Normal Ovarian Cells Research Articles

Molecular and functional profiling of primary normal ovarian cells defines insights into cancer development and drug responses

Patients with ovarian cancer, especially the high-grade serous (HGSOC) subtype, face poor prognosis due to late diagnosis and treatment resistance. Owing to the high heterogeneity of HGSOC, identifying the origin of the disease and optimal treatments is difficult. Here, we characterized two primary immortalized human ovarian cell lines, HOSE1C and HOSE2C, comparing their molecular profiling with representative HGSOC cells. We identified molecular features associated with normal and malignant phenotype of ovarian cells by applying single-cell transcriptomics, high-content imaged-based Cell Painting, and high-throughput drug testing. Our findings reveal distinct transcriptomic and morphological profiles for the two HOSEs, with a stromal phenotype. Moreover, their responses to the tumor microenvironment differ, exemplified by STAT1 and GREM1 upregulation in HOSE1C and HOSE2C, respectively. We identified selective activation of ERK/MEK targeted inhibitors in cancer cells compared to HOSEs. This study offers insights into the normal and malignant ovarian cells, shedding light on cancer development and drug responses.

Epirubicin/folic acid and meropenem loaded on graphene oxide-gelatin can be used as a novel candidate for anti-cancer and antibacterial drug development

Resistance to meropenem and epirubicin poses a significant global threat, particularly in developing nations with constrained health resources. To overcome this problem, nanotechnology provides several promising solutions, including drug delivery systems that can improve the effectiveness of drugs. The objectives of this work is to characterize the anticancer mechanism of Graphene Oxide (GO) coated with Gelatin (Gel) and conjugated with the anticancer drug Epirubicin (EPi), along with functionalization with Folic Acid in SK-OV3 cancer cell lines for the first time. Furthermore, meropenem was loaded onto Graphene Oxide-Gelatin (GO-Gel) to improve its efficacy. The nanocomposites were characterized using FT-IR, XRD, FESEM and EDX. The viability of the ovarian cancer cell lines (SKOV3) and normal ovarian cell lines (HUVEC) after treatment with GO-Gel, Graphene Oxide-Gelatin-Folic acid (GO-Gel-FA), free Epi and Graphene Oxide-Gelatin-Folic acid/ Epirubicin (GO-Gel–FA/Epi) nanocomposite, was studied by the MTT assay. Expression of the TNFα, Bax, Bcl-2, and NF-κB in the GO-Gel–FA/Epi nanocomposite treated cells, were investigated by qRT-PCR. Disc diffusion assay was utilized to assess the antimicrobial activity of free mer and GO-Gel-Mer nanocomposite against two gram-positive bacteria and two gram-negative bacteria. Results demonstrated that The GO-Gel–FA/Epi nanocomposite showed greater cytotoxic effects on SKOV3cells than normal HUVEC cells. The expression of the Bax was upregulated, while the expression of the Bcl-2, TNFα and NF-κB was reduced in GO-Gel–FA/Epi nanocomposite-treated cells. The Graphene Oxide-Gelatin-Meropenem (GO-Gel-Mer) nanocomposite showed a controlled release within 45 h. GO-Gel-Mer nanocomposite showed much more activity against bacteria in comparison to free Mer. GO-Gel-FA/Epi nanocomposite possesses strong anti-proliferative properties against SK-OV3 cancer cells and indicated promising inhibitory candidate for anticancer therapy. The novel synthesized GO-Gel-Mer nanocomposite can be used as an effective antimicrobial nanomaterial against a range of microbial pathogens, including gram-negative and gram-positive bacteria.

High-throughput drug screening identifies novel therapeutics for Low Grade Serous Ovarian Carcinoma

Low grade serous carcinoma (LGSOC) is a rare epithelial ovarian cancer with unique molecular characteristics compared to the more common tubo-ovarian high-grade serous ovarian carcinoma. Pivotal clinical trials guiding the management of epithelial ovarian cancer lack sufficient cases of LGSOC for meaningful subgroup analysis, hence overall findings cannot be extrapolated to rarer chemo-resistant subtypes such as LGSOC. Furthermore, there is a need for more effective therapies for the treatment of relapsed disease, as treatment options are limited. To address this, we conducted the largest quantitative high-throughput drug screening effort (n = 3436 compounds) in 12 patient-derived LGSOC cell lines and one normal ovary cell line to identify unexplored therapeutic avenues. Using a combination of high-throughput robotics, high-content imaging and novel data analysis pipelines, our data set identified 60 high and 19 moderate confidence hits which induced cancer cell specific cytotoxicity at the lowest compound dose assessed (0.1 µM). We also revealed a series of known (mTOR/PI3K/AKT) and novel (EGFR and MDM2-p53) drug classes in which LGSOC cell lines showed demonstrable susceptibility to.

