Ovarian Clear Cell Carcinoma Cell Research Articles

ARID1A gene silencing reduces the sensitivity of ovarian clear cell carcinoma to cisplatin.

In ovarian clear cell carcinoma (OCCC), the mutation rate of the AT-rich interaction domain 1A (ARID1A) gene is 46-57%. However, the effects of ARID1A gene silencing by small interfering RNA (siRNA) on the sensitivity of OCCC to cisplatin have not been investigated. Thus, this study aimed to elucidate the association between ARID1A gene silencing and drug resistance in OCCC. Three pairs of ARID1A gene siRNA fragments (siRNA-1, siRNA-2 and siRNA-3) were designed and transiently transfected into ES2 OCCC cells using RNAi Max reagent. Western blotting results demonstrated that the transfection reduced ARID1A protein expression levels, with the siRNA-3 group having the lowest levels. The IC50 value, determined using a Cell Counting kit-8 assay, was significantly increased by siRNA-3 transfection compared with that in blank control and negative control groups. The cell survival rate following treatment with 50 µM cisplatin for 48 h was significantly increased in the siRNA-3 group compared with the blank control and negative control groups. Flow cytometric analysis revealed that the apoptosis rate for cisplatin-treated cells was significantly lower in cells with siRNA-3 transfection than in those without, and the apoptosis rate in siRNA-3-transfected cells was lower than that in the negative control group. Western blot analysis showed that the expression level of AKT in cisplatin-treated cells was significantly decreased compared with that in the negative control group, and the AKT expression level in cisplatin-treated cells was significantly higher with siRNA-3 transfection than without. Therefore, the results demonstrated that ARID1A siRNA efficiently decreased ARID1A expression, which reduced cisplatin chemosensitivity and cell apoptosis in ES2 OCCC cells via the regulation of AKT expression.

Functional redundancy of the Notch pathway in ovarian cancer cell lines.

Epithelial ovarian cancer is the most lethal gynecologic malignancy, despite advances in treatment. The most common histological type, high-grade ovarian serous carcinoma (OSC) is usually diagnosed at an advanced stage, and although these types of tumors frequently respond to surgery and platinum-based chemotherapy, they usually recur. Ovarian clear cell carcinoma (OCCC) is an unusual histological type, which is known to be intrinsically chemoresistant and is associated with poor prognosis in advanced stages. In recent years, genetic alterations and epigenetic modulation of signaling pathways have been reported in OSC and OCCC, including the overexpression of Notch pathway elements and histone deacetylases. Histone deacetylase inhibitors (HDACis), including vorinostat (suberoylanilide hydroxamic acid), alter the transcription of genes involved in cell growth, survival and apoptosis, and have become an attractive therapeutic approach. However, no previous work has addressed the effect of HDACis, and in particular vorinostat, on Notch signaling in ovarian cancer. Therefore, the present study aimed to investigate the modulation of the Notch pathway by vorinostat in ovarian cancer. Using immunofluorescence and quantitative polymerase chain reaction, the present results revealed that vorinostat activated the Notch pathway in OCCC and OSC cell lines, through different Notch ligands. In OCCC, the activation of the Notch pathway appeared to occur through Delta-like (Dll) ligands 1, 2 and 3, whereas in OSC Dll1 and Jagged 1 and 2 ligands were involved. The activation of the Notch pathway by vorinostat, in OCCC and OSC cell lines, culminated in the increased expression of the same downstream transcription factors, hairy enhancer of split (Hes) 1 and 5, and Hes-related proteins 1 and 2. In conclusion, vorinostat modulates the expression of several downstream targets of the Notch pathway and independent Notch receptors and ligands that are expressed in OSC and OCCC. This upregulation of the Notch pathway may explain why vorinostat therapy fails in ovarian carcinoma treatment, as shown in certain clinical trials.

Preclinical Investigations of PM01183 (Lurbinectedin) as a Single Agent or in Combination with Other Anticancer Agents for Clear Cell Carcinoma of the Ovary.

