Mouse Ovarian Cancer Cell Line Research Articles

Acid-Sensitive Outwardly Rectifying Cl- Current in OV2944 Mouse Ovarian Cancer Cells.

Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3)but was slightly restored upon PS administration. These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.

Entinostat potentiates responsiveness to olaparib in both induced and intrinsic PARPi resistant mouse ovarian cancer cell lines (2308)

Entinostat potentiates responsiveness to olaparib in both induced and intrinsic PARPi resistant mouse ovarian cancer cell lines (2308)

Single CAR-T cell treatment controls disseminated ovarian cancer in a syngeneic mouse model

BackgroundTreatment of some blood cancers with T cells that express a chimeric antigen receptor (CAR) against CD19 have shown remarkable results. In contrast, CAR-T cell efficacy against solid tumors has...

Abstract 4907: Atovaquone-induced increase in reactive oxygen species leads to impaired glycolytic functions in ovarian cancer cells

Abstract Atovaquone (ATO) is an FDA-approved anti-malarial drug, which has also shown potent anti-cancer effects. We have previously demonstrated ATO as an effective chemotherapy against high-grade serous ovarian cancer in cell lines and mouse model. In the present study, we aim to determine if ATO is asserting its anti-cancer properties by altering cellular metabolism. Glycolysis and oxidative phosphorylation (OXPHOS) are the two major sources of intracellular energy. ATO reduces OXPHOS energy production by inhibiting mitochondrial complex III. We first determined whether there is a shift in energy production when OXPHOS is inhibited by assessing glycolytic function in various human (OVCAR3 and OVCAR5) and mouse (ID8) ovarian cancer cell lines. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed by the Seahorse XF Analyzer. First, we pretreated cells with ATO at IC70 concentrations for 1 hour prior to metabolic analysis. As expected, pretreatment with ATO reduces OCR in all cell lines. However, ATO treatment increased basal glycolysis but decreased glycolytic capacity and glycolytic reserve. This suggests that ATO-treated cells have an impaired ability to shift energy production towards glycolysis when there is an increase in energy demand. One of the initial events that occur in ATO-treated cells is a surge in reactive oxygen species (ROS) as measured by H2DCFDA ROS assay. In the human cell lines, we found that ATO significantly increased ROS by approximately 100-fold (MFI). Whereas, in the mouse cell line, the ATO-induced increase in ROS were comparatively attenuated. We hypothesized that the increase in ROS leads to inhibition of glycolysis. We tested this by pre-treating cells with 2mM N-acetylcysteine (NAC), an oxygen radical scavenger, for 1 hour before measuring the metabolic responses to ATO. We found that NAC attenuated the inhibition of glycolytic processes in human cell lines but not in the mouse cell line. Our lab has previously shown that ATO induced activation and increased expression of p53. Studies have shown that ROS activate p53, which in turn upregulates the TP53 induced glycolysis and apoptosis regulatory phosphatase (TIGAR), inhibiting the conversion of F6P to F-2,6-BP and ultimately leads to inhibition of glycolysis. Increases in ROS also shuttle G6P towards the phosphate pentose pathways through G6P dehydrogenase for antioxidant production. We found that long-term ATO treatment for 24 hours also resulted in increases in both TIGAR and G6PD mRNA expression. Taken together, these results suggest that ATO decreased plasticity of metabolic capacity in ovarian cancer cells through ROS and ROS associated glycolytic pathways. Further, this study provides mechanistic insights into ATO’s potential as a chemotherapeutic agent. Citation Format: Nicha Boonpattrawong, Sejal Sharma, Rob Schultz, Mayra A. Betancourt Ponce, Lisa M. Barroilhet, Manish Patankar. Atovaquone-induced increase in reactive oxygen species leads to impaired glycolytic functions in ovarian cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4907.

27 Entinostat restores responsiveness to olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 μM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 μM Entinostat, 10 μM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicletreated controls (p=0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p<0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer.

