Ovarian Cancer Tumorigenesis Research Articles

WNT7A Regulates Tumor Growth and Development Through the WNT/CTNNB1 Pathway in Ovarian Cancer.

The canonical WNT signaling pathway regulates embryonic ovarian development as well as normal ovarian function and folliculogenesis by governing cellular proliferation and differentiation. Alterations in the WNT system have been implicated in ovarian cancer, however, the contribution of specific ligand partners associated with WNT signaling to ovarian tumorigenesis have not been elucidated. The goal of the current study was to examine the role and functional mechanisms of WNT7A in ovarian cancer. WNT7A was expressed in 40-60 % of high grade serous ovarian malignant tumors, and 20-30 % of mucinous and clear cell adenocarcinomas, but was not detected in borderline/benign tumors, normal ovary or endometrioid carcinomas. Further, WNT7A was detected in only the highly invasive SKOV3.ip1 cell line in a cohort of normal and malignant ovarian epithelial cells. Using lentiviral transfection of shRNA, WNT7A was silenced in SKOV3.ip cells and two subclones, ip11 and ip12, were selected for further study after confirmation of WNT7A mRNA and protein suppression. To characterize the role of WNT7A in advanced ovarian cancer, nude mice were injected with SKOV3.ip1, ip11, and ip12 intraperitoneally or subcutaneously. In the i.p. group, mice receiving ip11 and ip12 cells had significantly lower tumor burden and implanted tumor number. While mice injected with SKOV3.ip1 parental cells failed to gain weight during the experiment (a sign of advanced illness), those with ip11 and ip12 cells displayed increased body weight throughout the study. Gross and histological examination further revealed greatly reduced invasion of ip11 and ip12 cells into intestinal mesentery and serosa compared to SKOV3.ip parental cells. Tumor growth of ip11 and ip12 was significantly decreased compared to SKOV3.ip controls in mice given s.c. injection as well, and western blot analysis of homogenates verified sustained reduction of WNT7A expression in ip11 and ip12 tumors. Immunohistochemical staining of s.c. tumors showed decreased expression of cyclin D1, a WNT7A transcriptional target and regulator of cell cycle and proliferation, in ip11 and ip12 tumors compared to SKOV3.ip1. In vitro analysis of cell function revealed significantly reduced proliferation, adhesion, and migration, as well as cell cycle arrest in ip11 and ip12 cells compared to SKOV3.ip1 parental cells. Further, expression of MMP7, a transcriptional target of WNT7A involved in tumor invasion, was reduced in ip11 and ip12 cells compared to SKOV3.ip1 cells. These results were verified by stable overexpression of WNT7A in ovarian cancer cells which lack endogenous WNT7A. Increased proliferation, adhesion, and migration were observed in these cells. Activation of the TopFlash (TCF/LEF) reporter confirmed that transfection of WNT7A activates the canonical WNT/CTNNB1 pathway. Further, co-transfection with FZD5 (WNT receptor) produced an even greater increase in TopFlash reporter activity, and this effect was inhibited by co-transfection with the WNT receptor antagonist SFRP2. These results indicate that WNT7A is a critical regulator of ovarian cancer tumorigenesis and disease progression mediated by intracellular signaling through the WNT/CTNNB1 pathway. Supported by American Cancer Society Illinois Division #139038. (platform)

Abstract LB-16: Inhibition of RalA as a novel strategy for targeting ovarian cancer

