PRC2 Epigenetically Silences Th1-Type Chemokines to Suppress Effector T-Cell Trafficking in Colon Cancer

Abstract

Infiltration of tumors with effector T cells is positively associated with therapeutic efficacy and patient survival. However, the mechanisms underlying effector T-cell trafficking to the tumor microenvironment remain poorly understood in patients with colon cancer. The polycomb repressive complex 2 (PRC2) is involved in cancer progression, but the regulation of tumor immunity by epigenetic mechanisms has yet to be investigated. In this study, we examined the relationship between the repressive PRC2 machinery and effector T-cell trafficking. We found that PRC2 components and demethylase JMJD3-mediated histone H3 lysine 27 trimethylation (H3K27me3) repress the expression and subsequent production of Th1-type chemokines CXCL9 and CXCL10, mediators of effector T-cell trafficking. Moreover, the expression levels of PRC2 components, including EZH2, SUZ12, and EED, were inversely associated with those of CD4, CD8, and Th1-type chemokines in human colon cancer tissue, and this expression pattern was significantly associated with patient survival. Collectively, our findings reveal that PRC2-mediated epigenetic silencing is not only a crucial oncogenic mechanism, but also a key circuit controlling tumor immunosuppression. Therefore, targeting epigenetic programs may have significant implications for improving the efficacy of current cancer immunotherapies relying on effective T-cell-mediated immunity at the tumor site.

Figure 1. Inverse correlation exists between Th1-type chemokines and the PRC2 complex in colon cancer

A: CXCL9 and CXCL10 mRNA in colitis tissue and colon cancer tissue. CXCL9 and CXCL10 mRNA levels were detected by real-time PCR in human colon cancer and colitis tissue (n=6 tissues, *p<0.05). B: CXCL9 and CXCL10 protein in colitis tissue and colon cancer tissue. CXCL9 and CXCL10 protein was detected by ELISA in 1 gram of human colon cancer and colitis tissue (n=6 tissues, *p<0.05). C, D: CXCL9 and CXCL10 mRNA in IFNγ-treated colitis tissue, colon cancer tissue and tissue adjacent to colon cancer. Single epithelial cells were prepared from colon cancer tissue (C, D), tissue adjacent to colon cancer (C) and colitis tissue (D), and treated with IFNγ (5ng/ml). CXCL9 and CXCL10 mRNA levels were detected by real-time PCR. One representative of 3 independent experiments is shown. E–I: Correlation between Th1-type chemokines and PRC2 components in patients with colon cancer. Numbers represent r-values with significance (p<0.05). Spearman analysis for correlation were conducted from Khambata-Ford Colon database from Oncomine (oncomine.org, n=80).

Figure 2. PRC2 represses Th1-type chemokine expression in colon cancer

A–F: Effects of DZNep on IFNγ-stimulated Th1-type chemokine production. CXCL9 and CXCL10 mRNA levels were detected by real-time PCR in C1 cells (A, B), DLD-1 (C, D), and SW480 (E, F) cells treated with IFNγ (0.5 or 10 ng/ml) and DZNep (0.5, 2, or 5 uM) (3 independent experiments shown, *p<0.05). G, H: Effects of shEZH2 on IFNγ-mediated chemokine production. C1 cells expressing shEZH2 or scr vector were cultured with IFNγ (0.5 ng/ml), and CXCL9 (G) and CXCL10 (H) mRNA was detected by real-time PCR (3 independent experiments shown, *p<0.05); fold change shown relative to scr with IFNγ.

Figure 3. Th1-type chemokine repression is mediated via H3K27 methylation

A–C: H3K27me3 occupancy in the promoter area of CXCL9 and CXCL10 in colon cancer cells. H3K27me3-ChIP assay was performed in DLD-1 colon cancer cells for the promoter areas of CXCL9 (A), the intergenic region of CXCL9 and CXCL10 (B), and the promoter areas of CXCL10 (C) (3 independent experiments shown, *p<0.01). kb: kilobase; TSS: transcription start site. D: ChIP positive and negative control. H3K27me3-ChIP assay was performed in DLD-1 colon cancer cells for HoxB1 and GAPDH (3 independent experiments shown). E: Effects of GSK126 on the IFNγ-mediated chemokine production. C1 cells were treated with IFNγ (5 ng/ml) and 0.5uM GSK126. CXCL10 mRNA was detected by real-time PCR (3 independent experiments shown,*p<0.05). F: Effects of JMJD3 overexpression on IFNγ -mediated chemokine production. DLD-1 cells were transfected with PKH3 (empty vector) and pCMV HA JMJD3 (JMJD3 OE) before IFNγ (0.5 ng/ml) was added and RT-PCR was performed for CXCL10 (4 independent experiments shown,*p<0.05). G,H: Effects of GSK-J4 on IFNγ-mediated chemokine production. C1 cells were treated with IFNγ (0.5 ng/ml) and 5uM or 10uM GSK-J4 and CXCL9 and CXCL10 mRNA was detected by real-time PCR (3 independent experiments shown,*p<0.05).

Figure 4. PRC2 affects CD8⁺ T cell migration toward colon cancer

A, B: Effect of shEZH2 on migration of CD4⁺ (A) and CD8⁺ (B) T cells. shEZH2 and control vector expressing DLD-1 cells were treated with 5 ng/ml IFNγ and the supernatants were collected for T cell migration assay. Activated T cells were subject to the migration toward the supernatants in the presence of anti-human CXCR3 or control. The migrated T cells were assessed by flow cytometry analysis. One representative of 3 independent experiments is shown. C: The association between CD8/CXCL9/CXCL10 transcripts and survival in patients with colorectal carcinoma (Smith colorectal, Smith colorectal 2, Jorissen Colorectal 3, and Staub colorectal). The analyses were conducted by combining multiple databases from Oncomine (oncomine.org) (n= 185, p=0.0089). D: The association between EZH2/SUZ12/EED transcripts and survival in patients with colorectal carcinoma (Smith colorectal, Smith colorectal 2, Jorissen Colorectal 3, and Staub colorectal). The analyses were conducted by combining multiple databases from Oncomine (oncomine.org) (n=105, p=0.037). E–G: Correlation between PRC2 components and CD4 and CD8A mRNA in patients with colorectal carcinoma. Numbers represent r-values with significance (p<0.05). Spearman analysis for correlation was conducted from multiple databases (Khambata-Ford Colon, Staub Colorectal, Smith Colorectal 2, TCGA, and Jorissen Colorectal 3) from Oncomine (oncomine.org). NS: not significant.