Hypoxia-driven metabolic reprogramming of adipocytes fuels cancer cell proliferation.

Obesity increases the risk of certain cancers, especially tumours that reside close to adipose tissue (breast and ovarian metastasis in the omentum). The obesogenic and tumour micro-environment share a common pathogenic feature, oxygen deprivation (hypoxia). Here we test how hypoxia changes the metabolome of adipocytes to assist cancer cell growth. Human and mouse breast and ovarian cancer cell lines were co-cultured with human and mouse adipocytes respectively under normoxia or hypoxia. Proliferation and lipid uptake in cancer cells were measured by commercial assays. Metabolite changes under normoxia or hypoxia were measured in the media of human adipocytes by targeted LC/MS. Hypoxic cancer-conditioned media increased lipolysis in both human and mouse adipocytes. This led to increased transfer of lipids to cancer cells and consequent increased proliferation under hypoxia. These effects were dependent on HIF1α expression in adipocytes, as mouse adipocytes lacking HIF1α showed blunted responses under hypoxic conditions. Targeted metabolomics of the human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes media revealed that culture with hypoxic-conditioned media from non-malignant mammary epithelial cells (MCF10A) can alter the adipocyte metabolome and drive proliferation of the non-malignant cells. Here, we show that hypoxia in the adipose-tumour microenvironment is the driving force of the lipid uptake in both mammary and ovarian cancer cells. Hypoxia can modify the adipocyte metabolome towards accelerated lipolysis, glucose deprivation and reduced ketosis. These metabolic shifts in adipocytes could assist both mammary epithelial and cancer cells to bypass the inhibitory effects of hypoxia on proliferation and thrive.

FOXC2 Promotes Vasculogenic Mimicry in Ovarian Cancer.

Simple SummaryTumors need a continuous supply of oxygen and nutrients to sustain growth. One coping mechanism is to secret factors that promote the development of new blood vessels. However, this may not be sufficient for the growth of highly aggressive tumors. Vasculogenic mimicry is another coping mechanism whereby cancer cells form vascular-like structures capable of carrying blood and nutrients. Expression of nuclear FOXC2 has been associated with aggressiveness and advanced stage in most cancers, including ovarian cancer. We confirmed most of the known mechanisms by which FOXC2 promotes cancer aggressiveness. Additionally, we found evidence that FOXC2 expression is associated with vasculogenic mimicry in ovarian cancer samples and that FOXC2 overexpression promotes vasculogenic mimicry in cell culture.FOXC2 is a forkhead family transcription factor that plays a critical role in specifying mesenchymal cell fate during embryogenesis. FOXC2 expression is associated with increased metastasis and poor survival in various solid malignancies. Using in vitro and in vivo assays in mouse ovarian cancer cell lines, we confirmed the previously reported mechanisms by which FOXC2 could promote cancer growth, metastasis, and drug resistance, including epithelial-mesenchymal transition, stem cell-like differentiation, and resistance to anoikis. In addition, we showed that FOXC2 expression is associated with vasculogenic mimicry in mouse and human ovarian cancers. FOXC2 overexpression increased the ability of human ovarian cancer cells to form vascular-like structures in vitro, while inhibition of FOXC2 had the opposite effect. Thus, we present a novel mechanism by which FOXC2 might contribute to cancer aggressiveness and poor patient survival.

Abstract 795: Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 µM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 µM Entinostat, 10 µM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicle-treated controls (p= 0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p&amp;lt;0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p&amp;lt; 0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer. Citation Format: Tyler Woodard, Viju Gupta, Simona Miceska, Bisiayo Fashemi, Wendy Zhang, Dineo Khabele. Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 795.

