The oncogenic role of the In1-ghrelin splicing variant in prostate cancer aggressiveness

Abstract

BackgroundThe Ghrelin-system is a complex, pleiotropic family composed of several peptides, including native-ghrelin and its In1-ghrelin splicing variant, and receptors (GHSR 1a/b), which are dysregulated in various endocrine-related tumors, where they associate to pathophysiological features, but the presence, functional role, and mechanisms of actions of In1-ghrelin splicing variant in prostate-cancer (PCa), is completely unexplored. Herein, we aimed to determine the presence of key ghrelin-system components (native-ghrelin, In1-ghrelin, GHSR1a/1b) and their potential pathophysiological role in prostate cancer (PCa).MethodsIn1-ghrelin and native-ghrelin expression was evaluated by qPCR in prostate tissues from patients with high PCa-risk (n = 52; fresh-tumoral biopsies), and healthy-prostates (n = 12; from cystoprostatectomies) and correlated with clinical parameters using Spearman-test. In addition, In1-ghrelin and native-ghrelin was measured in plasma from an additional cohort of PCa-patients with different risk levels (n = 30) and control-healthy patients (n = 20). In vivo functional (proliferation/migration) and mechanistic (gene expression/signaling-pathways) assays were performed in PCa-cell lines in response to In1-ghrelin and native-ghrelin treatment, overexpression and/or silencing. Finally, tumor progression was monitored in nude-mice injected with PCa-cells overexpressing In1-ghrelin, native-ghrelin and empty vector (control).ResultsIn1-ghrelin, but not native-ghrelin, was overexpressed in high-risk PCa-samples compared to normal-prostate (NP), and this expression correlated with that of PSA. Conversely, GHSR1a/1b expression was virtually absent. Remarkably, plasmatic In1-ghrelin, but not native-ghrelin, levels were also higher in PCa-patients compared to healthy-controls. Furthermore, In1-ghrelin treatment/overexpression, and to a much lesser extent native-ghrelin, increased aggressiveness features (cell-proliferation, migration and PSA secretion) of NP and PCa cells. Consistently, nude-mice injected with PC-3-cells stably-transfected with In1-ghrelin, but not native-ghrelin, presented larger tumors. These effects were likely mediated by ERK1/2-signaling activation and involved altered expression of key oncogenes/tumor suppressor genes. Finally, In1-ghrelin silencing reduced cell-proliferation and PSA secretion from PCa cells.ConclusionsAltogether, our results indicate that In1-ghrelin levels (in tissue) and circulating levels (in plasma) are increased in PCa where it can regulate key pathophysiological processes, thus suggesting that In1-ghrelin may represent a novel biomarker and a new therapeutic target in PCa.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-017-0713-9) contains supplementary material, which is available to authorized users.

Plasma ghrelin O‐acyltransferase (GOAT) enzyme levels: A novel non‐invasive diagnosis tool for patients with significant prostate cancer

“…When viewed as a whole, the results of the present manuscript indicating that GOAT levels are markedly elevated in Sig PCa and are associated to aggressiveness features in PCa (i.e. GS and presence of metastasis), together with the previous results showing a strong correlation of GOAT levels with In1‐ghrelin variant levels in PCa, invite to suggest that GOAT enzyme and In1‐ghrelin variants could be functionally linked in PCa, where In1‐ghrelin variant might be the primary target of GOAT, and that an autocrine/paracrine circuit involving these two components of the ghrelin system may possibly operate in PCa to increase the aggressiveness features of PCa cells, which set the stage for future investigations.…”

“…Moreover, plasma GOAT levels tended to be associated with an earlier diagnosis of CRPC, which might also indicate that this enzyme may serve to develop future therapeutic target for PCa. In line with this, we have recently demonstrated that GOAT enzyme is positively correlated in PCa with the levels of the In1‐ghrelin splicing variant, but not with those of native‐ghrelin, wherein the presence of In1‐ghrelin variant drastically increased the aggressiveness features of PCa, acting as a true oncogene in this pathology . In fact, this previous study demonstrated that In1‐ghrelin silencing diminished the aggressiveness of PCa cells (e.g.…”

Clinical Utility of Ghrelin-O-Acyltransferase (GOAT) Enzyme as a Diagnostic Tool and Potential Therapeutic Target in Prostate Cancer

“…DU145 and LNCaP cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), cultured according to the manufacturer's instructions, validated by analysis of short tandem repeats sequences using GenePrint 10 System (Promega, Barcelona, Spain) and checked for mycoplasma contamination by PCR as previously reported [8,16]. DU145 cells were selected for functional in vitro and in vivo analyses based on its high expression levels of In1-ghrelin, the main oncogenic element of the ghrelin axis in prostate cancer, which is also a putative target of GOAT [8]. The GOAT inhibitor "GO-CoA-Tat" (032-37; Phoenix Biotech, Burlingame, CA, USA) was resuspended in water and used at 10 µM since this dose has been previously reported to be effective reducing GOAT activity [17].…”

“…In this regard, a ghrelin system has emerged as a pivotal regulatory axis in PCa pathophysiology [5,6], as well as a source of potential PCa biomarkers, since certain peptides derived from this pleiotropic system (i.e., native ghrelin, In1-ghrelin splice variant) are secreted by PCa cells and associated to PCa aggressiveness [7,8]. Ghrelin axis is controlled, at least in part, by the Ghrelin O-Acyl Transferase (GOAT or MBOAT4), an enzyme involved in the acylation and, thus, activation of ghrelin.…”

“…However, to date, the clinical utility of GOAT as a diagnostic (or prognostic/aggressiveness) tool in urine samples (a body sample less invasive and with a more prostate-specific content) from PCa patients has not been explored. Furthermore, although the actions of other elements of the ghrelin system (ghrelin, In1-ghrelin) in PCa have been well defined [7,8], the putative pathophysiological role of GOAT in this cancer type remains unknown. For these reasons, in this study, we aimed to explore, for the first time, the clinical utility of urine GOAT levels (and compared with plasma levels) to diagnose PCa, using an ample cohort of patients (almost 1000 patients) and to determine the role of GOAT in the pathophysiology of PCa.…”