Loss of solute carrier family 7 member 2 exacerbates inflammation-associated colon tumorigenesis

Abstract

Solute carrier family 7 member 2 (SLC7A2, also known as CAT2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in wound repair. We have reported that both SLC7A2 expression and L-Arg availability are decreased in colonic tissues from inflammatory bowel disease patients and that mice lacking Slc7a2 exhibit a more severe disease course when exposed to dextran sulfate sodium (DSS) compared to wild-type (WT) mice. Here, we present evidence that SLC7A2 plays a role in modulating colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinogenesis (CAC). SLC7A2 was localized predominantly to colonic epithelial cells in WT mice. Utilizing the AOM-DSS model, Slc7a2^-/- mice had significantly increased tumor number, burden, and risk of high-grade dysplasia vs. WT mice. Tumors from Slc7a2^-/- mice exhibited significantly increased levels of the proinflammatory cytokines/chemokines IL-1β, CXCL1, CXCL5, IL-3, CXCL2, CCL3, and CCL4, but decreased levels of IL-4, CXCL9, and CXCL10 compared to tumors from WT mice. This was accompanied by a shift toward pro-tumorigenic M2 macrophage activation in Slc7a2-deficient mice, as marked by increased colonic CD11b⁺F4/80⁺ARG1⁺ cells with no alteration in CD11b⁺F4/80⁺NOS2⁺ cells by flow cytometry and immunofluorescence microscopy. The shift toward M2 macrophage activation was confirmed in bone marrow-derived macrophages from Slc7a2^-/- mice. In bone marrow chimeras between Slc7a2^-/- and WT mice, the recipient genotype drove the CAC phenotype, suggesting the importance of epithelial SLC7A2 in abrogating neoplastic risk. These data reveal that SLC7A2 has a significant role in the protection from CAC in the setting of chronic colitis, and suggest that the decreased SLC7A2 in inflammatory bowel disease (IBD) may contribute to CAC risk. Strategies to enhance L-Arg availability by supplementing L-Arg and/or increasing L-Arg uptake could represent a therapeutic approach in IBD to reduce the substantial long-term risk of colorectal carcinoma.

Figure 1.. SLC7A2 expression is increased in inflammation-associated tumorigenesis.

C57BL/6 WT and Slc7a2^–/– mice were exposed to the AOM-DSS protocol (AOM-DSS) or not (Ctrl) and colonic tissues were harvested at day 77. Colon sections were stained for SLC7A2 protein by immunohistochemistry. Scale bar = 100 μm. Representative images of at least 3 mice per group.

Figure 2.. Slc7a2^–/– mice exhibit increased tumorigenesis after exposure to the AOM-DSS model of CAC using 2.5% DSS.

(a) Tumor number was assessed by gross visual inspection, utilizing a dissecting microscope. (b) Tumor burden was determined by the addition of the calculated area of each identified tumor, as assessed with an electronic caliper for both length and width. (c) Histologic colitis score in nontumor area. In (a), (b), and (c), ***P < 0.001 versus WT Ctrl; §P < 0.05 and §§§P < 0.001 versus WT AOM-DSS by one-way ANOVA with Student-Newman-Keuls posthoc multiple comparisons test. (d) Survival curve assessed by log-rank (Mantel-Cox) test. In (a), (b), (c), and (d), n = 25–29 control, 48–55 DSS-treated, and 61–62 AOM-DSS-treated mice per genotype. (e) Percentage of cases with either no adenoma, low grade dysplasia, and high grade dysplasia determined by a gastrointestinal pathologist (M.K.W.) in a blinded manner. By Chi Square test, *P = 0.0161. The number of mice with each diagnosis is shown on the graph. (f) Representative H&E-stained images from AOM-DSS-treated mice. Scale bar = 100 μm.

Figure 3.. Slc7a2^–/– mice have significantly altered cytokine and chemokine production within colon tumors.

Protein levels were assessed by Luminex Multiplex Array from colonic tissues 77 days post-AOM injection. In all panels, **P < 0.01, ***P < 0.001 versus WT AOM DSS non-tumor; ^###P < 0.001 versus Slc7a2^–/– AOM-DSS non-tumor; and §P < 0.05, §§P < 0.01, and §§§P < 0.001 versus WT AOM-DSS tumor by one-way ANOVA with Student-Newman-Keuls posthoc multiple comparisons test. n = 11–15 control, 7–15 DSS-treated, and 13–18 AOM-DSS-tumors with paired non-tumor area per genotype.

Figure 4.. AOM-DSS exposure leads to increased M2 colonic macrophages in Slc7a2^–/– mice.

(a-b) Quantification of CD11b⁺F4/80⁺ARG1⁺ macrophages and CD11b⁺F4/80⁺NOS2⁺ macrophages per gram of colon tissue. (c) Ratio of CD11b⁺F4/80⁺ARG1⁺/CD11b⁺F4/80⁺NOS2⁺ in the same colon. (d) Representative flow plots showing the increase in CD11b⁺F4/80⁺ARG1⁺ macrophages in Slc7a2^–/– AOM-DSS colon compared to WT AOM-DSS colon. In a-c, §P < 0.05 versus WT AOM-DSS by one-way ANOVA with Student-Newman-Keuls posthoc multiple comparisons test. n = 2–3 control, 5–7 DSS-treated, and 7–9 AOM-DSS-treated per genotype.

Figure 5.. ARG1⁺CD68⁺ macrophages are increased in Slc7a2^–/– AOM-DSS tumors.

Representative images of immunofluorescence staining for (a) macrophage marker, CD68 (red); M2 marker, ARG1 (green); CD68⁺ARG1⁺ cells (yellow, merge). Cell nuclei, DAPI (blue). (b) Macrophage marker, CD68 (red); M1 marker, NOS2 (green); CD68⁺NOS2⁺ cells (yellow, merge). Cell nuclei, DAPI (blue). Representative merged images of at least 3 per group. Scale bars = 50 μm.

Figure 6.. Bone marrow-derived macrophages (BMmacs).

BMmacs were stimulated with classical M1 stimuli, IFN-γ (200 U/mL) and LPS (100 ng/mL), or M2 stimuli, IL-4 (10 ng/mL) and IL-10 (10 ng/mL), for 24 hours. Cells were collected for isolation of RNA and protein. The supernatants were collected. (a) mRNA levels of M1 markers, Nos2 and Il1b, were assessed by qRT-PCR in unstimulated (Ctrl) or stimulated cells. (b) mRNA levels of M2 markers, Arg1 and Chil3, were assessed by qRT-PCR in unstimulated (Ctrl) or stimulated cells. (c) Concentration of NO^–₂ in the supernatants assessed by Griess assay, and IL-1β in the supernatant assessed by ELISA, in unstimulated (Ctrl) or stimulated cells. n = 4–8 biological replicates per genotype. In (a-c), **P < 0.01 and ***P < 0.001 versus WT unstimulated; §§P < 0.01 and §§§P < 0.001 versus WT stimulated, by one-way ANOVA with Newman-Keuls post-test. (d) Representative Western blot of CHIL3 levels in BMmacs activated with M2 stimulus for 24 hours. n = 3 biological replicates. Combined densitometry shown. In (d), *P < 0.05 versus WT stimulated by Student’s t test.

Figure 7.. Bone marrow transplant between WT and Slc7a2^–/– mice.

Irradiated animals were given bone marrow-derived hematopoietic cells and were treated with AOM-DSS. (a) Tumor number was assessed by gross visual inspection, utilizing a dissecting microscope. (b) Tumor burden was determined by the addition of the calculated area of each identified tumor, as assessed with an electronic caliper for both length and width. (c) Histologic colitis was determined by a gastrointestinal pathologist (M.K.W.) in a blinded manner. In (a), (b), and (c), *P < 0.05 versus WT to WT AOM-DSS; §P < 0.05 and §§P < 0.01 versus Slc7a2^–/– to WT AOM-DSS, by one-way ANOVA with Student-Newman-Keuls posthoc multiple comparisons test. (d) Survival curve assessed by log-rank (Mantel-Cox) test. (e) Percentage of cases with either no adenoma, low grade dysplasia, and high grade dysplasia determined by a gastrointestinal pathologist (M.K.W.) in a blinded manner. By Chi Square test, *P < 0.05. n = 8–12 AOM-DSS-treated animals per transplant type.