Primary Epithelial Ovarian Cancer Cells Research Articles

Synergistic antitumor effect of liposomal-based formulations of olaparib and topotecan in primary epithelial ovarian cancer cells

BackgroundOlaparib is a PARP inhibitor inducing synthetic lethality in tumors with deficient homologous recombination (HRD) caused by BRCA1/2 mutations. The FDA has approved monotherapy for first-line platinum-sensitive, recurrent high-grade epithelial ovarian cancer. Combination therapy alongside DNA-damaging therapeutics is a promising solution to overcome the limited efficacy in patients with HRD. The present study was designed to develop topotecan- and olaparib-loaded liposomes (TLL and OLL) and assess the effectiveness of their combination in patient-derived ovarian cancer samples.MethodsWe used HEOC, four clear-cell tumors (EOC 1–4), malignant ascites, and an OCI-E1p endometrioid primary ovarian cancer cell line and performed NGS analysis of BRCA1/2 mutation status. Antiproliferative activity was determined with the MTT assay. The Chou-Talalay algorithm was used to investigate the in vitro pharmacodynamic interactions of TLLs and OLLs.ResultsThe OLL showed significantly higher efficacy in all ovarian cancer types with wild-type BRCA1/2 than a conventional formulation, suggesting potential for increased in vivo efficacy. The TLL revealed substantially higher toxicity to EOC 1, EOC 3, ascites and lower toxicity to HEOC than the standard formulation, suggesting better therapeutic efficacy and safety profile. The combination of studied compounds showed a higher reduction in cell viability than drugs used individually, demonstrating a synergistic antitumor effect at most of the selected concentrations.ConclusionsThe concentration-dependent response of different ovarian cancer cell types to combination therapy confirms the need for in vitro optimization to maximize drug cytotoxicity. The OLL and TLL combination is a promising formulation for further animal studies, especially for eliminating epithelial ovarian cancer with wild-type BRCA1/2.

Serum starvation-based method of ovarian cancer cell dormancy induction and termination in vitro.

Awakening and growth reinitiation by dormant cells may contribute to epithelial ovarian cancer (EOC) relapse. The links between these phenomena are loose because of the limited stock of compelling models of EOC dormancy. Here, we show a simple and convenient dormancy research protocol based on serum starvation. This study was conducted on established EOC cell lines A2780, OVCAR-3, and SKOV-3, as well as on primary EOC cells. Cell growth arrest and proliferation were monitored by assessing the Ki67 antigen, PKH26 fluorescence, and cell cycle distribution. In addition, cells were tested for ERK1/2/p38 MAPK activity ratio, apoptosis, and senescence. The study showed that 72-h serum starvation induces G0/G1 growth arrest of a significant fraction of cells, accompanied by reduced Ki67 and ERK1/2/p38 MAPK activity ratio, without signs of apoptosis or cellular senescence. Moreover, providing cells with 72 h of a medium enriched in 5% serum allows the culture to regain its proliferative potential. At the same time, we attempted to induce and terminate dormancy with Mitomycin C addition and withdrawal, which were unsuccessful. In conclusion, serum starvation is a convenient way to reliably induce dormancy in EOC cells, allowing them to be efficiently awakened for further mechanistic research in vitro.

TWIST1 induces proteasomal degradation of β-catenin during the differentiation of ovarian cancer stem-like cells

Ovarian cancer (OC) is one of the leading gynecologic cancers worldwide. Cancer stem-like cells are correlated with relapse and resistance to chemotherapy. Twist1, which is involved in ovarian cancer stem-like cell differentiation, is positively correlated with CTNNB1 in different differentiation stages of ovarian cancer cells: primary epithelial ovarian cancer cells (primary EOC cells), mesenchymal spheroid-forming cells (MSFCs) and secondary epithelial ovarian cancer cells (sEOC cells). However, the expression of β-catenin is inversed compared to CTNNB1 in these 3 cell states. We further demonstrated that β-catenin is regulated by the protein degradation system in MSFCs and secondary EOC but not in primary EOC cells. The differentiation process from primary EOC cells to MSFCs and sEOC cells might be due to the downregulation of β-catenin protein levels. Finally, we found that TWIST1 can enhance β-catenin degradation by upregulating Axin2.

