Weizhong Wu

Fudan University, Shanghai, Shanghai Shi, China

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Publications (13)50.12 Total impact

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    ABSTRACT: Background Organ site-specific metastasis is an ominous feature for most poor-prognostic hepatocellular carcinoma (HCC) patients. Cancer cell lines and animal models are indispensable for investigating the molecular mechanisms of organ specific tropism. However, till now, little is known about the drivers in HCC metastatic tropism, and also no effective way has been developed to block the process of tropistic metastasis. Methods In this study, we established several monoclonal HCC cell lines from HCCLM3-RFP together with their xenograft models, and then analyzed their metastatic potentials and tropisms using in-vitro and in-vivo assays, and finally elucidated the driving forces of HCC tropistic metastases. Results Six monoclonal cell lines with different organ site-specific tropism were established successfully. SPARC, VCAM1 and ANGPTL4 were found positively correlated with the potentials of lung metastasis, while ITGA1 had a positive relation to lymph node metastasis of enterocoelia. Conclusions By our powerful platforms, HCC metastatic tropisms in clinic could be easily mimicked and recapitulated for exploring the bilateral interactions between tumor and its microenvironment, elucidating the drivers of HCC metastatic tropisms, and testing anti-cancer effects of newly developed agent in pre-clinical stage.
    Preview · Article · Dec 2015 · BMC Cancer
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    ABSTRACT: The molecular mechanisms that control metastasis of hepatocellular cancer (HCC) are still poorly understood. It has been determined that microRNA (miRNA) expression has tissue and cell specific, and decreased expression of specific miRNA could induce tumor genesis or metastasis. In this study, we identified that miR-17-5p was expressed lower in high metastatic capability HCC cell lines HCCLM3 and MHCC97H than low metastatic HCC cell line HepG2 by real-time (RT)-PCR. Restoration of miR-17-5p could significantly repress the invasiveness and metastasis of MHCC97H cell line. Furthermore, we validated c-Myc as a downstream and functional target of miR-17-5p using luciferase reporter assay. Immunohistochemical assay revealed that the expression of c-Myc protein levels was significantly increased in cancerous tissues compared with para-tumor tissues. After clinical data analysis, we observed that the higher level of c-Myc was significantly associated with a reduced overall survival (p = 0.0209). Consistent with previous research, we also demonstrated that c-Myc could upregulate the expression of miR-17-5p. Taken together, our data indicated that there is a regulatory feedback loop between miR-17-5p and c-Myc, in which miR-17-5p could suppress some of the distinguishing features, invasion, and metastasis, of oncogenic c-Myc in HCC cells, and meanwhile, miR-17-5p is upregulated by c-Myc role as a transcription factor, although further studies are still needed.
    No preview · Article · Nov 2015 · Tumor Biology
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    ABSTRACT: Alpha-fetoprotein (AFP) is a liver cancer associated protein and has long been utilized as a serum tumor biomarker of disease progression. AFP is usually detected in HCC patients by an antibody based system. Recently, however, aptamers generated from systematic evolution of ligands by exponential enrichment (SELEX) were reported to have an alternative potential in targeted imaging, diagnosis and therapy. In this study, AFP-bound ssDNA aptamers were screened and identified using capillary electrophoresis (CE) SELEX technology. After cloning, sequencing and motif analysis, we successfully confirmed an aptamer, named AP273, specifically targeting AFP. The aptamer could be used as a probe in AFP immunofluorescence imaging in HepG2, one AFP positive cancer cell line, but not in A549, an AFP negative cancer cell line. More interesting, the aptamer efficiently inhibited the migration and invasion of HCC cells after in vivo transfection. Motif analysis revealed that AP273 had several stable secondary motifs in its structure. Our results indicate that CE-SELEX technology is an efficient method to screen specific protein-bound ssDNA, and AP273 could be used as an agent in AFP-based staining, diagnosis and therapy, although more works are still needed.
    Full-text · Article · Oct 2015 · Scientific Reports
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    ABSTRACT: Dysregulation of endocytosis was viewed as an emerging feature of cancer development and progression. A large GTPase dynamin2 plays a significant role in receptor tyrosine kinases (RTKs) endocytosis. The study was designed to investigate its roles in hepatocellular carcinoma (HCC) metastasis and its underlying mechanism. Dynamin2 expression in cancer tissues from HCC patients was assessed by immunohistochemistry and its prognostic significance for the patients was conducted using univariate and multivariate analysis. Its role in tumor invasion and metastasis was evaluated in vitro by gene silence using siRNA-mediated approach and the small molecule inhibitor of Dynasore. EGFR expression in HCC cell lines and EGFR downstream signaling ERK1/2 was evaluated by Western-blot and immunofluorescence analyses after Dynamin2 inhibition. Our data indicated that low expression of dynamin2 was well correlated with invasion characteristics and shorter overall survival. HCC cell migration, colony formation and invasion were significantly increased after the inhibition of dynamin2 in HCC cells. Internalization of EGFR was markedly reduced when dynamin2 was knock down or inhibition. In addition, we observed that dynamin2 regulated EGF mediated EGFR downstream Ras/ERK1/2 signaling and p-ERK1/2 accumulation in nucleus. The results demonstrate a possible mechanism of dynamin involved EGFR endocytosis and modulation of metastasis in HCC. Dynamin2 inhibits the invasion and metastasis of HCC cells by the promotion of EGFR endocytosis and downregulation of ERK1/2 phosphorylation.
    No preview · Article · May 2015 · American Journal of Cancer Research
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    ABSTRACT: Malic enzyme 1 (ME1) links the glycolytic and citric acid cycles and is important for NADPH production, glutamine metabolism, and lipogenesis. Recently, its deregulation has been implicated in the progression of various cancers. However, the role of ME1 in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we utilized short hairpin RNA-mediated gene silencing to investigate the biological effects of ME1 depletion in HCC and determined its prognostic significance in HCC. ME1 expression was examined by real-time (RT)-PCR and Western blot using five HCC cell lines and one normal liver cell line. We used polyethylenimine nanoparticles to deliver a short hairpin RNA to induce cessation of ME1 expression in HCC cells. Changes in NADPH production and reactive oxygen species (ROS) production were studied. Metastatic potentials of HCC cells were evaluated in vitro. Furthermore, we evaluated the protein level of ME1 in para-tumor and cancerous tissues of 65 HCC patients with detailed clinical, pathological, and clinical follow-up data. Patients' survivals were further assessed as well. Upregulated ME1 expression was observed in HCC cell lines. Downregulation of ME1 attenuated NADPH production and stimulated ROS production. Silencing ME1 was noted to inhibit migratory and invasive properties of HCC cells by inducing the E-cadherin expression and decreasing of N-cadherin and vimentin expression in a ROS-dependent pathway. Overexpression of ME1 was observed in a major fraction of HCC samples. Higher level of ME1 in tumors was significantly associated with reduced overall survival (Kaplan-Meier analysis, P = 0.024) and reduced progression-free survival (Kaplan-Meier analysis, P = 0.011). Inhibition of ME1 expression decreases HCC metastasis via suppression of epithelial-mesenchymal transition (EMT) processes in ROS-induced pathways. ME1 overexpression associates with unfavorable prognoses in patients with HCC, suggesting that ME1 is a poor prognostic predictor of hepatocellular carcinoma.
