Abstract 837: Copper efflux transporter-mediated resistance to cisplatin

Abstract

Abstract The copper (Cu) efflux transporters ATP7A and ATP7B individually mediate resistance to the Pt-containing chemotherapeutic agents and have been identified as a primary pathway for the sequestration and/or export of these drugs. While the structure and transport function of ATP7A and ATP7B have been well studied, the mechanism by which they mediate resistance to the Pt drugs remains unknown. The experimental system consisted of the human fibroblast cell line Me32a, which does not express either ATP7A or ATP7B, and sublines previously produced by transfection that express either ATP7A (MeMNK) or ATP7B (MeWND). The MeMNK sub-line was 2.2-fold resistant to cisplatin whereas the MeWND cells had a 1.8-fold increase in IC50. The MeMNK and MeWND cell lines also contained a small population of cells that survived lethal dosages of DDP (10 - 20%). The MeMNK and MeWND cells were also 1.6- and 1.9-fold, respectively, resistant to the specific vacuolar proton pump (V-ATPase) inhibitor bafilomycin (Baf). Further investigation revealed that cells expressing ATP7A or ATP7B also had increased expression of V-ATPase subunits A, D and H at the mRNA level that corresponded to 8.6 - 14-fold increases at the protein level indicating that ATP7A and ATP7B perturb the proton transport system that accounts for progressive acidification of vesicles in the secretory pathway. MeMNK and MeWND cells were found to be 34- and 22-fold resistant to brefeldin A (BFA) which reversibly disrupts transport of proteins in the vesicle secretory pathway and specifically targets the ADP-ribosylation factor-1 (Arf1). This led us to investigate the Arf family of proteins and we found that the levels of Arf1, Arf3 and Arf4 were markedly reduced in cells expressing ATP7A and ATP7B at the RNA level; the reduction in Arf1 appears to account for the resistance to BFA and indicates the that function of Arf1 has been taken over by some other protein. Furthermore, we found large perturbations in the level of proteins that constitute the regulatory machinery of the vesicle secretory pathway including guanine nucleotide exchange factors (GEFs) and coat protein complexes in the MeMNK and MeWND cells. These findings indicate that ATP7A and ATP7B both produce quite large perturbations in the expression of proteins whose function is essential to the formation and trafficking of vesicles in the secretory pathway. As ATP7A and ATP7B have previously been shown to be capable of sequestering cisplatin into the microvesicular fraction of the MeMNK and MeWND cells, we conclude that these changes are linked to the ability of these Cu transporters to mediate cisplatin resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 837. doi:1538-7445.AM2012-837