Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHSR). Previous work from our group demonstrated expression of ghrelin and GHSR in human endometrium during the menstrual cycle. It is suggested that the ghrelin axis is potentially involved in the extensive cyclical growth and development in endometrial tissues. In addition to regulating the normal growth and differentiation of the tissues, ghrelin has been implicated in the maintenance and progression of several human cancers. Ghrelin and/or the expression of GHSR has been demonstrated in a number of hormone-dependent cancers, including breast, testicular and prostate cancer, suggesting a possible autocrine and/or paracrine role for ghrelin in cancer cell growth. As ghrelin is expressed in normal endometrial tissues and ghrelin has functions in other endocrine-related cancers, we hypothesized that a similar system could exist in endometrial cancer. The aim of the present study was to investigate the expression of ghrelin and GHSR in human endometrial cancer and to examine the effect of ghrelin and GHSR in endometrial cancer cell progression.
Our results showed that ghrelin mRNA is expressed in a range of endometrial cancer tissues. Immunohistochemistry demonstrated GHSR1a protein expression in normal and cancerous glands in human endometrial tissue samples. Low to moderately invasive endometrial cancer cell lines were examined by RT-PCR and immunoblotting, demonstrating that ghrelin axis mRNA and protein expression correlate with differentiation status of Ishikawa (well-differentiated), HEC1B (moderately differentiated), and KLE (poorly differentiated) endometrial cancer cell lines. Moreover, treatment with ghrelin potently stimulated cell proliferation and inhibited cell death. Taken together, these data indicate that ghrelin promotes the progression of endometrial cancer cells in vitro, and may contribute to endometrial cancer pathogenesis.
To further investigate ghrelin-GHSR1a in endometrial cancer cell progression, we examined the effect of knocking down GHSR1a expression in the Ishikawa and KLE endometrial cancer cell lines by using RNA interference (RNAi) in vitro. The lentiviral short hairpin RNA targeting GHSR1a resulted in a stable reduction of GHSR1a mRNA and protein. GHSR1a knockdown Ishikawa and KLE cells showed less non-stimulated cell proliferation compared to the scrambled controls and proliferated less in response to ghrelin treatment than with controls. To examine whether the effects of GHSR1a knockdown on endometrial cancer cell growth inhibition are sustained in vivo, we next established a stable knockdown of GHSR1a in Ishikawa xenografts in NOD/SCID mouse model. Tumour volumes of GHSR1a-shRNA Ishikawa xenograft tumours were significantly reduced compared with scrambled control tumours as were tumour weights. These results suggested that both ghrelin and GHSR1a appear to be important in the growth of endometrial cancer cells.
In summary, this study demonstrated the possible role of ghrelin and particularly the ghrelin receptor, GHSR1a in the functional regulation of the human endometrial cancer. Our results indicated that decreased in GHSR1a protein level by RNAi significantly inhibits endometrial cancer cell line and xenograft tumour growth, suggesting that blocking GHSR1a activity may provide a possible therapeutic approach for endometrial cancer.