Therapeutic Strategy For Ovarian Cancer Research Articles

Abstract B46: Preclinical testing of TR3 activators in cisplatin-resistant ovarian cancer

Abstract Introduction: The vast majority of epithelial ovarian malignancies present as biologically aggressive, metastatic tumors. High incidence of recurrence following standard platinum-based therapy indicates an urgent need for new treatment strategies in advanced disease. We and others have shown that the immediate-early gene NR4A1/Nur77 (TR3) is linked with malignant tumors and is a potential novel target for therapy. Nucleus-to-mitochondria translocation of TR3, leading to cytochrome C release, is an important mechanism of pro-apoptotic chemotherapeutic agents, including cisplatin, in ovarian cancer cells. Furthermore, two distinct chemical classes of compound that activate TR3 by direct binding induce anti-tumor effects in certain cancer cell lines: C-DIM (an analog of diindoylmethane, a metabolite of the indole-3-carbinol chemopreventive agent found in cruciferous vegetables) and the fungal metabolite, cytosporone B (CsnB). Both classes are known to activate TR3-dependent transcription (transactivation) of pro-apoptotic genes, while CsnB also exclusively stimulates TR3 expression and cytoplasmic translocation. Objective: We hypothesized that targeted activation of TR3 will be an effective therapeutic strategy in ovarian cancer cells resistant to established cisplatin chemotherapy. To test this hypothesis, we determined the anti-tumor effects of C-DIM and CsnB in preclinical models of cisplatin-resistant ovarian cancer. Methods: The isogenic cell line pairs, OVCAR-8 and cisplatin-resistant NCI/ADR-Res, and A2780 parental (A2780PAR) and cisplatin-resistant A2780CP20 cells, were treated with C-DIM, CsnB or 0.1% DMSO as vehicle control. Sulforhodamine B colorimetric assays were used to assess cell growth and cytotoxicity in vitro. Western Blot and immunofluorescence assays were used to measure apoptotic markers (cleaved PARP, cleaved caspase 3 and cytochrome C release). TR3 DNA binding was measured by a luciferase reporter plasmid containing 3 copies of its consensus response element (NurRE). Results: C-DIM and CsnB reduced cell growth in all cell lines tested in a concentration-dependent manner. CsnB induced approximately 2-fold more potent effects than C-DIM (IC50 concentrations ranging from 20-25μM to 40-50μM, respectively). Strikingly, there were no statistically significant differences in IC50 concentrations for either drug in cisplatin-resistant cells compared to their respective isogenic counterpart. Consistent with their growth-inhibitory effects, both compounds induced p21 expression. C-DIM and CsnB treatment induced apoptosis, although different mechanisms likely underlie these effects. While both compounds stimulated TR3 transactivation, only CsnB also induced nucleus-to-mitochondrial translocation of TR3, accompanied by cytochrome C release. Conclusions: These studies highlight the potential of targeted activation of TR3, particularly by CsnB-like drugs, as a novel therapeutic strategy in cisplatin-resistant ovarian cancer. Citation Format: Andrew J. Wilson, Jeanette Saskowski, Dineo Khabele. Preclinical testing of TR3 activators in cisplatin-resistant ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B46.

Novel Recombinant Human B7-H4 Antibodies Overcome Tumoral Immune Escape to Potentiate T-Cell Antitumor Responses

