Normal Ovarian Epithelial Cell Line Research Articles

Abstract 2897: Preferential drug delivery to cancer cells than to normal cells by using the Niosome-Chitosan Thermo-responsive Double Package System (NCTR-DPS)

Abstract The main challenge of this research is to develop a localized, cost effective drug delivery system that can provide control over the release of drugs in ovarian cancer or brain tumor patients, in a manner that also reduces chemotherapeutic toxicity. We have designed a drug delivery system consisting of non-ionic surfactant vesicles (niosomes) packaged within a biodegradable, temperature sensitive hydrogel (chitosan) network (NCTR-DPS). Two chemotherapeutics, Paclitaxel and Carboplatin, conjugated to BODIPY 564/570 (RED fluorescence dye) were encapsulated in the NCTR-DPS. Their release was evaluated either in cell free system or following incubation with human cell lines: glioma (U373), ovarian carcinoma (OV2008), and two normal ovarian epithelial (MCC3 and I-Low) cell lines. Cellular localization of Paclitaxel-RED in live cells was evaluated using confocal microscopy and the level of direct fluorescence quantified with LAS AF software. Confocal imaging of the cells verified that the activity of the drugs remained intact within the drug delivery system. The cellular drug delivery was confirmed by cell's toxicity observed within two hours of incubation. The control study showed no toxicity of the niosome-chitosan alone (without the chemotherapeutics). Chitosan accumulation was greater on OV2008 cell surface than on MCC3 or I-Low cells. Also, significantly higher Paclitaxel-RED fluorescence was found in OV2008 compared to normal MCC3 cells, indicating a specific interaction between chitosan and Mucin1 (MUC1) receptors over expressed in ovarian carcinoma. These data were further confirmed by ATR-FTIR (Attenuated Total Reflectance- Fourier Transform Infra-Red) spectroscopy showing higher MUC1 levels in OV2008 than in normal epithelial cells. In addition, in-vivo examinations of the NCTR-DPS were carried out in 8-week old female mice (Strain: FVB/NJ) using Xenogen IVIS Spectrum to quantitatively monitor release of tracer molecules.In-vivo examinations revealed the release dependence on the packaging density; 25 - 40% of release occurred within 24 hours, followed by a steady release for over 14 days. By the 14th day, 98% of dye was released. Higher percentage release was observed for greater niosome chitosan ratios. Our results showed that the time release of the drugs could be finely controlled by varying packaging and crosslinking ratio in the chitosan network from 3:1 (loose network which translates into high release) to 5:1 (tight network which translates into low release). This NCTS-DPS drug delivery system can be injected directly to tumor sites preventing the need for surgical implantation. It may have positive implications in the treatment of ovarian cancer preferentially targeting tumor cells or brain tumors because of its controlled and localized release. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2897. doi:1538-7445.AM2012-2897

Abstract 1271: Aberrant activation of Spleen Tyrosine Kinase in ovarian cancer identified through a global phosphorylation profiling of protein tyrosine kinases

Abstract Aberrations in protein kinase activity and the consequent deregulation of downstream signaling pathways are considered a hallmark of oncogenesis. The purpose of this study was to investigate the phosphorylation state, the ultimate readout of dysregulated activity, of protein tyrosine kinases in ovarian tumors. An innovative Luminex multiplex platform was employed to systematically determine phosphorylation status of protein tyrosine kinases in a total of 69 protein lysates derived from normal human ovarian surface epithelial (HOSE) cells, ovarian tumors, and cancer cell lines. Significance Analysis of Microarrays (SAM) was performed to identify the signature of phosphorylated kinases with statistical significance. Profiling results were confirmed by using phosphor-specific kinase antibodies in immunoblot analysis of the profiled lysates and immunohistochemistry on formalin-fixed paraffin-embedded archived ovarian and fallopian tissues, as well as immunofluorescence of ovarian cancer cells growing as monolayer or 3D Matrigel cell cultures. A signature of twenty-two phosphorylated protein tyrosine kinases was identified by SAM analysis of the profiling data. Spleen Tyrosine Kinase (SYK), a novel tyrosine kinase included in the signature, was found significantly phosphorylated in lysates from ovarian cancer tissues compared to normal tissues by both Western blot analysis and immunohistochemistry on 94 clinical ovarian tissues (P=0.024). SYK phosphorylation was also identified in tubal intraepithelial carcinomas, the hypothesized precursors of high-grade serous ovarian carcinomas. However, SYK activation was not observed in ovarian cancer cell lines growing as attached monolayer cells, but was present when they grew as mouse xenografts or in 3D Matrigel in vitro cultures. We are the first to report that SYK, an extensively investigated tyrosine kinase in hematopoietic cells, is aberrantly phosphorylated and activated in ovarian cancer. SYK phosphorylation in ovarian cancer might be induced by extracellular stimuli that are exclusively present in the tumor microenvironment. These findings provide additional options in targeted therapy for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1271. doi:1538-7445.AM2012-1271

Dual inhibition of SRC and Aurora kinases induces postmitotic attachment defects and cell death