ObjectiveThe objective of this study was to evaluate the antitumor effects of lurbinectedin as a single agent or in combination with existing anticancer agents for clear cell carcinoma (CCC) of the ovary, which is regarded as an aggressive, chemoresistant, histological subtype.MethodsUsing human ovarian CCC cell lines, the antitumor effects of lurbinectedin, SN-38, doxorubicin, cisplatin, and paclitaxel as single agents were assessed using the MTS assay. Then, the antitumor effects of combination therapies involving lurbinectedin and 1 of the other 4 agents were evaluated using isobologram analysis to examine whether these combinations displayed synergistic effects. The antitumor activity of each treatment was also examined using cisplatin-resistant and paclitaxel-resistant CCC sublines. Finally, we determined the effects of mTORC1 inhibition on the antitumor activity of lurbinectedin-based chemotherapy.ResultsLurbinectedin exhibited significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. An examination of mouse CCC cell xenografts revealed that lurbinectedin significantly inhibits tumor growth. Among the tested combinations, lurbinectedin plus SN-38 resulted in a significant synergistic effect. This combination also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Everolimus significantly enhanced the antitumor activity of lurbinectedin-based chemotherapies.ConclusionsLurbinectedin, a new agent that targets active transcription, exhibits antitumor activity in CCC when used as a single agent and has synergistic antitumor effects when combined with irinotecan. Our results indicate that lurbinectedin is a promising agent for treating ovarian CCC, both as a first-line treatment and as a salvage treatment for recurrent lesions that develop after platinum-based or paclitaxel treatment.

Targeting FXYD2 by cardiac glycosides potently blocks tumor growth in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is an aggressive neoplasm with a high recurrence rate that frequently develops resistance to platinum-based chemotherapy. There are few prognostic biomarkers or targeted therapies exist for patients with OCCC. Here, we identified that FXYD2, the modulating subunit of Na+/K+-ATPases, was highly and specifically expressed in clinical OCCC tissues. The expression levels of FXYD2 were significantly higher in advanced-stage of OCCC and positively correlated with patients' prognoses. Silencing of FXYD2 expression in OCCC cells inhibited Na+/K+-ATPase enzyme activity and suppressed tumor growth via induction of autophagy-mediated cell death. We found that high FXYD2 expression in OCCC was transcriptionally regulated by the transcriptional factor HNF1B. Furthermore, up-regulation of FXYD2 expression significantly increased the sensitivity of OCCC cells to cardiac glycosides, the Na+/K+-ATPase inhibitors. Two cardiac glycosides, digoxin and digitoxin, had a great therapeutic efficacy in OCCC cells in vitro and in vivo. Taken together, our results demonstrate that FXYD2 is functionally upregulated in OCCC and may serve as a promising prognostic biomarker and therapeutic target of cardiac glycosides in OCCC.

Demethylation of HIN-1 reverses paclitaxel-resistance of ovarian clear cell carcinoma through the AKT-mTOR signaling pathway.

BackgroundMethylation of HIN-1 is associated with poor outcomes in patients with ovarian clear cell carcinoma (OCCC), which is regarded to be an aggressive, chemo-resistant histological subtype. This study aimed to evaluate whether 5-aza-2-deoxycytidine (5-aza-2-dC) can reverse methylation of the HIN-1 gene to restore chemo-sensitivity of OCCC and the possible mechanism.MethodsIn vitro flow cytometric analysis and evaluation of caspase-3/7 activity of paclitaxel-sensitive and resistant OCCC cell lines were performed. Methylation status and expression changes of HIN-1 in the OCCC cell lines treated with 5-aza-2-dC were evaluated, and immunohistochemical staining of HIN-1 in OCCC tissues was performed. In vivo tumor growth with or without 5-aza-2-dC treatment was analyzed, and Western blotting of AKT-mTOR signaling-related molecules was performed.ResultsG2-M phase arrest was absent in paclitaxel-resistant OCCC cells after treatment with the cytotoxic drug. The caspase activities of the chemo-resistant OCCC cells were lower than those of the chemo-sensitive OCCC cells when treated with paclitaxel. Methylation of HIN-1 was noted in paclitaxel-resistant OCCC cell lines and cancerous tissues. 5-aza-2-dC reversed the methylation of HIN-1, re-activated the expression of HIN-1, and then suppressed the in vivo tumor growth of paclitaxel-resistant OCCC cells. Immunoblotting revealed that phospho-AKT473 and phospho-mTOR were significantly increased in HIN-1-methylated paclitaxel-resistant OCCC cell lines. However, the expressions of phospho-AKT at Ser473 and Thr308 and phospho-mTOR decreased in the OCCC cells with a high expression of HIN-1.ConclusionsDemethylating agents can restore the HIN-1 expression in paclitaxel-resistant OCCC cells through the HIN-1-AKT-mTOR signaling pathway to inhibit tumor growth.

