Ghrelin-O-Acyltransferase (GOAT) Enzyme as a Novel Potential Biomarker in Gastroenteropancreatic Neuroendocrine Tumors

Abstract

Objectives: The association between the presence and alterations of the components of the ghrelin system and the development and progression of neuroendocrine tumors (NETs) is still controversial and remains unclear.

Methods: Here, we systematically evaluated the expression levels (by quantitative-PCR) of key ghrelin system components of in gastroenteropancreatic (GEP)-NETs, as compared to non-tumor adjacent (NTA; n = 42) and normal tissues (NT; n = 14). Then, we analyzed their putative associations with clinical-histological characteristics.

Results: The results indicate that ghrelin and its receptor GHSR1a are present in a high proportion of normal tissues, while the enzyme ghrelin-O-acyltransferase (GOAT) and the splicing variants In1-ghrelin and GHSR1b were present in a lower proportion of normal tissues. In contrast, all ghrelin system components were present in a high proportion of tumor and NTA tissues. GOAT was significantly overexpressed (by quantitative-PCR (qPCR)) in tumor samples compared to NTA, while a trend was found for ghrelin, In1-ghrelin and GHSR1a. In addition, expression of these components displayed significant correlations with key clinical parameters. The marked overexpression of GOAT in tumor samples compared to NTA regions was confirmed by IHC, revealing that this enzyme is particularly overexpressed in gastrointestinal NETs, where it is directly correlated with tumor diameter.

Conclusions: These results provide novel information on the presence and potential pathophysiological implications of the ghrelin system components in GEP-NETs, wherein GOAT might represent a novel diagnostic biomarker.

Fig. 1. Expression of ghrelin system components in normal GEP, adjacent non-tumor tissue and GEP-NETs.

The absolute mRNA expression of the different components of the ghrelin system was determined by qPCR in normal GEP controls, adjacent non-tumor tissue and GEP-NETs samples (values are adjusted by 18S expression). Data represent the mean ± SEM. Asterisks (*p < 0.05; **p < 0.01; ***p < 0.001) indicate significant changes by paired analysis between adjacent non-tumor and GEP-NETs and non-paired analysis between normal tissue and adjacent non-tumor or tumor tissues

Fig. 2. Immunohistochemical analysis of GOAT enzyme in adjacent non-tumor tissue and GEP-NETs.

The presence of GOAT by immunohistochemistry using a specific antibody was determined in a subset of samples, which included tumor and non-tumor regions from patients diagnosed with GEP-NETs. a Representative images of the IHC analysis of GOAT enzyme in a GEP-NET sample compared with the non-tumor adjacent tissue. b Absolute number of cases according to the intensity of GOAT IHC staining (0, 1 + , 2 + , 3 + ). c The graph indicates the percentage of tumor samples according to the intensity of GOAT expression by IHC, 0 and 1 + have been grouped as low expression while 2 + and 3 + have been grouped as high intensity by IHC. d Representative images of different GOAT staining in GEP-NETs. In the analysis, 0, 1 + , 2 + , 3 + stand for absent, low, moderate, and high intensities of the tumor region staining compared to the adjacent region with non-tumor tissue (3D1, 3D2, 3D3, 3D4, respectively). This analysis revealed that GOAT was present in the vast majority of tumor cells compared with non-tumor adjacent tissue, with different grades of staining. e Correlation between the absolute mRNA expression of GOAT determined by qPCR in GEP-NETs samples (values are adjusted by 18S expression) and the intensity of GOAT staining

Fig. 3. Correlations between epidemiological, clinical, histological, and molecular parameters in GEP-NETs.

The correlations between epidemiological, clinical, histological, and molecular parameters within GEP-NET samples were assessed by U-Mann–Whitney tests. Asterisks indicate significant associations (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 4. In vitro analysis of the consequences of GOAT inhibitor (GOATi) treatment in NET cell lines.

a Cell proliferation rate in BON-1 and QGP-1 cell lines after 24, 48, and 72 h of GOATi treatment determined by Alamar-blue assay. Paclitaxel (PAX) was used as inhibitory control in proliferation assays. b Cell migration rate in BON-1 after 24 h of treatment with GOAT inhibitor by wound-healing assay. Cell proliferation rate compared to control was assessed by multiple comparison tests while migration was assessed by U-Mann–Whitney test. Values represent the mean ± SEM of at least three individual experiments. Asterisks indicate significant differences (*p < 0.05; ***p < 0.001) compared with control (set at a 100%). Legend: ns means non-significant