Epithelial Ovarian Cancer Samples Research Articles

Abstract 4692: ARL4C, a novel ovarian cancer-related gene, its down-regulated predicts poor survival in ovarian cancer.

Abstract Purpose: To identify the genes involved in ovarian cancer progression and elucidate their biological actions in ovarian cancer. Experimental Design: Comparative genomic hybridization (CGH) was employed to screen the chromosome fragments with different copy numbers between the ovarian cancer cell line HO-8910 and its highly metastatic cell subline HO-8910PM. Fluorescent in situ hybridization (FISH) via biotin-labeled BAC clone probes was used to validate the results of CGH. Using gene expression microarray, differentially expressed genes on the target chromosome fragment were compared between the two cell lines. Lentiviral-mediated transfection was used to investigate the effects of candidate gene over-expression or knockdown on colony formation, proliferation and motility of ovarian cancer cells. Two hundred twelve epithelial ovarian cancer samples were analyzed for mRNA expression of candidate gene using RT-PCR. Associations of the gene expression with clinicopathological factors, progression free and overall survival were evaluated. Results: Chromosome fragment 2q36.1-37.3 was found hemizygous deletion in HO-8910PM cells by CGH and FISH. Comparing gene expression data, we identified ARL4C, located on chromosome 2q37.1, to be down-regulated in the HO-8910PM cells, compared with the HO-8910 cells. Western blot showed ARL4C expression was low/null in most ovarian cancer cell lines, including the HO-8910PM cells. ARL4C over-expression or knockdown had no obvious effect on colony formation and cell proliferation, but negatively regulated cell motility. ARL4C over-expression suppressed ovarian cancer cells migration and invasion. Inversely, ARL4C knockdown promoted ovarian cancer cells migration. Clinicopathologic studies further supported the relevance of ARL4C in ovarian cancers. Patients with medium or high levels of ARL4C mRNA were at lower risk for death, compared to those with low levels (HR, 0.45; 95% C.I., 0.27-0.76; P=0.003 for mid levels; and HR, 0.58; 95% C.I., 0.35-0.96; P = 0.035 for high levels) after adjusting for age, disease stage, tumor grade, histologic type and residual tumor size. Kaplan-Meier survival analysis demonstrated similar results. Conclusions: ARL4C is a novel ovarian cancer related gene that inhibits cell motility. It seems to play a protective role in cancer progression and is an independent prognostic factor for patient with ovarian cancer. More studies are needed to further elucidate the molecular mechanisms of ARL4C in ovarian cancer progression. Citation Format: Dan Su, Shenhau Xu, Haiyan Xu, Yun Gao, Jianguo Feng, Min Luo, Lisha Ying, Nengming Lin, Dionyssios Katsaros, Herbert Yu. ARL4C, a novel ovarian cancer-related gene, its down-regulated predicts poor survival in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4692. doi:10.1158/1538-7445.AM2013-4692

ATF5 Is Overexpressed in Epithelial Ovarian Carcinomas and Interference With Its Function Increases Apoptosis Through the Downregulation of Bcl-2 in SKOV-3 Cells

The activating transcription factor 5 (ATF5) is highly expressed in many kinds of tumors including glioblastoma and breast cancers, but its expression in epithelial ovarian neoplasms has not been investigated. Here, we show that ATF5 is highly expressed in the majority of epithelial ovarian cancer samples (43/60) as compared with benign ovarian tumor tissues (4/13) and normal ovarian tissues (1/10). Furthermore, we found that ATF5 expression significantly correlated with advanced clinical stage (P<0.05) and poor differentiation of epithelial ovarian carcinomas (P<0.05). Previous studies suggested that ATF5 is required for the survival of cancer cells, but the mechanisms by which ATF5 regulates genes and promotes cell survival are not clear. Our data additionally demonstrated that interference with the function of ATF5 could markedly increase the apoptosis of ovarian cancer cells and identified B-cell leukemia lymphoma-2 as an ATF5-targeted apoptosis-related gene. These findings may provide potential therapeutic application in epithelial ovarian cancer.

