Molecular and Cellular Pathobiology RhoJ Regulates Melanoma Chemoresistance by Suppressing Pathways That Sense DNA Damage

Melanomas resist conventional chemotherapeutics, in part, through intrinsic disrespect of apoptotic checkpoint activation. In this study, using an unbiased genome-wide RNA interference screen, we identified RhoJ and its effector PAK1, as key modulators of melanoma cell sensitivity to DNA damage. We find that RhoJ activates PAK1 in response to drug-induced DNA damage, which then uncouples ATR from its downstream effectors, ultimately resulting in a blunted DNA damage response (DDR). In addition, ATR suppression leads to the decreased phosphorylation of ATF2 and consequent increased expression of the melanocyte survival gene Sox10 resulting in a higher DDR threshold required to engage melanoma cell death. In the setting of normal melanocyte behavior, this regulatory relationship may facilitate appropriate epidermal melanization in response to UV-induced DNA damage. However, pathologic pathway activation during oncogenic transformation produces a tumor that is intrinsically resistant to chemotherapy and has the propensity to accumulate additional mutations. These findings identify DNA damage agents and pharmacologic inhibitors of RhoJ/PAK1 as novel synergistic agents that can be used to treat melanomas that are resistant to conventional chemotherapies. Cancer Res; 72(21); 5516–28. 2012 AACR. Introduction Melanoma, an aggressive and fatal malignancy resistant to current therapies, has increased in incidence and mortality over the last several decades (1). The DNA alkylating agents dacarbazine or temozolomide are still used to treat metastatic melanoma, but only 16% of patients respond with no improvement in overall survival (2). Melanomas are resistant to a spectrum of chemotherapies including cisplatin, paclitaxel, docetaxel, combination chemotherapy, immunotherapy, and even newly developed BRAF inhibitors (3, 4). Melanoma cells acquire the ability to invade adjacent tissues and resist chemotherapy early during their evolution (4), further underscoring the importance of developingmore effective treatments for this tumor. Melanoma cells upregulate multiple pathways that allow them to be intrinsically resistant to apoptosis (4)—this includes the activation of anti-apoptotic factors [inhibitor of apoptosis protein (IAP) family, FLIP], the downregulation of pro-apoptotic genes (APAF-1, BAD, BIM), and the activation of prosurvival pathways (NF-kB, AKT). Despite the fact that only 10% to 20% of melanomas contain p53 mutations, most melanoma tumors contain additionalmechanisms to suppress the function of p53, a central regulator of chemoresponsiveness (5). Transcriptional (6) and enzymatic regulators of melanogenesis (7) also modulate melanoma chemoresponsiveness. While extensive studies have identified multiple pathways that control melanoma chemoresistance, this information has not yet led to the development of more effective regimens to treat melanoma. Synthetic lethal functional genomics screening is an emerging strategy to identify drug targets for the rational design of synergistic agents with selective toxicity toward cancer cells (8, 9). A gene and a drug have a synthetic lethal relationship if mutation or depletion of that gene sensitizes cells to sublethal concentrations of a drug (8, 9). Synthetic lethal screening has been used to identify genes that regulate lung cancer, cervical cancer, and breast cancer chemoresistance (9–11). In this study, we use a systems-level screening approach to identify regulators of melanoma chemoresistance with the goal of discovering pathways that could be the molecular targets of new synergistic chemotherapy regimens. This screen identified RhoJ, a CDC42 homologue that regulates endothelial cell migration and angiogenesis (12), as a novel regulator of melanoma cell chemoresponsiveness. We find that RhoJ activates PAK1 in response to DNA damage, which then suppresses the ability of ATR to activate its downstream effectors Chk1 and ATF2. ATR suppression ultimately results in decreased DNA damage– induced apoptosis and the increased expression of prosurvival genes. Taken together, these studies uncover a new signaling pathway that coordinately regulates survival and chemoresistance. Authors' Affiliations: Departments of Dermatology and Biological Chemistry, University of California at Irvine, Irvine, California; and Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Anand K. Ganesan, Department of Dermatology andBiological Chemistry,University ofCalifornia at Irvine, Irvine,CA92697. Phone: 949-824-2926; Fax: 949-824-7454; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-0775 2012 American Association for Cancer Research. Cancer Research Cancer Res; 72(21) November 1, 2012 5516 on April 15, 2017. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst September 12, 2012; DOI: 10.1158/0008-5472.CAN-12-0775