Histone deacetylase inhibitors (HDI) have exhibited some efficacy in clinical trials, but it is clear that their most effective applications have yet to be fully determined. In this study, we show that HDIs influence the expression of a common polymorphic variant of the chemotherapy drug efflux transporter ABCG2, which contributes to normal tissue protection. As one of the most frequent variants in human ABCG2, the polymorphism Q141K impairs expression, localization, and function, thereby reducing drug clearance and increasing chemotherapy toxicity. Mechanistic investigations revealed that the ABCG2 Q141K variant was fully processed but retained in the aggresome, a perinuclear structure, where misfolded proteins aggregate. In screening for compounds that could correct its expression, localization, and function, we found that the microtubule-disrupting agent colchicine could induce relocalization of the variant from the aggresome to the cell surface. More strikingly, we found that HDIs could produce a similar effect but also restore protein expression to wild-type levels, yielding a restoration of ABCG2-mediated specific drug efflux activity. Notably, HDIs did not modify aggresome structures but instead rescued newly synthesized protein and prevented aggresome targeting, suggesting that HDIs disturbed trafficking along microtubules by eliciting changes in motor protein expression. Together, these results showed how HDIs are able to restore wild-type functions of the common Q141K polymorphic isoform of ABCG2. More broadly, our findings expand the potential uses of HDIs in the clinic. Cancer Res; 72(14); 3642–51. ©2012 AACR.

Background: Resistance to chemotherapy remains one of the principle obstacles to the treatment of colon cancer. In order to identify the molecular mechanism of this resistance, we investigated the role of the steroid and xenobiotic receptor (SXR) in the induction of drug resistance. Indeed, this nuclear receptor plays an important role in response to xenobiotics through the upregulation of detoxification genes. Following drug treatments, SXR is activated and interacts with the retinoid X receptor (RXR) to induce expression of some genes involved in drug metabolism such as phase I enzyme (like CYP), phase II enzymes (like UGT) and transporters (e.g. MDR1).Results: In this study, we have shown that endogenous SXR is activated in response to SN-38, the active metabolite of the anticancer drug irinotecan, in human colon cancer cell lines. We have found that endogenous SXR translocates into the nucleus and associates with RXR upon SN-38 treatment. Using ChIP, we have demonstrated that endogenous SXR, following its activation, binds to the native promoter of the CYP3A4 gene to induce its expression. RNA interference experiments confirmed SXR involvement in CYP3A4 overexpression and permitted us to identify CYP3A5 and MRP2 transporter as SXR target genes. As a consequence, cells overexpressing SXR were found to be less sensitive to irinotecan treatment.Conclusions: Altogether, these results suggest that the SXR pathway is involved in colon cancer irinotecan resistance in colon cancer cell line via the upregulation of select detoxification genes.

Histone deacetylase inhibitors (HDACi) induce hyperacetylation of histones by blocking HDAC catalytic sites. Despite regulatory approvals in hematological malignancies, limited solid tumor clinical activity has constrained their potential, arguing for better understanding of mechanisms of action (MOA). Multiple activities of HDACis have been demonstrated, dependent on cell context, beyond the canonical induction of gene expression. Here, using a clinically relevant exposure duration, we established DNA damage as the dominant signature using the NCI-60 cell line database and then focused on the mechanism by which hyperacetylation induces DNA damage. We identified accumulation of DNA–RNA hybrids (R-loops) following romidepsin-induced histone hyperacetylation, with single-stranded DNA (ssDNA) breaks detected by single-cell electrophoresis. Our data suggest that transcription-coupled base excision repair (BER) is involved in resolving ssDNA breaks that, when overwhelmed, evolve to lethal dsDNA breaks. We show that inhibition of BER proteins such as PARP will increase dsDNA breaks in this context. These studies establish accumulation of R-loops as a consequence of romidepsin-mediated histone hyperacetylation. We believe that the insights provided will inform design of more effective combination therapy with HDACis for treatment of solid tumors. Implications: Key HDAC inhibitor mechanisms of action remain unknown; we identify accumulation of DNA–RNA hybrids (R-loops) due to chromatin hyperacetylation that provokes single-stranded DNA damage as a first step toward cell death.