title: Resistance to arginine deiminase treatment in melanoma cells is associated with induced argininosuccinate synthetase expression involving c-Myc/HIF-1alpha/Sp4.
abstract: Arginine deiminase (ADI)-based arginine depletion is a novel strategy under clinical trials for the treatment of malignant melanoma with promising results. The sensitivity of melanoma to ADI treatment is based on its auxotrophy for arginine due to a lack of argininosuccinate synthetase (AS) expression, the rate-limiting enzyme for the de novo biosynthesis of arginine. We show here that AS expression can be transcriptionally induced by ADI in melanoma cell lines A2058 and SK-MEL-2 but not in A375 cells, and this inducibility was correlated with resistance to ADI treatment. The proximal region of the AS promoter contains an E-box that is recognized by c-Myc and HIF-1alpha and a GC-box by Sp4. Through ChIP assays, we showed that under noninduced conditions, the E-box was bound by HIF-1alpha in all the three melanoma cell lines. Under arginine depletion conditions, HIF-1alpha was replaced by c-Myc in A2058 and SK-MEL-2 cells but not in A375 cells. Sp4 was constitutively bound to the GC-box regardless of arginine availability in all three cell lines. Overexpressing c-Myc by transfection upregulated AS expression in A2058 and SK-MEL-2 cells, whereas cotransfection with HIF-1alpha suppressed c-Myc-induced AS expression. These results suggest that regulation of AS expression involves interplay among positive transcriptional regulators c-Myc and Sp4, and negative regulator HIF-1alpha that confers resistance to ADI treatment in A2058 and SK-MEL-2 cells. Inability of AS induction in A375 cells under arginine depletion conditions was correlated by the failure of c-Myc to interact with the AS promoter.
authors.affiliation: Department of Molecular Pathology, Unit 951, The University of Texas M. D. Anderson Cancer Center, 7435 Fannin Street, Houston, Texas 77030, USA.
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Tsai
authors.forename: Isamu
authors.initials: I
authors.lastname: Aiba
authors.forename: Soo-yong
authors.initials: SY
authors.lastname: Lee
authors.forename: Lynn
authors.initials: L
authors.lastname: Feun
authors.forename: Niramol
authors.initials: N
authors.lastname: Savaraj
authors.forename: Macus Tien
authors.initials: MT
authors.lastname: Kuo
mesh_terms: D001120:Arginine / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D019008:Drug Resistance, Neoplasm; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000494:pharmacology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011092:Polyethylene Glycols / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051706:Sp4 Transcription Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016271:Proto-Oncogene Proteins c-myc; C497124:SP4 protein, human; D051706:Sp4 Transcription Factor; D011092:Polyethylene Glycols; D001120:Arginine; D006867:Hydrolases; C019723:arginine deiminase; D001124:Argininosuccinate Synthase