Abstract 1713: Macrophage-induced bystander effect activates Wnt/β-catenin signaling and induces cellular dedifferentiation

Abstract Cancer stem cells (CSCs) in colorectal cancer (CRC) help maintain tumor heterogeneity, promote tumor growth, invasion, and metastasis, and produce resistance to therapeutic agents. The origin of colorectal CSCs, however, is unclear. Our recent studies showed that a macrophage-induced bystander effect (MIBE) induced gene expression of stem/progenitor cell markers including Ly6A/E and Dclk1 in murine colonic epithelial cells, implying that MIBE helps induce colorectal CSCs. In current studies we show that commensal-triggered MIBE activates Wnt/β-catenin signaling and induces dedifferentiation of colonic epithelial cells during colorectal carcinogenesis, thus contributing to the origin of colorectal CSCs. Exposure of murine primary epithelial cells (YAMC) to commensal-polarized macrophages induced phosphorylated Gsk3β (Ser9), active β-catenin, and Tcf4, suggesting activation of Wnt/β-catenin signaling by MIBE. In vivo, active β-catenin was evident in both stromal and epithelial cells for colon biopsies from E. faecalis-colonized Il10−/− mice compared to sham-colonized mice. In addition, we showed that 4-hydroxynonenal (4-HNE), a byproduct of lipid peroxidation of ω6 polyunsaturated fatty acids, and tumor necrosis factor α (TNFα) mediated activation of Wnt/β-catenin signaling. Epigenetic analysis revealed that MIBE activated Wnt/β-catenin signaling through DNA methylation in the CpG islands of secreted frizzled-related protein 2 (Sfrp2), a soluble modulator of Wnt. Next, we showed that exposure of YAMC cells to 4-HNE and TNFα induced expression of c-Myc, Klf4, Oct4, and Sox2―factors essential for pluripotent stem cells. Immunofluorescent staining of colon biopsies from E. faecalis-colonized Il10−/− mice showed increased expression of these factors in colonic epithelial cells, suggesting dedifferentiation induced by MIBE. This was confirmed by qRT-PCR and Western blots that showed increased expression of Dclk1 and CD44, two markers for colorectal CSCs. Furthermore, increased expression of DCLK1 was noted in human tubular adenomas and invasive CRCs compared to hyperplastic polyps. Finally, inhibition of β-catenin/TCF4 using FH535, and by silencing CTNNB1, decreased DCLK1 expression in HCT116 human colon cancer cells, supporting the notion that Wnt/β-catenin signaling induces cellular dedifferentiation leading to colorectal CSCs. In summary, these results demonstrate that commensal-polarized macrophages activate Wnt/β-catenin signaling and induce dedifferentiation of colonic epithelial cells through 4-HNE and TNFα-mediated bystander effects. These findings provide new evidence for the origin of CSCs in colorectal carcinogenesis and should lead to novel strategies for CRC prevention and therapy. Citation Format: Xingmin Wang, Yonghong Yang, Mark M. Huycke. Macrophage-induced bystander effect activates Wnt/β-catenin signaling and induces cellular dedifferentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1713.