Increased activity of SRC family kinases promotes tumor invasion and metastasis, and overexpression of the mitotic regulator Aurora kinase A (AURKA) drives tumor aneuploidy and chromosomal instability. These functions nominate SRC and AURKA as valuable therapeutic targets for cancer, and inhibitors for SRC and Aurora kinases are now being used in the clinic. In this study, we demonstrate potent synergy between multiple inhibitors of Aurora and SRC kinases in ovarian and colorectal cancer cell lines, but not in normal ovarian epithelial cell lines. Combination of Aurora and SRC inhibitors selectively killed cells that have undergone a preceding aberrant mitosis, and was associated with a postmitotic reattachment defect, and selective removal of aneuploid cell populations. Combined inhibition of Aurora kinase and SRC potentiated dasatinib-dependent loss of activated (Y(416)-phosphorylated) SRC. SRC and AURKA share a common interaction partner, NEDD9, which serves as a scaffolding protein with activities in cell attachment and mitotic control, suggesting SRC and AURKA might interact directly. In vitro, we observed physical interaction and mutual cross-phosphorylation between SRC and AURKA that enhanced SRC kinase activity. Together, these findings suggest that combination of SRC and Aurora-targeting inhibitors in the clinic may be a productive strategy.

Hedgehog signaling pathway regulates the growth of ovarian cancer spheroid forming cells

The hedgehog (Hh) pathway has been shown to be activated in numerous malignancies as well as in cancer stem cells. We sought to determine the importance of the Hh pathway in regulating growth and development of ovarian cancer spheroid-forming cells (SFCs). Ovarian cancer cell lines (ES2, TOV112D, OV90, and SKOV3) as well as a normal ovarian epithelial cell line (IOSE80) were grown in non-adherent growth conditions to form SFCs. Western blot analysis was used to determine the expression of Hh pathway proteins SMOH, PTCH, GLI1. SFCs were treated with Hh agonists (SHH and IHH) as well as an Hh inhibitor (cyclopamine) to determine changes in spheroid growth and survival. All ovarian cancer cell lines readily formed spheroids in non-adherent growth conditions while IOSE80 failed to form SFCs. Compared to IOSE80, ovarian cancer cell lines demonstrated significant activation of the Hh pathway as defined by increased expression of intranuclear GLI1. Both Hh agonists demonstrated significant increases in spheroid volume of at least 42-fold for SHH-treated cells and 46-fold for IHH-treated cells. With regard to survival, SFCs were 30-50% more resistant to cyclopamine than their corresponding monolayer cells. Despite this resistance, inhibition of the Hh pathway with cyclopamine prevented further growth of SFCs with a 10-, 5-, and 4-fold restriction in growth for ES2, SKOV3, and TOV112D, respectively. The hedgehog pathway appears to be important in regulating growth of ovarian cancer spheroid-forming cells. The activation and inhibition of this pathway demonstrates significant correlation to enhanced growth and growth restriction, respectively.

Abstract 4728: Functional analysis of candidate genes at ovarian cancer susceptibility loci

Abstract Genome-wide association studies (GWAS) have identified a plethora of common, low risk susceptibility alleles associated with multiple disease phenotypes. However, few studies have established the functional mechanisms underlying susceptibility at these loci. This is a challenging field. Evaluating the function of genetic variants that confer small relative disease risks (RRs=0.8-1.2) relies on robust models of normal and diseased tissues. We identified a low-penetrance susceptibility locus on chromosome 19p13.11 that confers susceptibility to serous epithelial ovarian cancer (EOC), breast cancer (BC) in BRCA1 carriers, and in triple negative (PGR-, ER-, HER2-) BC cases. There is also evidence that the region is associated with overall survival after a diagnosis of EOC. The SNPs that are most significantly associated with EOC/BC susceptibility lie in and around two candidate genes at this locus; MERIT40 and ANKLE1. ANKLE1 has no function that suggests it might be involved in cancer development; but MERIT40 is a particularly intriguing gene because the protein product stabilizes the Rap80/BRCA1/BRCC45/BRCC36 complex at double-stranded DNA breaks. We looked for evidence that MERIT40 and ANKLE1 were involved in EOC development using real-time PCR on a panel of 80 normal ovarian epithelial and EOC cell lines. We found MERIT40 to be significantly overexpressed in cancer cell lines (P = 5 × 10-9), but no change in ANKLE1 expression between the two (P = 0.54). MERIT40 is also upregulated in ∼70% of ovarian tumor specimens whereas there were no differences in gene expression for ANKLE1. These data suggests that MERIT40 expression increases during tumor development. We stably overexpressed GFP-tagged cDNAs of MERIT40 and ANKLE1 in normal ovarian, fallopian tube epithelia (the two EOC precursor cell types) and MCF10A breast epithelial cells. Neoplastic transformation was scored using migration, invasion and anchorage-independent growth assays. The phenotype of the MERIT40/ANKLE1-overexpressing cells in 3D cultures was evaluated by analysis of 3D cyst and spheroid architecture, and measurement of the proliferative indices of the cultures. Additionally, to investigate the mechanism by which MERIT40 is associated with EOC survival we knocked down MERIT40 expression in EOC cell lines using stable and doxycycline-inducible lentiviral shRNA vectors. Knockdown of MERIT40 in EOC lines did not significantly affect sensitivity to cisplatin or paclitaxel, but did induce cell cycle arrest during G1-phase in cells with MMR defects. This suggests that in the absence of MERIT40 EOC cells accumulate DNA damage and undergo cell cycle arrest. The in vitro modeling data so far suggests that MERIT40 is the likely target gene at the 19p13.11 locus associated with susceptibility to EOC and BC. We propose that MERIT40 overexpression in advanced tumors may enable cancer cells to tolerate DNA damage in the presence of somatic mutations in MMR genes. 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 4728. doi:10.1158/1538-7445.AM2011-4728