Abstract Abstract: The Ral (Ras like) GTPase guanyl nucleotide-binding proteins, RalA and RalB are direct effectors of pro-oncogene Ras. RalA and RalB are implicated in tumorigenesis and play distinct roles in mediating carcinogenesis. The critical role of RalA in the initiation and progression of ovarian cancer is entirely unclear. Here, we report that RalA is overactivated in human ovarian cancer cells and human ovarian cancer tissues. The RalA downstream effectors (RalBP-1 and CDC42) and the regulators (GalGDS, PP2A Aα and β, as well as Aurora Kinase) that modulate RalA activation during the post-translational process were found to be de-regulated in ovarian cancer cells. ShRNA-mediated knockdown of RalA inhibited ovarian cancer cell proliferation and invasion. Geranyl-Geranyl transferase inhibitors (GGTI-2147) or Aurora Kinase Inhibitor (AKI) that inhibit RalA activation at post-translational levels suppressed ovarian cancer cell growth and invasion in-vitro. Additionally, subcutaneous (SC) mouse model with these two inhibitors also showed effective inhibition of tumor growth in-vivo. Therefore, RalA is a key player in the biology and dispersal of ovarian cancer cells and is a promising target for the development of novel therapeutics. To the best of our knowledge, the role of RalA in ovarian cancer tumorigenesis has not been documented before, therefore our strategy in targeting RalA for treatment of ovarian cancer merits highly in terms of novelty. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-16. doi:10.1158/1538-7445.AM2011-LB-16

Osteopontin-c Splicing Isoform Contributes to Ovarian Cancer Progression

Ovarian carcinoma is one of the most aggressive gynecological diseases and generally diagnosed at advanced stages. Osteopontin (OPN) is one of the proteins overexpressed in ovarian cancer and is involved in tumorigenesis and metastasis. Alternative splicing of OPN leads to 3 isoforms, OPNa, OPNb, and OPNc. However, the expression pattern and the roles of each of these isoforms have not been previously characterized in ovarian cancer. Herein, we have evaluated the expression profiling of OPN isoforms in ovarian tumor and nontumor samples and their putative roles in ovarian cancer biology using in vitro and in vivo functional assays. OPNa and OPNb were expressed both in tumor and nontumor ovarian samples, whereas OPNc was specifically expressed in ovarian tumor samples. The isoform OPNc significantly activated OvCar-3 cell proliferation, migration, invasion, anchorage-independent growth and tumor formation in vivo. Additionally, we have also shown that some of the OPNc-dependent protumorigenic roles are mediated by PI3K/Akt signaling pathway. OPNc stimulated immortalized ovarian epithelial IOSE cell proliferation, indicating a role for this isoform in ovarian cancer tumorigenesis. Functional assays using OPNc conditioned medium and an anti-OPNc antibody have shown that most cellular effects observed herein were promoted by the secreted OPNc. According to our data, OPNc-specific expression in ovarian tumor samples and its role on favoring different aspects of ovarian cancer progression suggest that secreted OPNc contributes to the physiopathology of ovarian cancer progression and tumorigenesis. Altogether, the data open possibilities of new therapeutic approaches for ovarian cancer that selectively down regulate OPNc, altering its properties favoring ovarian tumor progression.

Identification of salt-inducible kinase 3 as a novel tumor antigen associated with tumorigenesis of ovarian cancer

Existence of humoral immunity has been previously demonstrated in malignant ascitic fluids. However, only a limited number of immunogenic tumor-associated antigens (TAAs) were identified, and few of which are associated with ovarian cancer. Here, we identified salt-inducible kinase 3 (SIK3) as a TAA through screening of a random peptide library in the phage display system. Overexpression of SIK3 markedly promoted cell proliferation, attenuated p21(Waf/Cip1) and p27(Kip) expressions in low-grade OVCAR3 cells, and permitted the cells to grow in mice. Decrease in SIK3 expression in high-grade SK-OV3 cells consistently demonstrated its tumorigenic potency by modulating the protein levels of cell cycle regulators. When the expressions of SIK3 and CA125 were compared in cancer tissues, immunohistochemical (IHC) studies indicated that cytoplasm-localized SIK3 was highly expressed in 55% of the ovarian cancer samples. In contrast, it was rarely detected in adenomyosis, leiomyoma and normal ovary tissues, showing its higher specificity (97%) to CA125 (65%) in ovarian cancer. Moreover, experiments using pharmacological inhibitors to block SIK3-induced p21(Waf/Cip1) expression revealed that activation of c-Src and phosphoinositide-3-kinase were critically required for its biological activity, suggesting that they are the downstream signaling mediators of SIK3. These data were further supported by IHC studies, showing coexpression of c-Src with SIK3 in 85% of the ovarian tumor samples stained positive for SIK3. Collectively, our findings indicate that SIK3 is a novel ovarian TAA. Overexpression of SIK3 promotes G1/S cell cycle progression, bestows survival advantages to cancer cells for growth and correlates the clinicopathological conditions of patients with ovarian cancer.

