Sa1977 Kynurenine Metabolic Pathway As a Rational Target for Colorectal Cancer: Defining Its Role in β-Catenin Signaling

Abstract

Background: Enzymes that initiate tryptophan metabolism along the kynurenine pathway (KP) are commonly overexpressed in colorectal cancer (CRC). One of these enzymes, IDO1, has recently been shown to promote tumorigenesis in a mouse model of colitis associated cancer and promote nuclear translocation of β-catenin and proliferation in CRC cells. The current investigation is aimed at identifying the signaling pathways activated by various KP metabolites and determining if a similar effect exists in non-neoplastic primary colonic epithelial cells (CEC). Methods: In vitro models of human CRC using various CRC cell lines (HT29, HCT116 and SW480) as well as human colon primary CECs cultured in growth factor (Wnt3a, Noggin, R-Spondin) enriched media were used. All experiments were performed in serum and growth factor free culture media with or without the addition of KP metabolites such as Kynurenine (Kyn), Quinolinic Acid (QA) and Picolinic Acid (PA). Expression of mRNA and protein as well as their phosphorylation status was quantified at multiple time points, and cell proliferation was measured by MTT assay. Results: Addition of Kyn, QA and PA (100 μM) rapidly induced (5 min) phosphorylation of AKT (Ser473) and LRP (Ser1490), a required co-receptor for activating Wnt/β-catenin signaling. The KP metabolites also rapidly inactivated GSK-3β as indicated by phosphorylation at Ser9 and induced expression of nuclear β-catenin (P-Ser552). These changes were followed by an induction of CyclinD1 expression at 12-36 hours along with an increase in CRC cell proliferation after Kyn and QA application. However, with PA, no functional changes in proliferation were observed. In contrast to CRC cells, KP metabolites treatment of primary human CECs led to comparatively moderate and delayed changes in AKT, GSK-3β and βcatenin phosphorylation. Additionally, no change in LRP phosphorylation or effect on primary CEC proliferation was observed. Conclusions: KP metabolites induce rapid and reversible changes in the activation state of cellular machinery linked to CRC cell proliferation and neoplastic growth. While similarities exist between initial signaling events of KP metabolites, important distinctions were observed in their ultimate effects on proliferation of CRC and primary colonic epithelial cells. Together these observations highlight the role of the KP metabolites in β-catenin signaling and form the basis of future interrogation of this pathway for therapeutic targeting of colorectal cancer that may spare the normal epithelium.