B7-H4 (VTCN1, B7x, B7s) is a ligand for inhibitory coreceptors on T cells implicated in antigenic tolerization. B7-H4 is expressed by tumor cells and tumor-associated macrophages (TAM), but its potential contributions to tumoral immune escape and therapeutic targeting have been less studied. To interrogate B7-H4 expression on tumor cells, we analyzed fresh primary ovarian cancer cells collected from patient ascites and solid tumors, and established cell lines before and after in vivo passaging. B7-H4 expression was detected on the surface of all fresh primary human tumors and tumor xenotransplants, but not on most established cell lines, and B7-H4 was lost rapidly by tumor xenograft cells after short-term in vitro culture. These results indicated an in vivo requirement for B7-H4 induction and defined conditions for targeting studies. To generate anti-B7-H4-targeting reagents, we isolated antibodies by differential cell screening of a yeast-display single-chain fragments variable (scFv) library derived from patients with ovarian cancer. We identified anti-B7-H4 scFv that reversed in vitro inhibition of CD3-stimulated T cells by B7-H4 protein. Notably, these reagents rescued tumor antigen-specific T-cell activation, which was otherwise inhibited by coculture with antigen-loaded B7-H4+ APCs, B7-H4+ tumor cells, or B7-H4- tumor cells mixed with B7-H4+ TAMs; peritoneal administration of anti-B7-H4 scFv delayed the growth of established tumors. Together, our findings showed that cell surface expression of B7-H4 occurs only in tumors in vivo and that antibody binding of B7-H4 could restore antitumor T-cell responses. We suggest that blocking of B7-H4/B7-H4 ligand interactions may represent a feasible therapeutic strategy for ovarian cancer.

Death Domain-associated Protein DAXX Promotes Ovarian Cancer Development and Chemoresistance

The role of DAXX in ovarian cancer development and metastasis has not been investigated before now. Overexpression of DAXX enhanced ovarian cancer cell proliferation, colony formation, and migration, whereas Daxx depletion had the opposite effects. DAXX promotes ovarian cancer cell proliferation and chemoresistance. ModulatingDAXXmay be an effective strategy for preventing the recurrence and chemoresistance of ovarian cancers. Understanding the genes involved in apoptosis and DNA damage responses may improve therapeutic strategies for ovarian cancer. The death domain-associated protein DAXX can be either a pro-apoptotic or an anti-apoptotic factor, depending on the cell type and context. In this study, we found that DAXX was highly expressed in human ovarian surface epithelial tumors but not in granulosa cell tumors. In cultured ovarian cancer cells, DAXX interacted with promyelocytic leukemia protein (PML) and localized to subnuclear domains (so-called PML nuclear bodies). A role for DAXX in ovarian cancer cell proliferation, metastasis, and radio/chemoresistance was examined. Overexpression of DAXX enhanced multiple ovarian cancer cell lines' proliferation, colony formation, and migration, whereas Daxx depletion by RNA interference had the opposite effects. When transplanted into nude mice, ovarian cancer cells that overexpressed DAXX displayed enhanced tumorigenesis capability in vivo, whereas Daxx depletion inhibited tumor development. Importantly, Daxx induced tumorigenic transformation of normal ovarian surface epithelial cells. Daxx also protected ovarian cancer cells against x-irradiation- and chemotherapy-induced DNA damage by interacting with PML. Taken together, our results suggest that DAXX is a novel ovarian cancer oncogene that promotes ovarian cancer cell proliferation and chemoresistance in ovarian cancer cells. Thus, modulating DAXX-PML nuclear body activity may be an effective strategy for preventing the recurrence and chemoresistance of ovarian cancers.

Abstract 1939: Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing.