EZH2 regulates expression of p57 and contributes to progression of ovarian cancer in vitro and in vivo

In order to determine the expression pattern of EZH2 in ovarian neoplasms and assess the functions and mechanism of EZH2 in tumorigenesis in vitro and in vivo, we detected the protein and mRNA expression of EZH2 and p57 in normal, benign and malignant ovarian tissues, used shRNA to knock down EZH2 expression in ovarian cancer cells and established a nude mouse xenograft model. We found EZH2 was overexpressed in ovarian tumor with the highest level expression in malignant ovarian tissues, and the variation of EZH2 expression at different pathological type/grade and International Federation of Gynecology and Obstetrics (FIGO) stages was statistically significant. Furthermore, the EZH2 expression was inversely correlated with the p57 mRNA level in ovarian tissues. Moreover, inhibition of endogenous EZH2 increased the expression of p57 and reduced proliferation and migration of ovarian cancer cells. Loss of EZH2 suppresses ovarian tumor formation in vivo. Our results indicate that the EZH2 gene acts as an oncogene in tumorigenesis of ovarian cancer with the possible mechanism to suppress the anti-oncogene p57. EZH2 is a potential therapeutic target for treatment of ovarian cancer.

Abstract 3208: Semaphorin 3F suppresses telomerase activity

Abstract Objectives: Telomerase is a ribonucleoprotein that is active in more than 90% of human tumors, including ovarian cancers. We have previously demonstrated that vascular endothelial growth factor (VEGF) stimulates telomerase activity in ovarian cancer cell lines through cellular VEGF receptors and the ERK1/2 pathway targeting Sp1 sites within the hTERT promoter. Since semaphorin 3F (S3F), a proposed tumor suppressor, has been shown to antagonize VEGF, the purpose of this study was to determine whether S3F inhibits telomerase Methods: Telomerase-positive ovarian cancer cell lines (OV2008, C-13, SW626) and telomerase-negative non-tumorigenic, SV-40 large-T antigen-transfected human ovarian surface epithelium (HOSE) cell lines were evaluated for endogenous RNA and protein expression of S3F, VEGF, and their shared receptors. Cultured cells were either treated with recombinant S3F protein or transiently transfected with cDNA to over-express S3F. Conversely, these cells were transiently transfected with siRNA targeting S3F ± specific receptor inhibitors. RT-PCR and western blot confirmed S3F over-expression, knock-down of S3F, or inhibition of receptors. Telomerase activity was measured by telomerase PCR-ELISA, while RT-PCR and western immunoblotting were utilized to detect S3F as well as to explore the signaling pathway involved in S3F-mediated telomerase regulation. Results: Telomerase activity was suppressed in ovarian cancer cell lines following treatment with S3F recombinant protein or S3F over-expression by transient transfection. Furthermore, siRNAs targeting S3F and NP-2 or treatment with the VEGF receptor inhibitor, CBO-P11, abrogated S3F mediated telomerase inhibition. Over-expression of S3F resulted in up-regulation of phosphorylation of Erk1/2. Conclusions: These data suggest that telomerase is a novel molecular target of S3F. Since, angiogenic and angiostatic factors play an important role in ovarian cancer tumorigenesis, S3F-mediated inhibition of telomerase represents a potential novel target for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3208.

