Context-dependent regulation of SIX1 by ETS1 within EMT-associated transcriptional networks

Epithelial-mesenchymal transition (EMT) is a fundamental process driving tumor plasticity, metastasis, and therapy resistance. Although E26 transformation-specific transcription factor 1 (ETS1) and Sine oculis homeobox homolog 1 (SIX1) are individually implicated in EMT-related transcriptional programs, the regulatory interplay and functional coordination of these proteins across tumor states remain unclear. Hence, this study aimed to investigate the mechanistic and clinical behavior of the ETS1-SIX1 axis, with a focus on hepatocellular carcinoma (HCC) and aggressive endothelial-like cancer phenotypes.

ETS1-SIX1 expression patterns were analyzed using real-time quantitative polymerase chain reaction in HCC-derived cell lines and the endothelial-like SK-HEP-1 cell line, whereas chromatin immunoprecipitation (ChIP) assays were performed in SK-HEP-1 cells to assess ETS1 binding to the SIX1 promoter. Clinical relevance was assessed using The Cancer Genome Atlas (TCGA) RNA-sequencing (RNA-seq) datasets (breast invasive carcinoma, colon adenocarcinoma, liver hepatocellular carcinoma (LIHC), and lung adenocarcinoma) with correlation and survival analyses, and validated using tumor cDNA panels and public transcriptomic data.

ChIP assays confirmed ETS1 binding to the SIX1 promoter. Expression analyses indicated an inverse relationship between ETS1 and SIX1 in HCC-derived cell lines, the endothelial-like SK-HEP-1 cell line, and tumor cDNA panels. Pan-cancer analyses showed decreased ETS1 and increased SIX1 expression in tumors, with stage-dependent heterogeneity. Elevated SIX1, but not ETS1, was associated with poorer overall survival, particularly in LIHC. Gene set enrichment analysis linked high-risk profiles to EMT, cell cycle, and immune-related pathways, while ETS1 expression decreased with increasing tumor grade.

This study supported a model in which ETS1 negatively regulates SIX1 expression within liver cancer-associated contexts. Integrating mechanistic and clinical analyses, the findings suggest that the ETS1-SIX1 axis contributes to EMT-driven tumor plasticity and aggressive tumor behavior, with potential relevance for future therapeutic investigation.