Ghrelin was first discovered in 1999 and was described as an endogenous ligand of the growth hormone secretagogue receptor (GHSR) (1). Ghrelin is produced in the fundic area of the stomach, where it is secreted directly into the bloodstream (2). Initially thought to regulate growth hormone (GH) secretion, ghrelin has been shown to serve various biological functions. A main role of ghrelin is in the gut-brain interaction (3), with expression in the hypothalamus and pituitary (4). This hormone is also known to serve a key role in appetite stimulation (5), gastric motility and acid secretion (6), stress and anxiety (7), and regulation of the circadian rhythm (8). Ghrelin is typically released when the stomach is empty, suggestive of its involvement in endocrine regulation of motility-dependent interdigestive gastric secretion (9).

Ghrelin has long been considered to be involved in tumorigenic proliferation and although its precise role is still uncertain, it has received increasing attention in research regarding gastrointestinal (GI) neoplasia. The immunohistochemical (IHC) expression of ghrelin has been documented in a number of endocrine and non-endocrine tumors (10,11), but it is still unclear whether an autocrine/paracrine loop or another type of interaction is involved. At present, it has not yet been reported if the expression of ghrelin and its receptors in tumor cells are protective against neoplasia or whether they stimulate carcinogenesis. Therefore, this review provides an assessment of the current literature, presenting the emerging trends and highlighting potential gaps in current knowledge regarding the implications of ghrelin from its production to the interaction with its known receptors (known as the ghrelin axis) in digestive malignancies.

Ghrelin is primarily produced in the stomach (12), where X/A-like cells are found in abundance in oxyntic glands, close to parietal cells; in addition, ghrelin displays rare colocalization with other hormones (13). Ghrelin cells are closed-type cells, having no contact with the lumen (2) and release their secretions within the vascular bed (14,15). However, ghrelin and its gene transcripts have also been identified in other enteroendocrine cells, namely enterochromaffin cells, S cells, I cells, L cells and M cells (16–20). Ghrelin is also produced in variable amounts in other segments of the GI tract (duodenum, jejunum, ileum, colon) (21–24), as well as in the lungs, kidneys, testes, prostate, ovaries, breasts, pituitary and hypothalamus (25–28). Ghrelin release is activated by a series of receptors, like β1-adrenergic, calcitonin gene-related peptide, glucose-dependent insulinotropic polypeptide and secretin receptors. Moreover, ghrelin is inhibited by both short-chain and long-chain fatty acids, lactate, extracellular calcium, amino acids and somatostatin receptors (9).

Genetic mapping has placed the ghrelin gene on the short arm of chromosome 3 (3p25-26) (27), with an initial gene product known as pre-proghrelin (29,30). Pre-proghrelin is a 117 amino acid pre-protein, containing a 23-amino acid N-terminal signal peptide that is cleaved to form the 94 amino acid peptide, proghrelin (31). Proghrelin is then split by a prohormone convertase to produce the 28 amino acid ghrelin peptide and a 66 amino acid C-terminal fragment (known as C-ghrelin), which gives rise to the hormone obestatin (32).

Acylated ghrelin, desacyl ghrelin and C-ghrelin, have been found in ghrelin-secreting cells and in the circulation, and have been previously described as peripheral forms of ghrelin (33–36). Desacyl ghrelin shows a remarkable predominance in peripheral blood (35,37), mainly owing to it being the most secreted and more stable form of ghrelin (34,38). Alternative ghrelin-related peptides and mRNA splice variants were subsequently identified (31,39). With multiple variants described to date, the intron 1 (In1)-ghrelin variant has an alternative C-terminal tail but displays high areas of conservation to ghrelin that are essential in the process of acylation and allow receptor binding (40). Ghrelin must be acylated with an octanoyl group to activate its receptor, which is achieved by a post-translational enzymatic change initiated by ghrelin-o-acyltransferase (GOAT) (41). Although this process is necessary for ghrelin to be highly physiologically active through its receptor (42,43), certain studies also suggest a metabolic role for desacyl ghrelin (44,45).

