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. 2022 May 4;28(5):764-774.
doi: 10.1093/ibd/izab306.

TIPE2 Promotes Tumor Initiation But Inhibits Tumor Progression in Murine Colitis-Associated Colon Cancer

Zienab Etwebi  1 , Jason R Goldsmith  1 , Mayassa Bou-Dargham  1 , Yuhua Tian  2 , Ryan Hood  1 , Nina Spitofsky  1 , Mingyue Li  3 , Honghong Sun  1 , Yunwei Lou  4 , Suxia Liu  5 , Christopher Lengner  2 , Youhai H Chen  1   6
Affiliations

TIPE2 Promotes Tumor Initiation But Inhibits Tumor Progression in Murine Colitis-Associated Colon Cancer

Zienab Etwebi et al. Inflamm Bowel Dis. .

Abstract

Background: Colorectal cancer (CRC) is the third leading cause of cancer in the United States, and inflammatory bowel disease patients have an increased risk of developing CRC due to chronic intestinal inflammation with it being the cause of death in 10% to 15% of inflammatory bowel disease patients. TIPE2 (TNF-alpha-induced protein 8-like 2) is a phospholipid transporter that is highly expressed in immune cells and is an important regulator of immune cell function.

Methods: The azoxymethane/dextran sulfate sodium murine model of colitis-associated colon cancer (CAC) was employed in Tipe2 -/- and wild-type mice, along with colonoid studies, to determine the role of TIPE2 in CAC.

Results: Early on, loss of TIPE2 led to significantly less numbers of visible tumors, which was in line with its previously described role in myeloid-derived suppressor cells. However, as time went on, loss of TIPE2 promoted tumor progression, with larger tumors appearing in Tipe2 -/- mice. This was associated with increased interleukin-22/STAT3 phosphorylation signaling. Similar effects were also observed in primary colonoid cultures, together demonstrating that TIPE2 also directly regulated colonocytes in addition to immune cells.

Conclusions: This work demonstrates that TIPE2 has dual effects in CAC. In the colonocytes, it works as a tumor suppressor. However, in the immune system, TIPE2 may promote tumorigenesis through suppressor cells or inhibit it through IL-22 secretion. Going forward, this work suggests that targeting TIPE2 for CRC therapy requires cell- and pathway-specific approaches and serves as a cautionary tale for immunotherapy approaches in general in terms of colon cancer, as intestinal inflammation can both promote and inhibit cancer.

Keywords: IL-22; TIPE2; colitis-associated colon cancer; tumor suppressor.

Plain language summary

TIPE2 (TNF-alpha-induced protein 8-like 2) regulates immune function. Here, we find that it differentially regulates the initiation and progression of its immunoregulatory properties affect murine colitis-associated colon cancer initiation and progression. Surprisingly, we found that TIPE2 a novel tumor suppressor in enterocytes, a cell compartment it was not previously known to directly regulate.

