Resistance to arginine deiminase treatment in melanoma cells is associated with induced argininosuccinate synthetase expression involving c-Myc/HIF-1α/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-1α and a GC-box by Sp4. Through ChIP assays, we showed that under noninduced conditions, the E-box was bound by HIF-1α in all the three melanoma cell lines. Under arginine depletion conditions, HIF-1α 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-1α 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-1α 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.

Prognostic and Therapeutic Impact of Argininosuccinate Synthetase 1 Control in Bladder Cancer as Monitored Longitudinally by PET Imaging

“…This is in contrast to recent work with ADI-PEG20 in ASS1-negative melanoma xenografts in which there was no effect on TK1 expression and FLT-PET metabolic responses were not detected (41). These and previous results using 2 [ 18 F]fluoro-2-deoxy-D-glucose tracers may be explained in part by the differential regulation of ASS1 promoter with HIF-1a-mediated repression in melanoma compared with epigenetic silencing identified in several other arginine auxotrophic cancers (9,16,42).…”

“…Arginine fuels an array of biosynthetic reactions, including proteins, nitric oxide (NO), polyamines, agmatine, and the amino acids proline and glutamate, and therefore may modulate tumorigenesis at multiple levels (7). Inactivation of the pleiotropic enzyme, argininosuccinate synthetase 1 (ASS1), with key roles in the urea cycle, citrulline-NO cycle, and arginine biosynthesis has emerged as a principal driver of tumoral arginine auxotrophy, with evidence for epigenetic silencing and hypoxia-inducible factor-1a (HIF-1a)-mediated transcriptional repression of ASS1 (5,8,9). Significantly, ASS1 loss has been associated with decreased overall survival in ovarian cancer and myxofibrosarcoma, and reduced metastasis-free survival in osteosarcoma, implicating a tumor-suppressor function for this metabolic gene (10)(11)(12).…”

Specificity Protein 1 (Sp1) Oscillation Is Involved in Copper Homeostasis Maintenance by Regulating Human High-Affinity Copper Transporter 1 Expression

“…Establishment of SCLC-hCtr1-wt and SCLC-hCtr1-DN stable cell lines has been described previously (Liang et al, 2009). Expression-recombinant DNA for Sp1-wt, and Sp2, Sp3, and Sp4 were described previously (Tsai et al, 2009). Recombinant LexA-VP16 and reporter pLG-(Gal4) 5 -(Lex4) 2 -E1B-Luc (LexA-Luc) were obtained from Mark Ptashne (Memorial Sloan-Kettering Cancer Center, New York, NY).…”

“…The ChIP assay was performed with a kit (Millipore, Billerica, MA) following the manufacturer's instructions. The hCtr1 and Sp1 promoter DNA sequences were determined by PCR using appropriate primer sets (available upon request) according to procedures described previously (Tsai et al, 2009).…”

Arginine enzymatic deprivation and diet restriction for cancer treatment

“…In vitro studies using both adherent and non-adherent ASS-deficient SCLC cell lines demonstrated that ADI-PEG20 caused dose-dependent anti-proliferative efficacy by inducing autophagy, followed by caspase-independent cell death (Kelly et al, 2012). Additionally, prolonged treatment with ADI-PEG20 may lead to the induction of ASS expression and the activation of other cellular pathways associated with resistance to apoptosis, possibly limiting the overall treatment window for ADI (Kim et al, 2009;Tsai et al, 2009). SCLCs xenografts treated continuously with 5 IU ADI-PEG20 study demonstrated a sustained response to ADI-PEG20 for the 90-day period of the study, suggesting that at least at this dose, profound resistance to the anti-proliferative effect of ADI-PEG20 had not been induced (Kelly et al, 2012).…”