Abstract 4306: KLF8 promotes human ovarian tumorigenesis in athymic mouse models

Abstract KLF8 is a dual transcription factor that plays a role in oncogenic transformation in mouse fibroblast cells and is highly over-expressed in human ovarian cancer. To test if KLF8 alone or together with each of the ovarian oncoprotein c-Myc, Stat3c or Akt plays a role in ovarian cell transformation, we used the immortalized normal human ovarian epithelial cell line T80 to generate stable cell lines that overexpress KLF8 alone or its combination with one of the other three oncoproteins. The Soft agar assay showed that T80/KLF8 formed significantly more colonies compared with the mock control cells, suggesting that KLF8 may play a role to transform T80 cells. Interestingly the cells expressing both KLF8 and c-Myc formed the largest amounts of colonies that were even greater than the sum of colonies formed by the cells expressing either KLF8 or c-Myc alone, suggesting a potential synergistic role of KLF8 with c-Myc. Surprisingly, however, overexpression of KLF8 alone was sufficient to induce tumorigenesis in athymic mice when the T80/KLF8 cells were injected subcutaneously (s.c.), intraperitoneally (i.p.) or orthotopically into the ovarian bursa. HE and IHC staining identified the tumors’ epithelial origin characteristic of human ovarian cancer. The s.c. growth rate of the T80/KLF8 tumors was greater than the T80/Stat3c tumors and similar to T80/Akt tumors although slower than the tumors formed by SKOV3ip1 and T80/c-Myc cells. The average survival time for the mice with the i.p. implantations was about six months for T80/KLF8 as compared to approximately two months for SKOV3ip1. These novel findings suggest that KLF8 plays a critical oncogenic role in synergy with c-Myc in the ovarian cancer progression. The mouse tumor models generated in this work provide important tools for the studies of mechanisms underlying human ovarian cancer 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 4306. doi:10.1158/1538-7445.AM2011-4306

Microcell-Mediated Chromosome Transfer Identifies EPB41L3 as a Functional Suppressor of Epithelial Ovarian Cancers

We used a functional complementation approach to identify tumor-suppressor genes and putative therapeutic targets for ovarian cancer. Microcell-mediated transfer of chromosome 18 in the ovarian cancer cell line TOV21 G induced in vitro and in vivo neoplastic suppression. Gene expression microarray profiling in TOV21 +19 hybrids identified 14 candidate genes on chromosome 18 that were significantly overexpressed and therefore associated with neoplastic suppression. Further analysis of messenger RNA and protein expression for these genes in additional ovarian cancer cell lines indicated that EPB41L3 (erythrocyte membrane protein band 4.1-like 3, alternative names DAL-1 and 4.1 B) was a candidate ovarian cancer-suppressor gene. Immunoblot analysis showed that EPB41L3 was activated in TOV21 +18 hybrids, expressed in normal ovarian epithelial cell lines, but was absent in 15 (78%) of 19 ovarian cancer cell lines. Using immunohistochemistry, 66% of 794 invasive ovarian tumors showed no EPB41L3 expression compared with only 24% of benign ovarian tumors and 0% of normal ovarian epithelial tissues. EPB41L3 was extersively methylated it ovarian cancer cell Hires and primary ovarian tumors compared with normal tissues (P = 00004), suggesting this may be the mechanism of gene inactivation it ovarian cancers. Constitutive reexpressior of EPB41L3 it a three-dimensional multicellular spheroid model of ovarian cancer caused significant growth suppressior and induced apoptosis. Transmission and scanning electron microscopy demonstrated many similarities between EPB41L3-expressing cells and chromosome 18 donor-recipient hybrids, suggesting that EPB41L3 is the gene responsible for reoplastic suppression after chromosome 18 transfer. Finally, ar irducible model of EPB41L3 expression it three-dimersioral spheroids confirmed that reexpression of EPB41L3 induces extensive apoptotic cell death it ovarian cancers.