Abstract 1948: Pvt1-encoded microRNA miR-1204 in tumorigenesis of human ovarian and breast cancer

Abstract Amplification at chromosome 8q24 is one of the most frequent genomic abnormalities in human cancers and is associated with reduced survival duration in ovarian and breast cancers. Recently, microRNAs (miRNAs) at 8q24 have been identified in the PVT1 locus. Herein, we explored the biological functions of one miRNA, miR-1204, residing within the exon1B of PVT1. Mature hsa-mir-1204 is over-expressed in PVT1 amplified ovarian and breast cancer cell lines (HEY, OVCAR8, SUM-159PT and MDA-MB231). In vitro treatment with antagomir against hsa-mir-1204 inhibited cell proliferation and induced apoptosis in the four PVT1 amplified ovarian and breast cancer lines. Conversely, over-expression of miR-1204 promoted cell growth in PVT1 non-amplified ovarian and breast cancer lines (OV90 and MCF10A) and cooperated with c-Myc to potentiate Rat1A cell colony growth in soft agar assays. Since an inverse expression pattern is often present between miRNA and its target genes, we used computational approaches (miRanda, TargetScan, Pita) to identify distinct miR-1204 candidate target genes from two independent expression microarray datasets, one with significantly down-regulated genes by PVT1 copy number gain in 147 breast tumors and the other involving genes significantly repressed by miR-1204 overexpression and derepressed by silencing of miR-1204 in ovarian and breast cancer cells. Thirty putative target genes of miR-1204 were empirically verified by Q-RT-PCR method, and this group of genes was enriched with genes regulating cell death, cell growth and proliferation, cell cycle, cellular assembly and organization. Combined, these data suggest that human miR-1204 contributes to ovarian and breast tumor proliferation and survival and down-regulates distinct targets including multiple tumor suppressor genes and serves as a potential therapeutic target against tumors with 8q24 amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1948.

Dendritic cells transduced with Rsf-1/HBXAP gene generate specific cytotoxic T lymphocytes against ovarian cancer in vitro

Recently, some studies have indicated that Rsf-1/HBXAP plays a role in chromatin remodeling and transcriptional regulation that may contribute to tumorigenesis in ovarian cancer. The present study demonstrates that using dendritic cells (DCs) from human cord blood CD34 + cells transduced with Rsf-1/HBXAP DNA plasmids by nucleofection generate specific cytotoxic T lymphocytes (CTL) against ovarian cancer in vitro. After transfection, DCs were analyzed for Rsf-1/HBXAP mRNA expression by RT-PCR and protein expression by Western blot. Then the DC phenotypes, T-cell stimulatory capacity, endocytic activity and migration capacity were explored by flow cytometry analysis, allogeneic mixed lymphocyte reaction, endocytosis and transwell chemotaxis assay, respectively. After transfection, Rsf-1/HBXAP expression was detected at mRNA and protein levels. Allogeneic T-cell proliferation induced by transfected DCs was obviously higher than non-transfected DCs, but the endocytosis capacity and migratory ability were not different. Rsf-1/HBXAP gene-transduced DCs could induce antigen-specific CTL and generate a very potent cytotoxicity to OVCAR3 cells. These data suggest that Rsf-1/HBXAP gene-transduced DCs may be a potential adjuvant immunotherapy for ovarian cancer in clinical applications.

Targeting insulin and insulin-like growth factor pathways in epithelial ovarian cancer.

Ovarian cancer is the most lethal of all gynecological malignancies, due in part to the diagnosis at an advanced stage caused by the lack of specific signs and symptoms and the absence of reliable tests for screening and early detection. Most patients will respond initially to treatment but about 70% of them will suffer a recurrence. Therefore, new therapeutic modalities are urgently needed to overcome chemoresistance observed in ovarian cancer patients. Evidence accumulates suggesting that the insulin/insulin growth factor (IGF) pathways could act as a good therapeutic target in several cancers, including ovarian cancer. In this paper, we will focus on the role of insulin/IGF in ovarian cancer tumorigenesis and treatment.