Pro-cancerogenic effects of spontaneous and drug-induced senescence of ovarian cancer cells in vitro and in vivo: a comparative analysis

BackgroundClinical outcomes of cancer cell senescence are still elusive. Here, we reveal and compare pro-cancerous activity of spontaneously and drug-inducible senescent ovarian cancer cells. Experiments were performed on tumors and tumor-derived primary epithelial ovarian cancer cells (pEOCs) that were obtained from chemotherapy-naïve patients and from patients who received carboplatin (CPT) and paclitaxel (PCT) before cytoreduction.ResultsThe analysis of tumors showed that senescent cancer cells are present in patients from both groups, albeit most frequently and covering a greater area in tissues from chemotherapy-positive women. This in vivo senescence of pEOCs translated to an expression of senescence markers in early-passage cells in vitro. A conditioned medium from senescent pEOCs fueled the cancer progression, including adhesion of non-senescent pEOCs to normal peritoneal cells, and their increased proliferation, migration, invasion, and EMT. Senescent pEOCs’ secretome promoted angiogenic activity of vascular endothelium, induced senescence of normal peritoneal cells, reprogrammed their secretome towards hypersecretion of cancer-promoting proteins, and stimulated motility of cancer cells subjected to a mesothelium- and fibroblast-derived medium. The most striking finding was, however, that spontaneously senescent pEOCs supported all the above pro-cancerous effects more efficiently than drug-inducible senescent cells, which was plausibly related to augmented release of several cancer spread mediators by these cells. The prevalence of spontaneously senescent pEOCs was most evident in experiments on mice when they were able, unlike the drug-inducible cells, to promote the development of drug-sensitive i.p. xenografts.ConclusionsOur study shows that spontaneous senescence of pEOCs should be treated as an independent pathogenetic factor of cancer progression.

Patient-Specific Variables Determine the Extent of Cellular Senescence Biomarkers in Ovarian Tumors In Vivo.

The mechanisms and clinical significance of the cellular senescence of tumor cells are a matter of ongoing debate. Recently, the triggers and molecular events underlying spontaneous, replicative senescence of primary epithelial ovarian cancer cells were characterized. In this study, we reanalyzed tumors obtained from ovarian cancer patients with respect to the expression of the senescence biomarkers SA-β-Gal and γ-H2A.X and the proliferative antigen Ki67. The results showed that the tumors displayed strong heterogeneity with respect to the expression of analyzed markers. The expression of SA-β-Gal and γ-H2A.X in the oldest patients (61–85 y.o.) was significantly higher than in the younger age groups. Conversely, the area of Ki67-positive cancer cells was greater in younger individuals. At the same time, there was a positive correlation between SA-β-Gal expression and calendar age in FIGO III–IV and malignant ascites-positive patients. The γ-H2A.X positively correlated with age in the whole group, FIGO III–IV, and ascites-positive patients. Ki67 levels correlated negatively with the age of patients among those same groups. Collectively, our study indicated that organismal aging may determine the development of the senescence phenotype in ovarian tumors, particularly in patients with advanced disease and those accumulating malignant ascites.

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166+ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166+ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.

Abstract A56: Novel L-sugar linked glycosylated antitumor ether lipids for killing platinum-resistant human epithelial ovarian cancer cells