Abstract 5405: Targeting STAT3 as a novel therapy for ovarian clear cell carcinoma

Abstract Objective: Advanced ovarian clear cell carcinoma (OCCC) carries a very poor prognosis in large part secondary to the high rate of resistance to standard platinum and taxane chemotherapy. STAT3 expression and activation has been shown to regulate tumor progression in various human cancers though has not been well studied in OCCC. Preliminary work in our lab has demonstrated constitutive activation of STAT3 (pSTAT3Tyr705 or pSTAT3727) in OCCC cell lines as well as human OCCC tumor tissue samples. Significantly, pSTAT3 is expressed in the absence of other forms of activated STAT (pSTAT1,2,6). Based on our preliminary data, we hypothesized that use of a novel STAT3 inhibitor, HO-3867, would be an effective agent against OCCC cell lines in vitro and in vivo using a novel orthotopic mouse model. Methods: Five OCCC cell lines (JHOC, OVISE, OVTOKO, RMGV, and ES2) were treated with HO-3867, cisplatin, or paclitaxel alone and/or in combination. Protein expression in tumor tissue and cell lines was determined by western blot (WB) and RT PCR was used to analyze RNA expression levels. MTT assay, BrdU assay, ANNEXIN V kit and flow cytometry were used to analyze cell viability, proliferation, apoptosis and cell cycle arrest. We use a novel orthotopic murine model using an ovarian bursal injection which was developed in our laboratory and has demonstrated primary ovarian tumor development and peritoneal carcinomatosis. Results: Treatment with HO-3867 decreased expression of pSTAT3 while total STAT3 remained constant. Treatment with cisplatin or paclitaxel resulted in a relative increase in pSTAT3. The inhibitory effect of HO-3867 on cell proliferation is associated with G2/M phase cell cycle arrest and apoptosis (>45%) within 24 hours of treatment. Treatment with HO-3867 resulted in a decrease in BCL2 and cleavage of caspase 3, caspase 7, and PARP confirming induction of apoptosis after treatment with HO-3867. Treatment with cisplatin or paclitaxel was less effective at decreasing cell viability, reducing proliferation, and inducing apoptosis. In vivo experiments using the orthotopic murine model are ongoing. Conclusion: HO-3867, a novel STAT3 inhibitor, appears to be efficacious against OCCC in vitro as compared to standard chemotherapeutics. Further investigation into this novel therapy is warranted given the generally poor response of OCCC to our standard chemotherapy regimens. Experiments to investigate the efficacy of HO-3867 in OCCC in vivo are underway. Citation Format: Kristin L. Bixel, Uksha Saini, Jack Fowler, Sneja Rajendran, Ross Wanner, Noriomi Matsumura, Kalman Hideg, Ikuo Konishi, David Cohn, Selvendiran Karrupaiyah. Targeting STAT3 as a novel therapy for ovarian clear cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5405. doi:10.1158/1538-7445.AM2015-5405

Transcriptional upregulation of HNF-1β by NF-κB in ovarian clear cell carcinoma modulates susceptibility to apoptosis through alteration in bcl-2 expression