Rapid enzyme-free detection of miRNA-21 in human ovarian cancerous cells using a fluorescent nanobiosensor designed based on hairpin DNA-templated silver nanoclusters

BackgroundCancer is known as one of the main non-communicable diseases and the leading cause of death in the new era. Early diagnosis of cancer requires the identification of special biomarkers. Currently, microRNAs (miRNAs) have attracted the attention of researchers as useful biomarkers for cancer early detection. Hence, various methods have been recently developed for detecting and monitoring miRNAs. Among all miRNAs, detection of miRNA-21 (miR-21) is important because it is abnormally overexpressed in most cancers. Here, a new biosensor based on silver nanoclusters (AgNCs) is introduced for detecting miR-21. ResultsAs a fluorescent probe, a rationally designed hairpin sequence containing a poly-cytosine motif was used to facilitate the formation of AgNCs. A guanine-rich sequence was also employed to enhance the sensing signal. It was found that in the absence of miR-21, adding a guanine-rich sequence to the detecting probe caused only a slight change in the fluorescence emission intensity of AgNCs. While in the presence of miR-21, the emission signal enhanced. A direct correlation was observed between the increase in the fluorescence of AgNCs and the concentration of miR-21. The performance of the proposed biosensor was characterized thoroughly and confirmed. The biosensor detected miR-21 in an applicable linear range from 9 pM to 1.55 nM (LOD: 2 pM). SignificanceThe designed biosensor was successfully applied for detecting miR-21 in human plasma samples and also in human normal and lung and ovarian cancer cells. This biosensing strategy can be used as a model for detecting other miRNAs. The designed nanobiosensor can measure miR-21 without using any enzymes, with fewer experimental steps, and at a low cost compared to the reported biosensors in this field.

A disulfidptosis-related lncRNAs cluster to forecast the prognosis and immune landscapes of ovarian cancer.

Disulfidptosis is a newly recognized form of regulated cell death that has been linked to cancer progression and prognosis. Despite this association, the prognostic significance, immunological characteristics and treatment response of disulfidptosis-related lncRNAs (DRLs) in ovarian cancer have not yet been elucidated. The lncRNA data and clinical information for ovarian cancer and normal samples were obtained from the UCSC XENA. Differential expression analysis and Pearson analysis were utilized to identify core DRLs, followed by LASSO algorithm. Random Survival Forest was used to construct a prognostic model. The relationships between risk scores, RNA methylation, immune cell infiltration, mutation, responses to immunotherapy and drug sensitivity analysis were further examined. Additionally, qRT-PCR experiments were conducted to validate the expression of the core DRLs in human ovarian cancer cells and normal ovarian cells and the scRNA-seq data of the core DRLs were obtained from the GEO dataset, available in the TISCH database. A total of 8 core DRLs were obtained to construct a prognostic model for ovarian cancer, categorizing all patients into low-risk and high-risk groups using an optimal cutoff value. The AUC values for 1-year, 3-year and 5-year OS in the TCGA cohort were 0.785, 0.810 and 0.863 respectively, proving a strong predictive capability of the model. The model revealed the high-risk group patients exhibited lower overall survival rates, higher TIDE scores and lower TMB levels compared to the low-risk group. Variations in immune cell infiltration and responses to therapeutic drugs were observed between the high-risk and low-risk groups. Besides, our study verified the correlations between the DRLs and RNA methylation. Additionally, qRT-PCR experiments and single-cell RNA sequencing data analysis were conducted to confirm the significance of the core DRLs at both cellular and scRNA-seq levels. We constructed a reliable and novel prognostic model with a DRLs cluster for ovarian cancer, providing a foundation for further researches in the management of this disease.

CRISPR/Cas9-based genome-wide screening for metastasis ability identifies FCGR1A regulating the metastatic process of ovarian cancer by targeting LSP1

BackgroundMetastasis is a main cause of death from ovarian cancer (OC). Identifying key markers involved in OC metastasis can aid in the effective detection of early postoperative metastasis. However, the role of FCGR1A in OC metastasis has yet to be fully established. A genome-wide CRISPR/Cas9-based screening system was used to identify regulatory factors involved in metastasis.MethodsThe expression of FCGR1A and LSP1 in ovarian cancer cell lines was examined by quantitative real-time polymerase chain reaction (qRT‒PCR). The functions of FCGR1A and LSP1 in OC cell migration, invasion and proliferation were determined using wound healing, Transwell invasion and CKK-8 assays. A transcription-activated library was used to identify the potential downstream genes of FCGR1A. FCGR1A expression was detected by immunohistochemistry and the immunity risk score (IRS) scores were calculated.ResultsFCGR1A was upregulated in OC cells compared with normal ovarian cells. Downregulation of FCGR1A inhibited metastasis, proliferation and epithelial–mesenchymal transition (EMT) progression in OC cells in vitro and intraperitoneal metastasis in vivo. Moreover, downregulation of FCGR1A was accompanied by decreased LSP1 expression. Overexpression of LSP1 partially reversed the tumor suppressive effect of FCGR1A downregulation. Higher FCGR1A expression was related to metastasis, higher grade, higher stage, and lymph node metastasis in OC. Survival analysis suggested that the group with higher FCGR1A expression had a lower tumor-free survival rate and a lower overall survival rate than did the group with low FCGR1A expression.ConclusionsFCGR1A enhances OC metastasis by regulating LSP1, and FCGR1A is associated with poor prognosis, suggesting that FCGR1A is a potential predictive factor for detecting early postoperative metastasis.

M6A-mediated hsa_circ_0061179 inhibits DNA damage in ovarian cancer cells via miR-143-3p/TIMELESS.

Ovarian cancer (OC) is among the most common and deadly solid malignancies in women. Despite many advances in OC research, the incidence of OC continues to rise, and its pathogenesis remains largely unknown. Herein, we elucidated the function of hsa_circ_0061179 in OC. The levels of hsa_circ_0061179, miR-143-3p, TIMELESS, and DNA damage repair-related proteins in OC or normal ovarian tissues and cells were measured using real-time quantitative polymerase chain reaction and immunoblotting. The biological effects of hsa_circ_0061179 and miR-143-3p on proliferation, clone formation, DNA damage, and apoptosis of OC cells were detected by the cell counting kit-8 assay, 5-methylethyl-2'-deoxyuridine, flow cytometry, the comet assay, and immunofluorescence staining combined with the confocal microscopy. The interaction among hsa_circ_0061179, miR-143-3p, and TIMELESS was validated by the luciferase reporter assay. Mice tumor xenograft models were used to evaluate the influence of hsa_circ_0061179 on OC growth in vivo. We found that human OC biospecimens expressed higher levels of hsa_circ_0061179 and lower levels of miR-143-3p. Hsa_circ_0061179 was found to bind with miR-143-3p, which directly targets TIMELESS. Hsa_circ_0061179 knockdown or miR-143-3p overexpression suppressed the proliferation and clone formation of OC cells and increased DNA damage and apoptosis of OC cells via the miR-143-3p/TIMELESS axis. Furthermore, we demonstrated that METTL3 could direct the formation of has_circ_0061179 through a specific m6A modification site. YTHDC1 facilitated the cytoplasmic transfer of has_circ_0061179 by directly binding to the modified m6A site. Our findings suggest that hsa_circ_0061179 acts as the sponge of miR-143-3p to activate TIMELESS signaling and inhibits DNA damage and apoptosis in OC cells.

Knockdown of CENPM activates cGAS-STING pathway to inhibit ovarian cancer by promoting pyroptosis.

