NOTCH3 signaling regulates MUSASHI-1 expression in metastatic colorectal cancer cells

MUSASHI-1 (MSI-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the NOTCH pathway through inactivation of NUMB, a NOTCH signaling repressor. To date, the mechanisms of regulation of MSI-1 levels remain largely unknown. Here, we investigated the regulation of MSI-1 by NOTCH signaling in colorectal cancer cell lines and in primary cultures of colorectal cancer metastases. Stimulation by the NOTCH ligand DLL4 was associated with an increase of MSI-1 mRNA and protein levels, and this phenomenon was prevented by the addition of an antibody neutralizing NOTCH2/3 but not NOTCH1. Moreover, forced expression of activated NOTCH3 increased MSI-1 levels, whereas silencing of NOTCH3 by short hairpin RNA reduced MSI-1 levels in both colorectal cancer cells and CRC tumor xenografts. Consistent with these findings, enforced NOTCH3 expression or stimulation by DLL4 increased levels of activated NOTCH1 in colorectal cell lines. Finally, treatment of colorectal cancer cells with anti-NOTCH2/3 antibody increased NUMB protein while significantly reducing formation of tumor cell spheroids. This novel feed-forward circuit involving DLL4, NOTCH3, MSI-1, NUMB, and NOTCH1 may be relevant for regulation of NOTCH signaling in physiologic processes as well as in tumor development. With regard to therapeutic implications, NOTCH3-specific drugs could represent a valuable strategy to limit NOTCH signaling in the context of colorectal cancers overexpressing this receptor.