CA1 contributes to microcalcification and tumourigenesis in breast cancer

Yabing Zheng, Bing Xu, Yan Zhao, He Gu, Chang Li, Yao Wang, Xiaotian Chang

Although mammary microcalcification is frequently observed and has been associated with poor survival in patients with breast cancer, the genesis of calcification remains unclear. Carbonic anhydrase I (CA1) has been shown to promote calcification by catalysing the hydration of CO2. This study aimed to determine whether CA1 was correlated with microcalcification and with other processes that are involved in breast cancer tumourigenesis. CA1 expression in breast cancer tissues and blood samples was detected using western blotting, real-time PCR, immunohistochemistry and ELISA. Calcification was induced in the cultured 4T1 cell line originating from mouse breast tumours, using ascorbic acid and β-glycerophosphate. Acetazolamide, a chemical inhibitor of CA1, was also added to the culture to determine the role of CA1 in calcification. The MCF-7 human breast cancer cell line was treated with anti-CA1 siRNA and was assessed using a CCK-8 cell proliferation assay, an annexin V cell apoptosis assay, transwell migration assay and a human breast cancer PCR array. The tag SNP rs725605, which is located in the CA1 locus, was genotyped using TaqMan® genotyping. Increased CA1 expression was detected in samples of breast carcinoma tissues and blood obtained from patients with breast cancer. A total of 15.3 % of these blood samples exhibited a 2.1-fold or higher level of CA1 expression, compared to the average level of CA1 expression in samples from healthy controls. Following the induction of calcification of 4T1 cells, both the number of calcium-rich deposits and the expression of CA1 increased, whereas the calcification and CA1 expression were significantly supressed in the presence of acetazolamide. Increased migration and apoptosis were observed in MCF-7 cells that were treated with anti-CA1 siRNA. The PCR array detected up-regulation of the androgen receptor (AR) and down-regulation of X-box binding protein 1 (XBP1) in the treated MCF-7 cells. Significant differences in the allele and genotype frequencies of rs725605 were detected in the cohort of patients with breast cancer but not in other tumours. The results of this study suggested that CA1 is a potential oncogene and that it contributes to abnormal cell calcification, apoptosis and migration in breast cancer.