While oestrogen, progesterone and growth factors, including growth hormone (GH), are clearly implicated in the pathogenesis of breast cancer, there is now evidence that the newly described ghrelin axis is also involved. The aims of this study were to investigate the expression of the ghrelin axis in breast cancer tissues and cell lines and to examine the effect of ghrelin on breast cancer cell proliferation in vitro. Ghrelin and its functional receptor, the growth hormone secretagogue receptor (GHSR) type 1a, were expressed in normal breast tissue and breast cancer specimens and cell lines. In contrast, the truncated GHSR type 1b isoform was exclusively expressed in breast carcinoma, suggesting that it has potential as a diagnostic marker. Ghrelin treatment significantly increases the proliferation of the MDA-MB-435 and MDA-MB-231 breast cancer cell lines in vitro. In addition, we have described the expression of a human preproghrelin isoform, exon 3-deleted preproghrelin, which encodes mature ghrelin plus a novel C-terminal peptide. Quantitative RT-PCR was used to demonstrate that this mRNA isoform is highly expressed in the MDA-MB-435 metastatic breast cancer cell line relative to the benign MCF-10A breast epithelial cell line. The unique C-terminal peptide of exon 3-deleted preproghrelin is expressed in the glandular epithelium of breast cancer tissues, with high-grade carcinoma exhibiting the strongest immunoreactivity. The data presented here suggest that components of the ghrelin axis may represent novel markers for breast cancer and potential therapeutic targets.

Purpose: There is evidence that the hormone ghrelin stimulates proliferation in the PC3 prostate cancer cell line although the underlying mechanism(s) remain to be determined. A novel, exon 3d eleted preproghrelin isoform has previously been detected in breast and prostate cancer cells; however, its characterization, expression, and potential function in prostate cancer tissues are unknown. Experimental Design: Expression of ghrelin and exon 3^deleted preproghrelin was investigated in prostate cancer cell lines and tissues by reverse transcription-PCR and immunohistochemistry. Proliferation and apoptosis assays were done in the LNCaP prostate cancer cell line to determine if ghrelin stimulates proliferation and/or cell survival. Stimulation of mitogenactivated protein kinase (MAPK) pathway activation by ghrelin was determined in PC3 and LNCaP cells by immunoblotting with antibodies specific for phosphorylated MAPKs. Results: Prostate cancer tissues display greater immunoreactivity for ghrelin and exon 3^deleted preproghrelin than normal prostate tissues, and prostate cancer cell lines secrete mature ghrelin into conditioned medium. Treatment with ghrelin (10 nmol/L), but not the unique COOH-terminal peptide derived from exon 3^deleted preproghrelin, stimulates proliferation in the LNCaP cells (45.0 F 1.7% above control, P < 0.01) and rapidly activates the extracellular signal-regulated kinase-1/2 MAPK pathway in both PC3 and LNCaP cell lines. Ghrelin, however, does not protect prostate cancer cells from apoptosis induced by actinomycin D (1 Ag/mL). The MAPK inhibitors PD98059 and U0126 blocked ghrelin-induced MAPK activation, as well as proliferation, in both cell lines. Conclusions: These data suggest that these components of the ghrelin axis may have potential as novel biomarkers and/or adjunctive therapeutic targets for prostate cancer.Ghrelin, a 28-amino-acid n-octanoylated peptide, acts via the growth hormone secretagogue receptor (GHS-R) to stimulate growth hormone release (1, 2) and has a range of other biological actions including stimulation of food intake, control of energy expenditure, modulation of insulin signaling and cardiovascular effects (3 -7). The finding that ghrelin has a proliferative effect was first described in the HepG2 hepatoma cell line (6) and prostate cancer cell lines (8). Subsequently, it has been shown that growth of other cell types is enhanced by ghrelin (9 -18). We have recently shown that ghrelin also stimulates proliferation in several breast cancer cell lines (19) in contrast to earlier studies (20).Both ghrelin and the GHS-R (a G protein -coupled receptor) are widely expressed in normal tissues (1, 21 -23) as well as in various tumors, including human pituitary adenomas and various endocrine neoplasms of the lung, stomach, and pancreas (24 -28). We have previously shown that components of the ghrelin/GHS-R axis, including an apparent human exon 3 -deleted preproghrelin mRNA variant, are expressed in prostate cancer cell lines (8). The exclusion of the third exon ...

The type 1 insulin-like growth factor receptor (IGF1R) is overexpressed by malignant melanomas compared with benign naevi, and mediates proliferation, motility and protection from apoptosis. However, the utility of IGF1R targeting as anti-cancer therapy may be limited by activating mutations in downstream signaling intermediates. We previously showed that IGF1R knockdown blocked survival of prostate cancer cells in which Akt activation was deregulated by PTEN loss. The current study investigated effects of IGF1R targeting in cells harboring activating RAS-RAF mutations, found in 70-80% of human melanomas. We assembled a panel of eight human melanoma cell lines: two expressing wild-type (WT) B-RAF and N-RAS, two with activating N-RAS mutations and four harboring V600E B-RAF. We also generated isogenic cell populations overexpressing WT or V600E B-RAF. Cells expressing V600E B-RAF were relatively resistant to apoptosis. However, IGF1R gene silencing was capable of inducing significant inhibition of survival, enhancement of apoptosis, and Btwo-fold sensitization to cisplatin and temozolomide. These effects were independent of mutation status and were associated with reduced activation of Akt and also, unexpectedly, of ERKs. These results support development of IGF1R targeting as therapy for melanoma, regardless of the presence of activating mutations in the RAS-RAF pathway.