Abstract Ovarian cancer is the most common cause of gynecologic malignancy related mortality in women. The combination of tumor reductive surgery and chemotherapy is the main therapeutic strategy for ovarian cancer. However, especially in the most of advanced cases, complete site reduction is impossible to achieve. In those cases, the sensitivity to chemotherapy strongly affects their prognoses. MicroRNAs (miRNAs), which are approximately 22-nucleotide non-coding RNAs, regulate protein expression by translational inhibition and degradation of their target mRNAs. The expression of miRNAs is dysregulated in various types of cancers, suggesting that miRNAs are involved in carcinogenesis and cancer progression. The purpose of present work was to identify miRNAs which are related to chemo-sensitivity in ovarian cancer. Using Next Generation Sequencing (NGS), miRNA profiling was performed in 38 advanced high-grade serous ovarian cancers (HGSOCs). RNAs including miRNAs were procured from the epithelial component of the laser microdissected tumor tissue removed from patients undergoing primary surgery without neo-adjuvant chemotherapy. A total of 1935 miRNAs and miRNA variants were detected by NGS and normalized expression data of each miRNAs and miRNA variants were used for further analyses. Comparing the copy numbers of each miRNAs between chemo-refractory group (patients with disease progression in the period of first line chemotherapy, n=7) and chemo-sensitive group (patients with disease progression in more than 6 months after completion of first line chemotherapy, n=13) by Mann-Whitney U test, 77 miRNAs and miRNA variants were found to be dysregulated with significant differences (p<0.05). The 10 most significant miRs identified by NGS were selected for further validation studies by real-time reverse transcription PCR (RT-PCR) analysis on the same sample set. To determine whether miRNAs could be used as a prognostic marker HGSOC, expression levels of those 10 miRNAs were quantified by RT-PCR using 95 HGSOC samples including the 38 samples used in the discovery set. The results showed that mir-625-3p expression levels were significantly reduced in the chemo-refractory group compared with the chemo-sensitive group as indicated by the NGS data (n=20, p=0.029), and by the RT-PCR data (n=63, p=0.004). Furthermore, Kaplan-Meier survival analysis with log-rank test indicated that low expression of mir-625-3p (less than mean expression) was significantly associated with shorter progression-free survival (n=95, p=0.007) and shorter over-all survival (n=95, p<0.001) as compared with the high expression group (more than mean expression). Our results strongly suggest that mir-625-3p might be a prognostic marker that can be used to predict chemo-sensitivity in patients with HGSOC. Further characterization of mir-625-3p as a therapeutic target for HGSOC is in progress. Citation Format: Tetsushi Tsuruga, Chi Lam Au Yeung, Cecilia S. Leung, Tsz-Lun Yeung, Kwong K. Wong, Rosemarie Schmandt, Ngai Na Co, Karen H. Lu, Samuel Mok. Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing. [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 1939. doi:10.1158/1538-7445.AM2013-1939

Characteristics of chemokine signatures elicited by EGF and TNF in ovarian cancer cells

BackgroundOvarian cancer, an inflammation-associated cancer, is the fifth leading cause of cancer deaths in women. The malignancy produces a large amount of tumor necrosis factor (TNF) which promotes a proinflammatory tumor microenvironment. In addition, the epidermal growth factor receptor (EGFR) is frequently overexpressed in high-grade ovarian cancer, which likely aggravates cancer progression. Since ovarian cancer progression is closely associated with chemokine networks driven by inflammation or EGFR activation, we investigated the chemokine signatures elicited by EGF and TNF in ovarian cancer cells to determine their individual profiles and if there was in fact some kind of synergy between their actions on the chemokine network.MethodsWe used a PCR array for the chemokine network to examine the signature of chemokines and their receptors elicited by EGF and TNF in four ovarian cancer cell lines (OVCAR-3, SKOV-3, CaOV-3 and TOV-21G).ResultsThe chemokine network revealed that ovarian cancer cells commonly expressed high levels of proinflammatory chemokines such as CCL20, CXCL1-3 and CXCL8 in response to EGF or TNF. However, the responsiveness to EGF or TNF displayed a cell line specific pattern. Although OVCAR-3 and SKOV-3 cells were responsive to either EGF or TNF, their TNF responsiveness was dominant. On the other hand, CaOV-3 and TOV-21G cells were responsive to EGF but less to TNF, probably due to the high levels of non-canonical nuclear factor (NF)-κB components such as IKKα and p52 in these cell lines compared to OVCAR-3 and SKOV-3 cells. Among chemokine receptors, only CXCR5 was responsive to EGF or TNF in CaOV-3 cells. Finally, CCL20 and CXCL8 responded synergistically in response to EGF and TNF in OVCAR-3 and SKOV-3 cells.ConclusionOur results indicate that CCL20, CXCL1-3 and CXCL8 are the primary chemokines induced by EGF or TNF and are elicited in these ovarian cancer cells via NF-κB, Akt and Erk signaling pathways. Of interest, there was a syngergistic response in terms of CCL20 and CXCL8 levels, when OVCAR-3 and SKOV-3 cells were exposed to EGF plus TNF. Targeting these proinflammatory chemokines may be a promising therapeutic strategy for ovarian cancer with abundant TNF and EGFR activation patterns.

