Yongxiu Yang

Therapeutic Potential of CLDN Family Proteins in Ovarian Cancer: Emerging Biomarkers and Targets

Background: Claudins (CLDNs), key components of tight junctions, are dysregulated in various cancers. However, the roles and therapeutic potential of specific CLDN family members-particularly CLDN6, CLDN9, and CLDN10-in ovarian cancer (OC) remain incompletely defined. To address this gap, we conducted a comprehensive analysis of the CLDN family to identify novel diagnostic and prognostic biomarkers as well as potential therapeutic targets for OC. Methods: Gene expression profiles and corresponding clinical data from The Cancer Genome Atlas ovarian cancer cohort (TCGA-OV) and two Gene Expression Omnibus (GEO) datasets (GSE18520, GSE26712) were analyzed. Differential expression of CLDN genes between OC and normal tissues was evaluated using R with appropriate bioinformatics packages (e.g., limma). Logistic regression models were employed to calculate odds ratios (ORs), and receiver operating characteristic (ROC) curves were generated across all datasets to identify consistently dysregulated CLDNs associated with OC. Prognostic hazard ratios (HRs) for these CLDNs were extracted from the Kaplan-Meier Plotter (KM Plotter) database and synthesized using a random-effects model to assess their associations with overall survival. Intersection analysis was performed to identify CLDNs exhibiting both significant differential expression and prognostic significance. Candidate targets underwent comprehensive validation, including single-cell RNA sequencing (scRNA-seq) to characterize cell-type-specific expression patterns. Notably, Key findings regarding CLDN6 were further validated by immunohistochemistry (IHC) on an independent tissue microarray (TMA), as well as functional assays in OC cell lines following siRNA-mediated knockdown. These included transwell invasion, wound healing (scratch) test, and measurements of mitochondrial depolarization, reactive oxygen species (ROS) accumulation, cell cycle arrest, and apoptosis. Results: CLDN6, CLDN9, and CLDN10 were consistently and significantly upregulated in OC compared to normal tissues across all datasets. Single-cell RNA sequencing revealed that CLDN6 and CLDN10 were predominantly expressed in malignant epithelial cell subsets, a pattern associated with aggressive tumor phenotypes. Meta-analysis of HRs showed that HR >1 in CLDN6 and HR <1 in CLDN10. Although CLDN10 is highly expressed in tumor cells, its hazard ratio (HR) is less than 1, and the underlying mechanism of this gene remains unclear. Experiments have confirmed that CLDN6 is closely associated with tumor invasion. Computational analysis, meta-analysis, and single-cell data collectively confirm that only CLDN6 is a clearly defined gene closely associated with tumor progression, a finding subsequently validated by experimental results. Notably, the combined signature comprising CLDN6, CLDN9, and CLDN10 exhibited superior diagnostic performance, with higher area under the curve (AUC) values in ROC analysis, compared to individual CLDNs or established OC biomarkers such as carbohydrate antigen 125 (CA125), human epididymis 4 (HE4), carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP). The signature also showed enhanced prognostic discrimination, as indicated by time-dependent ROC analysis. Protein overexpression of these targets was validated by IHC and Western blot. Functional assays further demonstrated that siRNA-mediated knockdown of CLDN6 significantly inhibited the proliferation of OC cells, promoted cell apoptosis, increased production of ROS, induced G1 phase arrest, inhibited cell invasion and migration in vitro. Furthermore, western blot analysis identified that knockdown of CLDN6 repressed the Wnt/β-catenin pathway. Nude mice experiments indicated that CLDN6 knockdown in OC cells dramatically suppresses the tumor growth and lung metastasis in vivo. Conclusions: CLDN6, CLDN9, and CLDN10 are critically involved in the pathogenesis and progression of OC. A biomarker panel combining these three claudins demonstrates superior diagnostic and prognostic performance compared to individual markers and established clinical biomarkers such as CA125 and HE4. Notably, functional evidence indicates that CLDN6 plays a pivotal role in regulating malignant phenotypes, highlighting its potential as a novel therapeutic target. These findings collectively support the clinical utility of the CLDN6/9/10 axis as both a non-invasive biomarker signature and a promising avenue for targeted intervention in ovarian cancer.

