Journal of Cellular and Molecular Medicine

IFI16 Induced by p53 Activates the NF‐κB Pathway to Counteract Cisplatin‐Induced Apoptosis in Cervical Cancer Cells

ABSTRACTCervical cancer ranks as the second most prevalent cancer among women worldwide, and the primary treatment for advanced cases involves cisplatin‐based chemotherapy. However, the duration of cisplatin treatment is typically short, with a median survival rate of approximately 1 year. This highlights the urgent need to enhance our understanding of cisplatin's mechanism of action in cervical cancer treatment. Our findings demonstrate that p53 induces the nuclear translocation of IFI16, leading to activation of the NF‐κB signalling pathway. This activation plays a crucial role in protecting cervical cancer cells against cisplatin‐induced apoptosis. The activation of NF‐κB is independent of STING, which is a downstream molecule of IFI16. STING signalling activation by cisplatin may not be associated with cisplatin‐induced apoptosis. To further validate this tumour‐promoting effect of IFI16 during cisplatin therapy, we established a subcutaneous implantation tumour model using mouse cervical cancer (U14) cells and conducted additional in vitro experiments. We examined the role and mechanism of IFI16 in cisplatin treatment of cervical cancer. The role of IFI16 in cervical cancer progression deserves further study. Targeted inhibition of IFI16 may be a new way to increase cisplatin sensitivity of cervical cancer cells.

Dedifferentiated Schwann cells promote perineural invasion mediated by the PACAP paracrine signalling in cervical cancer

AbstractPerineural invasion (PNI) has emerged as a key pathological feature and be considered as a poor prognostic factor in cervical cancer. However, the underlying molecular mechanisms are largely unknown. Here, PNI status of 269 cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) samples were quantified by using whole‐slide diagnostic images obtained from The Cancer Genome Atlas. Integrated analyses revealed that PNI was an indicative marker of poorer disease‐free survival for CESC patients. Among the differentially expressed genes, ADCYAP1 were identified. Clinical specimens supported that high expression of PACAP (encoded by ADCYAP1) contributed to PNI in CESC. Mechanistically, PACAP, secreted from cervical cancer cells, reversed myelin differentiation of Schwann cells (SCs). Then, dedifferentiated SCs promoted PNI by producing chemokine FGF17 and by degrading extracellular matrix through secretion of Cathepsin S and MMP‐12. In conclusion, this study identified PACAP was associated with PNI in cervical cancer and suggested that tumour‐derived PACAP reversed myelin differentiation of SCs to aid PNI.

BNIP3L/BNIP3‐Mediated Mitophagy Contributes to the Maintenance of Ovarian Cancer Stem Cells

ABSTRACTOvarian cancer remains the most lethal gynaecological malignancy, with tumour recurrence and chemoresistance posing significant therapeutic challenges. Emerging evidence suggests that cancer stem cells (CSCs), a rare subpopulation within tumours with self‐renewal and differentiation capacities, contribute to these hurdles. Therefore, elucidating the mechanisms that sustain CSCs is critical for improving treatment strategies. Mitophagy, a selective process for eliminating damaged mitochondria, plays a key role in maintaining cellular homeostasis, including CSC survival. Our study demonstrates that ovarian CSCs exhibit enhanced mitophagy, accompanied by elevated expression of the mitochondrial outer membrane receptors BNIP3 and BNIP3L. Knockdown of BNIP3 or BNIP3L significantly reduces mitophagy and impairs CSC self‐renewal, indicating that receptor‐mediated mitophagy is essential for CSC maintenance. Mechanistically, we identify that hyperactivated NF‐κB signalling drives the upregulation of BNIP3 and BNIP3L in ovarian CSCs. Inhibition of NF‐κB signalling, either via p65 knockdown or pharmacological inhibitors, effectively suppresses mitophagy. Furthermore, we demonstrate that elevated DNA‐PK expression contributes to the constitutive activation of NF‐κB signalling, thereby promoting mitophagy in ovarian CSCs. In summary, our findings establish that BNIP3/BNIP3L‐mediated mitophagy, driven by DNA‐PK‐dependent NF‐κB hyperactivation, is essential for CSC maintenance. Targeting the DNA‐PK/NF‐κB/BNIP3L‐BNIP3 axis to disrupt mitochondrial quality control in CSCs represents a promising therapeutic strategy to prevent ovarian cancer recurrence and metastasis.

CD81 Aggravates Ovarian Cancer Progression via p‐Cresyl Sulfate‐Mediated Mitophagy in Tim4+ Tumour‐Associated Macrophages

ABSTRACTOvarian cancer (OC) is characterised by widespread peritoneal metastasis. Tetraspanin CD81 is predominantly located at the cellular membrane and exhibits inconsistent roles in tumour progression. However, its precise function in OC remains unclear. We found that CD81 expression was significantly elevated in tumour tissues from OC patients with poor prognosis, and it directly promoted proliferation, and migration of OC cells. Stable knock‐down of CD81 expression ameliorated disease progression in a murine model of OC and induced metabolic responses in OC cells. Metabolomics and mass spectrometry identified the protein‐bound toxin p‐cresyl sulfate (PCS) as a key metabolite regulated by the CD81‐FAK signalling axis. One aspect is that PCS promoted the growth of OC cells. Furthermore, tumour‐derived PCS combined with Cdh1 to enhance Bnip3‐dependent mitophagy activity of Tim4 positive tumour‐associated macrophages (TAMs). Intraperitoneal injection of PCS reversed the therapeutic effects observed following CD81 knock‐down; the mitophagy of reprogrammed Tim4+ TAMs was also promoted, accompanied by alterations in antitumor immunity. In summary, we elucidated CD81 prompted Tim4+ TAMs mitophagy to induce OC progression via FAK/PCS/Cdh1 pathway, deepen our understanding of OC pathogenesis.

Assessing Auranofin for Second‐Line Use in Chemoresistant Ovarian Cancer: Effects on Tumour Spheroid and Primary Cell Growth

ABSTRACTOvarian cancer (OC) is the fifth leading cause of cancer‐related death among women and the most lethal gynaecological malignancy. The high mortality rate is primarily due to late diagnosis and the lack of targeted therapies. The gold standard treatment consists of debulking surgery followed by platinum/taxane‐based chemotherapy, which is initially effective in approximately 75% of patients. However, most women experience relapse and develop chemoresistance. To date, no therapy has proven to be decisive, underscoring the need for research into second‐line or alternative treatments to overcome chemoresistance and prevent relapses. Auranofin (AF) is a promising repositioned anticancer agent with a multifaceted mode of action both cancer cell type‐ and dose‐dependent. The current study evaluated AF's cytotoxicity on multicellular tumour spheroids derived from three ovarian cancer cell lines (SKOV3, A2780, and A2780 cisplatin‐resistant). Results demonstrated that AF inhibited spheroid formation and growth by inducing apoptosis. Furthermore, we showed that AF's mode of action involves the PI3K/Akt and NF‐κB pathways, and we highlighted differences in drug responses between cisplatin‐sensitive, resistant, and primary ovarian cancer cells. Finally, by examining the efficacy of AF and cisplatin in combination, we identified differential sensitivities among the cell lines and primary ovarian cancer cells.

Identification of Sphingosine Kinase 1 as a Novel Protein Regulated by High Molecular Weight Hyaluronan in Ovarian Cancer

ABSTRACT The effects of hyaluronan (HA) in cancer are widely studied; however, the role of different molecular weight HA is poorly understood. Identifying novel proteins regulated by different molecular weight HA may highlight novel therapeutic targets. Proteomics analysis was performed to identify novel proteins regulated by different molecular weight HA (27, 183 and 1000 kDa) in ES‐2 ovarian cancer cells over‐expressing Notch3 intra‐cellular domain. Our analyses identified sphingosine kinase 1 (SPHK1), a novel protein regulated by 183‐ and 1000‐kDa HA. Utilising online databases and high‐grade serous ovarian cancer (HGSOC) patient tissue microarray cohorts, we assessed the relationship between SPHK1 expression and ovarian cancer metastasis, recurrence and patient outcome. We assessed the effects of the HA synthesis inhibitor 4‐methylumbelliferone (4‐MU) on SPHK1 expression in ovarian cancer cells and HGSOC patient tissues using ex vivo tissue explant assays. SPHK1 was significantly increased in ovarian cancer compared to normal tissues, elevated in metastatic and recurrent HGSOC tissues and associated with poor patient outcome. 4‐MU significantly inhibited SPHK1 expression in ovarian cancer cells (ES‐2, CaOV3 and A2780) and HGSOC patient tissues. This study highlights a link between HA and SPHK1 expression in ovarian cancer. Our findings confirm an adverse effect on ovarian cancer prognosis. SPHK1 constitutes a novel promising target against ovarian cancer that warrants further investigation.

Deciphering Deleterious nsSNPs in MUC16's SEA Domain: Structural and Functional Implications in Cancer Metastasis via Computational Analysis

ABSTRACT MUC16 ranks among the top three genes exhibiting the highest mutation frequencies in various cancer types. It encodes transmembrane mucins present in the epithelial linings of the ocular, respiratory, gastric and female reproductive systems, serving to protect and maintain mucosal surfaces. Overexpression of MUC16 contributes to the differentiation, proliferation, invasion and metastasis of cancer cells in ovarian, endometrial, pancreatic, colon, breast and non‐small‐cell lung cancers. In this study, we analysed the structural and functional effects of pathogenic and potentially harmful non‐synonymous single nucleotide polymorphisms (nsSNPs) of MUC16, employing a blend of computational algorithms. Initially, SNPs data for MUC16 were gathered from the Ensembl database and refined using computational tools (PROVEAN, SIFT, PolyPhen‐2, SNAP‐2, MutPred, I‐Mutant3.0 and MUpro) to isolate four final pathogenic SNP variants (L151P, Y144N, C111Y and D108Y). Through evolutionary conservation analysis, we determined that these mutational variants originate from a highly conserved and stable domain. Our findings particularly emphasise the Y144N variant as a potentially highly deleterious mutation situated in the SEA5 domain. This variant could significantly impact stability, overall flexibility, compactness, expansion, glycosylation ability and metastatic potential when compared to both the wild‐type and other mutant variants. In summary, these findings shed light on missense mutational variants, providing insights into the vast array of disease susceptibilities associated with MUC16's glycosylation process. This understanding could aid in the development of effective drugs for diseases linked with these mutations.

PRMT3 and CARM1: Emerging Epigenetic Targets in Cancer

ABSTRACTThe family of protein arginine methyltransferases (PRMTs) occupies an important position in biology, especially during the initiation and development of cancer. PRMT3 and CARM1(also known as PRMT4), being type I protein arginine methyltransferases, are key in controlling tumour progression by catalysing the mono‐methylation and asymmetric di‐methylation of both histone and non‐histone substrates. This paper reviews the functions and potential therapeutic target value of PRMT3 and CARM1 in a variety of cancers. Studies have identified abnormal expressions of PRMT3 and CARM1 in several malignancies, closely linked to cancer progression, advancement, and resistance to treatment. Such as hepatocellular carcinoma, colorectal cancer, ovarian cancer, and endometrial cancer. These findings offer new strategies and directions for cancer treatment, especially in enhancing the effectiveness of conventional treatment methods.

Dual drug‐loaded polymeric mixed micelles for ovarian cancer: Approach to enhanced therapeutic efficacy of albendazole and paclitaxel

AbstractChemotherapy resistance remains a significant challenge in treating ovarian cancer effectively. This study addresses this issue by utilizing a dual drug‐loaded nanomicelle system comprising albendazole (ABZ) and paclitaxel (PTX), encapsulated in a novel carrier matrix of D‐tocopheryl polyethylene glycol 1000 succinate vitamin E (TPGS), soluplus and folic acid. Our objective was to develop and optimize this nanoparticulate delivery system using solvent evaporation techniques to enhance the therapeutic efficacy against ovarian cancer. The formulation process involved pre‐formulation, formulation, optimization, and comprehensive characterization of the micelles. Optimization was conducted through a 32 factorial design, focusing on the effects of polymer ratios on particle size, zeta potential, polydispersity index (PDI) and entrapment efficiency (%EE). The optimal formulation demonstrated improved dilution stability, as indicated by a critical micelle concentration (CMC) of 0.0015 mg/mL for the TPGS‐folic acid conjugate (TPGS‐FOL). Extensive characterization included differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and Fourier‐transform infrared spectroscopy (FTIR). The release profile exhibited an initial burst followed by sustained release over 90 h. The cytotoxic potential of the formulated micelles was superior to that of the drugs alone, as assessed by MTT assays on SKOV3 ovarian cell lines. Additionally, in vivo studies confirmed the presence of both drugs in plasma and tumour tissues, suggesting effective targeting and penetration. In conclusion, the developed TPGS‐Fol‐based nanomicelles for co‐delivering ABZ and PTX show promising results in overcoming drug resistance, enhancing solubility, sustaining drug release, and improving therapeutic outcomes in ovarian cancer treatment.

A Machine Learning Approach to Build and Evaluate a Molecular Prognostic Model for Endometrial Cancer Based on Tumour Microenvironment

ABSTRACTEndometrial cancer (EC) incidence and the associated tumour burden have increased globally. To build a molecular expression prognostic model based on the tumour microenvironment to guide personalised treatment using a machine learning approach. Two datasets were reviewed, including a training cohort (n = 698) and a testing cohort (n = 151). All patients underwent hysterectomy ± adnexectomy ± lymph nodes dissection between December 2014 and June 2020 at the PLA General Hospital First Medical Center and received necessary and regular follow‐up. We developed novel models using R software to predict factors that affect survival, such as progression‐free survival and overall survival. Then, the model was optimised by evaluating the prediction efficiency in multiple dimensions. Eight hundred and forty‐nine patients with EC were included in the study. Survival‐related influences on EC patients were identified by univariate analysis and cox regression equations. In addition, a nomogram was visualised in conjunction with demographic characteristics and the above meaningful clinicopathological variables. Ultimately, through a comprehensive assessment, a random forest model (RF16) was developed for complementing the findings of the molecular classification of EC. The RF16 not only specifically characterises tumour molecules, but also enhances the generalizability of the model by replacing gene sequencing with immunohistochemistry. This study showed that the machine learning model (RF16) is low‐cost, efficient, and clinically valuable in guiding treatment for EC patients.

The lncRNA TPTEP1 suppresses PI3K/AKT signalling and inhibits ovarian cancer progression by interacting with PTBP1

AbstractThe expression of the long noncoding RNA (lncRNA) TPTE pseudogene 1 (TPTEP1) is significantly downregulated in ovarian cancer (OC). However, the function and mechanism of the lncRNA TPTEP1 in OC have not been identified. To investigate the expression of the lncRNA TPTEP1, we analysed a publicly available dataset and 20 pairs of OC and normal ovarian samples tissue from the First Affiliated Hospital of Anhui Medical University. Functional assays were used to determine the role of the lncRNA TPTEP1 in OC progression. Furthermore, Western blot, FISH, RNA pull‐down, mass spectrometry and RNA immunoprecipitation approaches were used to determine the mechanism by which the lncRNA TPTEP1 affects OC progression. Animal experiments were used to determine the role of the lncRNA TPTEP1 in ovarian tumorigenicity in vivo. The expression of the lncRNA TPTEP1 in OC tissues was significantly lower than that in normal tissues and low expression of the lncRNA TPTEP1 was significantly correlated with advanced FIGO stage and the presence of malignant ascites in OC patients. In vitro and in vivo, regulation of the expression of the lncRNA TPTEP1 caused changes in OC cell proliferation, migration, invasion and apoptosis. Mechanistically, we found that TPTEP1 directly binds to the polypyrimidine tract‐binding protein 1 (PTBP1) protein and inhibits PI3K/AKT signalling. The lncRNA TPTEP1 inhibits PI3K/AKT signalling by directly binding PTBP1, possibly indicating the molecular mechanism underlying its biological function. With further research, these findings may aid in the development of clinically useful strategies for the treatment of OC.

High BMP7 expression is associated with poor prognosis in ovarian cancer

AbstractBone Morphogenetic Protein 7 (BMP7) is an extracellular signalling protein that belongs to the transforming growth factor‐β (TGF‐ β) superfamily. Previous transcriptomic data suggested that BMP7 expression may be disrupted in ovarian carcinoma and may play an important role in the aggressiveness of the disease. However, the protein expression in patient tumours has not been well studied. The current study aimed to assess BMP7 protein expression in a large cohort of ovarian carcinoma patient tumour samples to establish its associations with different clinical endpoints. Ovarian carcinoma tissue samples from 575 patients who underwent surgery for different subtypes of ovarian cancer were used. BMP7 protein expression was analysed by immunohistochemistry using tissue microarray and full face tumour sections. High BMP7 expression is associated with aggressive ovarian cancer clinicopathological variables including advanced FIGO stage, high grade, residual disease and poor overall survival. Elevated cytoplasmic and nuclear BMP7 expression was significantly associated with advanced FIGO stage, high tumour grade, presence of residual tumours and high‐grade serous carcinomas (p = 0.001, 0.005, 0.004, <0.001 and p < 0.001, <0.001, 0.002, 0.001 respectively). Increased cytoplasmic and nuclear BMP7 expression was also significantly associated with an adverse overall survival (p = 0.001 and 0.046 respectively). The study highlights the potential of BMP7 as a prognostic tool and as a potential novel target for ovarian cancer therapies to limit disease progression.

Machine learning‐derived identification of prognostic signature for improving prognosis and drug response in patients with ovarian cancer

AbstractClinical assessments relying on pathology classification demonstrate limited effectiveness in predicting clinical outcomes and providing optimal treatment for patients with ovarian cancer (OV). Consequently, there is an urgent requirement for an ideal biomarker to facilitate precision medicine. To address this issue, we selected 15 multicentre cohorts, comprising 12 OV cohorts and 3 immunotherapy cohorts. Initially, we identified a set of robust prognostic risk genes using data from the 12 OV cohorts. Subsequently, we employed a consensus cluster analysis to identify distinct clusters based on the expression profiles of the risk genes. Finally, a machine learning‐derived prognostic signature (MLDPS) was developed based on differentially expressed genes and univariate Cox regression genes between the clusters by using 10 machine‐learning algorithms (101 combinations). Patients with high MLDPS had unfavourable survival rates and have good prediction performance in all cohorts and in‐house cohorts. The MLDPS exhibited robust and dramatically superior capability than 21 published signatures. Of note, low MLDIS have a positive prognostic impact on patients treated with anti‐PD‐1 immunotherapy by driving changes in the level of infiltration of immune cells. Additionally, patients suffering from OV with low MLDIS were more sensitive to immunotherapy. Meanwhile, patients with low MLDIS might benefit from chemotherapy, and 19 compounds that may be potential agents for patients with low MLDIS were identified. MLDIS presents an appealing instrument for the identification of patients at high/low risk. This could enhance the precision treatment, ultimately guiding the clinical management of OV.

Dual Targeting EZH 2 and Histone Deacetylases in Human Uterine Sarcoma Cells Under Both 2 D and 3 D Culture Conditions

ABSTRACT Uterine sarcoma is strongly associated with poor prognosis. However, its treatment options remain limited. Tazemetostat is a potent and selective EZH2 inhibitor with limited clinical application. Entinostat is one of the strong inhibitors for HDAC1 and HDAC3. This study aimed to assess the effect of dual targeting of EZH2 and HDACs on the phenotype of uterine sarcoma cells in both 2D and 3D culture systems. The uterine sarcoma cell line (MES‐SA) was treated with varying concentrations of tazemetostat and/or entinostat for 24, 48 and 72 h. For 3D culture conditions, the cells were combined with Matrigel and seeded in V‐bottom plates and incubated for 5 days. Cell proliferation, cell cycle progression and apoptosis were evaluated. Additionally, the RNA expression, IHC staining, wound healing assay, DNMT and HDAC activity measurements were conducted. Our data showed that single‐inhibitor treatment with entinostat or tazemetostat significantly increased the cytotoxicity and significantly enhanced apoptosis concomitantly. Furthermore, both inhibitors induced cell cycle arrest in 2D and 3D culture conditions. We also demonstrated that entinostat, but not tazemetostat, suppressed the wound healing in the 2D culture. The combination treatment showed a significantly superior effect compared to single‐agent treatment. Our studies demonstrate that treatment with either entinostat or tazemetostat alone showed a potent anti‐uterine sarcoma effect in 2D and 3D culture conditions. Importantly, the combination of entinostat and tazemetostat produced superior therapeutic effects, suggesting that dual targeting EZH2 and HDACs may provide a promising treatment option for this aggressive cancer.