Abstract Glycosylated antitumor ether lipids (GAELs) are a promising class of investigational anticancer agents with potent antitumor activity against a range of cancers, including platinum-resistant high-grade serous ovarian cancer (HGSOC). We previously demonstrated that epithelial ovarian cancer (EOC) cell lines (HGSOC and endometrioid) and primary cells derived from patient ascites (HGSOC and clear cell) were sensitive to the cell-killing effects of D-glucosamine-derived GAELs via an apoptosis-independent mechanism. However, D-linked carbohydrates are metabolized by endogenous glucosidases in vivo, rendering the GAEL inactive. Thus, the aim of the current study was to synthesize a novel class of GAELs with L-linked carbohydrates and to evaluate their efficacy in EOC cell lines and patient samples. Out of seven novel compounds tested, L-rhamnose-GAEL was identified as the compound with the greatest efficacy for killing EOC cell lines and patient cells that were grown under adherent or nonadherent (3D) conditions. The drug-sensitive and drug-resistant syngeneic endometrioid EOC cell lines, A2780s and A2780cp, respectively, as well as the NIH:OVCAR-3 and COV362 HGSOC lines, were incubated with L-rhamnose-GAEL for 48 hours and the CC50 determined as 15 μM for A2780s, 22.5 μM for A2780cp, and 5 μM for NIH:OVCAR-3 and COV362 cells. Similar experiments were conducted with primary EOC cells, chemo-naïve EOC126 (clear-cell adenocarcinoma), and platinum-resistant EOC 183I (HGSOC). Cell viability decreased in a dose-dependent manner with CC50 of 12 μM for EOC126 and 22.5 μM for EOC183l. For comparison, when EOC126 and EOC183I were grown as 3D cultures the CC50for cisplatin was 20 μM and 40 μM, respectively. To evaluate the effect of using low-dose treatment of L-rhamnose-GAEL on primary EOC cells (chemo-naive and chemoresistant), cells were exposed to L-rhamnose-GAEL for 96 hours. These studies revealed that prolonged incubation led to low CC50 of 1-5 μM. These results showed that primary EOC cells derived from chemo-naïve and platinum-resistant patients were sensitive to the L-rhamnose-GAEL and offer a novel drug class capable of killing chemotherapy-resistant EOC cells. Citation Format: Mark W. Nachtigal, Frank Schweizer, Gilbert Arthur. Novel L-sugar linked glycosylated antitumor ether lipids for killing platinum-resistant human epithelial ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A56.

Abstract A02: CBX7 binds TWIST-1’s E-box to inhibit TWIST-1 function and curtail tumorigenecity and metastatic potential in ovarian cancer

Abstract Introduction: Ovarian cancer metastasizes via the transcoelomic route and yields overwhelmingly numerous micrometastases. This clinical presentation limits the value of standard of care and contributes to patient mortality. Whereas EMT is required in the acquisition of a migratory phenotype, MET has been shown to occur on secondary sites leading to the establishment of metastasis. The nature of the ovarian cancer cells that result after MET is, however, yet to be fully characterized. Whether these secondary epithelial ovarian cancer cells (sEOC) are required to repeat EMT in order to generate additional metastatic sites is not known. The objectives of this study are twofold: to determine if full MET occurs on secondary sites, and to determine mechanisms that confer to disseminated ovarian cancer cells the ability to rapidly spread and form the characteristic micrometastatic disease. Materials and Methods: Ovarian cancer spheroids were derived from three epithelial ovarian cancer (EOC) cell lines (OVCAR3, R182, R2615). sEOC were derived from the spheroids by plating in tissue culture-treated plates. Gene signature pattern was determined by RNAseq. mRNA and protein levels were quantified by RT-qPCR and Western blot, respectively. TWIST-1 activity was measured by a luciferase reporter system. Promoter binding was determined by anti-CBX7 chromatin IP followed by PCR. CBX7 was knocked out (KO) using CRISPR-Cas9. Tumorigenic potential was quantified by measuring resulting intraperitoneal burden (g) in nude mice. Results: PCA and clustering analysis of RNA-seq data showed sEOC do not fully recapitulate the molecular signature of the primary EOC cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintain a high expression of the classical mesenchymal effector, TWIST-1. TWIST-1 is, however, transcriptionally inactive in these cells as it is inhibited by the PcG protein, CBX7. KO of CBX7 in sEOC was sufficient to promote a mesenchymal morphology, significantly promote TWIST-1-induced transcription of mir-199A (p = 0.004, wt vs. CBX7KO), and induce higher protein levels of FOXC2. CBX7 precludes TWIST-1 from accessing its promoter by binding at or near the E box. Finally, KO of CBX7 was able to significantly enhance tumorigenicity and increase tumor load and metastatic potential in vivo (p=0.033, wt vs CBX7 KO). Conclusion: We demonstrate that full MET does not occur in ovarian cancer cells at secondary metastatic sites. Although sEOC display an epithelial morphology, they do not achieve the full molecular signature of the primary EOC cells from which they are derived. sEOC maintain high levels of TWIST-1 whose activity is inhibited by CBX7. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage to achieve a prompt reversal to a mesenchymal morphology and gain in tumorigenic potential upon perturbation of CBX7. This may explain ovarian cancer’s rapid progression and characteristic micrometastatic disease. Citation Format: Juanni Li, Ayesha B Alvero, Sudhakar Nuti, Roslyn Tedja, Cai M. Roberts, Mary Pitruzzello, Yimin Li, Qing Xiao, Sai Zhang, Yaqi Gan, Gil Mor, Gang Yin. CBX7 binds TWIST-1’s E-box to inhibit TWIST-1 function and curtail tumorigenecity and metastatic potential in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A02.