Abstract 3235: The role of DDR2 in chemoresistant ovarian cancer

Abstract Ovarian cancer is the fifth leading cause of cancer death for women in the United States. As many as 80% of ovarian cancer patients experience recurrent disease and the majority of recurrences become resistant to treatment. Once patients no longer respond to chemotherapy, there are minimal treatment options and they succumb to their disease. Therefore, there is a need to create new targeted therapies to overcome chemoresistance in ovarian cancer and improve patient survival. We have assembled a tumor microarray from 164 patient tumors to characterize the changes in protein expression that lead to resistance in clinical samples. We have found using IHC staining, that patients with high tyrosine kinase receptor discoidin domain receptor 2 (DDR2) protein expression (&amp;gt;70%) have significantly shorter overall survival times regardless of stage at diagnosis (HR= 2.108, p=0.002) as well as reduced platinum free interval (p=0.046). Additionally, platinum refractory tumors have higher DDR2 protein expression than platinum sensitive tumors (p=0.036). To further understand the mechanisms of chemoresistance, we created a novel, chemoresistant, syngeneic, mouse ovarian cancer cell line derived from ID8 trp53-/-BRCA2-/- GFP LUC cells from Walton et. al 2016, and have designated them CHRP5. DDR2 expression assessed via western blot increased significantly in the chemoresistance cells, suggesting that DDR2 is associated with chemoresistance. We knocked down DDR2 using a stable shRNA transfection in CHRP5 and used MTS survival assays to determine if decreased DDR2 expression increases sensitivity to chemotherapy. CHRP5 shDDR2 cells were significantly more sensitive to carboplatin than CHRP5 shControl cells (CHRP5 shControl IC50=213.6uM, CHRP5 shDDR2 IC50=128.5uM, p&amp;lt;0.000001), suggesting that DDR2 is involved in regulating chemoresistance in these cells. Additionally, we have submitted matched chemoresistant and chemosensitive mouse tumor samples for RNA-sequencing. We will use this data to determine which chemoresistant mechanisms are utilized, and if DDR2 is involved in regulating those mechanisms. In the future, we plan to rescue DDR2 in the knockdown cells to confirm that off target effects are not influencing the change in sensitivity. We also have access to a specific, allosteric inhibitor of DDR2 and plan to use this drug to determine if inhibiting DDR2 pharmacologically can overcome chemoresistance and reduce tumor burden in vivo and in vitro. In conclusion, DDR2 is more highly expressed in chemoresistant patients, and higher DDR2 expression correlates with reduced survival and platinum free interval. Additionally, knocking down DDR2 in chemoresistant cells increases sensitivity to chemotherapy. Therefore, DDR2 is a potential new target for ovarian cancer therapy and may be targeted to overcome chemoresistant disease and extend patient survival. Citation Format: Alyssa Oplt, Elizabeth Stock, Hollie Noia, Patrick Cannon, Gregory Longmore, Katherine C. Fuh. The role of DDR2 in chemoresistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3235.

Abstract 1564: Endothelial Notch activation is essential for the education of tumor associated macrophages