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

AbstractOestrogen 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) in vitro, and tumour growth and lung metastasis in vivo 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.

Expression of human dCTP pyrophosphatase 1 (DCTPP1) and its association with cisplatin resistance characteristics in ovarian cancer

AbstractCisplatin (DDP) resistance is a major challenge in treating ovarian cancer patients. A recently discovered enzyme called dCTP pyrophosphatase 1 (DCTPP1) has been implicated in regulating cancer characteristics, including drug responses. In this study, we aimed to understand the role of DCTPP1 in cancer progression and cisplatin response. Using publicly available databases, we analysed the expression and clinical significance of DCTPP1 in ovarian cancer. Our bioinformatics analysis confirmed that DCTPP1 is significantly overexpressed in ovarian cancer and is closely associated with tumour progression and poor prognosis after cisplatin treatment. We also found that DCTPP1 located in oxidoreductase complex and may be involved in various biological processes related to cisplatin resistance, including pyrimidine nucleotide metabolism, the P53 signalling pathway and cell cycle signalling pathways. We observed higher expression of DCTPP1 in cisplatin‐resistant cells (SKOV3/DDP) and samples compared to their sensitive counterparts. Additionally, we found that DCTPP1 expression was only enhanced in SKOV3/S cells when treated with cisplatin, indicating different expression patterns of DCTPP1 in cisplatin‐sensitive and cisplatin‐resistant cancer cells. Our study further supports the notion that cisplatin induces intracellular reactive oxygen species (ROS) and triggers cancer cell death through excessive oxidative stress. Knocking out DCTPP1 reversed the drug resistance of ovarian cancer cells by enhancing the intracellular antioxidant stress response and accumulating ROS. Based on our research findings, we conclude that DCTPP1 has prognostic value for ovarian cancer patients, and targeting DCTPP1 may be clinically significant in overcoming cisplatin resistance in ovarian cancer.

Comprehensive analysis of the interaction of antigen presentation during anti‐tumour immunity and establishment of AIDPS systems in ovarian cancer

AbstractThere are hundreds of prognostic models for ovarian cancer. These genes are based on different gene classes, and there are many ways to construct the models. Therefore, this paper aims to build the most stable prognostic evaluation system known to date through 101 machine learning strategies. We combined 101 algorithm combinations with 10 machine learning algorithms to create antigen presentation‐associated genetic markers (AIDPS) with outstanding precision and steady performance. The inclusive set of algorithms comprises the elastic network (Enet), Ridge, stepwise Cox, Lasso, generalized enhanced regression model (GBM), random survival forest (RSF), supervised principal component (SuperPC), Cox partial least squares regression (plsRcox), survival support vector machine (Survival‐SVM). Then, in the train cohort, the prediction model was fitted using a leave‐one cross‐validation (LOOCV) technique, which involved 101 different possible combinations of prognostic genes. Seven validation data sets (GSE26193, GSE26712, GSE30161, GSE63885, GSE9891, GSE140082 and ICGC_OV_AU) were compared and analysed, and the C‐index was calculated. Finally, we collected 32 published ovarian cancer prognostic models (including mRNA and lncRNA). All data sets and prognostic models were subjected to a univariate Cox regression analysis, and the C‐index was calculated to demonstrate that the antigen presentation process should be the core criterion for evaluating ovarian cancer prognosis. In a univariate Cox regression analysis, 22 prognostic genes were identified based on the expression profiles of 283 genes involved in antigen presentation and the intersection of genes (p < 0.05). AIDPS were developed by our machine learning‐based integration method, which was applied to these 22 genes. One hundred and one prediction models are fitted using the LOOCV framework, and the C‐index is calculated for each model across all validation sets. Interestingly, RSF + Lasso was the best model overall since it had the greatest average C‐index and the highest C‐index of any combination of models tested on the validated data sets. In comparing external cohorts, we found that the C‐index correlated AIDPS method using the RSF + Lasso method in 101 prediction models was in contrast to other features. Notably, AIDPS outperformed the vast majority of models across all data sets. Antigen‐presenting anti‐tumour immune pathways can be used as a representative gene set of ovarian cancer to track the prognosis of patients with cancer. The antigen‐presenting model obtained by the RSF + Lasso method has the best C‐INDEX, which plays a key role in developing antigen‐presenting targeted drugs in ovarian cancer and improving the treatment outcome of patients.

Histone modification‐linked prognostic model for ovarian cancer reveals LBX2 as a novel growth promoter

AbstractOvarian cancer (OC) is a deadly disease with limited treatment options and poor overall survival rates. This study aimed to investigate the role of histone modification‐related genes in predicting the prognosis of OC patients. Transcriptome data from multiple cohorts, including bulk RNA‐Seq data and single‐cell scRNA‐Seq data, were collected. Gene set enrichment analysis was used to identify enriched gene sets in the histone modification pathway. Differentially expressed genes (DEGs) between histone modification‐high and histone modification‐low groups were identified using Lasso regression. A prognostic model was constructed using five selected prognostic genes from the DEGs in the TCGA‐OV cohort. The study found enrichment of gene sets in the histone modification pathway and identified five prognostic genes associated with OC prognosis. The constructed risk score model based on histone modification‐related genes was correlated with immune infiltration of T cells and M1 macrophages. Mutations are more prevalent in the high‐risk group compared to the low‐risk group. Several drugs were screened against the model genes. Through in vitro experiments, we confirmed the expression patterns of the model genes. LBX2 facilitates the proliferation of OC. Histone modification‐related genes have the potential to serve as biomarkers for predicting OC prognosis. Targeting these genes may lead to the development of more effective therapies for OC. Additionally, LBX2 represents a novel cell proliferation promoter in OC carcinogenesis.

RPL35A drives ovarian cancer progression by promoting the binding of YY1 to CTCF promoter

AbstractOvarian cancer is one of the most common gynaecological malignancies with poor prognosis and lack of effective treatment. The improvement of the situation of ovarian cancer urgently requires the exploration of its molecular mechanism to develop more effective molecular targeted drugs. In this study, the role of human ribosomal protein l35a (RPL35A) in ovarian cancer was explored in vitro and in vivo. Our data identified that RPL35A expression was abnormally elevated in ovarian cancer. Clinically, high expression of RPL35A predicted short survival and poor TNM staging in patients with ovarian cancer. Functionally, RPL35A knock down inhibited ovarian cancer cell proliferation and migration, enhanced apoptosis, while overexpression had the opposite effect. Mechanically, RPL35A promoted the direct binding of transcription factor YY1 to CTCF in ovarian cancer cells. Consistently, RPL35A regulated ovarian cancer progression depending on CTCF in vitro and in vivo. Furthermore, RPL35A affected the proliferation and apoptosis of ovarian cancer cells through PPAR signalling pathway. In conclusion, RPL35A drove ovarian cancer progression by promoting the binding of YY1 and CTCF promoter, and inhibiting this process may be an effective strategy for targeted therapy of this disease.

ALKBH5 regulates ovarian cancer growth via demethylating long noncoding RNA PVT1 in ovarian cancer

AbstractThe long noncoding RNA PVT1 is reported to act as an oncogene in several kinds of cancers, especially ovarian cancer (OV). Abnormal levels of N6‐methyladenosine, a dynamic and reversible modification, are associated with tumorigenesis and malignancies. Our previous study reported that PVT1 plays critical roles in regulating OV. However, it is still largely unknown how m6A modification affects OV via PVT1. In this study, we aimed to investigate the regulation of ALKBH5 by affecting PVT1 in OV. We first found that the PVT1 RNA level was higher in OV cells than in IOSE80 cells, and conversely, the m6A modification level of PVT1 was lower in OV cells. By searching the HPA, ALKBH5, which is responsible for PVT1 demethylation, was found to be upregulated in OV tissues versus normal ovarian tissues. ALKBH5 binds to PVT1 RNA, and knockdown of ALKBH5 decreased PVT1 RNA levels. ALKBH5 also increased FOXM1 levels by upregulating PVT1, at least partially. Knockdown of ALKBH5 suppressed OV growth, colony formation, tumour formation and invasion, which were partially reversed by overexpression of PVT1. Moreover, ALKBH5 knockdown decreased FOXM1 levels by regulating PVT1 RNA expression, subsequently increasing the sensitivity to carboplatin, 5‐FU and docetaxel chemotherapy. Taken together, these results indicate that ALKBH5 directly regulates the m6A modification and stability of PVT1. Then, modified PVT1 further regulates FOXM1 and thus affects malignant behaviours and chemosensitivity in OV cells. All these results indicate that ALKBH5 regulates the malignant behaviour of OV by regulating PVT1/FOXM1.

Fluorescence in situ hybridization test for detection of endometrial carcinoma cells by non‐invasive vaginal swab

AbstractEndometrial cancer (EC) is the most common gynaecological malignancy with increasing incidence in developed countries. As gold standard, hysteroscopy confirms only 30% of suspected ECs. The detection of EC cells in the vagina by fluorescence in situ hybridization (FISH) after a smear test could reduce invasive procedures in the future. Using array‐based comparative genome hybridization (aCGH) on 65 endometrial carcinomas, most frequently imbalanced regions of the tumour genome were identified. Bacterial artificial chromosomes were used to generate FISH‐probes homologue to these human regions. The FISH test was hybridized on swabs specimens collected from the vaginal cavity. Samples from six patients without EC were selected as a negative control and on 13 patients with known EC as a positive control. To distinguish between benign and EC cases, the cut‐off value has been defined. A first validation of this EC‐FISH Test was performed with swabs from 41 patients with suspected EC. The most common genomic imbalances in EC are around the CTNNB1, FBXW7 and APC genes. The cut‐off is defined at 32% of analysed cells without diploid signal pattern. This differs significantly between the positive and negative controls (p < 0.001). In a first validation cohort of 41 patients with suspected EC, the EC‐FISH Test distinguishes patients with and without EC with a sensitivity of 91% and a specificity of 83%. The negative predictive value is 96%. This is the first report of a non‐invasive EC‐FISH Test to predict EC in women with suspected EC.

RAD51AP1 promotes progression of ovarian cancer via TGF‐β/Smad signalling pathway

AbstractOvarian cancer (OC) is one of the leading causes of female deaths. However, the molecular pathogenesis of OC has still remained elusive. This study aimed to explore the potential genes associated with the progression of OC. In the current study, 3 data sets of OC were downloaded from the GEO database to identify hub gene. Somatic mutation data obtained from TCGA were used to analyse the mutation. Immune cells were used to estimate effect of the hub gene to the tumour microenvironment. RNA‐seq and clinical data of OC patients retrieved from TCGA were used to investigate the diagnostic and prognostic values of hub gene. A series of in vitro assays were performed to indicate the function of hub gene and its possible mechanisms in OC. As a result, RAD51AP1 was found as a hub gene, which expression higher was mainly associated with poor survival in OC patients. Up‐regulation of RAD51AP1 was closely associated with mutations. RAD51AP1 up‐regulation accompanied by accumulated Th2 cells, but reduced CD4 + T cells and CD8 + T cells. Nomogram demonstrated RAD51AP1 increased the accuracy of the model. Down‐regulation of RAD51AP1 suppressed proliferation, migration and invasion capabilities of OC cells in vitro. Additionally, scatter plots showed that RAD51AP1 was positively correlated with genes in TGF‐β/Smad pathway. The above‐mentioned results were validated by RT‐qPCR and Western blotting. In conclusion, up‐regulation of RAD51AP1 was closely associated with mutations in OC. RAD51AP1 might represent an indicator for predicting OS of OC patients. Besides, RAD51AP1 might accelerate progression of OC by TGF‐β/Smad signalling pathway.

Tool Comparison for Detecting Tumour Cells in Endometrial Cancer via Single‐Cell Copy Number Variations Analysis

ABSTRACT Copy number variations (CNVs) are considered a hallmark of cancer and their inference from high‐resolution single‐cell transcriptome (scRNA‐seq) analyses may offer great opportunities for the study of tumor heterogeneity. We compared the results of four major tools (SCEVAN, CopyKAT, InferCNV and sciCNV) that use inferred CNVs to predict endometrial cancer (EC) cells, in order to assess their reliability and offer useful suggestions to researchers to improve the accuracy of their predictions. In this study, we identified EC cells from publicly available scRNA‐seq data using well‐established EC biomarkers reported in the literature. SCEVAN and CopyKAT tools have moderate sensitivity, but significantly overestimate the true number of true EC tumour cells. However, a comparative analysis of the different tumour subclones revealed that a lower number of false positives can be obtained by selecting only those that contain a high percentage of epithelial cells. In contrast, InferCNV and sciCNV do not directly predict tumour cells, but rather infer CNVs and compute CNV scores. However, the score distribution curves of the CNV scores did not clearly distinguish between malignant and non‐malignant cell populations, and therefore we were unable to evaluate the performance of either software. We highlight the lack of agreement between the tools and also towards the expected results. Our findings suggest exercising caution in the automated use of these tools. Until more accurate algorithms become available, we recommend filtering predictions ensuring that the necessary but not sufficient condition that the predicted tumour cells are at least epithelial is met.

SCENE: Signature Collection for Endometrial Cancer Prognosis

ABSTRACTEndometrial cancer (EC) is the most common malignancy of the female reproductive tract; its prognosis is difficult to predict. Despite the technique of single‐cell transcriptomic analysis (scRNA‐seq) returning single‐cell level expression data and promising to improve the accuracy of prognosis prediction, a tool that correlates transcriptomic signatures with survival is missing. To this aim, we have created SCENE, a database that collects information to correlate EC transcriptomic signatures with patient prognosis. We performed a review of the literature present in PubMed to collect transcriptomic signatures annotated with their characteristics, differential expression between healthy and sick patients, between patients with more and less favourable prognosis, and cellular pathways in which the genes are involved, as well as references to the original studies. The analysis of about 200 studies has allowed us to obtain 700 mRNA signatures, 60 microRNA (miRNA), and 150 long non‐coding RNA (lncRNA), involved in 60 molecular pathways. Each signature is annotated with its specific prognostic outcome that it influences, such as overall survival (OS), progression‐free survival (PFS), relapse‐free survival (RFS), and disease‐specific survival (DSS). The SCENE resource collects and annotates information that is widespread in the literature to facilitate the interpretation of transcriptomic data obtained with any technique in EC. In the case of scRNA‐seq data, SCENE may reveal cells predisposed to develop therapy resistance and metastasis.

METTL3‐induced FGD5‐AS1 contributes to the tumorigenesis and PD‐1/PD‐L1 checkpoint to enhance the resistance to paclitaxel of endometrial carcinoma

AbstractEndometrial cancer (EC), a widely occurring cancer in the uterus, is among the top four most frequent malignancies in women. To improve approaches for combating this disease, it is essential to gain a more comprehensive comprehension of the intricate causes of EC. Accumulating evidence highlight the essential role of long non‐coding RNA (LncRNA) in EC progression, while its biological and mechanical function has not been fully revealed. In this study, a LncRNA microarray analysis was performed using four pairs of paclitaxel (PTX) resistant EC cells, FGD5‐AS1 was identified as a significantly upregulated gene. Biologically, it was found that FGD5‐AS1 enhances chemoresistance of EC cells to PTX treatment and blocking immune escape via PD‐1/PD‐L1 checkpoint. Furthermore, FGD5‐AS1 exerted an oncogene role in EC cells via promoting cell proliferation and migration. Mechanically, METTL3 could upregulate FGD5‐AS1 expression via N6‐methyladenosine (m6A) modification. The biological roles of METTL3 were exerted via modulating FGD5‐AS1 expression in EC. Collectively, our research has shed light on the involvement of the METTL3/FGD5‐AS1 axis in the development of PTX resistance in EC. This finding offers a new avenue for further exploration of the underlying mechanisms of chemoresistance in EC and provides valuable insights for the development of potential therapeutic targets in the treatment of EC.

SNORA73B promotes endometrial cancer progression through targeting MIB1 and regulating host gene RCC1 alternative splicing

AbstractEndometrial cancer (EC) is a common gynaecological malignant tumour with unclear pathogenesis. Small nucleolar RNA (snoRNA) is involved in many biological processes, including those of cancers. Using the Cancer Genome Atlas (TCGA) database, the expression pattern of a snoRNA, SNORA73B, was analysed. The biological functions of SNORA73B were assessed by in vitro proliferation, apoptosis, migration, and invasion assays and in vivo by the xenograft model. RNA sequencing (RNA‐seq) and RNA immunoprecipitation assays were performed to determine the relationship between SNORA73B and its target genes. High‐performance liquid chromatography (HPLC) was performed to detect the pseudouridine content of the mindbomb E3 ubiquitin protein ligase 1 gene (MIB1). The stability of MIB1 mRNA was evaluated using a transcription inhibitor, actinomycin D. By performing co‐immunoprecipitation assays, the change in the ubiquitin levels of the Jagged canonical Notch ligand 1 (Jag 1), caused by SNORA73B and MIB1, was identified. RNA‐seq and qRT‐PCR were performed to detect the alternative splicing of the regulator of the chromosome condensation 1 gene (RCC1). The TCGA database analysis showed that SNORA73B was highly expressed in EC. SNORA73B promoted cell proliferation, migration, and invasion and inhibited apoptosis. SNORA73B modified the pseudouridine content in MIB1 and increased the stability of MIB1 mRNA and protein; thus, it affected Jag 1 ubiquitination and further activated the Notch pathway. SNORA73B also affected the alternative splicing of RCC1, increasing the number of transcripts, RCC1‐T2 and RCC1‐T3, which promoted cell proliferation, migration, and invasion. SNORA73B can be a potential target for EC.

Integrated analysis identifies RAC3 as an immune‐related prognostic biomarker associated with chemotherapy sensitivity in endometrial cancer

AbstractEndometrial cancer (EC) is one of the most common gynaecological malignant tumours with a high incidence, leading to urgent demands for exploring novel carcinogenic mechanisms and developing rational therapeutic strategies. The rac family of small GTPase 3 (RAC3) functions as an oncogene in various human malignant tumours and plays an important role in tumour development. However, the critical roles of RAC3 in the progression of EC need further investigation. Based on TCGA, single‐cell RNA‐Seq, CCLE and clinical specimens, we revealed that the RAC3 was specifically distributed in EC tumour cells compared to normal tissues and functioned as an independent diagnostic marker with a high area under curve (AUC) score. Meanwhile, the RAC3 expression in EC tissues was also correlated with a poor prognosis. In detail, the high levels of RAC3 in EC tissues were reversely associated with CD8+T cell infiltration and orchestrated an immunosuppressive microenvironment. Furthermore, RAC3 accelerated tumour cell proliferation and inhibited its apoptosis, without impacting cell cycle stages. Importantly, silencing RAC3 improved the sensitivity of EC cells to chemotherapeutic drugs. In this paper, we revealed that RAC3 was predominantly expressed in EC and significantly correlated with the progression of EC via inducing immunosuppression and regulating tumour cell viability, providing a novel diagnostic biomarker and a promising strategy for sensitizing chemotherapy to EC.

PPP1R14B is a diagnostic prognostic marker in patients with uterine corpus endometrial carcinoma

AbstractUterine corpus endometrial carcinoma (UCEC) is one of the most common malignancies of the female genital tract. A recently discovered protein‐coding gene, PPP1R14B, can inhibit protein phosphatase 1 (PP1) as well as different PP1 holoenzymes, which are important proteins regulating cell growth, the cell cycle, and apoptosis. However, the association between PPP1R14B expression and UCEC remains undefined. The expression profiles of PPP1R14B in multiple cancers were analysed based on TCGA and GTE databases. Then, PPP1R14B expression in UCEC was investigated by gene differential analysis and single gene correlation analysis. In addition, we performed gene ontology term analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, gene set enrichment analysis, and Kaplan–Meier survival analysis to predict the potential function of PPP1R14B and its role in the prognosis of UCEC patients. Then, a tool for predicting the prognosis of UCEC, namely, a nomogram model, was constructed. PPP1R14B expression was higher in UCEC tumour tissues than in normal tissues. The results revealed that PPP1R14B expression was indeed closely associated with tumour development. The results of Kaplan–Meier plotter data indicated that patients with high PPP1R14b expression had poorer overall survival, disease‐specific survival, and progression‐free interval than those with low expression. A nomogram based on the results of multifactor Cox regression was generated. PPP1R14B is a key player in UCEC progression, is associated with a range of adverse outcomes, and can serve as a prognostic marker in the clinic.