Abstract B13: Identification of a metalloprotease-chemokine signaling system in the ovarian cancer microenvironment: Implications for antiangiogenic therapy

Abstract Ovarian cancer is a lethal gynecologic malignancy with poor survival, hence it would benefit from therapies that block key signaling pathways involved in tumor-stromal interactions and tumor vascularity. We previously showed that Protease Activated Receptor 1 (PAR1) is an important regulator of angiogenesis, ascites and metastasis in peritoneal mouse models of ovarian cancer. In this study we tested the hypothesis that matrix metalloprotease1 (MMP-1) activates PAR1 which stimulates angiogenesis and tumorigenesis in ovarian cancer through its paracrine control of the endothelial CXCR1/2 receptors. We demonstrated that MMP-1-PAR1 stimulation leads to a 2–5 fold increase in several angiogenic factors including IL-8 and GRO from ovarian cancer cells. These secreted factors then act on endothelial CXCR1/2 receptors to cause endothelial cell proliferation, tube formation and migration. A cell penetrating pepducin, PZ-206, that targets the third intracellular loop of CXCR1 and CXCR2 receptors (also known as X1/2pal-i3) can significantly inhibit both endothelial cell proliferation and tube formation. Avastin-the VEGF blocking antibody was unable to inhibit IL-8 and GRO- stimulated tube formation. To validate our in vitro observations we injected matrigel plugs containing MMP-1 stimulated/unstimulated ovarian cancer (OVCAR-4) conditioned media into the flanks of nude mice. After 7 days, the matrigel plugs were harvested and stained for CD31 and whole mount confocal microscopy was done in a blinded manner. Mice injected with MMP-1 stimulated, OVCAR-4 CM showed an 8 fold increase in blood vessel formation which was significantly inhibited by injecting mice with CXCR1/2 inhibitor PZ206 (5mg/kg/d) as compared with control pepducin or vehicle. These studies will help us validate novel therapeutic targets such as CXCR1/2 pepducins for ovarian cancer that block angiogenesis and tumor progression to improve survival in this lethal disease. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B13.

BMP-2 signaling in ovarian cancer and its association with poor prognosis

BackgroundWe previously observed the over-expression of BMP-2 in primary cultures of epithelial ovarian cancer (EOC) cells as compared to normal epithelial cells based on Affymetrix microarray profiling [1]. Here we investigate the effect of BMP-2 on several parameters of ovarian cancer tumorigenesis using the TOV-2223, TOV-1946 and TOV-112D EOC cell lines.MethodsWe treated each EOC cell line with recombinant BMP-2 and assayed various parameters associated with tumorigenesis. More specifically, cell signaling events induced by BMP-2 treatment were investigated by western-blot using anti-phosphospecific antibodies. Induction of Id1, Snail and Smad6 mRNA expression was investigated by real time RT-PCR. The ability of cells to migrate was tested using the scratch assay. Cell-cell adhesion was analyzed by the ability of cells to form spheroids. We also investigated BMP-2 expression in tissue samples from a series of EOC patients.ResultsTreatment of these cell lines with recombinant BMP-2 induced a rapid phosphorylation of Smad1/5/8 and Erk MAPKs. Increased expression of Id1, Smad6 and Snail mRNAs was also observed. Only in the TOV-2223 cell line were these signaling events accompanied by an alteration in cell proliferation. We also observed that BMP-2 efficiently increased the motility of all three cell lines. In contrast, BMP-2 treatment decreased the ability of TOV-1946 and TOV-112D cell lines to form spheroids indicating an inhibition of cell-cell adhesion. The expression of BMP-2 in tumor tissues from patients was inversely correlated with survival.ConclusionThese results suggest that EOC cell secretion of BMP-2 in the tumor environment contributes to a modification of tumor cell behavior through a change in motility and adherence. We also show that BMP-2 expression in tumor tissues is associated with a poorer prognosis for ovarian cancer patients.

