New approaches to multidrug resistance in cancer

ED04-03 We first described the cloning of the human MDR1 (ABCB1) gene encoding P-glycoprotein (P-gp), a multidrug efflux pump, in 1986 (Chen et al., Cell 47: 381-389, 1986). Since then, the important function of this ABC transporter in conferring resistance to cytotoxic anti-cancer drugs and its role in normal pharmacokinetics has been described in some detail, but effective strategies to use this information to improve clinical treatment of cancer and enhance drug delivery to target organs such as the brain have yet to be devised. Two major discoveries have revolutionized our ability to use information about ABC transporters to improve therapy of cancer. The first was the finding that there are 48 human ABC transporters, many of which, including MRP (ABCC) family members (MRP1 or ABCC1 and several others) and ABCG2 can transport cytotoxic drugs out of cells. Thus, the possibility of predicting drug resistance based on expression of ABC transporters and other drug resistance genes may soon be realized (Gottesman et al., Nat. Rev. Cancer 2: 48-58, 2002 and Szakacs et al., Nat. Rev. Drug Discov. 5: 219-234, 2006). In addition, we have recently observed that a common haplotype form of ABCB1 that includes synonymous (silent) mutations can change the function of P-gp for some substrates and inhibitors without changing cellular protein levels or plasma membrane localization (Kimchi-Sarfaty et al., Science 315: 525-528, 2007 and Sauna et al., Cancer Res. 67: 9609-9612, 2007). This raises the possibility of predicting, based on a haplotype of P-gp and potentially of other multidrug-resistance genes, how cytotoxic drugs will be handled by the body and how best to develop inhibitors that can reverse multidrug resistance. Many different ABC transporters and other gene products contribute to multidrug resistance in cancer There are two major hypotheses relating to multidrug resistance in cancer. The first is that the majority of treatment failures relate to the intrinsic growth properties of the cancers. The second is that there are discrete genes, not directly related to the malignant transformation process, that confer resistance to anti-cancer drugs. These two ideas are not mutually exclusive since it is clear that the transformation process per se can turn on expression of multidrug-resistance genes such as ABCB1, and that development of resistance can occur during chemotherapy of tumors in which the growth properties do not change dramatically. Our work has focused on understanding the role of ABC transporters and other MDR genes in cancer cells in vitro and more recently in cancers obtained from patients. As noted, many other ABC transporters can be shown to protect cells from the cytotoxic effects of anti-cancer drugs (Szakacs et al., Nat. Rev. Drug Discov. 5: 219-234, 2006). These include 6 ABC transporters whose expression may increase as cancer cells are exposed to cytotoxic drug selection (ABCA2, ABCB1, ABCC1, ABCC2, ABCC4 and ABCG2). In addition, overexpression of 6 other ABC transporters by transfection results in drug resistance (ABCB11, ABCC3, ABCC5, ABCC6, ABCC10 and ABCC11). To determine whether other ABC transporters could contribute to resistance to cytotoxic agents, we correlated expression of transporters with patterns of drug resistance in the NCI-60 cancer cell lines (Szakacs et al., Cancer Cell 6: 129-37, 2004). Expression of up to half of the 48 known human transporters was shown to correlate with resistance to specific cytotoxic drugs. A similar analysis of solute carrier protein (SLC and SLCO) expression indicated that uptake transporters could also determine cancer cell sensitivity to certain drugs (M. Okabe, G. Szakacs, M. Hall, and M. M. Gottesman, in preparation). A review of the literature, and consultations with other labs that study multidrug resistance, as well as a tabulation of the many genes studied in our own laboratory that have been shown to contribute to multidrug resistance, resulted in a list of 380 gene products whose expression could plausibly be correlated with multidrug resistance in cancer. We sought to create a tool that could reliably be used to detect the presence of these gene products by measuring their mRNA levels in tissue samples from cancer patients. We settled on the ABI TaqMan® Low Density Array (TLDA) system, which uses a microfluidic chip containing probes specific for these 380 drug-resistance genes as well as several housekeeping genes as controls. The gene products include the transporters as well as metabolizing systems, conjugating systems, and the apoptosis pathway. The advantages of this system compared to standard microarrays are its increased sensitivity, the linearity of the assay over several logs, its reproducibility, and its reliability with various RNA samples. The major downside to this approach is that we are limited to genes that have already been associated with drug-resistance, making new gene discovery impossible. However, this approach allows us to test the credible hypothesis that many of the drug-resistance genes of consequence have already been discovered in cultured cancer cells. The TLDA assay has been applied to samples from several different cancers, with an emphasis on ovarian cancer, and the data are currently under analysis. Silent polymorphisms in the ABCB1 gene affect its function and drug efficacy For several years, reports have appeared in the literature linking altered drug pharmacokinetics to so-called "silent" or synonymous polymorphisms in the coding region of the ABCB1 gene (Sauna et al., Pharmacogenomics 8: 527-532, 2007 and Brinkmann et al., Drug Discov. Today 6: 835-839, 2001), especially the C3435T polymorphism. Subsequently, this polymorphism was shown to be part of a haplotype including C1236T and G2677T/A of which the former is also synonymous. In individual expression experiments, it was possible to show that none of these polymorphisms alone had a detectable effect on ABCB1 function, but altered substrate and inhibitor specificity could be demonstrated for the full haplotype or any two polymorphisms that included C3435T (Kimchi-Sarfaty et al., Science 315: 525-528, 2007). Detailed experiments indicated that for all polymorphisms and the full haplotype, equal amounts of mRNA and protein were made and the protein was properly localized to the plasma membrane. However, some substrates were not as well transported and some inhibitors were not as effective when tested with the haplotype ABCB1 transporter. Further analysis showed that although expressed on the cell surface, the haplotype transporter had an altered conformation as detected with a conformation-specific monoclonal antibody and by tryptic peptide analysis (Kimchi-Sarfaty et al., Science 315: 525-528, 2007). More recently, we have obtained evidence for a similar alteration in ABCB1 stably transfected transporting epithelia (LLC-PK1 cells). Ribosome progression through ABCB1 mRNA in vitro shows a strong toeprint at the position of the C3435T polymorphism. We interpret these results to suggest that change in mRNA structure, or possibly altered codon usage in the ABCB1 mRNA that transiently delays translation results in altered conformation in the ABCB1 (P-gp) protein. This is plausible since P-gp translation and folding are co-translationally controlled. There are many implications of these studies for personalizing cancer treatment. Patients whose cancers have these polymorphisms may be more or less resistant to certain anti-cancer drugs, and may be less responsive to inhibitors intended to reverse multidrug resistance. Studies using reversing agents have not to date examined the haplotype of the cancer P-gp and this may prove necessary in the future. Furthermore, drug pharmacokinetics can be affected by this haplotype, so drug dosage may have to be modified for patients carrying the haplotype form of P-gp. Whether or not similar polymorphisms occur for other transporters and drug resistance genes remains to be determined.