Hepatocyte nuclear factor-1β (HNF-1β) is a transcriptional factor that has an important role in endometriosis–ovarian clear cell carcinoma (OCCC) sequence by modulating cell kinetics and glucose metabolism. However, little is known about the detailed molecular mechanisms that govern its regulation and function. Herein, we focus on upstream and downstream regulatory factors of HNF-1β in OCCCs. In clinical samples, HNF-1β expression was positively correlated with the active form of NF-κB/p65 in OCCCs, and closely linked with a low nuclear grade and non-solid architecture. In cell lines, transfection of p65 resulted in increased HNF-1β mRNA and protein expression in TOV-21G cells (OCCC cell line with endogenous HNF-1β expression), in line with activation of the promoter, probably through interacting with the basic transcriptional machinery. Suppression of endogenous HNF-1β expression by siRNA increased apoptosis in TOV-21G cells, while treatment of Hec251 cells (endometrial carcinoma cell line with extremely low endogenous HNF-1β expression) stably overexpressing exogenous HNF-1β with doxorubicin abrogated apoptosis of the cells, along with increased ratio of bcl-2 relative to bax. Moreover, overexpression of HNF-1β led to upregulation of bcl-2 expression at the transcriptional level in TOV-21G cells, which provided evidence for a positive correlation between HNF-1β and bcl-2 expression in OCCCs. These data, therefore, suggest that association between HNF-1β and NF-κB signaling may participate in cell survival by alteration of apoptotic events, particularly in mitochondria-mediated pathways, through upregulation of bcl-2 expression in OCCCs.

Molecular changes in endometriosis-associated ovarian clear cell carcinoma

BackgroundEndometriosis is frequently associated with and thought of having propensity to develop into ovarian clear cell carcinoma (OCCC), although the molecular transformation mechanism is not completely understood. MethodsWe employed immunohistochemical (IHC) staining for marker expression along the potential progression continuum. Expression profiling of microdissected endometriotic and OCCC cells from patient-matched formalin-fixed, paraffin-embedded samples was performed to explore the carcinogenic pathways. Function of novel biomarkers was confirmed by knockdown experiments. ResultsPTEN was significantly lost in both endometriosis and invasive tumour tissues, while oestrogen receptor (ER) expression was lost in OCCC relative to endometriosis. XRCC5, PTCH2, eEF1A2 and PPP1R14B were significantly overexpressed in OCCC and associated endometriosis, but not in benign endometriosis (p⩽0.004). Knockdown experiments with XRCC5 and PTCH2 in a clear cell cancer cell line resulted in significant growth inhibition. There was also significant silencing of a panel of target genes with histone H3 lysine 27 trimethylation, a signature of polycomb chromatin-remodelling complex in OCCC. IHC confirmed the loss of expression of one such polycomb target gene, the serous ovarian cancer lineage marker Wilms’ tumour protein 1 (WT1) in OCCC, while endometriotic tissues showed significant co-expression of WT1 and ER. ConclusionsLoss of PTEN expression is proposed as an early and permissive event in endometriosis development, while the loss of ER and polycomb-mediated transcriptional reprogramming for pluripotency may play an important role in the ultimate transformation process. Our study provides new evidence to redefine the pathogenic programme for lineage-specific transformation of endometriosis to OCCC.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance.

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

Preclinical Efficacy for AKT Targeting in Clear Cell Carcinoma of the Ovary

The aim of this study was to determine the role of AKT as a therapeutic target in ovarian clear cell carcinoma (CCC), an aggressive, chemoresistant histologic subtype of ovarian cancer. AKT activation was assessed by immunohistochemistry (IHC) using human tissue microarrays of primary ovarian cancers, composed of both CCC and serous adenocarcinoma (SAC). The growth-inhibitory effect of AKT-specific targeting by the small-molecule inhibitor, perifosine, was examined using ovarian CCC cell lines in vitro and in vivo. Finally, the activity of perifosine was examined using in CCC-derived tumors that had acquired resistance to anti-VEGF or chemotherapeutics such as bevacizumab or cisplatin, respectively. Interestingly, AKT was frequently activated both in early-stage and advanced-stage CCCs. Treatment of CCC cells with perifosine attenuated the activity of AKT-mTORC1 signaling, inhibited proliferation, and induced apoptosis. The effect of perifosine was more profound under conditions of high AKT activity compared with low AKT activity. Increased AKT activation and enhanced sensitivity to perifosine were observed in the context of cisplatin-resistant CCC. Treatment with perifosine concurrently with cisplatin significantly enhanced the antitumor effect of cisplatin. Moreover, perifosine showed significant antitumor activity in CCC-derived tumors that had acquired resistance to bevacizumab or cisplatin. Collectively, these data reveal that AKT is frequently activated in ovarian CCCs and is a promising therapeutic target in aggressive forms of ovarian cancer. AKT-targeted therapy has value in a first-line setting as well as a second-line treatment for recurrent disease developing after platinum-based chemotherapy or bevacizumab treatment.

