A novel panel of differentially-expressed microRNAs in breast cancer brain metastasis may predict patient survival

Breast cancer brain metastasis (BCBM) is an area of unmet clinical need. MicroRNAs (miRNAs) have been linked to the metastatic process in breast cancer (BC). In this study, we aim to determine differentially-expressed miRNAs utilising primary BCs that did not relapse (BCNR, n = 12), primaries that relapsed (BCR) and their paired (n = 40 pairs) brain metastases (BM) using the NanoString™ nCounter™ miRNA Expression Assays. Significance analysis of microarrays identified 58 and 11 differentially-expressed miRNAs between BCNR vs BCR and BCR vs BM respectively and pathway analysis revealed enrichment for genes involved in invasion and metastasis. Four miRNAs, miR-132-3p, miR-199a-5p, miR-150-5p and miR-155-5p, were differentially-expressed within both cohorts (BCNR-BCR, BCR-BM) and receiver-operating characteristic curve analysis (p = 0.00137) and Kaplan-Meier survival method (p = 0.0029, brain metastasis-free survival; p = 0.0007, overall survival) demonstrated their potential use as prognostic markers. Ingenuity pathway enrichment linked them to the MET oncogene, and the cMET protein was overexpressed in the BCR (p < 0.0001) and BM (p = 0.0008) cases, compared to the BCNRs. The 4-miRNAs panel identified in this study could be potentially used to distinguish BC patients with an increased risk of developing BCBM and provide potential novel therapeutic targets, whereas cMET-targeting warrants further investigation in the treatment of BCBM.

and TPM1 tumour-suppressor genes, whereas it affects metastasis by directly targeting TIMP3, SERPIN5B, PDCD4 and BCL2 8,9 . To date, only three studies with very limited numbers of samples (n = 4-9), have compared miRNA expression between matched primary unselected BC and BCBM tissues [12][13][14] . The current study screened a larger cohort of unselected BC samples that recur (BCR) and their paired brain metastases (BM), as well as primary BC samples that did not recur (BCNR) to identify differentially-expressed miRNAs related to BCBM. Such information is vital if biomarkers or therapeutic targets are to be identified which could transform the prevention and treatment of BCBM.

Differentially expressed miRNAs between primary BCNR vs BCR and BCR vs BCBMs.
Assessment of the global variation using principal component analysis (PCA) applied to the miRNA counts indicated a molecular distinction between the primary BCNR (blue dots), the BCRs (red dots) that progress to brain metastasis and the BCBM (green dots) cases, with a small number of overlapping samples mainly between BCRs and BCBMs (Fig. 1A).
A signature of miRNAs may define distant metastasis to the brain. Four of the significantly differentially-expressed miRNAs identified by SAM, hsa-miR-132-3p, hsa-miR-199a-5p, hsa-miR-150-5p and hsa-miR-155-5p were present within both cohorts of BCNR vs BCR and BCR vs BCBM (Fig. 3A). Statistical analysis (t-test) on the individual miRNAs showed a p < 0.01 and p < 0.0001 for all these miRNAs between BCNR vs BCR and BCR vs BCBM respectively (Fig. 3A). Only the hsa-miR-199a-5p showed a significant p value (p < 0.0001) between BCNR and BCBM. The differential expression of the 4 miRNAs within BCNR vs BCR (irrespectively of BC subtype) and BCR vs BCBM cohorts leads us to focus and investigate them further. In order to discriminate BCNR from BCR and study the diagnostic accuracy of these miRNAs as biomarkers for brain metastasis, receiver-operating characteristic curve (ROC) analysis was performed (Fig. 3B) using their mean log 2 -expression values. The area under the curve (AUC) was 0.8194 (95% CI: 0.6932-0.9457, p = 0.00137), meaning that a patient (BCR) will have a more abnormal test result than 81.94% of the controls (BCNR). The cut-off level of the combined 4-miRNA was 7.892 with 76.7% sensitivity and 83.3% specificity. This cut-off was used to dichotomised the primary BC cases into high-risk (>7.892) and low-risk (<7.892) and was used to perform Kaplan-Meier (log-rank) analysis of BMFS and OS. The result showed that low-risk group had a higher BMFS rate (p = 0.0029, HR: 0.314, 95% CI: 0.146-0.673) and a better overall survival (p = 0.0007, HR: 0.267, 95% CI: 0.124-0.573) than the high-risk group (Fig. 3C,D respectively), confirming that the expression levels of the 4-miRNA signature were significantly correlated with patient outcome. The individual miRNA contribution in BMFS and OS is illustrated in Supplementary Fig. 5. High miR-132-3p expression confers a protective effect whereas high miR-150-5p, miR-155-5p and miR-199a-5p confer a risky effect (HR < 1 and HR > 1 respectively).
IPA's miRNA target-filter functionality with filtering for experimentally validated targets identified 147 genes linked to 4 major networks (Fig. 4A). MET (cMET, HGF human growth factor ligand) oncogene was one of the genes in the top identified network (ID1, Fig. 4A) and it was also present in the IPA core analysis of the differentially-expressed miRNAs between BCNR vs BCR (Supplementary Fig. 2A) and BCR vs BCBM ( Supplementary Fig. 4A). The cMET was overexpressed in primary BCRs and their paired BMs in comparison to the primary BCNRs (

