Abstract 3489: Elucidation of taxane resistance in prostate cancer through RNA-Seq analysis of circulating tumor cells

Abstract

Abstract Prostate cancer is the most commonly diagnosed male cancer in the United States. Taxanes are the only established chemotherapy drugs proven to be effective in improving survival of men with advanced prostate cancer through disruption of the AR-signaling axis downstream of microtubule stabilization. However, there is significant heterogeneity in how patients respond to taxanes and most patients ultimately become refractory due to the development of drug resistance. Currently, the molecular basis of clinical taxane resistance in PC is poorly understood. Prostate cancer circulating tumor cells (P-CTCs) are often found in the peripheral blood of patients suffering from metastatic prostate cancer and have been clinically used as prognostic biomarker for metastatic progression and treatment outcome. The objective of this study is to identify clinically relevant mechanisms of taxane resistance through conducting RNA-Seq analysis in P-CTCs isolated from patients before, during and after they become refractory to taxane chemotherapy. To show feasibility of RNA-Seq experiments with limiting samples such as CTCs and given the presence of contaminating leucocytes, a pilot experiment was performed in which limiting numbers of prostate cancer cells (LNCaP) either pure or enriched following spiking into healthy donor blood, were analyzed by RNA-Seq. Matching healthy donor blood processed with the same enrichment protocol was used as germline control as well as control for the presence of contaminating leucocytes following CTC enrichment. Trimmed reads were aligned to human reference genome (hg38) using STAR. Determination of Fragments Per Kilobase of exon per Million mapped fragments (FPKM) was performed using Cufflinks and heat map was built based on the value of log10(FPKM+1). Gene expression analysis showed that markers of prostate (such as AR, PSMA, KLK3, KLK2, and AMACR) or epithelial lineage (such as EpCAM, CDH1, KRT8 and KRT18) were detected in both pure LNCaP cells-regardless of amount- as well as limited number of captured LNCaP cells in the presence of contaminating leucocytes. In contrast, healthy donor blood was negative for the prostate and epithelial lineage markers and positive for the leucocyte specific markers (such as CD45 and CD16). Gene set enrichment analysis (GSEA) indicated significant enrichment for Andorgen response, MYC, MTOR and RB related pathways in pure and captured LNCap cells compared with healthy donor blood. These data clearly show that by using RNA-Seq we can detect the prostate and epithelial specific gene signatures of limited number of spiked prostate cancer cells using the microfluidic device. Ongoing work includes RNA-Seq analysis of P-CTCs isolated from patients before and after taxane treatment, in order to detect differentially expressed genes, pathways, and potentially driver somatic mutations associated with clinical taxane resistance. Citation Format: Jiaren Zhang, Ada Gjyrezi, Prashant Thakkar, Giuseppe Galletti, Akanksha Verma, Olivier Elemento, Paraskevi Giannakakou. Elucidation of taxane resistance in prostate cancer through RNA-Seq analysis of circulating tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3489.