Notch Receptor 2 Drives Epithelial-Mesenchymal Transition and M2-Like Macrophage Polarization in Human Colorectal Cancer Tissues and HCT116 Cells.

Abstract

Despite advances in understanding colorectal cancer (CRC) biology, how tumor-cell signaling coordinates epithelial-mesenchymal transition (EMT) with macrophage polarization remains incompletely defined. We investigated whether NOTCH2 expression is associated with EMT and with a GATA3/IL-4-dependent macrophage-polarizing phenotype in CRC. NOTCH2 expression and prognostic associations were analyzed using TCGA data and validated in 20 paired CRC and adjacent normal tissue (ANT) specimens. Gain- and loss-of-function experiments in HCT116 cells assessed proliferation, migration, invasion, EMT markers, GATA3, and IL-4. A Transwell co-culture system containing transfected HCT116 cells and peripheral blood mononuclear cells (PBMCs) was used to evaluate CD206-positive M2-like macrophage polarization, and IL-4 neutralization was used to test the contribution of IL-4. NOTCH2 was increased in CRC tissues, and higher NOTCH2 expression was associated with poorer overall survival. NOTCH2 overexpression increased CRC-cell proliferation, migration, invasion, Vimentin, GATA3, and IL-4, while reducing E-cadherin; NOTCH2 silencing produced the opposite changes. Clinical tissues showed increased NOTCH2, GATA3, IL-4, CD163, and Vimentin, together with reduced E-cadherin. In co-culture, NOTCH2 overexpression increased the proportion of CD206-positive M2-like cells, whereas NOTCH2 knockdown reduced it. IL-4 neutralization significantly attenuated the increase in CD206-positive cells induced by NOTCH2 overexpression. These findings support associations among NOTCH2-associated malignant phenotypes, EMT, altered GATA3/IL-4 expression, and IL-4-dependent M2-like macrophage polarization in CRC, while direct transcriptional regulation of the GATA3/IL-4 axis remains to be established.