Data‐independent acquisition for proteomic applications in early‐stage endometrial cancer progression

AbstractAimMost endometrial cancer (EC) patients are diagnosed at an early‐stage (FIGO stage I or II), with a favorable prognosis. However, some high‐grade, early‐stage EC patients have unexpected recurrences and undesirable results, the molecular alterations that underlie these tumors are far from being fully understood. Our goal was to use proteome analysis to examine dysregulated pathways in this specific subgroup of EC.MethodsWe used data‐independent acquisition (DIA) quantitative proteomics to analyze cancer and matched paracancerous tissues from 20 EC patients (10 high‐grade and 10 low‐grade). Immunohistochemistry (IHC) analysis was used to validate protein expression of six hub genes.ResultsIn total, 7107 proteins were quantified, while 225 downregulated and 366 upregulated proteins in high‐grade cancer tissues, 130 downregulated and 413 upregulated proteins in high‐grade paracancerous tissues. The pathway associated with oxidative phosphorylation (OXPHOS) was upregulated and have similar expression patterns in high‐grade EC tissues and matched paracancerous tissues. OXPHOS‐related protein, ATP5F1D showed the best classification and diagnostic ability in distinguishing high‐grade from low‐grade EC. In both cancer and paracancerous tissues, double‐label immunofluorescence demonstrated ITGA4 and COL4A1 co‐localized at the basal membrane.ConclusionsOur present works elucidate that metabolism reprogramming is associated with high‐grade, early‐stage EC, particularly OXPHOS is upregulated. Noticeably, the paracancerous tissues have undergone molecular changes similar to cancer tissues, maybe they have signal exchange via secreted proteins (ITGA4 and COL4A1). The upregulation of OXPHOS‐related proteins may be the potential biomarker for EC diagnosis, and targeting OXPHOS metabolism might be an effective treatment for high‐grade, early‐stage EC.

Correlation between malignant peritoneal cytology and survival in patients with uterine leiomyosarcoma and endometrial stromal sarcoma

This study aimed to examine the correlation between malignant peritoneal cytology and overall survival among patients with uterine leiomyosarcoma and endometrial stromal sarcoma. Patients with uterine leiomyosarcoma and endometrial stromal sarcoma between January 2010 and December 2016 were identified from the Surveillance, Epidemiology, and End Results database. The multiple imputation method was used to address missing values. Propensity score matching was conducted to balance baseline data between the malignant and negative peritoneal cytology groups. The prognostic significance of malignant peritoneal cytology was evaluated using Cox regression, random survival forest, and subgroup analyses. Among 733 eligible patients, 8% (59/733) had malignant peritoneal cytology, increasing to 20% (42/209) in advanced cases. Before and after propensity score matching, patients with malignant peritoneal cytology had significantly lower 5-year overall survival rates and shorter median survival time than patients with negative peritoneal cytology. Multivariate Cox regression revealed that malignant peritoneal cytology (hazard ratio 2.03, 95% confidence interval 1.29 to 3.20, p=0.002) was an independent prognostic factor for uterine leiomyosarcoma and endometrial stromal sarcoma. Random survival forest further indicated that, among the factors analyzed, peritoneal cytology status was second only to the International Federation of Gynecology and Obstetrics (FIGO) stage in terms of prognostic prediction. Finally, subgroup analyses substantiated the correlation between malignant peritoneal cytology and unfavorable overall survival in most subgroups. Malignant peritoneal cytology status was an important prognostic factor complementing FIGO stage and was associated with a reduction in overall survival. Peritoneal cytology evaluation during hysterectomy may be recommended for prognosis estimation for uterine leiomyosarcoma and endometrial stromal sarcoma.