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Figures

Figure 1.
Figure 1.
Colitis and tumor development in mice after azoxymethane and dextran sulfate sodium administration. A, Weight change over time; n = 17 for wild-type (WT) and 19 for Tipe2–/– mice, pooled across 3 independent experiments. Statistical analysis by 2-way analysis of variance; ∗∗∗∗P < .0001 for comparisons across time and group. B-D, Histolopathology score assessing colitis, colon length, and representative histological images at 70 days of colitis-associated colon cancer (CAC); n = 14 for WT and 18 for Tipe2–/– mice, pooled across 3 independent experiments. Scale bars=110 µm; inserts are 2× magnified. E, Survival curve over 70 days of CAC; n = 20 per group, pooled across 2 independent experiments. P = .1105 by Mantel-Cox test. F, Tumor burden per mouse; n = 18 for WT and 23 for Tipe2–/– mice pooled across 2 independent experiments. ∗∗∗∗P < .0001. G, Individual tumor diameter; n = 39 for WT and 7 for Tipe2–/– mice pooled across 2 independent experiments. H, Neoplasia index (histopathological analysis) at 70 days of CAC; n = 14 for WT and 18 for Tipe2–/– mice, pooled across 3 independent experiments. All graphs display mean±SEM unless otherwise indicated. Statistical analyses by Mann-Whitney nonparametric t test unless otherwise indicated.
Figure 2.
Figure 2.
Tipe2  –/– mice have similar levels of inflammatory cytokines to those of wild-type (WT) mice, but increased levels of interleukin (IL)-22 and associated signaling. A, Real-time polymerase chain reaction for tumor necrosis factor (Tnf), Il1b, and Il1b in distal colonic tissue taken from azoxymethane (AOM)/dextran sulfate sodium (DSS) mice after 70 days of treatment. n = 12 per group from Tnf and Il1b, and n = 9 per group for Il6. Results were pooled across 2 independent experiments. B, Luminex assay for serum cytokines in AOM/DSS mice after 70 days of treatment. n = 5 per group for TNF, n = 5 for WT, n = 3 for Tipe2 –/– for IL17a, n = 5 for WT and n = 10 for IL6 (samples without detectable levels were excluded from analysis, leading to the variable sample numbers). Samples were pooled across 2 independent experiments. C, Real-time polymerase chain reaction for Il22 in in distal colonic tissue taken from AOM/DSS mice after 70 days of treatment; n = 10 per group, pooled across 2 independent experiments. D, Luminex assay for serum IL22 in AOM/DSS mice after 70 days of treatment; n = 7 for WT and 10 for Tipe2 –/–. Samples were pooled across 2 independent experiments. E, IL-22 excretion by AOM/DSS colonic explants harvested at 70 days of treatment, measured by sandwich enzyme-linked immunosorbent assay. n = 4 mice for WT, 5 mice for Tipe2 –/–. Representative of 2 independent experiments. F and G, pSTAT3Y705 staining in AOM/DSS mice after 70 days of treatment; results pooled from 2 independent experiments. F, Quantification done by counting 3 random sections per slide from 5 different mice per group; n = 15 per group. G, Representative micrographs: bars=50 µm, inserts 2× multiplied. H-J, Proliferation as measured by Ki-67 staining. H Quantification by measuring length of proliferative zone in all longitudinal crypts from 3 images per slide. A total of 7 WT and 8 Tipe2 –/– slides were analyzed; total n = 183 for WT and 207 for Tipe2 –/–. G, Representative micrographs: bars=220 µm, inserts 2× multiplied. J, Representative tumor-specific micrographs; bars=110 µm. All graphs display mean±SEM unless otherwise indicated. Statistical analyses by Mann-Whitney nonparametric t test unless otherwise indicated. P as indicated; ∗∗∗∗P < .0001.
Figure 3.
Figure 3.
