides a means to visualize conveniently and quantitatively the three‐dimensional tumor microvascular architecture during tumorigenesis and treatment. In the present study, micro‐computed tomography was utilized to visualize the vasculature of hepatic tumors in SV‐40 transgenic rats. The rats were anesthetized and the vasculature of the livers was infused via an aortic cannula, first with heparinized 0.9% saline, then with 10% buffered formalin and finally with radiopaque silicone rubber (Microfil 122). The livers were first scanned by micro‐CT to visualize the microvasculature and then prepared for histology to visualize the parenchyma. At 10 months of age, all transgenic rats developed gross tumors in their livers. Histological examination revealed that the malignant hepatic lesions in the livers displayed mixed structures resembling hepatoblastoma, cholangiocellular or hepatocellular differentiation in human. Various sized vessels were visible extending to the tumors. Three‐dimensional micro‐computed tomography showed that those vessels were disorganized and tortuous with atypical branching patterns. Often vascular “lakes” were found in the mass of the large tumors. These results show the utility of micro‐computed tomography for studying tumor vasculogenesis. Supported in part by NIH grant EB 000305.

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor β (TGF‐β) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis‐promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain‐containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS‐containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF‐β1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF‐β1‐mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF‐β1‐induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF‐β1‐mediated HSC activation and transcription of genes encoding tumor‐promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet‐derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation‐real‐time quantitative PCR showed that canonical p300‐mediated acetylation of histones also facilitated transcription in response to TGF‐β1 stimulation. Interestingly, although both TGF‐β1‐mediated and stiffness‐mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF‐β1 were distinct from those of stiffness‐p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated‐HSC/myofibroblasts by C646, short hairpin RNA, or cre ‐mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF‐β1‐stimulated HSC activation by both noncanonical (cytoplasm‐to‐nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.

Abstract Introduction: Liver metastasis of pancreatic or colorectal tumors is a major cause of cancer-related death worldwide. When cancer cells reach the liver parenchyma and make a direct contact with the surrounding extracellular matrix (ECM), β1-integrin-FAK signaling is activated allowing the cells attach, migration, and proliferate. Thus, the β1-integrin-FAK signaling axis is key for cancer cells to colonize the liver. However, mechanisms governing β1-integrin activation and how this pathway promotes liver metastasis remain incompletely understood. Aim: We sought to test the hypothesis that vasodilator-stimulated phosphoprotein (VASP), an actin binding protein, regulates the β1-integrin-FAK signaling axis, thereby, promoting liver metastasis of GI cancer. Methods: We employed lentiviruses encoding different VASP shRNAs to knockdown (KD) VASP in human GI cancer cells. We used a Matrigel-based 3-dimensional (3D) cell culture model and intrasplenic tumor injection mouse model to assess the role of VASP for cancer cells in vitro and in vivo. Immunofluorescence (IF) was used to study localization of VASP and active β1-integrin (using Huts-4 antibody), and Western blot analysis (WB) was used to quantitate protein levels of active β1-integrin and YAP1/TAZ. Additionally, we performed VASP immunohistochemistry (IHC) to analyze VASP immunoreactivity in 116 colorectal or pancreatic adenocarcinomas of patients. Results: In the Matrigel-based 3D-culture model, a single cancer cell gave arise to a 3D-cancer spheroid after being cultured for 6-8 days. Using KM12L4, HCT116, or HT29 colorectal cancer cells, and L3.6 or MIA PaCa-1 pancreatic cancer cells, we found that VASP KD reduced the sizes of the 3D-cancer spheroids grown on Matrigel (P<0.05 by t-test for each cell line). IF and WB show that VASP KD reduced levels of Huts-4 (active β1-integrin) in KM12L4, HCT116 colorectal cancer cells and in L3.6, MIA PaCa-1 pancreatic cancer cells. VASP KD also reduced the protein levels of P-FAK and YAP1/TAZ in the 4 cell lines tested. Additionally, YAP1/TAZ protein levels were reduced by PF-228, a FAK inhibitor that blocks the β1-integrin-FAK signaling. In the intrasplenic tumor implanation mouse model, VASP KD suppressed liver metastasis of GI cancer as well as reduced Huts-4 and YAP1 signals of their liver metastases in mice (P<0.05 by t-test). IHC of patient's samples reveals that VASP protein levels were upregulated in cancer cells in 81% of colorectal tumors (51/63) and pancreatic tumors (43/53), with higher VASP levels being associated with liver metastasis of colorectal cancer (P<0.01 by Fisher exact test). Conclusions: VASP promotes ECM-mediated β1-integrin-FAK-YAP1/TAZ signaling axis of GI cancer. We propose that VASP and the β1-integrin-FAK-YAP1/TAZ signaling axis represent potential new therapeutic targets for liver metastasis of pancreatic or colorectal cancer. Citation Format: Xiaoyu Xiang, Jinhua Piao, Selvaraj Muthusamy, Lei Wang, Yibin Deng, Shengbing Huang, Raul Urrutia, Jinping Lai, Ningling Kang. Vasodilator-stimulated phosphoprotein promotes β1-integrin-FAK-YAP1/TAZ signaling axis that is required for liver metastasis of GI cancer. [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 1633.

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

Abstract Cancer-associated fibroblasts (CAFs), the most abundant cells in the tumor microenvironment (TME), are a key source of the extracellular matrix (ECM) that constitutes the desmoplastic stroma. Through remodeling of the reactive tumor stroma and paracrine actions, CAFs regulate cancer initiation, progression, and metastasis, as well as tumor resistance to therapies. The CAFs found in stroma-rich primary hepatocellular carcinomas (HCC) and liver metastases of primary cancers of other organs predominantly originate from hepatic stellate cells (HSTC), which are pericytes associated with hepatic sinusoids. During tumor invasion, HSTCs transdifferentiate into myofibroblasts in response to paracrine signals emanating from either tumor cells or a heterogeneous cell population within the hepatic tumor microenvironment. Mechanistically, HSTC-to-myofibroblast transdifferentiation, also known as, HSTC activation, requires cell surface receptor activation, intracellular signal transduction, gene transcription, and epigenetic signals, which combined ultimately modulate distinct gene expression profiles that give rise to and maintain a new phenotype. The current review defines a paradigm that explains how HSTCs are activated into CAFs to promote liver metastasis. Furthermore, a focus on the most relevant intracellular signaling networks and epigenetic mechanisms that control HSTC activation is provided. Finally, we discuss the feasibility of targeting CAF/activated HSTCs, in isolation or in conjunction with targeting cancer cells, which constitutes a promising and viable therapeutic approach for the treatment of primary stroma-rich liver cancers and liver metastasis. Mol Cancer Res; 13(4); 604–12. ©2014 AACR.

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.