Turning off the ferroptosis switch: ACAA1-Driven PI3K/AKT/Nrf2 signaling as a novel driver of endometrial cancer progression

Endometrial carcinoma (EC) is a prevalent gynecologic malignancy with rising global incidence. Dysregulated lipid metabolism promotes EC progression through estrogen synthesis, metabolic reprogramming, and tumor microenvironment remodeling. Ferroptosis, an iron-dependent cell death driven by lipid peroxidation, represents a potential therapeutic strategy, yet its resistance mechanisms in EC remain unclear. We identify Acetyl-CoA Acetyltransferase 1 (ACAA1), a key enzyme in mitochondrial fatty acid β-oxidation, as an oncogenic factor in EC. We demonstrate that ACAA1 is significantly upregulated in EC tissues via bioinformatic analysis and clinical samples. Functionally, ACAA1 overexpression enhances tumor cell proliferation, migration, energy metabolism, and lipid droplet synthesis in vitro, while accelerating tumor growth in vivo in xenograft models. Mechanistically, ACAA1 activates the PI3K/AKT pathway, leading to nuclear translocation of the transcription factor Nrf2. This ACAA1/PI3K/AKT/Nrf2 axis suppresses ferroptosis by regulating redox homeostasis and lipid peroxidation, thereby promoting EC progression. Our findings reveal ACAA1 as a novel regulator of ferroptosis resistance and tumorigenesis in EC, highlighting its potential as a promising therapeutic target for EC treatment.

Therapeutic potential of targeting PCLAF in endometrial cancer: Insights from Wnt/β-catenin and P53 regulatory mechanisms

Endometrial cancer (EC), a widespread gynecological malignancy, has limited therapeutic options, particularly in patients with advanced, metastatic, or recurrent disease. PCLAF, a proliferation-related protein, is overexpressed in several tumors; however, its role and mechanism in EC remain largely unknown. Ten hub genes in EC were identified and predicted through bioinformatics analyses. The expression of PCLAF in EC was validated using molecular biology technology, while its function was investigated using in vitro proliferation, cell cycle, migration, and apoptosis assays. Mechanistically, the transcriptome and proteome sequencing revealed potential regulatory pathways for PCLAF in EC. Finally, experiments on animals were executed to test the Ishikawa cell growth in vivo. PCLAF exhibited high expression in both EC cells and tissues. Silencing PCLAF significantly impaired cell proliferation, migration and invasion, blocked G1 phase progression of the cell cycle and activated apoptosis. Sequencing results and western blot analysis confirmed that knockdown of EC cells inhibits the Wnt/β-catenin pathway and activates the p53 pathway. The in vivo results confirmed that PCLAF knockdown effectively inhibited EC cell growth. In conclusion, our findings suggest that silencing PCLAF hinders EC progression by suppressing the Wnt/β-catenin pathway and activating the p53 signaling pathway. This insight suggested that targeting PCLAF may offer a new therapeutic avenue for EC treatment.

Maackiain induces apoptosis and autophagy via ROS-mediated endoplasmic reticulum stress in endometrial cancer

Endometrial cancer (EC) is a common gynecological cancer, characterized by increasing incidence and mortality rates. Maackiain (MA), a natural flavonoid compound, has multiple biological activities, but little is known about how it affects EC cells. In the present study, CCK-8, EdU, colony formation, and flow cytometry assays were used to evaluate the effects of MA on EC cell proliferation, apoptosis, and reactive oxygen species (ROS) levels. The effect of MA on autophagy in EC cells were examined through the observation of cell morphology and ultrastructure, and cells were transfected with AdPlus-mCherry-GFP-LC3B for further analysis. Transcriptomic and western blot analyses revealed the underlying mechanism. To evaluate the anti-EC effect of MA in vivo, a xenograft model was established. The results demonstrated that MA inhibited KLE and Ishikawa cell growth in a dose-dependent manner. Furthermore, MA significantly suppressed EC xenograft tumor growth in vivo while exhibiting low toxicity. In addition, EC cells treated with MA exhibited pro-apoptotic and pro-autophagic responses, with the latter exhibiting cytoprotective properties. MA also induced the accumulation of ROS, which promoted endoplasmic reticulum (ER) stress. Notably, the use of the N-acetyl-L-cysteine (NAC) ROS scavenger and the 4-phenylbutyric acid (4-PBA) ER stress inhibitor effectively mitigated the autophagy and apoptosis induced by MA. These results collectively implied that MA triggers autophagy and apoptosis in EC cells through ROS-mediated ER stress, highlighting its potential as a therapeutic agent against EC.

