Ubiquitin-specific protease 7 accelerates p14(ARF) degradation by deubiquitinating thyroid hormone receptor-interacting protein 12 and promotes hepatocellular carcinoma progression.

UNLABELLED: The prognosis for hepatocellular carcinoma (HCC) remains dismal in terms of overall survival (OS), and its molecular pathogenesis has not been completely defined. Here, we report that expression of deubiquitylase ubiquitin-specific protease 7 (USP7) is higher in human HCC tissues than in matched peritumoral tissues. Ectopic USP7 expression promotes growth of HCC cells in vivo and in vitro. Mechanistically, USP7 overexpression fosters HCC cell growth by forming a complex with and stabilizing thyroid hormone receptor-interacting protein 12 (TRIP12), which induces constitutive p14(ARF) ubiquitination. Clinically, USP7 overexpression is significantly correlated with a malignant phenotype, including larger tumor size, multiple tumor, poor differentiation, elevated alpha-fetoprotein, and microvascular invasion. Moreover, overexpression of USP7 and/or TRIP12 correlates with shorter OS and higher cumulative recurrence rates of HCC. CONCLUSION: USP7 stabilizes TRIP12 by deubiquitination, thus constitutively inactivating p14(ARF) and promoting HCC progression. This represents a novel marker for predicting prognosis and a potential therapeutic target for HCC.

PI-88 inhibits postoperative recurrence of hepatocellular carcinoma via disrupting the surge of heparanase after liver resection.

Phosphomannopentaose sulfate (PI-88), an effective inhibitor of heparanase (HPSE), exhibited anti-recurrence and anti-metastasis activity in preliminary clinical trials of hepatocellular carcinoma (HCC); however, the underlying mechanisms remain uncertain. Our aim was to reveal the mechanism by which PI-88 inhibits recurrence and intrahepatic metastasis. A tissue microarray containing samples from 352 HCC patients was used to determine HPSE expression. We performed enzyme-linked immunosorbent assay (ELISA) to detect plasma levels of HPSE in 40 HCC patients. We also used quantitative polymerase chain reaction, western blot analysis, and immunohistochemical staining to assess HPSE expression of HCC cell lines and tissues. The in vitro effects of PI-88 were examined by cell proliferation and migration assays. In vivo PI-88 activity was assessed using murine orthotopic HCC models. Intratumoral HPSE was an independent prognostic marker for postsurgical overall survival (P = 0.001) and time to recurrence (P < 0.001) of HCC patients with hepatectomy. Elevated levels of HPSE were detected both in postsurgical plasma of HCC patients and an orthotopic mouse model after hepatectomy. PI-88 inhibited tumor recurrence and metastasis after liver resection in the mouse model. In vitro expression of HPSE was up-regulated by overexpression of early growth response 1 (EGR1), which is induced after hepatectomy. Up-regulation of HPSE enhanced the sensitivity of HCC cells to PI-88 and the inhibitive effect of PI-88 on cell proliferation and migration. Our data show that PI-88 effectively inhibits postoperative recurrence and intrahepatic metastasis of HCC, providing an experimental basis for the clinical application of PI-88 in HCC patients who have undergone hepatectomy.

Generation and characterization of a tetraspanin CD151/integrin α6ß1-binding domain competitively binding monoclonal antibody for inhibition of tumor progression in HCC.

Our previous studies revealed that tetraspanin CD151 plays multiple roles in the progression of hepatocellular carcinoma (HCC) by forming a functional complex with integrin α6ß1. Herein, we generated a monoclonal antibody (mAb) that dissociates the CD151/integrin α6ß1 complex, and we evaluated its bioactivity in HCCs. A murine mAb, tetraspanin CD151 (IgG1, called CD151 mAb 9B), was successfully generated against the CD151-integrin α6ß1 binding site of CD151 extracellular domains. Co-immunoprecipitation using CD151 mAb 9B followed by Western blotting detected a 28 kDa protein. Both immunofluorescent and immunohistochemical staining showed a good reactivity of CD151 mAb 9B in the plasma membrane and cytoplasm of HCC cells, as well as in liver cells. In vitro assays demonstrated that CD151 mAb 9B could inhibit neoangiogenesis and both the mobility and the invasiveness of HCC cells. An in vivo assay showed that CD151 mAb 9B inhibited tumor growth potential and HCC cells metastasis. We successfully produced a CD151 mAb 9B targeting the CD151/integrin α6ß1-binding domain, which not only can displayed good reactivity to the CD151 antigen but also prevented tumor progression in HCC.

Transfection of thymidine phosphorylase cDNA to human hepatocellular carcinoma cells enhances sensitivity to fluoropyrimidine but augments endothelial cell migration.