NotI Microarrays: Novel Epigenetic Markers for Early Detection and Prognosis of High Grade Serous Ovarian Cancer

Chromosome 3-specific NotI microarray (NMA) containing 180 clones with 188 genes was used in the study to analyze 18 high grade serous ovarian cancer (HGSOC) samples and 7 benign ovarian tumors. We aimed to find novel methylation-dependent biomarkers for early detection and prognosis of HGSOC. Thirty five NotI markers showed frequency of methylation/deletion more or equal to 17%. To check the results of NMA hybridizations several samples for four genes (LRRC3B, THRB, ITGA9 and RBSP3 (CTDSPL)) were bisulfite sequenced and confirmed the results of NMA hybridization. A set of eight biomarkers: NKIRAS1/RPL15, THRB, RBPS3 (CTDSPL), IQSEC1, NBEAL2, ZIC4, LOC285205 and FOXP1, was identified as the most prominent set capable to detect both early and late stages of ovarian cancer. Sensitivity of this set is equal to (72 ± 11)% and specificity (94 ± 5)%. Early stages represented the most complicated cases for detection. To distinguish between Stages I + II and Stages III + IV of ovarian cancer the most perspective set of biomarkers would include LOC285205, CGGBP1, EPHB1 and NKIRAS1/RPL15. The sensitivity of the set is equal to (80 ± 13)% and the specificity is (88 ± 12)%. Using this technique we plan to validate this panel with new epithelial ovarian cancer samples and add markers from other chromosomes.

Nerve Growth Factor Induces the Expression of Chaperone Protein Calreticulin in Human Epithelial Ovarian Cells

Epithelial ovarian cancer is highly angiogenic and high expression of Nerve Growth Factor (NGF), a proangiogenic protein. Calreticulin is a multifunctional protein with anti-angiogenic properties and its translocation to the tumor cell membrane promotes recognition and engulfment by dendritic cells. The aim of this work was to evaluate calreticulin expression in human normal ovaries, benign and borderline tumors, and epithelial ovarian cancer samples and to evaluate whether NGF regulates calreticulin expression in human ovarian surface epithelium and in epithelial ovarian cancer cell lines. Calreticulin mRNA and protein levels were analyzed using RT-PCR, Western blot and immunohistochemistry in 67 human ovarian samples obtained from our Institution. Calreticulin expression induced by NGF stimulation in cell lines was evaluated using RT-PCR, Western blot and immunocytochemistry. We found a significant increase of calreticulin mRNA levels in epithelial ovarian cancer samples as compared to normal ovaries, benign tumors, and borderline tumors. Calreticulin protein levels, evaluated by Western blot, were also increased in epithelial ovarian cancer with respect to benign and borderline tumors. When HOSE and A2780 cell lines were stimulated with Nerve Growth Factor, we found an increase in calreticulin protein levels compared to controls. This effect was reverted by GW441756, a TRKA specific inhibitor. These results suggest that NGF regulates calreticulin protein levels in epithelial ovarian cells through TRKA receptor activation.

Nerve Growth Factor Stimulates Cellular Proliferation of Human Epithelial Ovarian Cancer

Due to its ability to induce vascular endothelial growth factor expression and proliferation, migration, and vasculogenesis of endothelial cells, nerve growth factor (NGF) has been considered as an angiogenic factor in epithelial ovarian cancer (EOC). In this work, we evaluated the angiogenic and proliferative mRNA expression profiles of EOC and addressed the responsiveness of EOC explants to NGF stimulation. Twenty EOC samples were obtained from Obstetrics and Gynecology Department, University of Chile's Clinical Hospital. Global gene expression profiles of selected poorly differentiated serous EOC samples were obtained with DNA oligonucleotide microarrays. In addition, EOC explants were subjected to NGF stimulation and levels of p-AKT, BAX, BCL2, Ki-67, c-MYC, and FOXL2 proteins were determined by immunohistochemistry. Results showed that mRNAs coding for specific transcriptional regulators and antiapoptotic components of the NGF signaling pathway were upregulated in EOC cells. At the protein level, key members of the NGF pathway including p-AKT, BCL2/BAX, Ki-67, and c-MYC were found increased, while FOXL2 was decreased in response to NGF stimulation. These findings strongly suggest that NGF stimulates cellular proliferation of human EOC.

Chromosome 17q25 genes, RHBDF2 and CYGB, in ovarian cancer

It has been proposed that the frequent loss of heterozygosity (LOH) of an entire chromosome 17 contig in epithelial ovarian cancers (EOC) is the consequence of the inactivation of multiple tumour suppressor genes on this chromosome. We report the characterization of a 453 Kb 17q25 locus shown previously to exhibit a high frequency of LOH in EOC samples. LOH analysis further defined the minimal region of deletion to a 65 Kb interval flanked by D17S2239 and D17S2244, which contains RHBDF2, CYGB and PRCD as tumour suppressor gene candidates. Tissue specific expression excluded PRCD as a candidate. RHBDF2 was expressed at low levels in the majority of benign and low malignant potential (LMP) tumours, and in a subset of malignant ovarian tumour samples, as compared with primary cultures of normal ovarian surface epithelial cell (NOSE) samples. CYGB was expressed at low levels in the majority of LMP and malignant samples compared with benign and NOSE samples. In contrast to CYGB expression, RHBDF2 was expressed at low or undetectable levels in EOC cell lines exhibiting tumourigenic characteristics and up-regulated in a genetically modified EOC cell line rendered non-tumourigenic. DNA sequence analysis identified variants but no apparent deleterious mutations in either gene. Methylation-specific PCR analysis suggested that promoter methylation of CYGB but not RHBDF2 occurred in 6 of 31 malignant samples. The results combined suggest that RHBDF2 and CYGB may play distinctive roles in ovarian cancer and could be added to the growing roster of chromosome 17 genes implicated in this disease.