Systematic analysis and validation of differential gene expression in ovarian serous adenocarcinomas and normal ovary

Cancer of the ovary confers the worst prognosis among women with gynecological malignancies, primarily because most ovarian cancers are diagnosed at late stage. Hence, there is a substantial need to develop new diagnostic biomarkers to enable detection of ovarian cancer at earlier stages, which would confer better prognosis. In addition, the identification of druggable targets is of substantial interest to find new therapeutic strategies for ovarian cancer. The expression of 22,500 genes in a series of 67 serous papillary carcinomas was compared with 9 crudely enriched normal ovarian tissue samples by RNA hybridization on oligonucleotide microarrays. Multiple genes with near-uniformly expression were elevated in carcinomas of varying grade and malignant potential, including several previously described genes (e.g., MUC-1, CD9, CD24, claudin 3, and mesothelin). We performed immunohistochemical staining with antibodies against several of the proteins encoded by differentially expressed genes in an independent cohort of 71 cases of paraffin-embedded ovarian cancer samples. We found striking differences in EpCAM (p < 0.005), CD9 (p < 0.001), MUC-1 (p < 0.001), and claudin 3 proteins (p < 0.001) but not for mesothelin (p > 0.05) using the Mann-Whitney U test. Protein expression of a majority of the differentially expressed genes tested was found to be elevated in ovarian carcinomas and, as such, define potential new biomarkers or targets.

Secreted Stress-Induced Phosphoprotein 1 Activates the ALK2-SMAD Signaling Pathways and Promotes Cell Proliferation of Ovarian Cancer Cells

Stress-induced phosphoprotein 1 (STIP1), a cochaperone that organizes other chaperones, heat shock proteins (HSPs), was recently shown to be secreted by human ovarian cancer cells. In neuronal tissues, binding to prion protein was required for STIP1 to activate the ERK (extracellular-regulated MAP kinase) signaling pathways. However, we report that STIP1 binding to a bone morphogenetic protein (BMP) receptor, ALK2 (activin A receptor, type II-like kinase 2), was necessary and sufficient to stimulate proliferation of ovarian cancer cells. The binding of STIP1 to ALK2 activated the SMAD signaling pathway, leading to transcriptional activation of ID3 (inhibitor of DNA binding 3), promoting cell proliferation. In conclusion, ovarian-cancer-tissue-secreted STIP1 stimulates cancer cell proliferation by binding to ALK2 and activating the SMAD-ID3 signaling pathways. Although animal studies are needed to confirm these mechanisms in vivo, our results may pave the way for developing novel therapeutic strategies for ovarian cancer.

Smoothened Antagonists Reverse Taxane Resistance in Ovarian Cancer

The hedgehog pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether hedgehog signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the hedgehog receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of hedgehog pathway members was assessed in three pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using quantitative PCR and Western blot analysis. Cell lines were exposed to increasing concentrations of two different Smo antagonists (cyclopamine, LDE225) alone and in combination with carboplatin or paclitaxel. Selective knockdown of Smo, Gli1, or Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay. A2780cp20 and SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel, or combination therapy. Chemoresistant cell lines showed higher expression (>2-fold, P < 0.05) of hedgehog signaling components compared with their respective parental lines. Smo antagonists sensitized chemotherapy-resistant cell lines to paclitaxel, but not to carboplatin. LDE225 treatment also increased sensitivity of ALDH-positive cells to paclitaxel. A2780cp20 and SKOV3TRip2 xenografts treated with combined LDE225 and paclitaxel had significantly less tumor burden than those treated with vehicle or either agent alone. Increased taxane sensitivity seems to be mediated by a decrease in P-glycoprotein (MDR1) expression. Selective knockdown of Smo, Gli1, or Gli2 all increased taxane sensitivity. Smo antagonists reverse taxane resistance in chemoresistant ovarian cancer models, suggesting combined anti-hedgehog and chemotherapies could provide a useful therapeutic strategy for ovarian cancer.