Abstract Epithelial ovarian cancer (EOC) is one of the most lethal gynecological cancers worldwide. Notch1 receptors (N1ICD) are frequently activated in Endothelial cells (ECs) of human tumors to facilitate metastasis. Using VE-Cadherin-CreERT2 transgenic mice to induce gene recombination exclusively in ECs, we explored the role of Notch signaling in ECs in vivo. RbpjiΔEC (VE-Cad-CrERT2xRbpjflox) mice inhibit the canonical Notch signaling upon tamoxifen administration. We modeled metastatic epithelial ovarian cancer (EOC) by injecting the mouse ovarian cancer cell line ID8 intraperitoneally. After four weeks of tumor growth, we observed a significant decreased tumor burden and reduced infiltration of monocyte-derived macrophages into the TME upon EC-specific Rbpj deletion. In vitro, we also confirmed a decreased infiltration of human monocytes through an EC barrier upon Rbpj deletion in human ECs. We isolated tumor-associated macrophages (TAMs) from RbpjΔEC tumor bearing mice and their littermate controls and performed microarray analysis. TCs educate TAMs through CD44-mediated cholesterol depletion. Several bioinformatic analyses showed that TAM from RbpjiΔEC mice were not able to induce the expected signature of TAMs in metastatic EOC because cholesterol depletion cannot take place. This indicates that the TC-induced macrophage education requires the presence of Rbpj in ECs. In vitro experiments showed that CD44 expression was downregulated in monocytes co-cultured with ECs lacking RBPJ. Analysis of the angiocrine factors regulated by Notch signaling revealed CXCL2 as a novel Rbpj-mediated Notch target gene: activation of Notch1 in ECs in vitro induced the production of the chemotactic cytokine CXCL2, whereas Rbpj-deletion (CRISPR-Cas9 technology) in N1ICD transduced ECs abolished CXCL2 expression. In addition, CXCL2 stimulation increased CD44 expression. In keeping with this, the analysis of publicly available ovarian cancer patient’s datasets we found that CXCL2 expression correlates with CD44 expression, suggesting that CXCL2 induces CD44 also in patients. CD44 is a co-receptor of a complex involving CXCR2 and CD74, which without CD44 it is not possible to signal. CD74 expression has been associated with worse prognosis in metastatic ovarian cancer and associated with an immunosuppressive phenotype in macrophages. Bioinformatic analyses of publicly available data showed that Rbpj in ECs is necessary for the immunosuppressive role of CD74. Finally, T cells sorted from RbpjiΔEC tumor bearing mice showed stronger cytotoxic activity (LDH-cytotoxicity assay) than those sorted from their littermate controls. In summary, ovarian cancer cells employ the tumor endothelium to secrete CXCL2 in order to facilitate an immunosuppressive microenvironment. Citation Format: Ronja Mülfarth, Elisenda Alsina-Sanchis, Iris Moll, Sarah Böhn, Adrian Stögbauer, Francesca De Angelis Rigotti, Lena Wiedmann, Jacqueline Taylor, Andreas Fischer, Juan Rodriguez Vita. Endothelial Notch activation is essential for the education of tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1564.

Abstract 1153: Targeting CA125 transcription for ovarian cancer treatment

Abstract CA125 coded by MUC16 gene is highly expressed in most ovarian cancer cells and serves as a tumor marker to monitor disease progression or response to treatment of ovarian cancer patients. However, targeting CA125 for ovarian cancer treatment has never been successful. We performed multiple steps of high-fidelity PCR and obtained a 4.2kb DNA fragment upstream of the human MUC16 gene, which included a region upstream from the transcription start site (TSS) and a region downstream from the TSS that contains both the 5' untranslated region and the first 136bp of the open reading frame. Reporter assays showed that this DNA fragment possesses strong transactivation activity in CA125-high cancer cells, but not in CA125-low cells, indicating that the DNA fragment we cloned contains the transactivation region that controls specific expression of MUC16 gene in ovarian cancer cells. We further refined the promoter and found a 1040bp fragment with similar transcriptional activity and specificity. We used this refined MUC16 promoter to replace the E1A promoter in the adenovirus type 5 genome DNA to control expression of E1A, an essential gene for adenovirus replication, and successfully generated a conditionally replication-competent adenovirus that can replicate in and lyse CA125-high-expressing cancer cells, including CAOV3, Kuramochi, MADH2774, OVCAR3, OVCAR4, and TOV112D cells, but not in CA125-low or -negative cancer cell lines, such as A2780, OVCAR5, PEO4, or SKOV3 cells. In vivo studies showed that intraperitoneal virus injection prolonged survival of NSG mice inoculated intraperitoneally with MADH2774 cells. Preliminary experiment showed that a mouse ovarian cancer cell line, ID8, infected with the virus might elicit a protective immune response to rechallenge of parental cancer cells. Our data indicate that targeting MUC16 transactivation for ovarian cancer treatment by conditionally replicative adenovirus development is feasible and practical. Citation Format: Er Yue, Guangchao Yang, Yuanfei Yao, Guangyu Wang, Yanqiao Zhang, Edward W. Wang. Targeting CA125 transcription for ovarian cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1153.

