Cell division cycle 20 promotes tumor progression and predicts poor clinical outcome in childhood and adult adrenocortical carcinoma

Figures

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  Fig. 1. Workflow of this study.

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  Fig. 2. Differentially expressed genes (DEGs) between ACC and normal tissues. (A-B) Batch correction of expression data from GSE10927 and GSE19750 to generate the GSE10927-19750 cohort. (C) Heatmap of DEGs between ACC and normal tissues. (D) Enrichment analysis results of upregulated genes using Gene Ontology (GO). (E) Enrichment analysis results of downregulated genes using GO.

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  Fig. 3. Identification of core gene CDC20 in ACC. (A) By integrating DEGs, genes with AUC values > 0.7, HR > 1, and P-values < 0.05 in the GSE10927-19750 dataset, and genes with AUC values > 0.7, HR > 1, and P-values < 0.05 in TCGA datasets for overall survival (OS), progression-free interval (PFI), we identified 8 core genes. (B) Univariate Cox regression analysis of the 8 core genes across all cohorts. (C) AUC values of the 8 core genes across all cohorts. (D) Expression levels of CDC20 between ACC and normal tissues in GSE10927, GSE19750, and GSE10927-19750 cohorts. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Fig. 4. Validation of CDC20 as an independent prognostic factor for ACC. (A–H) Kaplan-Meier survival analysis of CDC20 across all cohorts. (I) Univariate Cox regression analysis of CDC20, age, gender, and tumor stage across all cohorts. (J) Multivariate Cox regression analysis of CDC20 across all cohorts.

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  Fig. 5. The gene set enrichment analysis (GSEA) results for the TCGA and GSE76019 datasets. (A) GSEA results of CDC20-related GO terms in TCGA dataset. (B) GSEA results of CDC20-related GO terms in GSE76019 dataset.

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  Fig. 6. Immunohistochemistry (IHC) staining results of CDC20 in ACC and adrenocortical adenoma. (A–C) Microscopic images (10×, 20×, 40×) of CDC20 IHC staining in adrenocortical adenoma. (D–F) Microscopic images (10×, 20×, 40×) of CDC20 IHC staining in ACC. (G) IHC scoring comparison between ACC (n = 6) and adrenocortical adenoma (n = 5) tissues.

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  Fig. 7. Cell experiments. (A) Western blotting to assess the impact of 3 siRNAs on CDC20 expression in ACC cells. (B–C) Quantitative analysis of CDC20 expression after CDC20 gene knockdown in NCI-H295R (B) and SW-13 (C) cell lines. (D) Colony formation assay results in NCI-H295R and SW-13 cell lines. (E–F) Quantification of proliferating cells in NCI-H295R (E) and SW-13 (F) cell lines. (G) Transwell assay results in NCI-H295R and SW-13 cell lines. (H–I) Quantification of migrating cells in NCI-H295R (H) and SW-13 (I) cell lines. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistical significance.

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  Fig. 8. Immune infiltration and immunotherapy. (A–D) Correlation analysis between CDC20 expression and CD8+ T cell infiltration levels using TIMER and Xcell algorithms in TCGA and GSE76019 datasets. (E–F) Assessment of tumor immune dysfunction and exclusion (TIDE) score correlation with CDC20 expression levels in TCGA and GSE76019 datasets. (G–H) Subclass mapping (Submap) algorithm evaluation of expression similarity between two CDC20 expression subgroups and different immunotherapy response patients in TCGA and GSE76019 datasets. (I–L) Kaplan-Meier survival analysis of high and low CDC20 expression subgroups among patients receiving anti-PD-1 therapy in four cohorts.

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  Fig. 9. Analysis of CDC20-related mutation and copy number variation (CNV). (A) Landscape of top 20 frequent mutated genes. (B) Differential expression of CDC20 between wild-type and mutated groups among the top 20 frequent mutated genes. (C) Landscape of top 10 chromosomal amplifications and top 10 chromosomal deletions. (D) Differential expression of CDC20 between variant and non-variant groups among the 10 chromosomal amplifications and top 10 chromosomal deletions. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no statistical significance.