PHF6 promotes the progression of endometrial carcinoma by increasing cancer cells growth and decreasing T‐cell infiltration

AbstractUterine corpus endometrial carcinoma (UCEC) is the most common cancer of the female reproductive tract. The overall survival of advanced and recurrent UCEC patients is still unfavourable nowadays. It is urgent to find a predictive biomarker and block tumorgenesis at an early stage. Plant homeodomain finger protein 6 (PHF6) is a key player in epigenetic regulation, and its alterations lead to various diseases, including tumours. Here, we found that PHF6 expression was upregulated in UCEC tissues compared with normal tissues. The UCEC patients with high PHF6 expression had poor survival than UCEC patients with low PHF6 expression. PHF6 mutation occurred in 12% of UCEC patients, and PHF6 mutation predicted favourable clinical outcome in UCEC patients. Depletion of PHF6 effectively inhibited HEC‐1‐A and KLE cell proliferation in vitro and decreased HEC‐1‐A cell growth in vivo. Furthermore, high PHF6 level indicated a subtype of UCECs characterized by low immune infiltration, such as CD3+ T‐cell infiltration. While knockdown of PHF6 in endometrial carcinoma cells increased T‐cell migration by promoting IL32 production and secretion. Taken together, our findings suggested that PHF6 might play an oncogenic role in UCEC patients. Thus, PHF6 could be a potential biomarker in predicting the prognosis of UCEC patients. Depletion of PHF6 may be a novel therapeutic strategy for UCEC patients.

Boric Acid Suppresses Cell Survival by Triggering Endoplasmic Reticulum Stress‐Induced Autophagy in Cervical Cancers

ABSTRACTCervical cancer ranks as the fourth most common cancer amongst women globally. This study aimed to investigate boric acid's effects on endoplasmic reticulum (ER) stress and autophagy signalling pathways in cervical cancer cells. We first assessed boric acid's effects on cell viability and proliferation in HUF and HeLa cell lines. Subsequently, we analysed cyclin D1 and CDK4 levels and boric acid‐induced nuclear morphology changes. We then examined autophagosome formation and mRNA expression of autophagy/ER stress markers (Beclin1, p62, LC3‐I/II, GRP78, p‐IRE1α, p‐PERK, CHOP and cleaved‐caspase‐3) in HeLa cells. The findings revealed that boric acid's IC50 was 3.17 mM for HUF cells but significantly lower (641.2 μM) for HeLa cells, indicating cancer cell sensitivity. In HeLa cells, boric acid‐induced a dose‐dependent decrease in cyclin D1 and CDK4 levels (associated with G1 phase arrest), which we did not observe in HUF cells. Additionally, boric acid treatment caused nuclear abnormalities in HeLa cells. Boric acid promoted autophagy by enhancing autophagosome formation and upregulating Beclin1, p62, and LC3‐I/II expression. Concurrently, it induced ER stress by increasing GRP78, p‐IRE1α, p‐PERK and CHOP expression. Furthermore, boric acid increased cleaved‐caspase‐3 expression and apoptotic cell counts. In conclusion, this study underscores boric acid's potential therapeutic effects in cervical cancer through ER stress and autophagy regulation.

Circ_0067835 sponges miR‐324‐5p to induce HMGA1 expression in endometrial carcinoma cells

AbstractEndometrial cancer is a common gynaecological malignant tumour among women across the world. Circular RNAs (circRNAs) are a novel kind of non‐coding RNAs, and they can play a crucial role in multiple cancers. Nevertheless, the mechanisms of circRNAs in regulating gene expression in endometrial cancer are still unclear. Here, our work sought to focus on the role that circ_0067835 exert in progression and development of endometrial cancer cells. We observed circ_0067835 was markedly elevated in endometrial cancer. Then, changes in endometrial cancer cell (RL95‐2 and HEC‐1B) function were determined after circ_0067835 knockdown. Loss‐of‐functional assays revealed that circ_0067835 down‐regulation significantly repressed RL95‐1 and HEC‐1B cell proliferation, migration and invasion. Bioinformatics analysis, luciferase reporter experiment and RNA pull‐down assay were employed to predict and validate circ_0067835 can bind to miR‐324‐5p. Increase in miR‐324‐5p remarkably depressed the proliferation, migration and invasion of endometrial cancer cells via inhibiting high mobility group A1 (HMGA1). HMGA1 is identified as a vital prognostic biomarker in endometrial cancer. Currently, we reported circ_0067835 was positively correlated with HMGA1 in endometrial cancer. We implied that circ_0067835 was capable of sponging miR‐324‐5p and inducing its downstream target HMGA1 in vitro and in vivo. In conclusion, circ_0067835 can compete with miR‐324‐5p, resulting in HMGA1 up‐regulation, and therefore induce the development of endometrial cancer.

Long non‐coding RNA DLX6‐AS1 mediates proliferation, invasion and apoptosis of endometrial cancer cells by recruiting p300/E2F1 in DLX6 promoter region

AbstractEndometrial cancer features abnormal growth of cells of the inner lining of the uterus with the potential to invade to other organs. Accumulating evidence suggests that aberrant expression of long non‐coding RNA (lncRNA) may facilitate cancer progression. The aim of the present study was to identify the molecular mechanisms of the lncRNA known as DLX6 antisense RNA 1 (DLX6‐AS1) in endometrial cancer. Microarray‐based analysis was utilized to predict expression profile and possible function pattern of DLX6‐AS1 and DLX6 in endometrial cancer, and their expression was quantified in 78 clinically obtained endometrial cancer tissues and also in cell lines. We next assessed the effects of DLX6‐AS1 and DLX6 on proliferation, invasion and apoptosis of endometrial cancer cells. A mouse xenograft model was established to confirm DLX6‐AS1 functions and explore its underlying regulatory mechanisms in vivo. DLX6‐AS1 and DLX6 were highly expressed in endometrial cancer tissues and cells, and their silencing weakened the proliferative and invasive abilities of endometrial cancer cells and tumours, while promoting apoptosis. Mechanistic investigations indicated that DLX6‐AS1 formed a triplex structure with DLX6 via interaction with p300/E2F1 acetyltransferase. Thus, we find that functional up‐regulation of DLX6‐AS1 can promote endometrial cancer progression via a novel triplex mechanism that may prove to be great clinical significance for future treatments of endometrial cancer.

CircPSMC3 alleviates the symptoms of PCOS by sponging miR‐296‐3p and regulating PTEN expression

AbstractPolycystic ovary syndrome (PCOS), the most common female endocrine disease that causes anovulatory infertility, still lacks promising strategy for the accurate diagnosis and effective therapeutics of PCOS attributed to its unclear aetiology. In this study, we determined the abnormal reduction in circPSMC3 expression by comparing the ovarian tissue samples of PCOS patients and normal individuals. The symptom relief caused by up‐regulation of circPSMC3 in PCOS model mice suggested the potential for further study. In vitro functional experiments confirmed that circPSMC3 can inhibit cell proliferation and promote apoptosis by blocking the cell cycle in human‐like granular tumour cell lines. Mechanism study revealed that circPSMC3 may play its role through sponging miR‐296‐3p to regulate PTEN expression. Collectively, we preliminarily characterized the role and possible insights of circPSMC3/miR‐296‐3p/PTEN axis in the proliferation and apoptosis of KGN cells. We hope that this work provides some original and valuable information for the research of circRNAs in PCOS, not only to better understand the pathogenesis but also to help provide new clues for seeking for the future therapeutic target of PCOS.

Utilises Machine Learning Techniques to Deeply Analyse the Role of Lysosome‐Dependent Cell Death in Endometrial Cancer and Its Interactions With the Tumour Microenvironment

ABSTRACT By integrating gene expression data, clinical features and multimodal data, we constructed a machine learning model capable of accurately predicting the prognosis of endometrial cancer patients. The study found that key genes related to lysosome‐dependent cell death exhibit significant expression pattern heterogeneity in endometrial cancer and are closely associated with immune cell infiltration and metabolic characteristics within the tumour microenvironment. Patients in the high‐risk group tend to have lower immune scores and a higher prevalence of immunosuppressive cell types, such as regulatory T cells and M2 macrophages, which may be linked to poorer prognosis and resistance to immunotherapy. Additionally, we discovered that the expression of lysosome‐dependent cell death‐related genes correlates with patients' sensitivity to chemotherapeutic drugs, providing new perspectives for personalised treatment of endometrial cancer. Through this study, we characterised the prognostic relevance of lysosome‐dependent cell death–related genes in endometrial cancer, and identified biomarkers with potential utility for risk assessment and therapeutic stratification.

Prognostic significance of immune landscape in tumour microenvironment of endometrial cancer

AbstractTumour microenvironment (TME) is crucial to tumorigenesis. This study aimed to uncover the differences in immune phenotypes of TME in endometrial cancer (EC) using Uterine Corpus Endometrial Carcinoma (UCEC) cohort and explore the prognostic significance. We employed GVSA enrichment analysis to cluster The Cancer Genome Atlas (TCGA) EC samples into immune signature cluster modelling, evaluated immune cell profiling in UCEC cohort (n = 538) and defined four immune subtypes of EC. Next, we analysed the correlation between immune subtypes and clinical data including patient prognosis. Furthermore, we analysed the expression of immunomodulators and DNA methylation modification. The profiles of immune infiltration in TCGA UCEC cohort showed significant difference among four immune subtypes of EC. Among each immune subtype, natural killer T cells (NKT), dendritic cells (DCs) and CD8+T cells were significantly associated with EC patients survival. Each immune subtype exhibited specific molecular classification, immune cell characterization and immunomodulators expression. Moreover, the expression immunomodulators were significantly related to DNA methylation level. In conclusion, the identification of immune subtypes in EC tissues could reveal unique immune microenvironments in EC and predict the prognosis of EC patients.

CircRNA WHSC1 targets the miR‐646/NPM1 pathway to promote the development of endometrial cancer

AbstractCircular RNAs (circRNAs) play important roles in human cancer progression. Their high stability and tissue specificity make circRNAs important molecular targets for clinical diagnosis, treatment and prognosis. However, the functions and molecular mechanisms of circRNA WHSC1 in endometrial cancer are unknown. CircWHSC1 expression in normal endometrial and endometrial cancer tissues was detected using PCR. Overexpression or knockdown of circWHSC1 in endometrial cancer cell lines HEC‐1B or Ishikawa, respectively, cell function experiments were used to detect the impact of circWHSC1 on endometrial cancer cells. A nude mouse xenograft model was used to detect changes in tumorigenesis of HEC‐1B cells after circWHSC1 overexpression. Bioinformatics and dual luciferase reporter gene technology were used to predict and validate the sponging ability of circWHSC1 on microRNAs. Gene expression changes were detected by using Western blotting. CircWHSC1 expression was increased in endometrial cancer tissues. CircWHSC1 overexpression promoted the proliferation, migration and invasion of endometrial cancer cells and decreased apoptosis. CircWHSC1 knockdown had the opposite effect. CircWHSC1 overexpressed nude mice showed increased tumorigenicity. Bioinformatics predicted that circWHSC1 binds to miR‐646, which was confirmed using luciferase reporter gene assays. High expression of miR‐646 could reverse the effect of circWHSC1 on endometrial cancer cells. Western blotting showed increased or decreased levels of nucleophosmin 1 (NPM1), an miR‐646 downstream target, after circWHSC1 overexpression or knockdown, respectively. CircWHSC1 promotes endometrial cancer development through sponging miR‐646 and targeting NPM1.

Circ_PUM1 promotes the development of endometrial cancer by targeting the miR‐136/NOTCH3 pathway

Abstract Endometrial cancer is one of the most common gynaecological malignancies and the sixth most common cause of cancer‐related death among women. Here, we define the role and molecular mechanism of circ_0000043 (hereafter referred to as circ_PUM1) in the development and progression of endometrial carcinoma. QRT‐PCR was used to detect the expression of circ_PUM1 in normal endometrial tissue and endometrial carcinoma tissues. Changes in cell function and tumorigenicity in nude mice were examined after circ_PUM1 overexpression or knockdown. Bioinformatic analysis and dual‐luciferase reporter assay were used to predict and analyse the miRNAs that circ_PUM1 binds. Gene expression changes were analysed using Western blot. Circ_PUM1 was expressed at significantly higher levels in endometrial cancer tissues than in normal tissues. Up‐regulation of circ_PUM1 promoted the proliferation, migration and invasion of endometrial carcinoma cells. Opposite results were observed with circ_PUM1 knockdown, and the tumorigenic ability of endometrial cancer cells after circ_PUM1 knockdown was reduced compared to control cells. Circ_PUM1 is capable of binding to miR‐136, and up‐regulating its target gene NOTCH3, which can be reversed by overexpression of miR‐136. Circ_PUM1 can compete with miR‐136, leading to up‐regulation of NOTCH3, and thereby promote the development of endometrial cancer.

Relationship between polycomb‐group protein BMI‐1 and phosphatases regulating AKT phosphorylation level in endometrial cancer

AbstractThe PI3K/AKT pathway is frequently activated in endometrial carcinoma. BMI‐1 (B‐lymphoma Mo‐MLV insertion region 1) protein affects expression of PTEN (phosphatase and tensin homolog) in some cancers, but its significance for endometrial tumorigenesis is not known. The objective of this study was to determine the relationship between BMI‐1 and expression of factors affecting AKT (protein kinase B) phosphorylation level in endometrial cancer. The expression of proteins and mRNAs was investigated in endometrial cancer specimens and samples of non‐neoplastic endometrial tissue by Western blot and RT‐PCR, respectively. The impact of BMI‐1 down‐regulation on AKT phosphorylation and expression of genes coding for several phosphatases were studied in HEC1A cells. The results showed that BMI‐1 depletion caused increase in PHLPP1 and PHLPP2 (PH domain and leucine‐rich repeat protein phosphatases 1/2) expression and decrease in phospho‐AKT (pAKT) level. In more advanced tumours with higher metastatic potential, the expression of BMI‐1 was lower compared to tumours less advanced and without lymph node metastasis. There were significant inverse correlations between BMI‐1 and PHLPPs, especially PHLPP1 in normal endometrial samples. The inverse correlation between BMI‐1 and PHLPP1/PHLPP2 expression was observed in PTEN positive but not PTEN negative cancers. Low PHLPP2 expression in tumours predicted poorer overall survival. BMI‐1 impacts on AKT phosphorylation level in endometrial cells by regulation of PHLPP expression.

Single‐Cell RNA Sequencing Analysis Reveals Correlation Between Immune Cell Composition and Gene Expression in Cervical Cancer

ABSTRACT Cervical cancer has become a glaring concern for women's health globally. The use of single‐cell RNA sequencing (scRNA‐seq) contributes to a comprehensive understanding of cellular heterogeneity and the immune cell landscape in the TME of cervical cancer. This study is to investigate the distribution pattern of immune cell subsets and their correlation with some gene expression based on single‐cell RNA sequencing (scRNA‐seq) data in patients with cervical cancer. We collected cervical cancer single‐cell RNA sequencing data and explored the quality of the data using the violin plots, scatter plots, variance plots and elbow plots, as well as a search for highly variable genes. We clustered cells with UMAP and t‐SNE clustering analyses and then labelled cell populations via flow cytometry and immunohistochemistry. We also analysed the biological functions of critical genes using GO enrichment analysis, and the expression patterns of individual genes at the single‐cell level. Lastly, we calculated the shift of immune cell proportion and explored the relationship between key genes like TNFRSF18 and immune cell subgroups. We identified 12 unique cell populations in cervical cancer samples and stained positive for epithelial cells, T cells and macrophages. Functional enrichment analysis revealed the gene expression pattern associated with multiple biological processes and molecular interactions in the tumour microenvironment. Certain genes, such as 16 FOXP3 and CD8A, displayed different expression patterns across the immune cell subsets. Additionally, the expression of TNFRSF18 was directly related to the proportions of most of the immune cells and inversely related to a few T and B lymphocyte subsets. This study offers a comprehensive landscape of immune cell proportions within the cervical cancer TME and uncovers a complexity in the relationships between gene expression and tumour‐infiltrating immune cell subsets. These results will provide valuable clues for the study of the immune microenvironment in cervical cancer and will shed some light into novel therapeutic approaches.

Identification of Glutathione Synthetase as a Therapeutic Target for Cervical Cancer via Combining Bioinformatics and Experimental Validation

ABSTRACT Cervical cancer remains a leading cause of cancer‐related mortality among women worldwide, posing a severe threat to female health. Previous research indicates that cuproptosis is a copper‐dependent form of regulated cell death, holding potential as a therapeutic avenue. This study aimed to identify and validate Cuproptosis‐Related Genes (CRGs) as biomarkers and therapeutic targets in cervical cancer. Transcriptomic data from TCGA and GTEx databases were analysed alongside curated literature data, leading to the identification of 67 pivotal CRGs. Diagnostic and prognostic models were constructed using machine learning algorithms and LASSO‐Cox regression, respectively. Glutathione synthetase (GSS) was selected for subsequent functional validation in cellular assays. Drug sensitivity analysis, mechanistic investigations and in vivo experiments were conducted to evaluate therapeutic potential. Statistical analyses were performed using R and GraphPad Prism. Our analysis identified GSS as a core gene. Functional experiments showed that GSS promotes cervical cancer cell proliferation and invasion under cuproptosis‐inducing conditions. Drug sensitivity analysis linked GSS to vorinostat, which inhibits tumour growth by suppressing the PI3K/Akt pathway and downregulating GSS. These findings were confirmed in both in vitro and in vivo studies. This study identifies GSS as a key cuproptosis regulator and a promising therapeutic target in cervical cancer, suggesting a novel precision medicine strategy.

Impact of High‐Risk HPV Infection on PI3K, MALAT1, H19 and LINC00460 Expression in Cervical Cells

ABSTRACT High‐risk human papillomavirus (HPV) is a central factor in cervical cancer development, largely due to its E6 and E7 oncoproteins that disrupt normal cellular regulation. This study explored the influence of high‐risk HPV on the expression of PI3K and the long non‐coding RNAs (lncRNAs) MALAT1, H19 and LINC00460 in cervical cells. Using a case–control design, cervical liquid samples from 50 HPV‐positive patients and 20 healthy controls were analysed via quantitative real‐time PCR, with statistical methods employed to assess correlations between viral oncoproteins and target gene expression. Results demonstrated a significant upregulation of PI3K (24.59‐fold change, p  < 0.036), MALAT1 (9.75‐fold change, p  < 0.005), LINC00460 (1.15‐fold change, p  < 0.013) and H19 (7.1‐fold change, p  < 0.018) in HPV‐infected samples, indicating their potential role in HPV‐mediated oncogenesis. Although correlation analysis revealed trends between E6/E7 and certain lncRNAs, these were not statistically significant. Overall, these findings deepen our understanding of the molecular changes linked to high‐risk HPV infections and identify PI3K, MALAT1 and H19 as promising biomarkers and therapeutic targets for cervical cancer. Future studies should further investigate these interactions to enhance early detection and improve treatment strategies for HPV‐associated malignancies.

HDAC7 promotes ovarian cancer malignancy via AKT/mTOR signalling pathway

AbstractOvarian cancer is of the most lethal malignancy and causes serious threat to women health worldwide. A deep understanding of molecular mechanisms underlying ovarian cancer progression is critical for the development of promising therapeutic strategies. In this study, we aimed to employ immunohistochemistry to determine the protein level of HDAC7 in patient tissues, our data showed HDAC7 levels are upregulated in tumour tissues. In addition, we also performed Kaplan–Meier survival analysis to investigate the association between HDAC7 expression and clinical prognosis, and found that HDAC7 expression was associated with poor prognosis in ovarian cancer patients. Inhibition of HDAC7 cells resulted in lower cell proliferation, invasion and colony formation. Furthermore, we also found that HDAC7 inhibition suppressed PI3K/AKT/mTOR pathway. In contrast, exogenous HDAC7 expression activated the PI3K/AKT/mTOR pathway in HDAC7 knockout cells and rescued the cell proliferation, invasion and colony formation. However, inhibition of p‐AKT induced lower cell proliferation, metastasis and colony formation abilities. In murine model, HDAC7 KO significantly decreased the tumour burden. These data indicate that HDAC7 is involved in regulation of PI3K/AKT/mTOR pathway and targeting of HDAC7 could be potential therapeutic strategy in the treatment of ovarian cancer.