Expression of 67-kDa laminin receptor was associated with tumor progression and poor prognosis in epithelial ovarian cancer

Objective67-kDa laminin receptor (67LR) has been identified as a prognostic biomarker for a variety of human cancers. We investigated the clinical significance of 67LR expression and its functional role in epithelial ovarian cancer (EOC). Methods67LR expression was evaluated by immunohistochemistry in 62 patients with EOC. We assessed the correlation of 67LR expression with clinical characteristics. In vitro experiment was performed for 67LR with inhibition using siRNA to evaluate its role in cell survival, apoptosis, and invasion in EOC cells. Results67LR was predominantly expressed on the cell membrane in the majority of EOC samples (45/62, 73%). 67LR expression was significantly correlated with advanced stage (P=0.001). Patients with 67LR expression had shorter progression-free survival among all the patients (P=0.010) and in particular among patients with advanced stages (P=0.046). When 67LR expression was inhibited by siRNA in EOC cells (HeyA8 and A2780), there was a significant decrease of cell proliferation and invasion as well as increase of apoptosis. ConclusionThese findings suggest that 67LR expression may play an important role in tumor progression into advanced stage with poor prognosis in EOC and down-regulation of 67LR on tumor cells may be a therapeutic target in those patients.

Low MAD2 expression levels associate with reduced progression‐free survival in patients with high‐grade serous epithelial ovarian cancer

Epithelial ovarian cancer (EOC) has an innate susceptibility to become chemoresistant. Up to 30% of patients do not respond to conventional chemotherapy [paclitaxel (Taxol®) in combination with carboplatin] and, of those who have an initial response, many patients relapse. Therefore, an understanding of the molecular mechanisms that regulate cellular chemotherapeutic responses in EOC cells has the potential to impact significantly on patient outcome. The mitotic arrest deficiency protein 2 (MAD2), is a centrally important mediator of the cellular response to paclitaxel. MAD2 immunohistochemical analysis was performed on 82 high-grade serous EOC samples. A multivariate Cox regression analysis of nuclear MAD2 IHC intensity adjusting for stage, tumour grade and optimum surgical debulking revealed that low MAD2 IHC staining intensity was significantly associated with reduced progression-free survival (PFS) (p = 0.0003), with a hazard ratio of 4.689. The in vitro analyses of five ovarian cancer cell lines demonstrated that cells with low MAD2 expression were less sensitive to paclitaxel. Furthermore, paclitaxel-induced activation of the spindle assembly checkpoint (SAC) and apoptotic cell death was abrogated in cells transfected with MAD2 siRNA. In silico analysis identified a miR-433 binding domain in the MAD2 3′ UTR, which was verified in a series of experiments. Firstly, MAD2 protein expression levels were down-regulated in pre-miR-433 transfected A2780 cells. Secondly, pre-miR-433 suppressed the activity of a reporter construct containing the 3′-UTR of MAD2. Thirdly, blocking miR-433 binding to the MAD2 3′ UTR protected MAD2 from miR-433 induced protein down-regulation. Importantly, reduced MAD2 protein expression in pre-miR-433-transfected A2780 cells rendered these cells less sensitive to paclitaxel. In conclusion, loss of MAD2 protein expression results in increased resistance to paclitaxel in EOC cells. Measuring MAD2 IHC staining intensity may predict paclitaxel responses in women presenting with high-grade serous EOC. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ALDH1-Bright Epithelial Ovarian Cancer Cells Are Associated with CD44 Expression, Drug Resistance, and Poor Clinical Outcome

