Granulocyte colony-stimulating factor receptor signalling via Janus kinase 2/signal transducer and activator of transcription 3 in ovarian cancer.

BACKGROUND: Ovarian cancer remains a major cause of cancer mortality in women, with only limited understanding of disease aetiology at the molecular level. Granulocyte colony-stimulating factor (G-CSF) is a key regulator of both normal and emergency haematopoiesis, and is used clinically to aid haematopoietic recovery following ablative therapies for a variety of solid tumours including ovarian cancer. METHODS: The expression of G-CSF and its receptor, G-CSFR, was examined in primary ovarian cancer samples and a panel of ovarian cancer cell lines, and the effects of G-CSF treatment on proliferation, migration and survival were determined. RESULTS: G-CSFR was predominantly expressed in high-grade serous ovarian epithelial tumour samples and a subset of ovarian cancer cell lines. Stimulation of G-CSFR-expressing ovarian epithelial cancer cells with G-CSF led to increased migration and survival, including against chemotherapy-induced apoptosis. The effects of G-CSF were mediated by signalling via the downstream JAK2/STAT3 pathway. CONCLUSION: This study suggests that G-CSF has the potential to impact on ovarian cancer pathogenesis, and that G-CSFR expression status should be considered in determining appropriate therapy.

10mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin.

OBJECTIVE: Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects. DESIGN: HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells. RESULTS: Ten mM glucosamine and 5-10mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn(387) and Asn(440), abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells. CONCLUSIONS: Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

The expression of the ADAMs proteases in prostate cancer cell lines and their regulation by dihydrotestosterone.

The ADAMs are a multi-functional gene family, some of which have been shown to play a role in diverse biological processes such as fertilization, myogenesis, neurogenesis and the activation of growth factors/immune regulators such as TNF-alpha. So-named because they possess both A Disintegrin And Metalloprotease domain, the ADAMs have potential implications for the metastasis of human tumour cells via cell adhesion and protease activities. However, no studies have yet comprehensively examined the expression or regulation of ADAMs in solid tumours. Therefore, the aim of this study was to examine the expression of the ADAMs in human prostate cancer cell lines and to examine their possible regulation by androgen, a primary hormonal regulator of prostate cancer cell proliferation and metastasis. Applying RT-PCR, ADAM-9, -10, -11, -15 and -17 mRNA expression was found in the androgen-dependent prostate cancer cell lines, LNCaP and ALVA-41 and the androgen-independent cell lines, DU-145 and PC-3. Northern blotting of LNCaP cell total RNA revealed transcripts for ADAM-9 (3.8 kb), ADAM-10 (4.4, 3.2 and 0.54 kb), ADAM-15 (3 kb) and ADAM-17 (4 and 2.6 kb). ADAM-11 transcript was not detected by Northern blotting possibly due to low levels of ADAM-11 mRNA expression. This is the first report of ADAM expression in prostate cancer cell lines. Since androgens are implicated in prostate cancer cell growth and maintenance, the regulation of ADAMs by dihydrotestosterone (DHT) was investigated in the androgen-dependent cell line LNCaP. It was shown by quantitative RT-PCR using continuous fluorescence monitoring that ADAM-10 mRNA expression was regulated in a bell shaped, dose-dependent manner by DHT. Maximum stimulation was observed at 1.0 nM DHT (5-fold significant increase). For ADAM-9 mRNA, a significant upregulation was found at 1.0 and 10 nM (1.5-1.7-fold increase). In contrast, ADAM-17 mRNA, was significantly inhibited at 0.1 and 1.0 nM (1.7-fold decrease). This is the first report, to our knowledge, illustrating hormonal regulation of ADAM mRNA. The novel data described here also provide a strong stimulus to the development of specific quantitative and functional assays for particular ADAMs. These assays, which are not yet available, are required to enable subsequent investigation, both in vitro and in vivo, of the specific roles of each ADAM in prostate cancer cell proliferation, cell motility and invasion.