The ghrelin receptor, GHSR, belongs to the G-protein coupled receptor family (46). There are two isoforms of the GHSR, GHSR1a and GHSR1b, both of which are widely expressed throughout the body. First identified in the pituitary and hypothalamus, GHSR1a is expressed in a broad range of tissues (bone; adipose tissue; lymphoid tissue) and organs, including the stomach, intestine, pancreas, spleen, thyroid gland, prostate, ovaries, testes, adrenal glands, kidneys, heart and lungs (47–50). GHSR1a is considered to be a functional receptor, as it mediates several of the effects of ghrelin, whereas GHSR1b is to be a considered a non-functional receptor, devoid of signal transduction activity (51). The expression of GHSR1b is mostly similar to that of its splicing variant with regard to localization, albeit it is different in intensity, with higher detectable levels of mRNA (11,25,50).

Acylated ghrelin preferentially binds to GHSR1a, leading to GH secretion. Both the acylated and desacylated forms have been identified as functionally active in local and systemic processes, such as adipogenesis and lipid retention (52–55). However, desacyl ghrelin does not bind to GHSR1a, suggesting its effects are GHSR-independent, with its cognate receptor remaining unknown (56,57). Desacyl ghrelin mainly exerts nonendocrine activities, with involvement in vascular remodeling, cardiovascular stability, muscle atrophy, cachexia and lipolysis (38,58–62). A schematic illustration of the main steps in the ghrelin circuit, from production to interaction with its receptor, is presented in Fig. 1.

Ghrelin is a hormone involved in a number of physiological processes outside of the GI tract; it is a key participant in the interaction between the enteric and the central nervous systems, with implications in physical, mental and behavioral changes owing to the regulation of the circadian rhythm, and stress and anxiety levels (3,7,60). In the GI tract, ghrelin interacts with all organs, stimulating gastric acid secretion, increasing GI motility (6) and modulating glucose levels through an increase of pancreatic insulin release and through insulin sensitivity (63). In the liver, ghrelin promotes gluconeogenesis and lipogenesis (64,65), the latter also being its main action in adipose tissue (66).

Even though the physiological roles of ghrelin are yet to be fully revealed, it has been demonstrated that ghrelin contributes to the pathology of a multitude of diseases. Its orexigenic role is well established (5,67,68); however, ghrelin also serves a function in inflammatory processes (69). The presence of ghrelin and its receptors on human leucocytes (T lymphocytes and macrophages) was noted as early as 2004 (70), with proven anti-inflammatory effects in both animal and human studies, in acute (71–74) and chronic settings (75–78), as well as in a neoplastic context (79). Ghrelin receptors have also been identified in cardiac tissue (80), where they modulate cardiac function and healing by influencing myocardial contraction (81), perfusion (82) and post-myocardial infarction-changes (41,72,83). In the lungs, ghrelin can attenuate pulmonary blood pressure through vascular remodeling and mitigate the changes induced by acute lung injury (84,85). Ghrelin also serves a role in the intricate pathologies of anorexia and cachexia (86,87) as well as neurodegenerative disorders, such as dementia (88), multiple sclerosis (89) and amyotrophic lateral sclerosis (90). The pleiotropic effects of the ghrelin axis are summarized in Fig. 2.

Present in both endocrine and non-endocrine tumors, such as breast, prostate, testicular, GI, pancreatic, renal, thyroid, lung and adrenal tumors (11,91–94), ghrelin and its receptors may give rise to a complex set of interactions that contribute to neoplastic development at various stages. Although the precise role of ghrelin is still uncertain, numerous studies have used different experimental approaches to document the involvement of the ghrelin axis in neoplasia.