Activated networking of platelet activating factor receptor and FAK/STAT1 induces malignant potential in BRCA1-mutant at-risk ovarian epithelium

ObjectivesIt is essential to understand the molecular basis of ovarian cancer etiology and tumor development to provide more effective preventive and therapeutic approaches to reduce mortality. Particularly, the molecular targets and pathways involved in early malignant transformation are still not clear. Pro-inflammatory lipids and pathways have been reported to play significant roles in ovarian cancer progression and metastasis. The major objective of this study was to explore and determine whether platelet activating factor (PAF) and receptor associated networking pathways might significantly induce malignant potential in BRCA1-mutant at-risk epithelial cells.MethodsBRCA1-mutant ovarian epithelial cell lines including (HOSE-636, HOSE-642), BRCA1-mutant ovarian cancer cell (UWB1.289), wild type normal ovarian epithelial cell (HOSE-E6E7) and cancerous cell line (OVCA429), and the non-malignant BRCA1-mutant distal fallopian tube (fimbria) tissue specimens were used in this study. Mutation analysis, kinase microarray, western blot, immune staining, co-immune precipitation, cell cycle, apoptosis, proliferation and bioinformatic pathway analysis were applied.ResultsWe found that PAF, as a potent pro-inflammatory mediator, induced significant anti-apoptotic effect in BRCA1-mutant ovarian surface epithelial cells, but not in wild type HOSE cells. With kinase microarray technology and the specific immune approaches, we found that phosphor-STAT1 was activated by 100 nM PAF treatment only in BRCA1-mutant associated at-risk ovarian epithelial cells and ovarian cancer cells, but not in BRCA1-wild type normal (HOSE-E6E7) or malignant (OVCA429) ovarian epithelial cells. Co-immune precipitation revealed that elevated PAFR expression is associated with protein-protein interactions of PAFR-FAK and FAK-STAT1 in BRCA1-mutant ovarian epithelial cells, but not in the wild-type control cells.ConclusionPrevious studies showed that potent inflammatory lipid mediators such as PAF and its receptor (PAFR) significantly contribute to cancer progression and metastasis. Our findings suggest that these potent inflammatory lipids and receptor pathways are significantly involved in the early malignant transformation through PAFR-FAK-STAT1 networking and to block apoptosis pathway in BRCA1 dysfunctional at-risk ovarian epithelium.

A conditionally replicative adenovirus, CRAd-S-pK7, can target endometriosis with a cell-killing effect

Novel therapeutic approaches for endometriosis based on molecular strategies may prove to be useful. Conditionally replicative adenoviruses (CRAds) are designed to exploit key differences between target and normal cells. The wild-type adenovirus (Adwt) promoter can be replaced by tissue-specific promoters, allowing viral replication only in target cells. Viral infectivity can be enhanced by altering Ad tropism via fiber modification. We investigated whether CRAds can be used to target endometriosis and determined the most efficient transcriptional- and transductional-targeting strategy. An in vitro study was carried out using human endometriotic cell lines, 11Z (epithelial) and 22B (stromal), normal human ovarian surface epithelial cell line (NOSE006) and primary human endometriosis cells. A total of 9 promoters and 12 Ad tropism modifications were screened by means of a luciferase reporter assay. From this screening data, three CRAds (CRAd-S-pK7, CRAd-S-RGD, CRAd-S-F5/3sigma1, all incorporating the survivin promoter but with different fiber modifications) were selected to perform experiments using Adwt and a replication-deficient virus as controls. CRAds were constructed using a plasmid recombination system. Viral-binding capacity, rates of entry and DNA replication were evaluated by quantitative real-time PCR of viral genome copy. Cell-killing effects were determined by crystal violet staining and a cell viability assay for different concentrations of viral particles per cell. Comparison of promoters demonstrated that the survivin promoter exhibited the highest induction in both endometriotic cell lines. Among the fiber-modified viruses, the polylysine modification (pK7) showed the best infection enhancement. CRAd-S-pK7 was validated as the optimal CRAd to target endometriosis in terms of binding ability, entry kinetics, DNA replication and cell-killing effect. CRAd-S-pK7 also exhibited a high level of DNA replication in primary endometriosis cells. CRAd-S-pK7 has the best infection and cell-killing effect in the context of endometriosis. It could prove to be a useful novel method to target refractory cases of endometriosis.

Abstract 4233: Dysregulated expression of stem cell transcription factor Nanog in development and progress of ovarian cancers

