CDC25A inhibition sensitizes melanoma cells to doxorubicin and NK cell therapy

Abstract

Cell division cycle 25 (CDC25) phosphatases serve as crucial regulators of cell cycle phase transitions and essential components of the checkpoint machinery involved in DNA damage response. Emerging evidence indicates the oncogenic potential of CDC25 family members across various cancers. However, comprehensive insights into the expression pattern and function of the CDC25 family in diverse cancers remain unexplored. In our study, we investigated CDC25 family using multiple databases, including gene expression levels, molecular signatures, diagnosis value, and prognostic value in pan-cancer. Furthermore, we focused on melanoma and systematically explored CDC25A expression and its clinical correlations. As a result, the expression of CDC25 family members is significantly abnormal in most cancers, correlating with poorer prognosis. CDC25 family members are differently regulated by DNA methylation and genetic alterations across various cancers. In addition, CDC25 family plays a critical role in the malignant progression of melanoma. Functional investigation reveals that CDC25A inhibition suppresses the proliferation of melanoma cells and sensitizes melanoma cells to chemotherapy and NK cell therapy. In conclusion, our study suggests that CDC25 family may serve as a significant biomarker for diagnosis and prognosis across multiple cancers, with CDC25A as a promising therapeutic target for melanoma.

Fig. 1. Differential expression of CDC25 family genes across various cancers.

A The heatmap illustrates the average expression of genes across each cell type within each slice at the spatial transcriptomic level. B Differential expression of genes between tumor and normal tissues across a pan-cancer cohort. C Immunohistochemical images results of CDC25 family members in normal and corresponding cancer tissues, as provided by the HPA database. D Differential expression of CDC25 family members in breast cancer, liver cancer, and melanoma cell lines. E Immunofluorescence images depicting CDC25 family members in melanoma cell line. Scale bars: 10 μm. F Survival prognosis analysis of cancers with CDC25 family members expression in Kaplan–Meier and Cox proportional hazard regression analysis.

Fig. 2. Genetic alteration features of CDC25 family members in pan-cancer.

A Survival prognosis analysis of cancers based on CDC25 methylation levels using Cox proportional hazard regression analysis. B, C Frequency and types of mutation in CDC25 family members.

Fig. 3. The expression and clinical association analysis of CDC25 in SKCM tumor tissue.

A Differential expression of CDC25 family members between the GSE46517 and GSE98394 cohorts. B Differential expression of CDC25 family members between the Ki67++ and Ki67− groups in the GSE22153 cohort. C Differential expression of CDC25 family members between the primary and metastatic groups in the GSE46517 cohort. D Survival prognosis analysis of cancer with CDC25 expression in Kaplan–Meier analysis. E Comparison of mean AUC values at 1, 3, and 5 years for prognostic models constructed by different algorithms. This heatmap shows the average AUC values for each time point. F Deconvolution analysis was employed to accurately assess cell composition at each point on the 10x Visium slide. Each dot represents a microregion (spot) sequenced by spatial transcriptomics, with darker red indicating a higher cell count. G Based on deconvolution results, the cell type with the highest composition in each microregion is calculated and visualized. Each dot represents a spot from spatial transcriptome sequencing, with different colors denoting different cell types. H Wilcoxon rank-sum tests were used to evaluate the statistical significance of gene expression differences between the three groups. I Spearman correlation analysis was performed to assess the correlation between cell counts and gene expression in all spots. The red line represents a positive correlation, the green line a negative correlation, and the gray line indicates non-significance. The thickness of the line corresponds to the absolute value of the correlation coefficient. In the triangular region, the color depth and square size represent the correlation strength, with red indicating positive correlation and blue indicating negative correlation. The darker the color and the larger the square, the more significant the correlation.

Fig. 4. The promoter methylation features of CDC25 family members in SKCM.

A Spearman correlation between the methylation level of CDC25 family members and its mRNA expression in SKCM. Each scatter represents a sample. B The methylation levels at different sites of CDC25 family members promoter were sequenced. Each scatter represents a different sample. C Kaplan–Meier survival analysis for OS and DSS of SKCM patients based on CDC25 family members expression and methylation. The expression levels of CDC25 family members are standardized using a z-score with respect to the methylation degree in specific regions. A z-score being greater than 0, defined as high expression or hypermethylated groups, otherwise as low expression or hypomethylated groups.

Fig. 5. CDC25A plays a crucial role in melanoma cells.

A The synergistic effects of CDC25 family member expression were analyzed. The left panel shows scatter plots representing the z-scores of the samples, where each point represents a sample. Different colors indicate distinct subgroups and the horizontal/vertical axes correspond to the z-scores of the two genes, respectively. A z-score ≤ 0 indicates low expression, while a z-score > 0 indicates high expression. The right panel shows a Kaplan–Meier survival analysis of melanoma patients based on the expression of CDC25 family members. B The expression levels of mCherry were analyzed in A375 and MUM2B CDC25A-KD cells by flow cytometry. C Western blot assessed the protein level of CDC25A in A375 CDC25A-KD cells. D Representative images of A375 and MUM2B CDC25A-KD cells were shown. Scale bars: 100 μm.

Fig. 6. The inhibition of CDC25A suppresses the proliferation of melanoma cells.

A, B Dose-response curves for A375 and MUM2B cells exposed to NSC663284 or menadione for 12 and 24 h. C Increased phosphorylation of CDK1/2 was detected by western blot, confirming that NSC663284 effectively inhibited the function of CDC25A phosphatase in A375 and MUM2B cells. The β-actin was used as a loading control. D CCK-8 assays demonstrated that NSC663284-induced CDC25A inhibition suppressed proliferation in A375 and MUM2B cells. E, F Cell cycle assays were performed to evaluate the effects of varying concentrations of NSC663284 on A375 cells. G Expression of cell cycle molecules upon CDC25A inhibition detected by western blot. The β-actin was used as a loading control. H Expression of EMT-related molecules following CDC25A inhibition was detected by Western blot analysis. The β-actin was used as a loading control.

Fig. 7. CDC25A inhibition sensitizes melanoma cells to doxorubicin and NK cell killing.

A, B Dose-response curves of A375 and MUM2B cells exposed to Doxorubicin or paclitaxel for 24 and 48 h. C Spearman correlation analysis of the relationship between CDC25A expression levels and drug sensitivity (IC50 values). Each scatter point represents a sample. The horizontal and vertical of the scatter point denote the gene expression level and the drug IC50 value of the sample respectively. Samples were classified into high-expression (red) and low-expression (blue) groups based on the median gene expression level. D, E CCK-8 assays evaluated the susceptibility of NSC663284 and doxorubicin in A375 and MUM2B cells. The synergistic effects of CDC25A expression and activated NK cell (F) and resting NK cell (G) infiltration on survival were analyzed. The left panels are scatter diagrams of z-scores of the samples, each scatter represents a sample, and different colors represent different subgroups. Z-scores ≤ 0 represent low expression, while z-scores > 0 indicate high expression. The right panels show the survival analysis of melanoma patients in each subgroup in Kaplan–Meier analysis. H–J CDC25A inhibition sensitized A375 and MUM2B cells to NK cell killing, as shown by 7-AAD staining and CCK-8 assay.