    No preview · Article · Mar 2015 · Tumor Biology
  • Weiwei Li · Ying Liang · Biwei Yang · Huichuan Sun · Weizhong Wu
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    ABSTRACT: Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is a transcriptional regulator and member of the basic helix-loop-helix/Per-ARNT-SIM (bHLH/PAS) superfamily. Recently, evidence of that ARNT is involved in carcinogenesis and cancer progression has emerged. The aim of current study was to investigate the role of ARNT2, a homologue of ARNT, in tumor growth, invasion and prognosis of hepatocellular carcinoma (HCC). METHODS: Tissue microarray and immunohistochemical staining were used to examine the expression of ARNT2 in 195 HCC tissues. Factors associated with ARNT2 levels were assessed by univariate and multivariate Cox regression analyses. Cell proliferation, migration, and invasion assays was performed by using ARNT2 silencing and overexpressing HCCLM6 cell line. Orthotopic xenograft HCC model was used to elucidate the effects of ARNT2 on HCC progression in vivo. High intratumoral of ARNT2 level was well correlated with longer overall survival (OS) and lower tumor to recurrence (TTR) of HCC patients after resection. Multivariate analysis revealed that intratumoral ARNT2 overexpression was an independent prognostic factor for both OS and TTR. Knockdown of ARNT2 in HCCLM6 cells was significantly enhanced while overexpression of ARNT2 significantly inhibited the ability of cell proliferation, invasion and migration. In animal studies, down-regulation of ARNT2 in HCCLM6 cells promoted, whereas up-regulation of ARNT2 in HCCLM6 cells reduced HCCLM6 growth in vivo. Our data demonstrate that ARNT2 plays an inhibitory role in HCC progression and suggest that ARNT2 may be a potential prognostic predictor and therapeutic target for HCC. This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2015 · Journal of Gastroenterology and Hepatology
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    ABSTRACT: Matrix stiffness as a novel regulation factor involves in modulating the pathogenesis of hepatocellular carcinoma (HCC) invasion or metastasis. However, the mechanism by which matrix stiffness modulates HCC angiogenesis remains unknown. Here, using Buffalo rat HCC models with different liver matrix stiffness backgrounds and an in vitro cell culture system of mechanically tunable Collagen1 (COL1)-coated polyacrylamide gel, we investigated the effects of different matrix stiffness levels on vascular endothelial growth factor (VEGF) expression in HCC cells and explored its regulatory mechanism for controlling HCC angiogenesis. Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing COL1 and lysyl oxidase (LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin β1 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to matrix stiffness simulation. After integrin β1 was blocked in HCC cells using specific monoclonal antibody, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were accordingly suppressed and downregulated in the treatment group as compared with those in the control group. All data suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin β1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression. This study unveils a new paradigm in which matrix stiffness as initiators to modulate HCC angiogenesis.
    No preview · Article · Jan 2014 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Recently, a phase II clinical trial in hepatocellular carcinoma (HCC) has suggested that the combination of sorafenib and 5-fluorouracil (5-FU) is feasible and side effects are manageable. However, preclinical experimental data explaining the interaction mechanism(s) are lacking. Our objective is to investigate the anticancer efficacy and mechanism of combined sorafenib and 5-FU therapy in vitro in HCC cell lines MHCC97H and SMMC-7721. Drug effects on cell proliferation were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle. Cell cycle distribution was measured by flow cytometry. Expression levels of proteins related to the RAF/MEK/ERK and STAT3 pathways and to cell cycle progression (cyclin D1) were determined by western blot analysis. Sorafenib and 5-FU alone or in combination showed significant efficacy in inhibiting cell proliferation in both cell lines tested. However, a schedule-dependent combined effect, associated with the order of compound treatments, was observed. Efficacy was synergistic with 5-FU pretreatment followed by sorafenib, but it was antagonist with the reverse treatment order. Sorafenib pretreatment resulted in a significant increase in the half inhibitory concentration (IC50) of 5-FU in both cell lines. Sorafenib induced G1-phase arrest and significantly decreased the proportion of cells in S phase when administrated alone or followed by 5-FU. The RAF/MEK/ERK and STAT3 pathways were blocked and cyclin D1 expression was down regulated significantly in both cell lines by sorafenib; whereas, the kinase pathways were hardly affected by 5-FU, and cyclin D1 expression was up regulated. Antitumor activity of sorafenib and 5-FU, alone or in combination, is seen in HCC cell lines. The nature of the combined effects, however, depends on the particular cell line and treatment order of the two compounds. Sorafenib appears to reduce sensitivity to 5-FU through down regulation of cyclin D1 expression by inhibiting RAF/MEK/ERK and STAT3 signaling, resulting in G1-phase arrest and reduction of the S-phase cell subpopulation when 5-FU is administrated after sorafenib, in which situation, combination treatment of the two agents results in antagonism; on the other hand, when sorafenib is administrated afterward, it can continue to work since it is not cell cycle specific, as a result, combination treatment of the two agents shows an additive-to-synergistic effect.
