Abstract P5-06-12: Roscovitine Confers Tumor Suppressive Effect on Therapy-Resistant Breast Tumor Cells

Abstract

Abstract Background: Current clinical strategies for treating hormonal breast cancer involve the use of antiestrogens (AE) that block ERα functions, and aromatase inhibitors (AI) that decrease local and systemic estrogen production. Both of these strategies improve outcomes for ER+ve breast cancer; however development of therapy resistance is a major clinical problem. Emerging data implicate ER-signaling cross talk, and alteration in ER subtypes as the major causes of resistance. Current evidence suggest that ERa increases proliferation, while ERα decreases cell proliferation and the ratio between ERα and ERα is the driving force for tumor cell proliferation. Further, most downstream events in the resistance pathways converge upon modulation of cell cycle regulatory proteins; the most conspicuous of which is the activation of Cyclin Dependent Kinase 2 (CDK2) pathway. Roscovitine is one of the most frequently studied andused CDK2 inhibitor. In this study, we examined whether Roscovitine confers tumor suppressive advantage to therapy resistant breast epithelial cells by inhibiting CDK functions. Methods: We tested the effect roscovitine using three different therapy resistant model cells; (a) MCF-7-Tam (acquired Tamoxifen resistance model); (b) MCF-7-LTLT-ca (acquired Letrozole resistance model); (c) MCF-7-HER2 that exhibit Tamoxifen resistance (ER-growth factor signaling cross talk model). Cells were treated with Roscovitine (0-30 uM) for 24 hours and proliferation was measured after 48h using a luminescent cell viability assay (Promega). CDK activity, expression of ERα, β subtypes, ER-coregulators, and expression of ER target genes was measured by Western and PCR assays. To test the efficacy of Roscovitine in vivo, pre-clinical nude mice models bearing xenografts of therapy resistant cells were subjected to orally available Roscovitine (100mg/kg/tid/oral) for 10 days or treated with vehicle and tumor growth was monitored for a further 15 days. Results: The results showed that Roscovitine (10uM) significantly inhibited the growth of all three therapy resistant cells in cell proliferation and foci formation assays (P<0.05). FACS analysis revealed that Roscovitine treatment increased proportion of cells in G2-M phase. Therapy resistant cells treated with Roscovitine showed decreased CDK activity and low cyclin D levels indicating decreased cell proliferation. Interestingly, these studies also revealed an unexpected discovery that CDK inhibitor Roscovitine has potential to alter the ratio of Estrogen receptors with preferential upregulation of ERα with concomitant down regulation of ERα, ER-coregulators including AIB1 and PELP1. Results from xenograft studies showed that Roscovitine substantially reduced growth of therapy resistant tumors. Conclusions: Our results suggest that Roscovitine can be used to inhibit the growth of therapy resistant cells. Since many advanced and therapy resistant tumors exhibit loss or reduced expression of ERα, dual action of Roscovitine will provide a novel drug to inhibit both CDKs and to increase the expression of tumor suppressor ERα, thus has potential to serve as a double-edged sword to interfere with the resistance mechanisms. Supported by DOD Fellowship W81XWH-09-1-0010 (BCN) and NIH grant CA095681(RV). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-12.