Abstract 3318: Phenotypic heterogeneity and instability of tumor-initiating cells in high-grade serous cancer

Abstract Serous ovarian cancer (SOC) is the leading cause of morbidity/mortality from gynecologic malignancy. Current therapies significantly increase survival, yet 70-90% of SOC patients recur within five years and die of their disease. The cancer stem cell (CSC) hypothesis holds that only some tumor cells have the potential to initiate and maintain tumors. Moreover, CSC might be resistant to many therapeutic agents that are effective against bulk tumor cells, which could contribute to treatment failure. We have established a large collection of primary human SOC samples and developed a robust, quantitative assay for SOC tumor-initiating cells (TIC). Using this assay, we found that TIC from primary SOC can be identified based on their expression of the surface marker CD133. However, we also observed heterogeneity and instability of the TIC phenotype with disease progression. We examined whether instability in the TIC phenotype may be due to the acquisition of additional genetic alterations in the CD133- compared to the CD133+ fraction. Preliminary analysis suggests that most genetic alterations are common in both fractions; however, in 3/6 cases, additional genetic alterations were observed in the CD133- fraction, suggesting the emergence of CD133- TIC might be, at least in part, genetically driven. The mechanism of genetic instability is currently under investigation. In addition, we have used expression microarrays to examine differences between CD133+ and CD133- populations. We find that &amp;gt;1000 probes are differentially expressed in these two groups. We have identified several pathways with altered expression in TIC-enriched fractions. The biological role of these pathways in HG-SOC is under investigation. Our ability to identify and characterize TIC in this disease may facilitate development of new and more effective therapeutic strategies for ovarian cancer, thereby benefitting patients with this devastating disease. 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 3318. doi:1538-7445.AM2012-3318

TGF-β: friend or foe? The role of TGF-β/SMAD signaling in epigenetic silencing of ovarian cancer and its implication in epigenetic therapy

Importance of the field: The TGF-β signaling pathway plays an important role in regulating numerous cellular processes including growth inhibition of ovarian surface epithelial (OSE) cells. However, epithelial ovarian cancer is refractory to the inhibitory functions of TGF-β, and yet TGF-β induces metastasis or epithelial–mesenchymal transition (EMT) in advanced ovarian cancer. How TGF-β plays a certain role in one cell but a different role in its malignant counterpart is not fully understood.Areas covered in this review: The role of TGF-β/SMAD signaling both in normal OSE cells and ovarian cancer as well as how dysregulation of this signaling pathway leads to epigenetic silencing of its downstream targets in ovarian neoplasias are reviewed. The therapeutic implication of this signaling pathway in epigenetic therapy of ovarian cancer are also discussed.What the reader will gain: The reader will gain insight on how dysregulation of TGF-β signaling alters promoter methylation and histone modifications of TGF-β downstream targets in ovarian cancer.Take home message: Disruption of TGF-β/SMAD signaling leads to epigenetic silencing of its target genes transiently through histone modifications but permanently by promoter hypermethylation. Targeting the TGF-β signaling pathway may be a novel therapeutic strategy in ovarian cancer.

