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 ...
Ghrelin, an n-octanoylated 28-amino-acid peptide capable of inducing GH secretion and food intake in humans and rats, is the endogenous ligand for the GH secretagogue receptor (GHS-R). Here we describe the expression and tissue distribution of the ghrelin/GHS-R axis in the mouse. We also report for the first time the identification of a novel mouse ghrelin mRNA variant in which there is a complete deletion of exon 4. Translation of this variant mRNA yields a protein containing ghrelin and an alternative C-terminal domain with a unique C-terminal peptide sequence. RT-PCR with primers specific for mouse ghrelin was used to demonstrate the mRNA expression of the full preproghrelin transcript and the exon 4-deleted variant in multiple mouse tissues. Real-time PCR was also employed to quantitate mRNA expression of ghrelin, the novel isoform and a previously reported ghrelin gene variant, ghrelin gene-derived transcript. We also demonstrated the tissue expression of the functional GHS-R in the mouse. Immunohistochemistry, employing antibodies raised against the mature human n-octanoylated ghrelin peptide and the putative C-terminal peptide encoded by the exon 4-deleted proghrelin variant, was used to demonstrate protein expression of ghrelin and the variant in multiple mouse tissues including stomach, kidney, and reproductive tissues. The coexpression of ghrelin and its receptor in a wide range of murine tissues suggests varied autocrine/paracrine roles for these peptides. Exon 4-deleted proghrelin, a novel mouse proghrelin isoform with a unique C-terminal peptide sequence, is also widely expressed in the mouse and thus may possess biological activity in these tissues.
Although the identification of menin-interacting partners and other evidence support a role for menin, the multiple endocrine neoplasia type 1 gene (MEN1) product, in regulating gene expression, little is known about the cellular pathways dysregulated by menin loss during tumorigenesis. The mouse models of MEN1 accurately mimic the human syndrome and provide an opportunity to assess the transcriptional effects of Men1 deletion in different endocrine tumor types to identify common pathway aberrations underlying tumorigenesis in MEN1-affected tissues. We compared the global gene expression profiles of pituitary adenomas and pancreatic islet tumors with control tissues from wild-type littermates. Amongst the 551 differentially expressed genes was significant over-representation of genes associated with chromatin remodelling, transcription and cell cycling, including some genes known to encode menin-binding partners, e.g., Rhox5 and Mll1. Consistent with increased cell-cycle transition from G1 to S phase was an elevation of Cdc7 expression in the tumors, which was confirmed by qRT-PCR using independent samples. In support of previous findings in islet tumors, we found down-regulation of the cell-cycle regulator, p18, in both the pancreatic islet and pituitary adenomas, suggesting that reduced p18 levels may be important for Men1-related tumorigenesis in multiple tissues. Surprisingly, we identified increased p16 transcript in pancreatic islet and pituitary tumors. This was accompanied by increased cytoplasmic localization p16 protein in tumor cells. The specific genes and general pathways we have found to be commonly dysregulated in MEN1 tumors, provide a platform for determining their roles in endocrine tumorigenesis. ' 2007 Wiley-Liss, Inc.
To identify possible genetic interactions between the mechanisms of tumor suppression of menin and pRb, we intercrossed mice with targeted deletions of Men1 and Rb1, and compared tumor development in cohorts of animals carrying single or dual mutations of these tumorsuppressor genes. In mice lacking one copy of Men1, pancreatic islet and anterior pituitary adenomas are common. In animals lacking one copy of Rb1, intermediate pituitary and thyroid tumors occur at high frequency, with less frequent development of pancreatic islet hyperplasia and parathyroid lesions. In mice heterozygous for both Men1 and Rb1, pancreatic hyperplasia and tumors of the intermediate pituitary and thyroid occurred at high frequency. Serum measurements of calcium and glucose did not vary significantly between genotypic groups. Loss of heterozygosity at the Rb1 locus was common in pituitary and thyroid tumors, whereas loss of menin was observed in pancreatic and parathyroid lesions. The tumor spectrum in the double heterozygotes was a combination of pathologies seen in each of the individual heterozygotes, without decrease in age of onset, indicating independent, non-additive effects of the two mutations. Together with the lack of increased tumor spectrum, this suggests that menin and pRb function in a common pathway of tumor suppression.
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