Zong-Tao Chai

MicroRNA-26a Inhibits Angiogenesis by Down-Regulating VEGFA through the PIK3C2α/Akt/HIF-1α Pathway in Hepatocellular Carcinoma

Background & Aims microRNAs (miRNAs) have been reported to regulate angiogenesis by down-regulating the expression of pro-angiogenic or anti-angiogenic factors. The aims of this study were to investigate whether miR-26a inhibited angiogenesis by down-regulating vascular endothelial growth factor A (VEGFA) and its clinical relevance in hepatocellular carcinoma (HCC). Methods The expression of miR-26a was modified in HepG2 and HCCLM3 cell lines respectively, and a panel of angiogenic factors was measured by real-time PCR in the cells. A luciferase reporter assay was used to validate the target gene of miR-26a. Specific inhibitors of signal transduction pathway and siRNA approaches were used to explore the regulatory mechanism of miR-26a. Migration and tube forming assays were conducted to show the changes of angiogenesis induced by miR-26a and its target genes. Finally animal studies were used to further validate those findings. Results Ectopic expression of miR-26a exhibited decreased levels of VEGFA in HepG2 cells. Migration and tube forming of human umbilical vein endothelial cells (HUVECs) were decreased in the conditioned medium from ectopic expression of miR-26a in HepG2 cells compared to control HepG2 cells. The pro-angiogenic effects of the conditioned medium of HepG2 cells on HUVECs were specifically decreased by LY294002, YC-1, and bevacizumab. Integrated analysis disclosed PIK3C2α as a downstream target gene of miR-26a. Ectopic expression of miR-26a suppressed ectopic and orthotopic tumor growth and vascularity in nude mice. The results in HCCLM3 were consistent with those in HepG2. miR-26a expression was inversely correlated with VEGFA expression in HCC patients. Conclusions miR-26a modulated angiogenesis of HCC through the PIK3C2α/Akt/HIF-1α/VEGFA pathway. The expression of VEGFA was inversely correlated with miR-26a expression in HCC tumors.

MiR-146a enhances angiogenic activity of endothelial cells in hepatocellular carcinoma by promoting PDGFRA expression

Endothelial cells (ECs) are critical for angiogenesis, and microRNAs play important roles in this process. We investigated the regulatory role of microRNAs in ECs of hepatocellular carcinoma (HCC) by examining the microRNA expression profile of human umbilical vein endothelial cells (HUVECs) in the absence or presence of human HCC cells, and identified miR-146a as the most highly upregulated microRNA. Furthermore, we revealed that miR-146a promoted the expression of platelet-derived growth factor receptor α (PDGFRA) in HUVECs, and this process was mediated by BRCA1. Overexpression of PDGFRA in the ECs of HCC tissues was associated with microvascular invasion and predicted a poorer prognosis. These results suggest that miR-146a plays a key role in regulating the angiogenic activity of ECs in HCC through miR-146a-BRCA1-PDGFRA pathway. MiR-146a and PDGFRA may emerge as potential anti-angiogenic targets on ECs for HCC therapy.

microRNA-26a suppresses recruitment of macrophages by down-regulating macrophage colony-stimulating factor expression through the PI3K/Akt pathway in hepatocellular carcinoma.

BackgroundmicroRNAs (miRNAs) have been reported to modulate macrophage colony-stimulating factor (M-CSF) and macrophages. The aim of this study was to find whether miR-26a can suppress M-CSF expression and the recruitment of macrophages.MethodsHepatocellular carcinoma (HCC) cell lines with decreased or increased expression of miR-26a were established in a previous study. M-CSF expression by tumor cells was measured by enzyme-linked immunosorbent assay, and cell migration assays were used to explore the effect of HCC cell lines on macrophage recruitment in vitro. Real-time PCR measured a panel of mRNAs expressed by macrophages. Xenograft models were used to observe tumor growth. Immunohistochemistry was conducted to study the relation between miR-26a expression and M-CSF expression and macrophage recruitment in patients with HCC.ResultsEctopic expression of miR-26a reduced expression of M-CSF. The conditioned medium (CM) from HepG2 cells that overexpressed miR-26a reduced the migration ability of THP-1 cells stimulated by phorbol myristate acetate (PMA) increased expression of interleukin (IL)-12b or IL-23 mRNA and decreased expression of chemokine (C-C motif) ligand (CCL)22, CCL17, and IL-10 mRNA, in comparison to the medium from the parental HepG2 cells. These effects could be interrupted by the PI3K/Akt pathway inhibitor LY294002. Ectopic expression of miR-26a in HCC cells suppressed tumor growth, M-CSF expression, and infiltration of macrophages in tumors. Similar results were also found when using HCCLM3 cells. Furthermore, the expression of miR-26a was inversely correlated with M-CSF expression and macrophage infiltration in tumor tissues from patients with HCC.ConclusionsmiR-26a expression reduced M-CSF expression and recruitment of macrophages in HCC.

Antiangiogenic therapy promoted metastasis of hepatocellular carcinoma by suppressing host-derived interleukin-12b in mouse models

Antiangiogenic therapy, specially sorafenib, has become the standard of care for patients with advanced hepatocellular carcinoma (HCC), however, the improvement in survival time is not satisfactory. Previous studies have found that, in some circumstances, antiangiogenic therapy promoted tumor metastasis and the mechanistic studies were mainly focus on cancer-cell-autonomous manners. In two experimental metastasis models with tail-vein injection with hepatoma cells and an orthotopic HCC mouse model, we found that pretreatment with two vascular endothelial growth factor receptor (VEGFR) inhibitors, sunitinib and sorafenib, facilitated tumor cell survival in blood stream and promoted lung metastasis from tumors that were subsequently incubated after drug discontinuation, indicating that host response joined into the pro-metastatic effects. An antibody microarray identified that interleukin (IL)-12b was decreased in the peripheral blood of the mice treated with the two VEGFR inhibitors. IL-12b suppression in macrophages and dendritic cells from host organs was found to play a crucial role in treatment-induced metastasis. Supplement with recombinant mouse IL-12b or restoration of IL-12b expression in the host by zoledronic acid, which was previously reported to enhance IL-12 expression in vitro and in vivo, alleviated the metastasis-promoting effects of sunitinib and sorafenib. These studies suggest that host response to VEGFR inhibitors facilitates HCC metastasis and restoration of IL-12b expression could translate into clinical benefits.

Colony-Stimulating Factor 1 Receptor Blockade Inhibits Tumor Growth by Altering the Polarization of Tumor-Associated Macrophages in Hepatocellular Carcinoma

Colony-stimulating factor-1 (CSF-1) and its receptor, CSF-1R, regulate the differentiation and function of macrophages and play an important role in macrophage infiltration in the context of hepatocellular carcinoma. The therapeutic effects of CSF-1R blockade in hepatocellular carcinoma remain unclear. In this study, we found that CSF-1R blockade by PLX3397, a competitive inhibitor with high specificity for CSF-1R tyrosine kinase, significantly delayed tumor growth in mouse models. PLX3397 inhibited the proliferation of macrophages in vitro, but intratumoral macrophage infiltration was not decreased by PLX3397 in vivo. Gene expression profiling of tumor-associated macrophages (TAM) showed that TAMs from the PLX3397-treated tumors were polarized toward an M1-like phenotype compared with those from vehicle-treated tumors. In addition, PLX3397 treatment increased CD8+ T-cell infiltration, whereas CD4+ T-cell infiltration was decreased. Further study revealed that tumor cell–derived CSF-2 protected TAMs from being depleted by PLX3397. In conclusion, CSF-1R blockade delayed tumor growth by shifting the polarization rather than the depletion of TAMs. CSF-1R blockade warrants further investigation in the treatment of hepatocellular carcinoma. Mol Cancer Ther; 16(8); 1544–54. ©2017 AACR.

Robo1 promotes angiogenesis in hepatocellular carcinoma through the Rho family of guanosine triphosphatases’ signaling pathway

Robo1 is a member of the Robo immunoglobulin superfamily of proteins, and it plays an important role in angiogenesis and cancer. In this study, we investigate the role of roundabout 1 (Robo1) in tumor angiogenesis in hepatocellular carcinoma (HCC). Firstly, the relationship between Robo1 expression on tumors and patient’s survival and endothelial cells in tumor blood vessels and patient’s survival was studied. Secondly, Robo1 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). Cell proliferation, motility, and tube formation were compared in HUVEC with different Robo1 expression. Also, HUVECs with different Robo1 expression were mixed with HCCLM3 and HepG2 hepatoma cells and then implanted in a nude mouse model to examine the effects of Robo1 in endothelial cells on tumor growth and angiogenesis. Cell motility-related molecules were studied to investigate the potential mechanism how Robo1 promoted tumor angiogenesis in HCC. The disease-free survival of the patients with high Robo1 expression in tumoral endothelial cells was significantly shorter than that of those with low expression (P = 0.021). Overexpression of Robo1 in HUVECs resulted in increased proliferation, motility, and tube formation in vitro. In the implanted mixture of tumor cells and HUVECs with an increased Robo1 expression, tumor growth and microvessel density were enhanced compared with controls. Robo1 promoted cell division cycle 42 (Cdc42) expression in HUVECs, and a distorted actin cytoskeleton in HUVECs was observed when Robo1 expression was suppressed. In conclusion, Robo1 promoted angiogenesis in HCC mediated by Cdc42.

Reduced expression of CD109 in tumor-associated endothelial cells promotes tumor progression by paracrine interleukin-8 in hepatocellular carcinoma

Tumor-associated endothelial cells (TEC) directly facilitate tumor progression, but little is known about the mechanisms. We investigated the function of CD109 in TEC and its clinical significance in hepatocellular carcinoma (HCC). The correlation between CD109 expressed on tumor vessels and the prognosis after surgical resection of HCC was studied. The effect of human umbilical vein endothelial cells (HUVEC) with different CD109 expression on hepatoma cell proliferation, migration, and invasion was compared in co-culture assay. Associated key factors were screened by human cytokine antibody array and validated thereafter. HUVEC with different CD109 expression were co-implanted with HCCLM3 or HepG2 cells in nude mice to investigate the effect of CD109 expression on tumor growth and metastasis. Reduced expression of CD109 on tumor vessels was associated with large tumor size, microvascular invasion, and advanced tumor stage. CD109 was an independent risk factor for disease-free survival (P = 0.001) after curative resection of HCC. CD109 knockdown in HUVEC promoted hepatoma cell proliferation, migration, and invasion. Interleukin-8 (IL-8) was a key tumor-promoting factor secreted from CD109 knockdown HUVEC. CD109 knockdown upregulated IL-8 expression through activation of TGF-β/Akt/NF-κB pathway in HUVEC. Co-implantation with CD109 knockdown HUVEC accelerated tumor growth and metastasis in mice models. In conclusion, CD109 expression on tumor vessels is a potential prognostic marker for HCC, and its reduced expression on TEC promoted tumor progression by paracrine IL-8.

Insufficient Radiofrequency Ablation Treated Hepatocellular Carcinoma Cells Promote Metastasis by Up-Regulation ITGB3

Radiofrequency ablation (RFA) is one of the standards of care for early stage hepatocellular carcinoma (HCC). However, rapid progression of residual tumor after RFA has been confirmed. The aim of this study was to investigate the underlying mechanism of this phenomenon. Human HCC cell lines HCCLM3 and HepG2 were employed to establish insufficient RFA models in vivo and in vitro, respectively. The effects of insufficient RFA on metastatic potential of residual tumors were evaluated. The molecular changes after insufficient RFA were evaluated by PCR array, western blot, immunofluorescence, and immunohistochemistry. Results showed that insufficient RFA significantly promoted lung and intrahepatic residual tumor cells in vivo, and heat intervention promoted migration and invasion of hepatoma cells in vitro. PCR array revealed that the expression of integrin β3 (ITGB3) and MMP2 were up-regulated in the residual tumors of HCCLM3 xenograft model. The up-regulation of ITGB3 was confirmed by qRT-PCR, Western blot and immunohistochemistry. Knockdown ITGB3 expression in HCCLM3 cells by shRNA significantly lowered the pro-metastatic effects of insufficient RFA. Mechanism studies indicated that ITGB3 mediated the expression of MMP2 by activing FAK/PI3K/AKT signaling pathway. The up-regulation of ITGB3 contributed to enhanced metastatic potential of residual cancer in HCCLM3 model after insufficient RFA. Targeting ITGB3 expression may further improve the clinical effects of RFA.

Abstract 4168: Robo1 promotes angiogenesis through CDC42/Rho GTPases signaling pathway in hepatocellular carcinoma

Angiogenesis is essential for cancer growth and metastasis. Since present anti-angiogenesis strategies could not fulfill the expected clinical efficacy in hepatocellular carcinoma (HCC), novel key regulatory molecules involving this process are still under investigation. It has been reported that the axon guidance receptor gene Robo1 is involved in tumor angiogenesis. Here, we report the function of Robo1 in human umbilical vein endothelial cells (HUVEC) as well as the hepatocellular carcinoma endothelial cells. High Robo1 expression in the tumor vessels associated with a poor survival in patients. Over-expression of Robo1 in HUVEC cell lines results in increased proliferation, migration and tube formation in HUVECs. These changes could be reversed by knockdown of Robo1. Moreover, we show that Robo1 expression promoted CDC42 and Rho expression in HUVEC. We demonstrated that Robo1/CDC42/Rho signaling axis in HUVECs, which is important for function of HUVECs. Taken together, our results showed that Robo1 promoted angiogenesis in hepatocellular carcinoma and provide new target for anti-angiogenesis therapy in HCC Note: This abstract was not presented at the meeting. Citation Format: Hui-Chuan Sun, Jian-Yang Ao, Zong-Tao Chai, Yuan-Yuan Zhang. Robo1 promotes angiogenesis through CDC42/Rho GTPases signaling pathway in hepatocellular carcinoma. [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 4168. doi:10.1158/1538-7445.AM2015-4168

Abstract 1095: microRNA-26a expression may suppress tumor growth by modulating macrophage infiltration in tumor through down-regulation of M-CSF in hepatocellular carcinoma

Purpose To Study molecular changes specific to tumor-derived endothelial cells. Materials and methods Tumor tissue and adjacent nontumor tissue were obtained from 27 hepatocellular carcinoma (HCC) patients. Primary cultured CD31+ and CD105+ tumor endothelial cells (TECs) and nontumor endothelial cells (NECs) were isolated by magnetic-activated cell sorting. Affymetrix GeneChip Human Gene ST Arrays were used to determine gene expression profiles of HCC cells and matched TECs and NECs. A capillary tube formation assay was used to evaluate tube formation by the endothelial cells. The tumorigenicity of isolated TECs was monitored in nonobese diabetic/severe combined immunodeficient mice. Results The isolated CD31+ and CD105+ TECs and NECs displayed features of endothelial cells. Both cultured HCC cells and TECs induced capillary tube formation in vitro and were incorporated into tumor vasculature in vivo. The TEC isolates had a gene signature resembling that of HCC cells, and TEC-specific genes were associated with poorer HCC survival. Conclusions A subset of TECs may originate from tumor cells, and they express a unique set of genes linked to HCC prognosis. Note: This abstract was not presented at the meeting. Citation Format: Zong-Tao Chai, Xiao-Dong Zhu, Jian-Yang Ao, Ling-Qun Kong, Yuan-Yuan Zhang, Hui-Chuan Sun. microRNA-26a expression may suppress tumor growth by modulating macrophage infiltration in tumor through down-regulation of M-CSF in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1095. doi:10.1158/1538-7445.AM2014-1095