Relationship of miR-101 and targeted epigenetic silencing of EzH2 on cell growth and invasiveness in ovarian cancer cells.

e15538 Background: Enhancer of zeste homolog 2 (EzH2), the catalytic subunit of Polycomb group (PcG) of the chromatin modifying complex, is abnormally elevated in several cancers. High levels of EzH2 correlate strongly with high-grade malignancies that are invasive, poorly differentiated and of advanced stage at presentation. miR-101 belongs to a family of micro-RNAs (miRNAs) that are short non-coding RNA sequences involved in epigenetic regulation of gene expression. miR-101 has been shown to bind to EzH2 mRNA inhibiting protein translation. Though recent reports demonstrate significant down regulation of microRNA-101 (miR-101) in several solid tumors suggesting that it might serve as a solid tumor signature, its role in ovarian cancer is not well defined. Methods: MDAH-2774 and SKoV3 cell lines were transfected with miR-101 expression plasmid (miR-101 sequence is as follows: 5' UAC AGU ACU GUG AUA ACU GAA G 3') using effectene (Qiagen Inc.,Valencia,CA). Standard protocols were followed for western blot analysis of EzH2 levels after EzH2 knockdown with miR-101 plasmid transfection. Triplicate growth curves were generated using Cell Counting Kit-8 (CCK-8) (Dojindo, Gaithersburg, MD). Eight week-old CB17/ICr-SCID mice were injected with 3 ×106 MDAH-2774 cells that were transfected with miR-101 plasmid. Results: miR-101 achieved more than 70% knockdown of the EzH2 protein 48 hrs after transfection in both SKoV3 and MDAH-2774 cell lines, demonstrating efficient inhibition of EzH2. Further, significant reduction in cell proliferation in both MDAH-2774 and SKoV3 cell lines was observed in response to EzH2 knock down. MDAH-2774 cells transfected with miR-101 showed 15% less growth than the control on day 3 after transfection, versus 48% for SKOV3 cells. EzH2 knockdown in CB17/ICr-SCID mice was associated with marked reduction in palpable tumor generation by MDAH-2774 cells, indicating that EzH2 promotes the growth of MDAH-2774 cell line in vivo. Conclusions: The results indicate that miR-101 mediated epigenetic silencing of EzH2 may be a potential therapeutic strategy for ovarian cancer. No significant financial relationships to disclose.

Proliferation suppression and apoptosis of ovarian carcinoma cells induced by small interfering RNA against vascular endothelial growth factor

The aim of this study was to investigate whether RNA interference (RNAi) targeting vascular endothelial growth factor (VEGF) could inhibit the proliferation and induce apoptosis of ovarian carcinoma cells. Human epithelial ovarian carcinoma cell line CaoV3 was transfected with VEGF-targeted small interfering RNA (siRNA) for 48 h. The down-regulation of VEGF expression was determined by real-time polymerase chain reaction and Western blot. The proliferation of CaoV3 cells was detected by a colorimetric BrdU assay. Caspase-3 activity and TUNEL assay were also detected to study the apoptosis of ovarian carcinoma cells induced by VEGF siRNA. The protein levels of survivin, MMP2 and MMP9 were measured by Western blot. mRNA and protein of VEGF were significantly down-regulated by VEGF siRNA. Down-regulation of VEGF expression dramatically suppressed the proliferation of CaoV3 cells, and increased the Caspase-3 activity. Nearly 100% of cells indicated TUNEL-positive. The expression of anti-apoptotic protein survivin was decreased, and the invasive related protein MMP2 and MMP9 were also significantly reduced by VEGF siRNA. Our study implied that siRNA targeting VEGF could effectively inhibit cell proliferation, induce cell apoptosis, and decrease the cell invasive potential. These findings suggest that the RNAi approach targeting VEGF may be an effective therapeutic strategy for ovarian cancer.