NSCs are permissive to oncolytic Myxoma virus and provide a delivery method for targeted ovarian cancer therapy.

Despite the development of many anticancer agents over the past 20 years, ovarian cancer remains the most lethal gynecologic malignancy. Due to a lack of effective screening, the majority of patients with ovarian cancer are diagnosed at an advanced stage, and only ~20% of patients are cured. Thus, in addition to improved screening methods, there is an urgent need for novel anticancer agents that are effective against late-stage, metastatic disease. Oncolytic virotherapy is a promising approach; unfortunately, systemic delivery of viruses to tumors remains a major challenge. In this regard, neural stem/progenitor cells (NSCs) with well-established tumor-homing properties may serve as an effective delivery platform for oncolytic viruses. In this study, we tested the efficacy of myxoma virus (MYXV), a rabbit-specific poxvirus that has demonstrated efficacy against a variety of tumors, using human and mouse ovarian cancer cell lines. We showed that MYXV effectively lysed ovarian cancer cells in vitro, reducing their viability. We also demonstrated that MYXV can infect human NSCs, specifically the clonal HB1.F3.CD21 NSC line. Taken together, these results suggest that NSC-mediated delivery of MYXV may be a promising strategy for achieving more selectively targeted anti-tumor efficacy.

Oxidative stress induced by the anti-cancer agents, plumbagin, and atovaquone, inhibits ion transport through Na+/K+-ATPase

Oxidative stress inhibits Na+/K+-ATPase (NKA), the ion channel that maintains membrane potential. Here, we investigate the role of oxidative stress-mediated by plumbagin and atovaquone in the inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit the proliferation of three human (OVCAR-3, SKOV-3, and TYKNu) and one mouse (ID8) ovarian cancer cell lines. The oxygen radical scavenger, N-acetylcysteine (NAC), attenuates the chemotoxicity of plumbagin and atovaquone. Whole-cell patch clamping demonstrates that plumbagin and atovaquone inhibit outward and the inward current flowing through NKA in SKOV-3 and OVCAR-3. Although both drugs decrease cellular ATP; providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either drug significantly decreases the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA, resulting in the inability to maintain ion transport. Therefore, when evaluating compounds that induce oxidative stress, it is important to consider the contribution of NKA inhibition to their cytotoxic effects on tumor cells.

Abstract 3309: Cardioprotective paraoxonase 2 enhances the tumoricidal activity of natural killer cells in a paracrine manner

