Platinum-resistant Ovarian Cancer Cell Lines Research Articles

Abstract 7209: Overcoming platinum resistance of high-grade serous ovarian cancer targeting the activated JAK/STAT pathways via extracellular vesicles

Abstract High-grade serous ovarian cancer (HGSOC) is the most common subtype of ovarian cancer. While HGSOC usually responds to initial treatment, most cases eventually relapse and develop platinum-resistant ovarian cancer (PROC). The molecular definition of PROC is still unclear, and overcoming PROC has been the biggest challenges. Extracellular vesicles (EVs) have an essential role in cell-to-cell communication within both the primary and distant microenvironments. In this study. we aimed to identify molecular mechanisms of PROC via EVs to suggest novel therapeutic strategies. Platinum responses were categorized by primary treatment-free interval of platinum less than 6 months as PROC, and more than 36 months as platinum good response. Ascites and tumor tissues have been collected prior to initial chemotherapy in patients with HGSOC for analysis according to platinum response. mRNA sequencing in tumor tissues identified significantly upregulated JAK/STAT pathways in PROC. The JAK/STAT family was highly expressed in PROC than the good response group, and also increased than adjusted normal ovaries and fallopian tubes. In addition, PROC cell lines and PROC patient-derived xenograft model tumor tissues showed high JAK expression. Small RNA sequencing for HGSOC tissues and ascites-EVs revealed that miR-135a-5p and miR-221-5p were highly expressed in both PROC tissues and ascites, and the expression of the two miRNAs had a positive correlation between tissues and ascites. Overexpression of the two miRNAs in HGSOC cell lines resulted in higher JAK family expression and resistance to cisplatin. JAK inhibitors (JAKi) had equivalent efficacy for platinum-sensitive/resistant HGSOC cell lines and JAKi highly suppress tumor growth in the PROC mouse model. In addition, suppression of JAK family expression in PROC cell lines significantly increased sensitivity to cisplatin. JAKi also had a synergistic effect with cisplatin for PROC cell lines. IPA analyses in mRNA sequencing in tumor tissues, TGF-β was highly expressed in upstream regulators in PROC, and TGF-β stimulation resulted in the upregulation of the two miRNAs in mesothelial cells, but not in HGSOC cell lines and fibroblasts. In summary, we here demonstrate novel molecular mechanisms of PROC via EVs and provided the rationale for JAK-targeted therapy for PROC. EV-miRNAs can regulate the PROC activated JAK/STAT pathway and can be diagnostic and therapeutic biomarkers for PROC definition or JAKi indications, suggesting that patient stratification in precision medicine for initial treatment of HGSOC. Citation Format: Kazuhiro Suzuki, Akira Yokoi, Kousuke Yoshida, Yusuke Yamamoto, Hiroaki Kajiyama. Overcoming platinum resistance of high-grade serous ovarian cancer targeting the activated JAK/STAT pathways via extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7209.

Abstract B095: Tenascin C promotes ovarian cancer stem cells interactions with the niche

Abstract Background: Effective treatment of ovarian cancer (OC) is greatly impacted by advanced, late-stage diagnosis which usually presents with intra-peritoneal metastases, chemoresistance and high recurrence. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids disseminated in the peritoneal fluid. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components that stiffen the tumor stroma and promote the aberrant activation of oncogenic processes, including survival, invasion, and stemness. Tenascin C (TNC) is one of the molecules secreted in the ECM and correlates with migration and metastatic progression in a variety of carcinomas. However, the functional role of TNC in spheroid formation and in the maintenance of the platinum resistant ovarian cancer stem phenotype has not been investigated yet. We tested the hypothesis that the increased expression of TNC and its secretion in the ECM compartment promotes survival of OCSCs, supports OC spheroid formation and tumor progression. Methods: We compared TNC expression in OC cells grown as spheroids and monolayers and in chemoresistant vs sensitive OC cells by realtime-PCR and Western Blot (WB). TNC blockade by siRNA knockdown (KD) was evaluated in combination with carboplatin on spheroid assay, colony formation, and by qPCR of stemness related gene expression. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the TNC promoter. Results: OC cells grown as spheroids showed a significant increase in TNC expression compared to monolayers. Our results also demonstrated an increase in TNC expression levels in platinum resistant OC cell lines when compared to the platinum sensitive counterpart. A comparison of normal adjacent ovarian epithelium and primary OC tumors on a multitissue array revealed that TNC expression coincided with a pro-tumorigenic phenotype (n=88, P<0.001), correlating with poor outcome. TNC-KD in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. Mechanistically, b-catenin-KD decreased TNC expression levels and β-catenin coimmunoprecipitated with the TNC promoter, suggesting that TNC is a direct β-catenin target. Additionally, OC spheroids treated with the Wnt ligand WNT-3A showed increased TNC expression compared to control untreated cells, as demonstrated by WB. Importantly, the enhanced expression of TNC was followed by its secretion in the ECM compartment, as observed via immunofluorescence imaging, implying a direct role of TNC in the ECM-rearrangement which is responsible for CSC maintenance and spheroid formation. Conclusions: TNC is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that strategies aimed at interfering with TNC expression and/or secretion along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Amber Rogers, Maya Shebeshi, Morganna Gordon, Rula Atwani, Virginie Lazar, Salvatore Condello. Tenascin C promotes ovarian cancer stem cells interactions with the niche [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B095.

