Modulation of indoleamine 2, 3-dioxygenase 1 expression by activated Tcells in breast cancer is controlled by epigenetic mechanisms

Abstract

Tumor infiltrating lymphocytes (TILs) secrete cytokines that modulate immune responses at the tumor microenvironment. Tumor suppressor activity of interferon gamma (IFNy) cytokine also activates expression of immune suppressive factors such as IDO and PD-L1 in tumor cells. However, there is still much to learn about how tumor cells counter the immune cells at the gene expression level. In this study, RNA-seq analysis of breast cancer cells after in-vitro co-culture with anti-CD3/CD28 activated human T -cells revealed that the IFNy induced immune response gene signature is common to both triple negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor positive (ER+) MCF7 cells. However, IDOl expression was differentially upregulated with significantly higher expression in MDA-MB-231 compared to MCF7 cells. Analysis of the TCGA breast invasive carcinoma dataset revealed subtype specific mRNA expression and IDOl promoter DNA methylation. We observed that IDOl mRNA expression and promoter methylation followed inverse correlation. TNBC/Basal subtype was hypomethylated at the IDOl promoter with higher mRNA expression compared to the ER+ subtype that was hypermethylated with relatively lower IDOl mR.NA expression. The IDOl promoter methylation was confirmed by pyrosequencing analysis of a panel of breast cancer cell lines and patient tumors. IFNy treatment of MDA-MB-231 and MCF7 breast cancer cells revealed no difference in terms of upstream signaling and IDOl mRNA stability. Treatment with demethylating agent, 5-azadeoxycytidine, synergistically up-regulated IDOl mRNA expression in ER+ MCF7 cells highlighting that CpG methylation controls !DO 1 gene expression. We also found a positive correlation between !DO I and CDBA expression and better relapse free survival in TNBC/basal subtype patients suggesting that !DO 1 expression is driven by intrinsic immune surveillance of TILs. These findings provide evidence that !DO 1 promoter methylation regulates anti-immune responses by tumor cells towards TILs and it could be used as a predictive biomarker for IDO inhibitor-based immunotherapy of breast cancer.