To investigate the expression of the second form of GnRH (GnRH-II) in tumor tissue and peripheral blood mononuclear cells (PBMCs) in malignant and benign ovarian tumors in humans.
Sixty-six women were studied: 24 with epithelial ovarian carcinomas, 22 with benign ovarian tumors and 20 in the control group undergoing surgery. Malignant, benign and normal ovarian tissue and PBMCs were obtained for measurement of GnRH-II mRNA levels using quantitative real-time RT-PCR.
The expression of GnRH-II was found to be 1.5 times higher in malignant ovarian tumors compared with benign ovarian tumors and the control group in post-menopausal patients (P<0.01). In the post-menopausal patient group with malignant ovarian tumors, there were significant positive correlations between serum FSH level and ovarian tissue GnRH-II mRNA expression (r=0.68; P=0.03), and serum LH level and ovarian tissue GnRH-II mRNA expression (r=0.71; P=0.02). Controls, benign and malignant groups were similar in terms of GnRH-II expression in PBMCs in the pre- and post-menopausal periods. There was no significant correlation between ovarian tissue GnRH-II mRNA expression vs. PBMC GnRH-II mRNA expression in patient and control groups.
We have shown increased GnRH-II expression in human ovarian cancer tissue in post-menopausal women in vivo. Expression of GnRH-II in PBMCs did not reflect the local GnRH-II expression levels in ovarian tissue. These preliminary data suggest that local GnRH-II may participate in the regulation of ovarian tumor growth in post-menopausal women.
"A good visualization of GnRH2 cell bodies and their projections will contribute to our understanding of the GnRH2 system. Even though GnRH2 expression has been detected in multiple normal1213141516 and malignant tissues/sites171819202122 and has been characterized during different biological processes232425, a clear description of GnRH2 neuronal groups and their local and far-ranging projections is still lacking. To accomplish this objective, we chose zebrafish as our research model. "
[Show abstract][Hide abstract] ABSTRACT: The presence and conservation of GnRH2 across vertebrate species suggest important biological roles. However, the function of GnRH2 remains unclear. A good research model for GnRH2 functional studies is still lacking largely due to the absence of GnRH2 in the widely used mouse model. Hence, we used the zebrafish, for which powerful genetic tools are available, and developed a transgenic (Tg) line expressing enhanced green fluorescence protein (eGFP). The high sensitivity of eGFP, which can diffuse throughout the neuron, enables us to document the complete projectome of GnRH2 neurons at different developmental stages. Fine projection structures were observed without sacrificing the fish. Crossed with the GnRH3:tdTomato Tg line, the GnRH2:eGFP Tg line provides us with an opportunity to visualize the entire GnRH system simultaneously in one organism. This work will provide a framework to understand the function of the highly-conserved GnRH2 system.
[Show abstract][Hide abstract] ABSTRACT: GnRH-II enhances ovarian cancer cell invasion in an autocrine manner. We have now found that GnRH-II increases 37-kDa laminin receptor precursor (LRP) production in GnRH receptor (GnRHR)-positive OVCAR-3 and CaOV-3 ovarian cancer cells, while small interfering RNA (siRNA)-mediated depletion of GnRH-II or GnRHR mRNA abrogates this. The invasiveness of ovarian cancer cells is also reduced >85% by siRNA-mediated knockdown of LRP levels and >50% by pretreatment of Matrigel with a synthetic peptide that blocks interactions between laminin and the 67-kDa nonintegrin laminin receptor which comprises two LRP subunits. Conversely, overexpressing LRP in CaOV-3 cells increases their invasiveness 5-fold, while overexpressing LRP with a nonfunctional laminin-binding site does not. Depletion of LRP by siRNA treatment reduces CaOV-3 cell attachment to laminin-coated plates by ∼80% but only reduces their binding to Matrigel by ∼20%. Thus, while LRP influences CaOV-3 cell adhesion to laminin, LRP must act in other ways to enhance invasion. Matrix metalloproteinases (MMPs) are key mediators of invasion, and LRP siRNA treatment of OVCAR-3 and CaOV-3 cells inhibits MMP-2 but not MMP-9 mRNA levels. Overexpressing LRP in these cells increases MMP-2 production specifically, while a laminin-binding deficient LRP does not. Importantly, LRP siRNA treatment abolishes GnRH-II-induced MMP-2 production, and invasion in OVCAR-3 and CaOV-3 cells, which was also seen after MMP-2 siRNA treatment. These results suggest that GnRH-II-induced LRP expression increases the amount of the 67-kDa nonintegrin laminin receptor, which appears to interact with laminin in the extracellular matrix to promote MMP-2 expression and enhance ovarian cancer cell invasion.
[Show abstract][Hide abstract] ABSTRACT: GnRH-II is produced by ovarian cancer cells and enhances their invasiveness in vitro. In our studies of OVCAR-3 and CaOV-3 ovarian cancer cell lines, GnRH-II treatment induced phosphorylation of Akt and glycogen synthase kinase (GSK)3β, as well as β-catenin accumulation in the nucleus, and the latter was reduced by small interfering RNA (siRNA)-mediated depletion of the GnRH receptor. The phosphatidylinositol 3 kinase (PI3K)/Akt pathway is involved in β-catenin-dependent signaling, and pretreatment of these human ovarian cancer cells with a PI3K/Akt inhibitor, LY294002, attenuated GnRH-II-stimulated phosphorylation of GSK3β and inhibited GnRH-II-induced invasion. It also attenuated GnRH-II induced trans-activation of a β-catenin-dependent reporter gene, most likely because GSK3β phosphorylation promotes translocation of β-catenin to the nucleus. Membrane type I matrix metalloproteinase (MT1-MMP) contributes to tumor progression directly, or by processing the latent MMP-2 zymogen, and is a known target of β-catenin signaling. When OVCAR-3 and CaOV-3 cells were treated with GnRH-II, MT1-MMP levels increased approximately 3-fold, whereas siRNA-mediated depletion of GnRH receptor or pretreatment with LY294002 abrogated this. In addition, lithium chloride, which increases GSK3β phosphorylation and the nuclear translocation of β-catenin, increased MT1-MMP levels in these ovarian cancer cells. By contrast, depletion of β-catenin by siRNA treatment abolished GnRH-II-induced MT1-MMP synthesis and reduced their invasive potential. Furthermore, siRNA-mediated reduction of MT1-MMP levels reduced GnRH-II-induced invasion in ovarian cancer cells. We therefore conclude that GnRH-II stimulates the PI3K/Akt pathway, and the phosphorylation of GSK3β, thereby enhancing the β-catenin-dependent up-regulation of MT1-MMP production, which contributes to ovarian cancer metastasis.
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