Platinum-Resistant Ovarian Cancer Is Vulnerable to the cJUN-XRCC4 Pathway Inhibition.

DNA double-strand breaks (DSBs) caused by platinum drugs are dangerous lesions that kill cancer cells in chemotherapy. Repair of DSB by homologous recombination (HR) and nonhomologous end joining (NHEJ) is frequently associated with platinum resistance in ovarian cancer. While the role of the HR pathway and HR-targeting strategy in platinum resistance is well studied, dissecting and targeting NHEJ machinery to overcome platinum resistance in ovarian cancer remain largely unexplored. Here, through an NHEJ pathway-focused gene RNAi screen, we found that the knockdown of XRCC4 significantly sensitized cisplatin treatment in the platinum-resistant ovarian cancer cell lines. Moreover, upregulation of XRCC4 is observed in a panel of platinum-resistant cell lines relative to the parental cell lines, as well as in ovarian cancer patients with poor progression-free survival. Mechanistically, the increased sensitivity to cisplatin upon XRCC4 knockdown was caused by accumulated DNA damage. In cisplatin-resistant ovarian cancer, the JNK-cJUN complex, activated by cisplatin, translocated into the nucleus and promoted the transcription of XRCC4 to confer cisplatin resistance. Knockdown of XRCC4 or treatment of the JNK inhibitor led to the attenuation of cisplatin-resistant tumor growth in the xenograft mouse models. These data suggest targeting XRCC4 is a potential strategy for ovarian cisplatin resistance in ovarian cancer.

The effect of the triazene compound CT913 on ovarian cancer cells in vitro and its synergistic interaction with the PARP-inhibitor olaparib

ObjectivesExtending the therapeutic spectrum of PARP-inhibitors (PARPi) beyond BRCA1-deficiency and/or overcoming PARPi-resistance is of high clinical interest. This is particularly true for the identification of innovative therapeutic strategies for ovarian cancer, given the recent advances in the use of PARPi in clinical practice. In this regard, the combination of PARPi with chemotherapy is a possible strategy for defining new therapeutic standards. In this study, we analyzed the therapeutic effect of novel triazene derivatives, including the drug CT913 and its metabolite CT913-M1 on ovarian cancer cells and describe their interaction with the PARPi olaparib. MethodsIn vitro assays for drug characterization including RNA-Seq were applied in a selected panel of ovarian cancer cell lines. ResultsCT913 treatment conferred a dose-dependent reduction of cell viability in a set of platinum-sensitive and platinum-resistant ovarian cancer cell lines with an IC50 in the higher micromolar range (553–1083 μM), whereas its metabolite CT913-M1 was up to 69-fold more potent, especially among long-term treatment (IC50 range: 8–138 μM). Neither of the drugs sensitized for cisplatin. CT913 conferred synthetic lethality in BRCA1-deficient ovarian cancer cells, indicating that its effect is augmented by a deficiency in homologous recombination repair (HR). Furthermore, CT913 showed a synergistic interaction with olaparib, independently of BRCA1 mutational status. CT913 strongly induced CDKN1A transcription, suggesting cell cycle arrest as an early response to this drug. It moreover downregulated a variety of transcripts involved in DNA-repair pathways. ConclusionsThis is the first study, suggesting the novel triazene drug CT913 as enhancer drug for extending the therapeutic spectrum of PARPi.

RETRACTED ARTICLE: The role of apatinib combined with paclitaxel (aluminum binding type) in platinum-resistant ovarian cancer