Abstract Introduction: Ovarian cancer is the most lethal of all gynecological malignacies due to its lack of symptoms at early stages. In addition, widespread intraperitoneal metastases and malignant ascites frequently develop. Therefore, it is very important to identify novel prognostic and therapeutic targets for this cancer. Many researchers believe that only a small subset of cancer cells are endowed with stem cell properties, which are responsible for tumor growth, metastatic progression and recurrence. Nanog is one of core transcription factors essential for the maintaining self-renewal and pluripotency in stem cells. This study investigated the clinical significance, functional roles and putative downstream targets of Nanog in ovarian cancer. Methods and Results: Differential protein expression profile of Nanog in clinical samples, including benign (n=6), borderline (n=7), carcinomas (n=97) and metastatic foci (n=24) were determined by immunohistochemistry. Differential mRNA expression pattern of Nanog in 3 normal ovarian epithelial cell lines, 16 ovarian cancer cell lines was assessed by quantitative real-time polymerase chain reaction. Subcellular localization of Nanog was performed by Western blotting. Nanog was found to be localized in the nucleus of ovarian cancers. High Nanog expression in ovarian cancers was significantly associated with high grade tumors, serous histological type and chemoresistance (p<0.05). Importantly, Nanog was found to be highly expressed in ovarian cancer cell lines with metastasis-associated property and in clinical samples of metastatic foci. Elevated Nanog expression was associated with poor overall and disease-free survival in the univariate analysis. Nanog expression became one of the independent prognostic factors in the multivariate analysis for overall but not disease-free survival. By Transwell migration and invasion assays, stable knockdown of Nanog in ovarian cancer cell line, OVCAR-3, impeded cell migration and invasion. Stable knockdown of Nanog also increased mRNA expression of E-cadherin (an essential epithelial mesenchymal transition marker, thus related to metastasis) and FOXO1A (an important gene related to chemoresistance). Conclusion: Nanog was associated with progression of ovarian tumors, patients’ overall survival and chemoresistance, suggesting that Nanog can be one of the potential prognostic markers and therapeutic molecular targets in ovarian cancer. 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 4233.

Abstract 133: Effects of iASPP on paclitaxel-mediated mitotic catastrophe and chemoresistance in ovarian cancers

Abstract Purpose: In this study, for the first time, we aimed to investigate the expression profile of iASPP in ovarian epithelial tumor in association with clinicopathological parameters. In vitro effect of iASPP on paclitaxel chemosensitivity will also be investigated. Materials and Methods: Expression and amplification status of iASPP was examined in 140 clinical samples and 16 ovarian cell lines using immunohistochemistry, immunoblotting and quantitative real-time PCR, and correlated with clinicopathological parameters. Changes in proliferation, mitotic catastrophe and apoptosis in ovarian cancer cell lines of different p53 status following paclitaxel exposure were also analyzed using TUNEL assay, MTT assay and immunofluorescence. Results: By immunohistochemistry and quantitative PCR, significantly higher protein and mRNA levels of iASPP was found in ovarian cancers when compared with borderline and benign ovarian tumour samples. Higher iASPP expression was also found in ovarian cancer cell lines than normal ovarian epithelial cell lines. Higher iASPP expression was significantly associated with carboplatin and paclitaxel chemoresistance (P=0.04), shorter overall (P=0.003) and disease-free survival (P=0.001). Multivariate analysis confirmed iASPP expression as an independent prognostic factor. Increased iASPP mRNA expression was significantly correlated with its gene amplification (P=0.018), suggesting that gene amplification contributes to overexpression of iASPP in ovarian caner. By in vitro assay, we demonstrated that iASPP overexpression in ovarian cancer cells conferred resistance to paclitaxel by reducing mitotic catastrophe in a p53-independent manner via activation of separase whilst knockdown of iASPP enhanced paclitaxel-mediated mitotic catastrophe through inactivating separase. Both securin and cyclin B1/CDK1complex were involved in regulating separase by iASPP. Conversely, overexpressed iASPP inhibited apoptosis in a p53-dependent mode. Conclusion: Our findings suggested that iASPP may be explored as a potential prognostic marker and a novel molecular target for chemotherapy of ovarian cancers, especially in patients with chemoresistant or recurrent cancers. 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 133.

Abstract 3183: Dysregulated expression and function of Plexin-B1 in ovarian cancers

Abstract Background: Ovarian cancer has a tendency to spread within the peritoneal cavity early in the disease course. Understanding the metastatic pathways of ovarian cancer is thus of high importance for devising ovarian cancer treatment. Plexin-B1 is a cognate receptor of Semaphorin 4D (Sema4D). This study aims at investigating the role of Plexin-B1 in ovarian cancer. Materials and Methods: Expression of Plexin-B1 and its ligand in ovarian epithelial cell lines was examined by western blot and qPCR. Their expression in benign, borderline, and malignant tumors was revealed by immunohistochemistrty of paraffin embedded tissues. High-resolution melting (HRM) curve analysis was used to screen for mutation in Rac binding region of PLXNB1. Recombinant Sema4D was purified and used to treat OVCA420 cells which express high level of Plexin-B1. siRNA was used to knock-down Plexin-B1 in OVCA420. Cell migration and invasion ability was assayed by transwell migration and invasion assay. Results: Plexin-B1 was most highly overexpressed in serous ovarian cancer cell lines OVCA420, OVCA429, and SW626, whereas Sema4D was ovexpressed in OVCAR3, TOV21G, and PAI. In contrast, Plexin-B1 and Sema4D were not expressed in immortalized normal ovarian epithelial cell lines. Immunohistochemical studies also revealed high Plexin B1 expression in ovarian borderline tumors and invasive cancers while no Plexin B1 expression was detectable in benign tumor. On the other hand, downregulation of Plexin-B1 was observed in the metastatic foci compared with their corresponding primary tumors. There is also a tendency of higher Plexin-B1 expression in serous histotype than in other histotypes. A preliminary HRM screening of Plexin-B1 cDNA from 28 ovarian cancers detected no mutation in the Rac binding region. Knock-down of Plexin-B1 by siRNA caused increased migration and invasion of ovarian cancer cells. In contrast to Sema4D, treatment of ovarian cancer cells OVCA420 by follicle-stimulating hormone which enhances ovarian cancer growth, led to Plexin-B1 downregulation. Conclusion: Our study suggested that Plexin-B1 may contribute to early ovarian carcinogenesis. Its dyregulation in ovarian cancers is paradoxically associated with altered cancer cell invasion and migration. The role of Plexin B1 in ovarian cancer progression is complex. 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 3183.

