NRF2-regulated genes SLC7A11 and NQO1 confer chemoresistance in mesotheliomas

3250 Mesothelioma is an aggressive and treatment-resistant tumor with increasing prevalence throughout the world. Expression of genes encoding antioxidants ( e.g ., glutathione) and xenobiotic detoxification enzymes has been shown to increase in mesothelioma cells, which may play an important role in anticancer drug resistance. SLC7A11 (xCT) is an amino acid transporter designated as system x c - , responsible for the cystine entry in exchange for intracellular glutamate. Once inside the cell, cystine is rapidly reduced to cysteine, the rate limiting amino acid for glutathione biosynthesis. Our previous studies showed that SLC7A11 overexpression in lung and ovarian cancers maintained a high intracellular glutathione level and conferred chemoresistance to multiple anticancer agents. The present study investigated the expression and activity of SLC7A11 and related gene NADP[H] quinine oxidoreductase 1 (NQO1), both of which are positively regulated by the transcriptional factor NRF2 and negatively regulated by the NRF2 suppressor gene KEAP1. We examined the mRNA and protein levels of SLC7A11, NQO1, NRF2 and KEAP1 in eight mesothelioma cell lines using real-time RT-PCR, dot blot, Western blot and immunocytochemistry techniques. We also inhibited the transport activity of SLC7A11 using glutamate or (S)-4-carboxyphenylglycine and examined the effects of SLC7A11 inhibition on the sensitivity of tumor cells to anticancer drugs geldanamycin and L-alanosine. Our results indicated that SLC7A11 and NQO1 are expressed at different levels in mesothelioma cell lines and their expression levels correlated with the degree of drug resistance in these cells. In cell lines with high levels of SLC7A11 expression (e.g., H2461), SLC7A11 inhibition significantly increased the growth inhibitory potency of geldanamycin, while the potency of L-alanosine, an amino acid analog and potential substrate of SLC7A11, was reduced. Although we hypothesized that constitutive activation of NRF2-regulated SLC7A11 and NQO1 may be due to the loss of function of KEAP1, no somatic mutation(s) in exon-coding regions of the KEAP1 gene have been detected in the eight cell lines by sequencing analysis of the tumor genomic DNA. These findings suggest a potential role of NRF2-regulated genes such as SLC7A11 and NQO1 in chemoresistance of mesothelioma. The mechanisms for overexpression of SLC7A11 and NQO1 in the subset of mesotheliomas need further investigation. Understanding the function of these genes and their regulatory pathways may lead to effective pharmacological treatment for this malignancy.