The ANXA2 gene is located on the long arm of chromosome 15 (15q21-q22), is approximately 40 kb in length, and contains 13 exons and 12 introns. The length of its mRNA is approximately 1665 bp, and the coding region spans base pairs 161 to 1180. The ANXA2 protein comprises 339 amino acids with a molecular weight of 36 kDa, and is accordingly referred to as p36. It is also known by several other names, including Annexin II, P39, calpactin Ⅰ heavy chain, and Lipocortin Ⅱ . ANXA2 is a calcium-dependent membrane phospholipid-binding protein, and is one of the most important members of the Annexin family; it is closely associated with the occurrence, development, invasion, and metastasis of many tumors, including breast cancer.
Because of the reduced evolutionary constraints on somatic cells, somatic cell mutations continue to accumulate and eventually lead to tumorigenesis. At the same time, persistent somatic cell evolution promotes genetic heterogeneity in the development of tumors [38–40]. It has been reported that many gene mutations are associated with breast cancer, including the polymorphisms of rs2046210 and rs3803662, single-nucleotide polymorphisms (SNPs) of the FGFR1 gene, the SNP of rs10800708 within the KIF14 miRNA, and SNPs of the CD44 gene [41–44]. However, whether ANXA2 mutation is associated with breast cancer has not been reported up to now.
In the present study, we identified ANXA2 mutations in breast cancer patients.
Breast cancer tissues exhibited more mutations (80/112) than adjacent normal tissues (10/112) (P < 0.05). Moreover, most mutations (8/10) in adjacent normal tissues were found in breast cancer tissues, few mutations (2/10) in adjacent normal tissues were different from the breast cancer tissues. The presence of mutations in normal tissues may be due to sequencing artifacts, presence of tumor cells in the normal bulk or preneoplastic evolution of the tumor-adjacent tissue. However, all the mutations of ANXA2 in adjacent normal tissues still need further study and confirmation.
We detected a high incidence of missense mutation in the tumor samples (40.18%) and 8.04% of normal tissues carrying a mutation, which was higher than known data: a combined analysis of 6995 breast cancer patients selected in 19 studies in cbioportal hub (https://www.cbioportal.org/) showed that ANXA2 mutation incidence was only 0.7% (less than 5%). The difference may lie in: Firstly, the limitation of samples in this paper involved; Secondly, data in the cbioportal hub were based on DNA level, while our results were based on mRNA level. The function of mRNA is active, which is related to the on / off of promoter, RNA degradation and post transcriptional silencing, etc. Because of this, it is more likely and easier to be changed than DNA in the state of breast cancer. At the same time, mRNA can also better reflect the molecular changes caused by breast cancer.
Previous reports mainly focused on the relationship between expression of ANXA2 and cancer genesis and prognosis of patients, and found that the high expression of ANXA2 was associated with the occurrence and development of tumors, distant metastasis, higher clinical stages, and poor outcomes and prognosis [4–16]. While, our results showed that ANXA2 mutations may be associated with the occurrence, molecular subtype, clinical stage, ER, PR and lymph node metastasis of breast cancer patients (P < 0.05) (Tables 4–5). The consistency of the results suggested that the high expression of ANXA2 may be associated with dysfunction or abnormality caused by mutations. However, this still needs further verification.
Because of the high heterogeneity and lack of typical receptors (ER, PR, HER-2+) and effective targeted therapies, TNBC is often considered to be synonymous with an increased risk of relapse and shorter survival time. Pearson Chi-Square results showed that TNBC patients with higher rate of ANXA2 mutations (16/22: 16 patients occurred ANXA2 mutations in 22 TNBC patients) than Luminal A (7/32), Luminal B (10/34) and HER-2+ (10/22), and the difference was significant (χ2 = 16.149, P = 0.001). Results of LSD-t showed that ANXA2 mutations frequency in TNBC (37/22: 37mutaions in 22 patients) was significantly different from three other groups (P < 0.05). These data implied that patients with TNBC molecular subtype were more likely to occur ANXA2 mutations, ANXA2 mutations may be potential diagnostic and therapeutic targets for TNBC.
In this study, Kaplan-Meier univariate survival analysis for 5-year OS results showed that c.(350TC > CT), c.(375G > A) and c.(693C > T) were meaningful (χ2 = 7.499, P = 0.006) (Fig. 5C-F, Table 8), though significant mutaiton and factor was not identified in multivariate Cox proportional risk regression model analysis for 5-year OS, (Tables 5–7), it still provided a reference for future early diagnoses and prognoses evaluation of breast cancer, and may be potential target for gene therapy of breast cancer.
Owing to an insufficient number of samples, we were unable to fully elucidate other mutations, and further investigations are necessary to determine whether there are false-positive and currently unidentified mutations. In this study, we only examined the mutation of ANXA2 coding area, but not in its non-coding area. Secondly, we only chose invasive ductal carcinoma as the research object, which does not reflect other types of breast cancer. Finally, the follow-up time was short, which can only reflect the survival rate within 5 years. Whether there is a difference between ANXA2 mutation and non-mutation patients after 5 years cannot be predicted.