We aimed to screen novel gene signatures for ovarian cancer (OC) and explore the role of biomarkers in OC via regulating pyroptosis using bioinformatics analysis. Differentially expressed genes (DEGs) of OC were screened from GSE12470 and GSE16709 datasets. Hub genes were determined from protein-protein interaction networks after bioinformatics analysis. The role of Centromeric protein M (CENPM) in OC was assessed by subcutaneous tumor experiment using hematoxylin-eosin and immunohistochemical staining. Tumor metastasis was evaluated by detecting epithelial-mesenchymal transition-related proteins. The proliferation, migration, and invasion were determined using cell counting kit and transwell assay. Enzyme-linked immunosorbent assay was applied to measure inflammatory factors. The mRNA and protein expression were detected using real-time quantitative PCR and western blot. We determined 9 hub genes (KIFC1, PCLAF, CDCA5, KNTC1, MCM3, OIP5, CENPM, KIF15, and ASF1B) with high prediction value for OC. In SKOV3 and A2780 cells, the expression levels of hub genes were significantly up-regulated, compared with normal ovarian cells. CENPM was selected as a key gene. Knockdown of CENPM suppressed proliferation, migration, and invasion of OC cells. Subcutaneous tumor experiment revealed that CENPM knockdown significantly suppressed tumor growth and metastasis. Additionally, pyroptosis was promoted in OC cells and xenograft tumors after CENPM knockdown. Furthermore, CENPM knockdown activated cGAS-STING pathway and the pathway inhibitor reversed the inhibitory effect of CENPM knockdown on viability, migration, and invasion of OC cells. CENPM was a novel biomarker of OC, and knockdown of CENPM inhibited OC progression by promoting pyroptosis and activating cGAS-STING pathway.

Characterization of immortalized ovarian epithelial cells with BRCA1/2 mutation.

We aimed to elucidate the mechanism underlying carcinogenesis by comparing normal and BRCA1/2-mutated ovarian epithelial cells established via Sendai virus-based immortalization. Ovarian epithelial cells (normal epithelium: Ovn; with germline BRCA1 mutation: OvBRCA1; with germline BRCA2 mutation: OvBRCA2) were infected with Sendai virus vectors carrying three immortalization genes (Bmi-1, hTERT, and SV40T). The immunoreactivity to anti-epithelial cellular adhesion molecule (EpCAM) antibodies in each cell line and cells after 25 passages was confirmed using flow cytometry. Chromosomes were identified and karyotyped to detect numerical and structural abnormalities. Total RNA extracted from the cells was subjected to human transcriptome sequencing. Highly expressed genes in each cell line were confirmed using real-time polymerase chain reaction. Immortalization techniques allowed 25 or more passages of Ovn, OvBRCA1, and OvBRCA2 cells. No anti-EpCAM antibody reactions were observed in primary cultures or after long-term passages of each cell line. Structural abnormalities in the chromosomes were observed in each cell line; however, the abnormal chromosomes were successfully separated from the normal structures via cloning. Only normal cells from each cell line were cloned. MMP1, CCL2, and PAPPA were more predominantly expressed in OvBRCA1 and OvBRCA2 cells than in Ovn cells. Immortalized ovarian cells derived from patients with germline BRCA1 or BRCA2 mutations showed substantially higher MMP1 expression than normal ovarian cells. However, the findings need to be validated in the future.

Abstract 2810: Intracellular tumor microbiota promotes metastasis and drives an immunosuppressive phenotype in ovarian cancer

Abstract Background: The tumor microenvironment has immense potential to change a patient’s prognosis and therapeutic response. One of the emerging components of interest in the tumor microenvironment is the presence of intracellular microbes. Numerous studies have focused on colon and pancreatic cancer because of their proximity to the commensals in the gut. The female reproductive system is another site in our body where the rich vaginal microbiome has the potential to affect nearby organs and impact diseases. However, there is still little investigation done to understand the impact of microbes on Ovarian Cancer (OC). Objective: To understand the role of the microbiome in OC metastasis, to develop new therapies to target the tumor microbiome Methods: We used a co-culture study, the parameters of which were optimized to study the infection of microbes in different cell lines. Using a wide array proteome profiler, we looked for cytokines and chemokines to understand changes in signaling post-infection. Protein levels in the conditioned media using ELISA. Changes in actin, paxillin, and LC3B (autophagy marker) were monitored using immunofluorescence microscopy and western blots. Migration was studied by using transwell assay and spheroid cultures. Results: We have identified two species of bacteria preferentially enriched in human ovarian tumors based on recent findings in the literature. We have demonstrated that these microbes are better at infecting OC cells in vitro over non-specific bacteria, can reside intracellularly, and are metabolically active. Upon infection, this intracellular uptake drives the release of pro-inflammatory cytokines and chemokine signaling by cancer and normal ovarian surface cells. We have also observed increased autophagy, changes in the actin cytoskeleton and reduced Paxillin foci on cancer cells infected with OC-associated microbes. This has led to increased migration in them; however, inactivated microbes did not change their migratory behavior. The infection also caused individual tumor cells to form more numerous and larger colonies when seeded into soft agar, suggesting an effect on cancer cell migration. Furthermore, we have shown that these microbes also elicit a more robust reaction in immune cells like Dendritic cells and macrophages, causing them to secrete chemokines like CCL 2,3,4,5, which also cause the cancer cells to migrate and have the potential to recruit neutrophils to drive the tumor microenvironment towards an immunosuppressive phenotype. Conclusion: Most ovarian cancer patients are diagnosed at Stage III/IV, where cancer has metastasized, and is unresponsive to immunotherapy as the cancer is considered “cold”. Through functional studies, we were able to gain insights into the role of the microbiome, which is associated with the clinical outcomes of OC, hoping to potentiate the existing therapies by probing the tumor microbiome. Citation Format: Ashutosh Kumar, Neelkanth Manoj Bardhan, Angela M. Belcher. Intracellular tumor microbiota promotes metastasis and drives an immunosuppressive phenotype in ovarian cancer [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 2810.