Network pharmacology and metabolomics elucidate the underlying effects and mechanisms of maackiain against endometrial cancer

Endometrial carcinoma (EC), a prevalent gynecological cancer, is characterized by rising incidence and mortality rates, highlighting the need for novel treatments to improve patient outcomes. Maackiain (MA) is a natural compound isolated from common herbal medicines, that has been reported to have anti-cancer effects. However, the underlying roles and mechanisms concerning EC remain unclear. This study focused on deeply exploring the potential roles and mechanisms of MA against EC by network pharmacology, experimentally validated, metabolomics, and molecular docking. A total of 86 potential targets of MA against EC were identified by network pharmacology. In vitro experiments further confirmed network pharmacology' predictions. In addition to suppressing EC cell proliferation, MA also paused the cell cycle at the G2/M phase in a dose-dependent manner. This effect is accompanied by increased p21 and phospho-p53 expression, as well as reduced expression of CDK1 and CCNB1. Furthermore, cell metabolomics analysis revealed that 285 metabolites were changed after MA administration, which majorly affects glycerophospholipid metabolism, nucleotide metabolism, choline metabolism in cancer, and purine metabolism. Combination network pharmacology, metabolomics, and molecular docking, PLA2G10, PDE4D, and PDE5A were found to be potential targets for therapeutic intervention. These findings underlined that MA has anti-EC potential by modulating multiple targets including PLA2G10, PDE4D, and PDE5A, inhibiting EC cell proliferation, inducing G2/M phase arrest, and causing metabolic shifts. This study provides theoretical support for advanced experimental research on its clinical applications.

CircRNA-regulated glucose metabolism in ovarian cancer: an emerging landscape for therapeutic intervention

Ovarian cancer (OC) has the highest mortality rate among female reproductive system tumours, with limited efficacy of traditional treatments and 5-year survival rates that rarely exceed 40%. Circular RNA (circRNA) is a stable endogenous circular RNA that typically regulates protein expression by binding to downstream miRNA. It has been demonstrated that circRNAs play an important role in the proliferation, migration, and glucose metabolism (such as the Warburg effect) of OC and can regulate the expression of glucose metabolism-related proteins such as GLUT1 and HK2, promoting anaerobic glycolysis of cancer cells, increasing glucose uptake and ATP production, and affecting energy supply and biosynthetic substances to support tumour growth and invasion. This review summarises the formation and characteristics of circRNAs and focuses on their role in regulating glucose metabolism in OC cells and their potential therapeutic value, providing insights for identifying new therapeutic targets.

SIK2: A critical glucolipid metabolic reprogramming regulator and potential target in ovarian cancer

AbstractAimTo explore the role of salt‐inducible kinase 2 (SIK2) on glucose and lipid metabolism in ovarian cancer (OC), so as to increase the understanding of potential inhibitors targeting SIK2 and lay a foundation for future precision medicine in OC patients.MethodsWe reviewed and summarized the regulation effect of SIK2 on glycolysis, gluconeogenesis, lipid synthesis, and fatty acids β‐oxidation (FAO) in OC, as well as the potential molecular mechanism and the prospects of potential inhibitors targeting SIK2 in future cancer treatments.ResultsMany pieces of evidence show that SIK2 is closed associated with glucose and lipid metabolism of OC. On the one hand, SIK2 enhances the Warburg effect by promoting glycolysis and inhibiting oxidative phosphorylation and gluconeogenesis, on the other hand, SIK2 regulates intracellular lipid metabolism through promoting lipid synthesis and FAO, all of which ultimately induces growth, proliferation, invasion, metastasis, and therapeutic resistance of OC. On this basis, SIK2 targeting may become a new solution for the treatment of a variety of cancer types including OC. The efficacy of some small molecule kinase inhibitors has also been demonstrated in tumor clinical trials.ConclusionSIK2 displays significant effects in OC progression and treatment through regulating cellular metabolism including glucose and lipid metabolism. Therefore, future research needs to further explore the molecular mechanisms of SIK2 in other types of energy metabolism in OC, based on this to develop more unique and effective inhibitors.

