SLC45A3-ELK4 is a novel and frequent erythroblast transformation-specific fusion transcript in prostate cancer

Abstract

Chromosomal rearrangements account for all erythroblast transformation-specific (ETS) family member gene fusions that have been reported in prostate cancer and have clinical, diagnostic, and prognostic implications. Androgen-regulated genes account for the majority of the 5' genomic regulatory promoter elements fused with ETS genes. TMPRSS2-ERG, TMPRSS2-ETV1, and SLC45A3-ERG rearrangements account for roughly 90% of ETS fusion prostate cancer. ELK4, another ETS family member, is androgen regulated, involved in promoting cell growth, and highly expressed in a subset of prostate cancer, yet the mechanism of ELK4 overexpression is unknown. In this study, we identified a novel ETS family fusion transcript, SLC45A3-ELK4, and found it to be expressed in both benign prostate tissue and prostate cancer. We found high levels of SLC45A3-ELK4 mRNA restricted to a subset of prostate cancer samples. SLC45A3-ELK4 transcript can be detected at high levels in urine samples from men at risk for prostate cancer. Characterization of the fusion mRNA revealed a major variant in which SLC45A3 exon 1 is fused to ELK4 exon 2. Based on quantitative PCR analyses of DNA, unlike other ETS fusions described in prostate cancer, the expression of SLC45A3-ELK4 mRNA is not exclusive to cases harboring a chromosomal rearrangement. Treatment of LNCaP cancer cells with a synthetic androgen (R1881) revealed that SLC45A3-ELK4, and not endogenous ELK4, mRNA expression is androgen regulated. Altogether, our findings show that SLC45A3-ELK4 mRNA expression is heterogeneous, highly induced in a subset of prostate cancers, androgen regulated, and most commonly occurs through a mechanism other than chromosomal rearrangement (e.g., trans-splicing).

Figure 1

Schematic of chromosome 1q32.1 (Chr.1q32.1) demonstrating the orientation and relative distance of SLC45A3 and ELK4. Red arrows and bar indicate the SLC45A3-ELK4 Taqman assay primers and probe, respectively.

Figure 2

Taqman expression data of SLC45A3-ELK4 and ELK4 mRNA levels in 31 prostate cancer samples (red bar) relative median of the values obtained from the 6 benign samples (green bar) in which cases yielding higher than 10 fold relative SLC45A3-ELK4 mRNA levels are indicated in dark red; and 10 cell lines (9 cancer and 1 benign, HK-2) relative to RWPE-1 (purple) (a). Schematic of the sequencing results obtained from PCR (primers are indicated in red) and 5’RACE (primer indicate in blue) that correspond to the different SLC45A3-ELK4 mRNA variants (v, see Supplemental Information for junction sequence, b). Taqman assay results from RNA extracted from 5 samples (C08, C03, C33 and C30 corresponding to cancer positive biopsies and C13 corresponded to cancer negative biopsy (c)). Contingency table of the 14 samples that yielded adequate TCFL1 values (inset).

Figure 3

Schematic of the region Chr.1q32 demonstrating the position of the primer pairs (blue boxes, a). SLC45A3 exon 5 and ELK4 exon 1 positions are indicated. qPCR results obtained for 16 prostate cancer samples (ordered from left to right as a function of the level of SLC45A3-ELK4 mRNA levels) and from LNCaP cells (b). Colored bars indicates samples with over 10-fold higher (red) or benign-like (light red) SLC45A3-ELK4 mRNA levels. All qPCR experiments were run in triplicate. Bars indicate the average calibrated values.

Figure 4

Median fold induction of SLC45A3-ELK4 (a), ELK4 (b) and KLK3 (PSA, c) mRNA in LNCaP cells treated with 1nM R1881 in the absence or presence of 10 µM flutamide at the indicated time points. All experiments were run in triplicate (SEM indicated by the error bars).