NLRC5/MHC class I transactivator is a target for immune evasion in cancer Sayuri Yoshihamaa,b, Jason Roszikc, Isaac Downsa, Torsten B. Meissnerd, Saptha Vijayana, Bjoern Chapuye, Tabasum Sidiqa, Margaret A. Shippe, Gregory A. Lizeec,f, and Koichi S. Kobayashia,1 aDepartment of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, College Station, TX 77843; bDepartment of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; cDepartment of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77054; dDepartment of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115; eDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; and fDepartment of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX 77054 Edited by Jeremy Boss, Emory University School of Medicine, Atlanta, GA, and accepted by the Editorial Board April 11, 2016 (received for review February 5, 2016) Cancer cells develop under immune surveillance, thus necessitating but also activates other critical genes involved in the MHC class I immune escape for successful growth. Loss of MHC class I expression antigen-presentation pathway, including the immunoproteasome provides a key immune evasion strategy in many cancers, although component LMP2 (PSMB9), peptide transporter TAP1,andB2M the molecular mechanisms remain elusive. MHC class I transactivator (14, 17), thus regulating most of the key components in the MHC (CITA), known as “NLRC5” [NOD-like receptor (NLR) family, caspase class I antigen-presentation machinery. Nlrc5-deficient mice ex- recruitment (CARD) domain containing 5], has recently been identified hibit impaired constitutive and inducible expression of MHC as a critical transcriptional coactivator of MHC class I gene expression. class I genes in vivo (18–22). In addition, Nlrc5-deficient cells + Here we show that the MHC class I transactivation pathway mediated display an impaired ability to elicit CD8 T-cell activation, as by CITA/NLRC5 constitutes a target for cancer immune evasion. In all evidenced by impaired IFN-γ production and diminished cyto- the 21 tumor types we examined, NLRC5 expression was highly cor- lytic activity (18, 19, 21). related with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation Results pathway, including LMP2/LMP7, TAP1,andβ2-microglobulin. Epige- Expression of NLRC5 and MHC Class I Genes Is Correlated in Human netic and genetic alterations in cancers, including promoter methyl- Cancers. Because of the prominent role of NLRC5 in orches- ation, copy number loss, and somatic mutations, were most prevalent trating the expression of MHC class I and class I-related genes, we in NLRC5 among all MHC class I-related genes and were associated examined gene-expression profiles of biopsy samples from the co- with the impaired expression of components of the MHC class I path- hort of 7,747 solid cancer patients in The Cancer Genome Atlas way. Strikingly, NLRC5 expression was significantly associated with (TCGA) database. The expression of HLA-B was highly correlated + NLRC5 = the activation of CD8 cytotoxic T cells and patient survival in multiple with the level of expression in the entire cohort (rs 0.753) A cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker (Fig. 1 ). Correlation analysis for gene expression among 14 cancer HLA-B NLRC5 and potential therapeutic target of cancers. types demonstrated that and expression showed high positive correlation (rs > 0.70) in nine cancer types and in- > MHC class I | CITA | cancer | immune evasion | NLRC5 termediate positive correlation (rs 0.50) in five cancer types (Fig. 1 B and C), with the highest correlation observed in melanoma. In addition to HLA-B, the expression of HLA-A, HLA-C, B2M, LMP2, uring cancer progression, neoplastic cells accumulate nu- LMP7 (PSMB8), and TAP1 was also highly correlated with NLRC5 Dmerous mutations that constitute potentially immunogenic expression in melanoma and other cancers (Fig. 1D and Fig. S1A). neo-epitopes. Thus, most tumors concurrently need to use mechanisms that enable escape from immune surveillance for Significance successful growth and progression (1). It has been demonstrated that cancer cells use multiple strategies of immune evasion, in- + Tumor antigen presentation to CD8 T cells by MHC class I cluding increased resistance to cytotoxic T-cell killing, induction of molecules is crucial for immune responses against cancers, anergy in activated T cells, elimination of effector T cells, recruit- whereas the loss of MHC class I is a common immune evasion ment of regulatory immune cell subsets, and reduced recognition of strategy used by cancers. However, the molecular mechanisms tumor-associated antigens by effector T cells (2). Impaired MHC