ObjectiveTo assess the anti-tumor activity and side effects of different dosages of paclitaxel (albumin binding type) (hereinafter referred to as nab-P) combined with Apatinib (hereinafter referred to as AP) in platinum-resistant ovarian cancer cell line and xenograft models.MethodsSKOV-3/DDP cell line was selected as the research object in cytology experiment. Firstly, we divided it into three groups for experiments to explore the individual effects of nab-P and AP. a): Control group, blank control, no drug intervention; b): nab-P group, nab-P 40 μmol/l; c): AP group, AP 50 μmol/l (Drug doses were IC-50 values that detected by MTT assay). Apoptosis related protein (Bax, bcl-2), vascular related protein(p-VEGFR-2), invasion related protein (MMP-2) expression were detected by Western blot and Cellular immunofluorescence, the invasion ability of tumor cells were detected by Transwell and Cell scratch test. Based on these dates, secondly, establishing different doses of nab-P combined with Ap to explore the curative effect of combination therapy. a): Control group, blank control, no drug intervention; b): Group-1, nab-P 5 μmol/l + AP 10 μmol/l, c): Group-2, nab-P 4.5 μmol/l + AP 10 μmol/l, d): Group-3, nab-P 4 μmol/l + AP 10 μmol/l, e): nab-P group, nab-P 5 μmol/l, f): AP group, AP 10 μmol/l (MTT assay). The combination index was analyzed by Compusyn software, Western blot, Immunofluescence, Transwell and Cell scratch test also were also chose to observe of inhibition effect. Thirdly, we used xenograft models to verify the results of cytological experiments. Tumor-forming BALB/c female nude mice were randomly divided into 4 groups, a): Control group, no drug intervention, only saline injection, b): nab-P 20 mg/kg + AP 150 mg/kg, c): nab-P 18 mg/kg + AP 150 mg/kg, d): nab-P 16 mg/kg + AP 150 mg/kg (The doses were guided by the pharmaceutical manufacturers). The tumor growth curve was analyzed during the experiment. And the apoptosis related protein (Bax, bcl-2), angiogenesis related protein (CD31, p-VEGFR-2) and invasion related protein (MMP-2) were observed by Western blot, Immunofluescence and Immunohistochemistry to analysis the ant-tumor effects. The quality of life in nude mice were observed to analysed the drug-induced side effects.ResultIn the separate medication section, (1) The IC-50 value of nab-P was 45.53 ± 4.06 μmol/l, while the AP was 50.66 ± 4.96 umol/L (48 h). (2) The expressions of bcl-2 (nab-P group, AP group), p-VEGFR-2 (AP group), MMP-2(nab-P group, AP group) were higher than Control group, while Bax (nab-P group, AP group) lower (P < 0.01). (3) The cell invasive ability was decreased after the nab-P and AP intervation (P < 0.01). In the combination medication section, (1) Compusyn showed the Combination index (Cl) were all below 1 (Cl < 1), that means nab-P and AP are synergism. (2) The combination IC-50 value was nab-P 5.28 μmol/l + AP 10.56 μmol/l (48 h). (3) In the detection of related protein expression, the combination of drugs can improve the anti-tumor effect, otherwise, after combined with AP, when nab-P were reduced dose in proper quantity, there were no obvious different in drug effect. (4) After reducing the doses of nab-P, the average food intake of nude mice increased from 4.50 g ± 0.17 to 5.55 g ± 0.13, and the one-hour activity increased from 6.11 min ±0.16 to 6.34 min ±0.13.Conclusionnab-P, a chemotherapeutic agent, can play an anti-tumor role in platinum-resistant ovarian cancer, but it can cause adverse effects that increase with dose. When combined with AP, the two drugs have synergistic effect, which can improve the anti-tumor effects of single drug. In addition, when combined with AP, the doses of nab-P can be appropriately reduced under the standard of recommended to reduce the toxicity of chemotherapy drugs, without affecting the anti-tumor effect.

Abstract A65: Combination of a thioxodihydroquinazolinone compound with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential

Abstract Cancer stem cell-like cells (CSC-LCs) contribute to drug resistance and recurrence of ovarian cancer. Strategies that can eradicate CSC-LCs are expected to substantially improve the outcome of ovarian cancer treatments. We have previously identified a class of thioxodihydroquinazolinone small molecules, which have strong synergistic antitumor activity with platinum drugs, the standard chemotherapeutic agents for ovarian cancer treatment. In the current study, using the activity of aldehyde dehydrogenase (ALDH) as a marker of CSC-LCs, we demonstrated that the combination of thioxodihydroquinazolinone compound 19 with cisplatin is able to deplete ALDH-high CSC-LC population in both established platinum-resistant ovarian cancer cell lines and primary ovarian cancer cells from metastatic ascites of a cisplatin-resistant patient. Compound 19 enhanced the accumulation of intracellular cisplatin in ALDH-high ovarian cancer cells. The combination of compound 19 with cisplatin was also able to reduce the sphere-forming capability of cisplatin-resistant ovarian cancer cells. Using a spheroid-based in vitro metastasis model of ovarian cancer, we demonstrated that the combination of compound 19 with cisplatin prevents spheroid ovarian cancer cells from attaching to substratum and spreading. In vivo, the treatment by the combination of compound 19 with cisplatin significantly reduced tumor burden in a cisplatin-resistant intraperitoneal ovarian cancer xenograft mouse model, as compared to cisplatin-alone treatment. Taken together, our study demonstrated that the thioxodihydroquinazolinone compounds are a new class of agents that in combination with cisplatin are capable of eliminating CSC-LCs in ovarian cancer. Further development of thioxodihydroquinazolinone small molecules may yield a more effective treatment for cisplatin-resistant metastatic ovarian cancer. Citation Format: Jing Ma, Joseph Salamoun, Peter Wipf, Robert Edwards, Bennett Van Houten, Wei Qian. Combination of a thioxodihydroquinazolinone compound with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A65.

