Expression analysis of prostate cancer Hoechst 33342 side populations identifies the differing origins of CRPC

Abstract

Introduction & Objectives: We have isolated Hoechst 33342 side population cells with stem like (Distal Side Population (DSP)), transiently amplifying (Proximal Side Population (PSP)) and terminally differentiated (Non Side Population (NSP)) characteristics from hormone sensitive and castrate resistant prostate tumours with the aim of determining from where the tumour initiating cells derive and to identify potential therapeutic targets. Materials & Methods: Prostate epithelial cells were sub-fractionated by FACS using Hoechst 33342 and CD133 to generate DSP (Hoechstlow , CD133-ve), PSP (Hoechstlow , CD133+ve) and NSP (Hoechsthigh) subpopulations from hormone unmanipulated (n=2) and hormone manipulated patients (n=2). Sub-populations were characterised on a Hu133 plus 2 micro-array and compared with Hoechst 33342 sub-populations from patients with benign hyperplasia or normal prostate tissue from men aged <40. Differential gene expression (DGE) was assessed in the Partek Genomic Suite with targetable pathway analysis by Ingenuity Pathway Analysis (IPA (Qiagen)) and QUADrATiC tool (Queens University Belfast) packages. Results: Hierarchical cluster analysis of DGEs showed DSPs from PCa patients are genetically distinct from BPH and normal prostate samples. PCa DSP samples based on hormone manipulation shows differential gene expression; with DSP from hormone manipulated samples forming a distinct genetic subgroup whilst those from hormone unmanipulated samples share genetic features with PCa, BPH and normal samples. Ingenuity pathway analysis (IPA) highlighted significant changes in 34 pathways between DSP and PSP in hormone manipulated samples as compared with 1 in the unmanipulated samples. QUADrATiC exploration of gene expression connectivity mapping towards FDA approved drugs targets identifies potential activity specifically associated with stem cell survival pathways for hormone manipulated DSP and insulin secretagogues for unmanipulated DSP. Conclusions: Our differential expression pathway and drug target analysis suggests that the tumour initiating cell for primary prostate cancer is within the Hoechst 33342low CD133+ve PSP population but following hormone manipulation, the tumour initiating cell arises from the stem like Hoechst 33342low CD133-ve DSP.