Endogenous Human CaMKII Inhibitory Protein Suppresses Tumor Growth by Inducing Cell Cycle Arrest and Apoptosis through Down-regulation of the Phosphatidylinositide 3-Kinase/Akt/HDM2 Pathway

Inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII) results in hypophosphorylation of CaMKII substrates and in some cases suppresses cell growth. We previously presented the first report of the human CaMKII inhibitory protein, hCaMKIINbeta. Here we report the functional characterization of hCaMKIINbeta in ovarian cancer cells. We showed that hCaMKIINbeta was highly expressed in normal ovarian tissues but was not detected in human ovarian adenocarcinoma, indicating that decreased expression of hCaMKIINbeta may be involved in the pathogenesis of human ovarian adenocarcinoma. As an endogenous CaMKII inhibitor, hCaMKIINbeta could significantly inhibit the growth of human ovarian cancer cells in vitro. In vivo, hCaMKIINbeta decreased the tumorigenicity and growth of HO-8910PM human ovarian cancer cells and prolonged the survival of tumor-bearing mice. hCaMKIINbeta blocked cell cycle progression and induced apoptosis of HO-8910PM cells, which was correlated with the up-regulation of p21, p53, and Bax and the down-regulation of cyclin A, cyclin D1, cyclin E, CDK2, phosphorylated retinoblastoma, and Bcl-2. We further demonstrated that hCaMKIINbeta-mediated CaMKII inhibition suppressed Akt activation, leading to the down-regulation of HDM2, which was responsible for the up-regulation of p53 and p21 in human ovarian cancer cells. The tumor-suppressive effect and the negative expression in human ovarian cancer tissues suggest that hCaMKIINbeta may play an important role in the regulation of tumor cell growth, possibly contributing to the development of new therapeutic strategies for ovarian cancer.

Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro

BackgroundIQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells.MethodsWe used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay.ResultsIQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAi-mediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration.ConclusionOur findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.

Cisplatin Induces p53-Dependent FLICE-Like Inhibitory Protein Ubiquitination in Ovarian Cancer Cells

Understanding the mechanism of cisplatin (CDDP) action may improve therapeutic strategy for ovarian cancer. Although p53 and FLICE-like inhibitory protein (FLIP) are determinants of CDDP sensitivity in ovarian cancer, the interaction between p53 and FLIP remains poorly understood. Here, using two chemosensitive ovarian cancer cell lines and various molecular and cellular approaches, we show that CDDP induces p53-dependent FLIP ubiquitination and degradation, and apoptosis in vitro. Moreover, we showed that Itch (an E3 ligase) forms a complex with FLIP and p53 upon CDDP treatment. These results suggest that p53 facilitates FLIP down-regulation by CDDP-induced FLIP ubiquitination and proteasomal degradation.

Expression and biological role of the prostaglandin D synthase/SOX9 pathway in human ovarian cancer cells

New therapeutic strategies for ovarian cancer include the identification of involved signaling pathways that could potentially serve as a source of biomarkers for early stages of the disease. In this study, we show that the embryonic male prostaglandin D synthase (Pgds)/SOX9 pathway is expressed at both the RNA and protein levels in different types of human ovarian tumors, pointing to Pgds and SOX9 as possible diagnostic markers for ovarian carcinomas. Using ovarian cancer cell lines, we found, first, that components of the Pgds/SOX9 pathway are expressed in these cells, and second, that treatment of these cells with prostaglandin D2 (PGD2) can inhibit their growth via its DP1 receptor and induce apoptosis. Finally, using siRNA and overexpression strategies, we demonstrate that SOX9 expression is induced by PDG2 and is responsible for PDG2-mediated growth inhibition. Accordingly, as stimulating the PGD2/DP1 signal transduction pathway upregulates SOX9 expression, either activators of this pathway or DP1 agonists may be useful as new therapeutic agents.

Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines

To develop a novel therapeutic strategy for ovarian cancer, we constructed a recombinant adenovirus which highly expresses pro-apoptotic Bax protein and examined its therapeutic effect on a series of ovarian cancer cell lines: A2780, A2780/cDDP, OVCAR-3 and SK-OV-3. A recombinant adenovirus carrying the Bax-α gene (AxCALNKYbax) induced high expression of the Bax-α protein in all the cell lines. The cytotoxic effect of Bax was observed in three ovarian cancer cell lines: the per cent reduction in the number of cells was 40.0% for cisplatin-sensitive A2780, 50.0% for cisplatin-resistant A2780/cDDP, and 64.8% for marginally cisplatin-resistant OVCAR-3. In contrast, it was only 12.3% for cisplatin-resistant SK-OV-3. Cisplatin-resistant A2780/cDDP had a p53 mutation and exhibited attenuated Bax induction after cisplatin treatment, which may explain why supplementation of Bax was effective in this chemoresistant ovarian cancer. Combination with cisplatin or paclitaxel enhanced the cytotoxic effect of Bax induction in all but one cell line including cisplatin-resistant A2780/cDDP. It appears that adenovirus-mediated Bax induction, with or without combination with conventional chemotherapy, useful strategy for the treatment of ovarian cancer.

Prognostic factors in ovarian carcinoma in complete histologic remission at second-look surgery.

Prognosis of ovarian carcinoma in complete histologic remission (CHR) at second-look surgery is still controversial. In a series of 83 patients in CHR we studied retrospectively several prognostic factors (age, stage, histologic grade, histologic type, initial residual disease after surgery, CA 125 normalization period) to determine which patients present a high risk of relapsing after CHR and could be included in therapeutic protocols for consolidation treatment. Univariate analysis showed that the combination of CA 125 normalization < 8 weeks with absence of macroscopic tumoral residue after initial surgery permits the definition of a group with a very good prognosis, while for patients with CA 125 normalization period > 8 weeks and an initial macroscopic residual tumor, the prognosis is relatively poor (progression-free survival 100% vs. 47%, at 2 years P < 0.05). Using the Cox multivariate analysis, only the initial tumoral residue is of prognostic significance for progression-free survival; there is no prognostic significance for overall survival. The therapeutic strategy for ovarian cancer may be improved for patients in CHR after second-look surgery by determining those at high risk, making it possible to confine consolidation treatment trials to such a group.

Gamma-interferon gene transfer as a therapeutic strategy for ovarian cancer.

Ovarian carcinoma is the fourth most frequent cause of cancer-related death in women. This cancer have a bad prognosis because usually diagnosed at a late stage when neoplasic ascite occurs. Furthermore, it is usually resistant to chemotherapy when relapses occur. So, it appears necessary to develop new treatments, such as gene therapy, to try to better eradicate the disease.KeywordsOvarian CancerGene TherapyOvarian CarcinomaThyroid Follicular CellIntraabdominal OrganThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Analysis of antigen expression at multiple tumor sites in epithelial ovarian cancer

The question of whether the antigenic phenotype of human epithelial ovarian cancer varies in a given patient between the primary tumor and metastatic sites or among metastatic sites themselves is an important issue in planning potential therapeutic strategies for ovarian cancer. We have obtained tumor specimens from at least two separate sites during operations on 12 patients with epithelial ovarian cancer, and we have typed these specimens with a group of 18 monoclonal antibodies that react with cell-surface glycoprotein and carbohydrate antigens, including blood group antigens. Antibodies with relative specificity for malignant cells as well as those that detect more widely distributed epithelial antigens were used. A total of 31 specimens from 12 patients with advanced adenocarcinoma (8 serous, 3 undifferentiated, 1 endometrioid) of the ovary were studied, including fresh ascites cells in two patients. Frozen sections of tumor specimens were stained with the antibodies by the indirect immunoperoxidase technique and graded semiquantitatively. Little difference was seen in antigenic expression of tumors that were obtained from various sites in the same patient for either the epithelial cell markers or blood group markers. Intratumoral antigenic heterogeneity was seen, but this was generally quite consistent within a given patient's specimens. As anticipated, variations in antigen expression were seen among specimens from different patients. The antigenic phenotype of the tumor specimens in a given patient, as determined immunohistochemically by our group of antibodies, showed only minor variation among primary and metastatic sites.