Experimental studies have investigated the connection between ghrelin and neoplasia, in attempts to uncover a novel prognostic factor or to find missing links in the processes of tumorigenesis, invasion and metastasis. The majority of in vitro studies have focused on ghrelin and GHSR splice variants expression in various tumor cell lines, investigating the effects of ghrelin on tumor development. A summary of these findings regarding GI tumor cells and highlighting the diversity of results published on this topic can be found in Table I. Ghrelin has also been reported to be expressed in experimental studies using cell lines of leukemia (95), breast carcinoma (96), pulmonary adenocarcinoma (97), pancreatic ductal adenocarcinoma (98), prostate carcinoma (99), gastric adenocarcinoma (100) and colorectal adenocarcinoma (101,102). and it has shown pleiotropic effects in the vast majority of these tumor cell lines (11).

Ghrelin expression is less variable compared with the expression of its receptors, which is highly variable between different types of cancer. GHSR1a and GHSR1b are most often co-expressed, with GHSR1a expression levels demonstrated to be higher compared with those for GHSR1b in oral squamous cell carcinoma (SCC) and gastric cancer (GC) cell lines (100,103). For breast cancer cell lines, an initial study reported that lines MDA-MB-231, MCF-7, MDA-MB-435 and T47D have positive IHC expression of the ghrelin/GHSR axis (27), whereas GHSR1a mRNA could not be detected in lines MDA-MB231, MCF7 and T47D, in spite of specific binding of ghrelin in these cells, implying that an unidentified interaction or binding site modulates these effects (96). High GHSR1b expression was identified in breast cancer cell line MDA-MB-231 (39). A similar difference in expression was demonstrated in colon cancer cell lines (101).

The effects of ghrelin can vary according to the type of cancer. Previous studies have found ghrelin can have a proliferative effect on a number of cancer cell lines including breast (27), prostate (28), gastric (100), colorectal (101,102,104), oral (103), pancreatic (98), adrenocortical (105) and endometrial (106). Conversely, D-Lys-growth hormone releasing peptide 6 or other ghrelin-specific antibodies can antagonize ghrelin and inhibit this proliferative effect (101). Certain studies attribute this proliferative effect to the desacyl form (27,91,95), suggesting that in these cell lines ghrelin can stimulate cell proliferation via an autocrine pathway independent of GHSR1a (95). However, ghrelin has also been shown to inhibit proliferation and exhibit an antiapoptotic effect in cell lines of the same cancer type, as is the case for breast carcinoma cell lines MCF7 and MDA-MB231 (96) and prostate carcinoma cell lines PC-3 and DU-145 (91). Both acylated and deacylated ghrelin inhibit DU-145 cell proliferation (91,99,107). A recent study on chemosensitive ovarian cells demonstrated that acylated ghrelin promotes cell proliferation and survival, and inhibit apoptosis through its interaction with GHRS1a via the PI3K/Akt signaling pathway, thereby rendering the cells resistant to chemotherapy, even at very low levels (108).

The ghrelin splice variant, In1-ghrelin, can increase the proliferation rate of the MDA-MB-231 breast cancer cell line (39) and stimulate proliferation and migration of MCF-7 and MDA-MB-231 cells (109). Expression of this variant was also found in pancreatic adenocarcinoma cell lines, which do not express ghrelin, wherein it enhanced the proliferation and migration of tumor cells (110).

To better document and understand these effects, the activation pathway of ghrelin has been examined extensively, and numerous pathway activation mechanisms have been identified. Through the interaction of ghrelin and GHRS the PI3-K/Akt/mTOR signaling pathway is able to mediate the migration and invasiveness of pancreatic adenocarcinoma cells (98). One study demonstrated that NF-κB signaling pathway activation can contribute to ghrelin-induced cell migration in glioma cells (111). Furthermore, Lin et al (94) determined ghrelin has diverse implications in tumorigenesis and tumor spread, with the ability to promote metastasis in the case of renal cell carcinoma (RCC) via Snail, a transcriptional repressor of E-cadherin, and invasion through the upregulation of the kinase Aurora A, an activating mechanism which was also associated with a poor prognosis (112). Similarly, invasion in GC was determined to be a result of GHSR/NF-κB signaling pathway activation (100).