Loss of TIPE2 results in bigger tumors as disease progresses. A, Weight change over time. n = 9 for wild-type (WT) and 8 for Tipe2 –/– mice, pooled across 2 independent experiments. Statistical analysis by 2-way analysis of variance. No significant differences between groups were found. B-D, Histolopathology score assessing colitis, colon length, and representative histological images at 91 days of colitis-associated colon cancer (CAC). For histology, n = 8 for WT and 7 for Tipe2 –/–, pooled across 2 independent experiments; for colon length, n = 22 for Wt and 24 for Tipe2 –/–, pooled across 3 independent experiments. Scale bars=220 µm; inserts are 2× magnified, and statistical analyses were by Student’s t test. E, Representative (n = 5 independent experiments) whole mount images of colons after 91 days of CAC. Arrows point to individual tumors. F, Survival curve over 70 days of CAC. n = 20 per group, pooled across 2 independent experiments. ∗∗P = .006 by Mantel-Cox test. G, Tumor burden per mouse; n = 23 for WT and 26 for Tipe2 –/– mice pooled across 5 independent experiments. H, Individual tumor diameter; n = 55 for WT, and 41 for Tipe2–/– mice pooled across 5 independent experiments; ∗∗∗∗P < .0001. I, Neoplasia index (histopathological analysis) at 91 days of CAC. n = 8 for WT and 7 for Tipe2–/– mice, pooled across 2 independent experiments. J, Serum cytokines in azoxymethane (AOM)/dextran sulfate sodium (DSS) mice after 91 days of treatment, measured by sandwich enzyme-linked immunosorbent assay. n = 7 for WT and 10 for Tipe2  –/– mice, except for IL-22, where n = 9 for Tipe2– /– mice. Samples were pooled across 2 independent experiments; statistical analyses by Student’s t test. K, Real-time polymerase chain reaction of colonic tissue at 91 days for Tnf, Il6, Il22, and Il1b. n = 20 per group for Tnf, 17 per group for Il6, 18 per group for Il22, and 20 per group for Il1b, with individual replicates pooled across 2 individual experiments comprising ≥8 mice per genotype. Analysis was by Mann-Whitney nonparametric t test unless otherwise described, with significance as indicated on each graph or as described. All graphs display mean±SEM unless otherwise indicated.
Figure 4.
Figure 4.
Tipe2 –/– mice with colitis-associated colon cancer have increased numbers of CD11c+CD8a+ T cells. A-C, Flow cytometry for CD11c+ and CD11c+CD8a+ cells isolated from the lamina propria and intraepithelial colonic compartments of mice subjected to 91 days of colitis-associated colon cancer; n = 7 for wild-type (WT) and 8 for Tipe2–/– mice, pooled between 2 independent experiments. C, Sample flow gates. Gating is on lymphocytes, live cells, and CD45, and sample gates are percentages of CD45+ cells (see Figure S3). D, Real-time polymerase chain reaction of colonic tissue at 91 days for Itgae, Il23, and Il23r; n = 8 for WT and 6 for Tipe2–/– mice, pooled between 2 independent experiments. Analysis by Mann-Whitney nonparametric t test, with significance as indicated on each graph. For all panels, all graphs display mean±SEM unless otherwise indicated. Statistical analyses by Student’s t test unless otherwise specified with significance as indicated on each graph.
Figure 5.
Figure 5.
TIPE2 is a colonic tumor suppressor. A, Real-time polymerase chain reaction of isolated tumor and adjacent tumor colonocytes at 91 days of colitis-associated colon cancer (CAC), for Tnfaip8l2 and Ptprc. Each data point represents 2 to 3 pooled mice, and results are pooled across 3 independent experiments. n = 4 all groups, except Tipe2 –/– mice where n = 6 for Tnfaip8l2 and n = 7 for Ptprc. Analysis by Mann-Whitney nonparametric t test, with significance as indicated on each graph. B and C, RNAScope for Tipe2 and Ptprc (CD45). B and C, Quantification, using automated image analysis to measure the number of positive RNA probe signals per slide, with representative photomicrographs. For colonic tissue, n = 4 mice per group, except for Tipe2 –/–, where n = 7. For gut-associated lymphoid tissue (GALT), n = 7 for wild-type (WT) and 5 for Tipe2 –/– mice; bars=50 µm. D and E, Proliferation assay and organoid growth assessment € in shCtrl (Ctrl) and short hairpin RNA–mediated Tipe2-knockdown (T2KD) APC-mutant (∆A) and APC/p53-mutant (∆AP) murine colonoids that represent progressively advanced stages of colonic adenoma or carcinoma. n = 3 per group, representative of 2 independent experiments, and analyzed by parametric t test. All graphs display mean±SEM unless otherwise indicated. Statistical analyses by Mann-Whitney nonparametric t test unless otherwise indicated. P as indicated; ∗∗∗∗P < .0001.
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