CBX7 binds the E-box to inhibit TWIST-1 function and inhibit tumorigenicity and metastatic potential.

Deaths from ovarian cancer usually occur when patients succumb to overwhelmingly numerous and widespread micrometastasis. Whereas epithelial-mesenchymal transition is required for epithelial ovarian cancer cells to acquire metastatic potential, the cellular phenotype at secondary sites and the mechanisms required for the establishment of metastatic tumors are not fully determined. Using in vitro and in vivo models we show that secondary epithelial ovarian cancer cells (sEOC) do not fully re-acquire the molecular signature of the primary epithelial ovarian cancer cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintains a high expression of the mesenchymal effector, TWIST-1. TWIST-1 is however transcriptionally non-functional in these cells as it is precluded from binding its E-box by the PcG protein, CBX7. Deletion of CBX7 in sEOC was sufficient to reactivate TWIST-1-induced transcription, prompt mesenchymal transformation, and enhanced tumorigenicity in vivo. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage of prompt reversal to a mesenchymal phenotype upon perturbation of CBX7. We also describe a sub-classification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

Preclinical Activity of Sacituzumab Govitecan, an Antibody-Drug Conjugate Targeting Trophoblast Cell-Surface Antigen 2 (Trop-2) Linked to the Active Metabolite of Irinotecan (SN-38), in Ovarian Cancer.

Background: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Sacituzumab govitecan (SG) is a novel antibody-drug-conjugate (ADC) targeting trophoblast-antigen-2 (Trop-2), a cell surface glycoprotein highly expressed in many epithelial tumors, to deliver SN-38, the active metabolite of irinotecan. This study aimed to evaluate Trop-2 expression in EOC tissues and the preclinical activity of SG against primary EOC cell lines and xenografts.Methods: Trop-2 expression was assessed in 90 formalin-fixed-paraffin-embedded tumors and nine primary tumor cell lines by immunohistochemistry (IHC) and flow cytometry, respectively. Trop-2 expression and cell viability after exposure to SG in primary tumor cell lines, non-targeting control ADC, and SG-parental antibody hRS7 were evaluated using flow-cytometry-based-assays. Antibody-dependent-cell-cytotoxicity (ADCC) against Trop-2+ and Trop-2– EOC cell lines was tested in vitro using 4 h Chromium-release-assays. In vivo activity of SG was evaluated against Trop-2+ EOC xenografts.Results: Moderate-to-strong staining was seen in 47% (42/90) of ovarian tumors by IHC while 89% (8/9) of the primary EOC cell lines overexpressed Trop-2 by flow cytometry. EOC Trop-2+ were significantly more sensitive to SG compared to control ADC (p < 0.05). Both SG and hRS7 mediated high ADCC activity against Trop-2+ cell lines. SG also induced significant bystander killing of Trop-2– tumor cells admixed with Trop-2+ EOC cells. In in vivo experiments SG treatment demonstrated impressive anti-tumor activity against chemotherapy-resistant EOC xenografts.Conclusion: SG demonstrates remarkable preclinical activity against biologically aggressive and chemotherapy-resistant EOC cell lines and a significant bystander effect against EOC cell lines with heterogenous Trop-2 expression. Clinical trials are warranted.

Deciphering the Molecular Mechanism of Spontaneous Senescence in Primary Epithelial Ovarian Cancer Cells.

