DNA hypermethylation driven by DNMT1 and DNMT3A favors tumor immune escape contributing to the aggressiveness of adrenocortical carcinoma

Abstract

Background: Adrenocortical carcinoma is rare and aggressive endocrine cancer of the adrenal gland. Within adrenocortical carcinoma, a recently described subtype characterized by a CpG island methylator phenotype (CIMP) has been associated with an especially poor prognosis. However, the drivers of CIMP remain unknown. Furthermore, the functional relation between CIMP and poor clinical outcomes of patients with adrenocortical carcinoma stays elusive.

Results: Here, we show that CIMP in adrenocortical carcinoma is linked to the increased expression of DNA methyltransferases DNMT1 and DNMT3A driven by a gain of gene copy number and cell hyperproliferation. Importantly, we demonstrate that CIMP contributes to tumor aggressiveness by favoring tumor immune escape. This effect could be at least partially reversed by treatment with the demethylating agent 5-azacytidine.

Conclusions: In sum, our findings suggest that co-treatment with demethylating agents might enhance the efficacy of immunotherapy and could represent a novel therapeutic approach for patients with high CIMP adrenocortical carcinoma.

Keywords: 5-azacytidine; Adrenocortical carcinoma; CIMP; DNA methylation; DNA methyltransferases; DNMTs; Demethylating agents; Immune escape.

Fig. 1

DNMT1 and DNMT3A expression levels are augmented in hCIMP ACC and associated with increased copy numbers. a Expression levels of DNMT1, DNMT3A, DNMT3B, TET1, TET2, and TET3 in TCGA patients exhibiting lCIMP (n = 32), iCIMP (n = 26), and hCIMP (n = 21) (two-sided t test, ***p < 0.005). b Kaplan–Meier estimates of overall survival for ACC patients, as a function of DNMT1 or DNMT3A expression (divided as high, medium and low) for the TCGA dataset. c Pattern of copy number alterations in hCIMP and lCIMP ACC in chromosomes containing DNMT1, DNMT3A, and DNMT3B genes. d Expression levels of DNMT1 and DNMT3A in TCGA patients according to copy number status of the genes

Fig. 2

Increased cell proliferation in hCIMP ACC tumors is a likely second driver of augmented DNMT1 and DNMT3A expression, along with copy number aberrations in the DNMT genes. a and b Correlation between DNMT1 (a) or DNMT3A (b) expression and cell proliferation; hCIMP samples (n = 21, orange), iCIMP (n = 26, yellow), and lCIMP (n = 32, blue). c Modeling of inter-dependencies between genomic copy number status of DNMT genes, cell proliferation and gene expression using partial correlations for the TCGA data set. R_partial stands for the Pearson partial correlation coefficient calculated when controlling for the third factor. d and e Effect of a 48 h treatment with the proliferation inhibitor AZD-5438 (Azd) at 1 or 5 µM on the expression of DNMT1, DNMT3A, DNMT3B, and the proliferation marker MKI67 in the hCIMP and lCIMP ACC cell lines H295R (d) and MUC-1 (e). DMSO was used as the vehicle. Data represent the means from at least four independent experiments ± SEM (Wilcoxon test, *p < 0.05, **p < 0.01)

Fig. 3

hCIMP ACC tumors are characterized by lower abundance of tumor-infiltrating immune cells. a Pathway enrichment analysis on genes repressed by DNA methylation in hCIMP tumors from TCGA dataset. The complete KEGG pathways database was used for the analysis and only the significantly enriched pathways are depicted on the figure. b Relative abundance of tumor-infiltrating immune and non-immune stromal cell populations computed using MCP-counter [19] in lCIMP (n = 32), iCIMP (n = 26), and hCIMP (n = 21) samples from TCGA dataset. Comparison of the relative abundances of each population using the Kruskal–Wallis test followed by Dunn's test with Benjamini–Hochberg corrections. ***p < 0.001. c Left: quantification of the percentage of tumor-infiltrating CD3 + T cells. Data represent the means from at least six independent experiments ± SEM. p value of the Mann–Whitney U Test: p = 0.12. Right: representative screenshot of a highly infiltrated lCIMP sample and a lowly infiltrated hCIMP sample

Fig. 4

Demethylating agent 5-azacytidine (AZA) can partially revert DNA hypermethylation of CpG islands in the hCIMP ACC cell line H295R restoring activity of immune response genes. a Overlap between genes significantly up- or down-regulated upon AZA treatment (FDR < 0.05) in H295R (5242 genes), MUC-1 (125 genes), and HUVEC (254 genes) cell lines. b Unsupervised hierarchical clustering of 3 cell lines (H295R, MUC-1 and HUVEC) treated or not with AZA 5 μM was based on their promoter CpG islands methylation profile. The clustering was obtained using the CpG with the most variable methylation level (s.d. > 0.4). c Global percentage of methylated CpG among the CpG covered by RRBS reads. AZA induces a decrease in CpG methylation in all three cell lines, but this effect is less pronounced in the MUC-1 cell line that has the lowest basal level of DNA methylation. d Pathway enrichment analysis on genes induced by AZA in H295R through promoter demethylation. Pathways related to the immune response are highlighted in red. e Examples of differentially methylated region (DMRs) upon AZA treatment in H295R located in promoters of two immune response genes

Fig. 5

Suggested model of the establishment of the high CIMP in ACC. Augmented expression of DNA methyltransferases DNMT1 and DNMT3A, driven by genomic aberrations and hyperproliferation, results in an increase of promoter DNA methylation of hundreds of genes; consequently, methylation-dependent silencing of genes related to immune response leads to lower infiltration of the tumor by immune cells