PURPOSE: To investigate the effects on sensitivity to fluoropyrimidine and endothelial cell (EC) migration by transfection with thymidine phosphorylase (TP) cDNA to a hepatocellular carcinoma (HCC) cell line SMMC-7721. METHODS: SMMC-7721 was transfected with pcDNA3.1/zeo (+) with human TP cDNA. TP mRNA expression was determined by RT-PCR. Sensitivity to fluoropyrimidine was determined by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Induction of EC migration was detected by Boyden chamber assay. RESULTS: The construction of pcDNA3.1/zeo(+)-TP was verified by digestion with restriction endonuclease Apa1. When comparison was made between SMMC-7721 cell clone transfected with pcDNA3.1/zeo(+)-TP (S-TP) and control clone transfected with pcDNA3.1/zeo(+) (S-vector), we found that TP mRNA expression level was much higher in S-TP, being 2.09+/-0.16 vs 0.48+/-0.06 in S-vector (P < 0.01), sensitivity to 5'-deoxy-5-fluorouridine (5'-dFUrd, a prodrug of 5-fluorouracil) in S-TP was significantly enhanced compared with that in S-vector (IC(50); 56.81+/-9.85 micromol/l vs 162.25+/-11.03 micromol/l, P < 0.01), and the culture medium of S-TP possessed more potential to induce EC migration than that of S-vector (the number of ECs appearing on the outer surfaces of the membrane was 275+/-29 vs 122+/-35 per field, P < 0.01). CONCLUSION: Sensitivity to 5'-dFUrd could be enhanced by transfection with TP cDNA for SMMC-7721 cells. However, EC migration was also promoted at the same time. Therefore, transfection with TP alone might have no potential to enhance anti-tumoral effects of fluoropyrimidine in HCC.

[Up-regulation of thymidine phosphorylase and anti-angiogenesis by interferon alpha in human hepatocellular carcinoma cell line and xenograft].

OBJECTIVE: To further study the impact of interferon-alpha (IFN-alpha) on thymidine phosphorylase (TP) expression and angiogenesis. METHODS: Human hepatocellular carcinoma cells of the line SMMC-7721 were cultured and added with IFN-alpha of different doses: 0 (as control group), 1000, 5000 and 10,000 U/ml. Twenty-four hours later RT-PCR was used to detect the TP mRNA expression. Boyden chamber method was used to examine the endothelial cells migration. Suspension of SMMC-7721 cells was inoculated subcutaneously into 30 male BALB/c-nu/nu mice, the mice were randomly divided into 5 equal groups to be subcutaneously injected with IFN-alpha of different doses: 0 (as control group), 1.0 x 10(6), 3.0 x 10(6), 9.0 x 10(6), and 1.5 x 10(7) U.kg(-1).d(-1) for 3 weeks. The eating behavior, activity, body weight, and tumor size were observed. The rats were killed 2 days after the drug injection was stopped. The subcutaneous tumors were taken out to undergo histological examination and TP protein expression by ELISA. The microvessel density (MVD) was detected using anti-CD34 monoclonal antibody. RESULTS: The TP mRNA expression of the SMMC-7721 cells induced by IFN-alpha of the doses of 5000 U/ml and 10,000 U/ml significantly increased in comparison with the un-treated SMMC-7721 cells (0 U/ml, P < 0.05). The endothelial cell migration significantly increased in the IFN-alpha 1000 U/ml group compared with the control group (P < 0.001), and then decreased along with the increase of IFN-alpha dose; there were no significant differences in the epithelial migration among the groups of 0, 5000, and 10,000 U/ml IFN-alpha doses (all P > 0.05). The TP protein expression levels of the tumor in the rats treated with IFN-alpha of the doses of 9.0 x 10(6), and 1.5 x 10(7) U.kg(-1).d(-1) were 48 ng/mg +/- 24 ng/mg and 60 ng/mg +/- 6 ng/mg respectively, 1.9 and 2.4 times that of the control group (both P < 0.01). The MVD of the tumors was 6.0 +/- 1.8 in the 9.0 x 10(6) U.kg(-1).d(-1) IFN-alpha group, significantly higher than that of the control group (P < 0.01); and was 4.0 +/- 1.5 in the 1.5 x 10(7) U.kg(-1).d(-1) group, significantly lower than that of the 9.0 x 10(6) U.kg(-1).d(-1) group (P < 0.05) and not significantly different from that of the control group. The tumor inhibiting rate of the 1.5 x 10(7) U.kg(-1).d(-1) IFN-alpha group was 68%, significantly higher than that of the untreated group (P < 0.05). CONCLUSION: IFN-alpha of certain doses up-regulate the TP expression, and inhibit the angiogenesis induced by TP as well.

High expressions of vascular endothelial growth factor and platelet-derived endothelial cell growth factor predict poor prognosis in alpha-fetoprotein-negative hepatocellular carcinoma patients after curative resection.

PURPOSE: To evaluate the prognosis value of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in alpha-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC) patients after curative resection. METHODS: Tumor tissue microarrays (TMAs) were used to detect the expressions of VEGF and PD-ECGF in consecutive 162 AFP-negative HCC patients undergoing curative resection between 1997 and 2000 in our institute. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Multivariate study with Cox's proportional hazard model was used to evaluate the prognosis-related aspects. RESULTS: The positive rates of VEGF and PD-ECGF in tumor tissues were 59.9% (97/162) and 62.3% (101/162), respectively. Univariate analysis showed that VEGF and PD-ECGF were prognostic factors for relapse-free survival (P = 0.034 and P = 0.033, respectively). Multivariate analyses demonstrated that the co-index (VEGF/PD-ECGF) was an independent prognostic factor for overall survival and relapse-free survival (P = 0.002 and P = 0.000, respectively). CONCLUSION: The co-index of VEGF and PD-ECGF is a promising independent predictor for recurrence and survival of AFP-negative HCC patients after curative resection.