Abstract 706: The serine/threonine kinase Pim-1 promotes drug resistance mediated by the ATP-binding cassette multidrug resistance protein breast cancer resistance protein (BCRP, ABCG2) by stabilizing higher-order BCRP multimers
Authors: Natarajan Karthika , Burcu Mehmet , Baer Maria R. 
Published: June 2012

Abstract
Background: The ATP-binding cassette (ABC) multidrug resistance protein breast cancer resistance protein (BCRP, ABCG2) is a half-transporter that was thought to function as a homodimer, but has been shown to exist as a homotetramer in the membranes of drug resistant cells and to also form higher-order multimers. The serine/threonine kinase Pim-1, expressed in both hematologic malignancies and solid tumors, phosphorylates BCRP at T362 and promotes its multimerization and drug efflux function. Inhibiting Pim-1 sensitizes BCRP-overexpressing cells to BCRP substrate chemotherapy drugs. Pim-1 phosphorylation has been previously shown to stabilize some of its substrate proteins, but the mechanism of its effect on BCRP is unknown. We sought to delineate the mechanism of Pim-1 effect on BCRP. Methods: BCRP-overexpressing 8226/MR20 myeloma cells were treated with the protein translation inhibitor cycloheximide (10µg/ml) in the presence and absence of the Pim-1 inhibitor SGI-1776 (SuperGen, Inc., Dublin, CA) (1μM) for 0, 2, 4, 6 and 8 hours, and BCRP levels were measured by densitometric analysis, normalized for GAPDH levels, following Native PAGE as well as SDS-PAGE electrophoresis and immunoblotting. 8226/MR20 cells were also treated with the proteasome inhibitor MG-132 (2 μM) or the lysosome inhibitor bafilomycin A1 (10 nM) in the presence of cycloheximide and in the presence and absence of SGI-1776. Results: Under Native PAGE electrophoresis, BCRP migrated predominantly as a tetramer, with lower levels of higher-order multimers. Half-lives were approximately 6 and 2 hours, respectively. In the presence of SGI-1776, the half-life of higher-order BCRP multimers decreased to approximately 1 hour, while BCRP tetramer levels increased, consistent with destabilization of higher-order BCRP multimers and conversion to tetramers. Proteasomal inhibition with MG-132 and to a lesser extent lysosomal inhibition with bafilomycin A1 increased the half-life of both BCRP tetramers and higher order multimers. However, on co-incubation with SGI-1776, bafilomycin A1 caused marked stabilization of higher-order BCRP multimers, with a lesser effect on tetramers, while proteasomal inhibition with MG-132 had no effect, consistent with Pim-1 protecting high-order multimers from lysosomal degradation. Finally, BCRP dimers measured by SDS-PAGE electrophoresis under non-reducing conditions increased with time during cycloheximide treatment and the increase was more rapid in the presence of SGI-1776, consistent with more rapid destabilization of BCRP multimers when Pim-1 kinase was inhibited. Conclusion: Phosphorylation of BCRP by Pim-1 stabilizes BCRP higher-order multimers, protecting them from dissociation and degradation in the lysosome.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 706. doi:10.1158/1538-7445.AM2011-706