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Changes in mRMA/protein expression and signaling pathways in in vivo passaged mouse ovarian cancer cells

ABSTRACT: The cure rate for late stage epithelial ovarian cancer (EOC) has not significantly improved over several decades. New and more effective targets and treatment modalities are urgently needed. RNA-seq analyses of a syngeneic EOC cell pair, representing more and less aggressive tumor cells in vivo were conducted. Bioinformatics analyses of the RNA-seq data and biological signaling and function studies have identified new targets, such as ZIP4 in EOC. Many up-regulated tumor promoting signaling pathways have been identified which are mainly grouped into three cellular activities: 1) cell proliferation and apoptosis resistance; 2) cell skeleton and adhesion changes; and 3) carbohydrate metabolic reprogramming. Unexpectedly, lipid metabolism has been the major down-regulated signaling pathway in the more aggressive EOC cells. In addition, we found that hypoxic responsive genes were at the center stage of regulation and detected functional changes were related to cancer stem cell-like activities. Moreover, our genetic, cellular, biochemical, and lipidomic analyses indicated that cells grown in 2D vs. 3D, or attached vs. suspended had dramatic changes. The important clinical implications of peritoneal cavity floating tumor cells are supported by the data proved in this work. Overall, the RNA-seq data provide a landscape of genetic alterations during tumor progression.

ORGANISM(S): Mus musculus

PROVIDER: GSE111630 | GEO | 2018/05/09

REPOSITORIES: GEO

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Project description:Background: In previous studies we showed that IL-27 shares several effects with IFN-γ in human cancer cells. To identify novel extracellular mediators, potentially involved in epithelial ovarian cancer (EOC) biology, we analyzed the effect of IL-27 or IFN-γ on the secretome of EOC cells in culture. Methods: The secretome of cytokine-treated or untreated SKOV3 cells was analyzed by nano-UHPLC-MS/MS and eluting peptides by an Orbitrap Fusion Tribrid mass spectrometer. Extracellular GBP1 was studied by ELISA, immunoprecipitation, GTP-agarose pull-down, and Western blotting. GBP and STAT proteins were analysed on cell lysates by Western blot. GBP1 was transfected using lipofectamine reagent and cell viability measured by MTT assay. Results: IL-27 and IFN-γ modulate the extracellular release of a limited fraction of proteins that were also induced in the whole cell. Among these proteins we focused our attention on GBP1, a guanylate-binding protein and GTPase, which is a mediator of several biological activities of IFNs. Interestingly GBP1, 2, and 5 were induced by cytokine treatment in EOC cells, but only GBP1 was secreted. ELISA and immuno-blotting analyses showed that cytokine-stimulated EOC cells release full-length GBP1 molecule in vitro, through non-classical pathways, not involving microvesicles. Moreover, soluble, full-length GBP1 accumulates in the ascites of most EOC patients, suggesting a potential role of extracellular GBP1 in the tumor environment. EOC cells enriched from ascites showed constitutive tyrosine-phosphorylated STAT1 and STAT3 proteins and GBP1 expression, supporting a role of STAT-activating cytokines in GBP1 induction in vivo. Data from the TCGA dataset of EOC indicate that high GBP1 gene expression correlates with a better overall survival. Accordingly GBP1 transfection reduced SKOV3 cell proliferation, suggesting a potential anti-tumor activity of GBP1. Conclusions: Our data show for the first time that soluble GBP1 is released by cytokine-stimulated EOC cells in vitro and accumulates in EOC patients’ ascites. GBP1 expression may have anti-tumor effects, in EOC, as suggested by TCGA dataset analysis and in vitro transfection experiments. Further studies to identify the biological role of soluble GBP1 in the tumor environment of EOC are warranted.

Project description:Previously, we have identified the RUNX2 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from epithelial ovarian cancer (EOC) patients, when compared to primary cultures derived from matched primary (prior to CT) tumors. However, we found no differences in the RUNX2 methylation in primary EOC tumors and EOC omental metastases, suggesting that DNA methylation-based epigenetic mechanisms have no impact on RUNX2 expression in advanced (metastatic) stage of the disease. Moreover, RUNX2 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. Knockdown of the RUNX2 expression in EOC cells led to sharp decrease of cell proliferation and significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as various genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX2 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the RUNX2 gene in EOC progression and suggest that RUNX2 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX2 and other members of the RUNX gene family in ovarian tumorigenesis. To better understand the molecular mechanisms of RUNX2 gene action in ovarian cancer cells, we employed the Agilent Whole Human Genome microarrays, containing ~ 44,000 genes to identify global gene expression changes upon RUNX2 suppression in SKOV3 cells. We compared the gene expression of the previously selected clone shRNA- RUNX2-knockdown clone 3 (cl3) against the corresponding control clone. The microarray experiments were performed in duplicates, as two hybridizations were carried out for the RUNX2-suppressing cell clone against the corresponding control, using a fluorescent dye reversal (dye-swap) technique.

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Changes in mRMA/protein expression and signaling pathways in in vivo passaged mouse ovarian cancer cells

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