Crosstalk between PI3K and Ras pathways via protein phosphatase 2A in human ovarian clear cell carcinoma

Hypoxia-inducible factor-1 (HIF-1) is one of the most promising pharmacological targets for all types of cancer, including ovarian cancer. Ovarian clear cell carcinoma (OCCC) has poor prognosis because of its insensitivity to chemotherapy. To elucidate the characteristics of this troublesome cancer, we examined HIF-1α expression under normoxia or hypoxia in various ovarian cancer cell lines. HIF-1α was highly expressed under normoxia only in RMG-1, an OCCC cell line. To examine whether HIF-1 is involved in the tumorigenesis of RMG-1 cells, we established HIF-1α-silenced cells, RMG-1HKD. The proliferation rate of RMG-1HKD cells was faster than that of RMG-1 cells. Furthermore, the activity of MEK/ERK in the Ras pathway increased in RMG-1HKD cells, whereas that of mTOR in the PI3K pathway did not change. Activation of the Ras pathway was attributable to the increase in phosphorylated MEK via PP2A inactivation. To confirm the crosstalk between the PI3K and Ras pathways in vivo, RMG-1 or RMG-1HKD cells were transplanted into the skin of nude mice with rapamycin (an inhibitor of mTOR), PD98059 (an inhibitor of MEK), or both. RMG-1HKD cells showed higher sensitivity to PD98059 than that observed in RMD-1 cells, whereas the combination therapy resulted in synergistic inhibition of both cells. These findings suggest that inhibition of HIF-1, a downstream target of mTOR in the PI3K pathway, activates the Ras pathway on account of the increase in MEK phosphorylation via PP2A inactivation, and the crosstalk between the 2 pathways could be applied in the combination therapy for HIF-1-overexpressing cancers such as OCCC.

151PD - Arid1A Role in Cell Cycle Regulation and Proliferation in Mouse and Human Gynaecological Tissues Reveals Potential Therapeutic Targets

ABSTRACT Aim: Ovarian clear cell carcinoma (OCCC) arises from endometriosis and can be considered an ectopic uterine cancer. ARID1A mutations occur as an early event in 50% of OCCC and 30% of high-grade endometrial cancers. We aimed to characterise the function of ARID1A in the normal uterus and ovary using an Arid1a mouse model and immortalised human ovarian surface epithelium cells. Methods: Arid1a expression during the mouse estrus cycle and its relationship to estrogen (ER), progesterone receptor and proliferation were assayed in a cohort of 28 mice. Illumina Beadchip arrays and MetaCore pathway analysis were used to assay ARID1A-driven transcriptional programmes in mouse uterus and, after siRNA-mediated knockdown, in mouse embryonic fibroblasts (MEF), immortalised human ovarian surface epithelium (IOSE) and JHOC-5 OCCC cells. SRB proliferation assays were performed in MEFs, IOSE and OCCC cell lines. Proliferation in the uterus was assessed 28 days after tamoxifen-induced knockout in Arid1afl/fl ROSA26Cre-ERT2 or wild type (WT) mice. Results: Arid1a expression was not hormonally regulated in the mouse uterus at either the mRNA or protein level, but Arid1a expression was correlated with stromal ER expression (r = 0.6, p = 0.0003). ARID1A-regulated genes in mouse uterus, MEFs and IOSE cells overlapped significantly (p = 1.7x10-13). Pathways involved in the G2/M checkpoint were significantly enriched in all 3 systems. The conserved core transcriptional program included AURKA, PLK1, PLK4, CCNB1 and CCND1. Conversely, genes involved in inflammation and cell adhesion pathways were ARID1A-regulated in JHOC-5 cells. ARID1A knockdown increased proliferation in MEFs, IOSE cells in an HNF1B-dependent manner and in 4 of 6 ARID1A-expressing OCCC cell lines. In vivo knockout in the uterine epithelium resulted in a dramatic increase in Ki67 staining in Arid1afl/fl compared to WT mice. Conclusions: A core ARID1A-driven transcriptional programme, conserved accross normal tissues and species appears to exist, centred around regulation of genes involved in the G2/M checkpoint. ARID1A loss promotes proliferation in normal tissues, providing important clues as to the mechanisms of ARID1A-driven carcinogenesis. Mitotic kinases emerge as potential therapeutic targets in ARID1A mutant tumours, an observation that is not evident from studies in cancer cell lines. Disclosure: All authors have declared no conflicts of interest.

