Colorectal cancer (CRC) progression is driven by cancer stem cells (CSCs) that evade treatment through dynamic phenotype modulation. Our previous research identified BEX2 as a significant regulator of CRC malignancy involving the Hedgehog (Hh) pathway. This study aimed to elucidate the influence of BEX2 on CRC stemness and the interaction with the Hh signaling pathway, potentially uncovering innovative therapeutic strategies for combating CRC.

TCGA and GEO data were analyzed to correlate BEX2 expression with clinical outcomes and stemness markers in CRC. Functional assays, including spheroid formation, flow cytometry, extreme limiting dilution assay (ELDA), Transwell, wound healing, and cell viability assay, were performed in DLD1 and HCT116 cell lines. Immunoblotting and qRT-PCR assessed BEX2 expression with in vivo validation in NOD/SCID mice.

The findings revealed a negative correlation between BEX2 expression and the levels of stemness-associated genes with a significant association with CRC patient’ prognosis. Overexpression of BEX2 diminished CRC stemness potential, whereas BEX2 knockdown led to a pronounced enhancement of these stem-like characteristics. Further investigation revealed that BEX2 inhibited the Hh pathway. BEX2 interacted with MCL1, promoting ubiquitination and degradation, thereby decreasing MCL1 stability. Low BEX2 expression stabilized MCL1, which enhanced stemness potential. These results suggested BEX2 modulates CRC stemness via MCL1 downregulation.

Taken together, the current study findings highlight BEX2 and MCL1 as potential therapeutic targets in CRC with BEX2 emerging as a key regulator of stemness, chemoresistance, and invasiveness. These findings advance our understanding of CRC and pave the way for more effective therapies.