Spontaneous senescence of cancer cells remains a puzzling and poorly understood phenomenon. Here we comprehensively characterize this process in primary epithelial ovarian cancer cells (pEOCs). Analysis of tumors from ovarian cancer patients showed an abundance of senescent cells in vivo. Further, serially passaged pEOCs become senescent after a few divisions. These senescent cultures display trace proliferation, high expression of senescence biomarkers (SA-β-Gal, γ-H2A.X), growth-arrest in the G1 phase, increased level of cyclins D1, D2, decreased cyclin B1, up-regulated p16, p21, and p53 proteins, eroded telomeres, reduced activity of telomerase, predominantly non-telomeric DNA damage, activated AKT, AP-1, and ERK1/2 signaling, diminished JNK, NF-κB, and STAT3 pathways, increased formation of reactive oxygen species, unchanged activity of antioxidants, increased oxidative damage to DNA and proteins, and dysfunctional mitochondria. Moreover, pEOC senescence is inducible by normal peritoneal mesothelium, fibroblasts, and malignant ascites via the paracrine activity of GRO-1, HGF, and TGF-β1. Collectively, pEOCs undergo spontaneous senescence in a mosaic, telomere-dependent and telomere-independent manner, plausibly in an oxidative stress-dependent mechanism. The process may also be activated by extracellular stimuli. The biological and clinical significance of pEOC senescence remains to be explored.

Targeting the Formyl Peptide Receptor type 1 to prevent the adhesion of ovarian cancer cells onto mesothelium and subsequent invasion

BackgroundThe biological behavior of epithelial ovarian cancer (EOC) is unique since EOC cells metastasize early to the peritoneum. Thereby, new anti-target agents designed to block trans-coelomic dissemination of EOC cells may be useful as anti-metastatic drugs. The Urokinase Plasminogen Activator Receptor (uPAR) is overexpressed in EOC tissues, and its truncated forms released in sera and/or ascitic fluid are associated with poor prognosis and unfavorable clinical outcome. We documented that uPAR triggers intra-abdominal dissemination of EOC cells through the interaction of its 84–95 sequence with the Formyl Peptide Receptor type 1 (FPR1), even as short linear peptide Ser-Arg-Ser-Arg-Tyr (SRSRY). While the pro-metastatic role of uPAR is well documented, little information regarding the expression and role of FPR1 in EOC is currently available.MethodsExpression levels of uPAR and FPR1 in EOC cells and tissues were assessed by immunofluorescence, Western blot, or immunohystochemistry. Cell adhesion to extra-cellular matrix proteins and mesothelium as well as mesothelium invasion kinetics by EOC cells were monitored using the xCELLigence technology or assessed by measuring cell-associated fluorescence. Cell internalization of FPR1 was identified on multiple z-series by confocal microscopy. Data from in vitro assays were analysed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons. Tissue microarray data were analyzed with the Pearson’s Chi-square (χ2) test.ResultsCo-expression of uPAR and FPR1 by SKOV-3 and primary EOC cells confers a marked adhesion to vitronectin. The extent of cell adhesion decreases to basal level by pre-exposure to anti-uPAR84–95 Abs, or to the RI-3 peptide, blocking the uPAR84–95/FPR1 interaction. Furthermore, EOC cells exposed to RI-3 or desensitized with an excess of SRSRY, fail to adhere also to mesothelial cell monolayers, losing the ability to cross them. Finally, primary and metastatic EOC tissues express a high level of FPR1.ConclusionsOur findings identify for the first time FPR1 as a potential biomarker of aggressive EOC and suggests that inhibitors of the uPAR84–95/FPR1 crosstalk may be useful for the treatment of metastatic EOC.

An Ex-Vivo Culture System of Ovarian Cancer Faithfully Recapitulating the Pathological Features of Primary Tumors.

