Molecular characterization of neuroendocrine prostate cancer and identification of new drug targets

Abstract

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer that most commonly evolves from preexisting prostate adenocarcinoma (PCA). Using Next Generation RNA-sequencing and oligonucleotide arrays, we profiled 7 NEPC, 30 PCA, and 5 benign prostate tissue (BEN), and validated findings on tumors from a large cohort of patients (37 NEPC, 169 PCA, 22 BEN) using IHC and FISH. We discovered significant overexpression and gene amplification of AURKA and MYCN in 40% of NEPC and 5% of PCA, respectively, and evidence that that they cooperate to induce a neuroendocrine phenotype in prostate cells. There was dramatic and enhanced sensitivity of NEPC (and MYCN overexpressing PCA) to Aurora kinase inhibitor therapy both in vitro and in vivo, with complete suppression of neuroendocrine marker expression following treatment. We propose that alterations in Aurora kinase A and N-myc are involved in the development of NEPC, and future clinical trials will help determine from the efficacy of Aurora kinase inhibitor therapy.

Significance: We report on the largest in-depth molecular analysis of NEPC and provide new insight into molecular events involved in the progression of prostate cancer.

Keywords: aurora kinase A; drug targets; n-myc; neuroendocrine prostate cancer.

Figure 1

Characterization of NEPC: (A) Tumor with mixed features of NEPC and PCA. Hematoxylin and eosin (H&E) staining, immunohistochemical analysis for androgen receptor (AR) and ERG, and FISH for ERG breakapart (indicating gene fusion) (B) Gene expression of the 936 of 25932 genes showing differential expression between 7 NEPC cases and 30 PCAs, after a Benjamini-Hochberg correction for multiple hypothesis testing of <0.001 (Red =High Expression, Green = Low Expression). (C) Gene Expression of select genes comparing NEPC and PCA, including neuroendocrine associated genes (SYP, CHGB, CHGA), EZH2, MIB1 (Ki67), PSMA, AR, and androgen regulated genes (NKX3-1, KLK3 (PSA), TMPRSS2). (D) Graphical representation of the genomic landscape of PCA (in order of increasing Gleason Score) and NEPC, as determined by Affymetrix 6.0 oligonucleotide array (Red= Copy Number Gain, Blue= Copy Number Loss, White= No Change).

Figure 2

Evaluation of Aurora Kinase and N-Myc: (A) Gene expression of AURKA in Benign Prostate Tissue, PCA, and NEPC, as measured by RNA-Seq. RPKM= Reads per kilobase of exon per million mapped reads. (B) Gene expression of AURKA in Benign Prostate Tissue, PCA, and NEPC, as measured by RNA-Seq. RPKM= Reads per kilobase of exon per million mapped reads (C) Table summarizing IHC and FISH data from tumors from large cohort of PCA, NEPC, and benign prostate (D) Representative example of positive Aurora kinase A overexpression by IHC, and MYCN and AURKA amplification by FISH in human NEPC. Green= Centromeric Control Probes, Red = AURKA and MYCN loci as labeled in NEPC.

Figure 3

(A) Immunoblot analysis for protein expression of Aurora kinase A, Phosphorylated histone 3 (P04-H3), neuron specific enolase (NSE) and synaptophysin (SYP) after transient transfection of MYCN, AURKA, or Empty Vector (EV) in RWPE-1 cells. BE(2)N is a neuroblastoma cell line as positive control for NSE. (B) Stable LNCaP cell line over-expressing N-myc compared to empty vector (EV) : Immunoblot analysis for protein expression of N-myc, Aurora kinase A, P04-H3, NSE, SYP, PSA, AR, beta actin. qRT-PCR and microarray (MA) data showing induction of NSE (qRT-PCR) and EZH2 (MA) gene expression and suppression of AR (qRT-PCR) and androgen regulated genes (NKX3-1, TMPRSS2 (MA)) (C) Left: Immunoprecipitation of LNCaP-n-Myc cell lysates using antibodies directed against N-Myc or control IgG antibodies and Western blot using antibodies directed against Aurora kinase A (Aurora A) or control IgG antibodies. Right: LNCaP control (LNCaP E.V.) and LNCaP-n-Myc cells were treated with cycloheximide (CHX) for the indicated time (in minutes) and Aurora kinase A or beta-tubulin levels were assessed by immunoblotting. The normalized percent of Aurora kinase A relative to beta-tubulin and to time point 0 for LNCaP E.V. (gray line) or LNCaP-n-Myc (black line). (D) N-Myc directly binds to the SYP, NSE, and AR promoters in LNCaP-n-Myc cells and not LNCaP-EV. Not-to-scale schematic representation of SYP, NSE, and AR promoters showing the E-box sites (grey and black circles) indicated for each. The transcription start site for each gene is indicated with an arrow. Below each schematic are bar graphs showing the amount of enriched DNA (relative to input chromatin preparation) for each E-box site in the indicated cell lines following ChIP using either anti-N-Myc (right) or anti-IgG (left) antibodies. IMR-32 is a MYCN amplified neuroblastoma cell line. In IMR-32 cells, Nmyc binds promoters of SYP and NSE, but not AR.

Figure 4

NEPC demonstrates enhanced sensitivity to Aurora Kinase Inhibitor therapy compared to PCA (A) Viability assay of LNCaP cells transfected with MYCN or Empty Vector (EV) at 72 hours after treatment with vehicle or indicated doses of the pan-Aurora kinase inhibitor PHA-739358. (B) Viability assay of RWPE (blue circles), VCaP (gray diamonds), DU145 (gray triangles), and NCI-H660 (orange triangles) at 72 hours after treatment with vehicle or indicated doses of PHA-739358. (C) Percent tumor size after treatment of LNCaP (gray) and NCI-H660 (red) xenografts with vehicle (dotted lines) or PHA-739358 30 mg/kg IP BID (solid lines) twice a day for 5 days relative to day 0. Luciferase imaging at day 8 and tumor photographs at day 17 of representative tumors following treatment with either vehicle or PHA-73935. (D) Percent tumor size after treatment of LTL-362 xenografts with vehicle (dotted lines) or PHA-739358 30 mg/kg IP BID (solid lines) twice a day for 5 days relative to day 0. (E) Immunohistochemical staining for phosphorylated histone 3 (PO4-H3) in NCI-H660 or LNCaP tumors at day 4 of treatment with either vehicle or PHA-739358. (F) Immunohistochemistry for the neuroendocrine marker, synaptophysin, in NCI-H660 xenografts treated with vehicle (positive) and PHA-739358 (negative).