Abstract Background: Immune system can recognize the neoplastic cells and target them for destruction through tumoricidal process. Further, immune cell dysfunction leads to escape of cancer cells from immune recognition, lead to the initiation and progression of tumor formation. Clinical shreds of evidence suggest that the presence of intratumoral immune cells correlates with improved progression free and overall survival among patients with ovarian carcinoma and provide evidence of activation of antitumor mechanisms. Hence identification of drugs that target the cancer cells as well as activate the immune cells is a sound strategy for cancer therapy. Paraoxonase 2 (PON2) is a membrane-associated lactonase with lipid transport, anti-oxidant and phagocytosis/efferocytosis properties. PON2 deficiency aggravates systemic inflammation, and causes the metabolic associated diseases. Recently we have reported that overexpression of PON2 reduces the ovarian cells proliferation and tumor formation by reducing IGF-1 production and its signaling pathway. Further, using microarray based experiment, we have identified several pathways that modulate the immune cells function such as activation of Natural Killer (NK) cells, dendritic cell maturation, B cells activation, and communication between innate and an adaptive cells signaling pathway were regulated by PON2. Hypothesis: PON2 enhances tumoricidal activity in a paracrine fashion. Methods and Results: NK cells were isolated from control C57BL/6 and purity was assessed with flow cytometry using FITC anti mouse CD49B and results revealed that 95 % of NK cells population. NK cells treated with condition medium from ID8hPON2 (human PON2 overexpressed in mouse ovarian cancer cell line) show an increase in tumoricidal activity compared to condition medium from ID8EV. Neither ID8hPON2 nor ID8EV condition medium affects the viability of NK cells. Moreover, there is no difference in CD49B positive staining in the tumor microenvironment between ID8EV and ID8hPON2 cells receiving mice. Condition medium obtained from ID8hPON2 ID8EV group shows increases the interleukin 18 level and neutralizing antibody against IL18 fail to induce the tumoricidal activity indicating mechanistically PON2 enhances the tumoricidal activity via IL18 dependent manner. Furthermore, IL18 levels are increased in tumor micro environment. PON2 increase the IL18 through transactivation of c-Myc gene that are independent of mitochondrial function. Conclusion: Taken together, our study for the first time showed that PON2 enhances the tumerocidal activity via paracrine manner by upregulating IL18 and PON2 could be used as conventional as well as immunotherapy for ovarian cancer. Citation Format: Asokan Devarajan, Dawoud Sulaiman, Feng Su, Ekambaram Ganapathy, Robin Farias-Eisner, Srinivasa Reddy. Cardioprotective paraoxonase 2 enhances the tumoricidal activity of natural killer cells in a paracrine manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3309.

Combining DNMT and HDAC6 inhibitors increases anti-tumor immune signaling and decreases tumor burden in ovarian cancer

Novel therapies are urgently needed for ovarian cancer, the deadliest gynecologic malignancy. Ovarian cancer has thus far been refractory to immunotherapies that stimulate the host immune system to recognize and kill cancer cells. This may be because of a suppressive tumor immune microenvironment and lack of recruitment and activation of immune cells that kill cancer cells. Our previous work showed that epigenetic drugs including DNA methyltransferase inhibitors and histone deacetylase 6 inhibitors (DNMTis and HDAC6is) individually increase immune signaling in cancer cells. We find that combining DNMTi and HDAC6i results in an amplified type I interferon response, leading to increased cytokine and chemokine expression and higher expression of the MHC I antigen presentation complex in human and mouse ovarian cancer cell lines. Treating mice bearing ID8 Trp53−/− ovarian cancer with HDAC6i/DNMTi led to an increase in tumor-killing cells such as IFNg+ CD8, NK, and NKT cells and a reversal of the immunosuppressive tumor microenvironment with a decrease in MDSCs and PD-1hi CD4 T cells, corresponding with an increase in survival. Thus combining the epigenetic modulators DNMTi and HDAC6i increases anti-tumor immune signaling from cancer cells and has beneficial effects on the ovarian tumor immune microenvironment.

Bovine HDL and Dual Domain HDL-Mimetic Peptides Inhibit Tumor Development in Mice.

A growing body of literature supports the role of apolipoproteins present in HDL in the treatment of pro-inflammatory diseases including cancer. We examined whether bovine HDL (bHDL) and three dual-domain peptides, namely AEM-28 and its analog AEM-28–2, and HM-10/10, affect tumor growth and development in mouse models of ovarian and colon cancer. We demonstrate that bHDL inhibits mouse colorectal cancer cell line CT26-mediated lung tumor development, and mouse ovarian cancer cell line ID8-mediated tumor burden. We also demonstrate that, although to different degrees, dual-domain peptides inhibit cell viability of mouse and human ovarian and colon cancer cell lines, but not that of normal human colonic epithelial cells or NIH3T3 mouse fibroblasts. Dual-domain peptides administered subcutaneously or in a chow diet decrease CT26 cell-mediated tumor burden, tumor growth, and tumor dissemination in BALB/c mice. Plasma levels of lysophosphatidic acid (LPA) are significantly reduced in mice that received bHDL and the dual-domain peptides, suggesting that reduction by effecting accumulation and/or synthesis of pro-inflammatory lipids may be one of the mechanisms for the inhibition of tumor development by bHDL and the dual-domain peptides. Our studies suggest that therapeutics based on apolipoproteins present in HDL may be novel agents for the treatment of epithelial adenocarcinomas of the ovary and colon.