Abstract 6657: Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer

Abstract Objective: Epithelial Ovarian cancer (EOC) has the highest mortality rate among gynecologic malignancies, necessitating novel therapeutic approaches. Viral oncolytic immunotherapy with recombinant oncolytic vaccinia virus (OVV) offers promise for advanced-stage cancer treatment. OPCML is a GPI-anchored 3 Ig-domain tumor suppressor inactivated by somatic methylation in 87% of EOC. OVV with OPCML (OVV-OPCML) gene insertion may have potential as a therapeutic agent. This study explores the oncolytic, signaling-inhibitory, anti-angiogenic and synergistic immunotherapeutic properties of OVV-OPCML in EOC treatment, evaluating its therapeutic potential. Methods: To create OVV-OPCML, the virulent vaccinia virus Copenhagen strain F3L site was modified with OPCML cDNA, confirmed by qPCR and western blot. Oncolytic potential was assessed through in vitro and in vivo EOC cell infection with OVV-OPCML. Real-time cell analysis (RTCA) assessed cell proliferation. Xenograft tumor models in nude mice were used to evaluate in vivo oncolytic activity, while OVV-OPCML combined with anti-PD-1 treatment was tested using a syngeneic orthotopic EOC mouse model (ID8) in C57BL/6 immunocompetent mice. Serum IFN-γ and TNF-α were quantified via ELISA. Receptor Tyrosine Kinases, angiogenesis and phosphorylation together with immune markers were examined by western blotting and by multiplex immunohistochemistry (mIHC) analysis of tumor sections. Results: OVV-OPCML selectively infected OC cells, leading to viral replication and tumor cell death, while sparing normal ovarian cells in-vitro. OPCML protein expression increased post-infection. OVV-OPCML reduced ERBB2, EGFR, and FGFR1 expression in SKVO3 OV cells. Intratumoral or intraperitoneal (IP) OVV-OPCML administration in human ovarian cancer nude mouse models reduced tumor growth and improved survival. IP in-vivo OVV-OPCML in combination with anti-PD-1 treatment in vivo, in the IP ID8 syngeneic model demonstrated synergy with survival improvement. H&E staining showed tumor cell damage without significant harm to normal tissues. Protein expression of ERBB2, EGFR, FGFR1, and their phosphorylated forms decreased. CD31, VEGFR2, p-VEGFR2 (Tyr1175), and p-VEGFR2 (Tyr1214) downregulation suggested tumor angiogenesis disruption. OVV-OPCML treatment elevated serum TNF-α and INF-γ levels and increased CD8+ T cells and PD-L1 expression in OVV-OPCML treated syngeneic tumors. Conclusions: OPCML gene enhances OVV selectivity against OC cells through direct oncolysis and OPCML protein production, inhibiting specific RTKs, suppressing angiogenesis, and activating antitumor immune responses synergistically with anti-PD-1. OVV-OPCML therapy shows promise as a targeted strategy for ovarian cancer treatment. Citation Format: Yong Tang, Yingzhao Liu, Mingjing Deng, Fei Huang, Jiahu Wang, Paul Blake, Hani Gabra, Qi Wang. Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer [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 6657.

