IDO1 and Kynurenine Pathway Metabolites Activate PI3K-Akt Signaling in the Neoplastic Colon Epithelium to Promote Cancer Cell Proliferation and Inhibit Apoptosis

Abstract

The tryptophan-metabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) is frequently overexpressed in epithelial-derived malignancies, where it plays a recognized role in promoting tumor immune tolerance. We previously demonstrated that the IDO1-kynurenine pathway (KP) also directly supports colorectal cancer growth by promoting activation of β-catenin and driving neoplastic growth in mice lacking intact adaptive immunity. In this study, we sought to delineate the specific role of epithelial IDO1 in colon tumorigenesis and define how IDO1 and KP metabolites interact with pivotal neoplastic signaling pathways of the colon epithelium. We generated a novel intestinal epithelial-specific IDO1 knockout mouse and utilized established colorectal cancer cell lines containing β-catenin-stabilizing mutations, human colorectal cancer samples, and human-derived epithelial organoids (colonoids and tumoroids). Mice with intestinal epithelial-specific knockout of IDO1 developed fewer and smaller tumors than wild-type littermates in a model of inflammation-driven colon tumorigenesis. Moreover, their tumors exhibited reduced nuclear β-catenin and neoplastic proliferation but increased apoptosis. Mechanistically, KP metabolites (except kynurenic acid) rapidly activated PI3K-Akt signaling in the neoplastic epithelium to promote nuclear translocation of β-catenin, cellular proliferation, and resistance to apoptosis. Together, these data define a novel cell-autonomous function and mechanism by which IDO1 activity promotes colorectal cancer progression. These findings may have implications for the rational design of new clinical trials that exploit a synergy of IDO1 inhibitors with conventional cancer therapies for which Akt activation provides resistance such as radiation.Significance: This study identifies a new mechanistic link between IDO1 activity and PI3K/AKT signaling, both of which are important pathways involved in cancer growth and resistance to cancer therapy.

Figure 1:. Epithelial IDO1 promotes tumor growth and neoplastic proliferation in the colon.

Mice with intestinal epithelial specific Ido1 deletion (IDO1-iKO) and WT (Cre-) littermate controls were examined for gene expression and propensity for colon tumorigenesis when exposed to the AOM/DSS protocol. Mice received 10mg/kg Azoxymethane (AOM) followed by two, week-long cycles of 2.25% DSS in drinking water separated by sterile water for 2 weeks. A) Schematic for generating intestinal knockout (iKO) mice. B) Immunohistochemistry for IDO1 on AOM/DSS induced tumors in iKO and WT mice. C) and D) mRNA and IDO1 protein expression from isolated tracts of non-epithelial and epithelial cellular compartments of mice 48 hours after IDO1 induction with 20 µg IP of CpG DNA E) Measurement of kynurenine production in the supernatants of colon organoid culture from WT and iKO mice. F) Representative colon morphology with tumors highlighted by topically applied Alcian blue. Magnified image of tumors shown in inserts to highlight the difference in the sizes of tumors G) Tumor quantification showing reduced tumor size and number in IDO1-iKO mice. Statistical comparison by two-way ANOVA (D) or Student’s T-test (F). N=5-6 mice/group, 2-4 tumors counted/mouse.

Figure 2:. Epithelial IDO1 promotes nuclear B-Catenin activation, increases epithelial proliferation and protects from apoptosis.

A) Decreased nuclear β-catenin staining in IDO1-iKO tumors compared to WT. Magnified images of β-Catenin staining by immunohistochemistry in tumors and adjacent normal areas in WT and iKO mice. B) Representative immunohistochemistry images of β-Catenin target gene Cyclin D1. C) Epithelial proliferation by BrdU staining with its quantitation represented as epithelial proliferation index in tumors and adjacent normal epithelium. D) Apoptosis by Tunel assay including representative IF images quantitation. E) Representative images of Survivin immunohistochemistry.

Figure 3:. Kynurenine rapidly induces β-catenin nuclear translocation and activates Akt in CRC cells.

Experiments were completed with HT29 cells treated with kynurenine at 100 µM or at concentrations shown. The impact of kynurenine on β-catenin cellular location was assessed by A) Western blot and B) Immunofluorescence. Kyn did not activate Wnt pathway as evidenced by C) No observed change in the frizzled co-receptor activation as measured by pLRP5/6 S1490 levels and D) Kyn increasing EdU incorporation as a marker of proliferation in the presence of Wnt signaling inhibitor DKK1. *p<0.05. E) Kynurenine did induce dose dependent activation of Akt (pS473) and Akt phosphorylation target PRAS40 at physiological concentrations of kynurenine as measured by F) Intracellular and extracellular levels in CRC cell lines.

Figure 4:. Kyurenine metabolites rapidly activate β-catenin via intracellular activation of PI3K/Akt pathway.

A) HT29, DLD1 and HCT116 colon cancer cells were treated with Kyn or QA for specified time points followed by whole cell protein extraction. Western blots were performed to analyze Akt-Ser473 (activated), pPRAS40-T246 (activated), GSK3β-Ser9 (inactivated) and βCatenin Ser552 (activated) phosphorylation levels. B) Inhibition of the LAT1 amino acid transporter by either BCH or JPH203 blocks kynurenine mediated Akt pathway activation. C) Inhibition of Akt (MK2206) and PI3K (GDC0941) blocks kynurenine mediated phosphorylation of Akt at S473 and prevents downstream targets GSK3β from inactivation (pSer9) and β-catenin from activation (pSer552) in HT-29 cells. D) Inhibition of Aryl hydrocarbon receptor with CH-223191 does not prevent Akt activation in HT-29 CRC cells.

Figure 5:. IDO1 and activated Akt co-localize in neoplastic epithelium.

A) Tumors and normal epithelium of IDO1-iKO mice demonstrate diminished pAkt S473 immunostaining verses WT. Representative figure from immunostaining in 5 mice/group. B) Representative images of pAkt S473 co-staining with IDO1 immunostaining in human colon tumors. Arrows indicate dual negative crypts (yellow) and dual positive crypts (red). figure from immunostaining for 5 tumor tissues. C) Positive correlation between pAkt and IDO1 immunostaining based co-localization in human CRC. 5 CRCs with a total of 141 crypts compared.

Figure 6:. Kynurenine protects CRC cells from apoptosis and promotes viability during stress.

Apoptosis was induced in HT29 cells by TNFα (100 ng/ml) in the presence and absence of kynurenine (100 µM) and the Akt inhibitor MK-2206. Apoptosis was measured by cleaved PARP expression 12 hours after listed treatment A) Western blot. B,C) Immunofluorescence staining images and quantitation of cleaved PARP as a percentage of DAPI positive cells. D) Cell viability 24 hours after treatement measured by WST-8 assay. *P<0.05, **P<0.01, ***P<0.001

Figure 7:. Kynurenine mediated rapid activation of Akt and β-catenin differentiates tumor cells from normal cells.

Western blot was used to detect total and phosphorylated proteins in A) Normal human colon crypt derived organoids (colonoids) and tumoroids derived from colon cancer tissue derived from patients with FAP. B) Proliferation in human colonoids and tumoroids measured by WST assay after 72 hours of treatment with Kyn or QA. C) Representative western blot of tumoroid cyclin D1 expression 12 hours after Kyn or QA treatment with mean densitometry quantitation. D) Model for KP metabolites induced PI3K/Akt activation and its effect on tumor cells and nuclear β-catenin activation. Data shown are a representative data from 3 experiments. ***P<0.001.