Abstract A33: Characterization of signaling pathways associated with docetaxel resistance in prostate cancer

Abstract

Abstract Docetaxel (DTX) is the only treatment option for patients diagnosed with metastatic castration-refractory prostate cancer (CRPC). However, approximately 50% of patients do not respond to DTX, but are exposed to significant toxicity without direct benefit. This study aims to use mass spectrometry (MS) based techniques to characterize the perturbed intracellular signaling, particularly tyrosine phosphorylation, which contributes to the chemoresistant phenotype in CRPC patients, and thereby identify novel therapeutic targets. Two DTX-resistant prostate cancer cell lines (DU145-Rx & PC3-Rx) were established from their parental DTX-sensitive cell lines, DU145 & PC3, respectively, by a dose-escalation protocol. Phosphotyrosine profiling was performed by immunoaffinity coupled LC-MS/MS in combination with stable isotope labeling with amino acid in cell culture (SILAC). The chemosensitive and resistant cells were cultured in “light” and “heavy” SILAC media, respectively, and their isotopic labels were switched in a replicate experiment. Phosphotyrosine peptides were analysed on the state-of-the-art High Performance Liquid Chromatography MS/MS (Orbitrap Velos ETD). Raw MS spectra were quantified using MaxQuant software. DU145-Rx & PC3-Rx displayed 44 and 35-fold higher IC50 values, respectively, and significantly greater colony forming ability, in the presence of DTX, compared to the parental lines. Phosphotyrosine profiling of these cell lines identified a total of 365 phosphosites in the chemoresistant lines, corresponding to 227 proteins. 3 kinases and 3 cytoskeletal proteins showed a significant increase in tyrosine phosphorylation in both DTX-resistant models. These candidate proteins will be further characterized by gain- and loss-of-function approaches, both in vitro and in xenograft models, to elucidate the intracellular mechanism of chemoresistance, and to design therapeutic strategies to reverse the chemoresistant phenotype in CRPC. Citation Format: Brian Yong Lee, Falko Hochgrafe, Mark Raftery, Lisa G. Horvath, Roger J. Daly. Characterization of signaling pathways associated with docetaxel resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A33.