Abstract 1529: Pim-1 kinase protects P-glycoprotein from degradation and enables its glycosylation and cell surface expression
Authors: Maria R. Baer, Yingqiu Xie, Mehmet Burcu, Yun Qiu
Journal: Cancer Research (2010)

Abstract The ATP-binding cassette (ABC) protein P-glycoprotein (Pgp; ABCB1) is overexpressed in a broad range of malignancies, and its overexpression consistently correlates with adverse treatment outcome. The oncogenic serine/threonine kinase Pim-1 is also overexpressed in diverse malignances and phoshorylates multiple substrate proteins that are important in key cellular processes. Pim-1 plays a role in drug resistance, as we recently demonstrated that it phosphorylates the ABC transporter breast cancer resistance protein (BCRP; ABCG2) at threonine residue 362 and thereby promotes its multimerization and cell surface translocation. Pgp also contains a Pim-1 phosphorylation consensus sequence, QDRKLS, located at S683, between nucleotide-binding domain 1 and transmembrane domain 1. We therefore hypothesized that Pim-1 also regulates Pgp. Pgp is exported from the endoplasmic reticulum (ER) as a 150 kDa core-glycosylated species that undergoes glycosylation to mature 170 kDa Pgp, then translocates to the cell surface and mediates drug efflux; alternatively, 150 kDa Pgp is cleaved to a 130 kDa proteolytic product by ER proteases or undergoes ubiquitination and proteasomal degradation. Pgp+ HL60/VCR, 8226/Dox6 and OVCAR-8-Pgp cells were studied. Pim-1 and Pgp interaction was studied in a GST pull-down assay and phosphorylation in an in vitro kinase assay. Pim-1 knockdown and kinase inhibitor (quercetagetin) effects on Pgp expression were studied by immunoblotting and flow cytometry and effects on Pgp stability by immunoblotting following cycloheximide treatment. Drug sensitivity was studied in a colony growth assay. Pim-1 directly interacted with and phosphorylated Pgp both in intact cells and in vitro. Pim-1 knockdown or inhibition decreased both cellular and cell surface expression of 170 kDa Pgp, in association with accumulation of 130 kDa Pgp and increased ubiquitination and proteasomal degradation. Of note, total cellular Pgp increased when new protein synthesis was inhibited in conjunction with inhibition of both Pim-1 and proteasomal degradation, suggesting that Pim-1 stabilizes previously synthesized core-glycosylated Pgp and enables its glycosylation. Accumulation of core-glycosylated Pgp in the presence of the glycosylation inhibitor 2-deoxy-D-glucose was abrogated when Pim-1 kinase activity was inhibited, consistent with this hypothesis. Finally, Pim-1 inhibition sensitized Pgp-overexpressing cells to doxorubicin. Thus Pim-1 regulates Pgp expression by protecting core-glycosylated Pgp from protease- and proteasome-mediated degradation and enabling Pgp glycosylation and cell surface translocation and thus Pgp-mediated drug resistance. Pim-1 inhibitors are entering clinical trials and may provide a novel approach to abrogating Pgp-mediated drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1529.