A Neutrophil Extracellular Traps–Related Signature Predicts Clinical Outcomes and Identifies Immune Landscape in Ovarian Cancer

ABSTRACTOvarian cancer (OvCa) is the most lethal gynaecology malignancies worldwide. Neutrophil extracellular traps (NETs), net‐like protein structures produced by activated neutrophils and DNA‐histone complexes, have a central role in tumours, though haven't been fully explored in OvCa. We obtained transcriptome data from TCGA‐OvCa database (n = 376) as training, ICGC‐OvCa database (n = 111) as validation and GTEx database (n = 180) as controls. Through LASSO‐COX Regression analysis, we identified an eight‐gene signature among 87 NETs‐related genes, which was significantly related to poor prognosis in both TCGA‐OvCa and ICGC‐OvCa cohorts (Log‐rank p‐value = 0.0003 and 0.0014). Next, we constructed and validated a prognostic nomogram, consist of NETs‐related signature and clinical features (C‐index = 0.82). We evaluated 22 typical immune cell infiltration through CIBERSORT analysis, which implied upregulation of memory CD4 + T cells, follicular helper T cells and neutrophils in high‐risk group. Additionally, we predicted therapy sensitivity through TIDE algorithm, indicating that high NETs‐riskscore exhibited more sensitivity towards Sorafenib and less sensitivity towards immunotherapy. We initially reported that RAC2 upregulation was associated with NETs formation and poor prognosis (p‐value < 0.05) through IHC analysis of tissue microarrays (n = 125). Conclusively, NETs‐related signature was reliable for OvCa prognosis prediction and therapy assessment. Especially, RAC2 was predominantly related to NETs formation, thus providing hints towards anti‐tumour mechanism of NETs in OvCa.

Ether‐Linked Glycerophospholipids Are Potential Chemo‐Desensitisers and Are Associated With Overall Survival in Carcinoma Patients

ABSTRACTLipid reprogramming in carcinoma is reported to have a role in carcinogenesis, prognosis and therapy response. The lipid reprogramming could be contributed by either autonomous or nonautonomous resources. Since the nonautonomous lipid resources contributed by lipoproteins and their receptors have been reported in epithelial ovarian cancer (EOC), the impact of autonomous lipid metabolites was unknown. This report revealed a unique lipid class, ether‐linked phosphatidyl‐ethanolamine (PE O–), which enhances chemo‐insensitivity and progression in EOC and potentially cross carcinomas. Analysis of CCLEC/GDSCC database and in‐house cell line lipidomes identified PE O– as the major lipid associated with cisplatin/paclitaxel sensitivity. In the testing of PE O– effect on cancer phenotypes, it enhanced cell growth, migratory activities and promoted cisplatin/paclitaxel insensitivity. In addition, treating AGPS inhibitor‐sensitised chemo‐cytotoxic upon cisplatin/paclitaxel treatments. Treating PE O– could reverse AGPS inhibitor chemosensitisation effect on EOC cells. At last, using TCGA‐EOC transcriptome database, the PE O– related gene expressions were positive correlated with patient prognosis in general, or in whom were treated with platin‐ or taxel‐based chemotherapies. The expressions of genes for the synthesis of PE O– aggravates therapy response in EOC patients. PE O– facilitates human carcinoma cell line growth, mobility and chemo‐insensitivity.

ATP13A2 as a prognostic biomarker and its correlation with immune infiltration in cervical cancer: A retrospective study

Abstract While the oncogene ATP13A2 is reportedly involved in colorectal cancer, its role in cervical cancer (CC) has yet to be fully characterized. In this study, we investigated ATP13A2 as a potential prognostic biomarker of CC. To this end, we compared CC tissues with normal tissues to identify differentially expressed genes, identifying ATP13A2 as a potential marker of CC. Elevated ATP13A2 expression levels were identified in CC samples compared to noncancerous samples across various data sets, with further immunohistochemical validation. Functional enrichment analysis revealed that ATP13A2 plays an essential role in the CXCL12‐activated CXCR4 signalling pathway and chemotaxis regulation, which may alter immune infiltration. Notably, increased ATP13A2 levels were associated with poor overall survival. Furthermore, multiple clinical characteristics were significantly associated with ATP13A2 expression. Additionally, tumour bacterial infiltration was assessed using weighted co‐expression network analysis, revealing a relationship between ATP13A2 expression and bacteria in the CC tumour microenvironment. Our results suggest that ATP13A2 is a promising diagnostic and prognostic marker for CC. However, further large‐scale studies are needed to fully elucidate the mechanisms underlying the involvement of ATP13A2 in CC.

Glutamine metabolism prognostic index predicts tumour microenvironment characteristics and therapeutic efficacy in ovarian cancer

AbstractMounting evidence has highlighted the multifunctional characteristics of glutamine metabolism (GM) in cancer initiation, progression and therapeutic regimens. However, the overall role of GM in the tumour microenvironment (TME), clinical stratification and therapeutic efficacy in patients with ovarian cancer (OC) has not been fully elucidated. Here, three distinct GM clusters were identified and exhibited different prognostic values, biological functions and immune infiltration in TME. Subsequently, glutamine metabolism prognostic index (GMPI) was constructed as a new scoring model to quantify the GM subtypes and was verified as an independent predictor of OC. Patients with low‐GMPI exhibited favourable survival outcomes, lower enrichment of several oncogenic pathways, less immunosuppressive cell infiltration and better immunotherapy responses. Single‐cell sequencing analysis revealed a unique evolutionary trajectory of OC cells from high‐GMPI to low‐GMPI, and OC cells with different GMPI might communicate with distinct cell populations through ligand‐receptor interactions. Critically, the therapeutic efficacy of several drug candidates was validated based on patient‐derived organoids (PDOs). The proposed GMPI could serve as a reliable signature for predicting patient prognosis and contribute to optimising therapeutic strategies for OC.

TAB2 Promotes Immune Escape and Chemoresistance Through NF‐κB Pathway Activation in Cervical Cancer

ABSTRACTCervical cancer (CC) remains a major health challenge with high mortality rates due to chemoresistance and immune escape. However, the underlying mechanisms remain unclear. We investigated the role of TAB2 in CC using cisplatin‐resistant and parental cell lines. Cell proliferation, migration, sphere formation and T cell‐mediated killing assays were performed. Western blot and qRT‐PCR analysed protein and mRNA expression. NF‐κB pathway involvement was examined using the BAY 11–7082 inhibitor. TAB2 expression was significantly elevated in cisplatin‐resistant CC cells. TAB2 overexpression promoted chemoresistance and immune escape through NF‐κB pathway activation. Conversely, TAB2 knockdown or NF‐κB inhibition sensitised resistant cells to cisplatin and enhanced T cell‐mediated killing. The resistant phenotype could be rescued by restoring PD‐L1 expression. Our findings reveal TAB2 as a critical regulator of both chemoresistance and immune escape in CC through NF‐κB pathway activation. This suggests TAB2 as a potential therapeutic target for overcoming treatment resistance in CC.

Environmental‐relevant bisphenol A exposure promotes ovarian cancer stemness by regulating microRNA biogenesis

AbstractBisphenol A (BPA) is a ubiquitous environmental xenobiotic impacting millions of people worldwide. BPA has long been proposed to promote ovarian carcinogenesis, but the detrimental mechanistic target remains unclear. Cancer stem cells (CSCs) are considered as the trigger of tumour initiation and progression. Here, we show for the first time that nanomolar (environmentally relevant) concentration of BPA can markedly increase the formation and expansion of ovarian CSCs concomitant. This effect is observed in both oestrogen receptor (ER)‐positive and ER‐defective ovarian cancer cells, suggesting that is independent of the classical ERs. Rather, the signal is mediated through alternative ER G‐protein‐coupled receptor 30 (GPR30), but not oestrogen‐related receptor α and γ. Moreover, we report a novel role of BPA in the regulation of Exportin‐5 that led to dysregulation of microRNA biogenesis through miR‐21. The use of GPR30 siRNA or antagonist to inhibit GPR30 expression or activity, respectively, resulted in significant inhibition of ovarian CSCs. Similarly, the CSCs phenotype can be reversed by expression of Exportin‐5 siRNA. These results identify for the first time non‐classical ER and microRNA dysregulation as novel mediators of low, physiological levels of BPA function in CSCs that may underlie its significant tumour‐promoting properties in ovarian cancer.

EPAS1, a hypoxia‐ and ferroptosis‐related gene, promotes malignant behaviour of cervical cancer by ceRNA and super‐enhancer

AbstractHypoxia and Ferroptosis are associated with the malignant behaviour of cervical cancer. Endothelial PAS domain‐containing protein 1 (EPAS1) contributes to the progression of cervical cancer. EPAS1 plays important roles in hypoxia and ferroptosis. Using the GEO dataset, machine‐learning algorithms were used to screen for hypoxia‐ and ferroptosis‐related genes (HFRGs) in cervical cancer. EPAS1 was identified as the hub gene. qPCR and WB were used to investigate the expression of EPAS1 in normal and cervical cancer tissues. The proliferation, invasion and migration of EPAS1 cells in HeLa and SiHa cell lines were detected using CCK8, transwell and wound healing assays, respectively. Apoptosis was detected by flow cytometry. A dual‐luciferase assay was used to analyse the MALAT1‐miR‐182‐5P‐EPAS1 mRNA axis and core promoter elements of the super‐enhancer. EPAS1 was significantly overexpressed in cervical cancer tissues. EPAS1 could increase the proliferation, invasion, migration of HeLa and SiHa cells and reduce the apoptosis of HeLa and SiHa cell. According to the double‐luciferase assay, EPAS1 expression was regulated by the MALAT1‐Mir‐182‐5p‐EPAS1 mRNA axis. EPAS1 is associated with super‐enhancers. Double‐luciferase assay showed that the core elements of the super‐enhancer were E1 and E3. EPAS1, an HFRG, is significantly overexpressed in cervical cancer. EPAS1 promotes malignant behaviour of cervical cancer cells. EPAS1 expression is regulated by super‐enhancers and the MALAT1‐miR‐182‐5P‐ EPAS1 mRNA axis. EPAS1 may be a target for the diagnosis and treatment of cervical cancer.

ISG15 is downregulated by KLF12 and implicated in maintenance of cancer stem cell‐like features in cisplatin‐resistant ovarian cancer

AbstractDrug resistance is often developed during clinical chemotherapy of ovarian cancers. The ubiquitin‐like protein interferon‐stimulated gene 15 (ISG15) is possibly dependent on tumour context to promote or suppress progression of various tumours. The ubiquitin‐like protein interferon‐stimulated gene 15 (ISG15) was decreased in cisplatin‐resistant ovarian cancer cells. The current study identified that both ectopic wild type and nonISGylatable mutant ISG15 expression inhibited CSC‐like phenotypes of cisplatin‐resistant ovarian cancer cells. Moreover, ectopic ISG15 expression suppressed tumour formation in nude mice. In addition, ISG15 downregulation promoted CSC‐like features of cisplatin‐sensitive ovarian cancer cells. Furthermore, low ISG15 expression was associated with poor prognosis in patients with ovarian cancer. Transcriptional repressor Krüppel‐like factor 12 (KLF12) downregulated ISG15 in cisplatin‐resistant cells. Our data indicated that downregulating ISG15 expression, via weakening effect of KLF12, might be considered as new therapeutic strategy to inhibit CSC phenotypes in the treatment of cisplatin‐resistant ovarian cancer.

NANOG regulates epithelial–mesenchymal transition via AMPK/mTOR signalling pathway in ovarian cancer SKOV‐3 and A2780 cells

AbstractNANOG engages with tumour initiation and metastasis by regulating the epithelial–mesenchymal transition (EMT) in epithelial ovarian cancer (EOC). However, its role in association with pAMPKα, and its clinical significance in EOC have not been elucidated even though AMPK is known to degrade NANOG in various human cancers. Hence, we investigated the role of pAMPKα and its association with NANOG as potential prognostic biomarkers in EOC. Both NANOG and pAMPKα expression were significantly overexpressed in EOCs comparing nonadjacent normal epithelial tissues, benign tissues, and borderline tumours. NANOG overexpression was significantly associated with poor disease‐free survival (DFS) and overall survival (OS), whereas pAMPKα overexpression was associated with good DFS and OS. Importantly, multivariate analysis revealed that the combination of high NANOG and low pAMPKα expression was a poor independent prognostic factor for DFS and was associated with platinum resistance. In ovarian cancer cell lines, siRNA‐mediated NANOG knockdown diminished migration and invasion properties by regulating the EMT process via the AMPK/mTOR signalling pathway. Furthermore, treatment with AMPK activator suppressed expression of stemness factors such as NANOG, Oct4 and Sox2. Collectively, these findings established that the combination of high NANOG and low pAMPKα expression was associated with EOC progression and platinum resistance, suggesting a potential prognostic biomarker for clinical management in EOC patients.

CRB2 Facilitates Epithelial Ovarian Cancer Progression by Inducing Polarity Changes via Activation of the Wnt/ PCP Signalling Pathway

ABSTRACT Ovarian cancer exhibits high molecular heterogeneity and metastatic potential, contributing to its status as a leading cause of gynecologic cancer mortality. Cell polarity is essential in tumorigenesis, yet the role of Crumbs family proteins (CRBs), key regulators of apical–basal polarity, in epithelial ovarian cancer (EOC) remains unclear. In this study, we analysed CRB expression profiles using TCGA and GEO datasets, and validated our findings through immunohistochemical staining of ovarian tumour tissue microarrays. Among CRBs, CRB2 was significantly overexpressed in EOC tissues and correlated with poor patient prognosis. Functional assays revealed that CRB2 enhances ovarian cancer cell proliferation, migration, and invasion, while suppressing apoptosis. Immunofluorescence staining of planar cell polarity markers demonstrated that CRB2 induces polarity alterations in EOC cells. Furthermore, Western blot analysis suggested that CRB2 may activate the Wnt/planar cell polarity (PCP) signalling pathway, contributing to tumour progression. These findings identify CRB2 as a key modulator of cell polarity and a potential driver of EOC aggressiveness. CRB2 may serve as a novel prognostic biomarker and therapeutic target for epithelial ovarian cancer.

SNORD99 promotes endometrial cancer development by inhibiting GSDMD‐mediated pyroptosis through 2'‐O‐methylation modification

AbstractEukaryotic cells possess multiple mechanisms of self‐destruction, including pyroptosis and necroptosis. Pyroptosis is a type of programmed cell death characterized by cellular rupture and linked to inflammation. SnoRNA, a small non‐coding RNA in the nucleolus, can dysregulate specific RNAs through 2'‐O‐methylation, contributing to tumorigenesis. Our StarBase and qRT‐PCR analysis revealed SNORD99 upregulation in endometrial cancer (EC) tissue compared to normal tissue, suggesting its role in pathogenesis. SNORD99 overexpression enhanced migration and proliferation of EC cells, while ASO‐mediated suppression reduced malignant cell spread and division. RNA‐seq and base‐comparing analysis identified GSDMD's differential expression upon SNORD99 overexpression, forming the SNORD99‐FBL RNP complex. RTL‐P experiments showed SNORD99 increased GSDMD's 2'‐O‐methylation. SNORD99 reduced GSDMD, caspase‐1, and NLRP3 protein levels, implicating its role in pyroptosis. Optical and electron microscopy confirmed enhanced pyroptosis features. In summary, SNORD99 modifies GSDMD via 2'‐O‐methylation, suppressing pyroptosis and promoting EC progression. Developing pyroptosis‐inducing drugs may offer new cancer treatment avenues.

Atractylodes macrocephalaIII suppresses EMT in cervical cancer by regulating IGF2BP3 through ETV5

AbstractAtractylodes macrocephala III (ATL III), with anti‐inflammatory and antitumor effects, is the main compound of Atractylodes macrocephala. Whether ATL III has an effect on cervical cancer and the specific mechanism are still unclear. Here, we investigated the effects of ATL III on cervical cancer cells at different concentrations and found that ATL III downregulates insulin‐like growth factor 2 mRNA‐binding protein 3 (IGF2BP3), which was found to be highly expressed in cervical cancer tissue by RNA‐Seq. In this study, we found that ATL III promotes apoptosis and regulates epithelial–mesenchymal transition (EMT) in cervical cancer cells (HeLa and SiHa cells) and that IGF2BP3 is a common target gene of ATL III in HeLa and SiHa cells. The expression level of IGF2BP3 in cervical cancer cells was proportional to their migration and invasion abilities. This was verified by transfection of cells with a small interfering RNA and an IGF2BP3 overexpression plasmid. After ATL III treatment, the migration and invasion abilities of cervical cancer cells were obviously reduced, but these effects were attenuated after overexpression of IGF2BP3. In addition, the transcription factor IGF2BP3 was predicted by the JASPAR system. After intersection with our sequencing results, we verified the promotional effect of ETV5 (ETS translocation variant 5) on IGF2BP3 and found that ALT III inhibited ETV5. In general, our research showed that ATL III inhibits the migration and invasion of cervical cancer cells by regulating IGF2BP3 through ETV5.

Synergistic effect of PARP inhibitor and BRD4 inhibitor in multiple models of ovarian cancer

AbstractOvarian cancer has the highest facility rate among gynaecological tumours. Current therapies including PARP inhibitors have a defect that ovarian tumour is easy to recurrent and become resistant to therapy. To solve this problem, we found that BRD4 inhibitor AZD5153 and PARP inhibitor olaparib had a widespread synergistic effect in multiple models with different gene backgrounds. AZD5153 sensitizes cells to olaparib and reverses the acquired resistance by down‐regulating PTEN expression levels to destabilize hereditary materials. In this study, we used the following multiple ovarian cancer models PDX, PDO and 3D/2D cell lines to elucidate the co‐effect of AZD5153 and olaparib in vivo and in vitro. The similar results of these models further proved that the mechanism identified was consistent with the biological process occurring in ovarian cancer patients after drug treatment. This consistency between the results of different models suggests the possibility of translating these laboratory research findings into clinical studies towards developing treatments.

EphA2 promotes tumorigenicity of cervical cancer by up‐regulating CDK6

AbstractErythropoietin‐producing hepatocellular receptor A2 (EphA2) receptor tyrosine kinase plays an important role in tissue organization and homeostasis in normal organs. EphA2 is overexpressed in a variety of types of solid tumours with oncogenic functions. However, the role of EphA2 in cervical cancer (CC) is still needed to be further explored. Here, we examined the role of EphA2 by establishing a stable EphA2 knock‐down CC cell lines or a stable EphA2‐overexpressed CC cells lines. Overexpression of EphA2 increased cell proliferation and migration of CC while EphA2 knock‐down decreased the CC tumorigenicity. In addition, EphA2 knock‐down suppressed CC tumour development in the xenograft mouse model. Inhibition of EphA2 by AWL‐II‐41‐27, EphA2‐specific tyrosine kinase inhibitor, or knock‐down of EphA2 decreased mRNA and protein expression of cyclin‐dependent kinase (CDK) 6 in CC cells, which increased cellular susceptibility to epirubicin (EPI), an anti‐cancer chemotherapy drug. A clinicopathological study of EphA2 was conducted on a cohort of 158 human CC patients. EphA2 protein expression was positively correlated with CDK6 protein expression, invasion depth, lymph node metastasis and clinicopathological stage (P < .05). This study demonstrates the oncogenic activity of EphA2 in vitro and in vivo, which provides insights into the relevant mechanisms that might lead to novel treatments for CC.

USF1 regulated circPRDM4 modulates tumorigenesis and immune escape in chemoresistant cervical cancer

AbstractCervical cancer (CC) represents a major global health concern, characterized by chemoresistance and immune evasion mechanisms. Circular RNAs (circRNAs), which play a crucial role in cancer pathogenesis, particularly in the case of CC, have gained significant attention. The primary objective of this study was to investigate the functional significance of circRNAs in chemoresistant CC. A significant upregulation of circPRDM4 expression in chemoresistant CC cells. To investigate the functional consequences, we conducted circPRDM4 knockdown experiments, which resulted in the effective blockade of immune escape mechanisms employed by chemoresistant CC cells. Furthermore, circPRDM4 knockdown demonstrated a significant suppression of tumorigenesis in CC cells, highlighting its contribution to the oncogenic potential of CC. Investigating the regulatory mechanisms involved, we found that the transcriptional factor upstream stimulatory factor 1 (USF1) acts as an inducer of circPRDM4 expression. Remarkably, USF1 was found to effectively modulate CC cell immune escape via its interaction with circPRDM4. Moreover, our results revealed that USF1 is intricately involved in CC cell tumorigenesis through the regulation of circPRDM4. Collectively, our study elucidates the significant roles of circPRDM4 and its upstream regulator USF1 in chemoresistant CC cells. These findings underscore the importance of circRNAs in CC pathogenesis and provide valuable insights into the mechanisms underlying immune escape and tumorigenesis.