The success rate of establishing human cancer cell lines is not satisfactory and the established cell lines often do not preserve the molecular and histological features of the original tissues. In this study, we developed a novel culture method which can support proliferation of almost all primary epithelial ovarian cancer cells, as well as primary normal human oviductal epithelial cells. Cancer cells from fresh or frozen specimens were enriched by the anti-EpCAM antibody-conjugated magnetic beads, plated on Matrigel-coated plate and cultivated under the optimized culture conditions. Seventeen newly established ovarian cancer cell lines, which included all four major histotypes of ovarian cancer, were confirmed to express histotype-specific markers in vitro. Some of the cell lines from all the four histotypes, except mucinous type, generated tumors in immune-deficient mice and the xenograft tumor tissues recapitulated the corresponding original tissues faithfully. Furthermore, with poorly tumorigenic cell lines including mucinous type, we developed a novel xenograft model which could reconstruct the original tissue architecture through forced expression of a set of oncogenes followed by its silencing. With combination of the novel culture method and cell-derived xenograft system, virtually every epithelial ovarian cancer can be reconstituted in mice in a timely fashion.

Artemisinin derivatives inhibit epithelial ovarian cancer cells via autophagy-mediated cell cycle arrest.

Epithelial ovarian cancer (EOC) is the most fatal gynecologic malignancy due to its late diagnosis and lack of curative therapy. The antimalaria compound artemisinin and its derivatives, such as artesunate (ART) and dihydroartemisinin (DHA), have proven to be potent anticancer drugs and act through various anticancer mechanisms. To identify novel targets of artemisinin derivatives in EOC cells, we investigated the effects of ART and DHA on SKOV3 and primary EOC cell growth via CCK-8 assay. Both ART and DHA inhibited EOC cell growth. A cell cycle distribution analysis showed that ART and DHA caused G2/M cell cycle arrest. Moreover, ART and DHA induced autophagy in EOC cells, whereas autophagy inhibitors reversed the cell growth inhibition and cell cycle arrest induced by ART and DHA. Western blot analysis showed that ART and DHA also suppressed the cell cycle-related NF-κB-signaling pathway in EOC cells. These data suggest that artemisinin derivatives induce autophagy, block the cell cycle, and inhibit cell growth in EOC cells. Our research provides new targets for artemisinin derivatives for EOC treatment.

Pharmacological Inhibition of p38 MAPK by SB203580 Increases Resistance to Carboplatin in A2780cp Cells and Promotes Growth in Primary Ovarian Cancer Cells.

Chemoresistance renders current chemotherapy regimens ineffective against advanced epithelial ovarian cancer (EOC). Carboplatin (the first-line chemotherapeutic agent to treat EOC) induces cell death by regulating multiple signaling pathways. The objective of this study is to identify the signaling pathways that contribute to carboplatin resistance in EOC. To this end, we performed a proteome profiler human phospho-kinase array experiment and compared the phosphorylation profiles between the cisplatin-sensitive A2780s versus its derivative cisplatin-resistant A2780cp cells. The phospho-kinase array revealed that A2780s and A2780cp cells displayed different profiles in basal and carboplatin-induced phosphorylation. Phosphorylation of p38 MAPK was increased by carboplatin more markedly in A2780s cells compared to A2780cp cells. Inhibition of p38 MAPK activity by its specific inhibitor SB203580 increased resistance to carboplatin in A2780cp cells, but not in A2780s cells or in ascites-derived high-grade serous EOC cells. Interestingly, SB203580 increased the number of viable cells in the primary EOC cells, which was concomitant with an increase in survivin expression. In conclusion, inhibition of p38 MAPK by SB203580 increases resistance to carboplatin in A2780cp cells and the number of viable cells in the primary EOC cells, suggesting that pharmacological inhibition of p38 MAPK might not be an effective therapeutic strategy for EOC.

SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows promising antitumor activity in epithelial ovarian carcinoma with HER2/neu expression.

