Hedgehog-GLI and Notch Pathways Sustain Chemoresistance and Invasiveness in Colorectal Cancer and Their Inhibition Restores Chemotherapy Efficacy

SIMPLE SUMMARY: Colorectal cancer is a leading cause of cancer-related deaths, mainly caused by resistance to therapy and metastatic spread, in turn sustained by the activation of mechanisms such as the epithelial-to-mesenchymal transition (EMT). We investigate here the role of the Hedgehog-GLI and NOTCH signaling pathways, already associated with poor prognosis in CRC, in the mechanism of chemoresistance and EMT, using monolayer and organoids from two models of common mutations in CRC: KRAS and BRAF. Our results show that treatment with the chemotherapeutic drug 5-fluorouracil activated both pathways in the investigated contexts. However, we observed a different behavior in the investigated models: in KRAS-mutated CRC, the inhibition of both the HH-GLI and NOTCH pathways is necessary to enhance chemosensitivity, while in BRAF-mutated CRC the inhibition of HH-GLI is sufficient to impair both signaling pathways and promote chemosensitivity. ABSTRACT: Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC.