Wei-Zhong Wu

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.

Depletion of Tumor-Associated Macrophages Enhances the Effect of Sorafenib in Metastatic Liver Cancer Models by Antimetastatic and Antiangiogenic Effects

Purpose: To investigate the role of macrophages in tumor progression under sorafenib treatment and to explore whether combination of drugs that deplete macrophages improved the antitumor effect of sorafenib. Experimental Design: Tumor growth, lung metastasis, and tumor angiogenesis were observed in HCCLM3-R and SMMC7721, two human hepatocellular carcinoma xenograft nude mouse models, when treated with sorafenib (30 mg/kg daily, n = 6 per group) or a vehicle as control. Macrophage infiltration was measured in the peripheral blood and in sorafenib-treated tumor by immunohistochemistry and flow cytometry with F4/80 antibody and CD11b antibody. The effect of macrophage depletion on tumor angiogenesis and metastasis after sorafenib treatment, using two drug target macrophages, zoledronic acid (ZA) and clodrolip, was measured in the two models of hepatocellular carcinoma. Results: Although sorafenib significantly inhibited tumor growth and lung metastasis, it induced a significant increase in peripheral recruitment and intratumoral infiltration of F4/80- and CD11b-positive cells, which was accompanied with elevation of colony-stimulating factor-1, stromal-derived factor 1α, and vascular endothelial growth factor in the tumor and elevation of plasma colony-stimulating factor-1 and mouse vascular endothelial growth factor in peripheral blood, suggesting the role of macrophages in tumor progression under sorafenib treatment. Depletion of macrophages by clodrolip or ZA in combination with sorafenib significantly inhibited tumor progression, tumor angiogenesis, and lung metastasis compared with mice treated with sorafenib alone. ZA was more effective than clodrolip. Conclusions: Macrophages may have an important role in tumor progression under sorafenib treatment. ZA is promising when combined with sorafenib to enhance its antitumor effect. Clin Cancer Res; 16(13); 3420–30. ©2010 AACR.

High expression levels of putative hepatic stem/progenitor cell biomarkers related to tumour angiogenesis and poor prognosis of hepatocellular carcinoma

Background/aims To investigate the prognostic values of putative hepatic stem/progenitor cell (HSC/HPC) biomarkers in patients with hepatocellular carcinoma (HCC). Methods Fourteen biomarkers related to HSCs/HPCs or tumour angiogenesis were assessed by qRT-PCR and then validated by tissue microarrays (TMAs) in three independent cohorts of patients with HCC undergoing curative resection (n=67, 314 and 73). Results Most of the biomarkers were found to be overexpressed in patients with recurrent HCC by quantitative reverse transcription–PCR (qRT–PCR). The HSC/HPC biomarkers cytokeratin 19, ATP-binding cassette subfamily G member 2 (ABCG2), CD133, Nestin and CD44, and the markers of angiogenesis microvessel density (MVD, determined by CD34 immunostaining), vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) were confirmed as significant predictors for overall survival (OS) and/or relapse-free survival (RFS) in TMA analysis. As compared with the low HSC/HPC profile group, patients with a high HSC/HPC profile who had higher VEGF levels (p=0.012) and MVD (p=0.030) in tumours had significantly lower OS and RFS (p<0.0001). Based on Cox regression, a simplified model including CD133, CD44, Nestin and MVD was constructed and confirmed as an independent predictor for OS (p<0.0001) and RFS (p<0.0001), regardless of α-fetoprotein level, tumour stage and recurrence time (p<0.0001 for all). Conclusion High expression levels of HSC/HPC biomarkers are related to tumour angiogenesis and poor prognosis of HCC. The simplified model based on the HSC/HPC and tumour angiogenesis profile can be used to classify patients with HCC with a high risk of tumour recurrence after surgery.

