Wen‐Wei Chang

ELF4/TRIB3/CDK6 Axis Promotes Cancer Stem Cell Activity in Endometrial Cancer

ABSTRACT Endometrial cancer (EC) is the most prevalent gynecological malignancy globally. Here, we explored the role of E74‐like ETS transcription factor 4 (ELF4) in EC progression. Using the TISIDB web tool to analyze TCGA data, we found that elevated ELF4 expression correlates with higher histological grades and reduced overall survival in EC patients. Tissue microarray analysis confirmed a grade‐dependent increase in ELF4 protein levels. Knockdown of ELF4 in EC cell lines (AN3CA, HEC‐1A) and patient‐derived EC cells suppressed proliferation, cell cycle progression, and cancer stem cell (CSC) activity. Database analysis and RNA interference identified cyclin‐dependent kinase 6 (CDK6) as a downstream target of ELF4. ELF4 silencing reduced CDK6 mRNA and protein expression, while chromatin immunoprecipitation revealed direct binding of ELF4 to the CDK6 promoter. Conversely, ELF4 overexpression upregulated CDK6. Knockdown of CDK6 or treatment with the CDK4/6 inhibitor Palbociclib diminished tumorsphere formation and expression of stemness markers (OCT4, NANOG, c‐MYC) in both conventional and patient‐derived EC cells. We previously reported that the tribbles pseudokinase 3 (TRIB3)/ELF4 complex transactivates CTNNB1 expression; here, we show that TRIB3 knockdown also downregulates CDK6 at mRNA and protein levels, suggesting cooperative regulation of CDK6 by ELF4 and TRIB3. In EC specimens, ELF4, TRIB3, and CDK6 expression positively correlated, and Kaplan‐Meier analysis indicated that high co‐expression of these genes predicted the poorest overall survival. Collectively, our findings establish the ELF4/TRIB3/CDK6 axis as a critical regulator of EC progression and CSC maintenance, highlighting its potential as a therapeutic target for EC.

Ovatodiolide inhibits endometrial cancer stemness via reactive oxygen species-mediated DNA damage and cell cycle arrest

Endometrial cancer (EC) is a common gynecological cancer worldwide, often associated with a poor prognosis after recurrence or metastasis. Ovatodiolide (OVA) is a macrocyclic diterpenoid derived from Anisomeles indica that shows anticancer effects in various malignancies. This study aimed to evaluate the cytotoxic effects of OVA on EC cell proliferation and cancer stem cell (CSC) activity and explore its underlying molecular mechanisms. OVA treatment dose-dependently reduced the viability and colony formation of three EC cell lines (AN3CA, HEC-1A, and EMC6). It induced G2/M phase cell cycle arrest, associated with decreased cell division cycle 25C (CDC25C) expression and reduced activation of cyclin-dependent kinases 1 (CDK1) and 2 (CDK2). OVA also increased reactive oxygen species (ROS) production and DNA damage, activating the DNA damage-sensitive cell cycle checkpoint kinases 1 (CHK1) and 2 (CHK2) and upregulating the DNA damage marker γ-H2A.X variant histone (H2AX). It also suppressed the activation of mechanistic target of rapamycin kinase (mTOR) and nuclear factor kappa B (NF-κB) and downregulated glutathione peroxidase 1 (GPX1), an antioxidant enzyme counteracting oxidative stress. Moreover, OVA reduced the self-renewal capacity of CSCs, reducing the expression of key stemness proteins Nanog homeobox (NANOG) and octamer-binding transcription factor 4 (OCT4). The ROS inhibitor N-acetylcysteine attenuated the anti-proliferative and anti-CSC effects of OVA. Our findings suggest that OVA acts via ROS generation, leading to oxidative stress and DNA damage, culminating in cell cycle arrest and the suppression of CSC activity in EC. Therefore, OVA is a promising therapeutic agent for EC, either as a standalone treatment or an adjunct to existing therapies.