Abstract 194: Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L

Abstract Purpose: To identify mechanisms responsible for the development of adaptive resistance to anti-vascular endothelial growth factor (VEGF) therapy, and to develop novel agents to reverse its occurrence. Methods: Gene expression profiling was performed on endothelial cells isolated from tumors sensitive or resistant to anti-VEGF therapy in a syngeneic ovarian cancer mouse model. A series of in vitro (lentiviral transfection, tube formation, siRNA knockdown, luciferase promoter assays) and in vivo (including Tie2; PPARγ KO mice) experiments were then used to elucidate the upstream and downstream effectors of CD5L, as well as the efficacy of CD5L inhibition in pre-clinical orthotopic mouse models. Results: We identified substantially increased CD5L expression in endothelial cells isolated from anti-VEGF therapy resistant vs. sensitive tumors. Ectopic expression of CD5L in RF24 endothelial cells resulted in increased tube formation, cell migration, and cell proliferation, whereas siCD5L treatment resulted in an overall decrease. Observing that a PPARγ binding site was present in the CD5L promoter, we found that ectopic expression of PPARγ into RF24 cells results in increased CD5L expression, whereas siPPARγ treatment decreased it. Ectopic PPARγ expression increased luciferase activity but decreased activity with siPPARγ treatment when using a reporter assay for CD5L promoter. Conversely, mutation of the PPARγ binding site in the promoter construct lacked any increase in activity after ectopic PPARγ expression. IPA analysis identified HIF1α as playing an integral role in the PPARγ /CD5L activation pathway. Indeed, hypoxic conditions resulted in an increase of both PPARγ and CD5L in RF24 cells. HIF1α stabilizers (cobalt chloride) and inhibitors (YC-1 and topotecan) also showed an increase and decrease in PPARγ and CD5L expression, respectively. RPPA analysis of anti-VEGF resistant tumor endothelial cells implicated an upregulation of the PI3K pathway. While exogenous CD5L treatment led to increased p-AKT, the addition of the PI3K inhibitor LY294002 negated this effect and also decreased tube formation and cell migration. Next, we injected both WT B6 and Tie2:PPARγ KO mice with the ID8 mouse ovarian cancer cell line and treated with B20 until moribund. Mice harboring the PPARγ KO had significantly increased survival as well as decreased tumor weight, number of nodules, Ki67 expression, and blood vessel density. Our non-syngeneic model was then treated with B20 and either CD5L RNA aptamer or vehicle. The aptamer group treated with CD5L aptamer displayed significantly decreased tumor weight, number of nodules, Ki67 expression, and blood vessel density. Conclusions: This study identifies a pathway centered on CD5L as an important driver of adaptive resistance to anti-angiogenic therapy. Citation Format: Christopher J. LaFargue, Paola Amero, Kyunghee Noh, Selanare Mangala, Chunhua Lu, Minsoon Cho, Sunila Pradeep, Yihong Wan, Wei Hu, Rajesha Rupaimoole, Robert L. Coleman, Ningyan Zhang, Zhiqiang An, Gabriel Lopez-Bernstein, Vittorio de Franciscis, Anil K. Sood. Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 194.

