We are investigating the role of the Steroid and Xenobiotic Receptor (SXR) in the induction of drug resistance. SXR is an orphan nuclear receptor that is activated by a diverse class of compounds including steroids, bile acids, and xenobiotics. Ligand binding allows SXR to heterodimerize with the Retinoid X Receptor (RXR) and bind to response elements in promoters of its target genes, such as CYP3A4, CYP2C8, MRP2, and MDR1, upregulating their transcription. As a result, SXR may play a central role in induction of drug resistance by turning on transcription of genes required for drug detoxification. As previously reported (AACR abstract #534, 2003), we stably transfected a human SXR cDNA into MCF-7 cells, which do not express SXR. However, despite high levels of expression, SXR was only modestly functional as a ligand-dependent inducer of MDR1 expression in selected clones. We hypothesized that SXR may not be translocating into the nucleus to activate transcription of its target genes. To determine if the lack of MDR1 induction was due to aberrant localization of SXR, we performed immunofluorescence for SXR in the MCF-7/SXR clones. Our results show cytoplasmic localization of SXR in the absence of ligand and nuclear localization in the presence of ligand, indicating that SXR is able to translocate into the nucleus with the addition of an activator. Therefore, overexpression of functional SXR in cells that do not normally express the gene is not sufficient to gain full ligand-inducible expression of target genes seen in permissive cell types expressing even lower levels of endogenous SXR. We are investigating factors that might account for cell-type specificity. Further, to determine if SXR is necessary for induction of drug resistance genes in a cell line that expresses the functional endogenous receptor, we are knocking down SXR expression in LS180 colon carcinoma cells using RNA interference (RNAi). After designing three pol III-driven siRNAs against different regions of SXR, we found two sites that downregulate SXR expression in transient transfections. We have stably transfected LS180 cells with one of these siRNA constructs or with empty vector as a control. Once we have confirmed downregulation of SXR in these clones by Western analysis, we will treat the cells with known SXR activators and analyze the expression of MDR1 by Northern, Western, and flow cytometry. If MDR1 induction is dependent on SXR, then we expect that MDR1 expression will not increase with the addition of SXR ligands in our siRNA transfectants. Since overexpression of MDR1 is known to cause resistance to a variety of drugs, we believe that SXR may be an integral player in induction of drug resistance. Using a colony forming assay, we will determine if LS180 cells have increased resistance to MDR1/Pgp substrates (paclitaxel) when treated with SXR activators (rifampicin) and if downregulation of SXR prevents this drug resistance from occurring.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]