Targeting Cancer Stem Cells and Hedgehog Pathway: Enhancing Cisplatin Efficacy in Ovarian Cancer With Metformin

ABSTRACTOvarian cancer (OC) remains a leading cause of gynaecological cancer deaths due to late diagnosis and the emergence of resistance to platinum‐based chemotherapy, like cisplatin (Cis). Here, we investigated the potential of metformin (Met), a drug commonly used for type 2 diabetes, to overcome Cis resistance in OC. Our findings revealed a synergistic effect of Met with Cis in inhibiting cell viability, proliferation and colony/sphere formation capacity in both cisplatin‐sensitive (A2780) and ‐resistant (A2780/CDDP) ovarian cancer cell lines. This synergistic action triggered apoptosis through DNA damage, S‐phase cell cycle arrest and modulation of autophagy. Met also significantly decreased the expression of pluripotency transcription factors (Oct‐4, Sox2 and Nanog), indicating its potential to target cancer stem cells (CSCs). Furthermore, the combination therapy downregulated multidrug resistance protein 1 (MDR1) and excision repair cross‐complementation group 1 (ERCC1) expression, thereby sensitising resistant cells to Cis‐induced cytotoxicity. Additionally, the combination treatment suppressed the Hedgehog (Hh) signalling pathway, which is an important factor in inhibiting CSCs. Our study highlights the potential of the Met signalling pathway to synergise with Cis, overcoming therapeutic resistance in OC by targeting diverse cellular processes, including CSCs, and warrants further investigation in preclinical models.

RETRACTED: LBX2‐AS1 promotes ovarian cancer progression by facilitating E2F2 gene expression via miR‐455‐5p and miR‐491‐5p sponging

Abstract LBX2‐AS1 is a long non‐coding RNA that facilitates the development of gastrointestinal cancers and lung cancer, but its participation in ovarian cancer development remained uninvestigated. Clinical data retrieved from TCGA ovarian cancer database and the clinography of 60 ovarian cancer patients who received anti‐cancer treatment in our facility were analysed. The overall cell growth, colony formation, migration, invasion, apoptosis and tumour formation on nude mice of ovarian cancer cells were evaluated before and after lentiviral‐based LBX2‐AS1 knockdown. ENCORI platform was used to explore LBX2‐AS1‐interacting microRNAs and target genes of the candidate microRNAs. Luciferase reporter gene assay and RNA pulldown assay were used to verify the putative miRNA‐RNA interactions. Ovarian cancer tissue specimens showed significant higher LBX2‐AS1 expression levels that non‐cancerous counterparts. High expression level of LBX2‐AS1 was significantly associated with reduced overall survival of patients. LBX2‐AS1 knockdown significantly down‐regulated the cell growth, colony formation, migration, invasion and tumour formation capacity of ovarian cancer cells and increased their apoptosis in vitro. LBX2‐AS1 interacts with and thus inhibits the function of miR‐455‐5p and miR‐491‐5p, both of which restrained the expression of E2F2 gene in ovarian cancer cells via mRNA targeting. Transfection of miRNA inhibitors of these two miRNAs or forced expression of E2F2 counteracted the effect of LBX2‐AS1 knockdown on ovarian cancer cells. LBX2‐AS1 was a novel cancer‐promoting lncRNA in ovarian cancer. This lncRNA increased the cell growth, survival, migration, invasion and tumour formation of ovarian cancer cells by inhibiting miR‐455‐5p and miR‐491‐5p, thus liberating the expression of E2F2 cancer‐promoting gene.

Long noncoding RNA ATB promotes ovarian cancer tumorigenesis by mediating histone H3 lysine 27 trimethylation through binding to EZH2

AbstractOvarian cancer (OC) remains one of the most lethal gynecological malignancies. The unfavourable prognosis is mainly due to the lack of early‐stage diagnosis, drug resistance and recurrence. Therefore, it needs to investigate the mechanism of OC tumorigenesis and identify effective biomarkers for the clinical diagnosis. It is reported that long noncoding RNAs (lncRNAs) play important roles during the tumorigenesis of OC. Therefore, the present study aimed to study the role and clinical significance of LncRNAs ATB (lnc‐ATB) in the development and progression of OC. In our research, lnc‐ATB expression in OC tissues was elevated compared with adjacent normal tissues and high expression of lnc‐ATB was associated with poor outcomes of OC patients. The silencing of lnc‐ATB blocked cell proliferation, invasion and migration in SKOV3 and A2780 cells. RNA immunoprecipitation and RNA pull‐down results showed that lnc‐ATB positively regulated the expression of EZH2 via directly interacting with EZH2. Besides, the overexpression of EZH2 partly rescued lnc‐ATB silencing‐inducing inhibition of cell proliferation, invasion and migration. Chromatin immunoprecipitation assay results demonstrated that the silencing of lnc‐ATB reduced the occupancy of caudal‐related homeobox protein 1, Forkhead box C1, Large tumour suppressor kinase 2, cadherin‐1 and disabled homolog 2 interacting protein promoters on EZH2 and H3K27me3. These data revealed the oncogenic of lnc‐ATB and provided a novel biomarker for OC diagnosis. Furthermore, these findings indicated the mechanism of lnc‐ATB functioning in the progression of OC, which provided a new target for OC therapy.

Targeting UDP‐glucose dehydrogenase inhibits ovarian cancer growth and metastasis

AbstractMore than 70% of patients with ovarian cancer are diagnosed in advanced stages. Therefore, it is urgent to identify a promising prognostic marker and understand the mechanism of ovarian cancer metastasis development. By using proteomics approaches, we found that UDP‐glucose dehydrogenase (UGDH) was up‐regulated in highly metastatic ovarian cancer TOV21G cells, characterized by high invasiveness (TOV21GHI), in comparison to its parental control. Previous reports demonstrated that UGDH is involved in cell migration, but its specific role in cancer metastasis remains unclear. By performing immunohistochemical staining with tissue microarray, we found overexpression of UGDH in ovarian cancer tissue, but not in normal adjacent tissue. Silencing using RNA interference (RNAi) was utilized to knockdown UGDH, which resulted in a significant decrease in metastatic ability in transwell migration, transwell invasion and wound healing assays. The knockdown of UGDH caused cell cycle arrest in the G0/G1 phase and induced a massive decrease of tumour formation rate in vivo. Our data showed that UGDH‐depletion led to the down‐regulation of epithelial‐mesenchymal transition (EMT)‐related markers as well as MMP2, and inactivation of the ERK/MAPK pathway. In conclusion, we found that the up‐regulation of UGDH is related to ovarian cancer metastasis and the deficiency of UGDH leads to the decrease of cell migration, cell invasion, wound healing and cell proliferation ability. Our findings reveal that UGDH can serve as a prognostic marker and that the inhibition of UGDH is a promising strategy for ovarian cancer treatment.

Dihydrotanshinone I inhibits ovarian cancer cell proliferation and migration by transcriptional repression of PIK3CA gene

Abstract Dihydrotanshinone I (DHTS), extracted from Salvia miltiorrhiza, was found to be the most effective compound of tanshen extracts against cancer cells in our previous studies. However, the therapeutic benefits and underlying mechanisms of DHTS on ovarian cancer remain uncertain. In this study, we demonstrated the cytocidal effects of DHTS on chemosensitive ovarian cancer cells with or without platinum‐based chemotherapy. DHTS was able to inhibit proliferation and migration of ovarian cancer cells in vitro and in vivo through modulation of the PI3K/AKT signalling pathways. Combinatorial treatment of DHTS and cisplatin exhibited enhanced DNA damage in ovarian cancer cells. Overall, these findings suggest that DHTS induces ovarian cancer cells death via induction of DNA damage and inhibits ovarian cancer cell proliferation and migration.

The identification of six risk genes for ovarian cancer platinum response based on global network algorithm and verification analysis

AbstractOvarian cancer is the most lethal gynaecological cancer, and resistance of platinum‐based chemotherapy is the main reason for treatment failure. The aim of the present study was to identify candidate genes involved in ovarian cancer platinum response by analysing genes from homologous recombination and Fanconi anaemia pathways. Associations between these two functional genes were explored in the study, and we performed a random walk algorithm based on reconstructed gene‐gene network, including protein‐protein interaction and co‐expression relations. Following the random walk, all genes were ranked and GSEA analysis showed that the biological functions focused primarily on autophagy, histone modification and gluconeogenesis. Based on three types of seed nodes, the top two genes were utilized as examples. We selected a total of six candidate genes (FANCA, FANCG, POLD1, KDM1A, BLM and BRCA1) for subsequent verification. The validation results of the six candidate genes have significance in three independent ovarian cancer data sets with platinum‐resistant and platinum‐sensitive information. To explore the correlation between biomarkers and clinical prognostic factors, we performed differential analysis and multivariate clinical subgroup analysis for six candidate genes at both mRNA and protein levels. And each of the six candidate genes and their neighbouring genes with a mutation rate greater than 10% were also analysed by network construction and functional enrichment analysis. In the meanwhile, the survival analysis for platinum‐treated patients was performed in the current study. Finally, the RT‐qPCR assay was used to determine the performance of candidate genes in ovarian cancer platinum response. Taken together, this research demonstrated that comprehensive bioinformatics methods could help to understand the molecular mechanism of platinum response and provide new strategies for overcoming platinum resistance in ovarian cancer treatment.

Novel gene signatures for prognosis prediction in ovarian cancer

AbstractOvarian cancer (OV) is one of the leading causes of cancer deaths in women worldwide. Late diagnosis and heterogeneous treatment result to poor survival outcomes for patients with OV. Therefore, we aimed to develop novel biomarkers for prognosis prediction from the potential molecular mechanism of tumorigenesis. Eight eligible data sets related to OV in GEO database were integrated to identify differential expression genes (DEGs) between tumour tissues and normal. Enrichment analyses discovered DEGs were most significantly enriched in G2/M checkpoint signalling pathway. Subsequently, we constructed a multi‐gene signature based on the LASSO Cox regression model in the TCGA database and time‐dependent ROC curves showed good predictive accuracy for 1‐, 3‐ and 5‐year overall survival. Utility in various types of OV was validated through subgroup survival analysis. Risk scores formulated by the multi‐gene signature stratified patients into high‐risk and low‐risk, and the former inclined worse overall survival than the latter. By incorporating this signature with age and pathological tumour stage, a visual predictive nomogram was established, which was useful for clinicians to predict survival outcome of patients. Furthermore, SNRPD1 and EFNA5 were selected from the multi‐gene signature as simplified prognostic indicators. Higher EFNA5 expression or lower SNRPD1 indicated poorer outcome. The correlation between signature gene expression and clinical characteristics was observed through WGCNA. Drug‐gene interaction was used to identify 16 potentially targeted drugs for OV treatment. In conclusion, we established novel gene signatures as independent prognostic factors to stratify the risk of OV patients and facilitate the implementation of personalized therapies.

High neuropilin and tolloid‐like 1 expression associated with metastasis and poor survival in epithelial ovarian cancer via regulation of actin cytoskeleton

AbstractAbnormal expression of neuropilin and tolloid‐like 1 (NETO1) has been detected in some human carcinomas. However, the expression of NETO1 and the underlying mechanism in epithelial ovarian cancer (EOC) remain unknown. In this study, we found that a higher NETO1 expression in EOC tissue samples compared to normal ovarian tissue samples was significantly correlated with worse overall survival. Additionally, Cox regression analysis suggested that NETO 1 was independently associated with overall survival. NETO1 overexpression enhanced the EOC cells’ migration and invasion capability in vitro via regulation of actin cytoskeleton. Mechanistically, silencing NETO1 reduced the expression of β‐tubulin, F‐actin and KIF2A. In conclusion, our results demonstrated the critical role of NETO1 in EOC invasion, and therapies aimed at inhibiting its expression or activity might significantly control EOC growth, invasion and metastatic dissemination.

PCNP promotes ovarian cancer progression by accelerating β‐catenin nuclear accumulation and triggering EMT transition

AbstractEver reports showed that PCNP is associated with human cancers including neuroblastoma and lung cancer. However, the role and underlying molecular mechanism of PCNP in ovarian cancer have not been plenty elucidated. Herein, we first investigated the expression of PCNP in ovarian cancer tissues and cells, the effects of PCNP in ovarian cancer proliferation, apoptosis, migration and invasion, and determined the molecular mechanism of PCNP in ovarian cancer progression. The results indicated that PCNP was significantly overexpressed in human ovarian cancer tissues and cells, and related to poor prognosis in ovarian cancer patients. In addition, we also detected that PCNP promoted ovarian cancer cells growth, migration and invasion, as well as inhibited ovarian cancer cells apoptosis. Mechanistically, PCNP binding to β‐catenin promoted β‐catenin nuclear translocation and further activated Wnt/β‐catenin signalling pathway. Moreover, PCNP regulated the expression of genes involved in EMT and further triggered EMT occurrence. Conclusionally, PCNP may promote ovarian cancer progression through activating Wnt/β‐catenin signalling pathway and EMT, acting as a novel and promising target for treating ovarian cancer.

Identification of metabolism‐associated molecular subtype in ovarian cancer

AbstractOvarian cancer (OC) is the most lethal gynaecological cancer with genomic complexity and extensive heterogeneity. This study aimed to characterize the molecular features of OC based on the gene expression profile of 2752 previously characterized metabolism‐relevant genes and provide new strategies to improve the clinical status of patients with OC. Finally, three molecular subtypes (C1, C2 and C3) were identified. The C2 subtype displayed the worst prognosis, upregulated immune‐cell infiltration status and expression level of immune checkpoint genes, lower burden of copy number gains and losses and suboptimal response to targeted drug bevacizumab. The C1 subtype showed downregulated immune‐cell infiltration status and expression level of immune checkpoint genes, the lowest incidence of BRCA mutation and optimal response to targeted drug bevacizumab. The C3 subtype had an intermediate immune status, the highest incidence of BRCA mutation and a secondary optimal response to bevacizumab. Gene signatures of C1 and C2 subtypes with an opposite expression level were mainly enriched in proteolysis and immune‐related biological process. The C3 subtype was mainly enriched in the T cell‐related biological process. The prognostic and immune status of subtypes were validated in the Gene Expression Omnibus (GEO) dataset, which was predicted with a 45‐gene classifier. These findings might improve the understanding of the diversity and therapeutic strategies for OC.

Dopamine and cAMP‐regulated phosphoprotein 32kDa (DARPP‐32), protein phosphatase‐1 and cyclin‐dependent kinase 5 expression in ovarian cancer

AbstractDopamine and cyclic‐AMP activated phosphoprotein Mr32kDa (DARPP‐32) is a central signalling protein in neurotransmission. Following DARPP‐32 phosphorylation by protein kinase A (PKA), DARPP‐32 becomes a potent protein phosphatase 1 (PP1) inhibitor. DARPP‐32 can itself inhibit PKA following DARPP‐32 phosphorylation by cyclin‐dependent kinase 5 (Cdk5). Increasing evidence indicates a role for DARPP‐32 and its associated signalling pathways in cancer; however, its role in ovarian cancer remains unclear. Using immunohistochemistry, expression of DARPP‐32, PP1 and Cdk5 was determined in a large cohort of primary tumours from ovarian cancer patients (n = 428, 445 and 434 respectively) to evaluate associations between clinical outcome and clinicopathological criteria. Low cytoplasmic and nuclear DARPP‐32 expression was associated with shorter patient overall survival and progression‐free survival (P = .001, .001, .004 and .037 respectively). Low nuclear and cytoplasmic DARPP‐32 expression remained significantly associated with overall survival in multivariate Cox regression (P = .045, hazard ratio (HR) = 0.734, 95% confidence interval (CI) = 0.542‐0.993 and P = .001, HR = 0.494, 95% CI = 0.325‐0.749, respectively). High cytoplasmic and nuclear PP1 expression was associated with shorter patient overall survival and high cytoplasmic PP1 expression with shorter progression‐free survival (P = .005, .033, and .037, respectively). High Cdk5 expression was associated with shorter progression‐free survival (P = .006). These data suggest a role for DARPP‐32 and associated signalling kinases as prognostic markers with clinical utility in ovarian cancer.

Polysaccharides derived from Balanophora polyandra significantly suppressed the proliferation of ovarian cancer cells through P53‐mediated pathway

AbstractOvarian cancer (OC) is ranked the first among the cancers threatening women's health. It attracts tremendous attention of cancer researchers because of its extremely high mortality rate. Recent studies have indicated that traditional herbal medicines (THMs) can play a pivotal role in cancer prevention and treatment. THMs are gaining popularity as a source of anti‐cancer agents. The plant of Balanophora polyandra, which has been used as a traditional herbal medicine, has been known for exhibiting potential haemostatic, analgesic, anti‐inflammatory and anti‐cancer properties. However, few studies on inhibitory effect of B. polyandra on OC have been performed. In the present study, we found that B. polyandra polysaccharides (BPP) induced cell cycle arrest at S phase, triggered apoptosis and inhibited migration and invasion of OC cells. Furthermore, we also found that there was a potential and close relationship between BPP and P53‐mediated pathway. Overall, these findings suggest that BPP can be a potential therapeutic agent for the treatment of OC.

Long non‐coding RNA SNGH7 Is activated by SP1 and exerts oncogenic properties by interacting with EZH2 in ovarian cancer

Abstract Long non‐coding RNAs (lncRNAs) are key regulators or a range of diseases and chronic conditions such as cancers, but how they function in the context of ovarian cancer (OC) is poorly understood. The Coding‐Potential Assessment Tool was used to assess the likely protein‐coding potential of SNHG7. SNHG7 expression was elevated in ovarian tumour tissues measured by qRT‐PCR. The online database JASPAR was used to predict the transcription factors binding to SNHG7. Twenty‐four‐well Transwell plates were used for invasion assays. RNA immunoprecipitation was performed to determine RNA‐protein associations. EdU assay was introduced to detect cell proliferation. Chromatin immunoprecipitation was performed to confirm the directly interaction between DNA and protein. We discovered that in the context of OC there is a significant up‐regulation of the lncRNA SNHG7. Knocking down this lncRNA disrupted both OC cell invasion and proliferation, while its overexpression had the opposite effect. SP1 binding sites were present in the SNHG7 promoter, and chromatin immunoprecipitation (ChIP) confirmed direct SP1 binding to this region, activating SNHG7 transcription. We found that at a mechanistic level in OC cells, KLF2 is a probable SNHG7 target, as we found that SHNCCC16 directly interacts with EZH2 and thus represses KLF2 expression. In summary, this research demonstrates that lncRNA SNHG7 is an SP1‐activated molecule that contributes to OC progression by providing a scaffold whereby EZH2 can repress KLF2 expression.

AMPH1 functions as a tumour suppressor in ovarian cancer via the inactivation of PI3K/AKT pathway

Abstract AMPH1, an abundant protein in nerve terminals, plays a critical role in the recruitment of dynamin to sites of clathrin‐mediated endocytosis. Recently, it is reported to be involved in breast cancer and lung cancer. However, the impact of AMPH1 on ovarian cancer is unclear. In this study, we used gain‐of‐function and loss‐of‐function methods to explore the role of AMPH1 in ovarian cancer cells. AMPH1 inhibited ovarian cancer cell growth and cell migration, and promoted caspase‐3 activity, resulting in the increase of cell apoptosis. In xenograft mice model, AMPH1 prevented tumour progression. The anti‐oncogene effects of AMPH1 on ovarian cancer might be partially due to the inhibition of PI3K/AKT signalling pathway after overexpression of AMPH1. Immunohistochemistry analysis showed that the staining of AMPH1 was remarkably reduced in ovarian cancer tissues compared with normal ovarian tissues. In conclusion, our study identifies AMPH1 as a tumour suppressor in ovarian cancer in vitro and in vivo. This is the first evidence that AMPH1 inhibited cell growth and migration, and induced apoptosis via the inactivation of PI3K/AKT signalling pathway on ovarian cancer, which may be used as an effective strategy.