The role of aldehyde dehydrogenase 1 (ALDH1) as an ovarian cancer stem cell marker and its clinical significance have rarely been explored. We used an Aldefluor assay to isolate ALDH1-bright (ALDH1(br)) cells from epithelial ovarian cancer cell lines and characterized the properties of the stem cells. ALDH1(br) cells were enriched in ES-2 (1.3%), TOV-21G (1.0%), and CP70 (1.2%) cells. Both ALDH1(br) and ALDH1(low) cells repopulated stem cell heterogeneity, formed spheroids, and grew into tumors in immunocompromised mice, although these processes were more efficient in ALDH1(br) cells. In the ES-2 and CP70 cells, ALDH1(br) cells conferred more chemoresistance, and were more enriched in CD44 (by 1.74-fold and 5.18-fold, respectively) than in CD133 (by 1.39-fold and 1.17-fold, respectively), compared with ALDH1(low) cells. Immunohistochemical staining for ALDH1 on a tissue microarray containing 84 epithelial ovarian cancer samples revealed that patients with higher ALDH1 expression (>50%) had poor overall survival, compared with those with lower ALDH1 (P = 0.004) and yielded an odds ratio of death of 2.43 (95% CI = 1.12 to 5.28) by multivariate analysis. The results did not support ALDH1 alone as an ovarian cancer stem cell marker, but demonstrated that ALDH1 is associated with CD44 expression, chemoresistance, and poor clinical outcome. The use of a combination of ALDH1 with other stem cell markers may help define ovarian cancer stem cells more stringently.

Establishing a molecular taxonomy for epithelial ovarian cancer (EOC) from 363 formalin-fixed paraffin embedded (FFPE) specimens.

5000 Background: Despite histological heterogeneity and evidence of some molecular subtypes (e.g. BRCA1/2-deficient) EOC continues to be treated as a single disease. Using unsupervised hierarchical clustering of DNA microarray expression data we aimed to identify molecular subgroups with a view to personalized therapy. Methods: Microarray expression analysis was performed on 363 FFPE EOC samples (by far the largest dataset to date) with linked prospectively collected clinical data. Unsupervised clustering was applied to the most variable genes. This was followed by computational classification in the serous samples only. Results: Unsupervised analysis of all samples identified 6 subgroups which were most significantly related to histology (p=3.65 x 10-27) and had different overall survival (OS; p<0.0001). Clear cell tumors were present in one group, mucinous in another, serous and endometrioid were spread across 4 groups each. When a similar approach was applied to pure serous samples (no mixed tumors; n=199) three subgroups were identified with different OS (p<0.0001). Intriguingly, the dominant discriminatory biology within the serous cohort was related to vasculature development (FDR p.vasc.dev.=7.31 x 10-7) and more specifically to angiogenic processes (FDR p.Angiogenesis= 8.59 x 10-5). Up-regulation of these angiogenesis-related genes predominated in only one of the 3 serous subgroups (58 out of 199 tumours). Class labels were assigned to these samples based upon expression of these angiogenesis-related genes and a gene expression signature was developed which could identify the novel angiogenesis molecular subgroup. Conclusions: 1) It is possible to consistently perform transcriptomic analysis from small FFPE ovarian tumor sections and cluster similar tumors. 2) We have identified 6 main clusters of EOC, strongly associated with histology and survival. 3) In the pure serous subgroup, 3 clusters were identified with significantly differing survival; one of these subgroups was defined by upregulation of multiple angiogenesis genes. In light of the recently reported activity of bevacizumab in EOC (GOG 218 and ICON7) this subgroup may explain the observed response.

MicroRNA let-7a: A potential marker for selection of paclitaxel in ovarian cancer management

Objectives Let-7 is a family of small non-coding RNAs regulating the expression of many genes that control important cellular activities. Let-7 is shown in vitro to sensitize cancer cells to platinum, but induce ovarian cancer resistance to paclitaxel. This study aims to investigate the effect of let-7a expression on survival outcomes of epithelial ovarian cancer (EOC) patients treated with different chemotherapy. Methods Let-7a expression was measured with qRT-PCR in ovarian tumors of 178 EOC patients who received platinum-based chemotherapy with and without paclitaxel after surgery. Survival analysis was performed to assess the effects of let-7a and chemotherapy on disease outcomes. Results Let-7a expression was detectable in the EOC samples, but the expression was not associated with disease stage, tumor grade, histology and debulking results. Patients who responded to platinum with paclitaxel had significantly lower let-7a than those who did not. Survival analyses showed that patients with high let-7a had better survival compared to those with low let-7a when they were treated with platinum without paclitaxel. The hazards ratios (HRs) for death and disease progression were 0.52 (95% CI: 0.29–0.96) and 0.48 (0.26–0.89) for high let-7a when compared to low let-7a, respectively. However, when patients were treated with platinum and paclitaxel, high let-7a was associated with worse progression-free and overall survival. The HRs for death and disease progression were 3.87 (95% CI: 1.28–11.66) and 3.48 (95% CI: 1.25–9.67) for high let-7a when compared to low let-7a, respectively. Further studies showed that among patients with low let-7a, those treated with paclitaxel in addition to platinum survived better than those treated without paclitaxel [adjusted-HRs were 0.31 (95% CI: 0.15–0.66) for death and 0.40 (95% CI: 0.22–0.75) for disease], while among those with high let-7a, the two types of treatment made no difference in patient survival. Conclusions The study suggests that the beneficial impact of the addition of paclitaxel on EOC survival was significantly linked to let-7a levels, and that miRNAs such as let-7a may be a useful marker for selection of chemotherapeutic agents in EOC management.

