NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG

The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression.KEAP1-NRF2通路（KEAP1-NRF2 pathway）可通过转录诱导参与抗氧化合成与解毒过程的基因，响应氧化应激信号，进而调控细胞氧化还原稳态。既往研究表明，KEAP1突变可使人黑色素瘤发生组成型NRF2激活，并使其对顺铂（cisplatin, CDDP）和达卡巴嗪（dacarbazine, DTIC）产生耐药性。本研究旨在明确热休克蛋白90抑制剂（HSP90 inhibitor）17-AAG是否可有效清除携带KEAP1突变的黑色素瘤，因NRF2靶基因NQO1是17-AAG生物活化的关键酶。在携带或不携带KEAP1突变的黑色素瘤及非小细胞肺癌细胞系中，NQO1的表达水平与17-AAG敏感性呈负相关。尽管存在野生型KEAP1，NQO1仍在正常黑素细胞及多株黑色素瘤细胞系中呈高表达，且其表达依赖于NRF2的激活。鉴于顺铂或达卡巴嗪均可产生活性氧（reactive oxygen species, ROS）以激活NRF2，本研究探究了上述药物能否使NQO1低表达的黑色素瘤细胞对17-AAG增敏。实验结果显示，在5株NQO1低表达细胞系中的4株内，可检测到17-AAG与顺铂联用的协同细胞毒性，但在携带KEAP1突变的细胞系中未观察到此协同效应。上述数据表明，17-AAG有望成为治疗携带KEAP1突变或高表达NQO1的黑色素瘤的潜在化疗药物。