Aberrant Regulation of the MRP3 Gene in Non-small Cell Lung Carcinoma

Introduction: Multidrug-resistant protein-3 (MRP3), a membrane-bound transporter, facilitates efflux of toxic compounds, including certain chemotherapies, out of cells. Aberrant MRP3 expression has been linked to drug resistance in non-small cell lung carcinoma (NSCLC). We sought to determine if tumor MRP3 expression patterns correlate with the mutational status of upstream regulators, including nuclear factor erythroid-2-related factor 2 (Nrf2) and its functional repressor Keap1 in NSCLC cell lines and patient samples. Methods: To identify putative Nrf2-binding sites in the MRP3 promoter and to evaluate Keap1, Nrf2, and p53 mutation status in four cell lines and 33 NSCLC surgically resected tumor specimens with regard to their impact on MRP3 levels. Results: Chromatin immunoprecipitation analysis of the MRP3 promoter revealed an almost threefold increase in Nrf2 binding to the third putative Nrf2-binding sequence distal to the start site, demonstrating direct regulation of MRP3 by Nrf2. In NSCLC cell lines, elevated Nrf2 protein was observed in cell lines with increased MRP3 RNA expression. In patient tumor specimens, the presence of mutations in Keap1/Nrf2 correlated with MRP3 RNA levels (p < 0.05). p53 mutations were observed in 33% of cases, and all Keap1 mutant-positive tumors possessed a p53 mutation (n = 5; p = 0.0019). Conclusions: We demonstrate direct involvement between the transcription factor Nrf2 and the MRP3 promoter, which leads to the up-regulation of the MRP3 gene. In addition, we found a statistically significant correlation between the presence of Keap1/Nrf2 mutations and increased MRP3 messenger RNA levels in our NSCLC patient samples.