FOXF2 may inhibit esophageal squamous cell carcinoma growth and metastasis by regulating the EZR-ERBB2 axis.

Background: FOXF2, a member of the transcription factor FOX family proteins, plays a key role in tumorigenesis and tumor aggressiveness. However, the potential molecular mechanism of FOXF2 in esophageal squamous cell carcinoma (ESCC) remains largely unknown. Exploring its role and mechanism in ESCC progression may help identify new diagnostic markers and therapeutic targets. The aim of this study is to investigate the potential functions of the FOXF2 gene within the context of ESCC and to elucidate the underlying molecular pathways involved.

Methods: Using the GoMiner database, GeneCard database, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and the COMPARTMENTS subcellular localization database, we identified the most likely downstream molecule of the FOXF2 gene, EZR; the subcellular locations of FOXF2 and EZR; the possible biological pathways [Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)]; and the protein interactions networks of the EZR gene enriched from the OMICS datasets via Metascape. We also used The Cancer Genome Atlas database to analyze the correlation between EZR and ERBB signaling pathway. In addition, we verified the RNA and protein expression of the target genes using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we employed Western blot analysis and plasmid transfection and lentiviral infection techniques to gene edit FOXF2 and EZR in different EC cells to obtain stable overexpression or knockdown of the cell lines. This was followed by ex vivo and in vivo experiments including migration assay, cell scratch assay, clone formation assay, and a xenotransplantation mouse model to validate the functional phenotype of the gene-edited cells.

Results: We found that knockdown of FOXF2 expression significantly enhanced the growth, invasion, and metastasis of ESCC cells both in vitro and in vivo. Moreover, we demonstrated that FOXF2 was predominantly expressed in the nucleus and directly interacted with EZR, thereby inhibiting EZR transcriptional expression, resulting in suppressed ERBB2 signal function, ultimately halting ESCC growth and metastasis.

Conclusions: Taken together, these results reveal the tumor-suppressive functions of FOXF2 in inhibiting EZR-mediated ERBB2 activation, suggesting that FOXF2 could serve as a potential novel predicting prognostic biomarker for ESCC.