METTL3-mediated maturation of miR-126-5p promotes ovarian cancer progression via PTEN-mediated PI3K/Akt/mTOR pathway

Methyltransferase-like 3 (METTL3) functions as an RNA methyltransferase that controls the modification of N(6)-methyladenosine (m6A) to influence the biosynthesis, decay, and translation of mRNAs. This study aims to investigate the regulation of METTL3-mediated promotion of microRNA-126-5p (miR-126-5p) in the progression of ovarian cancer and to identify the mechanisms in relation to phosphatase and tensin homolog (PTEN) and the PI3K/Akt/mTOR pathway. We found high expression of miR-126-5p in ovarian cancer samples compared to paired adjacent samples, and also in ovarian cancer cell lines. Gain-of-function experiments demonstrated that overexpression of miR-126-5p promoted ovarian cancer cell proliferation, migration, and invasion, and inhibited their apoptosis. Luciferase reporter assay identified that miR-126-5p could directly bind to PTEN. By targeting PTEN, miR-126-5p could activate the PI3K/Akt/mTOR pathway. Furthermore, the RNA methyltransferase METTL3 promoted the maturation of miR-126-5p via the m6A modification of pri-miR-126-5p. Finally, in vitro and in vivo experiments substantiated that silencing of METTL3 impeded the progression and tumorigenesis of ovarian cancer by impairing the miR-126-5p-targeted inhibition of PTEN and thus blocking the PI3K/Akt/mTOR pathway. Coherently, knockdown of METTL3 inhibited the effect of miR-126-5p to upregulate PTEN, and thus prevents PI3K/Akt/mTOR pathway activation, thereby suppressing the development of ovarian cancer. These findings highlight potential targets for the future ovarian cancer treatment as well as tumorigenic mechanisms mediated by m6A modification.

In vivo and in vitro studies of Alloimperatorin induced autophagy in cervical cancer cells via reactive oxygen species pathway

Alloimperatorin (Alloi) has been shown to have anti-proliferative effects in our previous studies. we aimed to investigate whether Alloimperatorin induces autophagy through the reactive oxygen species (ROS) pathway and anticancer activity in vivo. The anti-proliferative effect of Alloimperatorin was evaluated using a cell counting kit (CCK-8 kit). Apoptosis was detected using flow cytometry. Confocal microscopy, immunofluorescence, and mRFP-GFP-LC3 lentivirus transfection were used to verify autophagy. Electron microscopy detection of autophagosomes was induced by Alloimperatorin. Western blotting was used to detect autophagy proteins in HeLa and SiHa cells. A xenograft model was used to monitor the inhibitory effect of Alloimperatorin on tumor growth in nude mice. The results showed that Alloimperatorin induced ROS production and inhibited the proliferation of HeLa and SiHa cells. Furthermore, Alloimperatorin increased the apoptosis rate in HeLa and SiHa cells. Confocal microscopy fluorescence indicated that Alloimperatorin increased autophagy fluorescence of HeLa and SiHa cells. mRFP-GFP-LC3 lentivirus transfection and electron microscopy demonstrated that Alloimperatorin increased autophagy in HeLa and SiHa cells. Western blotting showed that Alloimperatorin induced the expression of autophagy proteins in HeLa and SiHa cells. However,

Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

Abstract Epithelial ovarian cancer (EOC) is the leading cause of death among gynecological malignancies, and the tumors with advanced-stage are frequently characterized by extensive metastasis. Although metabolic reprogramming of amino acids represents a hallmark of cancer, its specific role in the metastatic progression of EOC remains poorly understood. Here, we identified a critical metabolic vulnerability in omental metastasis of EOC. Despite defective endogenous synthesis, arginine accumulation depends on exogenous uptake. In vivo experiments demonstrated that dietary arginine deprivation suppressed tumor growth and metastasis, whereas supplementation or enhanced uptake of arginine promoted tumor cell proliferation, invasion, and migration in vitro. Mechanistically, increased arginine binds to the RNA helicase DDX3X, inducing nuclear retention of DDX3X and further promoting the transcription of DNA damage response (DDR)-related genes, thereby facilitating DDR through activating the ATM/CHK2/P53 axis to enable cancer cells to survive under metastatic stress. Notably, arginine restriction or pharmacological inhibition of DDX3X did effectively suppress both primary tumor growth and omental metastasis in mouse models. Collectively, our findings reveal that arginine is a metabolic vulnerability in omental metastasis of EOC, indicating that arginine restriction and DDX3X inhibition represent promising therapeutic strategies.