Abstract 4871: Genomic amplifications in ovarian carcinoma are associated with specific histone modifications

Abstract Genomic amplification is a frequent molecular genetic alteration in many cancer types that results in upregulation and thus activation of oncogenes located in the amplified regions. However, the mechanism by which amplified genes are upregulated is poorly understood. We hypothesized that epigenetic regulation of gene expression was important for activation in addition to the increased DNA copy number for genes within amplified genomic loci. To test this hypothesis, we used ovarian cancer as an example because it is a highly lethal disease and, more importantly, ovarian cancer cells are characterized by a high level of genomic instability as reflected by widespread DNA copy number changes. We analyzed the data from chromatin immunoprecipitation array (Chip-chip) and single nucleotide polymorphism (SNP) array to determine the correlation between specific histone modifications and genome-wide DNA copy number alterations in OVCAR3 ovarian cancer cells and ovarian surface epithelial cell culture. Among 8 histone marks examined, representing both heterochromatic and euchromatic chromatin, we identified the strongest correlation between increased DNA copy number and the active histone marks, histone H4 acetylation at lysine 5 (p-value 7.8×10^-6; FDR 5.2×10^-5) and lysine 12 (p-value 7.6×10^-5; FDR 2.6×10^-4). In contrast, there was no significant correlation between DNA amplification and repressive histone marks including histone H3 methylation at lysines 9 and 27. These correlations were validated by quantitative real-time PCR of ChIP samples at confirmed amplified regions on chromosomes 11 and 19, which harbor the RSF1 and AKT2 oncogenes. Furthermore, all genes within the amplicons were overexpressed in the OVCAR3 cell line harboring the amplicons as compared to a normal ovarian surface epithelial cell line. By using ovarian cancer as a disease model, our results indicate that an amplicon-associated specific histone modification pattern facilitates gene expression and suggest that both genetic (DNA copy number gain) and epigenetic (active histone marks) events contribute to upregulation of amplified genes which may ultimately contribute to the development of human cancer. 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 4871.

Abstract 4789: Functional characterization and clinical correlation of the tumor antigen casein kinase 1-epsilon in ovarian cancer

Abstract OBJECTIVES: Prior studies have identified antibodies to casein kinase I epsilon (CKI-ε) in the serum of patients with ovarian cancer. We investigated the expression pattern, functional roles, and clinical correlation of CKI-ε in ovarian cancer. METHODS: The expression of CKI-ε in 8 healthy ovaries and 68 ovarian tumor samples was determined by IHC and scored on intensity of antibody expression (scale 0-3) and the percentage of the area stained (scale 0-3). The average from both scores was assigned to each patient. Clinical characteristics for each patient were retrieved from electronic medical records. Statistical significance was determined using the Fisher exact test. Univariate analysis was performed by constructing probability curves according to the Kaplan-Meier method. Immortalized normal human ovarian surface epithelial (HOSE) cell lines with ectopic expression of CKI-ε were established by transfection with a CKI-ε expression construct. CKI-ε-targeting shRNAs were used to generate ovarian cancer cell lines that have stably suppressed CKI-ε expression. Protein expression was determined by Western blot. Growth rate was determined by cell counting. Three-dimensional spheroid culture was performed on Matrigel. The growth of ovarian cancer cell lines harboring either a control shRNA or a CKI-ε-shRNA was evaluated in a mouse xenograft model. The effects of IC261, an inhibitor of CKI-ε, on ovarian cancer cells were evaluated by MTT assay. RESULTS: CKI-ε was overexpressed in 67% of invasive ovarian tumors (P-value=0.001) and in 14 of 17 ovarian cancer cell lines. Normal HOSE cells that have ectopic expression of CKI-ε have accelerated growth rate and formed spheroids in Matrigel that were two to four times larger than those formed by control HOSE. In contrast, cancer cell lines harboring specific CKI-ε shRNA had reduced growth rate and 6-fold reduced tumor weight in the xenograft model (P-value=0.007). The cancer cell lines also showed CKI-ε-dependent sensitivity to IC261. The median CKI-ε expression level among the ovarian cancer tissue samples was 3. For patients whose score was ≤ 3 and > 3, the mean overall survival was 91 months and 48 months, respectively. Similarly, the mean disease free survival interval was 43 months and 22 months for patients whose score was ≤ 3 and > 3, respectively. Patients who scored > 3 tended to have late stage disease (61%) compared to patients who scored ≤ 3 (39%), (p=0.06). CONCLUSIONS: CKI-ε is highly expressed in ovarian tumors and cancer cell lines. Studies of cell lines with alterations in CKI-ε expression showed that this signaling protein regulates cell growth and survival. 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 4789.

