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.

Mutant KRAS, the principal isoform of RAS, plays a pivotal role in the oncogenesis of colorectal cancer by constitutively activating the RAF/MEK/ERK and PI3K/AKT pathways. Effective targeted therapies are urgently needed. We investigated whether rigosertib, a benzyl styryl sulfone RAS signaling disruptor, could selectively kill KRAS-mutant colorectal cancer cells.

CCK-8 was used to determine the cell viability. Patient-derived tumor and cancer cell xenograft models were used to detect the inhibitory efficacy of rigosertib. Flow cytometry was used to evaluate the apoptosis and cell cycle progression. Apoptosis and cell cycle arrest markers were detected by Western blot. DCFH-DA was used to determine the reactive oxygen species. Immunohistochemistry staining and Western blot were performed to characterize RAS signaling markers in colorectal cancer tissues and cells.

Rigosertib (RGS) exhibited a cytotoxic effect against colorectal cancer cells, which was greater in KRAS-mutant cells. Furthermore, RGS induced mitotic arrest and oxidative stress-dependent apoptosis in KRAS-mutant DLD1 and HCT116 cells. Besides, RGS disrupted RAS signaling, and the inhibition of RAS/MEK/ERK was independent of cellular oxidative stress. Using patient-derived xenograft models, the response and tumor inhibition of RGS were significantly higher in the KRAS-mutant subgroup, while p-MEK, p-ERK, and p-AKT levels of RGS-treated tumors were significantly decreased. Finally, in a KRAS-mutant, chemotherapy-resistant patient-derived xenograft model, RGS showed a stronger therapeutic effect than the combination standard therapy involving fluoropyrimidine + oxaliplatin/irinotecan + bevacizumab.

These data showed that targeting RAS signaling using RGS could be a therapeutic treatment for KRAS-mutant colorectal cancer patients.