RNA demethylase ALKBH5 promotes ovarian carcinogenesis in a simulated tumour microenvironment through stimulating NF‐κB pathway

AbstractMethylation is the main form of RNA modification. N6‐methyladenine (m6A) regulates the splicing and translation of mRNA. Alk B homologue 5 (ALKBH5) participates in the biological regulation of various cancers. However, its role in ovarian carcinogenesis has not been unveiled. In the present study, ALKBH5 showed higher expression in ovarian cancer tissue than in normal ovarian tissue, but lower expression in ovarian cancer cell lines than in normal ovarian cell lines. Interestingly, Toll‐like receptor (TLR4), a molecular functioning in tumour microenvironment (TME), demonstrated the same expression trend. To investigate the effect of abnormal TME on ovarian carcinogenesis, we established an in vitro model in which macrophages and ovarian cancer cells were co‐cultured. In the ovarian cancer cells co‐cultured with M2 macrophages, the expression of ALKBH5 and TLR4 increased. We also verified that TLR4 up‐regulated ALKBH5 expression via activating NF‐κB pathway. Depending on transcriptome sequencing, m6A‐Seq and m6A MeRIP, we found that NANOG served as a target in ALKBH5‐mediated m6A modification. NANOG expression increased after mRNA demethylation, consequently enhancing the aggressiveness of ovarian cancer cells. In conclusion, highly expressed TLR4 activated NF‐κB pathway, up‐regulated ALKBH5 expression and increased m6A level and NANOG expression, all contributing to ovarian carcinogenesis. Our study revealed the role of m6A in ovarian carcinogenesis, providing a clue for inventing new target therapy.

RETRACTED: Long non‐coding RNA FEZF1‐AS1 induced progression of ovarian cancer via regulating miR‐130a‐5p/SOX4 axis

Abstract Emerging studies have revealed the critical role of long non‐coding RNAs (lncRNAs) in epithelial ovarian cancer (EOC) development and progression. Till now, the roles and potential mechanisms regarding FEZF1 antisense RNA 1 (FEZF1‐AS1) within ovarian cancer (OC) remain unclear. The objective of this study was to uncover the biological function and the underlying mechanism of LncRNA FEZF1‐AS1 in OC progression. FEZF1‐AS1 expression levels were studied in cell lines and tissues of human ovarian cancer. In vitro studies were performed to evaluate the impact of FEZF1‐AS1 knock‐down on the proliferation, invasion, migration and apoptosis of OC cells. Interactions of FEZF1‐AS1 and its target genes were identified by luciferase reporter assays. Our data showed overexpression of FEZF1‐AS1 in OC cell lines and tissues. Cell migration, proliferation, invasion, wound healing and colony formation were suppressed by silencing of FEZF1‐AS1. In contrast, cell apoptosis was promoted by FEZF1‐AS1 knock‐down in vitro. Furthermore, online bioinformatics analysis and tools suggested that FEZF1‐AS1 directly bound to miR‐130a‐5p and suppressed its expression. Moreover, the inhibitory effects of miR‐130a‐5p on the OC cell growth were reversed by FEZF1‐AS1 overexpression, which was associated with the increase in SOX4 expression. In conclusion, our results revealed that FEZF1‐AS1 promoted the metastasis and proliferation of OC cells by targeting miR‐130a‐5p and its downstream SOX4 expression.

Identification of immune‐enhanced molecular subtype associated with BRCA1 mutations, immune checkpoints and clinical outcome in ovarian carcinoma

Abstract Ovarian carcinoma has the highest mortality among the malignant tumours in gynaecology, and new treatment strategies are urgently needed to improve the clinical status of ovarian carcinoma patients. The Cancer Genome Atlas (TCGA) cohort were performed to explore the immune function of the internal environment of tumours and its clinical correlation with ovarian carcinoma. Finally, four molecular subtypes were obtained based on the global immune‐related genes. The correlation analysis and clinical characteristics showed that four subtypes were all significantly related to clinical stage; the immune scoring results indicated that most immune signatures were upregulated in C3 subtype, and the majority of tumour‐infiltrating immune cells were upregulated in both C3 and C4 subtypes. Compared with other subtypes, C3 subtype had a higher BRCA1 mutation, higher expression of immune checkpoints, and optimal survival prognosis. These findings of the immunological microenvironment in tumours may provide new ideas for developing immunotherapeutic strategies for ovarian carcinoma.

Mutually exclusive antiproliferative effect of cell line‐specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV‐3 vs RMUG‐S

Abstract We aimed to discover cell line‐specific overexpressed HOX genes responsible for chemoresistance and to identify the mechanisms behind HOX‐induced cell line‐specific chemoresistance in EOC. Ten HOX genes and eight EOC cell lines were tested for any cell line‐specific overexpression that presents a mutually exclusive pattern. Cell viability was evaluated after treatment with cisplatin and/or siRNA for cell line‐specific overexpressed HOX genes. Immunohistochemical (IHC) staining for HOXB9 was performed in 84 human EOC tissues. HOXA10 and HOXB9 were identified as cell line‐specific overexpressed HOX genes for SKOV‐3 and RMUG‐S, respectively. Inhibiting the expression of cell line‐specific HOX genes, but not of other HOX genes, significantly decreased cell viability. In SKOV‐3 cells, cell viability decreased to 46.5% after initial 10 µM cisplatin treatment; however, there was no further decrease upon additional treatment with HOXA10 siRNA. In contrast, cell viability did not significantly decrease upon cisplatin treatment in RMUG‐S cells, but decreased to 65.5% after additional treatment with HOXB9 siRNA. In both cell lines, inhibiting cell line‐specific HOX expression enhanced apoptosis but suppressed the expression of epithelial‐mesenchymal transition (EMT) markers such as vimentin, MMP9, and Oct4. IHC analysis showed that platinum‐resistant cancer tissues more frequently had high HOXB9 expression than platinum‐sensitive cancer tissues. HOXB9, which is overexpressed in RMUG‐S but not in SKOV‐3 cells, appeared to be associated with cell line‐specific platinum resistance in RMUG‐S. Inhibiting HOXB9 overexpression in RMUG‐S cells may effectively eliminate platinum‐resistant ovarian cancer cells by facilitating apoptosis and inhibiting EMT.

Comprehensive analysis of mRNA‐level and miRNA‐level subpathway activities for identifying robust ovarian cancer prognostic signatures

AbstractOvarian cancer (OvCa) causes the highest mortality among all gynaecologic cancers. A large number of mRNA‐ or miRNA‐based signatures were identified for OvCa patient prognosis. However, the comprehensive analysis of function‐level prognostic signatures is currently not considered in OvCa. In the present study, we respectively inferred subpathway activities from mRNA and miRNA levels based on high‐throughput expression profiles and reconstructed subpathways. Firstly, the activities of two tumour pathways were calculated and the difference between normal and tumour samples were analysed using multiple tumour types. Then, we calculated subpathway activities for OvCa based on the expression profiles from both mRNA and miRNA levels. Furthermore, based on these subpathway activity matrices, we performed bootstrap analysis to obtain sub‐training sets and utilized univariate method to identify robust OvCa prognostic subpathways. A comprehensive comparison of subpathway results between these two levels was performed. As a result, we observed subpathway mutual exclusion trend between the levels of mRNA and miRNA, which indicated the necessary of combining mRNA‐miRNA levels. Finally, by using ICGC data as testing sets, we utilized two strategies to verify survival predictive power of the mRNA‐miRNA combined subpathway signatures and performed comparisons with results from individual levels. It was confirmed that our framework displayed application to identify robust and efficient prognostic signatures for OvCa, and the combined signatures indeed exhibited advantages over individual ones. In the study, we took a step forward in relevant novel integrated functional signatures for OvCa prognosis.

IL‐8 promotes cell migration through regulating EMT by activating the Wnt/β‐catenin pathway in ovarian cancer

AbstractInterleukin‐8 (IL‐8), as an inflammatory chemokine, has been previously shown to contribute to tumorigenesis in several malignancies including the ovarian cancer. However, little is known about how IL‐8 promotes the metastasis and invasion of ovarian cancers cells. In this study, we found that IL‐8 and its receptors CXCR1 and CXCR2 were up‐regulated in advanced ovarian serous cancer tissues. Furthermore, the level of IL‐8 and its receptors CXCR1 and CXCR2 expression were associated with ovarian cancer stage, grade and lymph node metastasis. In vitro, IL‐8 promoted ovarian cancer cell migration, initiated the epithelial‐mesenchymal transition (EMT) program and activated Wnt/β‐catenin signalling. However, when treated with Reparixin (inhibitor of both IL‐8 receptors CXCR1 and CXCR2), effect of both endogenous and exogenous IL‐8 was reversed. Together, our results indicated that IL‐8 triggered ovarian cancer cells migration partly through Wnt/β‐catenin pathway mediated EMT, and IL‐8 may be an important molecule in the invasion and metastasis of ovarian cancer.

MCM10: An effective treatment target and a prognostic biomarker in patients with uterine corpus endometrial carcinoma

AbstractMolecular profiling has been applied for uterine corpus endometrial carcinoma (UCEC) management for many years. The aim of this study was to explore the role of MCM10 in UCEC and construct its overall survival (OS) prediction models. Data from TCGA, GEO, cbioPotal and COSMIC databases and the methods, such as GO, KEGG, GSEA, ssGSEA and PPI, were employed to bioinformatically detect the effects of MCM10 on UCEC. RT‐PCR, Western blot and immunohistochemistry were used to validate the effects of MCM10 on UCEC. Based on Cox regression analysis using the data from TCGA and our clinical data, two OS prediction models for UCEC were established. Finally, the effects of MCM10 on UCEC were detected in vitro. Our study revealed that MCM10 was variated and overexpressed in UCEC tissue and involved in DNA replication, cell cycle, DNA repair and immune microenvironment in UCEC. Moreover, silencing MCM10 significantly inhibited the proliferation of UCEC cells in vitro. Importantly, based on MCM10 expression and clinical features, the OS prediction models were constructed with good accuracy. MCM10 could be an effective treatment target and a prognostic biomarker for UCEC patients. The OS prediction models might help establish the strategies of follow‐up and treatment for UCEC patients.

A Systematic Review of the Molecular Mechanisms Involved in the Association Between PCOS and Endometrial and Ovarian Cancers

ABSTRACTPolycystic ovary syndrome (PCOS), a major cause of female infertility, affects 4%–20% of reproductive‐age women. Metabolic and hormonal alterations are key features of PCOS, potentially raising the risk of endometrial (EC) and ovarian (OVCA) cancers. This systematic review aims to summarise the proposed molecular mechanisms involved in the association between PCOS and EC or OVCA. This is achieved by conducting a thorough literature review and utilising specific search terms to identify all relevant studies published in English from 2010 to December 2022. PRISMA was followed, and the protocol was registered on PROSPERO (CRD42022375461). The QUADAS‐2 tool and Review Manager Software were employed to evaluate study quality and risk of bias respectively. Forty‐five eligible studies were selected with molecular signatures based on genomic, transcriptomic, metabolomic, proteomic and epigenetic analyses. Genes and their products deregulated in EC and/or OVCA were identified, including BRCA1, MLH1, NQO1 and ESR1, which were also deregulated in PCOS. Serum levels of IGF1, IGFBP1, SREBP1 and visfatin in women with PCOS were also identified as potential biomarkers of enhanced EC risk. Salusin‐β serum levels in individuals with PCOS were identified as a potential biomarker for increased risk of OVCA. Gene signature–based drug repositioning identified several drug candidates: metformin, fenofibrate, fatostatin, melatonin, resveratrol and quercetin, some already established and prescribed for PCOS. In conclusion, this study provides a strong basis for further research to confirm the identified molecular signatures and associated causal links for potential therapeutic prevention strategies for EC and OVCA in women with PCOS.

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

AbstractEpithelial 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.

Interplay of precision therapeutics and MD study: Calocybe indica's potentials against cervical cancer and its interaction with VEGF via octadecanoic acid

AbstractThe evolving landscape of personalized medicine necessitates a shift from traditional therapeutic interventions towards precision‐driven approaches. Embracing this paradigm, our research probes the therapeutic efficacy of the aqueous crude extract (ACE) of Calocybe indica in cervical cancer treatment, merging botanical insights with advanced molecular research. We observed that ACE exerts significant influences on nuclear morphology and cell cycle modulation, further inducing early apoptosis and showcasing prebiotic attributes. Characterization of ACE have identified several phytochemicals including significant presence of octadeconoic acid. Simultaneously, utilizing advanced Molecular Dynamics (MD) simulations, we deciphered the intricate molecular interactions between Vascular Endothelial Growth Factor (VEGF) and Octadecanoic acid to establish C.indica's role as an anticancer agent. Our study delineates Octadecanoic acid's potential as a robust binding partner for VEGF, with comprehensive analyses from RMSD and RMSF profiles highlighting the stability and adaptability of the protein–ligand interactions. Further in‐depth thermodynamic explorations via MM‐GBSA calculations reveal the binding landscape of the VEGF–Octadecanoic acid complex. Emerging therapeutic innovations, encompassing proteolysis‐targeting chimeras (PROTACs) and avant‐garde nanocarriers, are discussed in the context of their synergy with compounds like Calocybe indica P&C. This convergence underscores the profound therapeutic potential awaiting clinical exploration. This study offers a holistic perspective on the promising therapeutic avenues facilitated by C. indica against cervical cancer, intricately woven with advanced molecular interactions and the prospective integration of precision therapeutics in modern oncology.

A novel mitochondria‐related core gene signature to predict the prognosis and evaluate tumour microenvironment in CESC single‐cell validation

AbstractMitochondria and their related genes (MTRGs) are pivotal in the tumour microenvironment (TME) of cervical cancer, influencing prognosis and treatment response. This study developed a prognostic model using MTRGs to predict overall survival (OS) in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), aiming for personalized therapy. Analysing 14 MTRGs like ISCU and NDUFA11 through techniques such as univariate Cox regression, we found that a low mitochondrial (MT) score is associated with better survival, while a high MT score predicts poorer outcomes. The TME score, particularly influenced by CD8 T cells, also correlates with prognosis, with a high score indicating favourable outcomes. The interplay between MT and TME subtypes revealed that the best prognosis is seen in patients with a low MT and high TME score. Our findings highlight the role of MTRGs as potential biomarkers and therapeutic targets in cervical cancer, offering a novel approach to improving patient outcomes through a more nuanced understanding of mitochondrial function and immune interactions within the TME. This model presents a promising avenue for enhancing the precision of prognostic assessments in CESC.

Adenosine receptor A2b confers ovarian cancer survival and PARP inhibitor resistance through IL‐6‐STAT3 signalling

AbstractOvarian cancer is the deadliest gynecologic cancer worldwide, and the therapeutic options are limited. PARP inhibitor (PARPi) represents an effective therapeutic strategy and has been approved for maintenance therapy. However, the intrinsic or acquired resistance to PARPi becomes a big challenge. To investigate the mechanisms for PARPi resistance, we analysed public databases and established Olaparib‐resistant ovarian cancer cells for exploration. Our results showed that the inflammatory pathway and adenosine receptor A2b (Adora2b/A2B) expression were significantly increased in Olaparib‐resistant cells. A2B was highly expressed in recurrent ovarian tumours and negatively correlated with the clinical outcomes in cancer patients. Olaparib treatment enhanced A2B expression through NF‐κB activation. The elevated A2B contributed to Olaparib resistance by sensing adenosine signal and promoting tumour cell survival, growth and migration via IL‐6‐STAT3 signalling. Therefore, inhibition of A2B‐IL‐6‐STAT3 axis could overcome Olaparib resistance and synergize with Olaparib to reduce cancer cell growth and lead to cell death. Our findings reveal a critical role of A2B signalling in mediating PARPi resistance independent of DNA damage repair, providing insights into developing novel therapies in ovarian cancers.

Novel miRNA markers for the diagnosis and prognosis of endometrial cancer

AbstractAs endometrial cancer (EC) is a major threat to female health worldwide, the ability to provide an accurate diagnosis and prognosis of EC is promising to improve its treatment guidance. Since the discovery of miRNAs, it has been realized that miRNAs are associated with every cell function, including malignant transformation and metastasis. This study aimed to explore diagnostic and prognostic miRNA markers of EC. In this study, differential analysis and machine learning were performed, followed by correlation analysis of miRNA‐mRNA based on the miRNA and mRNA expression data. Nine miRNAs were identified as diagnostic markers, and a diagnostic classifier was established to distinguish between EC and normal endometrium tissue with overall correct rates >95%. Five specific prognostic miRNA markers were selected to construct a prognostic model, which was confirmed more effective in identifying EC patients at high risk of mortality compared with the FIGO staging system. This study demonstrates that the expression patterns of miRNAs may hold promise for becoming diagnostic and prognostic biomarkers and novel therapeutic targets for EC.

TGFA expression is associated with poor prognosis and promotes the development of cervical cancer

AbstractCervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) are the second most common cancers in women aged 20–39. While HPV screening can help with early detection of cervical cancer, many patients are already in the medium to late stages when they are identified. As a result, searching for novel biomarkers to predict CESC prognosis and propose molecular treatment targets is critical. TGFA is a polypeptide growth factor with a high affinity for the epidermal growth factor receptor. Several studies have shown that TGFA can improve cancer growth and progression, but data on its impact on the occurrence and advancement of CESC is limited. In this study, we used clinical data analysis and bioinformatics techniques to explore the relationship between TGFA and CESC. The results showed that TGFA was highly expressed in cervical cancer tissues and cells. TGFA knockdown can inhibit the proliferation, migration and invasion of cervical cancer cells. In addition, after TGFA knockout, the expression of IL family and MMP family proteins in CESC cell lines was significantly reduced. In conclusion, TGFA plays an important role in the occurrence and development of cervical cancer. Therefore, TGFA may become a new target for cervical cancer treatment.

Prognostic and immunotherapeutic potential of regulatory T  cell‐associated signature in ovarian cancer

Abstract Tumour‐induced immunosuppressive microenvironments facilitate oncogenesis, with regulatory T cells (Tregs) serving as a crucial component. The significance of Treg‐associated genes within the context of ovarian cancer (OC) remains elucidated insufficiently. Utilizing single‐cell RNA sequencing (scRNA‐Seq) for the identification of Treg‐specific biomarkers, this investigation employed single‐sample gene set enrichment analysis (ssGSEA) for the derivation of a Treg signature score. Weighted gene co‐expression network analysis (WGCNA) facilitated the identification of Treg‐correlated genes. Machine learning algorithms were employed to determine an optimal prognostic model, subsequently exploring disparities across risk strata in terms of survival outcomes, immunological infiltration, pathway activation and responsiveness to immunotherapy. Through WGCNA, a cohort of 365 Treg‐associated genes was discerned, with 70 implicated in the prognostication of OC. A Tregs‐associated signature (TAS), synthesized from random survival forest (RSF) and Least Absolute Shrinkage and Selection Operator (LASSO) algorithms, exhibited robust predictive validity across both internal and external cohorts. Low TAS OC patients demonstrated superior survival outcomes, augmented by increased immunological cell infiltration, upregulated immune checkpoint expression, distinct pathway enrichment and differential response to immunotherapeutic interventions. The devised TAS proficiently prognosticates patient outcomes and delineates the immunological milieu within OC, offering a strategic instrument for the clinical stratification and selection of patients.

Knockdown of long non‐coding RNA LINC00176 suppresses ovarian cancer progression by BCL3‐mediated down‐regulation of ceruloplasmin

AbstractOvarian cancer is a common malignancy among women with some clinically approved diagnostic coding gene biomarkers. However, long non‐coding RNAs (lncRNAs) have been indicated to play an important role in controlling tumorigenesis of ovarian cancer. Hereby, the aim of the study was to uncover the function of lncRNA LINC00176 in the development and progression of ovarian cancer by regulating ceruloplasmin (CP). Bioinformatics prediction in combination with RT‐qPCR analysis for the expression pattern of LINC00176 revealed that LINC00176 was highly expressed in ovarian cancer tissues as well as in ovarian cancer cell lines, respectively. LINC00176 was predominantly localized in the nucleus. Delivery of si‐LINC00176, oe‐LINC00176, si‐BCL3 and si‐CP plasmids was conducted to explore the effects of LINC00176 on ovarian cancer. Promoted proliferation, migration and invasion along with reduced apoptosis were observed in cells treated with oe‐LINC00176, while si‐BCL3 and si‐CP were able to block the promoting effects. Investigations with regard to the correlation between LINC00176 and promoter region of CP turned out to be positive via B‐cell CLL/lymphoma 3 (BCL3) by means of dual‐luciferase reporter gene assay, ChIP and RIP assays. Furthermore, oncogenic properties of the LINC00176/BCL3/CP axis were also demonstrated by tumour formation in vivo generated upon injecting cells in nude mice. Our results demonstrate that restored LINC00176 initiates tumorigenesis in ovarian cancer by increasing CP expression via recruiting BCL3, the mechanism of which represented a potential and promising therapeutic target for the disease.