Genome-wide gene expression profiles of ovarian carcinoma: Identification of molecular targets for the treatment of ovarian carcinoma

This study aimed to clarify the molecular mechanisms involved in ovarian carcinogenesis, and to identify candidate molecular targets for its diagnosis and treatment. The genome-wide gene expression profiles of 22 epithelial ovarian carcinomas were analyzed with a microarray representing 38,500 genes, in combination with laser microbeam microdissection. A total of 273 commonly up-regulated transcripts and 387 down-regulated transcripts were identified in the ovarian carcinoma samples. Of the 273 up-regulated transcripts, only 87 (31.9%) were previously reported as up-regulated in microarray studies using bulk cancer tissues and normal ovarian tissues for analysis. CHMP4C (chromatin-modifying protein 4C) was frequently overexpressed in ovarian carcinoma tissue, but not expressed in the normal human tissues used as a control. Our data should contribute to an improved understanding of tumorigenesis in ovarian cancer, and aid in the development of diagnostic tumor markers and molecular-targeting therapy for patients with the disease.

Enhanced Ovarian Cancer Tumorigenesis and Metastasis by the Macrophage Colony-Stimulating Factor

Coexpression of the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R) in metastatic ovarian cancer specimens is a predictor of poor outcome in epithelial ovarian cancer. This suggests that an autocrine loop is produced by which ovarian tumors can secrete CSF-1 stimulating the CSF-1R resulting in a more aggressive phenotype. Our current work sought to validate this autocrine stimulation model using stable transfection of a 4-kb CSF-1 construct into otherwise nonvirulent Bix3 ovarian cancer cells. A representative clone, Bix3T8.2, produced a 72-fold increase in CSF-1 gene transcription rate (by nuclear run-off assays) and a 57-fold increase in secreted CSF-1 protein (by sandwich ELISA), compared to parent cells. Comparison of Bix3T8.2 invasion, adhesion, and motility in vitro and metastasis in vivo were made to parental and transfectant controls. Up to 12-fold higher invasiveness was seen with Bix3T8.2 and 2- and 6-fold higher adhesion and motility, respectively, over controls in vitro. In nude mice, i.p. injection of Bix3T8.2 produced a wide array of visceral, nodal, and distant metastasis with a degree of enhanced tumor burden not seen in any of the 10 mice inoculated with transfectant control cells. Complete absence of tumor take distinguished 40% of mice implanted with transfectant control cells. Disruption of this autocrine loop using antisense oligomer therapy against CSF-1R and 3′ untranslated region knockdown of CSF-1 protein resulted in reversal of in vitro and in vivo tumor phenotypes. This CSF-1 feedback loop offers a model by which novel biologic therapies can potentially target multiple levels of this pathway.

PEA-15 Induces Autophagy in Human Ovarian Cancer Cells and Is Associated with Prolonged Overall Survival