Anti-Proliferation Effect of Theasaponin E₁ on the ALDH-Positive Ovarian Cancer Stem-Like Cells.

Ovarian cancer has the highest mortality rate of all gynecological malignancies and the five-year death rate of patients has remained high in the past five decades. Recently, with the rise of cancer stem cells (CSCs) theory, an increasing amount of research has suggested that CSCs give rise to tumor recurrence and metastasis. Theasaponin E1 (TSE1), which was isolated from green tea (Camellia sinensis) seeds, has been proposed to be an effective compound for tumor treatment. However, studies on whether TSE1 takes effect through CSCs have rarely been reported. In this paper, ALDH-positive (ALDH+) ovarian cancer stem-like cells from two platinum-resistant ovarian cancer cell lines A2780/CP70 and OVCAR-3 were used to study the anti-proliferation effect of TSE1 on CSCs. The ALDH+ cells showed significantly stronger sphere forming vitality and stronger cell migration capability. In addition, the stemness marker proteins CD44, Oct-4, Nanog, as well as Bcl-2 and MMP-9 expression levels of ALDH+ cells were upregulated compared with the original tumor cells, indicating that they have certain stem cell characteristics. At the same time, the results showed that TSE1 could inhibit cell proliferation and suspension sphere formation in ALDH+ cells. Our data suggests that TSE1 as a natural compound has the potential to reduce human ovarian cancer mortality. However, more research is still needed to find out the molecular mechanism of TSE1-mediated inhibition of ALDH+ cells and possible drug applications on the disease.

Combination of a thioxodihydroquinazolinone with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential.

Cancer stem cell-like cells (CSC-LCs) contribute to drug resistance and recurrence of ovarian cancer. Strategies that can eradicate CSC-LCs are expected to substantially improve the outcome of ovarian cancer treatment. We have previously identified a class of thioxodihydroquinazolinone small molecules, which have strong synergistic antitumor activity with platinum drugs, the standard chemotherapeutic agents for ovarian cancer treatment. In the current study, using the activity of aldehyde dehydrogenase (ALDH) as a marker of CSC-LCs, we demonstrated that the combination of thioxodihydroquinazolinone compound 19 with cisplatin is able to diminish ALDH-high CSC-LC populations in both platinum-resistant ovarian cancer cell lines and primary ovarian cancer cells from metastatic ascites of a cisplatin-resistant patient. Compound 19 enhanced the accumulation of intracellular cisplatin in ALDH-high ovarian CSC-LCs. The combination of compound 19 with cisplatin was also able to reduce the sphere-forming capability of cisplatin-resistant ovarian cancer cells. Using a spheroid-based in vitro metastasis model of ovarian cancer, we demonstrated that the co-administration of compound 19 with cisplatin prevents ovarian cancer spheroid cells from attaching to substratum and spreading. In a cisplatin-resistant in vivo intraperitoneal xenograft mouse model, the combination of compound 19 with cisplatin significantly reduced tumor burden, as compared to cisplatin alone. Taken together, our study demonstrated that thioxodihydroquinazolinones represent a new class of agents that in combination with cisplatin are capable of eliminating CSC-LCs in ovarian cancer. Further development of thioxodihydroquinazolinone small molecules may yield a more effective treatment for cisplatin-resistant metastatic ovarian cancer.

OvAd1, a Novel, Potent, and Selective Chimeric Oncolytic Virus Developed for Ovarian Cancer by 3D-Directed Evolution

Effective therapeutics for ovarian cancer continue to be urgently needed, particularly for chemotherapy-resistant cases. Here we present both a 3D-Matrigel culture-based expansion of our directed evolution method for generation of oncolytic virotherapies and two promising ovarian-cancer targeted oncolytic viruses, OvAd1 and OvAd2. OvAd1 was developed using Matrigel cell cultures, whereas OvAd2 was developed in parallel using traditional monolayer tissue culture methods. Both viruses are potent against a panel of platinum-resistant ovarian cancer cell lines and are attenuated on normal cells in vitro, resulting in therapeutic windows of ∼200-fold. We observed two benefits of the use of Matrigel-based cultures for directed evolution of these oncolytics: (1) use of Matrigel generated a bioselected pool that was more strongly attenuated on normal cells while retaining its potency against ovarian cancer cells, and (2) in an ovarian carcinomatosis model, the Matrigel-derived virus OvAd1 suppressed all tumor growth while the non-Matrigel-derived virus was 50% effective. Neither virus stimulated formation of peritoneal adhesions as seen for Ad5-based therapies. Consequently, these viruses are novel candidates for development as new effective treatments for aggressive ovarian cancer.