Predictive value of protease‐activated receptor‐2 (PAR2) in cervical cancer metastasis

AbstractMetastasis is the primary cause of an unfavourable prognosis in patients with malignant cancer. Over the last decade, the role of proteinases in the tumour microenvironment has attracted increasing attention. As a sensor of proteinases, proteinase‐activated receptor 2 (PAR2) plays crucial roles in the metastatic progression of cervical cancer. In the present study, the expression of PAR2 in multiple types of cancer was analysed by Gene Expression Profiling Interactive Analysis (GEPIA). Kaplan‐Meier plotter was used to calculate the correlation between survival and the levels of PAR2, Grb‐associated binding protein 2(Gab2) and miR‐125b. Immunohistochemistry (IHC) was performed to examine PAR2 expression in a tissue microarray (TMA) of CESCs. Empower Stats was used to assess the predictive value of PAR2 in the metastatic potential of CESC. We found that PAR2 up‐regulation was observed in multiple types of cancer. Moreover, PAR2 expression was positively correlated with the clinicopathologic characteristics of CESC. miR‐125b and its target Gab2, which are strongly associated with PAR2‐induced cell migration, are well‐characterized as predictors of the prognostic value of CESC. Most importantly, the Cancer Genome Atlas (TCGA) data set analysis showed that the area under the curve (AUC) of the PAR2 model was significantly greater than that of the traditional model (0.833 vs 0.790, P < .05), demonstrating the predictive value of PAR2 in CESC metastasis. Our results suggest that PAR2 may serve as a prognostic factor for metastasis in CESC patients.

Rad50 promotes ovarian cancer progression through NF‐κB activation

AbstractRad50 is a component of MRN (Mre11‐Rad50‐Nbs1), which participates in DNA double‐strand break repair and DNA‐damage checkpoint activation. Here, we sought to investigate the clinical and functional significance of Rad50 in high‐grade serous ovarian cancer (HGSOC). We found that Rad50 was frequently upregulated in HGSOCs and enhanced Rad50 expression inversely correlated with patient survival. In addition, ectopic expression of Rad50 promoted proliferation/invasion and induced EMT of ovarian cancer cells, whereas knockdown of Rad50 led to decreased aggressive behaviors. Mechanistic investigations revealed that Rad50 induced aggressiveness in HGSOC via activation of NF‐κB signaling pathway. Moreover, we identified CARD9 as an interacting protein of Rad50 in ovarian cancer cells and the activation of NF‐κB pathway by Rad50 is CARD9 dependent. Our findings provide evidence that Rad50 exhibits oncogenic property via NF‐κB activation in HGSOC.

Exosomal ANXA2 derived from ovarian cancer cells regulates epithelial‐mesenchymal plasticity of human peritoneal mesothelial cells

AbstractOvarian cancer, one of the malignant gynaecological tumours with the highest mortality rate among female reproductive system, is prone to metastasis, recurrence and chemotherapy resistance, causing a poor prognosis. Exosomes can regulate the epithelial‐mesenchymal plasticity of tumour cells, remodel surrounding tumour microenvironment, and affect tumour cell proliferation, invasion and metastasis. However, the function and mechanism of exosomes in the intraperitoneal implantation of ovarian cancer remain unclear. In this study, exosomal annexin A2 (ANXA2) derived from ovarian cancer cells was co‐cultured with human peritoneal mesothelial (HMrSV5) cells; functional experiments were conducted to explore the effects of exosomal ANXA2 on the biological behaviour of HMrSV5 and the related mechanisms. This study showed that ANXA2 in ovarian cancer cells can be transferred to HMrSV5 cells through exosomes, exosomal ANXA2 can not only promote the migration, invasion and apoptosis of HMrSV5 cells, but also regulates morphological changes and fibrosis of HMrSV5 cells. Furthermore, ANXA2 promotes the mesothelial‐mesenchymal transition (MMT) and degradation of the extracellular matrix of HMrSV5 cells through PI3K/AKT/mTOR pathway, finally affects pre‐metastasis microenvironment of ovarian cancer, which provides a new theoretical basis for the mechanism of intraperitoneal implantation and metastasis of ovarian cancer.

LncRNA PVT1 promotes the progression of ovarian cancer by activating TGF‐β pathway via miR‐148a‐3p/AGO1 axis

AbstractOvarian cancer is a lethal gynaecologic malignancy with poor diagnosis and prognosis. The long non‐coding RNA plasmacytoma variant translocation1 (PVT1) and argonaute 1 (AGO1) are associated with carcinogenesis and chemoresistance; however, the relationship between PVT1 and AGO1 and the downstream mechanisms in ovarian cancer remains poorly known. PVT1 and AGO1 expression was assessed through RT‐qPCR and Western blotting in both human tissues and cell lines. The viability and proliferation of ovarian cancer cells were determined by CCK‐8 assay and TUNEL assay in vitro and immunohistochemistry in vivo. Cell invasion and migration were investigated through transwell and wound‐healing assays. The roles and mechanisms of AGO1 on cell functions were further probed via gain‐ and loss‐of‐function analysis. We reveal that PVT1 expression was significantly increased in ovarian cancer tissues which is associated with advanced FIGO stage, lymph‐node metastasis, poor survival rate, and high expression of AGO1. PVT1 or AGO1 knockdown significantly reduced the cell viability and increased the cell apoptosis and inhibited ovarian tumour growth and proliferation. Furthermore, we discovered that PVT1 up‐regulated the expression of AGO1 and thus regulated the transforming growth factor‐β (TGF‐β) pathway to promote ovarian cancer progression through sponging miR‐148a‐3p. Additionally, the activation of ERK1/2, smad2 and smad4 is observed to be related to the PVT1/miR‐148a‐3p/AGO1/TGF‐β pathway‐induced cascades. Taken together, the present study reveals that PVT1/miR‐148a/AGO1 axis plays an important role in the progression of ovarian cancer and emphasize the potential as a target of value for ovarian cancer therapy.

Adipose‐derived mesenchymal stem cells induced PAX8 promotes ovarian cancer cell growth by stabilizing TAZ protein

AbstractOur previous studies have shown that the Adipose‐derived mesenchymal stem cells (ADSCs) can regulate metastasis and development of ovarian cancer. However, its specific mechanism has yet to be fully revealed. In this study, an RNA‐seq approach was adopted to compare the differences in mRNA levels in ovarian cancer cells being given or not given ADSCs. The mRNA level of paired box 8 (PAX8) changed significantly and was confirmed as an important factor in tumour‐inducing effect of ADSCs. In comparison with the ovarian cancer cells cultured in the common growth medium, those cultured in the medium supplemented with ADSCs showed a significant increase of the PAX8 level. Moreover, the cancer cell growth could be restricted, even in the ADSC‐treated group (P < .05), by inhibiting PAX8. In addition, an overexpression of PAX8 could elevate the proliferation of ovarian cancer cells. Moreover, Co‐IP assays in ovarian cancer cells revealed that an interaction existed between endogenous PAX8 and TAZ. And the PAX8 levels regulated the degradation of TAZ. The bioluminescence images captured in vivo manifested that the proliferation and the PAX8 expression level in ovarian cancers increased in the ADMSC‐treated group, and the effect of ADSCs in promoting tumours was weakened through inhibiting PAX8. Our findings indicate that the PAX8 expression increment could contribute a role in promoting the ADSC‐induced ovarian cancer cell proliferation through TAZ stability regulation.

Identification of immune microenvironment subtypes that predicted the prognosis of patients with ovarian cancer

AbstractOvarian cancer (OC) is associated with high mortality rate. However, the correlation between immune microenvironment and prognosis of OC remains unclear. This study aimed to explore prognostic significance of OC tumour microenvironment. The OC data set was selected from the cancer genome atlas (TCGA), and 307 samples were collected. Hierarchical clustering was performed according to the expression of 756 genes. The immune and matrix scores of all immune subtypes were determined, and Kruskal‐Wallis test was used to analyse the differences in the immune and matrix scores between OC samples with different immune subtypes. The model for predicting prognosis was constructed based on the expression of immune‐related genes. TIDE platform was applied to predict the effect of immunotherapy on patients with OC of different immune subtypes. The 307 OC samples were classified into three immune subtypes A‐C. Patients in subtype B had poorer prognosis and lower survival rate. The infiltration of helper T cells and macrophages in microenvironment indicated significant differences between immune subtypes. Enrichment analyses of immune cell molecular pathways showed that JAK–STAT3 pathway changed significantly in subtype B. Furthermore, predictive response to immunotherapy in subtype B was significantly higher than that in subtype A and C. Immune subtyping can be used as an independent predictor of the prognosis of OC patients, which may be related to the infiltration patterns of immune cells in tumour microenvironment. In addition, patients in immune subtype B have superior response to immunotherapy, suggesting that patients in subtype B are suitable for immunotherapy.

Development and verification of an immune‐related gene pairs prognostic signature in ovarian cancer

AbstractOvarian cancer (OV) is the most common gynaecological cancer worldwide. Immunotherapy has recently been proven to be an effective treatment strategy. The work here attempts to produce a prognostic immune‐related gene pair (IRGP) signature to estimate OV patient survival. The Gene Expression Omnibus (GEO) and Cancer Genome Atlas (TCGA) databases provided the genetic expression profiles and clinical data of OV patients. Based on the InnateDB database and the least absolute shrinkage and selection operator (LASSO) regression model, we first identified a 17‐IRGP signature associated with survival. The average area under the curve (AUC) values of the training, validation, and all TCGA sets were 0.869, 0.712, and 0.778, respectively. The 17‐IRGP signature noticeably split patients into high‐ and low‐risk groups with different prognostic outcomes. As suggested by a functional study, some biological pathways, including the Toll‐like receptor and chemokine signalling pathways, were significantly negatively correlated with risk scores; however, pathways such as the p53 and apoptosis signalling pathways had a positive correlation. Moreover, tumour stage III, IV, grade G1/G2, and G3/G4 samples had significant differences in risk scores. In conclusion, an effective 17‐IRGP signature was produced to predict prognostic outcomes in OV, providing new insights into immunological biomarkers.

MUC16 promotes EOC proliferation by regulating GLUT1 expression

AbstractAs a common malignancy in females with a higher incidence rate, epithelial ovarian cancer (EOC) is a heterogeneous disease with complexity and diversity in histology and therapeutic response. Although great progress has been made in diagnosis and therapeutic strategies, novel therapeutic strategies are required to improve survival. Although the promoting effect of mucin 16 (MUC16) on tumour progression has been reported, the potential mechanisms remain unclear. In our study, we reported that overexpression of MUC16 was significantly related to cell proliferation and disease progression in EOC. Results from clinical specimen analysis and cell experiment support this conclusion. Patients with a high MUC16 expression usually had a worse prognosis that those with a low expression. Cell proliferation ability was significantly decreased in EOC cell lines when the knockdown of MUC16. Further study shows that the function of MUC16 in cell proliferation is based on the regulation of glucose transporter 1 (GLUT1) expression. MUC16 can control glucose uptake by regulating GLUT1 in EOC cells, thereby promoting glycogen synthesis, so that tumour cells produce more energy for proliferation. This conclusion is based on two findings. First, the significant correlation between MUC16 and GLUT1 was verified by clinical specimen and TCGA data analysis. Then, alteration of MUC16 expression levels can affect the expression of GLUT1 and glucose uptake was also verified. Finally, this conclusion is further verified in vivo by tumour‐bearing mice model. To summarize, our results suggest that MUC16 promotes EOC proliferation and disease progression by regulating GLUT1 expression.

Systematic prediction of key genes for ovarian cancer by co‐expression network analysis

AbstractOvarian cancer (OC) is the most lethal gynaecological malignancy, characterized by high recurrence and mortality. However, the mechanisms of its pathogenesis remain largely unknown, hindering the investigation of the functional roles. This study sought to identify key hub genes that may serve as biomarkers correlated with prognosis. Here, we conduct an integrated analysis using the weighted gene co‐expression network analysis (WGCNA) to explore the clinically significant gene sets and identify candidate hub genes associated with OC clinical phenotypes. The gene expression profiles were obtained from the MERAV database. Validations of candidate hub genes were performed with RNASeqV2 data and the corresponding clinical information available from The Cancer Genome Atlas (TCGA) database. In addition, we examined the candidate genes in ovarian cancer cells. Totally, 19 modules were identified and 26 hub genes were extracted from the most significant module (R2 = .53) in clinical stages. Through the validation of TCGA data, we found that five hub genes (COL1A1, DCN, LUM, POSTN and THBS2) predicted poor prognosis. Receiver operating characteristic (ROC) curves demonstrated that these five genes exhibited diagnostic efficiency for early‐stage and advanced‐stage cancer. The protein expression of these five genes in tumour tissues was significantly higher than that in normal tissues. Besides, the expression of COL1A1 was associated with the TAX resistance of tumours and could be affected by the autophagy level in OC cell line. In conclusion, our findings identified five genes could serve as biomarkers related to the prognosis of OC and may be helpful for revealing pathogenic mechanism and developing further research.

SNORA70E promotes the occurrence and development of ovarian cancer through pseudouridylation modification of RAP1B and alternative splicing of PARPBP

AbstractThe present study demonstrated for the first time that SNORA70E, which belongs to box H/ACA small nucleolar noncoding RNAs (snoRNAs) who could bind and induce pseudouridylation of RNAs, was significantly elevated in ovarian cancer tissues and was an unfavourable prognostic factor of ovarian cancer. The over‐expression of SNORA70E showed increased cell proliferation, invasion and migration in vitro and induced tumour growth in vivo. Further research found that SNORA70E regulates RAS‐Related Protein 1B (RAP1B) mRNA through pseudouracil modification by combing with the pyrimidine synthase Dyskerin Pseudouridine Synthase 1 (DKC1) and increase RAP1B protein level. What's more, the silencing of DKC1/RAP1B in SNORA70E overexpression cells both inhibited cell proliferation, migration and invasion through reducing β‐catenin, PI3K, AKT1, mTOR, and MMP9 protein levels. Besides, RNA‐Seq results revealed that SNORA70E regulates the alternative splicing of PARP‐1 binding protein (PARPBP), leading to the 4th exon‐skipping in PARPBP‐88, forming a new transcript PARPBP‐15, which promoted cell invasion, migration and proliferation. Finally, ASO‐mediated silencing of SNORA70E could inhibit ovarian cancer cell proliferation, invasion, migration ability in vitro and inhibit tumorigenicity in vivo. In conclusion, SNORA70E promotes the occurrence and development of ovarian cancer through pseudouridylation modification of RAP1B and alternative splicing of PARPBP. Our results demonstrated that SNORA70E may be a new diagnostic and therapeutic target for ovarian cancer.

p53 as a unique target of action of cisplatin in acute leukaemia cells

AbstractAcute promyelocytic leukaemia (APL) occurs in approximately 10% of acute myeloid leukaemia patients. Arsenic trioxide (ATO) has been for APL chemotherapy, but recently several ATO‐resistant cases have been reported worldwide. Cisplatin (CDDP) enhances the toxicity of ATO in ovarian, lung cancer, chronic myelogenous leukaemia, and HL‐60 cells. Hence, the goal of this study was to investigate a novel target of CDDP action in APL cells, as an alternate option for the treatment of ATO‐resistant APL patients. We applied biochemical, molecular, confocal microscopy and advanced gene editing (CRISPR‐Cas9) techniques to elucidate the novel target of CDDP action and its functional mechanism in APL cells. Our main findings revealed that CDDP activated p53 in APL cells through stress signals catalysed by ATM and ATR protein kinases, CHK1 and CHK2 phosphorylation at Ser 345 and Thr68 residues, and downregulation and dissociation of MDM2‐DAXX‐HAUSP complex. Our functional studies confirmed that CDDP‐induced repression of MDM2‐DAXX‐HAUSP complex was significantly reversed in both nutilin‐3‐treated KG1a and p53‐knockdown NB4 cells. Our findings also showed that CDDP stimulated an increased number of promyelocytes with dense granules, activated p53 expression, and downregulated MDM2 in liver and bone marrow of APL mice. Principal conclusion of our study highlights a novel mode of action of CDDP targeting p53 expression which may provide a basis for designing new anti‐leukaemic compounds for treatment of APL patients.

Construction of miRNA‐lncRNA‐mRNA co‐expression network affecting EMT‐mediated cisplatin resistance in ovarian cancer

AbstractPlatinum resistance is one of the major concerns in ovarian cancer treatment. Recent evidence shows the critical role of epithelial–mesenchymal transition (EMT) in this resistance. Epithelial‐like ovarian cancer cells show decreased sensitivity to cisplatin after cisplatin treatment. Our study prospected the association between epithelial phenotype and response to cisplatin in ovarian cancer. Microarray dataset GSE47856 was acquired from the GEO database. After identifying differentially expressed genes (DEGs) between epithelial‐like and mesenchymal‐like cells, the module identification analysis was performed using weighted gene co‐expression network analysis (WGCNA). The gene ontology (GO) and pathway analyses of the most considerable modules were performed. The protein–protein interaction network was also constructed. The hub genes were specified using Cytoscape plugins MCODE and cytoHubba, followed by the survival analysis and data validation. Finally, the co‐expression of miRNA‐lncRNA‐TF with the hub genes was reconstructed. The co‐expression network analysis suggests 20 modules relating to the Epithelial phenotype. The antiquewhite4, brown and darkmagenta modules are the most significant non‐preserved modules in the Epithelial phenotype and contain the most differentially expressed genes. GO, and KEGG pathway enrichment analyses on these modules divulge that these genes were primarily enriched in the focal adhesion, DNA replication pathways and stress response processes. ROC curve and overall survival rate analysis show that the co‐expression pattern of the brown module's hub genes could be a potential prognostic biomarker for ovarian cancer cisplatin resistance.

Robust prognostic model based on immune infiltration‐related genes and clinical information in ovarian cancer

AbstractImmune infiltration of ovarian cancer (OV) is a critical factor in determining patient's prognosis. Using data from TCGA and GTEx database combined with WGCNA and ESTIMATE methods, 46 genes related to OV occurrence and immune infiltration were identified. Lasso and multivariate Cox regression were applied to define a prognostic score (IGCI score) based on 3 immune genes and 3 types of clinical information. The IGCI score has been verified by K‐M curves, ROC curves and C‐index on test set. In test set, IGCI score (C‐index = 0.630) is significantly better than AJCC stage (C‐index = 0.541, p < 0.05) and CIN25 (C‐index = 0.571, p < 0.05). In addition, we identified key mutations to analyse prognosis of patients and the process related to immunity. Chi‐squared tests revealed that 6 mutations are significantly (p < 0.05) related to immune infiltration: BRCA1, ZNF462, VWF, RBAK, RB1 and ADGRV1. According to mutation survival analysis, we found 5 key mutations significantly related to patient prognosis (p < 0.05): CSMD3, FLG2, HMCN1, TOP2A and TRRAP. RB1 and CSMD3 mutations had small p‐value (p < 0.1) in both chi‐squared tests and survival analysis. The drug sensitivity analysis of key mutation showed when RB1 mutation occurs, the efficacy of six anti‐tumour drugs has changed significantly (p < 0.05).

NF‐YA promotes the cell proliferation and tumorigenic properties by transcriptional activation of SOX2 in cervical cancer

AbstractNF‐YA is considered as a crucial regulator for the maintenance of cancer stem cell (CSC) and involved in various types of malignant tumours. However, the exact function and molecular mechanisms of NF‐YA in the progression of cervical cancer remains poorly understood. Here, the expression of NF‐YA detected by immunohistochemistry was gradually increased from normal cervical tissues, to the high‐grade squamous intraepithelial lesions, and then to cervical cancer tissues. NF‐YA promoted the cell proliferation and tumorigenic properties of cervical cancer cells as well as tumorsphere formation and chemoresistance in vitro. The luciferase reporter assay combined with mutagenesis analyses and Western blotting showed that NF‐YA trans‐activated the expression of SOX2 in cervical cancer. Furthermore, quantitative chromatin immunoprecipitation (qChIP) and electrophoretic mobility shift assay (EMSA) confirmed that NF‐YA protein directly bound to the CCAAT box region located upstream of the SOX2 promoter. Together, our data demonstrated that NF‐YA was highly expressed in cervical cancer and promoted the cell proliferation, tumorigenicity and CSC characteristic by trans‐activating the expression of SOX2.