The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

The selenoenzyme type I iodothyronine deiodinase (DIO1) catalyzes removal of iodine atoms from thyroid hormones. Although DIO1 action is reported to be disturbed in several malignancies, no work has been conducted in high-grade serous ovarian carcinoma (HGSOC), the most lethal gynecologic cancer. We studied DIO1 expression in HGSOC patients [The Cancer Genome Atlas (TCGA) data and tumor tissues], human cell lines (ES-2 and Kuramochi), normal Chinese hamster ovarian cells (CHO-K1), and normal human fallopian tube cells (FT282 and FT109). To study its functional role, DIO1 was overexpressed, inhibited [by propylthiouracil (PTU)], or knocked down (KD), and cell count, proliferation, apoptosis, cell viability, and proteomics analysis were performed. Lower DIO1 levels were observed in HGSOC compared to normal cells and tissues. TCGA analyses confirmed that low DIO1 mRNA expression correlated with worse survival and therapy resistance in patients. Silencing or inhibiting the enzyme led to enhanced ovarian cancer proliferation, while an opposite effect was shown following DIO1 ectopic expression. Proteomics analysis in DIO1-KD cells revealed global changes in proteins that facilitate tumor metabolism and progression. In conclusion, DIO1 expression and ovarian cancer progression are inversely correlated, highlighting a tumor suppressive role for this enzyme and its potential use as a biomarker in this disease.

FBXO28 promotes cell proliferation, migration and invasion via upregulation of the TGF-beta1/SMAD2/3 signaling pathway in ovarian cancer

BackgroundOvarian cancer is one of the most common gynecological malignancies due to the lack of early symptoms, early diagnosis and limited screening. Therefore, it is necessary to understand the molecular mechanism underlying the occurrence and progression of ovarian cancer and to identify a basic biomarker for the early diagnosis and clinical treatment of ovarian cancer.MethodsThe association between FBXO28 and ovarian cancer prognosis was analyzed using Kaplan‒Meier survival analysis. The difference in FBXO28 mRNA expression between normal ovarian tissues and ovarian tumor tissues was obtained from The Cancer Genome Atlas (TCGA), and Genotype-Tissue Expression (GTEx) cohorts. The expression levels of the FBXO28 protein in ovarian cancer tissues and normal ovarian tissues were measured via immunohistochemical staining. Western blotting was used to determine the level of FBXO28 expression in ovarian cancer cells. The CCK-8, the colony formation, Transwell migration and invasion assays were performed to evaluate cell proliferation and motility.ResultsWe found that a higher expression level of FBXO28 was associated with poor prognosis in ovarian cancer patients. Analysis of the TCGA and GTEx cohorts showed that the FBXO28 mRNA level was lower in normal ovarian tissue samples than in ovarian cancer tissue samples. Compared with that in normal ovarian tissues or cell lines, the expression of FBXO28 was greater in ovarian tumor tissues or tumor cells. The upregulation of FBXO28 promoted the viability, proliferation, migration and invasion of ovarian cancer cells. Finally, we demonstrated that FBXO28 activated the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer.ConclusionsIn conclusion, FBXO28 enhanced oncogenic function via upregulation of the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer.

The ORFIUS complex regulates ORC2 localization at replication origins.

High-grade serous ovarian cancer (HGSC) is a lethal malignancy with elevated replication stress (RS) levels and defective RS and RS-associated DNA damage responses. Here we demonstrate that the bromodomain-containing protein BRD1 is a RS suppressing protein that forms a replication origin regulatory complex with the histone acetyltransferase HBO1, the BRCA1 tumor suppressor, and BARD1, ORigin FIring Under Stress (ORFIUS). BRD1 and HBO1 promote eventual origin firing by supporting localization of the origin licensing protein ORC2 at origins. In the absence of BRD1 and/or HBO1, both origin firing and nuclei with ORC2 foci are reduced. BRCA1 regulates BRD1, HBO1, and ORC2 localization at replication origins. In the absence of BRCA1, both origin firing and nuclei with BRD1, HBO1, and ORC2 foci are increased. In normal and non-HGSC ovarian cancer cells, the ORFIUS complex responds to ATR and CDC7 origin regulatory signaling and disengages from origins during RS. In BRCA1-mutant and sporadic HGSC cells, BRD1, HBO1, and ORC2 remain associated with replication origins, and unresponsive to RS, DNA damage, or origin regulatory kinase inhibition. ORFIUS complex dysregulation may promote HGSC cell survival by allowing for upregulated origin firing and cell cycle progression despite accumulating DNA damage, and may be a RS target.

