High expression of DNMT1 in breast invasive carcinoma associated with tumor immune microenvironment and poor prognosis

Abstract (1) Background: Interleukin-6 (IL-6) is a multifunctional cytokine that was found to cause epigenetic changes by regulating DNA methyltransferase 1 (DNMT1). IL-6 has al-so been found to promote tumor progression and chemoresistance in numerous types of cancers. However, it remains unknown how IL-6 and DNMT1 may impact prognosis in different breast cancer types. (2) Methods: Patient survival rate in breast cancer patients and DNMT1 expression pattern was examined in TCGA datasets. Protein expression pattern of IL-6 and DNMT1 were investigated in the tissue microarray in our cohort (n=285, Show Chwan Memorial Hospital, Changhua) by immunohistochemistry (IHC). IL-6 and DNMT1 expressions were examined in breast cancer cell-lines. Cell invasion activity was compared between high and low IL-6/DNMT expressing breast cancer cell lines, and between cells treated with/without IL-6 antibody. (3) Results: DNMT1 mRNA level was significantly higher in the breast tumor tissues (p < 0.001). Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer patients with higher DNMT1 mRNA level showed a poorer overall survival compared to other breast cancer subtypes (P = 0.043). A positive correlation between IL-6 and DNMT1 protein expression levels was found in breast cancer tissue array. Concurrent overexpression of IL-6 and DNMT1 was associated with poor survival rate in our cohort examined by Kaplan-Meier survival curves. Cox regression further indicated that high expression of IL-6 and DNMT1 is an independent prognostic factor in HER2-positive patients. In addition, in breast cancer cell lines, HER2-positive MDA-MB-453 that had high IL-6 and DNMT1 expression exhibited higher invasiveness compared to that of the HER2-positive cell line SKBR3 with low IL-6 and DNMT1 expression. Finally, IL-6 recombinant protein (ng/ml) significantly promoted the invasion ability of low IL-6/low DNMT1 SKBR3 whereas IL-6 antibody (10 μg/ml) treatment significantly suppressed the invasion activity of high IL-6/high DNMT MDA-MB-453. Single cell sequencing data showed that DNMT1 was mainly expressed in malignant cells and innate and adaptive immune cells including monocytes, macrophages, fibroblasts, epithelial cells, CD4(+) T cells, and CD8(+) T cells. Besides, there existed a positive correlation between DNMT1 and the immune cells including CD8(+) T cells, CD4(+) T cells, and macrophages. (4) Conclusions: IL-6-DNMT1 axis could be responsible for HER2-positive breast cancer progression in tumor immune microenvironment. Our study suggests that IL-6 inhibition in combination with anti-HER2 therapy might be an effective approach in particular for HER2-positive patients with a potential to become a standard therapeutic strategy.