The E3 Ubiquitin Ligase Asb2α in T Helper 2 Cells Negatively Regulates Antitumor Immunity in Colorectal Cancer

ASB2 expression in human colorectal cancer biopsies correlated with shorter relapse-free survival. Microarray expression data (GSE39582) from human colorectal cancer biopsies containing tumor cells, stroma, and infiltrate (28) were analyzed for expression of ASB2, CD45/PTPRC, c-MAF, GATA3, and IRF4. A, Scatter plots showing correlation data for ASB2 and CD45/PTPRC, ASB2 and GATA3, ASB2 and c-MAF, and ASB2 and IRF4 transcripts in human colorectal cancer biopsies (n = 250). Linear regression-fit curves are shown as red lines. Correlations between nonparametric variables were evaluated using Spearman rank correlation test (r). B, ASB2 expression in the C1 (n = 55), C2 (n = 55), C3 (n = 27), C4 (n = 26), C5 (n = 61), and C6 (n = 26) subtypes of colorectal cancers as defined by Marisa and colleagues (28). Boxplots (with lower quartile, median, and upper quartile, Tukey whiskers) are shown. C, Kaplan–Meier curves showing relapse-free survival rates in patients with high (ASB2^high; n = 8), intermediate (ASB2^int; n = 138), and low (ASB2^low; n = 104) ASB2 expression. Relapse-free survival curves were assessed for statistically significant differences using the Log-rank (Mantel–Cox) test. D, Percentages of the C1–C6 subtypes in the ASB2^high, ASB2^int, and ASB2^low groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Deletion of Asb2 attenuated colitis-associated tumor formation and development. Control (ctrl) and cKO mice were injected with AOM, subsequently treated with the indicated cycles of DSS, and analyzed 69 (B–H) or 43 (I–K) days after AOM injection. A, Schematic representation of AOM/DSS-induced colorectal cancer treatment. B, Representative image of colons. Scale bar = 0.5 cm. C, Representative colon sections stained with alcian blue and nuclear fast red. Scale bar = 200 μm. D, Expression of Asb2α transcripts in spleen cells. E, Expression of Asb2α transcripts in sorted CD45⁺ and CD45⁻ cells isolated from colonic tissues of AOM/DSS-treated mice. F, Colon lengths after treatment. G, Tumor loads in the colon. H, Enumeration of total tumor burden and of tumors following classification according to their size. I, Colon lengths after treatment. J, Tumor loads in the colon. K, Enumeration of total tumor burden and of tumors following classification according to their size. L, Schematic representation of AOM/DSS-induced colorectal cancer treatment after bone marrow reconstitution of C57BL/6 CD45.1–irradiated mice with bone marrow cells from Mx1-Cre;Asb2^fl/fl or Mx1-Cre mice. Representative colon sections stained with alcian blue and nuclear fast red and tumor loads are shown. Scale bar = 200 μm. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM (sample size, Ctrl = 9 and cKO = 7 for D; Ctrl = 4 and cKO = 4 for E; Ctrl = 25 and cKO = 20 for F–H; Ctrl = 10 and cKO = 8 for I–K; and Mx1-Cre = 5 and Mx1-Cre;Asb2^fl/fl = 4 for L). *, P < 0.05; **, P < 0.01.

Emergency myelopoiesis in the bone marrow and the spleen of tumor-bearing mice. Control (ctrl) and cKO mice were left untreated (−) or treated with AOM and DSS (A/D). Cells were isolated from peripheral blood (A), bone marrow (B), and colon (C) 43 (d43) or 69 days (d69) after AOM injection and analyzed by flow cytometry. A, Peripheral blood parameters including numbers of lymphocytes, monocytes, granulocytes, platelets, and red blood cells. B, Cellularity of the bone marrow and numbers of the indicated bone marrow populations (see Materials and Methods or see below for definitions). C, Cellularity of the spleen and numbers of the indicated spleen populations (see Materials and Methods or see below for definitions). The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. In B and C, monocytes/macrophages are CD45⁺F4/80⁺CD11b⁺, dendritic cells are CD45⁺CD11c⁺, B cells are CD45⁺CD19⁺CD45R⁺, and T cells are CD45⁺CD3⁺. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Decreased Th2/Treg and increased Th1/Th17/CTL responses in cKO mice trigger antitumor immunity to inhibit colitis-associated tumorigenesis. Control (ctrl) and cKO mice were treated with AOM and DSS and the cells isolated from colons 43 (d43) or 69 days (d69) after AOM injection were analyzed by flow cytometry and qRT-PCR. A, Data represents the numbers of CD45⁺CD4⁺ cells. B, Data represents the numbers of CD45⁺CD4⁺IL4⁺ and CD45⁺CD4⁺IL13⁺ cells. C, Data represents the numbers of IL10⁺ Treg cells (CD45⁺CD4⁺FoxP3⁺IL10⁺). D, Representative flow cytometry plots for CD4 versus IFNγ within a CD45⁺CD4⁺ gate and numbers of CD45⁺CD4⁺IFNγ⁺ cells. Values inside the plots represent the percentages from the CD45⁺CD4⁺ gate. E, Data represents the numbers of Th17 cells (CD45⁺CD4⁺IL17A⁺). F, Data represents the numbers of CD45⁺CD8⁺ cells. G, Representative flow cytometry plots for CD8 versus IFNγ within a CD45⁺CD8⁺ gate and numbers of CD45⁺CD8⁺IFNγ⁺ cells. Values inside the plots represent the percentages from the CD45⁺CD8⁺ gate. H, Relative expression of perforin 1 and granzyme B mRNA. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. *, P < 0.05; **, P < 0.01.

Asb2α was expressed in Th2 cells and its loss impeded IL4 expression in Th2 cells. Naïve CD4⁺ T cells from ctrl and cKO mice were polarized ex vivo toward Th1 or Th2 and analyzed by flow cytometry and qRT-PCR. A, Relative expression of Gata3 and Tbet/Tbx21 mRNA. B, Data represent the percentages of Tbet⁺ and IFNγ⁺ in CD4⁺ cells. C, Data represent the percentages of Gata3⁺ in CD4⁺ cells. D, Data show representative flow cytometry plots for CD4 versus IL4, the percentages of IL4⁺ in CD4⁺ cells, and the IL4 geoMFI in CD4⁺ cells. Values inside the plots represent the percentages of IL4⁺ in CD4⁺cells. Naïve CD4⁺ T cells from control (ctrl) and cKO mice were polarized ex vivo toward Treg or Th17 and analyzed by flow cytometry. E, Data represents the percentage of FoxP3⁺ or IL10⁺ in CD4⁺ cells. F, Data represents the percentage of RORγt⁺ or IL17A⁺ in CD4⁺ cells. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. *, P < 0.05.