Phospho-enriched protein in astrocytes (PEA-15) is a 15-kDa phosphoprotein that slows cell proliferation by binding to and sequestering extracellular signal-regulated kinase (ERK) in the cytoplasm, thereby inhibiting ERK-dependent transcription and proliferation. In previous studies of E1A human gene therapy for ovarian cancer, we discovered that PEA-15 induced the antitumor effect of E1A by sequestering activated ERK in the cytoplasm of cancer cells. Here, we investigated the role of PEA-15 in ovarian cancer tumorigenesis, the expression levels of PEA-15 in human ovarian cancer, and whether PEA-15 expression correlated with overall survival in women with ovarian cancer. We overexpressed PEA-15 in low-PEA-15-expressing cells and knocked down PEA-15 in high-PEA-15-expressing cells and analyzed the effects on proliferation, anchorage-independent growth, and cell cycle progression. We then assessed PEA-15 expression in an annotated tissue microarray of tumor samples from 395 women with primary epithelial ovarian cancer and tested whether PEA-15 expression was linked with overall survival. PEA-15 expression inhibited proliferation, and cell cycle analysis did not reveal apoptosis but did reveal autophagy, which was confirmed by an increase in LC3 cleavage. Inhibition of the ERK1/2 pathway decreased PEA-15-induced autophagy. These findings suggest that the antitumor activity of PEA-15 is mediated, in part, by the induction of autophagy involving activation of the ERK1/2 pathway. Multivariable analyses indicated that the women with high-PEA-15-expressing tumors survived longer than those with low-PEA-15-expressing tumors (hazard ratio, 1.973; P = 0.0167). Our findings indicate that PEA-15 expression is an important prognostic marker in ovarian cancer.

Early diagnosis of epithelial ovarian cancer with cDNA microarry

Objective To investigate the roles of tumor suppressor genes-PTEN, nm23H1, VEGF165, Tiam1, MMP-2, Timp2, HE4 and S100A4 in tumorigenesis and progression of epithelial ovarian cancer(EOC) and develop a novel method for the early diagnosis of EOC.

Targeting Aurora Kinase Inhibits Tumorigenesis in Ovarian Cancer

Targeting Aurora Kinase Inhibits Tumorigenesis in Ovarian Cancer

Alternative Splicing of Breast Cancer Associated Gene BRCA1 from Breast Cancer Cell Line

Breast cancer is the most common malignancy among women, and mutations in the BRCA1 gene produce increased susceptibility to these malignancies in certain families. In this study, the forward 1-13 exons of breast cancer associated gene BRCA1 were cloned from breast cancer cell line ZR-75-30 by RT-PCR method. Sequence analysis showed that nine BRCA1 splice forms were isolated and characterized, compared with wild-type BRCA1 gene, five splice forms of which were novel. These splice isoforms were produced from the molecular mechanism of 5' and 3' alternative splicing. All these splice forms deleting exon 11b and the locations of alternative splicing were focused on two parts:one was exons 2 and 3, and the other was exons 9 and 10. These splice forms accorded with GT-AG rule. Most these BRCA1 splice variants still kept the original reading frame. Western blot analysis indicated that some BRCA1 splice variants were expressed in ZR-75-30 cell line at the protein level. In addition, we confirmed the presence of these new transcripts of BRCA1 gene in MDA-MB-435S, K562, Hela, HLA, HIC, H9, Jurkat and human fetus samples by RT-PCR analysis. These results suggested that breast cancer associated gene BRCA1 may have unexpectedly a large number of splice variants. We hypothesized that alternative splicing of BRCA1 possibly plays a major role in the tumorigenesis of breast and/or ovarian cancer. Thus, the identification of cancer-specific splice forms will provide a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention.

Expression of estrogen receptor-related receptors, a subfamily of orphan nuclear receptors, as new tumor biomarkers in ovarian cancer cells