    Full-text · Article · Jul 2013 · BMC Cancer
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    ABSTRACT: Background Postoperative interferon-α(IFN-α) treatment delays hepatocellular carcinoma(HCC) recurrence and prolongs patient survival, and may thus be an effective form of adjuvant therapy. However, clinical observations found that HCC recurs in some patients within 8 months of IFN-α treatment being discontinued. We investigated whether HCC regrowth appears after IFN-α is discontinued, whether re-initiated IFN-α is effective, and the underlying mechanisms of IFN-α treatment. Methods The human HCC nude mouse model LCI-D20 was used to study the effects of IFN-α treatment, discontinued IFN-α treatment, and re-initiated IFN-α treatment on tumor growth. Tumor weight, microvessel density(MVD), serum vascular endothelial growth factor (VEGF), and tumor cell apoptosis were analyzed. Angiogenesis-related factors were studied using cDNA microarray in different tumor samples and confirmed using reverse transcription–polymerase chain reaction(RT-PCR) and Western blotting assays. Finally, imatinib was added with re-initiated IFN-α treatment to improve efficacy. Results IFN-α (1.5×107 U/kg/day for 20 days) suppressed HCC growth by 60.3% and decreased MVD by 52.2% compared with the control. However, tumor regrowth occurred after IFN-α was discontinued, and re-initiated IFN-α treatment was not effective for inhibiting tumor growth or reducing MVD compared with a saline-treated group. cDNA microarray showed VEGF was down-regulated while platelet-derived growth factor-A (PDGF-A) was up-regulated when IFN-α treatment was re-initiated. These findings were further confirmed with RT-PCR and Western blotting assay. The combination of imatinib with re-initiated IFN-α reduced HCC weight by 30.7% and decreased MVD by 31.1% compared with IFN-α treatment only (P=0.003 and 0.015, respectively). Conclusion Tumor regrowth occurred after IFN-α treatment was discontinued. Re-initiated IFN-α treatment was not effective and was associated with up-regulation of PDGF-A, while the VEGF remained suppressed. The combination of a PDGF-receptor inhibitor with IFN-α improved the effect of the re-initiated treatment.
    Preview · Article · Oct 2012 · BMC Cancer
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    ABSTRACT: Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Metastasis is the major concern that causes death in HCC. The goal of this study was to identify tumor-derived proteins in serum during HCC metastasis using an orthotopic xenograft tumor model and explore the role of key protein in HCC metastasis. Serum samples collected from HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1 wk, 3 wks and 6 wks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. Twenty tumor-derived proteins were identified through human specific peptides. Secretory clusterin (sCLU), which was significantly upregulated during cancer progression and metastasis, was chosen for further study. The expression of sCLU was significantly higher in metastatic HCC cell lines and samples from metastatic HCC patients. ShRNA-mediated down-regulation of sCLU resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary metastasis in vivo. Overexpression of sCLU in HepG2 cell line showed increased cell migratory ability. Further study found that sCLU contributed to HCC migration and epithelial-mesenchymal transition (EMT) in vitro, and metastasis in vivo. In addition, sCLU also plays an important role in the regulation of TGF-β1-smad3 signaling. These findings suggest that sCLU may promote HCC metastasis via the induction of EMT process and may be a candidate target for HCC therapy.
    No preview · Article · Sep 2012 · The international journal of biochemistry & cell biology
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    ABSTRACT: Despite recent advances in the treatment of hepatocellular carcinoma (HCC), the chemotherapy efficacy against HCC is still unsatisfactory. The mammalian target of rapamycin (mTOR) has been emerged as an important cancer therapeutic target. However, HCC cells often resistant to rapamycin because of the paradoxical activation of Akt by rapamycin. In this study, we investigated whether bortezomib could enhance the antitumor effects of rapamycin. The effects of rapamycin and bortezomib on HCC proliferation, apoptosis, migration, and invasiveness in vitro were assessed by CCK-8 analysis, flow cytometry, Hoechst 33342 staining and transwell assays, respectively. Total and phosphorylated protein levels of Akt were detected by Western blotting. The effects of rapamycin and/or bortezomib on the mRNA expression levels of p53, p27, p21 and Bcl-2 family in HCCLM3 cells were evaluated by RT-PCR. The roles of rapamycin and bortezomib on HCC growth and metastasis in xenograft models were evaluated by tumor volumes and fluorescent signals. The effects of rapamycin and bortezomib on cell proliferation and apoptosis in vivo were test by PCNA and TUNEL staining. Bortezomib synergized with rapamycin to reduce cell growth, induce apoptosis, and inhibit cell mobility in vitro. Further mechanistic studies showed that bortezomib inhibited rapamycin-induced phosphorylated Akt, which in turn enhanced apoptosis of HCC cell lines. The alteration of the mRNA expression of cell cycle inhibitors p53, p27, p21 and apoptosis associated genes Bcl-2, Bax were also involved in the synergistic antitumor effects of rapamycin and bortezomib. P53 inhibitor PFT-α significantly attenuate the effect of rapamycin and bortezomib on cell apoptosis, which indicated that the pro-apoptotic effect of rapamycin and bortezomib may be p53-dependent. Treatment of HCCLM3-R bearing nude mice with rapamycin and bortezomib significantly enhanced tumor growth inhibition (72.4%), comparing with either rapamycin- (54.7%) or bortezomib-treated mice (22.4%). In addition, the lung metastasis was significantly suppressed in mice received the combination treatment (16.6%). The combination treatment of rapamycin and bortezomib significantly inhibited tumor cell proliferation and tumor angiogenesis in vivo. The combination of rapamycin with bortezomib could be a novel and promising therapeutic approach to the treatment of HCC.
