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,Carlos A Casiano,Alfonso M Durán,Charles Wang,Leanne Woods-Burnham,Sourav Roy,Juli Unternaehrer,Shannalee R Martinez,Evgeny Chirshev,Anamika Basu,Evelyn S Sanchez-Hernandez,Greisha L Ortiz-Hernández,Celine Greco,Joshua A Ramirez,Leslimar Ríos-Colón,Ubaldo Soto,Claude Boucheix,Xin Chen

doi:10.18632/oncotarget.25744

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.

Highlights

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second cause of cancer deaths among American men [1]
We demonstrated that these DTX-resistant cells overexpress the stress oncoprotein Lens Epithelium Derived Growth Factor of 75 kD (LEDGF/p75), and that depletion of this protein partially resensitized these cells to DTX [8, 18]
We confirmed that the DTXresistant PC3-DR and DU145-DR cell lines used in the RNA sequencing (RNA-seq) analysis and other experiments displayed significant upregulation of proteins previously implicated www.oncotarget.com by our group and others in PCa progression and DTX resistance [8, 9, 19,20,21,22,23] including LEDGF/p75, CLU, and ATP-binding cassette sub-family B member 1 (ABCB1), compared to the sensitive cells (Figure 1A-1C)

Summary

Introduction

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second cause of cancer deaths among American men [1]. For men diagnosed with advanced PCa, treatment with curative intent is no longer an option, and androgen deprivation therapy (ADT) remains the main therapeutic modality [2, 3]. New treatment options for mCRPC have been developed, including the next-generation androgen receptor-targeting agents abiraterone acetate and enzalutamide, therapeutic vaccines, and the second generation taxane cabazitaxel [6, 7]. These novel therapeutic agents, which are often administered sequentially or in combination with DTX, only moderately improve overall patient survival due to the development of therapy resistance

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