E6 hijacks KDM5C/lnc_000231/miR‐497‐5p/CCNE1 axis to promote cervical cancer progression

Abstract Emerging evidence suggests that long non‐coding RNA (lncRNA) plays an important role in disease development, particularly in cancers. Recent studies with genome‐wide sequencing on cervical squamous cell carcinoma and matched adjacent non‐tumour tissues showed that a newly identified lncRNA‐lnc_000231 was highly expressed in cervical cancers. However, the underlying mechanism through which it is activated and its role in cervical cancer progression is still unclear. In this study, first, we confirmed that lnc_000231 is up‐regulated in cervical cancer cells and tumour tissues. Mechanically, we demonstrated that E6 up‐regulates lnc_000231 expression through promoting its promoter region H3K4me3 modification by destabilizing KDM5C. In vitro and in vivo results showed that lnc_000231 promotes cervical cancer cell proliferation and tumour formation by acting as miR‐497‐5p sponge and maintaining cyclin E1 (CCNE1) expression. Thus, our studies identified a new signalling pathway through which E6 promotes cervical cancer progression. E6 hijacked KDM5C/lnc_000231/miR‐497‐5p/CCNE1 signalling pathway is a promising target for cervical cancer treatment in the future.

Involvement of a novel circularRNA, hsa_circ_0000520, attenuates tumorigenesis of cervical cancer cell through competitively binding with miR‐146b‐3p

AbstractThe implication of circular RNAs (circRNAs) in the pathogenesis of human cervical cancer (CC) has been demonstrated by numerous of researches, nevertheless, the whole regulatory network of circRNAs in CC remains unclear. In the present study, two GSE data sets (GSE113696 and GSE102686) were enrolled to analysed different expressed circRNA. We found that hsa_circ_0000520(circ_0000520) was decreased in CC tissues and cell lines. Functional studies indicated circ_0000520 overexpression in vitro repressed CC cell proliferation, invasion and migration, while promoted CC cell apoptosis. Moreover, circ_0000520 overexpression in vivo repressed CC tumour growth. Mechanismly, circ_0000520 and PAX5 were revealed to directly bind to miR‐146b‐3p, and circ_0000520 could indirectly regulate PAX5 by sponging miR‐146b‐3p. In conclusion, circ_0000520 repressed CC progression in vitro and in vivo by sponging miR‐146b‐3p to release PAX5.

LncRNA HAND2‐AS1 represses cervical cancer progression by interaction with transcription factor E2F4 at the promoter of C16orf74

AbstractCervical cancer is one of the major malignancies, the pathophysiology and progression of which remain to be scarcely understood. Long non‐coding RNAs (lncRNAs) have been previously implicated in the progression of cervical cancer. Here, the purpose of this study was to identify whether lncRNA heart‐ and neural crest derivative‐expressed 2‐antisense RNA 1 (HAND2‐AS1) affect the development of cervical cancer through regulation of chromosome 16 open reading frame 74 (C16orf74) by mediating a transcription factor E2F4. RT‐qPCR was performed to determine the expression of HAND2‐AS1 in cervical cancer cells. Then, cervical cancer cells were treated with HAND2‐AS1 or si‐E2F4 RNA, or C16orf74, after which the proliferation, colony formation, migration and invasion were detected. Moreover, the binding between HAND2‐AS1 and E2F4 or between E2F4 and C16orf74 was explored. Finally, the tumorigenesis of cervical cancer cells was measured in nude mice with altered HAND2‐AS1/E2F4/C16orf74 expression. HAND2‐AS1 exhibited poor expression in cervical cancer, and HAND2‐AS1 overexpression suppressed the proliferation, colony formation, migration and invasion of cervical cancer cells. In addition, HAND2‐AS1 was found to recruit transcription factor E2F4 to C16orf74 promoter region and down‐regulate C16orf74 expression. Lastly, HAND2‐AS1/E2F4/C16orf74 modulated the tumorigenesis of cervical cancer in nude mice. In conclusion, this study provided evidence on the inhibitory effect of HAND2‐AS1 on the development of cervical cancer through the suppression of C16orf74 expression by recruiting transcription factor E2F4. This study highlights the potential of lncRNA HAND2‐AS1 as a target in the treatment of cervical cancer.

A five‐mRNA signature associated with post‐translational modifications can better predict recurrence and survival in cervical cancer

AbstractHigh mortality of patients with cervical cancer (CC) stresses the imperative of prognostic biomarkers for CC patients. Additionally, the vital status of post‐translational modifications (PTMs) in the progression of cancers has been reported by numerous researches. Therefore, the purpose of this research was to dig a prognostic signature correlated with PTMs for CC. We built a five‐mRNA (GALNTL6, ARSE, DPAGT1, GANAB and FURIN) prognostic signature associated with PTMs to predict both disease‐free survival (DFS) (hazard ratio [HR] = 3.967, 95% CI = 1.985‐7.927; P < .001) and overall survival (HR = 2.092, 95% CI = 1.138‐3.847; P = .018) for CC using data from The Cancer Genome Atlas database. Then, the robustness of the signature was validated using GSE44001 and the Human Protein Atlas (HPA) database. CIBERSORT algorithm analysis displayed that activated CD4 memory T cell was also an independent indicator for DFS (HR = 0.426, 95% CI = 0.186‐0.978; P = .044) which could add additional prognostic value to the signature. Collectively, the PTM‐related signature and activated CD4 memory T cell can provide new avenues for the prognostic predication of CC. These findings give further insights into effective treatment strategies for CC, providing opportunities for further experimental and clinical validations.

RETRACTED: MicroRNA‐612 inhibits cervical cancer progression by targeting NOB1

Abstract Recently, many studies have shown that microRNA (miR)‐612 is involved in cancer progression. However, the role of miR‐612 in cervical cancer remains unclear. The present study aims to investigate the biological effects of miR‐612 on cervical cancer. The expression of miR‐612 in cervical cancer tissues and cell lines was analysed by quantitative reverse transcription‐polymerase chain reaction. The effect of miR‐612 cell proliferation, migration, invasion and apoptosis was studied by appropriate methods. Protein expression was determined by Western blot analyses. Bioinformatics analysis and luciferase reporter assays were performed to clarify the relationship between miR‐612 and nin one binding protein (NOB1). A xenograft model was established to examine the role of miR‐612 in vivo tumorigenesis. Cervical cancer tissues and cell lines showed down‐regulation of miR‐612 expression, which was associated with the Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stage and lymph node metastasis. Functional assays revealed that miR‐612 overexpression significantly suppressed cervical cancer cell proliferation, migration and invasion in vitro and delayed tumour growth in vivo. Mechanically, miR‐612 targeted NOB1 in cervical cancer cells, revealing a negative correlation between miR‐612 and NOB1in cervical cancer samples. NOB1 overexpression partially reversed the inhibitory effects of miR‐612 overexpression in cervical cancer cells. Taken together, these findings indicate that miR‐612 functions as a tumour suppressor in cervical cancer and suggest that miR‐612 may be a potential target in the therapeutic intervention of this malignancy.

IL‐6 expression promoted by Poly(I:C) in cervical cancer cells regulates cytokine expression and recruitment of macrophages

AbstractPoly(I:C) is a promising adjuvant for cancer treatment vaccines to enhance the host anti‐tumour immune response. However, the roles of poly(I:C) in the cervical cancer microenvironment and local immune reactions are not well understood. In this study, we investigated the roles of poly(I:C) in the cervical cancer. We analysed the cytokine transcription and secretion of cervical cancer cell lines and THP‐1–derived macrophages after poly(I:C) treatment, respectively. These results revealed that IL‐6 was significantly up‐regulated, and this up‐regulation was partly dose dependent. poly(I:C)‐stimulated supernatant of cervical cancer cells promoted M1‐type cytokine IL‐1β and IL‐6 expression of THP‐1–derived macrophages, but inhibited the expression of M2‐type cytokine, IL‐10 and CCL22. The recruitment of THP‐1–derived macrophages by poly(I:C)‐stimulated cervical cancer cell supernatant was also enhanced. Inhibition of IL‐6 expression in cervical cancer cells by siRNA transfection almost completely reversed the effects of poly(I:C) treatment. Finally, we found that phosphorylation of the NF‐κB signalling pathway in cervical cancer cells occurred quickly after poly(I:C) treatment. Moreover, the NF‐κB signalling pathway inhibitor PDTC significantly inhibited poly(I:C)‐induced IL‐6 expression. Taken together, these results suggest that poly(I:C) might regulate the effects of cervical cancer cells on tumour‐infiltrated macrophages, and subsequently promote a pro‐inflammatory tumour microenvironment.

RETRACTED: Celastrol inhibit the proliferation, invasion and migration of human cervical HeLa cancer cells through down‐regulation of MMP‐2 and MMP‐9

AbstractThe present study evaluated the anticancer potential of celastrol through down‐regulation of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. HeLa cells were incubated with different concentrations of celastrol (1, 10 and 100 µM) for 48h. Doxorubicin was used as a reference drug. Cancer cell migration, apoptosis, cell viability and mitochondrial fragmentation were evaluated following celastrol treatment. In addition, the expression level of MMP‐2, MMP‐9 and caspase‐3 was evaluated following celastrol treatment. HeLa cell viability was 94.1 ± 7, 53.4 ± 4 and 36.3 ± 2% at 1‐100 µM of celastrol, respectively. Apoptotic cell numbers were increased, and inhibition of larger wounds in cancer cells was observed following celastrol treatment. Celastrol‐treated cells showed condensed nuclei and clumped mitochondria. Reduced expression of MMP‐2 and MMP‐9 and increased expression of caspase‐3 were observed following celastrol treatment. Based on the experimental results, we are concluding that the celastrol was effective against HeLa cervical cancer cells.

DUSP7 inhibits cervical cancer progression by inactivating the RAS pathway

AbstractTo determine the differentially expressed proteins (DEPs) between paired samples of cervical cancer (CC) and paracancerous tissue by quantitative proteomics and to examine the effects of DUSP7 expression on the tumorigenesis and progression of CC. Proteomic profiles of three paired samples of CC and paracancerous tissue were quantitatively analysed to identify DEPs. The relationship between DEP expression and patient clinicopathological characteristics and prognosis was evaluated. The effects of the selected DEPs on CC progression were examined in SIHA cells. A total of 129 DEPs were found. Western blot and immunohistochemistry (IHC) staining analyses confirmed the results from quantitative proteomic analysis showing that the selected DEP, HRAS, P‐ERK1/2, and PLD1 levels were increased, whereas the DUSP7 level was decreased in CC tissue compared with the paired normal paracancerous tissues. The IHC results from the CC TMA analysis showed that the decreased expression of DUSP7 (p = 0.045 and 0.044) was significantly associated with a tumour size >2 cm and parametrial infiltration. In addition, the decreased expression of DUSP7 and increased expression of p‐ERK1/2 were adversely related to patient relapse (p = 0.003 and 0.001) and survival (p = 0.034 and 0.006). The expression of HRAS and p‐ERK1/2 was decreased in DUSP7‐SIHA cells compared with NC‐SIHA cells (p = 0.0003 and 0.0026). Biological functions in vitro, including invasion, migration and proliferation and tumour formation in vivo were decreased in DUSP7‐SIHA cells (all p < 0.05) but increased in shDUSP7‐SIHA cells (all p < 0.05). DUSP7 inhibits cervical cancer progression by inactivating the RAS pathway.

Lipid metabolism regulator human hydroxysteroid dehydrogenase‐like 2 (HSDL2) modulates cervical cancer cell proliferation and metastasis

AbstractHuman hydroxysteroid dehydrogenase‐like 2 (HSDL2) is a potent regulator in cancers and is also involved in lipid metabolism, but the role of HSDL2 in cervical cancer and whether it regulates the progress of cervical cancer through lipid metabolism remains unclear. In this study, we found that the overexpression of HSDL2 was in relation with cervical cancer progression including lymph nodes metastasis and recurrence. HSDL2 could serve as a novel marker of early diagnosis in cervical cancer. HSDL2 also gave impetus to tumorigenesis by initiating and promoting proliferation, invasion and migration of cervical cancer cells (Hela, C33A and SiHa) through EMT. Interestingly, we also searched that HSDL2 participated in oncogenesis by regulating lipid metabolism. In sum, our results gave the novel insight of HSDL2 functions which could be the potential for being the biomarker of prognosis and new target of therapy.

Human bone marrow mesenchymal stem cell‐derived extracellular vesicles impede the progression of cervical cancer via the miR‐144‐3p/CEP55 pathway

AbstractCervical cancer is the most common gynaecological malignancy, with a high incidence rate and mortality rate in middle‐aged women. Human bone marrow mesenchymal stem cells (hBMSCs) have been implicated in the initiation and subsequent development of cancer, along with the involvement of extracellular vesicles (EVs) mediating intracellular communication by delivering microRNAs (miRNAs or miRs). This study is aimed at investigating the physiological mechanisms by which EVs‐encapsulated miR‐144‐3p derived from hBMSCs might mediate the progression of cervical cancer. The expression profiles of centrosomal protein, 55 Kd (CEP55) and miR‐144‐3p in cervical cancer cell lines and tissues, were quantified by RT‐qPCR and Western blot analysis. The binding affinity between miR‐144‐3p and CEP55 was identified using in silico analysis and luciferase activity determination. Cervical cancer cells were co‐cultured with EVs derived from hBMSCs that were treated with either miR‐144‐3p mimic or miR‐144‐3p inhibitor. Cervical cancer cell proliferation, invasion, migration and apoptosis were detected in vitro. The effects of hBMSCs‐miR‐144‐3p on tumour growth were also investigated in vivo. miR‐144‐3p was down‐regulated, whereas CEP55 was up‐regulated in cervical cancer cell lines and tissues. CEP55 was targeted by miR‐144‐3p, which suppressed cervical cancer cell proliferation, invasion and migration and promoted apoptosis via CEP55. Furthermore, similar results were obtained by hBMSCs‐derived EVs carrying miR‐144‐3p. In vivo assays confirmed the tumour‐suppressive effects of miR‐144‐3p in hBMSCs‐derived EVs on cervical cancer. Collectively, hBMSCs‐derived EVs‐loaded miR‐144‐3p impedes the development and progression of cervical cancer through target inhibition of CEP55, therefore providing us with a potential therapeutic target for treating cervical cancer.

Phospholipase D1 promotes cervical cancer progression by activating the RAS pathway

AbstractThis study aimed to further investigate the effect of PLD1 on the biological characteristics of human cervical cancer (CC) cell line, CASKI and the potential related molecular mechanism. CRISPR/Cas9 genome editing technology was used to knock out the PLD1 gene in CASKI cells. Cell function assays were performed to evaluate the effect of PLD1 on the biological function of CASKI cells in vivo and in vitro. A PLD1‐overexpression rescue experiment in these knockout cells was performed to further confirm its function. Two PLD1‐knockout CASKI cell lines (named PC‐11 and PC‐40, which carried the ins1/del4 mutation and del1/del2/ins1 mutation, respectively), were constructed by CRISPR/Cas9. PLD1 was overexpressed in these knockout cells (named PC11‐PLD1 and PC40‐PLD1 cells), which rescued the expression of PLD1 by approximately 71.33% and 74.54%, respectively. In vivo, the cell function assay results revealed that compared with wild‐type (WT)‐CASKI cells, the ability of PC‐11 and PC‐40 cells to proliferate, invade and migrate was significantly inhibited. The expression of H‐Ras and phosphorylation of Erk1/2 (p‐Erk1/2) was decreased in PC‐11 and PC‐40 cells compared with WT‐CASKI cells. PC‐11 and PC‐40 cells could sensitize CASKI cells to cisplatin. More importantly, the proliferation, migration and invasion of PC11‐PLD1 and PC40‐PLD1 cells with PLD1 overexpression were significantly improved compared with those of the two types of PLD1 knockout cells. The sensitivity to cisplatin was decreased in PC11‐PLD1 and PC40‐PLD1 cells compared with PC‐11 and PC‐40 cells. In vivo, in the PC‐11 and PC‐40 tumour groups, tumour growth was significantly inhibited and tumour weight (0.95 ± 0.27 g and 0.66 ± 0.43 g vs. 1.59 ± 0.67 g, p = 0.0313 and 0.0108) and volume (1069.41 ± 393.84 and 1077.72 mm3 ± 815.07 vs. 2142.94 ± 577.37 mm3, p = 0.0153 and 0.0128) were significantly reduced compared to those in the WT‐CASKI group. Tumour differentiation of the PC‐11 and PC40 cells was significantly better than that of the WT‐CASKI cells. The immunohistochemistry results confirmed that the expression of H‐Ras and p‐Erk1/2 was decreased in PC‐11 and PC‐40 tumour tissues compared with WT‐CASKI tumour tissues. PLD1 promotes CC progression by activating the RAS pathway. Inhibition of PLD1 may serve as an attractive therapeutic modality for CC.

Genetic association study of intron variants in the forkhead box protein P3 gene in Chinese patients diagnosed with cervical cancer

Abstract The aim of this study was to investigate the effects of forkhead box protein P3 (FOXP3) intron single nucleotide variants (SNVs) in high‐risk human papilloma virus (HR‐HPV) infection and cervical cancer (CC) malignant lesions. We performed FOXP3 genotyping in 350 patients with CC and 350 healthy controls using the ImLDR multiple single nucleotide polymorphism genotyping technology. The heterozygous mutation TC in rs2294021 decreased the risk of HR‐HPV infection and CC malignant lesions (TC vs. TT: OR = 0.71, 95% CI = 0.51–0.99); the dominant model TC+CC and allele C in rs2294021 decreased the risk of CC malignant lesions (TC+CC vs. TT: OR = 0.69, 95% CI = 0.50–0.95; C vs. T: OR = 0.78, 95% CI = 0.63–0.97). The heterozygous mutation GA, dominant model GA+AA and allele A in rs3761549 also decreased the risk of HR‐HPV infection and CC malignant lesions (GA vs. GG: OR = 0.70, 95% CI = 0.51–0.96; GA+AA vs. GG: OR = 0.69, 95% CI = 0.51–0.94; A vs. G: OR = 0.75, 95% CI = 0.58–0.96). Patients with CC and HR‐HPV infection carrying rs2294021 TC and rs3761549 GA had lower expression of FOXP3 protein. Haplotype analysis revealed that T‐C‐A decreased the risk of HR‐HPV infection. Furthermore, we found a significant association between immune cells infiltration and prognosis in patients with CC. Our findings demonstrated that rs2294021 and rs3761549 variants may protect against HR‐HPV and CC malignant lesions by downregulating FOXP3 and that FOXP3 was associated with immune cells infiltration, which affected the prognosis of CC.

TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway

AbstractThe decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT‐PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin‐embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.

Development of a PANoptosis ‐Related Pathomics Prognostic Model in Ovarian Cancer: A Multi‐Omics Study

ABSTRACT Ovarian cancer (OC) is a high‐mortality gynaecological malignancy, and the role of PANoptosis, a comprehensive cell death mechanism, in its prognosis remains unexplored. This study aims to clarify it, potentially guiding OC diagnosis and treatment. We analysed the ovarian data from TCGA and GTEx, and the GSE184880 scRNA‐seq dataset from GEO. Spatial data and pathological images were sourced from the 10X Genomics website and GDC Portal. Features were extracted using CellProfiler and ResNet‐50, and a PANoptosis‐related pathomics prognostic model (PANPM) powered by deep learning was developed. The PANoptosis‐related hub gene STAT4 potentially served as a protective factor for patients with OC. A better prognosis in OC was found linked to higher PANoptosis. The PANPM, manifesting distinct advantages for clinical application by accurately extracting pathological features, performed excellently in validation and the high‐risk group indicated a poor prognosis. Additionally, STAT4 + T cells may inhibit OC, by activating the PANoptosis of epithelial cells through TNFSF12‐TNFRSF12A and TNF‐TNFRSF1A, which sheds light on potential therapeutic interventions involving STAT4 + T cells.