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

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.