Wnt/β-catenin pathway activation is associated with glucocorticoid secretion in adrenocortical carcinoma, but not directly with immune cell infiltration

Figures

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  Figure 1. Overview of the inactive and active Wnt/β-catenin pathway in ACC. In the inactive state, β-catenin is bound to its destruction complex consisting of GSK3β, APC, Axin and CKIα. This complex leads to the ubiquitinylation and phosphorylation of β-catenin and its subsequent destruction. Is the pathway activated, the Wnt ligand binds to the receptor frizzled (FZD) which leads to an activation of dishevelled (DVL) and inhibits the destruction complex. As a consequence, β-catenin is protected from degradation, accumulates in the cytoplasm and translocates to the nucleus. In the nucleus, β-catenin binds to the N terminus of TCF/LEF-1 transcription factor and recruits co-factors to activate the transcription of Wnt target genes like LRP receptors through inhibition of E3 ubiquitin ligases ZNRF3. ZNRF3/RNF43 translocate to the plasma membrane and lead to an ubiquitinylation of FZD through DVL and degradation of FZD. This step leads to an inactivation of the Wnt/β-catenin pathway. Tumour cells need to overcome this strong negative feedback loop by mutating ZNRF3, appearing in 21% of ACC patients or mutating β-catenin (CTNNB1) appearing in 16% of patients (21, 22). In addition to Wnt…

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  Figure 2. LEF-1 and CTNNB1 protein expression by immunohistochemistry in a healthy adrenal gland. Consecutive tissue slices were stained for LEF-1 (A–C) or CTNNB1 (D–F). While LEF1 expression is clearly expressed predominantly in the subcapsular zone and zona glomerulosa with exclusive nuclear expression, CTNNB1 is more diffusely expressed with both nuclear and cytoplasmic staining.

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  Figure 3. LEF-1 and CTNNB1 immunohistochemistry staining of full FFPE sections. Representative specimens of full ACC tissue sections stained for LEF-1 (A–I) or CTNNB1 (J–R) are shown with low (A–C, J–L) intermediate (D–F, M–O) and high (G–I, P–R) expression of LEF-1 and CTNNB1, respectively. Semi-quantitative assessment of expression was performed with H-score. (S) Correlation of semi-quantitatively assessed protein expression of nuclear LEF-1 and overall CTNNB1 (p value=0.0046).

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  Figure 4. Correlation of LEF-1 and immune infiltration levels in the tumour. Protein expression levels of LEF-1 as well as CD3+ (A), CD4+ (B), CD8+ (C), and FoxP3+ (D) levels (low: white; intermediate: green; high: purple) were divided into tertiles according to expression level and visualised in a stacked bar graph, respectively. Pearson correlation was used to show the linear relationship between the expression of LEF-1 (H-scores) and the immune cell infiltration (HPF), respectively. Testing for significance, fisher´s exact test was used.

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  Figure 5. Correlation of CTNNB1 and immune infiltration levels in the tumour. Protein expression levels of CTNNB1 as well as CD3+ (A), CD4+ (B), CD8+ (C), and FoxP3+ (D) levels (low: white; intermediate: green; high: purple) were divided into tertiles according to expression level and visualised in a stacked bar graph, respectively. Pearson correlation was used to show the linear relationship between the expression of CTNNB1 (H-scores) and the immune cell infiltration (HPF), respectively. Testing for significance, fisher´s exact test was used.

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  Figure 6. Correlation of LEF-1 (A), CTNNB1 (B), and GR (C) with the hormone status, respectively. Hormone status of tumour (inactive, glucocorticoid excess (GC) or other (GCA, A, GCAO) were correlated and visualised as a stacked bar graph correlating the hormone status with the protein expression levels of LEF-1 (A), CTNNB1 (B) or GR (C), respectively. H-score differences of LEF-1 (D), CTNNB1 (E) and GR (F) between patients with GC versus hormonal inactive tumours combined with androgen excess tumours are shown in (D–F). Statistical analysis was performed with fisher´s exact test.

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  Figure 7. Overall survival in patients with adrenocortical carcinoma according to LEF-1, CTNNB1 and GR determined by RNA analysis. Kaplan-Meier overall survival of all ACC patients with primary tumour samples (n=58) influenced by LEF-1 (A), CTNNB1 (B) and GR (C) expression.

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  Figure 8. Expressional differences between LEF-1-high and LEF-1-low tumours at RNA level. Expression of 354 immune-related genes were measured and quantified by Nanostring nCounter® from FFPE tissue to analyse expressional differences between LEF-1-high and -low tumours. Significant genes (p<0.05-0.01) are marked in green and highly significant genes (p<0.01) are marked in purple. Analysis performed by Nanostring nSolver® and Prism. Adjusted p value was calculated by Benjamini-Yekutieli.