Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin

Abstract

Background: Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified.

Methods: We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR.

Results: We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P < 0.05) in the PC3 prostate cancer cell line, however, ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines.

Conclusions: This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific. The expression of GOAT in prostate cancer supports the hypothesis that the ghrelin axis has autocrine/paracrine roles. We propose that the RWPE-1 prostate cell line and the PC3 prostate cancer cell line may be useful for investigating GOAT regulation and function.

Figure 1

Boxplot showing relative levels of GOAT/MBOAT4 mRNA expression. Expression in normal prostate tissue (denoted N) and prostate cancer tissue (stages II to IV) performed using an OriGene TissueScan qPCR Prostate Cancer panel. Data were normalised to β-actin and are represented as fold changes relative to expression of transcripts in a normal prostate sample (1.0). GOAT was not differentially expressed in any group (Kruskal-Wallis test; over all p = 0.56).

Figure 2

Real-time quantitative RT-PCR assays. Assays were performed to determine the relative mRNA expression levels of (A) GOAT (MBOAT4), (B) PC1/3 (PCSK1), (C) PC2 (PCSK2), and (D) furin (FURIN) in the RWPE-1 and RWPE-2 normal-prostate derived cell lines and the DU145, LNCaP and PC3 prostate cancer-derived cell lines. Data are expressed relative to the RWPE-1 cell line mRNA levels (set as 1) and are presented as means ± S.E.M. (n = 2, assays performed in duplicate) and compared by one-way ANOVA, followed by Tukey’s post-hoc analysis.* = P < 0.05 versus the RWPE-1 cell line.

Figure 3

Representative photomicrographs of ghrelin O-acyltransferase (GOAT) immunoreactivity in prostate cancer specimens and in benign prostatic hyperplasia (BPH). Diffuse cytoplasmic staining was detected in (A) poorly differentiated cancer specimens, (B) Gleason grade 4 + 5 and (C) Gleason grade 4+ 2. (D) No immunoreactivity was detected in some cancers (Gleason grade 3 +3) demonstrating the heterogeneity of GOAT expression in prostate cancer. More granular dot-like staining was present in (E, F) BPH sections. Scale bars are as indicated on micrographs.

Figure 4

Real-time quantitative RT-PCR analysis of GOAT gene expression in response to ghrelin and desacyl ghrelin treatments for 6 hours. GOAT mRNA expression in ghrelin treated (A) RWPE-1 normal prostate derived cell line, and the (C) DU145, (E) LNCaP and (G) PC3 prostate cancer cell lines and desacyl ghrelin-treated (B) RWPE-1, (D) DU145 and (F) LNCaP and (H) PC3 cell lines. Data is represented as means and standard error of two technical replicates from two independent replicate experiments (n = 2). * P < 0.05 indicates values that differ significantly (Student’s t-test) from vehicle-treated control (set at 1).