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

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].