ES-2 Cells Research Articles

COL6A3 Exosomes Promote Tumor Dissemination and Metastasis in Epithelial Ovarian Cancer.

Our study explores the role of cancer-derived extracellular exosomes (EXs), particularly focusing on collagen alpha-3 (VI; COL6A3), in facilitating tumor dissemination and metastasis in epithelial ovarian cancer (EOC). We found that COL6A3 is expressed in aggressive ES2 derivatives, SKOV3 overexpressing COL6A3 (SKOV3/COL6A3), and mesenchymal-type ovarian carcinoma stromal progenitor cells (MSC-OCSPCs), as well as their EXs, but not in less aggressive SKOV3 cells or ES2 cells with COL6A3 knockdown (ES2/shCOL6A3). High COL6A3 expression correlates with worse overall survival among EOC patients, as evidenced by TCGA and GEO data analysis. In vitro experiments showed that EXs from MSC-OCSPCs or SKOV3/COL6A3 cells significantly enhance invasion ability in ES2 or SKOV3/COL6A3 cells, respectively (both, p <0.001). In contrast, ES2 cells with ES2/shCOL6A3 EXs exhibited reduced invasion ability (p < 0.001). In vivo, the average disseminated tumor numbers in the peritoneal cavity were significantly greater in mice receiving intraperitoneally injected SKOV3/COL6A3 cells than in SKOV3 cells (p < 0.001). Furthermore, mice intravenously (IV) injected with SKOV3/COL6A3 cells and SKOV3/COL6A3-EXs showed increased lung colonization compared to mice injected with SKOV3 cells and PBS (p = 0.007) or SKOV3/COL6A3 cells and PBS (p = 0.039). Knockdown of COL6A3 or treatment with EX inhibitor GW4869 or rapamycin-abolished COL6A3-EXs may suppress the aggressiveness of EOC.

How Progesterone Receptor Expression Impacts Platinum Sensitivity in Ovarian Clear Cell Carcinoma: Insights from Clinical and Experimental Perspectives.

Ovarian clear cell carcinoma (OCCC) is often considered a relatively platinum-resistant malignancy. The aim of this study was to explore the influence of progesterone receptor (PR) expression levels on platinum sensitivity and survival outcomes in people with OCCC. A retrospective analysis was conducted with 80 people with OCCC who underwent surgery followed by adjuvant chemotherapy. PR expression was assessed via immunohistochemical (IHC) staining and quantified using the H score. The platinum sensitivity and survival outcomes of patients with weak and strong PR expression were compared. Additionally, cisplatin viability and migration experiments were conducted with OCCC cell lines (ES-2 and TOV-21G) with varying PR isoform expressions. Among the 80 patients, 62 were classified as having platinum-sensitive disease, while 18 had platinum-resistant disease. The mean total PR H- score of platinum-sensitive tumors was significantly higher than that of platinum-resistant tumors (p = 0.002). Although no significant differences in progression-free and overall survival were observed between patients with high and low PR expression, those with high PR expression tended to have longer survival. While PR protein was only weakly detectable in ES-2 and TOV-21G cells, a transfection of the PR-A or PR-B gene resulted in a strong expression of PR-A or PR-B, which led to significantly reduced proliferation and migration in ES-2 and TOV-21G cells. Furthermore, overexpression of PR-A or PR-B enhanced cisplatin cytotoxicity in these cell lines. In conclusion, strong PR expression was associated with improved platinum sensitivity and survival outcomes, consistent with our experimental findings. The potential of PR as a tumor sensitizer to cisplatin in OCCC warrants further investigation.

Tumour-derived exosome SNHG17 induced by oestrogen contributes to ovarian cancer progression via the CCL13-CCR2-M2 macrophage axis.

Oestrogen is known to be strongly associated with ovarian cancer. There was much work to show the importance of lncRNA SNHG17 in ovarian cancer. However, no study has revealed the molecular regulatory mechanism and functional effects between oestrogen and SNHG17 in the development and metastasis of ovarian cancer. In this study, we found that SNHG17 expression was significantly increased in ovarian cancer and positively correlated with oestrogen treatment. Oestrogen could promote M2 macrophage polarization as well as ovarian cancer cells SKOV3 and ES2 cell exosomal SNHG17 expression. When exposure to oestrogen, exosomal SNHG17 promoted ovarian cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) invitro, and tumour growth and lung metastasis invivo by accelerating M2-like phenotype of macrophages. Mechanically, exosomal SNHG17 could facilitate the release of CCL13 from M2 macrophage via the PI3K-Akt signalling pathway. Moreover, CCL13-CCR2 axis was identified to be involved in ovarian cancer tumour behaviours driven by oestrogen. There results demonstrate a novel mechanism that exosomal SNHG17 exerts an oncogenic effect on ovarian cancer via the CCL13-CCR2-M2 macrophage axis upon oestrogen treatment, of which SNHG17 may be a potential biomarker and therapeutic target for ovarian cancer responded to oestrogen.

Patient-Derived Exosomes as siRNA Carriers in Ovarian Cancer Treatment.

RNA interference is a powerful gene-silencing tool with potential clinical applications. However, its therapeutic use is challenging because suitable carriers are unavailable. Exosomes are stable small endogenous vesicles that can transport functional molecules to target cells, making them ideal small interfering RNA (siRNA) carriers. Herein, we elucidated the therapeutic potential of patient-derived exosomes as an siRNA carrier for ovarian cancer (OC) treatment. The exosomes were extracted from the culture medium of primary fibroblasts collected from the omentum of patients with OC during surgery. MET proto-oncogene, receptor tyrosine kinase (MET) was selected for gene silencing, c-Met siRNAs were synthesized and loaded into the exosomes (Met-siExosomes) via electroporation, and the treatment effect of the Met-siExosomes was assessed in vitro and in vivo. The Met-siExosomes downregulated the c-Met protein levels and inhibited OC cell proliferation, migration, and invasion. In xenograft experiments using SKOV3-13 and ES-2 cells, Met-siExosomes were selectively extracted from peritoneally disseminated tumors. Intraperitoneal treatment suppressed the c-Met downstream targets in cancer cells and prolonged mouse survival. The synthesized siRNAs were successfully and selectively delivered via the exosomes to intraperitoneally disseminated tumors. As patients with OC routinely undergo omentectomy and abundant fibroblasts can be easily collected from the omentum, patient-derived exosomes may represent a promising therapeutic siRNA carrier to treat OC.

Abstract 644: PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines

Abstract Ovarian cancer is the most lethal malignant disease of female. Treatment options for ovarian cancer are mostly the use of chemotherapeutics, surgery, or radiotherapy. However, due to resistance to drugs, relapse occurs and thus 5 years survival rate is only 47%. The tumor suppressor protein p53 works as a transcription factor to transcribe several proteins such as p21, p16 and PUMA that lead to halt cell cycle and also induce apoptosis when there is a damage in DNA. Mutation in several regions of p53 (mutp53) is quite common in cancers and can lead to the accumulation of p53 with loss of functions or sometimes gain of oncogenic functions. Thus there is a search for drugs that can degrade the mut53 and can restore the functions of normal p53. In this research, it was observed that the treatment of ovarian cancer cell lines (ES-2, HEY and OVCAR3) with atypical protein kinase C-iota (aPKCɩ) specific inhibitors ICA-1S can cause the degradation of mutp53 (53.6%, N=6 trials). Treatment with ICA-1S also lowered the level of phosphorylated p53 (ser15, 87%, N=3 trials). However, the level of MDM2 protein (murine double minute 2), a known p53 E3 ligase was not significantly (98%, N=2 trials) affected by ICA-1S in ES-2 cell line with mut p53 as well as p21 was also not affected. However level of PUMA was decreased both in ES-2 and HEY cell lines. Immunoprecipitation (IP) with aPKCɩ specific beads showed the aPKCɩ is associated with p53, though reverse IP with p53 beads was not able to show the association. Application of proteasome inhibitor MG-132 at a single dose of 0.5µM could not completely inhibit the degradation of mut53 and WTp53 in ICA-1S treated ES-2 cell line and HEY cell line, respectively, indicating the drug may be affecting the transcription of p53 as well. It is therefore postulated that aPKCɩ can phosphorylate p53, stabilizing it and may be affecting the transcription of p53 as well in those cell lines. Further research (kinase activity assay, immunoprecipitation, immunofluorescence) is going on to reveal the mode of this degradation. Citation Format: Mahfuza Marzan, Nuzhat Nowshin Oishee, Abigail Olatunji, Mildred Acevedo-Duncan. PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines [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 644.

Abstract A032: The role of ASIC2 and calcium influx in epithelial ovarian cancer pathogenesis

Abstract Objective: Inflammation associated with incessant ovulation plays a key role in epithelial ovarian cancer (EOC) pathogenesis. Ion channels, such as acid-sensing ion channel-2 or ASIC2 is known to be upregulated in inflammatory conditions and may play a role in calcium ion dysregulation. Here, we describe a potential relationship between ASIC2 and intracellular calcium levels in EOC development and progression. Methods: EOC cell lines OVCAR-8, ES-2 and SKOV-3 were grown to sub-confluence prior to experimentation. Global extracellular pH was changed with the use of hydrochloric acid (HCl) over various time points at hours (h): 2 h, 4 h, 8 h, 12 h and 24 h. ASIC2 expression was subsequently measured via western blot. Intracellular pH was measured with the use of pH-sensitive pHrodo red dye. Cytosolic pH was determined across all cell lines at baseline and after treatment with a known ASIC2 inhibitor: Diminazene (10 μM) over 30 minutes. Intracellular calcium levels were measured with a Fluo Calcium Indicator at baseline and with similar ASIC2 inhibition. Live cell imaging was utilized on a Nikon A1r confocal microscope. Results: Changing global, extracellular pH to 6.0 and 6.5 across various time points from 2-24 h did not affect ASIC2 protein expression via western blot analysis. We then utilized a pH-sensitive assay to determine whether intracellular pH changes were associated with ASIC2 expression. Interestingly, intracellular pH was noted to increase across most cell lines with ASIC2 inhibition via Diminazene treatment. There was a statistically significant increase in pH in ES2 and OVCAR8 cell lines. Because ASIC2 is known to result in influx of various ions, assessment of intracellular calcium levels was performed first after 30 minutes of Diminazene treatment. There was a corresponding significant increase in intracellular calcium levels in ES2 cells after ASIC2 inhibition. And there was a small trend, albeit not significant, toward a similar increase across SKOV-3 and OVCAR-8 cells. Conclusion: There seems to be a role for ASIC2 in EOC, particularly in the context of inflammation as noted by changes in intracellular pH levels. It is likely that ASIC2 may mediate changes in intracellular calcium signaling and promote tumorigenesis in ovarian cancer. Citation Format: Tanvi Joshi, Annelise Wilhite, Sagar Chokshi, Mary Howard Singleton, Jennifer Scalici, Kevin Lee. The role of ASIC2 and calcium influx in epithelial ovarian cancer pathogenesis [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 A032.

Abstract A020: NAD+ Boosting through NRH supplementation enhances treatment efficacy in EOC

Abstract Background: Dihydronicotinamide riboside (NRH), the reduced form of nicotinamide riboside (NR), is a recently identified, naturally occurring precursor of arguably the most crucial cofactor for cellular function, Nicotinamide Adenine Dinucleotide (NAD+). Recent investigation suggests that NRH is more adept at increasing NAD+ stores than traditional NAD precursors, and such extreme NAD+ boosting via NRH supplementation induces cytotoxicity in certain cellular contexts. It has also been shown that the lack of functional BRCA protein in epithelial ovarian cancer (EOC) directly impacts intracellular NAD+ levels. Given that altered cellular metabolism and DNA repair mechanisms are central alterations in EOC, and these processes are functionally dependent on NAD+, we sought to assess whether NRH supplementation in EOC cell lines enhanced cellular cytotoxicity alone and in combination with standard therapeutic agents. Methods: We supplemented 3 ovarian cancer cell lines (SKOV3, OVCAR8 and ES2) with NRH and traditional NAD precursor, NA (nicotinic acid) alone and in combination with carboplatin, paclitaxel, combination, and olaparib. Cytotoxicity was measured via CellTiter-Glo. RNA sequencing of EOC cell lines was performed to determine the differential expression of NAD+ anabolic and catabolic enzymes. Of note, all three cell lines are known to be BRCA1 &amp;2 wildtype while OVCAR8 cells are hypermethylated and harbor loss of heterozygosity (LOH high) at the BRCA1 loci. Results: Significant cytotoxicity was noted in NRH treated cells (~40 %) with minimal cell death in the NA treated lines. Strikingly, NRH supplementation enhanced carboplatin cytotoxicity by 25-50 % depending on cell line, paclitaxel cytotoxicity by 10-25 %, and the combination by 10-35 % whereas NA supplementation had no effect on the baseline treatment induced cytotoxicity. Interestingly, the effect of NRH supplementation with olaparib differed between the ES2 and OVCAR8 cell lines, with a significant 20 % increase in cell death in ES2 vs OVCAR8 cells with a negligible increase in cell death. PARP inhibition by olaparib requires NAD+. RNA sequencing revealed an increased expression in NAMPT, an enzyme involved in the NAD+ salvage pathway in the ES2 cell line relative to the OVCAR8, suggesting that the ES2 cells harbor a baseline NAD+ deficit rendering them more vulnerable to PARP inhibition following NAD+ boosting. This is also consistent with the OVCAR8 data suggesting that hypermethylated BRCA1 results in a relative increase in NAD+ to the extent that NRH supplementation does not enhance the efficacy of PARP inhibition. Conclusions: Here we show that in the context of EOC, exploitation of the NAD+ bioenergetic phenotype through NRH supplementation is a biologically feasible strategy to enhance the response of traditional therapy with potentially minimal toxicity. This data suggests several potential mechanisms by which cellular NAD+ availability belies treatment efficacy and resistance and highlights the potential utility of NAD+ metabolomics as a biomarker to guide treatment decisions. Citation Format: Kevin Lee, Tanvi Joshi, Catherine Lyons, Mary Howard Singleton, Elizabeth Catranis, Sagar Chokshi, Luciana Madiera da Silva, Marie Migaud, Jennifer Scalici. NAD+ Boosting through NRH supplementation enhances treatment efficacy in EOC [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 A020.

