Selenoprotein P influences colitis-induced tumorigenesis by mediating stemness and oxidative damage

Abstract

Patients with inflammatory bowel disease are at increased risk for colon cancer due to augmented oxidative stress. These patients also have compromised antioxidant defenses as the result of nutritional deficiencies. The micronutrient selenium is essential for selenoprotein production and is transported from the liver to target tissues via selenoprotein P (SEPP1). Target tissues also produce SEPP1, which is thought to possess an endogenous antioxidant function. Here, we have shown that mice with Sepp1 haploinsufficiency or mutations that disrupt either the selenium transport or the enzymatic domain of SEPP1 exhibit increased colitis-associated carcinogenesis as the result of increased genomic instability and promotion of a protumorigenic microenvironment. Reduced SEPP1 function markedly increased M2-polarized macrophages, indicating a role for SEPP1 in macrophage polarization and immune function. Furthermore, compared with partial loss, complete loss of SEPP1 substantially reduced tumor burden, in part due to increased apoptosis. Using intestinal organoid cultures, we found that, compared with those from WT animals, Sepp1-null cultures display increased stem cell characteristics that are coupled with increased ROS production, DNA damage, proliferation, decreased cell survival, and modulation of WNT signaling in response to H2O2-mediated oxidative stress. Together, these data demonstrate that SEPP1 influences inflammatory tumorigenesis by affecting genomic stability, the inflammatory microenvironment, and epithelial stem cell functions.

Figure 8. WNT signaling plays a pivotal role in the Sepp1^–/– phenotype.

(A) Survival curves in response to growth factor depletion in WT, Sepp1^+/–, and Sepp1^–/– enteroids. (B) Quantification of WNT proteins LEF-1, cyclin D1, and MMP-7 normalized to β-actin. Quantification is shown as fold change relative to WT and representative images of blots from 3 individual tumors from each genotype. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Newman-Keuls multiple comparison test.

Figure 7. Sepp1^–/– enteroids display increased stem cell characteristics as well as ROS production, proliferation, and decreased survival after oxidative stress.

(A) Sepp1 mRNA expression in tissue isolated from WT mice (n = 3 per group). Fold change expression normalized to Gapdh and relative to colonic Sepp1 expression. ***P < 0.001, 1-way ANOVA, Newman-Keuls multiple comparison test. (B) Percentage of surviving enteroids 1 day after plating, percentage of branching enteroids and stem spheroids counted 3 days after plating, and average number of branches per branching enteroid at 4 days after plating (n = 4 per group per experiment). *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed unpaired t test. (C) ROS quantification by carboxy-H₂DCFDA staining intensity, as measured 2 hours after treatment with H₂O₂. Fold change in intensity relative to WT enteroids treated with 0 μM H₂O₂ (n = 4 per group). Representative images of the single carboxy-H₂DCFDA channel and merged carboxy-H₂DCFDA and TO-PRO3 channels in WT and Sepp1^–/– enteroids treated with either 0 μM or 800 μM H₂O₂ (original magnification, ×100). *P < 0.05, 1-way ANOVA, Newman-Keuls multiple comparison test. (D) Proliferation, as determined by EdU⁺ cells per crypt area within WT and Sepp1^–/– enteroids after 2 hours of treatment with either 0 μM or 800 μM H₂O₂ (n = 4 per group with analysis of 10 enteroids per genotype) and representative images of EdU staining in WT and Sepp1^–/– enteroids after treatment with either 0 μM or 800 μM H₂O₂ (original magnification, ×100). *P < 0.05, **P < 0.01, 1-way ANOVA, Newman-Keuls multiple comparison test. (E) Survival curves for WT, Sepp1^+/–, and Sepp1^–/– enteroids after daily treatment with 400 μM H₂O₂.

Figure 6. Sepp1 haploinsufficiency-driven CAC persists in mice maintained on normal selenium diets.

(A) Endoscopic images of WT and Sepp1^+/– colons after the second cycle of DSS administration. (B) Quantitative assessment of endoscopic tumor burden (11, WT; 13, Sepp1^+/–). **P < 0.01, 2-tailed unpaired t test. (C) Gross representative images of tumors (D) and tumor counts at necropsy (11, WT; 13, Sepp1^+/–). ***P < 0.001, 2-tailed unpaired t test. (E) Tumor size distribution. *P < 0.05, unpaired t test with Welch’s correction. (F) Survival analysis of AOM/DSS-treated mice maintained on the indicated diets. ***P < 0.001, unpaired t test with Welch’s correction.

Figure 5. Both the selenium-rich region and the putative antioxidant domain of SEPP1 protect from inflammatory tumorigenesis.