Clinical significance of the NKG2D ligands, MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis.

To investigate the clinical significance of the expression of the NKG2D ligands MICA/B and ULBP2 in ovarian cancer. Eighty-two ovarian cancer patients and six patients without ovarian cancer from Department of Obstetrics and Gynecology of Kyoto University Hospital were enrolled in this study between 1993 and 2003. Expression of MICA/B, ULBP2, and CD57 in ovarian cancer tissue and normal ovary tissue was evaluated by immunohistochemical staining, and the relationship of these results to relevant clinical patient data was analyzed. Expression of MICs, ULBP2, and HLA-class I molecules in 33 ovarian cancer cell lines and two normal ovarian epithelial cell lines, as well as levels of soluble MICs and ULBP2 in the culture supernatants, were measured. Expression of MICA/B and ULBP2 was detected in 97.6 and 82.9% of ovarian cancer cells, respectively, whereas neither was expressed on normal ovarian epithelium. The expression of MICA/B in ovarian cancer was highly correlated with that of ULBP2. Strong expression of ULBP2 in ovarian cancer cells was correlated with less intraepithelial infiltration of T cells and bad prognoses for patients, suggesting that ULBP2 expression is a prognostic indicator in ovarian cancer. The expression of NKG2D ligands did not correlate with the levels of the soluble forms of the ligands. High expression of ULBP2 is an indicator of poor prognosis in ovarian cancer and may relate to T cell dysfunction in the tumor microenvironment.

Activation of Platelet-Activating Factor Receptor and Pleiotropic Effects on Tyrosine Phospho-EGFR/Src/FAK/Paxillin in Ovarian Cancer

Among the proinflammatory mediators, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a major primary and secondary messenger involved in intracellular and extracellular communication. Evidence suggests that PAF plays a significant role in oncogenic transformation, tumor growth, angiogenesis, and metastasis. However, PAF, with its receptor (PAFR) and their downstream signaling targets, has not been thoroughly studied in cancer. Here, we characterized the PAFR expression pattern in 4 normal human ovarian surface epithelial (HOSE) cell lines, 13 ovarian cancer cell lines, paraffin blocks (n = 84), and tissue microarrays (n = 230) from patients with ovarian cancer. Overexpression of PAFR was found in most nonmucinous types of ovarian cancer but not in HOSE and mucinous cancer cells. Correspondingly, PAF significantly induced cell proliferation and invasion only in PAFR-positive cells (i.e., OVCA429 and OVCA432), but not in PAFR-negative ovarian cells (HOSE and mucinous RMUG-L). The dependency of cell proliferation and invasion on PAFR was further confirmed using PAFR-specific small interfering RNA gene silencing probes, antibodies against PAFR and PAFR antagonist, ginkgolide B. Using quantitative multiplex phospho-antibody array technology, we found that tyrosine phosphorylation of EGFR/Src/FAK/paxillin was coordinately activated by PAF treatment, which was correlated with the activation of phosphatidylinositol 3-kinase and cyclin D1 as markers for cell proliferation, as well as matrix metalloproteinase 2 and 9 for invasion. Specific tyrosine Src inhibitor (PP2) reversibly blocked PAF-activated cancer cell proliferation and invasion. We suggest that PAFR is an essential upstream target of Src and other signal pathways to control the PAF-mediated cancer progression.

CAMP response element–binding protein is expressed at high levels in human ovarian adenocarcinoma and regulates ovarian tumor cell proliferation

Approximately 90% of human ovarian tumors result from transformation of ovarian surface epithelial cells. It has been hypothesized that repeated destruction of the epithelial cells during ovulation, followed by proliferation and migration of epithelial cells to restore the ovarian surface, renders these cells susceptible to mutagenic events. One of the proteins found to promote ovarian surface epithelial cell survival and proliferation was the transcription factor, cAMP response element-binding protein (CREB). Thus, the objective of this study was to determine whether CREB was also highly expressed in tumor cells originating from the ovarian epithelium. Using an ovarian cancer tissue array, it was observed that approximately 54% of the epithelial-derived human ovarian tumors displayed moderate or high levels of CREB immunostaining, while none of the normal ovarian samples did. Comparison of CREB levels in a human ovarian tumor cell line to those of a normal ovarian epithelial cell line revealed elevated levels of CREB and phosphorylated CREB in the ovarian tumor cells. To determine whether CREB regulated proliferation and/or apoptosis in the ovarian tumor cell line, CREB expression was suppressed using RNA interference. Decreased CREB expression significantly reduced ovarian tumor cell proliferation, while there was no effect on apoptosis in these cells. Finally, we showed that CREB is highly expressed in an in vivo murine model of ovarian tumorigenesis. Therefore, CREB is frequently overexpressed in ovarian cancer where it appears to promote cell proliferation.