Antitumor effects of redox-responsive nanoparticles containing platinum（Ⅳ）in ovarian cancer

Objectives: To explore the antitumor effects of redox-responsive nanoparticles containing platinum(Ⅳ)-NP@Pt(Ⅳ) in ovarian cancer. Methods: Redox-responsive polymer carriers were synthesized. Polymer carriers and platinum(Ⅳ)-Pt(Ⅳ) can self-assemble into NP@Pt(Ⅳ). Inductively coupled plasma mass spectrometry was performed to detect the platinum release from NP@Pt(Ⅳ) in reducing environment and the platinum content in ovarian cancer cells ES2 treated with cisplatin, Pt(Ⅳ) and NP@Pt(Ⅳ). The proliferation ability of the ovarian cancer cells were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was assessed by flow cytometry. Collection of primary ovarian cancer tissues from patients with primary high-grade serous ovarian cancer who were surgically treated at the Cancer Hospital of the Chinese Academy of Medical Sciences from October to December 2022. The high-grade serous ovarian cancer patient-derived xenograft (PDX) mice were intravenously injected with Cy7.5 labeled NP@Pt(Ⅳ) followed by in vivo imaging system. Mice were treated with PBS, cisplatin and NP@Pt(Ⅳ). Tumor volume and weight were measured in each group. Necrosis, apoptosis and cell proliferation of tumor tissues were detected by hematoxylin-eosin (HE) staining, TUNEL fluorescence staining and Ki-67 immunohistochemistry staining. Body weight and HE staining of heart, liver, spleen, lung and kidney of mice in each group were measured. Results: The platinum release of NP@Pt(Ⅳ) after 48 hours in reducing environment was 76.29%, which was significantly higher than that of 26.82% in non-reducing environment (P<0.001). The platinum content in ES2 cells after 4 hours and 7 hours of treatment with NP@Pt(Ⅳ) (308.59, 553.15 ng/million cells) were significantly higher than those of Pt(Ⅳ) (100.21, 180.31 ng/million cells) and cisplatin (43.36, 50.36 ng/million cells, P＜0.05). The half inhibitory concentrations of NP@Pt(Ⅳ) in ovarian cancer cells ES2, A2780, A2780DDP were 1.39, 1.42 and 4.62 μmol/L, respectively, which were lower than those of Pt(IV) (2.89, 7.27, and 16.74 μmol/L) and cisplatin (5.21, 11.85, and 71.98 μmol/L). The apoptosis rate of ES2 cells treated with NP@Pt(Ⅳ) was (33.91±3.80)%, which was significantly higher than that of Pt(Ⅳ) [(16.28±2.41)%] and cisplatin [(15.01±1.17)%, P＜0.05]. In high-grade serous ovarian cancer PDX model, targeted accumulation of Cy7.5 labeled NP@Pt(Ⅳ) at tumor tissue could be observed. After the treatment, the tumor volume of mice in NP@Pt(IV) group was (130±98) mm3, which was significantly lower than those in control group [(1 349±161) mm3, P<0.001] and cisplatin group [(715±293) mm3, P=0.026]. The tumor weight of mice in NP@Pt(IV) group was (0.17±0.09)g, which was significantly lower than those in control group [(1.55±0.11)g, P<0.001] and cisplatin group [(0.82±0.38)g, P=0.029]. The areas of tumor necrosis and apoptosis in mice treated with NP@Pt(Ⅳ) were higher than those in mice treated with cisplatin. Immunohistochemical staining revealed that there were low expressions of Ki-67 at tumor tissues of mice treated with NP@Pt(Ⅳ) compared with cisplatin. The change in body weight of mice in NP@Pt(Ⅳ) group was not significantly different from that of the control group [(18.56±2.04)g vs.(20.87±0.79)g, P=0.063]. Moreover, the major organs of the heart, liver, spleen, lung, and kidney were also normal by HE staining. Conclusion: Redox-responsive NP@Pt(Ⅳ), produced in this study can enhance the accumulation of cisplatin in ovarian cancer cells and improve the efficacy of ovarian cancer chemotherapy.

Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer

Although the pro-tumorigenic functions of hyaluronan (HA) are well documented there is limited information on the effects and targets of different molecular weight HA. Here, we investigated the effects of 27 kDa, 183 kDa and 1000 kDa HA on ES-2 ovarian cancer cells overexpressing the stem cell associated protein, Notch3. 1000 kDA HA promoted spheroid formation in ES-2 cells mixed with ES-2 overexpressing Notch3 (1:3). We report disabled-2 (DAB2) as a novel protein regulated by 1000 kDa HA and further investigated its role in ovarian cancer. DAB2 was downregulated in ovarian cancer compared to normal tissues but increased in metastatic ovarian tumors compared to primary tumors. High DAB2 expression was associated with poor patient outcome and positively correlated with HA synthesis enzyme HAS2, HA receptor CD44 and EMT and macrophage markers. Stromal DAB2 immunostaining was significantly increased in matched ovarian cancer tissues at relapse compared to diagnosis and associated with reduced survival. The proportion of DAB2 positive macrophages was significantly increased in metastatic ovarian cancer tissues compared to primary cancers. However, DAB2 overexpression significantly reduced invasion by both A2780 and OVCAR3 cells in vivo. Our research identifies a novel relationship between HA signalling, Notch3 and DAB2. We highlight a complex relationship of both pro-tumorigenic and tumor suppressive functions of DAB2 in ovarian cancer. Our findings highlight that DAB2 has a direct tumor suppressive role on ovarian cancer cells. The pro-tumorigenic role of DAB2 may be mediated by tumour associated macrophages and requires further investigation.