Abstract 937: Molecular dynamics of SrcS17 activation in chronic stress-induced tumor growth

Abstract Clinical studies have demonstrated that chronic stress can influence cancer progression. However, the underlying mechanisms are not fully understood. To determine the molecular drivers of downstream signaling networks activated in response to chronic stress, we performed a phosphoproteomic analysis and determined that the non-receptor tyrosine kinase, Src, was the key regulator of these networks. Since Src plays an important role in cancer biology, we examined the biological and clinical significance of Src in stress-mediated tumor growth. Norepinephrine (NE) rapidly activated SrcY419 in β-adrenergic receptor (ADRB) positive ovarian cancer cell lines, but not in ADRB-null cells. Confocal microscopy showed that Src was rapidly recruited to the cellular membrane after NE exposure in ADRB positive ovarian cancer cells. Furthermore, treatment with different ADRB agonists and blockers determined that ADRB2 is required for SrcY419 phosphorylation. Treatment with a cAMP agonist or PKA agonist/antagonists demonstrated that cAMP/PKA signaling is required for NE-induced Src activation. The unexpected Src activation via cAMP/PKA was found to be mediated by direct phosphorylation of SrcS17 following NE treatment. In Src-/- cells transiently expressing WT Src, NE caused SrcY419 phosphorylation, which was not observed when cells were transfected with a Src S17A construct. In order to investigate how S17 phosphorylation leads to Src activation, we performed molecular dynamic simulations and observed that upon SrcS17 phosphorylation, Src undergoes significant structural changes that expose its Y419 residue. To understand the functional consequences of stress-induced Src activation, we performed migration and invasion assays. Exposure to NE resulted in an increase in ovarian cancer cell migration and invasion that was completely abrogated by Src-targeted siRNA (P &amp;lt; 0.01). In various orthotopic mouse models of ovarian carcinoma, chronic restraint stress significantly increased tumor weights (P &amp;lt; 0.05). Increased tumor growth was completely blocked by Src silencing with Src siRNA-DOPC or by the non-specific beta-blocker, propranolol (P &amp;lt; 0.05). To test the clinical significance of our biological findings, we examined 91 epithelial ovarian cancer samples. Elevated pSrcY419 was associated with worse patient survival (P &amp;lt; 0.001), high tumoral NE levels (P &amp;lt; 0.001) and high scores on the Center for Epidemiologic Studies Depression Scale (P = 0.008). To examine the potential clinical impact of our findings, we investigated whether beta-blocker usage by patients affected cancer-related mortality. This analysis revealed that beta-blockers reduced mortality by 17% across all major cancer types and 14.6% among patients with ovarian and other gynecologic cancers. This work is the first to demonstrate that an ADRB-PKA-Src axis mediates the effect of chronic stress on tumor growth and progression. 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 937. doi:10.1158/1538-7445.AM2011-937

Coordinated Cancer Germline Antigen Promoter and Global DNA Hypomethylation in Ovarian Cancer: Association with the BORIS/CTCF Expression Ratio and Advanced Stage

Cancer germline (CG) antigens are frequently expressed and hypomethylated in epithelial ovarian cancer (EOC), but the relationship of this phenomenon to global DNA hypomethylation is unknown. In addition, the potential mechanisms leading to DNA hypomethylation, and its clinicopathologic significance in EOC, have not been determined. We used quantitative mRNA expression and DNA methylation analyses to determine the relationship between expression and methylation of X-linked (MAGE-A1, NY-ESO-1, XAGE-1) and autosomal (BORIS, SOHLH2) CG genes, global DNA methylation (5mdC levels, LINE-1, Alu, and Sat-α methylation), and clinicopathology, using 75 EOC samples. In addition, we examined the association between these parameters and a number of mechanisms proposed to contribute to DNA hypomethylation in cancer. CG genes were coordinately expressed in EOC and this was associated with promoter DNA hypomethylation. Hypomethylation of CG promoters was highly correlated and strongly associated with LINE-1 and Alu methylation, moderately with 5mdC levels, and rarely with Sat-α methylation. BORIS and LINE-1 hypomethylation, and BORIS expression, were associated with advanced stage. GADD45A expression, MTHFR genotype, DNMT3B isoform expression, and BORIS mRNA expression did not associate with methylation parameters. In contrast, the BORIS/CTCF expression ratio was associated with DNA hypomethylation, and furthermore correlated with advanced stage and decreased survival. DNA hypomethylation coordinately affects CG antigen gene promoters and specific repetitive DNA elements in EOC, and correlates with advanced stage disease. The BORIS/CTCF mRNA expression ratio is closely associated with DNA hypomethylation and confers poor prognosis in EOC.

