Evaluation of BRCA1-related molecular features and microRNAs as prognostic factors for triple negative breast cancers

Breast cancer susceptibility gene 1 (BRCA1) was the first tumor suppressor gene identified in breast and ovarian cancer. Located on chromosome 17 (17q21), it encodes a multifunctional protein that is involved in several cellular processes such as DNA repair and cell cycle control. BRCA1 is involved in large protein complexes and its interaction with other proteins, as BARD1, is required for its function.

BRCA1 seems to be associated with the triple negative breast cancer (TNBC) subtype because the histological features and clinical outcomes of TNBC sporadic tumors can be very similar to those found in the tumors of BRCA1 germline mutated patients. The traits that some sporadic cancers share with those occurring in BRCA1 mutation carriers were described and called ‘BRCAness’ by Turner et al. [1]. In particular, these cancers present a high rate of chromosomal alterations reflecting the absence of the BRCA1 DNA repair function.

TNBC has a poor prognosis and no targeted therapy is currently available. Because these cancers are heterogeneous in terms of therapeutic response, new therapeutic solutions are being sought. In this context, recent clinical data have shown that BRCA1-associated breast cancers appeared to be more sensitive to platinum agents in neoadjuvant chemotherapy than non-hereditary tumors [2–4]. In contrast, a phase II clinical trial found that poly (ADP-ribose) polymerase (PARP) inhibitors also showed promising activity in BRCA1-mutated breast cancer although there were no response in patients with TNBC regardless of BRCA1/2 mutation status [5], and phase III trials are ongoing in BRCA1/2-mutated BC and TNBC [6].

However, it is also becoming clear that germline BRCA1/2 mutations are neither necessary nor sufficient for patients to derive benefit from these agents [6]. This variability of response can be explained by different BRCA1 protein expression statuses inside the tumor, as several cases can be met : (i) germline BRCA1 is mutated in one allele, and the second is lost; thus, BRCA1 tumoral expression is missing [7, 8] (ii) germline BRCA1 is mutated in one allele, and the second is still active, so tumoral BRCA1 expression is normal; (iii) germline BRCA1 is mutated, but reversal somatic mutation occurs, and BRCA1 tumoral expression is restored, leading to PARP inhibitor treatment resistance [9, 10]; (iv) germline BRCA1 is normal, but tumoral expression is lost by promoter hypermethylation [11]; and (v) germline BRCA1 is normal, but tumoral expression is lost by post-transcriptional regulation, such as by miRNAs [12]. One could expect a greater likelihood of response for patients treated with platinum compounds or PARP inhibitors only if BRCA1 protein tumoral expression were lost. As a consequence, better characterization of BRCA1 expression status in TNBC would provide important knowledge to improve chemotherapy choices.

MicroRNAs are small non-coding RNAs that bind to the 3’ untranslated (3’UTR) region of target messenger RNAs (mRNAs), and they are known to regulate gene expression. They are deregulated in breast cancers: some of them are known to be oncogenic, and others are known as tumor suppressors. MiRNAs participate in a variety of biological processes, such as the immune response, as well as proliferation and metastasis, which are hallmarks of cancer [13, 14]. Many studies have implicated miRNAs in chemotherapy resistance, such as to cisplatin [15], and some of them have been used as prognostic biomarkers [16–18]. Moreover, some miRNAs could target BRCA1 mRNA expression, and, at the same time, their expression was affected by BRCA1 protein [12, 19–21].

Currently, the number of conventional breast cancer prognostic factors is limited (tumor size, histology and grade, hormone receptors status, lymph nodes invasion, proliferative index [Ki67], and tumor-infiltrating lymphocytes, as well as the age of the patient), and their use does not allow for accurate prediction of treatment resistance or relapse in TNBC. Defining new molecular prognostic factors to refine TNBC classification would be useful in facilitating a more adapted chemotherapy choice.

In this context, we quantified molecular parameters focusing on the BRCA1 gene expression regulation and function (BRCA1 promoter methylation, BRCA1 in situ mRNA expression, BRCA1 in situ protein expression and BRCA1 in situ interaction with BARD1) in 69 TNBC tumors from patients. The expression of 27 tumoral miRNAs was also measured. Those molecular parameters were associated with progression-free survival in uni- and multivariate statistical analyses to determine new prognostic factors.