Contribution of transcription factor, SP1, to the promotion of HB-EGF expression in defense mechanism against the treatment of irinotecan in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a worst histological subtype than other ovarian malignant tumor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. The aims of this study were to validate the efficacy of HB-EGF–targeted therapy for OCCC and to identify the transcription factor that contributed to the induction of HB-EGF by SN38 treatment in OCCC cells. HB-EGF was highly expressed in OCCC cells, and an increase of HB-EGF was induced by SN38 which had only antitumor effect among conventional anticancer agents on OCCC. A specific inhibitor of HB-EGF, a cross-reacting material 197 (CRM197), led to a synergistic increase in the number of apoptotic OCCC cells with the treatment of SN38. The luciferase assay with 5′-deletion promoter constructs identified a GC-rich element between −125 and −178 (the distal transcription start site was denoted +1) as a cis-regulatory region, and the treatment of SN38 induced luciferase activity in this region. An in silico and chromatin immunoprecipitation analysis estimated that SP1 bound to the cis-regulatory region of HB-EGF in OCCC cells. Real-time PCR and cell viability assays showed that the transfection of a small interfering RNA targeting SP1 suppressed the expression of HB-EGF induced by SN38, resulting in the enhanced sensitivity of SN38. Taken together, these results indicate that induction of HB-EGF expression contributed to defense mechanism against treatment of SN38 through the transcriptional activity of SP1 in OCCC cells.

Combination of therapy with 5-fluorouracil and cisplatin with electroporation in human ovarian carcinoma model in vitro

High electric field, applied to plasma membrane, affects organization of the lipid molecules, generating transient hydrophilic electropores. The application of the cell membrane electroporation in combination with cytotoxic drugs could increase the drug transport into cells. This approach is known as electrochemotherapy (ECT). Our work shows new data concerning the influence of electrochemical reaction with cisplatin or with 5-fluorouracil (5-FU) on cancer ovarian cells resistant to standard therapy with cisplatin, in comparison to ECT effect on human primary fibroblasts. We investigated the effect of electroporation and electrochemotherapy with 5-FU and cisplatin on human ovarian clear-cell carcinoma cell line (OvBH-1) and epithelial ovarian carcinoma cell line (SKOV-3) – both resistant to cisplatin typically used in ovarian cancers. As control cells, human gingival fibroblasts (HGF's) from primary culture were used. Electropermeabilization efficiency was determined by FACS analysis with iodide propidium. Efficiency of electrochemotherapy was evaluated with viability assay. The cytotoxic effect was dependent on the electroporation parameters and on drug concentration. Electroporation alone only insignificantly decreased cells proliferation in OvBH-1 line; SKOV-3 line was more sensitive to the electrical field. Electrochemotherapy with cisplatin and 5-FU showed promising effects on both ovarian cell lines with recovery of normal cells revealed after 72hours.

Bioenergetic analysis of ovarian cancer cell lines: profiling of histological subtypes and identification of a mitochondria-defective cell line.

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs.

