Improving radiotherapy in cancer treatment: Promises and challenges Improving radiotherapy in cancer treatment: Promises and challenges

Melanomas and other cancers that do not express argininosuccinate synthetase (AS), the rate-limiting enzyme for arginine biosynthesis, are sensitive to arginine depletion with pegylated arginine deiminase (ADI-PEG20). However, ADI resistance eventually develops in tumors because of AS upregulation. Although it has been shown that AS upregulation involves c-Myc, the underlying mechanisms remain unknown. Here we show that ADI-PEG20 activates Ras signaling and the effector extracellular signal–regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT/GSK-3β kinase cascades, resulting in phosphorylation and stabilization of c-Myc by attenuation of its ubiquitin-mediated protein degradation mechanism. Inhibition of the induced cell signaling pathways using PI3K/AKT inhibitors suppressed c-Myc induction and enhanced ADI-mediated cell killing. Notably, in an animal model of AS-negative melanoma, combination therapy using a PI3K inhibitor plus ADI-PEG20 yielded additive antitumor effects as compared with either agent alone. Taken together, our findings offer mechanistic insight into arginine deprivation metabolism and ADI resistance, and they illustrate how combining inhibitors of the Ras/ERK and PI3K/AKT signaling pathways may improve ADI-PEG20 anticancer responses. Cancer Res; 72(10); 2622–33. ©2012 AACR.

Three mechanisms have been proposed for the role of glutathione (GSH) in regulating cisplatin (CDDP) sensitivities that affects its ultimate cell-killing ability: (i) GSH may serve as a cofactor in facilitating multidrug resistance protein 2- (MRP2-) mediated CDDP efflux in mammalian cells, sinceMRP2-transfected cells were shown to confer CDDP resistance; (ii) GSH may serve as a redox-regulating cytoprotector based on the observations that many CDDP-resistant cells overexpress GSH andγ-glutamylcysteine synthesis (γ-GCS), the rate-limiting enzyme for GSH biosynthesis; (iii) GSH may function as a copper (Cu) chelator. Elevated GSH expression depletes the cellular bioavailable Cu pool, resulting in upregulation of the high-affinity Cu transporter (hCtr1) which is also a CDDP transporter. This has been demonstrated that overexpression of GSH by transfection withγ-GCS conferred sensitization to CDDP toxicity. This review describes how these three models were developed and critically reviews their importance to overall CDDP cytotoxicity in cancer cell treatments.

Recent studies have shown that the mammalian high-affinity copper transporter encoded by Ctr1 is involved in the uptake of cisplatin. However, the roles of hCtr1 in cisplatin-sensitive and cisplatin-resistant mammalian cells have not been investigated. Here, we show that, of five cisplatin-resistant cell lines, only one (SR2) exhibited substantial reduction in hCtr1 expression as compared with that in its sensitive line small cell lung cancers (SCLC), whereas copper efflux transporters ATP7A and ATP7B were not significantly altered. SR2 exhibited cross-resistance to carboplatin but not to oxaliplatin. Transfection of expression hemagglutinin-tagged hCtr1 cDNA into SCLC and SR2 cells enhanced the uptake of copper, cisplatin, carboplatin, and oxaliplatin, suggesting that hCtr1 transporter can transport these platinum-based drugs. Whereas increased sensitivities to all these platinum drugs were observed in hCtr1-transfected SCLC cells, increased sensitivities to cisplatin and carboplatin but not to oxaliplatin were observed in hCtr1-transfected SR2 cells. These results suggest that SR2 acquired an additional unique intracellular resistance mechanism to oxaliplatin. Finally, using hCtr1 deletion mutants, we showed that the NH2-terminal domain of hCtr1 was involved in transporting all these platinum-based antitumor agents. These results collectively show the importance of hCtr1 in the transport of platinum-based antitumor agents in cisplatin-sensitive and cisplatin-resistant variants.

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. [Mol Cancer Ther 2009;8(12):3223–33]