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. Author manuscript; available in PMC: 2024 Jan 5.
Published in final edited form as: Cancer Res. 2023 Jul 5;83(13):2123–2141. doi: 10.1158/0008-5472.CAN-22-2712

Figure 1: Differentiated, Wnt-active ACC subtype CIMP-high possesses aberrant PRC2 target hypermethylation with high EZH2 coupled to H3K27me3.

Figure 1:

A. Corticocapsular unit of adrenocortical homeostasis depicting peripheral mesenchymal cells (capsule) and human cortical populations zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR), that produce mineralocorticoids, glucocorticoids, and androgens, respectively. Differentiation in the cortex is centripetal, and zG, zF, and zR cells are supplied by peripheral capsular and cortical progenitors (arrow). The entire cortex is SF1 positive. ZG cells possess active Wnt/β-catenin signaling and lower zF/zR cells possess active ACTH signaling through protein kinase A (PKA). Wnt/β-catenin signaling fades in the upper zF, and these cells respond to ACTH/PKA with proliferation (Ki67). Mice do not have a zR.

B. GSVA was used on ACC-TCGA RNA-seq to calculate the expression score of genes that define zF differentiation or are regulated in a cell-cycle- or Wnt-dependent manner across ACC-TCGA. Score validation detailed in Methods and Supp Fig 1. Radar plot depicts average score for each ACC CIMP class, with values mapped onto an arbitrary scale along each axis. Heatmap below depicts p-value for comparisons.

C. 10 most significant gene sets from curated GSEA on genes with promoters targeted for hypermethylation in CIMP-high ACC.

D. Violin plot of PRC2 target CpGi methylation measured by Illumina 850k or 450k methylation array in fetal (n=3) or adult adrenal (zF, zR; n=4 each) and ACC from ACC-TCGA (n=79). Lines at median and quartiles.

E. EZH2 expression in ACC-TCGA by RNA-seq (n=78), left, and independent cohort by qPCR (n=102, FMUSP+UM classified by CIMP in (8)), right. CPM=counts per million. ACA=adrenocortical adenomas (benign adrenocortical tumors). Line at mean with 95% confidence interval (CI).

F-G. Representative images and scoring of tissue microarray (TMA) of human adult ACA (n=74) and primary ACC (n=74). TMA stained for EZH2 and H3K27me3 by immunohistochemistry (IHC). EZH2 quantified on 0–4 scale (%positive nuclei) by 2 independent observers. H3K27me3 quantified by MATLAB (20). EZH2 low=ACC with below median EZH2 expression (≤25% nuclei EZH2+), EZH2 high=ACC with above median EZH2 expression (>25% nuclei EZH2+). EZH2 mRNA/protein are correlated (Spearman r=0.5117, p<0.01, not shown). F, bar=100 μm. G, line at mean with 95% CI.

H. Disease-free (after R0/RX resection in patients without metastatic disease at diagnosis) and overall survival (all patients) stratified by ACC EZH2 expression.