Identification and Validation of Prognostic Markers for Endometriosis-Associated Ovarian Cancer.

Background: Growing evidence suggests that endometriosis (EMs) is a risk factor for endometriosis-associated ovarian cancer (EAOC). The aim was to identify and validate gene signatures associated with EMs that may serve as potential biomarkers for evaluating the prognosis of patients with EAOC. Methods: The data of EMs and control samples was obtained from GEO database. The weighted gene co-expression network analysis (WGCNA) identified modular genes significantly associated with EMs. The KEGG pathway and GO functional enrichment analyses were also performed. Univariate Cox regression analysis was conducted to screen marker genes associated with the prognosis of EAOC patients. Finally, RT-qPCR and immunohistochemical verified the expression of ADAMTS19 and TUBB in normal ovarian and EAOC tissues, and the biological functions of ADAMTS19 and TUBB were preliminarily explored by CCK8 and Transwell assays. Results: The WGCNA identified 2 co-expression modules, which in total included 615 genes, and 7642 differentially expressed genes (DEGs) were detected thorough analysis of the EAOC dataset. After taking the intersection of 615 modular genes and 7642 DEGs, 214 shared genes were obtained, and univariate COX regression analysis pointed 10 genes associated with the prognosis of EAOC. Moreover, it was demonstrated by RT-qPCR and immunohistochemical staining experiments that ADAMTS19 expression was elevated, while TUBB expression was reduced in EAOC compared with normal ovarian cells and tissues. Finally, cell experiments revealed that ADAMTS19 promoted the proliferation and invasion in EAOC cells, while overexpression of TUBB inhibited these processes. Conclusions: The present study identified and validated new EMs-associated gene markers, which could serve as potential biomarkers for assessing the prognostic risk of EAOC patients. In addition, some of these genes may have significance as novel therapeutic targets and could be used to guide clinical applications.

Engineered CAR-Tcells targeting the non-functional P2X purinoceptor 7 (P2X7) receptor as a novel treatment for ovarian cancer.

Recent studies have identified expression of the non-functional P2X7 (nfP2X7) receptor on various malignant cells including ovarian cancer, but not on normal cells, which makes it a promising tumour-associated antigen candidate for chimeric antigen receptor (CAR)-T-cell immunotherapies. In this study, we assessed the cytotoxic effects of nfP2X7-CAR-T cells on ovarian cancer using in vitro and in vivo models. We evaluated the effects of nfP2X7-CAR-T cells on ovarian cancer cell lines (SKOV-3, OVCAR3, OVCAR5), normal peritoneal cells (LP-9) and primary serous ovarian cancer cells derived from patient ascites invitro using monolayer and 3D spheroid assays. We also evaluated the effects of nfP2X7-CAR-T cells on patient-derived tissue explants, which recapitulate an intact tumour microenvironment. In addition, we investigated the effect of nfP2X7-CAR-T cells in vivo using the OVCAR-3 xenograft model in NOD-scid IL2Rγnull (NSG) mice. Our study found that nfP2X7-CAR-T cells were cytotoxic and significantly inhibited survival of OVCAR3, OVCAR5 and primary serous ovarian cancer cells compared with un-transduced CD3+ Tcells in vitro. However, no significant effects of nfP2X7-CAR-T cells were observed for SKOV3 or normal peritoneal cells (LP-9) cells with low P2X7 receptor expression. Treatment with nfP2X7-CAR-T cells increased apoptosis compared with un-transduced Tcells in patient-derived explants and correlated with CD3 positivity.Treatment with nfP2X7-CAR-T cells significantly reduced OVCAR3 tumour burden in mice compared with un-transduced CD3 cells for 7-8 weeks. This study demonstrates that nfP2X7-CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer.

Discovery of Tumor-Targeted 6-Methyl Substituted Pemetrexed and Related Antifolates with Selective Loss of RFC Transport.

