Bioinformatic-experimental screening uncovers multiple targets for increase of MHC-I expression through activating the interferon response in breast cancer

Expression of major histocompatibility complex I (MHC-I) on tumor cells is extremely important for antitumor immune response for its essential role in activating various immune cells including tumor-specific CD8+ T cells. Cancers of low MHC-I expression are commonly of little immune cell infiltration and exhibit poor prognosis in clinic. In this study, we conducted a bioinformatic-experimental screening to identify potential gene targets to enhance MHC-I expression in breast cancer. Through combination of MHC-I scoring, gene expression correlation analysis, and survival prognostication, we identify 243 genes negatively correlated with MHC-I expression in breast cancer, of which 144 genes were further identified as negative correlation factors for tumor-infiltrated lymphocytes. Finally, we verified partially according to KEGG or gene-dependency analysis in breast cancer MCF7 cells, and figured out multiple genes including PIP5K1A, NCKAP1, CYFIP1, DIS3, TBP and EXOC1 as potent gene targets for increase of MHC-I expression in breast cancer. Mechanistically, knockout each of these genes activates the intrinsic interferon response in breast cancer cells, which not only promoted MHC-I expression but also caused immunogenic death of breast cancer cells. Collectively, we identified multiple gene targets for increase of MHC-I expression in breast cancer. Overall design: The HEK-293T cells were purchased from the American Type Culture Collection (ATCC). The MCF7 cells were kindly provided by Dr. Cheguo Cai (Wuhan University). These cell lines were cultured in DMEM (GIBCO) medium supplemented with 10% FBS (Cellmax) and 1% penicillin-streptomycin (HyClone) under an atmosphere of 5% CO2 at 37 °C. All cells were negative for mycoplasma. CRISPR-Cas9 gRNA plasmids for the indicated 40 candidate genes were constructed by standard molecular biology technique. HEK-293T cells were seeded into plates for 20 hours, and then cells were transfected by standard calcium phosphate precipitation method at a density of 40%–50%. Gene knockout in MCF7 cells was achieved using a CRISPR-Cas9 system. Briefly, double-stranded oligonucleotides corresponding to the target sequences were cloned into the lenti-CRISPR-V2 vector, and then these lentiviral plasmids were co-transfected with two packaging plasmids into HEK-293T cells. Two days after transfection, the lentiviral supernatants were harvested, ultra-filtrated (0.45 mm filter, Millipore), and then infected MCF-7 cells in the presence of polybrene (8 mg/mL). The infected cells were selected with puromycin (2 mg/ml) for at least 6 days to establish stable cell lines.