Human Hepatocellular Carcinoma Tumor–derived Endothelial Cells Manifest Increased Angiogenesis Capability and Drug Resistance Compared with Normal Endothelial Cells

Purpose: Increasing evidence indicates that tumor-derived endothelial cells (TEC) possess a distinct and unique phenotype compared with endothelial cells (NEC) from adjacent normal tissue and may be able to acquire resistance to drugs. The aim of this study was to investigate the angiogenesis activity and response to drug treatment of TECs and NECs derived from human hepatocellular carcinoma (HCC). Experimental Design: TECs or NECs were isolated from HCC or adjacent normal liver tissue using anti-CD105 antibody coupled to magnetic beads. The phenotypic and functional properties of endothelial cells were characterized by testing the expression of CD105, CD31, CD144, vascular endothelial growth factor receptor-1, vascular endothelial growth factor receptor-2, and von Willebrand factor, and the ability of DiI-Ac-LDL-uptake and tube formations. CD105+ TECs were compared with CD105+ NECs and human umbilical vein endothelial cells (HUVEC) by examining their ability to proliferate, motility, ability to adhere to tumor cells, response to tumor conditioned medium, and reactions to the chemotherapy drugs Adriamycin and 5-fluorouracil and the antiangiogenic drug sorafenib. Results: Compared with CD105+ NECs and HUVECs, CD105+ TECs showed increased apoptosis resistance and motility and proangiogenic properties. Meanwhile, CD105+ TECs had a greater ability to adhere to tumor cells and survive in the tumor environment. Moreover, CD105+ TECs acquired more resistance to Adriamycin, 5-fluorouracil, and sorafenib than CD105+ NECs and HUVECs. Conclusions: TECs possessed enhanced angiogenic activity and resistance to chemotherapeutic drugs and an angiogenesis inhibitor, and may provide a better tool for studying tumor angiogenesis and antiangiogenesis drugs in HCC.

Overexpression of Platelet-Derived Growth Factor Receptor α in Endothelial Cells of Hepatocellular Carcinoma Associated with High Metastatic Potential

Purpose: Little information is available on the heterogeneity of the vascular endothelium in hepatocellular carcinoma. The aim of this study was to identify the differentially expressed genes in tumor endothelial cells from highly metastatic hepatocellular carcinoma. Experimental Design: Magnetic beads conjugated with anti-CD31 antibody were used to isolate vascular endothelial cells from hepatocellular carcinoma xenografts with different metastatic potentials in nude mice. Gene expression profiles for different endothelial cells were compared by use of cDNA microarray. The up-regulated gene was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. Results: cDNA microarray analysis revealed differential expression patterns in seven genes consistently presented in endothelial cells isolated from hepatocellular carcinoma with different metastatic potentials. Overexpression of platelet-derived growth factor receptor α was found only in the endothelium of highly metastatic hepatocellular carcinoma, which was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. Oral administration of STI571 (imatinib mesylate or Glivec), a protein tyrosine kinase inhibitor of platelet-derived growth factor receptor, combined with s.c. injection of IFN-α not only effectively reduced tumor weight (by 81.8%) and microvessel density (by 70.2%) but also inhibited lung metastasis (by 100%). Furthermore, immunohistochemical analysis of platelet-derived growth factor receptor α in human hepatocellular carcinoma tissues revealed its correlation with postoperative recurrence, especially in patients without microvessel invasion. Conclusions: The gene expression of hepatocellular carcinoma vascular endothelium is different between tumors with different metastatic potential. Platelet-derived growth factor receptor α, which is overexpressed in endothelium of highly metastatic hepatocellular carcinoma, may serve as a biomarker for predicting metastasis and a therapeutic target for highly metastatic hepatocellular carcinoma.

Mechanism of interferon alpha on inhibition of metastasis and angiogenesis of hepatocellular carcinoma after curative resection in nude mice

The aim of this study was to examine the mechanism of interferon alpha (IFN-α) on inhibition of metastasis and recurrence of hepatocellular carcinoma (HCC). Nude mice bearing human HCC xenografts with high metastatic potential (LCI-D20) underwent curative resection of tumors on postimplant day 11. IFN-α was begun the next day at different dosages given subcutaneously for 35 consecutive days; normal saline solution was injected into the control mice. The mice were killed 48 hours after the final treatment, and the parameters were evaluated. The HCC intrahepatic recurrence rate, the size of the recurrent lesions, the rate of lung metastasis, the serum vascular endothelial growth factor level, and the microvessel density (immunohistochemistry) were as follows: 100%, 2136 ± 794 mm3 (mean ± standard deviation), 100%, 265.7 ± 154.7 pg/ml, and 144 ± 37/HP, respectively, in the control mice, whereas these same values were 62.5%, 89 ± 45 mm3, 12.5%, 53.3 ± 9.9 pg/ml, and 86 ± 25/HP, respectively, in the IFN-α 1.5 × 107 U/kg treatment group (P < 0.05) and 26.7%, 46 ± 21 mm3, 0%, 65.2 ± 17.9 pg/ml, and 39 ± 14/HP in the IFN-α 3 × 107 U/kg treatment group, respectively (P < 0.05). However, a significant difference was not found in the serum levels of basic fibroblast growth factor among the control and IFN-α treatment groups. IFN-α inhibits metastasis and recurrence of human HCC after curative resection in nude mice mediated by antiangiogenesis through downregulating expression of vascular endothelial growth factor but not basic fibroblast growth factor.

