Human melanoma cells expressing V600E B-RAF are susceptible to IGF1R targeting by small interfering RNAs.

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 approximately two-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.

Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines.

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.

Expression of the ghrelin axis in the mouse: an exon 4-deleted mouse proghrelin variant encodes a novel C terminal peptide.

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.