Unknown,Transcriptomics,Genomics,Proteomics

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Overexpression of CD157 contributes to epithelial ovarian cancer progression by promoting mesenchymal differentiation.

ABSTRACT: Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little prospect for cure. Despite some advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, understanding the biological mechanisms underpinning EOC progression is critical for its treatment and to ameliorate patients survival. Recently, we reported that CD157 is expressed in EOC and controls tumor cell migration and invasion. Using stable overexpression and knockdown in OVCAR-3 and OV-90 ovarian cancer cell lines, we demonstrated that CD157 overexpression promotes morphological and functional changes, characterized by downregulation of epithelial marker and upregulation of mesenchymal ones. These are mediated at the transcriptional level by altering the expression of Snail and Zeb1 transcriptional repressors. The effects of CD157 overexpression on ovarian cancer phenotype translate into increased tumor cell motility and mesotelial invasion, while its knockdown significantly reduces the migratory potential, implying a direct correlation between CD157 expression levels and EOC aggressiveness. The analysis of the transcriptomic profiling highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing EOC cells. The overall picture deduced from the analysis of these modulated transcripts indicated that high CD157 expression results in strengthening of a number of biological functions that favour tumor progression (including cell differentiation, cell motility and migration), and weakening of selected biological processes that hinder the tumor progression (such as apoptosis, cell death and response to stress). Collectively, these data support a causal role of CD157 in the control of ovarian cancer progression motivating the existence of a direct correlation between the expression levels of CD157 and the adverse clinical outcome in EOC patients, and suggest that CD157 may represent a valuable therapeutic target. Gene expression analysisi of control cell lines (OVCAR-3/mock and OV-90/mock) and testing cell lines (OVCAR-3/CD157 and OV-90/CD157), with two replicates, with dye swap, performed for each sample.

ORGANISM(S): Homo sapiens

SUBMITTER: giovanna chiorino

PROVIDER: E-GEOD-36364 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little prospect for cure. Despite some advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, understanding the biological mechanisms underpinning EOC progression is critical for its treatment and to ameliorate patients survival. Recently, we reported that CD157 is expressed in EOC and controls tumor cell migration and invasion. Using stable overexpression and knockdown in OVCAR-3 and OV-90 ovarian cancer cell lines, we demonstrated that CD157 overexpression promotes morphological and functional changes, characterized by downregulation of epithelial marker and upregulation of mesenchymal ones. These are mediated at the transcriptional level by altering the expression of Snail and Zeb1 transcriptional repressors. The effects of CD157 overexpression on ovarian cancer phenotype translate into increased tumor cell motility and mesotelial invasion, while its knockdown significantly reduces the migratory potential, implying a direct correlation between CD157 expression levels and EOC aggressiveness. The analysis of the transcriptomic profiling highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing EOC cells. The overall picture deduced from the analysis of these modulated transcripts indicated that high CD157 expression results in strengthening of a number of biological functions that favour tumor progression (including cell differentiation, cell motility and migration), and weakening of selected biological processes that hinder the tumor progression (such as apoptosis, cell death and response to stress). Collectively, these data support a causal role of CD157 in the control of ovarian cancer progression motivating the existence of a direct correlation between the expression levels of CD157 and the adverse clinical outcome in EOC patients, and suggest that CD157 may represent a valuable therapeutic target.

Project description:Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC. Initially a total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression using RT-PCR analysis. The gene for claudin-7, CLDN7, was found to be significantly upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Two cell lines(OVCAR-2 and OVCA420) were chosen for microarray based gene expression analysis using small interfering RNA-mediated(SiRNA) knockdown of claudin-7. This treatment led to significant changes in gene expression that was validated by RT-PCR and immunoblotting. Ovarian cancer lines OVCA420 and OVCAR-2 were cultured in duplicate in McCoy’s 5A growth medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells cultured in 6-well plates were transfected with either Claudin-7 specific siRNA oligos duplexes (Ambion, Inc.Austin, TX) or control siRNA #1 (Dharmacon) using LipofectAMINE 2000 (Invitrogen). Cells were treated for 72 hours to allow maximum knockdown, after which they were harvested for RNA preparation. Total RNA, was extracted using Trizol, quality and quantity were checked using the Agilent 2100 Bioanalyzer with RNA 6000 Nano chip, (Agilent Technologies UK Ltd, West Lothian, UK). Biotinylated cRNA was generated using the Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX) starting with approximately 500 ng total RNA. Hybridization of the cRNA was to the Sentrix HumanRef-8 Expression BeadChip (Illumina, Inc., San Diego, CA). Microarray data processing and analysis was performed using Illumina Bead Studio software. Hierarchical clustering analysis of the significant genes was done using the JMP 6.0.0 software. Validation of the expression patterns of selected genes was performed using RT-PCR.