Two pathologic types of hepatocellular carcinoma with lymph node metastasis with distinct prognosis on the basis of CK19 expression in tumor

Few studies have investigated the pathologic types and prognosis of hepatocellular carcinoma (HCC) with lymph node metastasis (LNM). The purpose was to explore pathologic types and pertinent therapy of HCC with LNM.

Interferon alpha 2a downregulates VEGF expression through PI3 kinase and MAP kinase signaling pathways

Abstract An earlier report demonstrated that interferon alpha (IFN-α) inhibited tumor growth and recurrence in an MHCC97 xenograft model in nude mice by suppressing tumor angiogenesis rather than by inhibiting tumor cell proliferation. However, the underlying molecular mechanism was not fully elucidated. In this study, we demonstrated that IFN-α 2a could downregulate VEGF expression both in mRNA and in protein levels, as well as downregulating HIF-1α mRNA expression in MHCC97 cells in vitro. A cDNA micro array analysis followed by Northern and Western blot analysis revealed that PI3 kinase and MAP kinase signaling pathways might be inhibited by IFN-α 2a. Blocking the function of IFN-α receptor with a specific peptide could eliminate the inhibitory effects of IFN-α 2a on VEGF expression. In addition, wortmannin and PD098059, respective inhibitors of the PI3 kinase and the MAP kinase signaling pathways, when used independently or in combination, could also downregulate the VEGF synthesis and secretion in a similar pattern of IFN-α 2a. These observations may lead to the conclusion that IFN-α 2a could suppress VEGF synthesis and secretion by downregulating HIF-1α expression, via inhibition of the PI3 kinase and/or the MAP kinase signaling pathways.

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.

Coexpression of gene Oct4 and Nanog initiates stem cell characteristics in hepatocellular carcinoma and promotes epithelial-mesenchymal transition through activation of Stat3/Snail signaling

BackgroundOct4 and Nanog are key regulatory genes that maintain the pluripotency and self-renewal properties of embryonic stem cells. We previously reported that the two stemness markers were tightly associated with cancer progression and poor outcomes of hepatocellular carcinoma. In this study, we demonstrate that coexpression of Oct4/Nanog modulates activation of signal transducer and activator of transcription 3 (Stat3), an oncogenic transcription factor that is activated in many human malignancies including hepatocellular carcinoma (HCC), as well as the expression of Snail, a key regulator implicated in epithelial-mesenchymal transition and tumor metastasis.MethodsOct4 and Nanog were ectopic expressed in MHCC97-L cell lines via lentiviral gene transfection. The stemness characteristics including self-renewal, proliferation, chemoresistance, and tumorigenicity were assessed. The effect of coexpression of Oct4 and Nanog on epithelial-mesenchymal transition change, and the underlying molecular signaling was investigated.ResultsEctopic coexpression of Oct4 and Nanog empowered MHCC97-L cells with cancer stem cell (CSC) properties, including self-renewal, extensive proliferation, drug resistance, and high tumorigenic capacity. Significantly, Oct4 and Nanog encouraged epithelial-mesenchymal transition change contributing to tumor migration, invasion/metastasis in vitro and in vivo. Following molecular mechanism investigation indicated Oct4/Nanog-regulated epithelial-mesenchymal transition change through Stat3-dependent Snail activation. Moreover, silencing Stat3 abrogates Oct4/Nanog-mediated epithelial-mesenchymal transition (EMT) change and invasion/metastasis in HCC.ConclusionsWe delineate Oct4 and Nanog initiate stem cell characteristics in hepatocellular carcinoma and promote epithelial-mesenchymal transition through activation of Stat3/Snail signaling. Our findings propose Stat3/Snail pathway as a novel therapeutic target for the treatment of progression and metastasis of HCC with CSC-like signatures and epithelial-mesenchymal transition phenotype.