Copine 1 predicts poor clinical outcomes by promoting M2 macrophage activation in ovarian cancer.

Copine 1 (CPNE1), a membrane-binding protein, influences the prognosis of various cancers. According to cBioPortal, CPNE1 amplification is a prevalent genetic mutation in ovarian cancer but with unknown oncogenic mechanism. This study analysed the CPNE1 expression in ovarian cancer using online datasets, as validated by immunohistochemistry (IHC), quantitative polymerase chain reaction (qPCR) and western blotting. Concurrently, the prognostic value of CPNE1 was accessed. Cell Counting Kit-8, colony formation, transwells and xenograft experiments were performed to evaluate the functions of CPNE1 during ovarian cancer carcinogenesis. CPNE1 and its related genes were analysed by g:Profiler and Tumour Immune Estimation Resource. Furthermore, human monocytic THP-1 cells were co-cultured with ES2 cells to investigate the effect of CPNE1 on macrophage polarization. The results of bioinformatic analysis, IHC, qPCR and western blotting indicated a higher CPNE1 in ovarian cancer. CPNE1 overexpression demonstrated an association with a poor prognosis of ovarian cancer. Functionally, CPNE1 overexpression increased ES2 and SKOV3 cell proliferation, invasion and migration in vitro and promoted ovarian tumour xenograft growth in vivo, while CPNE1 knockdown led to opposite effects. Additionally, CPNE1 expression demonstrated an association with immune cell infiltration in ovarian cancer, especially macrophage. CPNE1 promoted protumour M2 macrophage polarization by upregulating cluster of differentiation 163 (CD163), CD206 and interleukin-10. Our study revealed that CPNE1 mediated M2 macrophage polarization and provided a therapeutic target for ovarian cancer.

Preparation and properties of tumor-targeting lentinan-oleic acid polymer micelles based on folic acid for adriamycin delivery

To develop a natural polysaccharide targeted nanomicelles drug delivery system loaded with the broad-spectrum anti-tumor drug adriamycin (ADM), FA-LNT-OA polymer was synthesized, the ADM/FA-LNT-OA polymer nanomicelles was prepared, and response surface approach was used to optimize the drug loading procedure of the ADM/FA-LNT-OA nanomicelles. Under the optimum conditions, the drug loading ratio was 14.36 ± 0.12% while the entrapment efficiency was 95.17 ± 0.13%. The drug-loaded micelles were spherical, with the size about 170 nm, and the size distribution was uniform. The CMC of ADM/FA-LNT-OA nanomicelles was 48 ± 0.46 μg/mL, their release was more sensitive to pH than that of ADM, and they had good stability. The FA-LNT-OA nanomicelles had good blood compatibility and cell compatibility. In vitro anti-tumor pharmacodynamic studies proved that the IC50 of ADM/FA-LNT-OA nanomicelles on Hela, ES-2 and A549 cells were 48.49 ± 0.25, 63.56 ± 0.68 and 105.93 ± 17.03 μg/mL respectively. Moreover, the plasma elimination half-life of ADM/FA-LNT-OA nanomicelles was 5.341 h, the Tmax was 0.083 h, the CL was 0.017 L/h/kg. Compared with the common ADM formulation, the ADM/FA-LNT-OA nanomicelles prolonged the clearance half-life of ADM, decreased the clearance rate, and extended the retention time of ADM in vivo. In conclusion, the above results laid the foundation for the increase of therapeutic effects and reduction of the clinical toxic and side effects of ADM.

Staphylococcal Enterotoxin C2 Mutant-Induced Antitumor Immune Response Is Controlled by CDC42/MLC2-Mediated Tumor Cell Stiffness.

As a biological macromolecule, the superantigen staphylococcal enterotoxin C2 (SEC2) is one of the most potent known T-cell activators, and it induces massive cytotoxic granule production. With this property, SEC2 and its mutants are widely regarded as immunomodulating agents for cancer therapy. In a previous study, we constructed an MHC-II-independent mutant of SEC2, named ST-4, which exhibits enhanced immunocyte stimulation and antitumor activity. However, tumor cells have different degrees of sensitivity to SEC2/ST-4. The mechanisms of immune resistance to SEs in cancer cells have not been investigated. Herein, we show that ST-4 could activate more powerful human lymphocyte granule-based cytotoxicity than SEC2. The results of RNA-seq and atomic force microscopy (AFM) analysis showed that, compared with SKOV3 cells, the softer ES-2 cells could escape from SEC2/ST-4-induced cytotoxic T-cell-mediated apoptosis by regulating cell softness through the CDC42/MLC2 pathway. Conversely, after enhancing the stiffness of cancer cells by a nonmuscle myosin-II-specific inhibitor, SEC2/ST-4 exhibited a significant antitumor effect against ES-2 cells by promoting perforin-dependent apoptosis and the S-phase arrest. Taken together, these data suggest that cell stiffness could be a key factor of resistance to SEs in ovarian cancer, and our findings may provide new insight for SE-based tumor immunotherapy.

BPA exposure enhances the metastatic aggression of ovarian cancer through the ERα/AKT/mTOR/HIF-1α signaling axis.

Long-term exposure to bisphenol A (BPA) in humans may promote ovarian cancer development. In present study, the mechanisms by which BPA mediates the aggression metastatic behavior of ovarian cancer were investigated in vitro/in vivo. The results showed that BPA (10μM) significantly promoted the proliferation, migration and invasion of human ovarian cancer cells (ES-2 and OVCAR-3cells); moreover, it promoted ES-2 and OVCAR-3cell glucose uptake, lactic acid release and intracellular ATP synthesis. After administration of 5μg/kg/day BPA, tumor volume was increased compared with that in control group. KEGG and GO enrichment analyses showed that the genes from ES-2cell in 10μM BPA-treated group were enriched mainly in central carbon metabolism and PI3K-AKT signaling pathway. Then, qRT‒PCR and western blotting results showed that BPA (10μM) increased the mRNA and protein expression levels of glycolysis-related genes and mTOR, p-AKT HIF-1α and ERα in vitro/vivo; whereas this effect was reduced after treatment with the ERα inhibitor methyl-piperidino-pyrazole. Furthermore, coimmunoprecipitation and mass spectrometry showed that BPA promoted the direct interaction of ERα with lactate dehydrogenase A. These results show that BPA directly promoted the proliferation, migration and invasion of ovarian cancer cells through the ERα/AKT/mTOR/HIF-1α signaling axis to enhance glycolysis.

Inhibiting eukaryotic initiation factor 5A (eIF5A) hypusination attenuated activation of the SIK2 (salt-inducible kinase 2)-p4E-BP1 pathway involved in ovarian cancer cell proliferation and migration.

Eukaryotic initiation factor 5A hypusine (eIF5AHyp) stimulates the translation of proline repeat motifs. Salt inducible kinase 2 (SIK2) containing a proline repeat motif is overexpressed in ovarian cancers, in which it promotes cell proliferation, migration, and invasion. Western blotting and dual luciferase analyses showed that depletion of eIF5AHyp by GC7 or eIF5A-targeting siRNA downregulated SIK2 level and decreased luciferase activity in cells transfected with a luciferase-based reporter construct containing consecutive proline residues, whereas the activity of the mutant control reporter construct (replacing P825L, P828H, and P831Q) did not change. According to the MTT assay, GC7, which has a potential antiproliferative effect, reduced the viability of several ovarian cancer cell lines by 20-35% at high concentrations (ES2 > CAOV-3 > OVCAR-3 > TOV-112D) but not at low concentrations. In a pull-down assay, we identified eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP1 (p4E-BP1) phosphorylated at Ser 65 as downstream binding partners of SIK2, and we validated that the level of p4E-BP1(Ser 65) was downregulated by SIK2-targeting siRNA. Conversely, in ES2 cells overexpressing SIK2, the p4E-BP1(Ser65) level was increased but decreased in the presence of GC7 or eIF5A-targeting siRNA. Finally, the migration, clonogenicity, and viability of ES2 ovarian cancer cells were reduced by GC7 treatment as well as by siRNA for eIF5A gene silencing and siRNA for SIK2 and 4E-BP1 gene silencing. Conversely, those activities were increased in cells overexpressing SIK2 or 4E-BP1 and decreased again in the presence of GC7. The depletion of eIF5AHyp by GC7 or eIF5A-targeting siRNA attenuated activation of the SIK2-p4EBP1 pathway. In that way, eIF5AHyp depletion reduces the migration, clonogenicity, and viability of ES2 ovarian cancer cells.

Establishment of highly metastatic ovarian cancer model with omental tropism via in vivo selection

Epithelial ovarian cancer (OC) is often diagnosed at an advanced stage with peritoneal metastasis, and preclinical models mimicking the natural course of OC peritoneal metastasis are essential to improve treatment. We implanted ES2 and ID8 cells in the ovaries of mice and obtained highly metastatic (HM) sublines from their omental metastases after three cycles invivo selection. Orthotopic xenografts derived from the HM sublines showed enhanced omental tropism and more extensive metastasis with earlier onset. The HM cells exhibited increased invitro migration and invasion properties, and RNA sequencing revealed that the genes related to epithelial-mesenchymal transition and extracellular matrix regulation were significantly altered in the HM cells. Among them, the upregulated genes were significantly associated with poorer survival in OC patients. In conclusion, these HM sublines can be leveraged to establish spontaneous metastatic OC mouse models, which may serve as ideal preclinical models for anti-metastasis therapy for OC patients.

Whole-exome mutational landscape and molecular marker study in mucinous and clear cell ovarian cancer cell lines 3AO and ES2

BackgroundOvarian cancer is one of the most lethal cancers in women because it is often diagnosed at an advanced stage. The molecular markers investigated thus far have been unsatisfactory.MethodsWe performed whole-exome sequencing on the human ovarian cancer cell lines 3AO and ES2 and the normal ovarian epithelial cell line IOSE-80. Molecular markers of ovarian cancer were screened from shared mutation genes and copy number variation genes in the 6q21-qter region.ResultsWe found that missense mutations were the most common mutations in the gene (93%). The MUC12, FLG and MUC16 genes were highly mutated in 3AO and ES2 cells. Copy number amplification occurred mainly in 4p16.1 and 11q14.3, and copy number deletions occurred in 4q34.3 and 18p11.21. A total of 23 hub genes were screened, of which 16 were closely related to the survival of ovarian cancer patients. The three genes CCDC170, THBS2 and COL14A1 are most significantly correlated with the survival and prognosis of ovarian cancer. In particular, the overall survival of ovarian cancer patients with high CCDC170 gene expression was significantly prolonged (P < 0.001). The expression of CCDC170 in normal tissues was significantly higher than that in ovarian cancer tissues (P < 0.05), and its expression was significantly decreased in advanced ovarian cancer. Western blotting and immunofluorescence assays also showed that the expression of CCDC170 in ovarian cancer cells was significantly lower than that in normal cells (P < 0.001, P < 0.01).ConclusionsCCDC170 is expected to become a new diagnostic molecular target and prognostic indicator for ovarian cancer patients, which can provide new ideas for the design of antitumor drugs.

Abstract 194: Ovarian cancer patient-derived organoids and tumor xenografts as preclinical models for precision medicine

Abstract Background: Patient-derived organoids (PDO) and patient-derived xenografts (PDX) has been investigated as preclinical models for drug development and precision medicine. In this study, we established ovarian cancer PDOs and PDX models from Aug 2020 to Nov 2022, and developed protocols. Methods: We obtained tumor tissue from several sites (ovary, omentum, diaphragm, and peritoneum) or ascites from surgery of ovarian cancer patients. The tissue samples were dissociated for single cells and ascitic samples were centrifuged and washed to concentrate the tumor cells. Then, it is embedded in matrigel or basement membrane extract (BME) and seeded on a 24-well plate supplemented with culture medium containing modified growth factors. The association with clinical characteristics with success of model establishment were evaluated. Drug screening for organoids were designed on the 384-well plate using Celltiter-Glo 3D cell viability assay (Promega). The list of drugs consists of alkylating agent, monoclonal antibody, taxane, tyrosine kinase inhibitor (TKI), and poly(ADP-ribose) polymerase inhibitor (PARPi). For increase of success rate for xenograft, we compared intrabursa and subcutaneous injection xenograft models using ES-2 cell line then patient tumors in NOG mouse. Results: A total of 397 organoids passed first passage, however 32 (8%) organoids were continuously cultured over 5 passages. The associated clinical factors for establishment of organoids were stage (p=0.020, low stage 20% of success vs. high stage 2.4% of success) and pathologic types (p=0.035, low grade serous cancer (LGSC) 30% and clear cell 16.7% of success vs. high grade serous cancer (HGSC) 5.6% of success). Thirteen drugs were evaluated and AUC represented various range according to patient from 0.0004 to 1.3876. Three sites (ovary, omentum and ascites) established organoids from one patient represented difference in drug sensitivity which suggest role of cancer microenvironment. The growth upto 100mm3 of intrabursa injection (12.33 ± 0.6667) was faster than that of subcutaneous injection model (15.00 ± 1.000, p-value = 0.09) using ES-2 cell. Then comparison of patients tumor (N=10) into intrabursa vs. subcutaneous are undergoing with one completed case showing fast intrabursa growing. Conclusions: During relative short period we developed many PDOs and PDXs for future study including drug development. Moreover, we focused to develop better model for PDXs and it will give a new approach for successful model establishment. (This work was supported by National Research Foundation of Korea grant, founded by the Korea government (MSIT) [No.2020R1A2C2010566]) Citation Format: Yebeen Yu, Hye Ju Park, Cha Yeon Kang, Myong Cheol Lim, Sang Yoon Park, Yo Han Woo, Yun-Hee Kim, Sun-Young Kong. Ovarian cancer patient-derived organoids and tumor xenografts as preclinical models for precision medicine [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 194.

Abstract 5023: PFKP contributes to ovarian cancer metastasis, stemness and anchorage-independent growth via α5 integrin and ERK/MMP9 signaling