    Full-text · Article · May 2012 · BMC Cancer
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    ABSTRACT: Hepatocellular carcinoma (HCC) is one of serious disorders with the highest morbidities and mortalities worldwide. Metastasis is the major concern that causes death in HCC. The goal of this study was to screen and identify potential serum proteins indicating HCC metastasis. Serum samples collected from control and HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1 wk, 3 wks and 6 wks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. A total of 554 proteins were identified and 80 proteins were differential expressed at least between one adjacent time points. Among them, expression level of transaldolase (TALDO) was validated in mouse and human serum. The level of TALDO protein was found to be higher in metastatic mice serum compared to that of non-metastatic mice. Human specific TALDO was then identified in mouse serum through human specific peptides. Immunohistochemical and western blot analysis showed that the expression of TALDO in human HCC tissues and HCC cell lines was associated with its metastatic behavior. Subsequent screening of TALDO expression in 72 clinical serum samples (comprising 36 non-metastatic HCC and 36 metastatic HCC samples) revealed higher TALDO level in the serum of metastatic HCC patients. A receiver operating characteristic (ROC) curve estimated a maximal sensitivity of 77.8% and 86.1% specificity for TALDO in detection of HCC metastasis. The present results demonstrated that the nude mouse xenograft model is an efficient system for performing metastasis-related biomarker discovery. TALDO may be useful biomarkers for the detection of HCC metastasis.
    No preview · Article · Dec 2011 · Cancer letters
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    ABSTRACT: Lymph node metastasis (LNM) is a chief cause of morbidity and mortality in patients with hepatocellular carcinoma (HCC) after hepatectomy. The aim of this study was to investigate the relationship between the expression of CXCR4 and vascular endothelial cell growth factor (VEGF)-C and the clinicopathological features of HCC with LNM. Immunohistochemical staining for CXCR4 and VEGF-C was performed on tissue microarrays that were constructed using tumor specimens from patients with HCC with (N = 123) or without (N = 145) LNM. The relationship between the clinicopathological features of HCC and the expression of CXCR4 and VEGF-C was analyzed using the Pearson chi(2) test, logistical regression analysis, and receiver operating characteristic analysis. Nuclear CXCR4 expression and VEGF-C expression were positively correlated with LNM and poor outcome in HCC. Moreover, nuclear CXCR4 expression was positively correlated with VEGF-C expression (correlation coefficient 0.256). Receiver operating characteristic analysis revealed that both factors were predictive of HCC LNM {CXCR4: area under the curve, 0.695 [95% confidence interval (CI), 0.630-0.759; VEGF-C: area under the curve, 0.629 (95% CI, 0.562-0.695]}. Patients with tumors exhibiting high nuclear CXCR4 expression or high VEGF-C expression had significantly poorer overall survival than those with low tumor expression of the corresponding factors. Multivariate analysis showed that UICC T stage [odds ratio (OR), 1.615, 95% CI, 1.306-1.997], nuclear CXCR4 expression (OR, 3.998; 95% CI, 2.706-5.907), and VEGF-C expression (OR, 1.903; 95% CI, 1.203-3.011) were independent risk factors for developing HCC LNM. These findings suggest that nuclear CXCR4 expression and VEGF-C expression can be used to identify patients with HCC at high risk for developing LNM.
    No preview · Article · Nov 2009 · The Cancer Journal