e14009 Background: Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. HER2 overexpression/amplification in EOC show considerable variation ranging from 8% to 66%. SYD985 (Synthon Biopharmaceuticals BV, Nijmegen, the Netherlands) is a novel HER2-targeting antibody-drug conjugate (ADC) composed of the monoclonal antibody (mAb) trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin). The objective of this study was to compare the anti-tumor activity of SYD985 to trastuzumab emtansine (T-DM1) in EOC. Methods: The cytotoxicity of SYD985 and T-DM1 was evaluated using ten primary EOC cell lines with 0/1+, 2+, and 3+ HER2/neu expression in antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability and bystander killing experiments. Finally, the in vivo activity of SYD985 and T-DM1 was also studied in ovarian cancer xenografts. Results: SYD985 and T-DM1 induced similar ADCC in the presence of effector cells [i.e., peripheral blood lymphocytes (PBL)] against EOC cell lines with high, moderate and low HER2/neu expression. In contrast, SYD985 was 3 to 42 fold more cytotoxic in the absence of PBL when compared to T-DM1. Specifically, in HER2/neu 1+ cell lines the mean IC50’s were 0.072 µg/mL and 3.035 µg/mL for SYD985 vs T-DM1 (p &lt; 0.0001), in HER2/neu 2+ cell lines 0.054 µg/mL and 1.168 µg/mL, (p &lt; 0.0001) and in HER2/neu 3+ cell lines 0.024 µg/mL and 0.088 µg/mL, respectively, (p &lt; 0.0001). Unlike T-DM1, SYD985 induced efficient bystander killing of HER2/neu 0/1+ EOC cells admixed with HER2/neu 3+ cells. In vivo studies confirmed that SYD985 is more active than T-DM1 in EOC and effective against HER2/neu 3+ xenografts. Additional (HER2/neu 2+ &amp; 1+) EOC xenograft studies are ongoing. Conclusions: SYD985 is a novel ADC with remarkable in vitro activity against EOC with strong (3+) as well as low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is more potent than T-DM1 in comparative experiments and unlike T-DM1, it may be active against EOC demonstrating moderate/low or heterogeneous HER2/neu expression. Clinical studies with SYD985 in EOC patients harboring chemo-resistant disease are needed.

Novel glycolipid agents for killing cisplatin-resistant human epithelial ovarian cancer cells

BackgroundChemotherapy resistance is one of the major factors contributing to mortality from human epithelial ovarian cancer (EOC). Identifying drugs that can effectively kill chemotherapy-resistant EOC cells would be a major advance in reducing mortality. Glycosylated antitumour ether lipids (GAELs) are synthetic glycolipids that are cytotoxic to a wide range of cancer cells. They appear to induce cancer cell death in an apoptosis-independent manner.MethodsHerein, the effectiveness of two GAELs, GLN and MO-101, in killing chemotherapy-sensitive and –resistant EOC cells lines and primary cell samples was tested using monolayer, non-adherent aggregate, and non-adherent spheroid cultures.ResultsOur results show that EOC cells exhibit a differential sensitivity to the GAELs. Strikingly, both GAELs are capable of inducing EOC cell death in chemotherapy-sensitive and –resistant cells grown as monolayer or non-adherent cultures. Mechanistic studies provide evidence that apoptotic-cell death (caspase activation) contributes to, but is not completely responsible for, GAEL-induced cell killing in the A2780-cp EOC cell line, but not primary EOC cell samples.ConclusionsStudies using primary EOC cell samples supports previously published work showing a GAEL-induced caspase-independent mechanism of death. GAELs hold promise for development as novel compounds to combat EOC mortality due to chemotherapy resistance.

MicroRNA-137 promotes apoptosis in ovarian cancer cells via the regulation of XIAP

Background:microRNAs (miRNAs) have regulatory roles in various cellular processes, including apoptosis. Recently, X-linked inhibitor of apoptosis protein (XIAP) has been reported to be dysregulated in epithelial ovarian cancer (EOC). However, the mechanism underlying this dysregulation is largely unknown.Methods:Using bioinformatics and a literature analysis, a panel of miRNAs dysregulated in EOC was chosen for further experimental confirmation from hundreds of miRNAs that were predicted to interact with the XIAP 3′UTR. A dual-luciferase reporter assay was employed to detect the interaction by cellular co-transfection of an miRNA expression vector and a reporter vector with the XIAP 3′UTR fused to a Renilla luciferase reporter. DAPI and TUNEL approaches were used to further determine the effects of an miR-137 mimic and inhibitor on cisplatin-induced apoptosis in ovarian cancer cells.Results:We identified eight miRNAs by screening a panel of dysregulated miRNAs that may target the XIAP 3′UTR. The strongest inhibitory miRNA, miR-137, suppressed the activity of a luciferase reporter gene fused with the XIAP 3′UTR and decreased the levels of XIAP protein in SKOV3 ovarian cancer cells. Furthermore, forced expression of miR-137 increased cisplatin-induced apoptosis, and the depressed expression of miR-137 decreased cisplatin-induced apoptosis in SKOV3 and primary EOC cells. Consistently, the disruption of miR-137 via CRISPR/Cas9 inhibited apoptosis and upregulated XIAP in A2780 cells. Furthermore, the effect of miR-137 on apoptosis could be rescued by XIAP in SKOV3 cells. In addition, miR-137 expression is inversely correlated with the level of XIAP protein in both ovarian cancer tissues and cell lines.Conclusions:Our data suggest that multiple miRNAs can regulate XIAP via its 3′UTR. miR-137 can sensitise ovarian cancer cells to cisplatin-induced apoptosis, providing new insight into overcoming drug resistance in ovarian cancer.

