Integrated analysis of promoter mutation, methylation and expression of AKT1 gene in Chinese breast cancer patients

Abstract

Background: As downstream mediators of PI3K /PTEN /AKT /mTORC1 pathway, the AKT isoforms play critical roles in tumorgenesis. Although the pleiotropic effects of AKT1 in breast cancer have been reported, the genetic and epigenetic characteristics of AKT1 promoter region in breast cancer remains to be identified. In this study we aimed to investigate the promoter mutation spectrum, methylation and gene expression pattern of AKT1 and their relationship with breast cancer.

Methods: By using PCR target sequence enrichment and next-generation sequencing technology, we sequenced AKT1 promoter region in pairs of breast tumor and normal tissues from 95 unselected Chinese breast cancer patients. The methylation of the promoter region and the expression profile of AKT1 in the same cohort were detected with bisulfite next-generation sequencing and qPCR, respectively.

Results: We identified 28 somatic mutations in 23 of the 95 (24.2%) breast cancer samples. And 19 of the 28 mutations were located in transcription factor (TF) binding sites. In the 23 patients with somatic mutations, no significant change of methylation or expression was found comparing with other patients. AKT1 promoter region was significantly hypo-methylated in tumor compared with matched normal tissue (P = 0.0014) in the 95 patients. The expression of AKT1 was significantly suppressed in tumor tissue (P = 0.0375). In clinicopathological factor analysis, AKT1 showed significant hypo-methylation (P = 0.0249) and suppressed expression (P = 0.0375) in HER2 negative subtype. And a trend of decrease in expression level (P = 0.0624) of AKT1 in the ER negative subtype was observed, which is significantly decreased in basal-like breast tumor (P = 0.0328).

Conclusions: Hypo-methylation and suppressed expression of AKT1 was observed to be associated with breast cancer in our cohort. The methylation and expression of AKT1 were both significantly associated with HER2 status. The promoter mutation of AKT1 did not show significant association with its methylation and expression status. These results suggested that the promoter mutation, methylation and gene expression of AKT1 may play distinct roles in tumorgenesis of breast cancer and the integrated analysis of methylation and expression of AKT1 might serve as potential biomarkers for diagnosis and classification of breast cancer.

Fig 1. The methylation and expression level of AKT1 in tumor and normal tissues from 95 breast cancer patients.

Boxplots show the average methylation (A) and expression (B) of AKT1 in breast tumor and normal tissues, and P values were calculated using the paired Wilcoxon signed rank test.

Fig 2. The methylation and expression level of AKT1 in breast cancer subtypes.

Boxplots show the average methylation (A) and expression (B) level of AKT1 in four different breast cancer subtypes, as well as methylation (C) and expression (D) in basal-like and non-basal-like breast tumor tissues. P values were calculated using the Kruskal—Wallis rank sum test for breast cancer subtypes and the Wilcoxon signed rank test for basal-like and non-basal-like breast tumor tissues.

Fig 3. The methylation and expression level of AKT1 in breast cancer patients according to ER/PR/HER2 status.

Boxplots show the average methylation (A, C, E) and expression (B, D, F) level of AKT1 between ER-positive and ER-negative subgroups, between PR-positive and PR-negative subgroups, as well as between HER2-positive and HER2-negative subgroups. P values were calculated using the Wilcoxon signed rank test.

Fig 4. The methylation and expression level of AKT1 in breast cancer patients with AKT1 mutations.

Boxplots show the average methylation (A) and expression (B) level of AKT1 in patient tumor tissues with or without AKT1 promoter mutations. P values were calculated using the Wilcoxon signed rank test.