Are platinum agents, paclitaxel and irinotecan effective for clear cell carcinoma of the ovary? DNA damage detected with γH2AX induced by anticancer agents

ObjectivesDifferences in the incidences and types of DNA damage induced by antitumor agents for clear cell carcinoma (CCC) were determined in 2 ovarian CCC cell lines using γH2AX.Material and methodsThe antitumor activity of anticancer agents, CDDP, CBDCA, PTX and SN-38, was examined using ovarian clear cell carcinoma cultured cell lines (OVISE and RMG-I). After culture, each cell line was treated with each anticancer agent, the cells were collected, fixed, and then reacted with the anti-γH2AX antibody. γH2AX and nuclear DNA were then simultaneously detected by flow cytometry using FITC and propidium iodide, respectively, to determine γH2AX in each cell cycle phase.ResultsAfter administration of CDDP, DNA damage was frequent in S-phase cells, while cell-cycle arrest occurred in the G1 and G2/M phases and γH2AX did not increase in CDDP-resistant cells. Sensitivities to CDDP and CBDCA differed between the two cell lines. The antitumor effect of PTX is induced by G2/M arrest, and combination treatment with CBDCA, inducing DNA damage in G2/M-phase cells, might be effective.ConclusionsThis is the first study in Japan to evaluate the antitumor activity of anticancer agents by focusing on the relationship between the cell cycle and DNA damage using γH2AX as an indicator. The immunocytochemical method used in this study detects γH2AX, which indicates DNA damage even at very low concentrations and with high sensitivity. Therefore, a promising method of easily and rapidly identifying agents potentially effective against CCC.

Fatty acid synthase expression associated with NAC1 is a potential therapeutic target in ovarian clear cell carcinomas

Background:This study examined the clinical significance of NAC1 and the expression level of its potential downstream target fatty acid synthase (FASN) in ovarian clear cell carcinomas (OCCCs), and evaluated the NAC1/FASN pathway as a potential therapeutic target.Methods:NAC1 and FASN expression and NACC1 gene amplification were assessed in ovarian cancers by immunohistochemistry, fluorescence in situ hybridisation, and clinical data collected by a retrospective chart review. C75, a FASN inhibitor, was used to assess whether this pathway represented a therapeutic target in OCCC.Results:High NAC1 expression was most frequent in clear cell tumours (40.0%:24/60). NACC1 gene amplification was identified in none of the 58 OCCCs. The frequency of NACC1 gene amplification was significantly higher in the high-grade serous histology than in the clear cell histology (P<0.01). NAC1 expression was significantly correlated with FASN expression in both OCCC samples and OCCC cell lines. Either high NAC1 expression or high FASN expression significantly correlated with shorter progression-free and overall survival (P=0.002 and 0.0048). NAC1 overexpression stimulated FASN expression, and NAC1 silencing using siRNA decreased FASN expression in OCCC cell lines. Profound growth inhibition was observed in C75-treated carcinoma cells with FASN overexpression when compared with the response in carcinoma cells without FASN expression.Conclusion:These findings indicate that NAC1/FASN overexpression is critical to the growth and survival of a subset of OCCC. The FASN silencing by the C75-induced phenotypes depends on the expression status of the targeted cell line. Therefore, NAC1/FASN pathway-targeted therapy may benefit selected OCCC patients.

Abstract 4225: Annexin A4 induces chemoresistance for multiple drugs in ovarian clear cell carcinoma

