Down-regulation of argininosuccinate synthetase is associated with cisplatin resistance in hepatocellular carcinoma cell lines: implications for PEGylated arginine deiminase combination therapy.

BACKGROUND: Many advanced human tumors, including hepatocellular carcinomas (HCC) are auxotrophic for arginine due to down-regulation of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine synthesis. The arginine-lowering agent PEGylated arginine deiminase (ADI-PEG 20) has shown efficacy as a monotherapy in clinical trials for treating arginine-auxotrophic tumors and is currently being evaluated in combination with cisplatin in other cancer types. Epigenetic silencing via methylation of the ASS1 promoter has been previously demonstrated in other cancer types, and a reciprocal relationship between ASS1 expression and cisplatin resistance has also been observed in ovarian cancer. However, the mechanism of ASS1 down-regulation, as well as the correlation with cisplatin resistance has not been explored in HCC. The present study investigates ADI-PEG 20 and cisplatin sensitivities in relation to ASS1 expression in HCC. In addition, we show how this biomarker is regulated by cisplatin alone and in combination with ADI-PEG 20. METHODS: ASS1 protein expression in both untreated and drug treated human HCC cell lines was assessed by western blot. The correlation between ASS1 protein levels, ADI-PEG 20 sensitivity and cisplatin resistance in these cell lines was established using a luminescence-based cell viability assay. Epigenetic regulation of ASS1 was analyzed by bisulfite conversion and methylation-specific PCR. RESULTS: A good correlation between absence of ASS1 protein expression, ASS1 promoter methylation, sensitivity to ADI-PEG 20 and resistance to cisplatin in HCC cell lines was observed. In addition, cisplatin treatment down-regulated ASS1 protein expression in select HCC cell lines. While, at clinically relevant concentrations, the combination of ADI-PEG 20 and cisplatin restored ASS1 protein levels in most of the cell lines studied. CONCLUSION: ASS1 silencing in HCC cell lines is associated with simultaneous cisplatin resistance and ADI-PEG 20 sensitivity which suggests a promising combination therapeutic strategy for the management of HCC.

Extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontin.

BACKGROUND: Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS: Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS: In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSIONS: The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies.