Human Epithelial Ovarian Cancer Cell Research Articles

AmphiregulinDownregulates E-cadherin Expressionby Activating YAP/Egr-1/Slug Signaling in SKOV3 Human Ovarian Cancer Cells.

Amphiregulin (AREG) stimulates human epithelial ovarian cancer (EOC) cell invasion by downregulating E-cadherin expression. YAP is a transcriptional cofactor that has been shown to regulate tumorigenesis. This study aimed to examine whether AREG activates YAP in EOC cells and explore the roles of YAP in AREG-induced downregulation of E-cadherin and cell invasion. Analysis of the Cancer Genome Atlas (TCGA) showed that upregulation of AREG and EGFR were associated with poor survival in human EOC. Treatment of SKOV3 human EOC cells with AREG induced the activation of YAP. In addition, AREG downregulated E-cadherin, upregulated Egr-1 and Slug, and stimulated cell invasion. Using gain- and loss-of-function approaches, we showed that YAP was required for the AREG-upregulated Egr-1 and Slug expression. Furthermore, YAP was also involved in AREG-induced downregulation of E-cadherin and cell invasion. This study provides evidence that AREG stimulates human EOC cell invasion by downregulating E-cadherin expression through the YAP/Egr-1/Slug signaling.

THUMPD3-AS1 inhibits ovarian cancer cell apoptosis through the miR-320d/ARF1 axis.

Ovarian cancer is one of the most common gynecologic malignancies that has a poor prognosis. THUMPD3-AS1 is an oncogenic long noncoding RNA (lncRNA) in several cancers. Moreover, miR-320d is downregulated and inhibited proliferation in ovarian cancer cells, whereas ARF1 was upregulated and promoted the malignant progression in epithelial ovarian cancer. Nevertheless, the role of THUMPD3-AS1 in ovarian cancer and the underlying mechanism has yet to be elucidated. Human normal ovarian epithelial cells (IOSE80) and ovarian cancer cell lines (CAVO3, A2780, SKOV3, OVCAR3, and HEY) were adopted for invitro experiments. The functional roles of THUMPD3-AS1 in cell viability and apoptosis were determined using CCK-8, flow cytometry, and TUNEL assays. Western blot was performed to assess the protein levels of ARF1, Bax, Bcl-2, and caspase 3, whereas RT-qPCR was applied to measure ARF1 mRNA, THUMPD3-AS1, and miR-320d levels. The targeting relationship between miR-320d and THUMPD3-AS1 or ARF1 was validated with dual luciferase assay. THUMPD3-AS1 and ARF1 were highly expressed in ovarian cancer cells, whereas miR-320d level was lowly expressed. THUMPD3-AS1 knockdown was able to repress cell viability and accelerate apoptosis of OVCAR3 and SKOV3 cells. Also, THUMPD3-AS1 acted as a sponge of miR-320d, preventing the degradation of ARF1. MiR-320d downregulation reversed the tumor suppressive function induced by THUMPD3-AS1 depletion. Additionally, miR-320d overexpression inhibited ovarian cancer cell viability and accelerated apoptosis, which was overturned by overexpression of ARF1. THUMPD3-AS1 inhibited ovarian cancer cell apoptosis by modulation of miR-320d/ARF1 axis. The discoveries might provide a prospective target for ovarian cancer treatment.

Injectable bio-multifunctional hyaluronic acid-based hydrogels loaded with poly ADP-ribose polymerase inhibitors for ovarian cancer therapy

The recent use of PARP inhibitors (PARPi) in the maintenance treatment of ovarian tumor has significantly improved the survival rates of cancer patients. However, the current oral administration of PARP inhibitors fails to realize optimal therapeutic effects due to the low bioavailability in cancerous tissues, and often leads to a range of systemic adverse effects including hematologic toxicities, digestive system reactions, and neurotoxicities. Therefore, the demand for an advanced drug delivery system that can ensure effective drug administration while minimizing these unfavorable reactions is pressing. Injectable hydrogel emerges as a promising solution for local administration with the capability of sustainable drug release. In this study, we developed an injectable hydrogel made from aminated hyaluronic acid and aldehyde-functionalized pluronic127 via Schiff base reaction. This hydrogel exhibits excellent injectability with short gelation time and remarkable self-healing ability, and is applied to load niraparib. The drug-loaded hydrogel (HP@Nir hydrogel) releases drugs sustainably as tested in vitro as well as displays significant anti-proliferation and anti-migratory properties on human epithelial ovarian cancer cell line. Notably, HP@Nir hydrogel effectively suppresses the growth of ovarian cancer, without significant adverse reactions as demonstrated in animal studies. Additionally, the developed hydrogel is gradually degraded in vivo for around 20 d, while maintaining good biocompatibility. Overall, the injectable hydrogel loaded with niraparib provides a secure and efficient strategy for the treatment and management of ovarian cancer.

Circ-RNF111 Promotes Proliferation of Ovarian Cancer Cell SKOV-3 by Targeting the MiR-556-5p/CCND1 Axis.

The aim of this study was to investigate the role and mechanism of circ-RNF111 in the human ovarian cancer cell line SKOV-3. First, qRT-PCR was used to detect circ-RNF111 and miR-556-5p expression levels in human normal ovarian epithelial cells IOSE80 and human ovarian cancer cells SKOV-3. CCK-8 and colony formation assays were adopted to determine the proliferation rate and cell viability of SKOV-3 cells, respectively. Additionally, in an attempt to reveal the mechanism of circ-RNF111, we predicted the targeting relationship between miR-556-5p and circ-RNF111 as well as miR-556-5p and CCND1 using the circinteractome and TargetScan databases, respectively, and validated their relationship by dual-luciferase reporter assay. The protein expression levels of CCND1 in SKOV-3 cells were detected by Western blot. Based on the above experiments, the expression of circ-RNF111 was found to be up-regulated in SKOV-3, and the knockdown of circ-RNF111 significantly inhibited the proliferation and viability of SKOV-3 cells. Then we confirmed that circ-RNF111 sponged miR-556-5p in SKOV-3 cells to up-regulate CCND1 expression. In addition, simultaneous inhibition of miR-556-5p or overexpression of CCND1 in SKOV-3 cells with knockdown of circ-RNF111 reversed the inhibitory effect of knockdown of circ-RNF111 on the protein expression level of CCND1, cell proliferation rate, and cell viability. In summary, circ-RNF111 promotes the proliferation of SKOV-3 cells by targeting the miR-556-5p/CCND1 axis. Circ-RNF111 may serve as a potential target for ovarian cancer therapy.

Mechanism of Ellagic Acid-Induced Autophagy in Cis-Diamminedichloroplatinum(II)-Resistant Ovarian Cancer Cells

This study investigated the effect of ellagic acid on autophagy in cisplatin-resistant human epithelial ovarian cancer cells. After the autophagy double-labeled mred fluorescence protein, green fluorescence protein, light chain 3 carrying adenovirus infected cisplatin-resistant human epithelial ovarian cancer cells, the changes of autophagy flux and autophagy-related proteins were detected. Cell counting kit-8 assay was employed to detect the viability of cisplatin-resistant human epithelial ovarian cancer cells. The results showed that the number of autophagosomes and autolysosomes in the ellagic acid group increased significantly in comparison with the in cisplatin-resistant human epithelial ovarian cancer group. With the increase in ellagic acid concentration, light chain 3-II/I ratio and Beclin1 expression increased, p62 expression and cell viability decreased, and caspase-3 activated, in cisplatin-resistant human epithelial ovarian cancer cells. Conversely, 3-methyladenine inhibited ellagic acid-induced autophagy and activated caspase-3 and enhanced the cell viability. Similarly, chloroquine significantly increased cell survival rate, light chain 3-II/I ratio, and activated caspase-3, but decreased p62 expression in the ellagic acid group. In conclusion, ellagic acid can induce autophagy to inhibit the proliferation of cisplatin-resistant human epithelial ovarian cancer cells.

Polystyrene nanoparticle exposure accelerates ovarian cancer development in mice by altering the tumor microenvironment

Microplastics and nanoplastics are ubiquitous pollutants, widely spread in the living and natural environment. Although their potential impact on human health has been investigated, many doubts remain about their effects in carcinogenic processes. We investigated the potential effects and its molecular mechanisms of polystyrene nanoplastics (PS-NPs) on epithelial ovarian cancer (EOC) using the human EOC cell line HEY as an in vitro cell model and mice as a mammalian model. In vivo exposure to PS-NPs (100 nm; 10 mg/L) via drinking water significantly accelerated EOC tumor growth in mice. In in vitro tests the PS-NPs reduced the relative viability of EOC cells in a dose-dependent manner. Histological analysis showed increased mitotic counts in EOC tumor tissues of PS-NP exposed mice. PS-NP exposure significantly affected gene expression and disturbed many metabolic pathways in both cultured EOC cells and EOC tumor tissue in mice. Gene functional and pathway analysis indicated that immune-related responses and the tumor microenvironment pathway were significantly enriched, which may be attributed to disturbed expression of thrombomodulin (THBD) and its regulators. It may be concluded that PS-NP exposure caused a significant acceleration of EOC tumor growth in mice and a dose-dependent decrease in the relative viability of EOC cells by altering the tumor growth microenvironment. This offers new insights into the mechanisms underlying PS-NP effects on EOC.

Endoplasmic Reticulum Stress-Related Ten-Biomarker Risk Classifier for Survival Evaluation in Epithelial Ovarian Cancer and TRPM2: A Potential Therapeutic Target of Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignant tumor. Endoplasmic reticulum (ER) stress plays an important role in the malignant behaviors of several tumors. In this study, we established a risk classifier based on 10 differentially expressed genes related to ER stress to evaluate the prognosis of patients and help to develop novel medical decision-making for EOC cases. A total of 378 EOC cases with transcriptome data from the TCGA-OV public dataset were included. Cox regression analysis was used to establish a risk classifier based on 10 ER stress-related genes (ERGs). Then, through a variety of statistical methods, including survival analysis and receiver operating characteristic (ROC) methods, the prediction ability of the proposed classifier was tested and verified. Similar results were confirmed in the GEO cohort. In the immunoassay, the different subgroups showed different penetration levels of immune cells. Finally, we conducted loss-of-function experiments to silence TRPM2 in the human EOC cell line. We created a 10-ERG risk classifier that displays a powerful capability of survival evaluation for EOC cases, and TRPM2 could be a potential therapeutic target of ovarian cancer cells.

NPFF stimulates human ovarian cancer cell invasion by upregulating MMP-9 via ERK1/2 signaling

Neuropeptide FF (NPFF) belongs to the RFamide peptide family. NPFF regulates a variety of physiological functions by binding to a G protein-coupled receptor (GPCR), NPFFR2. Epithelial ovarian cancer (EOC) is a leading cause of death among gynecological malignancies. The pathogenesis of EOC can be regulated by many local factors, including neuropeptides, through an autocrine/paracrine manner. However, to date, the expression and/or function of NPFF/NPFFR2 in EOC is undetermined. In this study, we show that the upregulation of NPFFR2 mRNA was associated with poor overall survival in EOC. The TaqMan probe-based RT-qPCR showed that NPFF and NPFFR2 were expressed in three human EOC cells, CaOV3, OVCAR3, and SKOV3. In comparison, NPFF and NPFFR2 expression levels were higher in SKOV3 cells than in CaOV3 or OVCAR3 cells. Treatment of SKOV3 cells with NPFF did not affect cell viability and proliferation but stimulated cell invasion. NPFF treatment upregulates matrix metalloproteinase-9 (MMP-9) expression. Using the siRNA-mediated knockdown approach, we showed that the stimulatory effect of NPFF on MMP-9 expression was mediated by the NPFFR2. Our results also showed that ERK1/2 signaling was activated in SKOV3 cells in response to the NPFF treatment. In addition, blocking the activation of ERK1/2 signaling abolished the NPFF-induced MMP-9 expression and cell invasion. This study provides evidence that NPFF stimulates EOC cell invasion by upregulating MMP-9 expression through the NPFFR2-mediated ERK1/2 signaling pathway.

Systematic high-throughput combination drug screen to enhance poly (ADP-ribose) polymerase (PARP) inhibitor efficacy in ovarian cancer.

5546 Background: PARP inhibitors currently offer the greatest benefit to patients with loss-of-function BRCA1 or 2 mutations or other causes of homologous recombination deficiency (HRD). We hypothesized that combination therapy, possibly by augmenting or inducing an HRD phenotype, would enhance the efficacy of PARP inhibition in epithelial ovarian cancer. Methods: We performed a high-throughput screen of an informer set containing FDA-approved drugs, clinical candidates, and small-molecule probes to identify synergistic partners with olaparib in representative HRD and non-HRD human epithelial ovarian cancer cells. Single agent activity was described by area under the curve of the Hafner growth rate index (AUC_GRI) and synergy was ranked by cumulative difference from Bliss (ΔBliss). Analysis of the drug screen was performed using algorithms developed by the Gulf Coast Consortia’s Combinatorial Drug Discovery Program at the Texas A&M Institute of Bioscience and Technology. MTT assays were then independently performed to verify and characterize synergy between CT7439, an agent selected following the drug screen, and olaparib. MTT data were analyzed using the SynergyFinder Plus web application. Results: In an initial single agent screen, we identified 62 compounds with a mean AUC_GRI <0.75, indicating inhibitory activity. Prioritizing more potent agents and those of mechanistic interest, we selected 40 hits to validate and advance to a combination screen. Diverse mechanisms of action were represented among the hits, including many small molecule inhibitors of various cell cycle pathways. Following a combination screen with olaparib, the compounds selected for final validation were: AZD 7762 (CHK1/2 inhibitor), dinaciclib (pan-CDK inhibitor), onalespib (HSP90 inhibitor), and SN-38 (topoisomerase 1 inhibitor). The ΔBliss for these agents are presented in the table. Due to mechanistic rationale and concern for overlapping toxicities among the other candidates, we focused further study on CDK inhibitors. In MTT assays, we identified robust synergy between olaparib and CT7439, a next-generation oral CDK12/13 inhibitor, with a mean Bliss synergy score of 1.31 and scores as high as 22.03 at certain concentrations in the non-HRD model OVCAR-5. These findings were recapitulated in the HRD cell lines OVCAR-4 (mean Bliss synergy score 1.56) and OVCAR-8 (mean Bliss synergy score 5.69). Results from an in vivo model will be presented as well. Conclusions: Pairing a PARP inhibitor and CDK inhibitor, a strategy identified by a high-throughput drug screen and known to impair homologous recombination, resulted in robust synergy in both HRD and non-HRD ovarian cancer models. Clinical translation of this combination may enhance the efficacy of PARP inhibitors in patients with ovarian cancer. [Table: see text]

Tripterygium glycosides reverse chemotherapy resistance in ovarian cancer by targeting the NRF2/GPX4 signal axis to induce ferroptosis of drug-resistant human epithelial ovarian cancer cells

Cisplatin resistance is the main cause of postoperative recurrence and difficulty in the treatment of ovarian cancer. It is urgently needed to identify therapeutic drugs with unique functions to overcome the current challenges in the treatment of ovarian cancer. In this study, we found that TG promoted the accumulation of ROS and MDA in A2780/DDP cells and downregulated the expression of key antioxidant molecules. In vivo, the survival rate of tumor-bearing nude mice was prolonged by TG without significant hepatotoxic reaction. The expression of key antioxidant molecules in tumor tissues was consistent with that in vitro. These findings revealed that TG disrupted homeostasis of redox reactions and induced ferroptosis in A2780/DDP cells, thereby enhancing cisplatin chemosensitivity of ovarian cancer. Overall, TG may be a novel potential therapeutic option for reversing resistance to cisplatin chemotherapy.

Tunicamycin induced inhibition of calpain 1 and 2 enzyme activity in ovarian cancer cells

Background: Tunicamycin (TN) is an antitumor agent and induced intracellular calcium levels in many cells, however its molecular mechanism is still needed to be explored. Calpeptin (Calp) is an inhibitor of both calpain 1 and 2 (CAPN-1/2) enzymes, and plays a fundamental role in tumor mechanism. In this study, the effects of TN and Calp were investigated on CAPN-1/2 enzyme activity in normal and ovarian cancer cells adhered to fibronectin. Methods: 24uM TN, 50uM Calp, and combined TN and Calp (TN+Calp) were applied for 1 and 12 hours to FN-bound (FN+) and non-FN-bound (FN-) normal human ovarian epithelial (IHOSE) and ovarian cancer (SKOV-3) cells. The activation of CAPN-1/2 was measured by the luminescent method and the significance of the results was analyzed with the t-test. Results: CAPN-1/2 enzyme activity (at 12 hour) was present in both cell lines, but the level of enzyme is higher in IHOSE cells compared to SKOV-3 cells. The results showed that 1 hour TN and TN+Calp applications stimulated CAPN-1/2 enzyme activity in IHOSE cells but did not show any stimulating effect in SKOV-3 cells. After 12-hour of treatment, the cells with TN, Calp or TN+Calp showed an inhibitory effect on CAPN-1/2 enzyme in both FN+IHOSE and SKOV-3 cells. At 12-hour TN+Calp administration was determined to be the most effective inhibitor in FN+ SKOV-3 since it inhibited CAPN1/2 activity statistically significantly more than both Calp and TN administrations. Conclusions: The effects of TN, Calp and TN+Calp applications on the CAPN-1/2 enzyme varied according to the cell type, normal or cancer cells, and whether the cell was bound to FN and the incubation period. 12 h administrations of TN, Calp or TN+Calp inhibited the CAPN-1/2 enzyme in both FN+ IHOSE and SKOV-3 cells.

Promoting action of long non-coding RNA small nucleolar RNA host gene 4 in ovarian cancer.

Long non-coding RNA (LncRNA) small nucleolar RNA host gene 4 (SNHG4) has been shown to be aberrantly expressed in a variety of cancers and involved in cancer development, but its role in ovarian cancer (OC) is unclear. The purpose of this study was to explore the biological function of SNHG4 in OC and reveal its potential downstream molecular targets. OC tumor tissue and normal tissue were collected; normal human ovarian epithelial cell line (IOSE80) and human ovarian cancer cell line (A2780, SKOV-3, OV-90 and CAOV3) were selected. RT-qPCR was used to detect SNHG4, miR-98-5p, and TMED5, while western blot was used to detect the protein expression levels of TMED5, Ki67, MMP-9, Bcl-2, Bax, Gsk3β, Wnt3a, and β-catenin. The subcellular localization of SNHG4 was assessed by nucleocytoplasmic separation assay. CCK-8, colony formation assay, flow cytometry, and Transwell were used to assess the biological behavior of OC cells. The targeting relationship between SNHG4, miR-98-5p and TMED5 was verified by dual luciferase reporter assay and RIP assay. In OC, SNHG4 and TMED5 were highly expressed, and miR-98-5p was underexpressed. Knockdown of SNHG4 inhibited OC cell proliferation, migration and invasion, promoted apoptosis, and prevented Wnt/β-catenin pathway activation. The effect of knockdown of SNHG4 was reversed by knockdown of miR-98-5p or overexpression of TMED5. Mechanistically, SNHG4 competitively adsorbed miR-98-5p to mediate TMED5 expression, thereby activating the Wnt/β-catenin pathway. SNHG4 accelerates OC development via mediating the miR-98-5p/TMED5 axis and activating the Wnt/β-Catenin pathway. SNHG4 gene silencing might be a novel option for OC treatment.

Indoxyl Sulfate Promotes Metastatic Characteristics of Ovarian Cancer Cells via Aryl Hydrocarbon Receptor–Mediated Downregulation of the Mas Receptor

Although platinum-combination chemotherapy shows a high response rate at the primary site, epithelial ovarian cancer (EOC) treatment remains challenging because of tumor recurrence and metastasis. Recent studies have revealed that chemotherapy paradoxically promotes cancer cell survival, proliferation, and metastasis, although the reason for this remains unclear. The underlying molecular mechanisms that contribute to chemotherapy-induced metastasis need to be elucidated to establish effective therapeutic strategies. Acute kidney injury is a known side effect of cisplatin treatment, and kidney dysfunction results in the accumulation of uremic toxins in the serum. The present study aimed to investigate whether indoxyl sulfate (IS), a representative uremic toxin, affects the pathophysiology of EOC. In this study, IS reduced the expression of Mas receptor (MasR) in cultured human EOC cells. Both knockdown of the aryl hydrocarbon receptor (AhR), which is an intracellular IS receptor, and inhibition of AhR function suppressed IS-mediated downregulation of MasR in SK-OV-3 cells. IS induced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in an AhR-dependent manner. Inhibition of the STAT3 pathway or reactive oxygen species production suppressed the IS-mediated reduction of MasR. IS stimulated cell migration and invasion of SK-OV-3 cells in an AhR-dependent manner. Cisplatin-nephropathy model mice exhibited elevated levels of serum IS accompanied by elevated levels of blood urea nitrogen and serum creatinine. Furthermore, intraperitoneal administration of IS in mice promoted tumor growth and metastasis. Finally, we found that the MasR agonist Ang-(1-7) suppressed the IS-mediated effects on cell proliferation, migration, and invasion of SK-OV-3 cells. However, the knockdown of MasR expression by specific small interfering RNA in the absence of IS resulted in only minimal promotion of cell migration and invasion. These findings demonstrate that IS promotes malignancy in ovarian cancer via AhR-mediated downregulation of MasR function, whereas Ang-(1-7) attenuates this effect, thereby suggesting that Ang-(1-7) could provide a future treatment strategy for this cancer type.

Drug repositioning of disulfiram induces endometrioid epithelial ovarian cancer cell death via the both apoptosis and cuproptosis pathways.

Various therapeutic strategies have been developed to overcome ovarian cancer. However, the prognoses resulting from these strategies are still unclear. In the present work, we screened 54 small molecule compounds approved by the FDA to identify novel agents that could inhibit the viability of human epithelial ovarian cancer cells. Among these, we identified disulfiram (DSF), an old alcohol-abuse drug, as a potential inducer of cell death in ovarian cancer. Mechanistically, DSF treatment significantly reduced the expression of the anti-apoptosis marker B-cell lymphoma/leukemia-2 (Bcl-2) and increase the expression of the apoptotic molecules Bcl2 associated X (Bax) and cleaved caspase-3 to promote human epithelial ovarian cancer cell apoptosis. Furthermore, DSF is a newly identified effective copper ionophore, thus the combination of DSF and copper was used to reduce ovarian cancer viability than DSF single treatment. Combination treatment with DSF and copper also led to the reduced expression of ferredoxin 1 and loss of Fe-S cluster proteins (biomarkers of cuproptosis). In vivo, DSF and copper gluconate significantly decreased the tumor volume and increased the survival rate in a murine ovarian cancer xenograft model. Thus, the role of DSF revealed its potential for used as a viable therapeutic agent for the ovarian cancer.

Cytotoxic Properties of C17 Polyacetylenes from the Fresh Roots of Panax ginseng on Human Epithelial Ovarian Cancer Cells.

Although C17 polyacetylenes from Panax ginseng exhibit cytotoxic properties against various tumor cells, there have been few experiments on epithelial ovarian carcinoma cells. This study aimed to investigate the cytotoxic effects of C17 polyacetylenes from P. ginseng against ovarian cancer cell lines. Four unreported (1–4) and fifteen known (5–19) C17 polyacetylenes were obtained from the roots of P. ginseng using repeated chromatography (open column, MPLC, and preparative HPLC). The chemical structures of all the compounds were determined by analyzing their spectroscopic data (NMR, IR, and optical rotation) and HR-MS. The structures of new polyacetylenes were elucidated as (3S,8S,9R,10R)-(-)-heptadeca-9,10-epoxy-4,6-diyne-3,8-diyl diacetate (1), (3S,8S,9R,10R)-(−)-heptadeca-1-en-9,10-epoxy-4,6-diyne-3,8-diyl diacetate (2), (−)-haptadeca-9,10-epoxy-8-methoxy-4,6-diyne-3,11-diol (3), and (3R,9R,10R)-(+)-3-acetoxy-9,10-dihydroxyheptadeca-1-en-4,6-diyne (4), named ginsenoynes O, P, and Q, and 3-acetyl panaxytriol, respectively. Subsequently, in vitro experiments on A2780 and SKOV3 human epithelial ovarian carcinoma cells were performed to assess the cytotoxic properties of the isolates. Among the isolates, panaquinquecol 4 (15) exhibited the most remarkable cytotoxic effects on both human ovarian cancer cells A2780 (IC50 value of 7.60 μM) and SKOV3 (IC50 value of 27.53 μM). Therefore, C17 polyacetylenes derived from P. ginseng may warrant further investigation for their therapeutic potential in epithelial ovarian cancer.

Extracellular vesicle-packaged miR-181c-5p from epithelial ovarian cancer cells promotes M2 polarization of tumor-associated macrophages via the KAT2B/HOXA10 axis.

The molecular mechanistic actions of tumor-derived extracellular vesicles (EVs) in modulating macrophage polarization in the tumor microenvironment of epithelial ovarian cancer (EOC) is largely unknown. The study was performed to clarify the effect and downstream mechanism of microRNA-181c-5p (miR-181c-5p)-containing EVs from EOC cells in the M2 polarization of tumor-associated macrophages (TAMs). EVs were isolated from normoxic and hypoxic human EOC cells SKOV3. Human mononuclear cell THP-1 was induced by phorbol-12-myristate-13-acetate to differentiate into TAMs. The targeting relationship between miR-181c-5p and KAT2B was verified by dual luciferase reporter gene assay. The interaction between KAT2B and HOXA10 was detected by immunofluorescence, Co-IP and ChIP assays. EdU staining, the scratch test and Transwell assay were used to assess the resultant cell proliferation, migration and invasion. The mouse xenograft model and the pulmonary metastasis model were developed through intraperitoneal injection of SKOV3 cells and tail vein injection of THP-1 cells, respectively. Hypoxic SKOV3 cell-derived EVs could be internalized by TAMs. SKOV3 cell-derived EVs induced by hypoxia (H-EVs) promoted M2 polarization of TAMs and facilitated the proliferation, migration and invasion of SKOV3 cells. miR-181c-5p was highly expressed in H-EVs and promoted the M2 polarization of TAMs. Further, miR-181c-5p targeted KAT2B, upregulated HOXA10 and activated the JAK1/STAT3 pathway, thereby promoting the M2 polarization of TAMs. In both mouse models, H-EV-derived miR-181c-5p promoted growth and metastasis of EOC cells. The miR-181c-5p-containing EVs from hypoxic EOC cells may upregulate HOXA10 by targeting KAT2B and activate the JAK1/STAT3 pathway to promote the M2 polarization of TAMs, ultimately promoting growth and metastasis of EOC cells in vitro and in vivo.

Ketogenic Diet Promotes Epithelial Ovarian Cancer Progression and Alters Tumor Gene Expression in-vivo (104)

<b>Objectives:</b> The ketogenic diet (KD) is hypothesized to impact tumor progression indirectly through altering the immune system as well as directly at the cellular level. Our studies were designed to assess changes in tumor resident immune cells and tumor gene expression in relation to epithelial ovarian cancer (EOC) progression in KD-fed mice. <b>Methods:</b> ID8 EOC cells, syngeneic with C57Bl/6J mouse strain, transfected with luciferase (ID8-Luc), were injected and monitored for tumor development via imaging with an <i>in vivo</i> Imaging System (IVIS) intraperitoneal D-luciferin. Tumor-bearing mice were fed a KD (10% protein, 0% carbohydrates, 90% fat), high fat diet (HFD) (10% protein, 15% carbohydrates, 75% fat), or control diet (CD) (10% protein, 77% carbohydrates, and 13% fat) (<i>n</i>=10 mice per group). EOC tumor growth was monitored weekly, and tumor burden was determined based on total flux recorded in photons/second measured. At the endpoint, tumors were collected and processed for flow cytometry and RNAseq analysis. Single-cell dissociated tumors (Papain System) were stained with an immune targeted antibody panel and analyzed on the BD Cytek Aurora. RNA was isolated (Qiagen), and sequencing was performed. Statistical tests included analysis of variance (ANOVA), permutational multivariate analysis (PERMANOVA), linear regression, and Wilcoxon test; a p-value of <0.05 was considered statistically significant. <b>Results:</b> Throughout the study, KD-fed mice exhibited a statistically significant increase in tumor progression. We observed a 9.1-fold increase in tumor volume from baseline in the KD-fed group compared to HFD and CD-fed groups (9.1 vs 2.0 vs 3.1-fold, respectively, p<0.001). At the endpoint, tumor-associated macrophages (TAM) were significantly enriched and activated lymphoid cells (NK cells) were significantly diminished in the KD group (p<0.001). There was a trend toward increased M2:M1 polarization in KD-fed mice. However, there was no significant difference in Treg cells or activated CD8+ cells among diet groups. EOC tumors in KD-fed mice exhibited a significant decrease in differential gene expression on RNA seq compared to CD- and HFD-fed tumors, with no significant difference between CD- and HFD-fed groups at the gene expression level. Gene set enrichment analysis (GSEA) demonstrated that EOC tumors from KD-fed mice had enhanced angiogenesis, inflammatory response, and epithelial to mesenchymal transition (EMT) phenotype, as well as altered lipid metabolism. Specifically, an increase in lipid catalytic activity and catabolism and decrease in lipid synthesis were noted among KD-fed mice versus HFD-fed mice. <b>Conclusions:</b> KD induced tumor progression in our murine EOC model compared to HFD and CD. KD was associated with an increase in TAM and a decrease in activated NK cells consistent with a protumorigenic immune environment. KD-fed mice exhibited markedly increased alterations in angiogenesis, inflammation, and EMT pathways with enhanced lipid catabolic activity. Further testing of KD in human EOC cells is needed to clarify the <i>in-vivo</i> effect of KD on EOC growth.Fig. 1

Ezrin Contributes to the Plasma Membrane Expression of PD-L1 in A2780 Cells.

Programmed death ligand–1 (PD–L1) is one of the immune checkpoint molecule localized on the plasma membrane of numerous cancer cells that negatively regulates T-cell-mediated immunosurveillance. Despite the remarkable efficacy and safety profile of immune checkpoint inhibitors (ICIs), such as anti-PD–L1 antibodies, restricted poor therapeutic responses to ICIs are often observed in patients with ovarian cancer. Because higher expression of PD–L1 in advanced ovarian cancer is associated with a decreased survival rate, identifying the potential molecules to regulate the plasma membrane expression of PD–L1 may provide a novel therapeutic strategy to improve the efficacy of ICIs against ovarian cancers. Here, we reveal the involvement of the ezrin/radixin/moesin (ERM) family, which crosslinks transmembrane proteins with the actin cytoskeleton by serving as a scaffold protein, in the plasma membrane expression of PD–L1 in the human epithelial ovarian cancer cell line A2780. Our results demonstrate that PD–L1 and all three ERMs were expressed at the mRNA and protein levels in A2780 cells, and that PD–L1 was highly colocalized with ezrin and moesin, but moderately with radixin, in the plasma membrane. Interestingly, RNA interference-mediated gene silencing of ezrin, but not of radixin or moesin, substantially reduced the plasma membrane expression of PD–L1 without altering its mRNA expression. In conclusion, our results indicate that ezrin may be responsible for the plasma membrane expression of PD–L1, possibly by serving as a scaffold protein in A2780 cells. Ezrin is a potential therapeutic target for improving the efficacy of ICIs against ovarian cancers.

Wnt5A and TGFβ1 Converges through YAP1 Activity and Integrin Alpha v Up-Regulation Promoting Epithelial to Mesenchymal Transition in Ovarian Cancer Cells and Mesothelial Cell Activation.

In this paper, we investigate whether Wnt5A is associated with the TGF-β1/Smad2/3 and Hippo-YAP1/TAZ-TEAD pathways, implicated in epithelial to mesenchymal transition (EMT) in epithelial ovarian cancer. We used 3D and 2D cultures of human epithelial ovarian cancer cell lines SKOV-3, OVCAR-3, CAOV-4, and different subtypes of human serous ovarian cancer compared to normal ovary specimens. Wnt5A showed a positive correlation with TAZ and TGFβ1 in high- and low-grade serous ovarian cancer specimens compared to borderline serous and normal ovaries. Silencing Wnt5A by siRNAs significantly decreased Smad2/3 activation and YAP1 expression and nuclear shuttling in ovarian cancer (OvCa) cells. Furthermore, Wnt5A was required for TGFβ1-induced cell migration and invasion. In addition, inhibition of YAP1 transcriptional activity by Verteporfin (VP) altered OvCa cell migration and invasion through decreased Wnt5A expression and inhibition of Smad2/3 activation, which was reverted in the presence of exogenous Wnt5A. We found that the activation of TGFβ1 and YAP1 nuclear shuttling was promoted by Wnt5A-induced integrin alpha v. Lastly, Wnt5A was implicated in activating human primary omental mesothelial cells and subsequent invasion of ovarian cancer cells. Together, we propose that Wnt5A could be a critical mediator of EMT-associated pathways.

Down-regulation of PSMD4 can attenuate autophagy, enhance the accumulation of intracellular ROS, and increase the sensitivity of epithelial ovarian cancer to carboplatin by inhibiting the NF-κB pathway.

BackgroundThe incidence of ovarian cancer ranks third among female gynecological cancers in the world, and more than 90% of patients are epithelial ovarian cancer (EOC). Carboplatin is the first-line chemotherapy drug for the treatment of EOC patients. However, patients who are resistant to carboplatin often do not benefit from it. Therefore, finding a key molecule that affects carboplatin sensitivity is expected to enhance the efficacy of carboplatin in EOC treatment.MethodsThe human EOC cell line SK-OV-3 and TOV-21G were used in this study. The second-generation sequencing technology was used to sequence the transcripts of carboplatin-resistant EOC cells and parental EOC cells. The bioinformatic analysis of the differentially expressed genes was performed by Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The EOC tissue chip in the Gene Expression Omnibus (GEO) database was also analyzed to screen the target gene. Flow cytometry, Hoechst staining, Western blot, MTS, mitochondrial specific reducing agent (Mito Tempo), and nuclear factor kappa-B (NF-κB) pathway inhibitor (BAY 11-7082) were used to explore the effect of proteasome 26S subunit, non-ATPase 4 (PSMD4) on the autophagy, apoptosis, and reactive oxygen species (ROS) accumulation of carboplatin-resistant EOC cells treated with carboplatin. In vivo, the carboplatin-resistant EOC cell lines treated with PSMD4 knockdown were injected subcutaneously into mice (twelve female BALB/c nude mice) to construct EOC subcutaneous xenograft tumor models.ResultsBased on second-generation sequencing technology and bioinformatics analysis, it was found that PSMD4 is the core molecule in the carboplatin resistance regulatory network in EOC, and its expression is up-regulated in EOC carboplatin-resistant tissues and cells. In vitro and in vivo experimental results show that the down-regulated expression of PSMD4 is closely related to the increase in sensitivity of EOC to carboplatin. Mechanically, we found that inhibiting PSMD4 expression may inhibit the activation of the NF-κB pathway, promote carboplatin-induced ROS accumulation in EOC cells, inhibit EOC cell autophagy, and enhance the sensitivity of EOC to carboplatin.ConclusionsDown-regulation of PSMD4 may inhibit the activation of the NF-κB pathway and autophagy, and up-regulate the level of intracellular ROS accumulation, thereby promoting carboplatin-mediated EOC cell apoptosis and enhancing carboplatin sensitivity.