RNA sequencing reveals upregulation of a transcriptomic program associated with stemness in metastatic prostate cancer cells selected for taxane resistance

// Christina K. Cajigas-Du Ross 1, 3 , Shannalee R. Martinez 1, 3 , Leanne Woods-Burnham 1, 3 , Alfonso M. Durán 1, 3 , Sourav Roy 2 , Anamika Basu 1 , Joshua A. Ramirez 1 , Greisha L. Ortiz-Hernández 1, 3 , Leslimar Ríos-Colón 1, 3 , Evgeny Chirshev 3 , Evelyn S. Sanchez-Hernandez 1, 3 , Ubaldo Soto 3 , Celine Greco 4, 5 , Claude Boucheix 4, 5 , Xin Chen 6 , Juli Unternaehrer 3 , Charles Wang 3, 6 and Carlos A. Casiano 1, 3, 7 1 Center for Health Disparities and Molecular Medicine, Loma Linda University School of Medicine, Loma Linda, CA, USA 2 Department of Entomology and Institute for Integrative Genome Biology, University of California Riverside, Riverside, CA, USA 3 Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, USA 4 Inserm, UMR-s935, Villejuif, France 5 University of Paris XI, Université Paris-Saclay, Orsay, France 6 Center for Genomics, Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, CA, USA 7 Department of Medicine, Loma Linda University School of Medicine, Loma Linda, CA, USA Correspondence to: Carlos A. Casiano, email: ccasiano@llu.edu Keywords: prostate cancer; RNA sequencing; docetaxel resistance; cancer stem cells; therapeutic targeting Received: April 18, 2018      Accepted: June 13, 2018      Published: July 13, 2018 ABSTRACT Patients with metastatic castration-resistant prostate cancer (mCRPC) develop resistance to conventional therapies including docetaxel (DTX). Identifying molecular pathways underlying DTX resistance is critical for developing novel combinatorial therapies to prevent or reverse this resistance. To identify transcriptomic signatures associated with acquisition of chemoresistance we profiled gene expression in DTX-sensitive and -resistant mCRPC cells using RNA sequencing (RNA-seq). PC3 and DU145 cells were selected for DTX resistance and this phenotype was validated by immunoblotting using DTX resistance markers (e.g. clusterin, ABCB1/P-gp, and LEDGF/p75). Overlapping genes differentially regulated in the DTX-sensitive and -resistant cells were ranked by Gene Set Enrichment Analysis (GSEA) and validated to correlate transcript with protein expression. GSEA revealed that genes associated with cancer stem cells (CSC) (e.g., NES, TSPAN8, DPPP, DNAJC12, and MYC ) were highly ranked and comprised 70% of the top 25 genes differentially upregulated in the DTX-resistant cells. Established markers of epithelial-to-mesenchymal transition (EMT) and CSCs were used to evaluate the stemness of adherent DTX-resistant cells (2D cultures) and tumorspheres (3D cultures). Increased formation and frequency of cells expressing CSC markers were detected in DTX-resistant cells. DU145-DR cells showed a 2-fold increase in tumorsphere formation and increased DTX resistance compared to DU145-DR 2D cultures. These results demonstrate the induction of a transcriptomic program associated with stemness in mCRPC cells selected for DTX resistance, and strengthen the emerging body of evidence implicating CSCs in this process. In addition, they provide additional candidate genes and molecular pathways for potential therapeutic targeting to overcome DTX resistance.