Although ghrelin serves a known role in the inflammatory processes of the GI (113,114), data regarding its promoting role in GI carcinogenesis is still controversial. Studies on malignant cell lines have shown conflicting results, as aforementioned. Therefore, research has now shifted from cell line experiments to analyzing tissue expression and circulating levels of ghrelin and its isoforms. The complex dynamics of ghrelin in GI tumors is described in Table II, which provides an integrated overview of the representative results in this research field.

As previously mentioned, ghrelin and its receptors are expressed in numerous endocrine and non-endocrine tumor cell types, such as pituitary, prostate, breast and lung (10,96,99,164). Since ghrelin acts not only as a local, but also as a systemic hormone, its potential role in cancer development in other organs has been investigated.

Oral SCC demonstrates the expression of the ghrelin axis, with ghrelin levels being negatively correlated with tumor invasiveness (165). Furthermore, decreasing ghrelin levels appear to be correlated with lower degrees of differentiation, indicating the potential value of tissue ghrelin expression as a prognostic marker (165). Another study demonstrated that expression of ghrelin and GHSR were gradually increased in oral tumors as benign cells developed cytological and architectural features of dysplasia and further progressed to becoming malignant (166).

An initial study on kidney tumors reported low to absent expression of ghrelin in RCC, with all 21 cases of conventional-subtype analyzed having no IHC ghrelin expression and partial-to-total loss of ghrelin expression when assessed through radioimmunoassays (167). However, certain kidney tumors, such as oncocytomas, did express ghrelin, albeit less than normal kidney tissue (167). Furthermore, Lin et al (94) assessed ghrelin expression levels in a cohort of 562 clear-cell type RCC cases and demonstrated that ghrelin levels were high and indicated a poor prognosis, with high ghrelin expression being associated with lymph node and distant metastases.

Ghrelin is expressed in both normal and tumoral breast tissue (96,168). While ghrelin expression was investigated in normal mammary epithelium (168), Grönberg et al (169) also investigated ghrelin expression and the potential role of this hormone in male breast carcinogenesis, revealing that ghrelin expression was associated with a lower risk of breast cancer death. A study on 144 female breast cancer specimens determined ghrelin to be moderately to strongly expressed, which was positively correlated with survival and disease-free interval (92).

Cassoni et al (91) demonstrated prostatic adenocarcinomas were positive for ghrelin expression when assessed by reverse transcription-PCR but showed no immunoreactivity when assessed by IHC (91). Serum levels of ghrelin are higher in patients with prostate cancer compared with patients with benign prostate hyperplasia (170). Increased levels of In1-ghrelin and GOAT were also observed in patients with prostate cancer compared with a healthy control group (109).

Ghrelin may also serve a role in endometrial carcinogenesis through coupling with its GHSR1a receptor, with hormone and receptor expression detectable in benign and malignant tissue specimens (171). Expression has also been identified in various histological subtypes of lung cancer, accompanied by the expression of GHSR1a (172,173).

The role of ghrelin in GI tract malignancies is still incompletely characterized. The complexity of the ghrelin axis and the interaction between this hormone and cancer cells is still unclear. Studies on cancer cell lines show a spectrum of ghrelin and ghrelin receptor expression, and new splice variants of ghrelin involved in carcinogenesis are emerging. The role of IHC to detect expression is unclear, as most papers do not focus on tumor histological subtype or other factors such as tumor heterogeneity. The use of ghrelin alone as a potential serological biomarker is debatable, as it is regulated by various metabolic factors and is linked with cancer-associated inflammation. Although certain of the effects of ghrelin can be quantified, there are still large discrepancies in this field owing to the variety of peripheral, circulating forms of ghrelin incompletely matched with a corresponding receptor. Further investigation is required to identify a potential desacyl ghrelin receptor that will facilitate the accurate identification of sites of action for this peptide and to elucidate its underlying mechanisms of action. The present review has demonstrated that our current understanding of the ghrelin system does not provide sufficient evidence to justify its role as a useful biomarker in GI malignancies. Therefore, large cohort studies with well-defined exclusion criteria and a complex framework, factoring in plasma levels, tissue expression and genetic alterations, are needed to establish direct correlations between ghrelin levels and GI malignancies.