The Hippo pathway transcriptional co-activator, YAP, is an ovarian cancer oncogene

The Salvador-Warts-Hippo (SWH) pathway was first discovered in Drosophila melanogaster as a potent inhibitor of tissue growth. The SWH pathway is highly conserved between D. melanogaster and mammals, both in function and in the mechanism of signal transduction. The mammalian SWH pathway limits tissue growth by inhibiting the nuclear access and expression of the transcriptional co-activator, Yes-associated protein (YAP). Mutation and altered expression of SWH pathway proteins has been observed in several types of human cancer, but the contribution of these events to tumorigenesis has been unclear. Here we show that YAP can enhance the transformed phenotype of ovarian cancer cell lines and that YAP confers resistance to chemotherapeutic agents that are commonly used to treat ovarian cancer. We find that high nuclear YAP expression correlates with poor patient prognosis in a cohort of 268 invasive epithelial ovarian cancer samples. Segregation by histotype shows that the correlation between nuclear YAP and poor survival is predominantly associated with clear cell tumors, independent of stage. Collectively our findings suggest that YAP derepression contributes to the genesis of ovarian clear cell carcinoma and that the SWH pathway is an attractive therapeutic target.

Tyrosine kinase A receptor (trkA): A potential marker in epithelial ovarian cancer

ObjectivesTo evaluate the role of trkA receptor as a potential tumor marker in serous epithelial ovarian cancer and its relationship with the angiogenic factors expression as vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). Additionally, to examine whether NGF and VEGF secreted by epithelial ovarian cancer (EOC) explants and from epithelial ovarian cancer cell line (A2780) are involved in the process of angiogenesis, such as cellular proliferation, migration and differentiation of the human endothelial cell line (EA.hy926). MethodsThe mRNA levels of VEGF, NGF and trkA receptors were measured using PCR in 60 ovarian samples. Cellular localization and semi-quantitative estimation of VEGF, NGF, total trkA and p-trkA was performed using IHC in epithelial cells. NGF, total trkA and p-trkA protein were also evaluated in endothelial cells from the same tissues. Human endothelial cell line EA.hy926 was cultured with conditioned media obtained from both EOC explants and from the A2780 cell line, with or without NGF stimulus. ResultsSignificantly higher levels of NGF, total trkA and p-trkA protein expressions were observed in epithelial and endothelial cells in poorly differentiated EOC versus normal ovary. Interestingly, the p-trkA receptor expression level showed the most significant difference and its presence was only found in borderline tumor and EOC samples indicating the importance of trkA receptor in EOC as a potential tumor marker.A significant increase in proliferation, migration and differentiation of EA.hy926 cells was observed with NGF, and this effect was significantly reverted when NGF was immuno-blocked and when a trkA inhibitor was used, showing that NGF is an important angiogenic factor in EOC by activating its trkA receptor. ConclusionThese results indicate that p-trkA may be considered as a new potential tumor marker in EOC, and that NGF may also act as a direct angiogenic factor in EOC.

Abstract 4653: Role of serum HE4 in studying ovarian cancer progression and response to therapy with ARCHITECT HE4 assay

Abstract Human epididymis protein 4 (HE4) is a whey acidic four-disulfide core protein. Serum HE4 has been shown to be a promising biomarker for epithelial ovarian cancer (EOC) with manual HE4 EIA assays. The ARCHITECT system is one of the major automated formats for cancer immunodiagnostics. This abstract describes the clinical performance of an investigational ARCHITECT HE4 assay in the measurement of serum HE4 from longitudinal samples of subjects with EOC and single-point samples from healthy subjects, and subjects with malignancy, benign diseases, EOC or other malignant diseases. PROCEDURES: The ARCHITECT HE4 Assay is a paramagnetic microparticle chemiluminescent assay adopting the sensitive CHEMIFLEX® technology. Single-point and longitudinal serum samples were tested with an investigational ARCHITECT HE4 Assay reagent lot on the ARCHITECT i2000SR. Single-point sera were collected from healthy subjects (N = 400), subjects with pregnancy (N = 50) and benign disease (N = 612), and subjects which were diagnosed with EOC (N = 314) and other cancers (N = 250) including endometrial, breast, gastrointestinal, lung, and bladder cancer. Longitudinal sera (N = 506), including initial time point and follow-up visits (average = 5.7 per subject), were collected from subjects (N = 76) which were diagnosed with EOC. All of the single-point and longitudinal samples were handled in accordance with IRB-approved and GCP-compliant protocols. RESULTS: Using an ARCHITECT HE4 cut-off value of &amp;gt;140 pmol/L, the percent distributions of ARCHITECT HE4 by cohort are: 3% of healthy subjects, 0% with pregnancy, 7.2% of subjects with benign diseases, 53.5% with EOC and 28.4% with other cancers. Using the upper 95% point of the distribution of the HE4 ratio at 14% to categorize the successive HE4 readings into those that were and were not significantly elevated for the longitudinal samples, the sensitivity, specificity and positive predictive value of HE4 elevation versus disease progression were 53.4% (95% CI: 44.1%-62.5%), 78.8% (95% CI: 74.7% - 82.4%) and 43.2% (95% CI: 32.9% - 54.1%) respectively. The total concordance between HE4 elevation and disease progression was 73%. Receiver Operator Characteristic analysis showed an area under the curve of 0.685 (SE = 0.033) for the diagnosis of progression from the ratio of successive HE4 readings. When comparing the change of the HE4 ratio with the diagnostic response to therapy, a decrease in HE4 reflected response to therapy with a sensitivity of 82% and a specificity of 53%. CONCLUSIONS: The study with single-point samples has shown the utility of ARCHITECT HE4 values as a serum biomarker for EOC. The longitudinal increase of ARCHITECT HE4 values in sera seemed to be effective in recognizing the progression of EOC. The longitudinal decrease of ARCHITECT HE4 values in sera seemed to correlate with the response of EOC subjects to therapy. 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 4653.