An accurate technique to determine BRCA1 tumoral expression status in TNBC would allow for informed decision and choosing platinum derivatives or PARP inhibitor treatments, because hypersensitivity to these agents has been described in cases of loss of BRCA1 expression [3, 5]. Thus, we developed alternative techniques to evaluate, in tumors, the expression status of BRCA1 at three different levels: mRNA, protein, and maintenance of BRCA1 interaction with BARD1. This multiple approach presented the advantage of incorporating different types of information, allowing for cross-control, and offering the possibility of combining the data. Several BRCA1 studies have described mRNA expression using RT-qPCR or protein expression by immunohistochemistry, but studies describing both mRNA and protein expressions has been very rare [28], despite BRCA1 expression being known to be subjected to multiple regulations [19]. The commercially available antibodies directed against BRCA1 lack the specificity required to identify the BRCA1 protein for clinical purpose because no immunohistochemical (IHC) differences in BRCA1 protein expression were found between cases with and without BRCA1 germline mutations by Pérez-Vallés and colleagues [29]. To improve the sensitivity and specificity of the BRCA1 detection compared with IHC, we used proximity ligation assays with two primary antibodies against the N- and C-terminus domains of BRCA1. The second advantage of this technique was that it only allowed for the measurement of the full-length proteins. BRCA1 must be ligated to its interacting protein BARD1 to repair DNA. Some BRCA1 variants, such as splicing variants [29], can be expressed in the tumor but can lose their interaction with their partners. To obtain a reflection of BRCA1 function maintenance in tumors, proximity ligation assay were performed to visualize the portion of BRCA1 ligated to BARD1.

Although the three levels of BRCA1 tumoral expression were correlated inside the same tumor, highly heterogenous intra-tumoral expression was observed, hampering accurate quantification. The lack of correlation between PFS and BRCA1 expression was probably a consequence of this high variability. We concluded that none of these three facets of the BRCA1 tumoral expression could be used for clinical decision purposes.

The TNBC cohort that we explored included six patients with a known germline BRCA1 mutation. However, no significant differences in BRCA1 expression at the levels of mRNA, protein, or ligation to BARD1 were observed in these cases, probably due to the small number of patients. Interestingly, two of these six BRCA1 mutated patients also presented a methylated form of the BRCA1 promoter, although Lips et al. described these events as mutually exclusive [30]. This combination of events could increase the risk of breast cancer because these patients are also the two youngest who developed breast cancer in our cohort of 69 patients, but this possibility will need to be confirmed on a larger cohort. The work of Ertuk and Cecener also stated that miRNAs expression can be different in BRCA1 mutated or normal TNBC tumors [31]. However, we could not observe similar effect, probably due to the small number of patients.

Statistical multivariate analysis demonstrated that miR-548c-5p was an independent prognostic factor for breast cancer. Patients with a good prognosis presented higher intratumoral expression of this microRNA. Although implicated in multiple biological processes including cancer, no role for miR-548c-5p has ever been reported in the breast cancer field. Mir-548 is a large, poorly conserved primate-specific miRNA gene family. Sixty-nine human mir-548 genes are located on almost all human chromosomes and its widespread distribution pattern and specific sequence indicate its evolutionary origin from the MADE1 transposable element [32, 33]. There are more than 3500 putative mir-548 target genes, but none have been experimentally demonstrated for miR-548c-5p.

The measurement of tumoral miR-548c-5p expression levels in combination with three conventional breast cancer prognostic factors (node invasion, tumor size and cytokeratin 5/6 expression), allowed for the relapse prediction of patients with an AUC = 0.96. A study in a larger cohort would be needed to confirm this observation, and to determine whether quantification of this microRNA expression in the tumor could be used to steer patients with poor predicted prognosis toward alternative chemotherapies.

We also showed that patients with poor predicted prognoses calculated by this model presented higher expression of miR-210, miR-503-5p and BRCA1 mRNA. Indeed, high miR-210 expression has already been reported by other teams to be correlated with relapse and short survival [19, 34]. miR-503-5p was already emphasized in our previous work: this microRNA is highly expressed in endothelial cells and, can be secreted in exosomes and transferred into breast cancer cell lines to inhibit tumor growth by targeting CCND2 and CCND3 [35]. Moreover, neoadjuvant chemotherapy for breast cancer leads to increased plasma levels of miR-503, as also observed for miR-34a, which could be implicated in the anti-tumor effects of chemotherapy in breast cancer patients [35, 36]. Concerning the higher expression of BRCA1 mRNA observed in the poor-prognosis tumors, we could hypothesize that patients expressing high levels of BRCA1 would present a lower response to chemotherapy because TNBC BRCA1 mutated patients are known to respond better to chemotherapy [37].

MiR-484 was reported by Dvinge et al. as a good potential housekeeping microRNA in breast cancer because its expression was homogenous among samples in all breast cancers subtypes [27]. However, Cox univariate analysis showed that high miR-484 expression was associated with a bad prognosis in our TNBC cohort. Volinia et al. also reported such an association [18]. In a high-throughput study aiming at better defining miRNA-mRNA interaction, BRCA1 was identified as interacting with miR-484. However, we did not observed any inverse correlation between those two parameters. Although, miR-484 expression was strongly associated with two other poor-prognosis miRNAs: miR-205 (Rho Spearman: 0.4, p-val :0.003) and miR-93 (Rho Spearman : 0.52, p-val = 0.0001), the Diana MiRPath database did not present any experimentally demonstrated common target gene of the three miRNAs [38].

BRCA1 was expressed in a spatially heterogeneous manner in TNBC, making very difficult any study correlating its expression or activity with prognosis. However, this study emphasized miR-548c-5p tumoral expression as a new independent prognostic factor that could improve the performance of relapse prediction models based on node invasion, tumor size and cytokeratin five and six expression status.