Abstract BACKGROUND: Platinum drugs play the key role for postoperative adjuvant therapy and treatment in progress and recurrent ovarian cancer. We have previously reported that ovarian clear cell carcinoma (CCC) has increased expression of Annexin A4 (Anx A4) in comparison with other subtypes of ovarian cancer. Enhanced expression of Anx A4 has been shown to induce chemoresistance via extracellular efflux of carboplatin. However, it remains unclear whether targeting Anx A4 is effective for improving the sensitization against chemotherapy, like platinum drugs. The aim of this study was to evaluate the role of Anx A4 in chemoresistance in ovarian CCC. MATERIAL AND METHODS: RMG-I cell lines were used to establish the stable knockdown of Anx A4. Plasmids encoding short hairpin RNAs (shRNAs) targeting Anx A4 were transfected. Chemoresistance was assessed by IC50 value in various drugs. RESULTS: Compared with control cells, knockdown of Anx A4 gene expression in RMG-I cells induced chemosentisitzation around 2-fold to cisplatin (IC50: 11.2 μM to 5.8 μM, P&amp;lt;0.01) and Carboplatin (IC50: 130.4 μM to 63.3 μM, P&amp;lt;0.01). Unexpectedly, knockdown of Anx A4 also improved the chemosentisitzation of epirubicin (IC50: 256.7 nM to 180.3 nM, P&amp;lt;0.01) and Paclitaxel (IC50: 5.5 nM to 2.9 nM, P&amp;lt;0.01). In consistent with the improved chemosensitivity, drugs induced p53 was markedly increased by knockdown of Anx A4 in RMG-I cells. In xenograft models, significant chemosensitization toward cisplatin was demonstrated by knockdown of Anx A4 in RMG-I cells. CONCLUSIONS: Inhibition of Anx A4 induced chemosensitization in ovarian CCC cells. Anx A4 blockade might be a novel therapeutic target of chemoresistance in patients with ovarian CCC. 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 4225. doi:1538-7445.AM2012-4225

Prognostic and therapeutic impact of the chromosome 20q13.2 ZNF217 locus amplification in ovarian clear cell carcinoma

The goal of this study was to examine the clinical significance of ZNF217 amplification and assess whether ZNF217 could be a potential therapeutic target in ovarian clear cell carcinoma (OCCC). ZNF217 expression and amplification in OCCC was assessed by immunohistochemistry, fluorescence in situ hybridization, and clinical data collected via a retrospective chart review. ZNF217 gene knockdown using silencing RNA (siRNA) was used to assess ZNF217 functions in OCCC cell lines. Gene amplification was identified in 12 of 60 (20.0%) OCCCs. ZNF217 copy number correlated significantly with ZNF217 protein expression (r = 0.341; P<.01). ZNF217 amplification correlated significantly with shorter progression-free (P = .0042) and overall (P = .0199) survival. There were nonsignificant trends between high ZNF217 protein expression and poor progression-free (P = .2594) and overall (P = .2199) survival. Multivariate analysis revealed ZNF217 gene amplification to be an independent prognostic factor for progression-free and overall survival after standard platinum agent-based chemotherapy (P = .0339 and P = .031, respectively). Profound growth inhibition and apoptosis were observed in ZNF217 siRNA-treated cancer cells with gene amplification compared with cancer cells with ZNF217 moderate expression without ZNF217 gene amplification or with low ZNF217 expression. These findings indicate that ZNF217 overexpression is critical to growth and survival of OCCCs with ZNF217 gene amplification. Furthermore, they suggest that ZNF217 siRNA-induced phenotypes depend on amplification status of OCCCs. Therefore, ZNF217-targeted therapy may benefit OCCC patients with ZNF217 amplification.

Glypican‐3 could be an effective target for immunotherapy combined with chemotherapy against ovarian clear cell carcinoma

Glypican-3 (GPC3) is useful not only as a novel tumor marker, but also as an oncofetal antigen for immunotherapy. We recently established HLA-A2-restricted GPC3(144-152) peptide-specific CTL clones from hepatocellular carcinoma patients after GPC3(144-152) peptide vaccination. The present study was designed to evaluate the tumor reactivity of a HLA-A2-restricted GPC3(144-152) peptide-specific CTL clone against ovarian clear cell carcinoma (CCC) cell lines. The GPC3(144-152) peptide-specific CTL clone could recognize HLA-A2-positive and GPC3-positive ovarian CCC cell lines on interferon (IFN)-γ enzyme-linked immunospot assay and showed cytotoxicity against KOC-7c cells. The CTL clone recognized naturally processed GPC3-derived peptide on ovarian CCC cells in a HLA class I-restricted manner. Moreover, we confirmed that the level of GPC3 expression was responsible for CTL recognition and that subtoxic-dose chemotherapy made tumor cells more susceptible to the cytotoxic effect of CTL. Thus, it might be possible to treat ovarian CCC patients by combining chemotherapy with immunotherapy. Our data suggest that GPC3 could be an effective target for immunotherapy against ovarian CCC.