Abstract Background: Ovarian cancer is one of the most aggressive malignancies threatening women’s health worldwide. Its high mortality rate is largely due to symptoms not appearing until late in the disease, usually after the cancer has metastasized. Despite current state-of-the-art treatments, the prognosis for patients remains poor. New effective treatments are urgently needed to improve patient survival. High lactate production and low glucose oxidation, independent of oxygen availability, is known as the Warburg effect and is common in cancer. Phosphofructokinase 1 (PFK-1) irreversibly converts fructose 6-phosphate to fructose 1,6-bisphosphate, the first step in the Warburg effect. The platelet isoform of PFK (PFKP) regulates glycolysis and plays roles in tumorigenesis in other human cancers, the precise mechanisms has yet to be defined. Moreover, PFKP in ovarian cancer remain unknown. We hypothesize that PFKP modulates metastasis and stemness in ovarian cancer. Methods: Expression of PFKP in clinical samples, ascites-derived tumor cells, normal ovarian epithelial cells (HOSEs) and ovarian cancer cell lines was determined by immunohistochemistry/immunofluorescence and qPCR/immunoblotting, respectively. The correlation between PFKP and clinicopathological parameters was analyzed. Ectopic expression or knockdown of PFKP followed by migration, invasion, sphere formation and soft agar assays were performed in ascites-derived tumor cells and ovarian cancer cell lines to investigate the effects on metastasis, stemness and malignant transformation respectively. Additionally, the related signaling pathway was explored by qPCR and immuoblotting. Results: Up-regulation of PFKP in clinical samples, ascites-derived tumor cells and ovarian cancer cell lines was found when compared to HOSEs. Higher PFKP was correlated to shorter overall and disease-free survival. Functionally, knockdown of PFKP by siRNA/shRNA approaches in ascites-derived tumor cells, ES-2 and HeyA8 cells decreased migration, invasion, sphere formation and colony formation in soft agar, which was accompanied by decreased expression α5 integrin, pERK and MMP9. Ectopic expression of PFKP in SKOV-3 cells showed opposite effects. Intriguingly, we found that MEK-1 inhibitor (U0126) or an anti-MMP9 neutralizing antibody could block PFKP-mediated migration/sphere formation abilities. Conclusion: PFKP was overexpressed in ovarian cancer and regulates metastasis, stemness and anchorage-independent growth. α5 integrin and ERK/MMP9 signaling were involved in such functional effects. PFKP could be a potential prognostic marker and therapeutic target for ovarian cancer. Citation Format: Ruiqian Zhang, Michelle K.Y. Siu, Xuetang Mo, Mingo M.H. Yung, Jingjing Wang, Yuxin Jiang, Annie N.Y. Cheung, Hextan Y.S. Ngan, Karen K.L. Chan. PFKP contributes to ovarian cancer metastasis, stemness and anchorage-independent growth via α5 integrin and ERK/MMP9 signaling. [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 5023.

Proteomic Study on Multi-Organ Metastases of Human Ovarian Clear Cell Carcinoma Cell Line in a Xenograft Mouse Model Based on a Novel Sequence-Specific Analysis Strategy.

To investigate the gene regulation of tumor cells in the process of different organ metastasis on a xenograft mouse model and screen the genes involved in the organ-target metastasis of tumor cells. A multi-organ metastasis model was constructed with a human ovarian clear cell carcinoma cell line (ES-2) based on a severe immunodeficiency mouse strain (NCG). Differentially expressed tumor proteins among multi-organ metastases were successfully characterized by microliter liquid chromatography-high-resolution mass spectrometry, sequence-specific data analysis and multivariate statistical data analysis. Liver metastases were selected as typical for subsequent bioinformatic analysis. Selected liver metastasis-specific genes in ES-2 cells were validated by sequence-specific quantitation including high resolution-multiple reaction monitoring quantification at protein level and quantitative real-time polymerase chain reaction at mRNA level. From the mass spectrometry data, a total of 4503 human proteins were identified using the sequence-specific data analysis strategy. Of them, 158 proteins were selected as specifically regulated genes in liver metastases for subsequent bioinformatics studies. Based on Ingenuity Pathway Analysis (IPA) pathway analysis and sequence-specific quantitation, Ferritin light chain (FTL), lactate dehydrogenase A (LDHA) and long-chain-fatty-acid-CoA ligase 1 (ACSL1) were finally validated as specifically upregulated proteins in liver metastases. Our work provides a new approach to analyze gene regulation in tumor metastasis in xenograft mouse model. In presence of a large number of mouse protein interference, we validated the up-regulation of human ACSL1, FTL and LDHA in ES-2 liver metastases, which reflects the adaptive regulation of tumor cells to the liver microenvironment through metabolic reprogramming.

Zinc Finger Protein 90 Knockdown Promotes Cisplatin Sensitivity via Nrf2/HO-1 Pathway in Ovarian Cancer Cell.

Our study discussed the role of Zfp90 in ovarian cancer (OC) cell lines' sensitivity to cisplatin. We used two OC cell lines, SK-OV-3 and ES-2, to evaluate their role in cisplatin sensitization. The protein levels of p-Akt, ERK, caspase 3, Bcl-2, Bax, E-cadherin, MMP-2, MMP-9 and other drug resistance-related molecules, including Nrf2/HO-1, were discovered in the SK-OV-3 and ES-2 cells. We also used a human ovarian surface epithelial cell to compare the effect of Zfp90. Our outcomes indicated that cisplatin treatment generates reactive oxygen species (ROS) that modulate apoptotic protein expression. The anti-oxidative signal was also stimulated, which could hinder cell migration. The intervention of Zfp90 could greatly improve the apoptosis pathway and block the migrative pathway to regulate the cisplatin sensitivity in the OC cells. This study implies that the loss of function of Zfp90 might promote cisplatin sensitization in OC cells via regulating the Nrf2/HO-1 pathway to enhance cell apoptosis and inhibit the migrative effect in both SK-OV-3 and ES-2 cells.