Identification of PRTFDC1 silencing and aberrant promoter methylation of GPR150, ITGA8 and HOXD11 in ovarian cancers

Methylated promoter CpG islands (CGIs) can be used to find novel tumor-suppressor genes and disease markers. In this study, to identify promoter CGIs aberrantly methylated in human ovarian cancers, we performed a genome-wide screening for differentially methylated DNA fragments using methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 185 DNA fragments specifically methylated in an ovarian cancer cell line (ES-2), compared with a normal human ovarian surface epithelial cell line (HOSE6-3), and 33 of them were derived from putative promoter CGIs. Ten ovarian cancer cell lines were analyzed by methylation-specific PCR, and seven ( GPR150, LOC222171, PRTFDC1, LOC339210, ITGA8, C9orf64 and HOXD11) of the 33 CGIs were methylated in one or more of the cell lines. Their downstream genes were barely expressed in cell lines without unmethylated DNA molecules by quantitative reverse-transcription-PCR. Demethylation of methylated cell lines with 5-aza-2′-deoxycytidine restored expression of two genes ( PRTFDC1 and C9orf64). In primary ovarian cancers, CGIs of GPR150 (in 4 of 15 cancers), ITGA8 (2/15), PRTFDC1 (1/15) , and HOXD11 (1/15) were methylated. Silencing of PRTFDC1 was revealed here for the first time, and aberrant methylation of GPR150, ITGA8 and HOXD11 could be candidate tumor markers.

Activin Receptor-Like Kinase 7 Induces Apoptosis through Up-Regulation of Bax and Down-Regulation of Xiap in Normal and Malignant Ovarian Epithelial Cell Lines

Transforming growth factor-beta superfamily has been implicated in tumorigenesis. We have recently shown that Nodal, a member of transforming growth factor-beta superfamily, and its receptor, activin receptor-like kinase 7 (ALK7), inhibit proliferation and induce apoptosis in human epithelial ovarian cancer cell lines. In this study, we further investigated the cellular mechanisms underlying the apoptotic action of ALK7 using an immortalized ovarian surface epithelial cell line, IOSE397, and an epithelial ovarian cancer cell line, OV2008. Infection of these cells with an adenoviral construct carrying constitutively active ALK7 (Ad-ALK7-ca) potently induced cell death; all cells died after 3 and 5 days of Ad-ALK7-ca infection in IOSE397 and OV2008 cells, respectively. ALK7-ca induced the expression of proapoptotic factor Bax but suppressed the expression of antiapoptotic factors Bcl-2, Bcl-XL, and Xiap. Silencing of Bax by small interfering RNA in IOSE397 cells significantly reduced ALK7-ca-induced apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay but partially blocked ALK7-ca-induced caspase-3 activation and did not affect the down-regulation of Xiap by ALK7-ca. Dominant-negative Smad2, Smad3, and Smad4 blocked ALK7-ca-regulated Xiap and Bax expression and caspase-3 activation. Thus, ALK7-induced apoptosis is at least in part through two Smad-dependent pathways, Bax/Bcl-2 and Xiap.

Methylation of death-associated protein kinase in ovarian carcinomas

Death-associated protein (DAP) kinase is a serine/threonine kinase that plays an integral role in apoptosis and metastasis. The purpose of our study was to determine the methylation status of DAP kinase in ovarian carcinomas. Thirty-one patients with histologically confirmed epithelial ovarian cancers treated at Roswell Park Cancer Institute, Buffalo, New York, between 1987 and 1999 were studied. Sixty-two samples were examined for DAP kinase methylation status: 1 normal human genomic DNA sample from a healthy individual, 1 transformed normal surface ovarian epithelial cell line (IOSE, from Dr Nancy Auersperg, Vancouver, Canada), 2 ovarian carcinoma cell lines (OVCAR3 and A2780), 1 ovarian serous cystadenoma, and 30 ovarian carcinomas. Additionally, peripheral blood DNA was examined from the patients with the serous cystadenoma and ovarian carcinomas. Methylation-specific polymerase chain reaction was performed using primers designed for the unmethylated and methylated promoter regions. The DAP kinase gene was unmethylated in both the normal human genomic DNA sample and the transformed normal surface epithelial ovarian cell line. The two ovarian cancer cell lines were methylated. In the 30 patients with malignant disease, methylation of DAP kinase was observed in 20 (67%). Peripheral blood DNA was available in 26 (87%) of the 30 patients. Comparison of the paired samples indicated that 14 (54%) were methylated and 12 (46%) were unmethylated. There was no correlation between the DAP kinase methylation status and stage, grade, histology, or survival. Methylation of CpG islands in the promoter region of the DAP kinase gene is common in peripheral blood DNA and tissue samples of patients with ovarian carcinomas. This molecular aberration may represent a potential target for therapeutic intervention.