Ghrelin as a Potential Molecular Marker of Adrenal Carcinogenesis

In Vivo and in Vitro Evidence

Hanna Komarowska; Marcin Rucinski; Marianna Tyczewska; Nadia Sawicka-Gutaj; Marta Szyszka; Aleksandra Hernik; Anna Klimont; Paulina Milecka; Laura Migasiuk; Mateusz Biczysko; Ilona Idasiak-Piechocka; Marek Karczewski; Marek Ruchala

Disclosures

Clin Endocrinol. 2018;89(1):36-45. 

In This Article

Abstract and Introduction

Abstract

Context: Adrenal tumours belong to one of the most prevalent neoplasms. It is a heterogeneous group with different aetiology, clinical manifestation and prognosis. Its histopathologic diagnosis is difficult and identification of differentiation markers for tumorigenesis is extremely valuable for diagnosis.

Design: To assess ghrelin expression and the relationship among ghrelin, IGF2 and the clinicopathological characteristics of adrenal tumours. To investigate the influence of ghrelin on ACC cell line proliferation.

Materials and methods: Expression of ghrelin and IGF2 in a total of 84 adrenal tissue samples (30 adenoma, 12 hyperplasia, 8 myelolipoma, 20 pheochromocytoma, 7 carcinoma and 7 unchanged adrenal glands) were estimated. Every operated patient from whom samples were obtained underwent clinicopathological analysis. All the parameters were compared among the groups examined and correlations between these were estimated.

H295R cell line was incubated with ghrelin to assess its effect on proliferation and migration rate.

Results: The highest ghrelin expression was observed in carcinoma samples and the lowest in the control group. Ghrelin expression was 21 times higher in carcinoma (P = .017) and 2.4 times higher in adenoma (P = .029) compared with controls. There were no statistically significant differences between myelolipoma (P = .093) and pheochromocytoma (P = .204) relative to the control. Ghrelin level was significantly higher in carcinoma compared to adenoma (P = .049) samples. A positive correlation between ghrelin and IGF2 expression was observed only in myelolipoma (P = .001).

Ghrelin at concentrations of 1 × 10−6 mol/L and 1 × 10−8 mol/L significantly stimulated proliferation and migration rate in the H295R cell line.

Conclusion: Ghrelin appears to be an essential factor in driving adrenal tumours development.

Introduction

Adrenal tumours are one of the most prevalent neoplasms comprising a heterogeneous group of malignancies with various clinical presentations and prognoses. The proper diagnosis of adrenal gland tumours is essential for subsequent management. Greater than 70% of tumours are discovered incidentally during radiologic examinations, and usually these lesions are benign, hormonally inactive, less than 3–4 cm in diameter, and do not require treatment. Other adrenal tumours may cause various symptoms depending on hormonal activity and malignancy. Aldosterone–producing tumours (APT), and equally frequent cortisol–producing tumours (CPT) are found in 2%–9% of all adrenal tumours, unlike androgen–producing tumours, which are relatively rare.[1–3] Tumours arising from the adrenal medulla are termed pheochromocytomas, and approximately 10%–15% of these tumours are of hereditary origin.[4] Other changes observed in the adrenals are myelolipomas that comprise about 8% of all adrenal tumours and are characterised by the predominance of mature adipocytes. Adrenal metastases mostly from kidney, lung, breast and colorectal cancers are present in up to 20% of patients with known malignant epithelial tumours at autopsy. Finally, adrenal hyperplasia refers to growth of the adrenal glands, which can be classified according to inheritance or morphology.[2]

Adrenal cortical carcinoma (ACC) is a rare and highly aggressive cancer. The clinical manifestations of this neoplasm depend on the hormonal activity of the tumour. It has been found that 50%–80% of adult patients manifest symptoms of hypercortisolemia, 40%–60% androgenisation, and extremely rarely hyperaldosteronism.[5–7] In hormonally inactive ACC cases, symptoms are related to the presence of a tumour mass or distant metastases. The histopathological diagnosis of ACC is problematic. Most often, the diagnosis is based on Weiss criteria with Aubert's modifications.[8] However, diagnostic pitfalls are very common, and 1 of 10 tumours is misclassified.[9]

There are 4 clinical stages of ACC. Complete surgical resection is still the most important curative treatment for adrenal cancer and mitotane is the only approved drug for ACC. However, the response to therapy is unpredictable and efficacy of treatment is unsatisfactory. As the prognosis for these patients is poor, emerging new drugs hold promise for ACC. Insulin–like growth factor 1 (IGF1) receptor inhibitors and multiple tyrosine kinase inhibitors directed at several intracellular and extracellular targets are of particular interest. As with mitotane, the efficacy of this treatment varies from patient to patient for unknown reasons.[10]

Knowledge of the molecular mechanisms involved in adrenocortical tumorigenesis is still insufficient. The most common molecular alterations in ACC are overexpression of insulin–like growth factor 2 (IGF2) and constitutive activation of the Wnt/b–catenin signalling pathway. Activation of the insulin–like growth factor 1 receptor (IGF1R) by IGF2 leads to cell proliferation by activation of the PI3K/AKT/mTOR cascade and the RAS–MAPK pathway.[11] Many studies have reported different expression levels of IGF1 and IGF2 in ACC compared to adrenocortical adenomas (ACA).[10,12] IGF2 expression is low or absent in the beginning of oncogenesis, which may suggest that other signalling pathways may also play a role in ACC tumorigenesis.[13,14]

Ghrelin is an endogenous ligand that stimulates the secretion of growth hormone.[15] Ghrelin is predominantly produced and secreted by endocrine cells of the gastric mucosa, and it circulates in 2 forms: acylated and unacylated.[16] Initially, unacylated ghrelin had been considered as the inactive form, but currently it is known that both forms exhibit biological activity. Ghrelin expression has also been demonstrated in a number of organs and tumours.[17,18] Widely distributed, ghrelin regulates different functions in the body and influences energy homeostasis and GH release.[19] Recent data indicate that ghrelin regulates a number of processes related to cancer progression and may be a critical factor in metastasis development.[20]

The development of ACC is probably caused by autocrine overexpression of different growth factors. It is known that overexpression of a single gene cannot act as the only driver of malignant adrenocortical tumorigenesis, and many accompanying factors may play a role in this process.

The aim of our study was to analyse the expression of ghrelin and IGF2 in a large group of adrenal tumours and healthy adrenals. Moreover, we investigated the relationship between ghrelin and IGF2 expressions and clinicopathological characteristics of adrenal gland tumours including their hormonal activity. To find a causal relationship between ghrelin overexpression and adrenal malignancy, we performed an in vitro study investigating the influence of ghrelin on adrenocortical cancer cell line proliferation (H295 R).

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