Generation of Cisplatin-Resistant Ovarian Cancer Cell Lines

Ovarian cancer is the most lethal gynecological cancer in which cisplatin-based treatment plays a fundamental role as the first line chemotherapy option. However, development of platinum-resistance is a critical and poorly understood problem in ovarian cancer treatment. Although in vitro generation of platinum-resistant ovarian cancer cell lines is a long established approach to uncover the molecular mechanisms underlying resistance development, the methodology of this resistance induction is poorly explained in publications. The aim of this study was to propose a method for induction of resistance in ovarian cancer cell lines. To reach this aim, A2780 human ovarian cancer cell line was continuously exposed to stepwise increasing concentrations of cisplatin (0.5–2.6µM) over a period of 6 months and three resistant sublines were collected. Cisplatin resistance was examined by clonogenic survival assay and growth curve analysis was carried out in order to evaluate the proliferation characteristics of the established sublines. The A2780 resistant sublines exhibited 5.1 to 11.7 fold resistance to cisplatin, compared to their parental cells, and although growth rate and plateau saturation density significantly decreased by cisplatin resistance enhancement, all three resistant sublines presented a typical growth curve even though they were cultured in the cisplatin containing medium. These results suggest that reliable drug resistant human ovarian cancer cell lines can be successfully established by this method.

Eribulin mesylate targets human telomerase reverse transcriptase in ovarian cancer cells.

Treatment of advanced ovarian cancer involves platinum-based chemotherapy. However, chemoresistance is a major obstacle. Cancer stem cells (CSCs) are thought to be one of the causes of chemoresistance, but the underlying mechanism remains elusive. Recently, human telomerase reverse transcriptase (hTERT) has been reported to promote CSC-like traits. In this study, we found that a mitotic inhibitor, eribulin mesylate (eribulin), effectively inhibited growth of platinum-resistant ovarian cancer cell lines. Eribulin-sensitive cells showed a higher efficiency for sphere formation, suggesting that these cells possess an enhanced CSC-like phenotype. Moreover, these cells expressed a higher level of hTERT, and suppression of hTERT expression by siRNA resulted in decreased sensitivity to eribulin, suggesting that hTERT may be a target for eribulin. Indeed, we found that eribulin directly inhibited RNA-dependent RNA polymerase (RdRP) activity, but not telomerase activity of hTERT in vitro. We propose that eribulin targets the RdRP activity of hTERT and may be an effective therapeutic option for CSCs. Furthermore, hTERT may be a useful biomarker to predict clinical responses to eribulin.

Abstract IA17: Targeting the methylome for epigenetic resensitization of ovarian cancer

Abstract Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. Although most patients respond to platinum-based chemotherapy, relapses are common, leading to platinum-resistant OC, which is uniformly fatal. OC progression is associated with accumulation of epigenetic alterations. In particular, deoxycytosine methylation of CpG islands in promoter regions of tumor suppressor genes (TSGs) plays a prominent role in the development and progression of drug-resistant epithelial OC. We recently demonstrated for the first time in a clinical trial that therapeutic interventions targeting the OC methylome reverse drug resistance and induce meaningful clinical responses. We showed that repetitive low-dose decitabine reactivated silenced genes and restored sensitivity to carboplatin, providing strong clinical and biological support for further study of hypomethylating agents in heavily pre-treated, platinum-resistant ovarian cancer patients. While the FDA-approved demethylating agent decitabine is prone to deamination by cytidine deaminase, SGI-110 (Astex Pharmaceuticals, Inc.), a dinucleotide analogue of decitabine, is more stable, less toxic, and a promising alternative to restoring silenced TSG expression in cancer cells by reversal of DNA methylation. Our preclinical evaluation demonstrated that SGI-110 resensitized platinum-resistant OC cell lines to cisplatin (CDDP) (3-fold reduction in IC50) and reduced the CDDP IC50. SGI-110 treatment induced significant demethylation and subsequent transcriptional derepression of tumor suppressors and differentiation-associated genes in OC cells. SGI-110 alone or in combination with CDDP was well tolerated in non-tumor bearing mice. Significant antitumor activity was observed in mice harboring subcutaneous OC tumors and treated with single SGI-110 and SGI-110 + CDDP treatment in both a biweekly and daily (QD5) regimen. In addition to reducing tumor growth in xenografts, SGI therapy was effective in causing global as well as TSG demethylation and gene reexpression in vivo. Furthermore, the antitumor activity of SGI-110 was associated with reduced chromatin compaction, allowing greater CDDP intercalation into DNA, as assessed by increased DNA platinum adducts in SGI-treated OC cells. The results of our preclinical study support our recently activated clinical trial NCT01696032 using SGI-110 in combination with carboplatin in patients with recurrent, platinum-resistant OC. Clinical specimens (tumor and plasma samples) will be analyzed for epigenetic biomarker changes. We seek to bring forward the new concept of epigenetic targeting in platinum resistant OC by priming the tumors with SGI-110 and set the stage for interventions targeting the OC epigenome, as well as guide and impact the design of future clinical investigations in OC. Citation Format: Kenneth P. Nephew, Daniela Matei, Pietro Taverna, Fang Fang, Jessica Tang, Gavin Choy, John Lyons, Mohammad Azab, Jay Pilrose, John Turchi. Targeting the methylome for epigenetic resensitization of 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 IA17.