Pemetrexed and related 5-substituted pyrrolo[2,3-d]pyrimidine antifolates are substrates for the ubiquitously expressed reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT) and folate receptors (FRs) which are more tumor-selective. A long-standing goal has been to discover tumor-targeted therapeutics that draw from one-carbon metabolic vulnerabilities of cancer cells and are selective for transport by FRs and PCFT over RFC. We discovered that a methyl group at the 6-position of the pyrrole ring in the bicyclic scaffold of 5-substituted 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidine antifolates 1-4 (including pemetrexed) abolished transport by RFC with modest impacts on FRs or PCFT. From molecular modeling, loss of RFC transport involves steric repulsion in the scaffold binding site due to the 6-methyl moiety. 6-Methyl substitution preserved antiproliferative activities toward human tumor cells (KB, IGROV3) with selectivity over IOSE 7576 normal ovary cells and inhibition of de novo purine biosynthesis. Thus, adding a 6-methyl moiety to 5-substituted pyrrolo[2,3-d]pyrimidine antifolates affords tumor transport selectivity while preserving antitumor efficacy.

LncRNA AOC4P impacts the differentiation of macrophages and T-lymphocyte by regulating the NF-κB pathways of KGN cells: Potential pathogenesis of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a multifactorial endocrine disease, which is an important cause of female infertility worldwide. PCOS patients are in a state of chronic low-grade inflammation, and immune imbalance is considered as a potential cause of its pathogenesis. The expression of AOC4P in PCOS and normal ovarian granulosa cells (GCs) was detected by real-time quantitative PCR. KGN cells were induced by dihydrotestosterone at 500ng/mL to construct the PCOS model. After lentivirus-infected, KGN cells were constructed with AOC4P overexpression cell lines, the proliferation and apoptosis levels of KGN cells in AOC4P and NC groups were detected. Human monocyte cell line (THP-1)-derived macrophages and peripheral blood mononuclear cells (PBMC) were co-cultured with KGN cells for 48h, respectively, and the differentiation of macrophages and CD4+ T cells were detected by flow cytometry. Decreased AOC4P expression was found in PCOS patients. After constructing the PCOS cell model, we observed that overexpression of AOC4P promoted KGN cell proliferation and inhibited apoptosis. After co-culture with AOC4P overexpressed KGN cells, M1 macrophages decreased, M2 macrophages increased, T helper cells type 1 (Th1)/Th2 ratio increased, and regulatory T cell (Treg) cells increased. Finally, we found that AOC4P inhibited the activation of the nuclear factor κ B (NF-κB) pathway in KGN cells. In this study, we found that AOC4P regulated the NF-κB signaling pathway by inhibiting the phosphorylation of P65, thereby affecting the proliferation and apoptosis of GCs, altering the differentiation of macrophages and T cells, thus contributing to the pathogenesis of PCOS.

Chromosome instability region analysis and identification of the driver genes of the epithelial ovarian cancer cell lines A2780 and SKOV3.

Epithelial ovarian cancer (EOC) is one of the most prevalent gynaecological cancers worldwide. The molecular mechanisms of serous ovarian cancer (SOC) remain unclear and not well understood. SOC cases are primarily diagnosed at the late stage, resulting in a poor prognosis. Advances in molecular biology techniques allow us to obtain a better understanding of precise molecular mechanisms and to identify the chromosome instability region and key driver genes in the carcinogenesis and progression of SOC. Whole-exome sequencing was performed on the normal ovarian cell line IOSE80 and the EOC cell lines SKOV3 and A2780. The single-nucleotide variation burden, distribution, frequency and signature followed the known ovarian mutation profiles, without chromosomal bias. Recurrently mutated ovarian cancer driver genes, including LRP1B, KMT2A, ARID1A, KMT2C and ATRX were also found in two cell lines. The genome distribution of copy number alterations was found by copy number variation (CNV) analysis, including amplification of 17q12 and 4p16.1 and deletion of 10q23.33. The CNVs of MED1, GRB7 and MIEN1 located at 17q12 were found to be correlated with the overall survival of SOC patients (MED1: p = 0.028, GRB7: p = 0.0048, MIEN1: p = 0.0051), and the expression of the three driver genes in the ovarian cell line IOSE80 and EOC cell lines SKOV3 and A2780 was confirmed by western blot and cell immunohistochemistry.

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10–250 μM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 μM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.