An Investigation into the role of the ghrelin axis in hormone-dependent cancer and characterisation of a novel Exon 3-deleted preproghrelin isoform and its murine homologue

Ghrelin is a 28 amino acid peptide hormone with a unique octanoic acid modification that has an extensive range of physiological effects, including stimulation of growth hormone (GH) release, appetite regulation, and modulation of reproductive functions. The cognate receptor for ghrelin is the growth hormone secretagogue receptor (GHS-R), a G protein-coupled receptor with two documented isoforms, the functional GHS-R type 1a and the C-terminally truncated GHS-R type 1b. Several ghrelin variants have also been identified in addition to the n-octanoylated form of ghrelin. In our laboratory, we have identified a novel exon 3-deleted preproghrelin variant that retains sequence for the mature ghrelin hormone and also encodes a novel C-terminal peptide (designated as C-terminal 3 peptide). There is emerging evidence to suggest that the ghrelin axis, encompassing ghrelin, several ghrelin variants and both forms of the GHS-R, is implicated in tumour growth. The objective of this project is to investigate the role of the ghrelin axis in hormone-dependent cancer and to further characterise the expression and function of the novel exon 3-deleted preproghrelin isoform. Hormone-dependent cancers, including prostate and breast cancers, are significant causes of morbidity and mortality in the Western world. Improved diagnoses and treatments earlier in the progression of the disease are urgently required to improve patient outcomes. Growth factors play an integral role in prostate and breast cancer, particularly in the emergence of aggressive, hormone-refractory disease that is resistant to standard therapies. We have previously identified ghrelin as being a novel growth factor for prostate cancer cells in vitro and have hypothesised that this may be extended to other hormone-dependent cancer types including breast cancer. In the current study, techniques including real-time quantitative RT-PCR, Western blot analysis and immunohistochemistry have been used to determine and quantitate ghrelin, exon 3-deleted preproghrelin and GHS-R expression in prostate and breast cancer. Ghrelin and exon 3-deleted preproghrelin are highly expressed in prostate cancer tissues compared to expression levels in normal prostate glands. Similarly, breast carcinoma specimens display greater immunoreactivity for ghrelin and exon 3-deleted preproghrelin than normal breast tissues. Expression of the exon 3-deleted preproghrelin mRNA isoform is upregulated in the oestrogen-independent, highly malignant MDA-MB-435 breast cancer cell line compared to the non-tumourigenic MCF-10A breast epithelial cell line, suggesting that augmented transcription of the isoform is associated with an increased malignant potential in breast cancer. The functional GHS-R type 1a is expressed in normal breast tissue and breast cancer specimens and cell lines. In contrast, the truncated GHS-R type 1b isoform is exclusively expressed in breast carcinoma. These data suggest that GHS-R type 1b, ghrelin and exon 3-deleted preproghrelin display potential as novel diagnostic markers for prostate and breast cancer. These studies have been the first to demonstrate that ghrelin may have an important role in cell proliferation in breast and prostate cancer. Functional assays demonstrated that (10nM) ghrelin stimulated proliferation in the LNCaP prostate cancer cell lines (45.0 ± 1.7% above control, P &lt0.01) and rapidly activated the ERK 1/2 mitogen-activated kinase (MAPK) pathway in both PC3 and LNCaP cell lines. It does not, however, protect these cells from chemically-induced apoptosis. The MAPK inhibitors PD98059 and U0126 blocked ghrelin-induced MAPK activation, as well as cell proliferation, in both cell lines. Prostate cancer cells secrete mature ghrelin in vitro, and may therefore stimulate MAPK pathways in an autocrine manner. Ghrelin also appears to act as a growth factor in breast cancer cell proliferation, as the growth of MDA-MB-435 and MDA-MB-231 breast cancer cell lines is significantly increased by ghrelin treatment. Our findings suggest that the ghrelin axis could provide an important new target for adjunctive therapies for both breast and prostate cancer. The C-terminal 3 peptide derived from exon 3-deleted preproghrelin may be an important new component of the ghrelin axis and studies into its function are currently in progress. Although it did not induce MAPK cascades or stimulate proliferation in prostate or breast cancer cell lines, the discovery of a murine counterpart, exon 4-deleted preproghrelin, indicates that it is highly conserved. Exon 4-deleted preproghrelin is expressed in all mouse tissues examined, with stomach being the predominant site of synthesis. Other components of the ghrelin axis were also found to be present in a wide-range of mouse tissues including brain, ovary and prostate. This comprehensive report has paved the way for future work with in vivo mouse models of cancer. This study has provided a substantial basis for the further evaluation of ghrelin, exon 3-deleted preproghrelin and the GHS-R type 1b as novel diagnostic/prognostic markers for prostate and breast cancer and supports the rationale for targeting the ghrelin axis for treatment of these tumours.