The potential role of miRNAs in therapy of breast and ovarian cancers associated with BRCA1 mutation

Cisplatin is particularly useful in the treatment of breast and ovarian cancers associated with causative BRCA1 variants [16]. Its mechanism of action is based on the formation of cross-links between two DNA strands, and also within the same strand. This inhibits DNA replication and cell division, culminating in apoptosis [17]. Byrski et al. found that 80% of BRCA1 variant carriers, who received cisplatin as neoadjuvant treatment of breast cancer, had a good response and progression-free survival (PFS) [18].

A relatively new and promising therapeutic approach for the treatment of breast cancer is the use of inhibitors of poly (ADP-ribose) polymerase (PARP), an enzyme involved in the repair of DNA breaks caused by different mechanisms, including homologous recombination. In wildtype cells, DSBs are removed by HR, but in the case of cells where BRCA1 is inactivated (by mutation or an epigenetic modification) these breaks are not repaired, resulting in DNA fragmentation and cell apoptosis [19]. The lack of properly functioning BRCA proteins can be partially compensated by PARP enzymes. If PARP inhibitors are used, the consequences of BRCA mutations are stronger – the possibility of repairing DBs is significantly decreased resulting in synthetic lethality [3, 4]. These data suggest the potential value of using PARP-1 inhibitors in cancer therapy. To date, Olaparib (Astra Zeneca) has been the only PARP-1 inhibitor approved for the therapeutic use. It is used in BRCA1 variant carriers – who have had a partial or complete remission (CR or PR) in the maintenance treatment of ovarian cancer sensitive to platinum drugs [11, 20]. According to the FDA recommendations, Olaparib may be used in patients who received 3 first lines of chemotherapy of cancers showing sensitivity to platins. The median progression-free survival as a result of the use of a PARP-1 inhibitor as maintenance therapy was 11.2 months compared to 4.3 months in the placebo group [21].

Sun et al. have searched for a group of miRNAs that target the BRCA1 3’UTR region. They used 6 algorithms to identify these miRNAs that were predicted to influence BRCA1 3’UTR. This led to the identification of 28 miRNAs that could potentially influence BRCA1 expression. Of these, miRNA-9 (miR-9) proved to be the most efficient in reducting BRCA1 activity in a luciferase reporter assay [22].

Sun et al. collected 58 patients with serous ovarian cancer (stage IIIc or IV). All of patients were treated with platinum/taxane- based chemotherapy and PFS was calculated. Samples of the tumors were used to construct the tissue microarrays. Then, they determined the levels of miR-9 expression by in-situ hybridization and the expression of BRCA1 by immuno-histochemistry.

The results of this study revealed that low BRCA1 expression and high expression of miRNA-9 was associated with platinum sensitivity and longer PFS (low vs high BRCA1 expression: median PFS = 37.3 months vs 15.2 months; high vs low miRNA-9 expression, median PFS = 26.4 months, vs 15.4 months).

These results were validated using another group of 113 ovarian tumors. Among them, 45 showed low expression of BRCA1 and 63 had elevated expression of miRNA-9. It was statistically significant that 34 tumors with low expression of BRCA1 were sensitive to platinum, as well as 43 cancers with high expression of miRNA-9.

The same group, in addition to the platinum sensitivity in patients with BRCA1 mutations, performed experiments on the response to treatment with a PARP-1 inhibitor in mice breast tumours that overexpressed miRNA-9. The results revealed that increased expression of miRNA-9 inhibits tumour growth in mice during the treatment with PARP1 AG014699 inhibitors: (PARP-1 inhibitor plus miRNA-9 agomiR: mean tumor volume = 14.3 mm3, vehicle alone: mean tumor volume = 178.8 mm3) [22].

Gu et al. used 317 ovarian cancers to find BRCA1/2 - directed miRNAs which influences sensitivity to platinum based therapies. Therefore, they divided patients into two groups: 218 samples with wild-type BRCA1/2 and 99 samples with BRCA1/2 somatic or germline mutations. Using TCGA portal data they obtained information about miRNA expression. Then, multiple algorithms were used (f.ex. TargetScan) to identify miRNAs that potentially regulate BRCA1/2. Next, they separated the group of 218 wild-type BRCA1/2 ovarian cancers into a training and test set. Among the training set they identified 57 miRNAs targeting BRCA1/2 and between them searched for those that were associated with better overall survival (OS) in patients with wild-type BRCA1/2. Three miRNAs: miRNA-146a, miRNA-148a, miRNA-545 were identified. Next, they separated patients into miRNA related high and low- risk groups, depending on OS and PFS, using the median risk score as the cut-off. Patients from the low-risk group of miRNAs had longer median overall survival than from high-risk group (median OS = 51.9 vs 35.8 months).