Abstract 3188: Forward and reverse signalings of EphB4/EfnB2 are active in epithelial ovarian cancer and are inhibited by dasatinib

Abstract Ephrin receptor (Eph) family constitutes the largest subfamily of trans-membrane tyrosine kinases. Their ligands, the ephrins (Efn), are membrane-anchored proteins grouped into two classes: class A ligands which are GPI-anchored proteins; class B ligands which contain a trans-membrane and a cytoplasmic tail. Eph-signaling is termed forward, Efn-signaling is termed reverse. Eph/Efn proteins are expressed in virtually all tissues and they are involved in a number of cellular events during embryonic development such as cell migration, repulsion versus adhesion, and cell to cell communication. In cancers, Eph/Efn involment is controversial. The Eph appeared to exert a suppressive role in colon and breast cancers, and the Eph/Efn signaling has been showed to drive or inhibit invasion via a reciprocal regulatory loop in glioblastomas and prostate cancer. In epithelial ovarian cancers (EOCs) the co-expression of EphB4 and EfnB2 in seventy-two EOC samples was associated to worst prognosis, highlighting a possible autocrine/paracrine activation loop in EOCs. We analyzed the signaling through EphB4/EfnB2 pair in EOC cell lines that express both the receptor and the ligand or only the receptor. To analyze the EphB4 activation we evaluated the EphB4 phosphorylation in growing cells and in cells upon binding of a soluble EfnB2-Fc molecule on cell in suspension. We found that EphB4 is phosphorylated in cells growing in presence of fetal calf serum and is further phosphorylated upon EfnB2-Fc stimulation. Accordingly, treatment of growing EOC cells with the soluble EfnB2-Fc induced a decrease in growth capability, meaning that the ligand in soluble form inhibited the binding of the cell membrane ligand to the receptor. In line with this results, silencing of EphB4 by a specific siRNA pool, inhibited cell growth by impairing cell survival. On the other site, silencing of EfnB2 lead to inhibition of EphB4 and src phosphorylations, followed by FAK phosphorylation and remodeling of focal adhesions. Therefore, in EOC cell lines expressing both the receptor and the ligand forward and reverse signaling of EphB4/EfnB2 were active. In addition, we showed that a panel of EOC cells are sensitive (IC50&amp;lt;10 nM) to the kinase inhibitor dasatinib which has been showed to have the same binding capacity on EphB4 and src. Indeed, dasatinib reduced both EphB4 and src phosphorylation in EOC cells. Ongoing experiments of dasatinib treatment in nude mice bearing OVCAR3 xenografts in the peritoneum showed an increase of the survival rate in the treated animals. These in vitro and in vivo experiments demonstrated for the first time that in EOC cells dasatinib targeted both EphB4 and src kinase thus suggesting dasatinib as a valuable therapy in EOC patients. Partially supported by FIRB and AIRC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3188.