(A) Quantification of colonic selenium in WT (n = 6) and Sepp1^{Δ240–361/Δ240–361} (n = 6) mice and (B) number of tumors per mouse (16, WT; 15, Sepp1^{Δ240–361/Δ240–361}). *P < 0.05, ***P < 0.001, 2-tailed unpaired t test. (C) Percentage of total tumors per genotype with either high-grade dysplasia (HGD) or low-grade dysplasia (LGD) (n = 15 per group, ***P < 0.001, χ² contingency analysis) and representative images from tumors of the given genotypes (original magnification, ×40). Scale bar: 200 μm. (D) Quantification of intratumoral proliferation, as measured by Ki67⁺ cells per tumor HPF (12, WT; 17, Sepp1^{Δ240–361/Δ240–361}). (E) Crypt DNA damage, as measured by 8-hydroxyguanine⁺ cells per crypt averaged from 20 crypts within each mouse (8, WT; 9, Sepp1^{Δ240–361/Δ240–361}). (F) Intratumoral DNA damage, as measured by 8-hydroxyguanine⁺ cells per tumor HPF (8, WT; 17, Sepp1^{Δ240–361/Δ240–361}). (G) Number of tumors and (H) average tumor size within either WT (n = 12) or Sepp1^U40S/U40S (n = 16) mice. (I) Quantification of crypt proliferation, as measured by Ki67⁺ cells per crypt averaged from 20 crypts within each mouse (8, WT; 8, Sepp1^U40S/U40S); (J) intratumoral proliferation, as measured by Ki67⁺ cells per tumor HPF (11, WT; 19, Sepp1^U40S/U40S); (K) crypt DNA damage, as measured by 8-hydroxyguanine⁺ cells per crypt averaged from 20 crypts within each mouse (8, WT; 8, Sepp1^U40S/U40S); and (L) intratumoral DNA damage, as measured by 8-hydroxyguanine⁺ cells per tumor HPF (9, WT; 8, Sepp1^U40S/U40S). *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed unpaired t test.

Figure 4. SEPP1 regulates protumorigenic M2 macrophage polarization.

Quantification of (A) intratumoral total macrophage staining, as determined by F4/80⁺ cells per tumor HPF; (B) M1 macrophage staining, as determined by F4/80⁺/IL-1β⁺ cells per tumor HPF; and (C) M2 macrophage staining, as determined by F4/80⁺/ARG1⁺ cells per tumor HPF (8, WT; 12, Sepp1^+/–; 8, Sepp1^–/–). (D) Inos (n = 5 per genotype) and Il1b (n = 6 per genotype) and (E) Ym1 mRNA expression (n = 6 per group) in WT, Sepp1^+/–, and Sepp1^–/– in vitro–activated bone marrow macrophages. Graphs demonstrate fold change in expression relative to unstimulated (US) WT macrophages normalized to Gapdh. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Newman-Keuls multiple comparison test.

Figure 3. Intratumoral apoptosis and DNA damage are increased in response to complete Sepp1 knockout, and proliferation is increased in Sepp1^+/– tumors.

(A) Quantification of intratumoral apoptosis, as determined by TUNEL⁺ cells per tumor HPF (original magnification, ×40; 6, WT; 10, Sepp1^+/–; 4 Sepp1^–/–), and quantification of cleaved caspase-3 protein normalized to caspase-3 and cleaved PARP protein normalized to β-actin. Quantification is shown as fold change relative to WT (n = 6 per group). (B) Quantification of intratumoral proliferation, as determined by Ki67⁺ cells per tumor HPF (6, WT; 10, Sepp1^+/–; 4, Sepp1^–/–). (C) Quantification of intratumoral DNA damage, as measured by 8-hydroxyguanine⁺ cells per tumor HPF (6, WT; 10, Sepp1^+/–; 4 Sepp1^–/–). *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Newman-Keuls multiple comparison test.

Figure 2. Sepp1 haploinsufficiency augments inflammatory carcinogenesis.

(A) Quantification of colonic selenium in WT (n = 8), Sepp1^+/– (n = 5), and Sepp1^–/– (n = 8) mice fed a selenium-supplemented (1.0 PPM) diet. (B) Representative gross colon images. (C) Tumor number (tumors per mouse) and (D) average tumor size (mm²) per mouse (17, WT; 15, Sepp1^+/–; 20, Sepp1^–/–). (E) Representative images of Swiss-rolled colons from each genotype (original magnification, ×25). Scale bar: 500 μm. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Newman-Keuls multiple comparison test.

Figure 1. Absence of SEPP1 exacerbates tumorigenesis in response to AOM and injury after chronic DSS treatment.

(A) Schematic of the AOM protocol used. Mice were injected with AOM and aged for 6 months before being sacrificed on day 180 after AOM treatment. (B) Schematic of the chronic DSS protocol used. Mice were subjected to three 5-day cycles of 3% DSS ad libitum. There were 16 days of recovery between each DSS administration, and mice were monitored for injury by endoscopy (black circles) 4 days after each DSS cycle (gray rectangles). (C) Immunofluorescent staining of SEPP1 (red) within the colons and small intestines of WT and Sepp1^–/– mice (original magnification, ×100). (D) ACF counts per mouse in WT (n = 4), Sepp1^+/– (n = 4), and Sepp1^–/– (n = 3) mice subjected to the AOM protocol. (E) Endoscopic colitis score, ACF counts, and Ki67 staining in mice subjected to the chronic DSS protocol (7, WT; 10, Sepp1^+/–; 10, Sepp1^–/–). *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Newman-Keuls multiple comparison test.