A subfamily of orphan receptors, estrogen receptor-related receptors (ERRs), has been demonstrated to modulate the transcription of some estrogen responsive genes via variant estrogen response elements (EREs). This study was conducted to determine whether human ERRalpha, ERRbeta, and ERRgamma might be involved in the tumorigenesis of ovarian cancer. RT-PCR was performed to analyze the expression of hERRalpha, hERRbeta, hERRbeta-2, and hERRgamma mRNA in five ovarian cancer cell lines as well as 33 samples of ovarian cancer and 12 samples of normal ovary. Serum CA-125 levels were also analyzed in all samples by ELISA. Progression-free survival and overall survival of patients with different expression of ERRs were analyzed by the Kaplan-Meier method. To analyze the subcellular localization of ERRalpha, a green fluorescent protein (GFP)-reporter plasmid of hERRalpha was constructed and transfected into the ovarian cancer cell line OVCAR-3. Expression of hERRalpha-GFP fusion protein was observed in the nucleus of OVCAR-3 ovarian cancer cell lines. We observed increased expression of hERRalpha mRNA (P = 0.020) and hERRgamma mRNA (P = 0.045) in ovarian cancers compared to normal ovaries. In contrast, hERRbeta was only observed in 9.1% of ovarian cancers. We found a positive correlation between the serum CA-125 levels and hERRalpha expression (P = 0.012), but not hERRbeta and hERRgamma expression. Survival analysis showed that the hERRalpha-positive group has a reduced overall survival (P = 0.015), and the ERRgamma-positive group has a longer progression-free survival (P = 0.020). In multivariate analysis, expression of hERRalpha was an independent prognostic factor for poor survival (relative risk, 3.032; 95% CI, 1.27-6.06). Based on our results, ERRs may play an important role in ovarian cancer. hERRalpha may represent a biomarker of poor prognosis, and hERRgamma may be a new therapeutic target in ovarian cancer.

Expression of RASSF1A and RASSF1C transcripts in human primary ovarian cancers

To investigate the expression of two major alternative transcripts of RASSF1 gene (RASSF1A and RASSF1C) in human primary ovarian cancers and their biological implication as a new tumor suppressor gene. Reverse transcription-polymerase chain reaction (RT-PCR) and laser capture microdissection (LCM) were used to determine mRNA expression of the two major alternative transcripts of RASSF1 gene (RASSF1A and RASSF1C) in 3 ovarian cancer cell lines and 80 cases of primary ovarian cancers. RASSF1A mRNA was undetectable in SK-OV-3 cell line. Expression of RASSF1A and RASSF1C in 80 primary ovarian cancers were 40.0% (32/80) and 91.3% (73/80) respectively. RASSF1A mRNA expression was detectable more frequently in stage I and II (71.4%, 10/14; 75.0%, 9/12) than in stage III and IV ovarian cancers (26.7%, 12/45; 14.1%, 1/9) (P < 0.05). The expression level was also higher in well and moderately differentiated tumor groups (58.6%, 17/29; 50.0%, 10/20) than in poorly differentiated tumor group (16.1%, 5/31) (P < 0.05). There is a preferential loss of RASSF1A expression in human ovarian cancers and its expression is correlated with the tumor stage and the degree of histological differentiation which, as a tumor suppressor gene, might play an important role in the tumorigenesis of human primary ovarian cancer.

Whole genome expression profiling of advance stage papillary serous ovarian cancer reveals activated pathways.

Ovarian cancer is the most lethal type of gynecologic cancer in the Western world. The high case fatality rate is due in part because most ovarian cancer patients present with advanced stage disease which is essentially incurable. In order to obtain a whole genome assessment of aberrant gene expression in advanced ovarian cancer, we used oligonucleotide microarrays comprising over 40,000 features to profile 37 advanced stage papillary serous primary carcinomas. We identified 1191 genes that were significantly (P < 0.001) differentially regulated between the ovarian cancer specimens and normal ovarian surface epithelium. The microarray data were validated using real time RT-PCR on 14 randomly selected differentially regulated genes. The list of differentially expressed genes includes ones that are involved in cell growth, differentiation, adhesion, apoptosis and migration. In addition, numerous genes whose function remains to be elucidated were also identified. The microarray data were imported into PathwayAssist software to identify signaling pathways involved in ovarian cancer tumorigenesis. Based on our expression results, a signaling pathway associated with tumor cell migration, spread and invasion was identified as being activated in advanced ovarian cancer. The data generated in this study represent a comprehensive list of genes aberrantly expressed in serous papillary ovarian adenocarcinoma and may be useful for the identification of potentially new and novel markers and therapeutic targets for ovarian cancer.