Project description:With the onset of resistance, ovarian cancer cells display almost unpredictable adaptive potential. This may derive from genetic ancestry of the tumor cells and can be additionally tailored by post translational protein modifications (PTMs). The latter are profoundly shaped by the physical and chemical cues of the cancer microenvironment which, for ovarian cancer, is primarily the abdominal cavity. In this study, we took advantage of high-end proteome and phosphoproteome analyses combined with multiparametric morphometric profiling for the description of the proteome signatures of high-grade serous carcinomas (OVCAR-3) and non-serous carcinomas (SKOV-3) ovarian cancer cells. For the functional experiments, we applied two different protocols, representing typical stimuli of the peritoneal cavity and of the growing tumor mass: on the one side hypoxia (oxygen 1%) which develops within the tumor mass or is experienced during migration/extravasation in non-vascularized tissues. For comparison, fluid shear stress (2.8 dyn/cm2, orbital shaker method) which characterizes the tumor surface in peritoneal cavity or metastases spread in the bloodstream. After 3 hours incubation, treatment groups were clearly distinguishable by PCA analysis. Whereas basal proteome profile of SKOV-3 and OVCAR-3 cells appeared almost unchanged, phosphoproteome analysis revealed multiple regulations. These affected primarily cellular structure and proliferative potential and consolidated after 24h treatment. Albeit maintaining their individuality, hypoxia modified cell metabolism and morphology of both OVCAR-3 and SKOV-3: upon oxygen reduction cell size increased in concerted regulation of pathways related to Rho-GTPases and/or cytoskeletal elements (proteome and phosphoproteome). Also shear stress stimulation sharpened the response profile of SKOV-3 and OVCAR-3: this retraced their pathophysiological behavior and actively modified structural proteins as well as metabolism (e.g. delta(14)-sterol reductase, Kinesin-like proteins (KIF-22/20A) and Actin-related protein 2/3 complex). In conclusion, we characterized a biochemical and structural fingerprinting describing the adaptive potential of ovarian cancer cells to physical/chemical stressors typical for the abdominal cavity.

Project description:We previously demonstrated that the osteopontin-c (OPNc) splice variant activates several aspects of the progression of ovarian cancers. The goal of the present study was to develop cell line model to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. Methods: OvCar-3 cells were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses; Results: Of 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34, were differentially expressed between OvCar-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Conclusions: Overall, the present study elucidated transcriptional changes of ovarian cancer cells in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features. Cell Culture, OPN Plasmids and Transfection; As a model to examine the signaling pathways modulated by OPNc overexpression in ovarian carcinoma, we used OvCar-3 cell line, which were provided by ATCC. The open reading frame of OPNc was cloned into the pCR3.1 mammalian expression vector as previously described [Tilli et al., 2012]. Transfections were performed using LipofectamineTM 2000 (Invitrogen, CA). Our previous data demonstrated that OvCar-3 stably transfected cells contain higher levels of protein and the mRNA OPNc splicing isoform in relation to their endogenous levels in empty vector-transfected cells. OvCar-3 transfected with empty vector (EV) were used as a negative control in these assays. Human Cancer Pathway Finder PCR Array The Human Cancer Pathway Finder SuperArray (PAHS-033A; Qiagen) was used to determine changes in the specific genes encoding proteins related to the main hallmarks of cancer in response to OPNc overexpression. The assay design criteria ensure that each qPCR reaction will generate single, gene-specific amplicons and prevent the co-amplification of non-specific products. The qPCR Assays used in PCR Arrays are optimized to work under standard conditions enabling a large number of genes to be assayed simultaneously. Their specificity is guaranteed by SABiosciences when RT2 SYBR Green qPCR Master Mixes are used as part of the complete PCR Array System protocol. Simultaneous gene expression analyses require similar qPCR efficiencies for accurate comparison among genes. RT2 qPCR Primer Assays are designed with an amplicon size ranging from 100 to 250 bp and with PCR efficiencies uniformly greater than 90%. Overall, more than 10 thermodynamic criteria are included in the design of each RT2 qPCR Primer Assay to ensure the most reliable and accurate results for pathway-based gene expression analysis in the PCR Array System. We analyzed mRNA levels of 84 genes related to cell cycle control, apoptosis and cell senescence, signal transduction molecules and transcription factors, adhesion, angiogenesis, invasion and metastasis; and also 5 housekeeping genes and genomic DNA contamination controls. The PCR plates were run using the CFX96 Real-Time System cycler (BioRad, Hercules, CA), following a superarray two-step cycling PCR protocol, in which each plate ran one cycle for 10 minutes at 95M-BM-0C, as well as 40 cycles of 95M-BM-0C for 15 seconds and 60M-BM-0C for 1 minute. After the superarray protocol was run for each plate, RT-PCR data analysis was performed using the website:http://www.SABiosciences.com/pcrarraydataanalysis.php, in order to compare gene expression of OPNc-overexpressing cells and empty vector transfected cells. Total RNA quality control, cDNA synthesis and the quantitative real-time RT-PCR (qRT-PCR) array were performed as recommended by the manufacturer (Qiagen). Data analysis of gene expression was performed using Excel-based PCR Array Data Analysis Software provided by manufacturer (Qiagen). Fold-changes in gene expression were calculated using the M-NM-^TM-NM-^TCT method, and five stably expressed housekeeping genes (M-NM-22 microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, GAPDH and M-NM-2-actin) were used to normalize the level of expression. The statistical analysis was performed to compare the gene expression values for the OPNc-overexpressing cells and those transfected with empty vector. P<0.05 was considered statistically significant. Only genes showing a 1.5-fold or greater change were considered for further analysis.

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Overexpression of CD157 contributes to epithelial ovarian cancer progression by promoting mesenchymal differentiation.

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