521 Cell Cycle Regulation Changes in Drug-Resistant Cancers

Cell cycle perturbation is a common event in tumorigenesis and the development of anticancer drug resistance. Acquired drug resistance is a significant hindrance in achieving effective cancer treatment. Thus, understanding of mechanisms underlying acquired drug resistance is vital to develop strategies to overcome those processes and to improve the survival rates of cancer patients. Moreover, understanding drug resistance can lead to the discovery of potential druggable targets that can contribute towards increasing success of cancer intervention. In this study, molecular mechanisms leading to acquired drug resistance in various cancer cell lines were investigated. Informed by cDNA microarray screens, the cell cycle regulators p57Kip2, Weel and Stratifin (SFN) were shown to be frequently deregulated in drug resistance. Using Western blotting and qPCR, p57Kip2 was shown to be downregulated in platinum-resistant ovarian cancer cell lines PEO1CarbR and PEOlCisR in comparison with the platinum-sensitive counterpart. p57Kip2 downregulation was due to epigenetic silencing (hypermethylation of gene promoter region). Thus, p57Kip2 silencing with siRNA in otherwise drug sensitive cells recapitulated platinum resistance. Interestingly, p57Kip2 downregulation conferred collateral sensitivity towards the synthetic CDK inhibitor drug Seliciclib (CYC202). A similar approach was adopted in the study of Weel and SFN in taxane resistance. Weel and SFN were downregulated in Taxol-resistant SK-OV-3 and A2780 cell lines compared to drug-sensitive counterparts. Silencing of Weel in Taxol-sensitive SK-OV-3 decreased the apoptotic response to Taxol treatment, but this effect was not seen using SFN silencing. I conclude that Weel plays an important role in response to Taxol. However, p53 status is also associated with this response. Additionally, examination of several drug resistant and sensitive pairs of cancer cell lines indicated that DNA damage checkpoint and cell cycle regulation are significant factors in mediating anticancer drug resistance. Based on the results, the ATR-Chkl-Weel pathway was shown to be the most deregulated pathway in drug-resistant cancer cells. I conclude that deregulation of cell cycle is essential to the adaptive responses and enabling characteristics that result in the drug resistant phenotype.

Costunolide induces apoptosis in platinum-resistant human ovarian cancer cells by generating reactive oxygen species

Objective The acquired resistance to platinum-based drugs has become an obstacle in the management of ovarian cancer. We investigated the apoptosis-inducing effect of costunolide, a natural sesquiterpene lactone, in platinum-resistant human ovarian cancer cells, along with the molecular mechanism of action. Methods Costunolide and cisplatin were examined in platinum-resistant human ovarian cancer cells. MTT assay for cell viability, PI staining for cell cycle profiling, and Annexin V assay for apoptosis analysis. ROS production and protein expression was assessed by H 2DCFDA staining and Western blotting, respectively. Combination effect was determined using the Combination Index (CI) method. Results It was found that costunolide is more potent than cisplatin in inhibiting cell growth in three platinum-resistant ovarian cancer cell lines (MPSC1 PT, A2780 PT, and SKOV3 PT). Costunolide induced apoptosis of platinum-resistant cells in a time- and dose-dependent manner and suppressed tumor growth in SKOV3 PT-bearing mouse model. In addition, costunolide triggered the activation of caspase-3, -8, and -9. Pretreatment with caspase inhibitors neutralized the pro-apoptotic activity of costunolide. We further demonstrated that costunolide induced a significant increase in intracellular reactive oxygen species (ROS). Additionally, the antioxidant N-acetyl-L-cysteine (NAC) significantly attenuated the costunolide-induced production of ROS, activation of caspases, down-regulation of Bcl-2, and apoptosis in platinum-resistant ovarian cancer cells. Moreover, costunolide synergized with cisplatin to induce cell death in platinum-resistant ovarian cancer cells. Conclusions Taken together, these data suggest that costunolide, alone or in combination with cisplatin, may be of therapeutic potential in platinum-resistant ovarian cancer.

Abstract 1777: Platinum sensitize ovarian carcinoma cells to ABT-737 in vitro and ex vivo through inhibition of Mcl-1

Abstract In ovarian carcinoma, chemoresistance is the main responsible for the poor 5-year overall survival, remaining under 30%. Protection against apoptotic cell death is particularly involved in this chemoresistance, and among the observed alterations, overexpression of anti-apoptotic Bcl-2 proteins is of first importance. Among them, Bcl-xL et Mcl-1 appeared to cooperate to protect ovarian cancer cells against apoptosis, thus constituting together pertinent targets. In this context, the use of BH3-mimetic molecule ABT-737, that targets Bcl-xL, could constitute an alternative strategy to reverse the platinum chemoresistance in ovarian carcinoma. However, ABT-737 remaining unable to efficiently inhibit Mcl-1 activity, its clinical use in ovarian carcinoma thus requires defining of another tool able to inhibit Mcl-1. In this study, we investigated in vitro and ex vivo the capacity of platinum derivatives (cisplatin and carboplatin) to inhibit Mcl-1 expression or activity and therefore sensitize ovarian carcinoma cells to ABT-737. SKOV3 and IGROV1-R10 platinum-resistant ovarian cancer cell lines were exposed to these drugs, as single agents or associated, using various exposure protocols in vitro or ex vivo (SKOV3 and IGROV1-R10 xenografted tumor nodes developed in nude mice and subsequently used for slicing and ex vivo treatment). We thus studied apoptosis induction as well as Mcl-1 and pro-apoptotic BH3-only proteins expression. In vitro study: whereas neither cisplatin nor ABT-737 alone presented any toxicity, the association of ABT-737 and platinum was highly cytotoxic in both cell lines. We tested several protocols and showed that ABT-737 must be present during the 24h following carboplatin exposure to allow cell death. These observations are in agreement with the needed down-regulation or inactivation of Mcl-1 by platinum prior or concomitant with ABT-737 exposure to induce cell death. As expected, cisplatin as well as carboplatin were able either to inhibit Mcl-1 expression or to induce BH3-only expression (particularly Noxa and Puma). Moreover, we observed a synergetic effect of the association ABT-737/platinum on BH3-only Bim and Noxa expression, these events being correlated to the ability of platinum to sensitize to ABT-737. Ex vivo study: we confirmed these observations in SKOV3 and IGROV1-R10 ex vivo tumor slices models. Whereas ABT-737 and platinum derivatives remained poorly cytotoxic or completely ineffective as single agents, their association was highly cytotoxic. In conclusion, this strategy associating ABT-737 to platinum appears as an attractive way to reverse resistance to platinum derivatives that remain the most active drugs in ovarian cancer. Moreover, this study presents platinum derivatives as pertinent sensitizers to ABT-737, through its direct or indirect Mcl-1 inhibition, opening new perspectives for the clinical use of this promising BH3-mimetic molecule. 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 1777. doi:10.1158/1538-7445.AM2011-1777

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 3072: Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer

Abstract Refractory to TGF-β is frequently observed in ovarian cancer, and disrupted TGF-β/SMAD4 signaling results in aberrant expression of downstream target genes in the disease. We hypothesized that aberrant expression of TGF-β/SMAD4 targets are mediated through epigenetic mechanism and also contribute to resistance to TGF-β meditated growth inhibition. Our previous report using chromatin immunoprecipitation microarray (ChIP-chip) identified FBXO32 as one of SMAD4 targets in immortalized ovarian surface epithelial cell (IOSE) (Qin et al., BMC Syst Biol, 17: 73, 2009). In the present study, we investigated the mechanism conferring FBXO32 down-regulation, its clinical significance, and its function in ovarian cancer. Our result showed that expression of FBXO32 was observed in normal ovarian surface epithelium but not in ovarian cancer cell lines (HeyC2, SKOV3, CP70, A2708, MCP2, MCP3) using real time RT-PCR. Promoter methylation of FBXO32 was seen in ovarian cancer cell lines, HeyC2 and SKOV3, that display constitutive TGF-β/SMAD4 signaling. Moreover, our finding that epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggested that epigenetic regulation of FBXO32 in ovarian cancer may be a common event. Re-expression of FBXO32 markedly impeded proliferation of a platinum-resistant ovarian cancer cell lines, HeyC2 and CP70 (colony number: HeyC2: 19.33 ± 3.06 vs 1 ± 0, P&amp;lt; 0.005; CP70: 46.5 ± 6.36 vs 1.5 ± 0.7, P&amp;lt; 0.001) by using colony formation assay, due to increased apoptosis of the cells (CP70; apoptotic cell%, control: 2.55 ± 0.17; FBXO32: 10.72 ± 1.07, P&amp;lt;0.05), and resensitized the cells to cisplatin (P &amp;lt; 0.05). Surprisingly, elevated apoptosis was not observed in HeyC2 cells which have much higher drug-resistance than CP70 cells (IC50, HeyC2: 5.6ug/ml, CP70: 2.2ug/ml). However, an increase in the G1 cell population was observed in FBXO32-transfected HeyC2 cells. In in vivo study, the FBXO32-transfecte CP70 showed smaller tumor size compared to vector control after day14 (P &amp;lt; 0.05) of tumor injection. In advanced stage ovarian cancer patients, significant methylation frequency (29.3%; P&amp;lt;0.05) of FBXO32 was observed by real-time qMSP and such an outcome further correlated with the protein expressed level of FBXO32 by IHC stain. Importantly, FBXO32 methylation was significantly associated with shorter progression-free survival, as determined by both Kaplan-Meier analysis (P&amp;lt;0.05) and multivariate Cox regression analysis (hazard ratio 1.003, P&amp;lt;0.05). In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-β/SMAD4 signaling, and FBXO32 methylation status predicts survival in ovarian cancer patients. Future regimen of treating ovarian cancer by ectopic expression of FBXO32 can be considered. 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 3072.

Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer

Resistance to TGF-β is frequently observed in ovarian cancer, and disrupted TGF-β/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-β/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-β/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; P<0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan–Meier analysis (P<0.05) and multivariate Cox regression analysis (hazard ratio: 1.003, P<0.05). Reexpression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer cell line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-β/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer.

Apoptotic and chemotherapeutic properties of iron (III)-salophene in an ovarian cancer animal model.

The cytotoxicity of organometallic compounds iron(III)-, cobalt(III)-, manganese(II)-, and copper(II)-salophene (-SP) on platinum-resistant ovarian cancer cell lines was compared. Fe-SP displayed selective cytotoxicity (IC(50) at ~1 muM) against SKOV-3 and OVCAR-3 cell lines while Co-SP caused cytotoxic effects only at higher concentrations (IC(50) at 60 muM) and Cu-SP effects were negligible. High cytotoxicity of Mn-SP (30-60 muM) appeared to be nonspecific because the Mn-chloride salt reduced cell viability similarly. The effect of Fe-SP at 1 muM proved to be ovarian cancer cell selective when compared to a panel of cell lines derived from different tumors. The first irreversible step in the induction of cell death by Fe-SP occurred after 3 hrs as indicated by the mitochondrial transmembrane potential (DeltaPsim) and was mainly linked to apoptotic, not necrotic events. To evaluate the toxicity of Fe-SP in vivo we conducted an acute toxicity study in rats. The LD(50) of Fe-SP is >2000 mg/kg orally and >5.5 mg/kg body weight by intraperitoneal injection. An ovarian cancer animal model showed that the chemotherapeutic relevant dose of Fe-SP in rats is 0.5-1 mg/kg body weight. The present report suggests that Fe-SP is a potential therapeutic drug to treat ovarian cancer.

Secreted protein acidic and rich in cysteine as a regulator of murine ovarian cancer growth and chemosensitivity

Secreted protein acidic and rich in cysteine (SPARC) influences the growth of several solid tumors. Our objectives were to determine the effect of SPARC on the growth and response to cisplatin therapy of platinum-resistant ovarian cancer. SPARC expression was determined in 4 platinum-resistant ovarian cancer cell lines. The effect of increasing SPARC on cell proliferation was determined in vitro. The effect of host-derived SPARC on tumor growth and response to therapy was determined in vivo using the murine ovarian cancer cell line, OSEID8, which was injected into the peritoneum of wild-type (WT) and SPARC-null (SP-/-) mice. Forced expression of SPARC decreased growth of platinum-resistant ovarian cancer cell lines in vitro. In vivo, tumor growth was more aggressive in the absence of host-derived SPARC resulting in decreased survival compared with WT mice (P = .005). Cisplatin did not improve survival of WT mice. In contrast, cisplatin therapy resulted in a significant survival advantage (P = .0048) and decreased tumor volume (P = .02) in SP-/- animals. We conclude that SPARC is an important extracellular matrix protein that regulates the growth and chemosensitivity of ovarian cancer. In general, SPARC appears to control tumor cell growth but also impede the efficacy of cisplatin therapy. Therefore, selective inhibition of SPARC may provide an attractive strategy for increasing the efficacy of therapy in platinum-resistant ovarian tumors.