Frequent Downregulation of miR-34 Family in Human Ovarian Cancers

The miR-34 family is directly transactivated by tumor suppressor p53, which is frequently mutated in human epithelial ovarian cancer (EOC). We hypothesized that miR-34 expression would be decreased in EOC and that reconstituted miR-34 expression might reduce cell proliferation and invasion of EOC cells. miR-34 expression was determined by quantitative reverse transcription-PCR and in situ hybridization in a panel of 83 human EOC samples. Functional characterization of miR-34 was accomplished by reconstitution of miR-34 expression in EOC cells with synthetic pre-miR molecules followed by determining changes in proliferation, apoptosis, and invasion. miR-34a expression is decreased in 100%, and miR-34b*/c in 72%, of EOC with p53 mutation, whereas miR-34a is also downregulated in 93% of tumors with wild-type p53. Furthermore, expression of miR-34b*/c is significantly reduced in stage IV tumors compared with stage III (P = 0.0171 and P = 0.0029, respectively). Additionally, we observed promoter methylation and copy number variations at mir-34. In situ hybridization showed that miR-34a expression is inversely correlated with MET immunohistochemical staining, consistent with translational inhibition by miR-34a. Finally, miR-34 reconstitution experiments in p53 mutant EOC cells resulted in reduced proliferation, motility, and invasion, the latter of which was dependent on MET expression. Our work suggests that miR-34 family plays an important role in EOC pathogenesis and reduced expression of miR-34b*/c may be particularly important for progression to the most advanced stages. Part of miR-34 effects on motility and invasion may be explained by regulation of MET, which is frequently overexpressed in EOC.

Trop-2 overexpression as an independent marker for poor overall survival in ovarian carcinoma patients

Background Prognostic factors currently available are insufficient to predict the clinical course of epithelial ovarian cancer (EOC). In a previous microarray study we identified the human trophoblast cell surface antigen Trop-2 as one of the top differentially expressed genes in serous papillary EOCs compared to normal human ovarian surface epithelial (HOSE) short-term cultures. The aim of the present investigation was to analyse Trop-2 expression at mRNA and protein level and to assess its prognostic significance in EOC. Methods Using quantitative real-time PCR we tested a total of 104 fresh-frozen EOC tissues and 24 HOSE for Trop-2 mRNA expression. Trop-2 protein expression was then examined by immunohistochemistry in matched formalin-fixed paraffin-embedded EOC samples and in 13 normal ovaries. Finally, we correlated Trop-2 expression to EOC conventional clinicopathological features and patient outcomes. Results We found a significant Trop-2 mRNA and protein upregulation in EOCs compared to normal controls ( p < 0.001). Trop-2 protein overexpression was significantly associated with the presence of ascites ( p = 0.04) and lymph node metastases ( p = 0.04). By univariate survival analysis, Trop-2 protein overexpression was significantly associated with decreased progression-free ( p = 0.02) and overall survival ( p = 0.01). Importantly, Trop-2 protein overexpression was an independent prognostic marker for shortened survival time in multivariate Cox regression analysis ( p = 0.04, HR = 2.35, CI 95% = 1.03–5.34). Conclusions Our results indicate, for the first time, that Trop-2 protein overexpression correlates with an aggressive malignant phenotype and may constitute a novel prognostic factor for EOC. The targeting of Trop-2 overexpression by immunotherapeutic strategies may represent an attractive and potentially effective approach in patients harbouring EOC.

RUNX3 Gene Methylation in Epithelial Ovarian Cancer Tissues and Ovarian Cancer Cell Lines

Methylation plays an important role in the regulation of gene expression in many cancer tissues. RUNX3 is an important tumor suppressor gene located on human chromosome 1p36.1, and many tumors do not express it due to methylation of the promoter region of the CpG island. The molecular mechanisms involved in RUNX3 gene expression and epithelial ovarian cancer are not fully understood. This study investigates the relationship between RUNX3 methylation and expression in ovarian cancer. The methylation of the RUNX3 gene promoter region was measured in 32 primary epithelial ovarian cancer samples and corresponding nonmalignant ovarian tissues, 36 benign epithelial ovarian tumor tissues, and 10 normal ovarian tissues by methylation-specific PCR (MSP) and RT-PCR. The relationships between RUNX3 methylation status, expression, and clinicopathologic characteristics were analyzed. RUNX3 methylation was further assessed by MSP and RT-PCR before and after 5-aza-2'-deoxycytidine (5-aza-dc) treatment in normal and cancer cell lines. We detected RUNX3 methylation in 53.1% of primary ovarian cancer tumors, 16.7% of benign ovarian tumors, and 28% of nonmalignant tissues surrounding ovarian cancers. No methylation was detected in normal ovarian tissues. No significant correlation between RUNX3 methylation and clinicopathological characteristics was observed. The RT-PCR results found RUNX3 expression in all normal ovarian tissues (10/10) and in most of the unmethylated ovarian cancer tissues (12/15); in contrast, it was not detected in most of the RUNX3-methylated ovarian cancer tissues (16/17). Our data suggest that methylation plays a critical role in the regulation of RUNX3 repression, and that it is significantly correlated with RUNX3 mRNA expression in ovarian cancer tissues (p = 0.006).