Additionally, Gu et al. divided 317 ovarian cancer patients into three groups: BRCA1/2 mutation carriers, miRNA high-risk and miRNA low-risk groups. Patients with BRCA1/2 alterations had significantly longer OS than patients from the high-risk group (median OS = 49.5 vs 34.1 months). Of note, patients with BRCA1/2 mutations and those from the low-risk miRNA group had similar survival (median OS = 49.5 vs 52.2 months).

These results imply that miRNA-146a, miRNA-148a, miRNA-545 interfered with the DNA damage repair pathway by reducing BRCA1 expression [23].

In another study of miRNA-146a, Shen et al. examined 42 patients with familial breast cancer and 82 with familial ovarian cancers. Using an in vitro assay, they found that the polymorphism of G to C in miRNA-146a increased miRNA-146a expression. Furthermore, the variant allele binds more strongly to the 3’UTR BRCA1 region than allele G, which is associated with reduced BRCA1 expression [24, 25].

Using prediction algorithms Moskwa et al. found miRNA-182 targets BRCA1 in breast cancer. In order to establish the sensitivity to IR (γ-irradiation) and PARP-1 inhibitors, they utilized up-regulated miRNA-182 in MDA-MB231 cells and downregulated miRNA-182 in 21NT cells. It was shown that MDA-MB231 cells with over-expression of miRNA-182 were significantly more sensitive to IR and PARP-1 inhibitors.

The same group also found that treatment with Olaparib (PARP-1 inhibitor) retarded growth of miR-182 expressing tumors in mice [25, 26].

Using micro-array analysis, He et al. identified 19 deregulated miRNAs in the MCF-7/DDP cell line, including 11 down-regulated and 8 up-regulated miRNAs. One of them was miRNA-218, of which expression was most reduced in cells resistant to cisplatin in patients with BRCA1 mutations. To prove this in clinical samples, He et al. examined 85 primary breast cancer samples with different chemosensitivity to cisplatin.

No relationship was found between the level of miRNA-218 expression and the age of onset, the presence of lymph node metastases or clinical stage (He et al. 2015).

However, they found a correlation between survival rate in breast cancer and expression of BRCA1. A total of 85 patients were divided into 2 groups, depending on the levels of expression of BRCA1 and miRNA-218.

The Kaplan-Meier estimator revealed a statistically significant relationship between miRNA218, the expression of BRCA1 and survival in patients with breast cancer: [expression of miRNA-218 – survival time in months: 30.9 for low vs. 42.2 for high; expression of BRCA1- survival time in months: 41.9 for low vs. 31.5 for high expression [27].

Zavala et al. used 39 formalin- fixed paraffin embedded (FFPE) samples of triple negative breast cancer to find the clinical relevance of miRNA-146a and miRNA-638 in association with BRCA1 expression. The presence of BRCA1 variants was assessed. Three patients carried BRCA1 germline mutations. They divided the tumours into two groups, depending on the level of expression of miRNAs and BRCA1. It has been demonstrated that women with high expression of miRNA-638, miRNA-146a and abnormal BRCA1 had a longer overall survival in comparison to those with the low expression of these miRNAs and normally expressed BRCA1 [28].

miRNA-638 is deregulated in many different cancers [26, 29]. Therefore, Tan et al., using FFPE breast cancer samples and several human breast cancer cell lines, wanted to determine how miRNA-638 influenced sensitivity to UV-light and cisplatin in triple- negative breast cancer (TNBC). Thanks to TARGETSCAN-VERT and miRanda they revealed that miRNA-638 regulates BRCA1 expression. The FFPE samples were divided into normal tissue and breast cancer tissue and subsequently isolated RNA. Identifying that overexpression of miRNA-638 can lead to an increased susceptibility of TNBC cells to DNA damaging agents, such as UV radiation and cisplatin treatment. Although 5-fluorouracil and epirubicin also belong to the group of DNA damaging agents, no relationship was reported between an improved response to treatment with these agents and miRNA-638 levels [30].