Abstract 891: Plumbagin and atovaquone inhibit Na+/K+-ATPase through the generation of oxidative stress

Abstract Plumbagin and atovaquone are chemotoxic to ovarian, breast and other tumors. Both molecules inhibit oxidative phosphorylation causing a rapid increase in intracellular oxygen radicals and apoptosis. Oxidative stress is also known to inhibit the activity of Na+/K+-ATPase (NKA), the ion channel that maintains the membrane potential. Here, we investigate if the oxidative stress mediated by plumbagin and atovaquone also leads to inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit proliferation of three human (OVCAR-3, SKOV-3 and TYKNu) and one mouse (ID8) ovarian cancer cell lines. Using SKOV-3 and OVCAR-3 as models for ovarian cancer, we demonstrate that oxygen radical scavenger, N-acetylcysteine (NAC) attenuated the cytotoxicity of plumbagin and atovaquone. Using whole cell patch clamping we demonstrate that plumbagin and atovaquone inhibit outward and inward current flowing through NKA in SKOV-3 and OVCAR-3. Both drugs decrease cellular ATP. Providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either of the drugs for 1-2 h resulted in a significant decrease in the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA and hence the membrane potential cannot be optimally maintained. Evaluation of compounds that induce oxidative stress should therefore consider contribution of NKA inhibition to the cytotoxic activity of such agents. Citation Format: Yousef Alharbi, Arvinder Kapur, Mildred Felder, Lisa Barroilhet, Bikash Pattnaik, Manish S. Patankar. Plumbagin and atovaquone inhibit Na+/K+-ATPase through the generation of oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 891.

Therapeutic targeting of the immunosuppressive axis in ovarian cancer using dietary supplements

Abstract Ovarian cancer is the deadliest gynecologic malignancy worldwide and mortality rates have remained steady despite advances in treatment. A major obstacle for current therapies is overcoming tumor immunosuppression, therefore targeting inhibitory factors produced in the tumor microenvironment could enhance antitumor responses. Natural Killer T (NKT) cells, a subset of T cells that recognize glycolipid antigens presented by CD1d, are important mediators of antitumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which binds with high affinity to CD1d, thereby suppressing NKT cell activation. In addition to GD3, we have identified vascular endothelial growth factor (VEGF) as another immunosuppressive factor secreted by ovarian cancers. We found that VEGF inhibition results in decreased GD3 expression in human ovarian cancer cell lines and restoration of NKT cell responses. In addition, we found that treatment with VEGF can directly impair NKT cell functional responses. To therapeutically target the VEGF-GD3 signaling cascade, mouse and human ovarian cancer cell lines were treated with triptolide, a phytochemical which has demonstrated potent antitumor activity in vitro and in vivo. Treatment with triptolide reduces VEGF secretion by ovarian cancer cell lines and results in a concomitant decrease in the expression of GD3 synthase. In addition, pre-treatment of ovarian cancer cells with triptolide improves the ability of NKT cells to recognize and lyse the cancer cells. Thus, we have identified a novel therapeutic strategy that can be used to block the production of immunosuppressive factors, thereby restoring NKT cell-mediated antitumor immune responses in ovarian cancer.

Experimental Observation on the Retransmission of Radiation Side Effects

[Objective] To observe the retransmission of radiation side effects between cells. [Materials and Methods] The mouse ovarian cancer cell line NUTU19 was irradiated with 6MV-X-rays, and the culture medium was prepared for the first-generation conditioned medium. The first-generation effector cells were used to detect the NUTU19 cell line and the intestinal mucosal epithelial IEC-6 cell line. The secretion of effector cells was a second-generation conditioned medium, and the second-generation effector cells NUTU19, IEC-6, and mouse lymphocytes were treated to measure cell viability and apoptosis. [Results] After treated with NUTU19 second-generation medium for 48 hours, the apoptosis rate of IEC-6 and NUTU19 cells was promoted (p>0.05), and the apoptosis rate of lymphocytes was decreased (p 0.05). After treatment with IEC-6 second-generation medium for 48h, the apoptosis of NUTU19 and IEC-6 was promoted (p 0.05). It promoted the apoptosis of NUTU19 (p<0.05). [Conclusion]: Under certain conditions, tumor cells, intestinal epithelial cells and lymphocytes can retransmit the damage of the side effect of radiation.