leading to MHC class I deficiency have remained poorly de- class I-mediated antigen presentation is a major immune evasion fined. We demonstrate here that MHC class I transactivator mechanism in cancer (3, 4), with MHC class I loss reported in (CITA)/NOD-like receptor (NLR) family, caspase recruitment cervical cancer (92%) (5), penile cancer (80%) (6), breast cancer (CARD) domain containing 5 (NLRC5) is a major target for (71%) (7), nonsmall cell lung cancer (64%) (8), and esophageal cancer immune evasion. Reduced expression of MHC class I squamous cell carcinoma (67%) (9), among others. Although a and related genes in cancer is frequently associated with genetic number of mechanisms have been described for HLA loss, includ- and epigenetic changes in NLRC5. The reduced NLRC5 expression ing the loss of heterozygosity, HLA gene methylation, nonsense/ + TAP1/2 β B2M islinkedtoimpairedCD8 T-cell activation and poor patient missense mutations, and loss of or 2-microglobulin ( ), prognosis. These data indicate that CITA/NLRC5 is a novel prog- the dominant underlying molecular mechanism seems to reside at nostic marker and potential therapeutic target of cancers. the transcriptional level (10). Transcriptional regulation of MHC class I genes remained largely undefined until the recent discovery Author contributions: S.Y., T.B.M., S.V., B.C., M.A.S., and K.S.K. designed research; S.Y., I.D., of CITA (MHC class I transactivator), known as NLRC5 [NOD- T.B.M., S.V., B.C., and T.S. performed research; S.Y., J.R., I.D., T.B.M., S.V., B.C., M.A.S., G.A.L., and like receptor (NLR) family, caspase recruitment (CARD) domain K.S.K. analyzed data; and S.Y., G.A.L., and K.S.K. wrote the paper. containing 5] (11, 12). NLRC5 is an IFN-γ–inducible nuclear pro- The authors declare no conflict of interest. tein (13–15) that specifically associates with and activates promoters This article is a PNAS Direct Submission. J.B. is a guest editor invited by the Editorial Board. of MHC class I genes by generating a CITA enhanceosome com- 1To whom correspondence should be addressed. Email: [email protected]. plex with other transcription factors (14, 16, 17). A striking feature This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. INFLAMMATION IMMUNOLOGY AND of CITA/NLRC5 is that it does not solely induce MHC class I genes 1073/pnas.1602069113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1602069113 PNAS | May 24, 2016 | vol. 113 | no. 21 | 5999–6004 Downloaded by guest on October 2, 2021 Preferential Methylation of NLRC5 in Cancer Is Associated with AB5 1 r ) =0.753 (p<0.001) r Impaired Cytotoxic T-Lymphocyte Activity. 0.8 Epigenetic changes in 4 0.6 0.4 cancer cells represent an important mechanism to alter gene 3 0.2 expression in favor of cancer growth and immune evasion (26). 0 (log10, TPM) 2 HLA-B expression n=7747 Abnormal methylation of CpG islands in promoter regions can 1 -2 -1 0 1 2 3 transcriptionally suppress genes that are unfavorable for cancer NLRC5 expression expression of NLRC5 x HLA-B (log10, TPM) growth (27). Treatment of various cancer cell lines with a DNA- C Melanoma Bladder Thyroid Breast Uterine Brain 5 methylation inhibitor, 5-azacitidine (5-Aza), resulted in the up- 5 r =0.849 (p<0.001) 5 r =0.848 (p<0.001) 5 r =0.839 (p<0.001) 5 r =0.800 (p<0.001) r =0.763 (p<0.001) 5 r =0.745 (p<0.001) 4 NLRC5 HLA-B 4 4 regulation of and expression, suggesting that 4 4 4 3 3 3 methylation of the NLRC5 promoter might play a role in the loss 3 3 3 2 2 2 (log10, TPM) of MHC class I expression in cancer (Fig. 2A). Therefore, the HLA-B expression 1 1 2 2 1 2 -1 0 1 2 -10123-1 0 1 2 -1 0 1 2 3 -10123 -1012 NLRC5 NLRC5 expression (log10, TPM) level of DNA methylation at a CpG island in the pro- Melanoma (n=471) moter in various cancer types was quantified using a methylation- D HLA-A HLA-C B2M LMP2 LMP7 TAP1 r =0.693 (p<0.001) r =0.805 (p<0.001) r =0.797 (p<0.001) r =0.885 (p<0.001) r =0.825 (p<0.001) 4 r =0.857 (p<0.001) 5 5 5 3 3 specific probe (Fig. 2B). Methylation of the NLRC5 promoter 4 2 3 4 4 2 was observed at higher frequency in multiple cancers than in the 3 1 2 3 3 1 corresponding normal tissues (excluding prostate, thyroid, and 2 0 1 ( log10, TPM) ( log10, Gene expression kidney, where high methylation was observed even in normal 2 1 2 -1 0 0 -1012-1012-1012-1012-1012-1012 NLRC5 expression (log10, TPM) tissues) (Fig. 2C and Fig. S2A). Furthermore, analysis of biopsy normal tumor samples from 6,523 solid cancer patients revealed that methyl- E 3 r =0.638 (p<0.001) F 3 r =0.580 (p<0.001) G 2 2 n=106 NLRC5 Prostate n=498 ** ation of the promoter was negatively correlated with 1 1 n=320 NLRC5 = − D 0 0 Lung n=1019 ** expression (rs 0.585) (Fig.