Mesothelial cells interact with tumor cells for the formation of ovarian cancer multicellular spheroids in peritoneal effusions.

Epithelial ovarian cancer (EOC) dissemination is primarily mediated by the shedding of tumor cells from the primary site into ascites where they form multicellular spheroids that rapidly lead to peritoneal carcinomatosis. While the clinical importance and fundamental role of multicellular spheroids in EOC is increasingly appreciated, the mechanisms that regulate their formation and dictate their cellular composition remain poorly characterized. To investigate these important questions, we characterized spheroids isolated from ascites of women with EOC. We found that in these spheroids, a core of mesothelial cells was encased in a shell of tumor cells. Analysis further revealed that EOC spheroids are dynamic structures of proliferating, non-proliferating and hypoxic regions. To recapitulate these in vivo findings, we developed a three-dimensional co-culture model of primary EOC and mesothelial cells. Our analysis indicated that, compared to the OVCAR3 cell line, primary EOC cells isolated from ascites as well as mesothelial cells formed compact spheroids. Analysis of heterotypic spheroid microarchitecture revealed a structure that grossly resembles the structure of spheroids isolated from ascites. Cells that formed compact spheroids had elevated expression of β1 integrin and low expression of E-cadherin. Addition of β1 integrin blocking antibody or siRNA-mediated downregulation of β1 integrin resulted in reduced tightness of the spheroids. Interestingly, the loss of MUC16 and E-cadherin expression resulted in the formation of more compact spheroids. Therefore, our findings support the heterotypic nature of spheroids from malignant EOC ascites. In addition, our data describe an unusual link between E-cadherin expression and less compact spheroids. Our data also emphasize the role of MUC16 and β1 integrin in EOC spheroid formation.

Establishment and Characterization of Primary Human Ovarian Cancer Stem Cell Line (CD44+ve)

Ovarian cancer is ranked as the 7th most lethal cancer worldwide with 239,000 new cases annually. The mortality rate is high because most ovarian tumors are diagnosed at advanced stages and are resistant to chemotherapy and thus incurable due to the lack of effective early detection of ovarian tumors. There is a small sub-population of ovarian tumor cells capable of self-renewal and differentiation into different cancer cell types, called cancer stem cells (CSCs), which might be responsible for cancer relapse. The CD44+ phenotype in ovarian tumor cells elucidates cancer initiating cell-like properties of promoting differentiation, metastasis, and chemotherapy-resistance. Increased expression of genes previously associated with CSCs promotes regenerative capacity by promoting stem cell function that can drive cancer relapse and metastasis. In this study we present a method to isolate the primary epithelial ovarian cancer cells from human solid tumor and establish CD44+ve primary ovarian cancer stem cell (OCSCCD44+ve) line using magnetic microbeads. Also we evaluated the expression of stemness genes Nanog, Sox2, Oct4, and Nestin by real-time qPCR analysis. Thequantitative analysis by real-time qPCRshows that OCSCCD44+ve overexpressed the embryonic stem cell marker genes Nanog, Oct4, Sox2, and Nestin when compared with ovarian cancer cells OCCCD44-ve as positive control and ovarian cells as negative control. We demonstrate that CD44 in malignant ovarian tumors is a critical molecule that exhibits cancer stem cell properties that enhance tumorigenicity and cancer metastasis. Our results provide a better understanding of ovarian CSCs, which is important for future in vivo studies with subsequent target therapy for preclinical studies.