Discussion
In this study, we investigated the differential-expression of miRNAs between primary BCNR vs primary BCR and primary BCR vs their paired BMs. Assessment of the global variation using PCA indicated a molecular distinction between the primary BCNR, the BCRs that progress to brain metastasis and the BCBM cases. The overlap observed between the primary BCRs and their paired BMs suggests that many of the miRNAs linked to metastasis are already present in a proportion of the matched primary breast tumours. Consistent with the literature, we observed hormone (ER and/or PgR) receptor loss in BCBM cases [15][16][17] . Since brain metastasis is a late event in hormone receptor-positive BC, cases losing hormone sensitivity may behave more like TNs that are known to have a higher rate of BM and a worse prognosis.
To identify miRNAs that may be associated with BCBM, we analysed a cohort of primary BCs that recur and their paired BMs. This revealed a signature of 11 differentially-expressed miRNAs with previously published in-vitro and in-vivo data supporting their relevance in the processes of invasion and metastasis [8][9][10][11][12]14,24,28,29 . For instance, higher expression of miR-155-5p has been reported in localised early breast cancer than in metastasis, including brain metastasis 28 . The miR-155-5p promotes cancer cell extravasation by regulating the function of the blood-brain endothelial barrier and its overexpression shifts the TGF-β response from growth inhibition to EMT transition, invasion and metastasis in breast cancer through loss of C/EBP-β 3,8,23,24 . Moreover, miR-199a-3p www.nature.com/scientificreports www.nature.com/scientificreports/ and miR-214-3p, both up-regulated in human BC stem cells, enhance stem cell behaviour and facilitate metastasis by targeting Ezh2, p53, PTEN, BIM, and Snail 8,26,30 . Within our current cohort of primary BCs and their paired BMs, miR-10b-5p, miR-150-5p were upregulated while, miR-132-3p, and miR-9-5p were downregulated. These miRNAs have been linked to the induction of mesenchymal markers early in the EMT transition and to neovascularisation, both facilitating metastatic growth 3,8-10,31 . Overexpression of miR-150 promotes growth, clonogenicity and reduces apoptosis in breast cancer cells 29,32 , whereas primary BCs with increased levels of miRNA-10b have been associated with lymph node metastasis, de novo metastatic disease, as well as brain metastasis 3,6,[8][9][10][11] . Consistent with our data, miR-9 has been reported to be upregulated in non-CNS metastatic disease when compared to the primary BC, supporting its role in metastasis and colonisation 14 . In comparison to normal tissue, miR-132-3p is downregulated in primary BC, whereas its overexpression in cell lines and animal models, significantly suppress BC cell proliferation, invasion, migration and metastasis [33][34][35] . Interestingly, miR-132-3p has been characterised as a 'neuro-immunomiR' , a class of miRNAs which operate within and between the neural and immune compartments supporting its potential importance in brain metastasis 36 . A recent study showed that over-expression of miR-212/132 in hypoxic mouse and human brain microvascular endothelial cells decreased the blood-brain-barrier properties by targeting the transcription of tight junction and tight junction-associated proteins, highlighting further the importance of this miRNA in the brain 37 . MiR-132, miR-155, miR-150 and miR-10b regulate both innate and adaptive immunity and in the context of cancer, their immunomodulatory effects can enable malignant cells to evade the immune system [21][22][23]25,38 . The 11 differentially-expressed miR-NAs within the primary BCR and their paired BMs were also linked by IPA to genes and pathways involved in inflammation and immunity, cell stemness and EMT transition. IPA target-filter analysis linked them to ILs, PI3K and neuroinflammation signalling pathways ( Supplementary Fig. 6B), highlighting further the activation of an inflammatory network. The current data demonstrate the potential importance of immuno-regulation in the biology of BCBM and the potential for the use of immuno-oncology agents in their treatment 39,40 .
Four of the differentially-expressed miRNAs within the primary BCRs and their paired BMs, miR-132-3p, miR-150-5p, miR-199a-5p, miR-155-5p, were also differentially-expressed in the cohort of primary BCNRs as compared to primary BCRs. This observation lead us to focus and investigate them further. To evaluate their prognostic significance, we calculated the AUC of the ROC curve for development of brain metastasis using the BCNR patients as non-metastatic controls. The high AUC value indicated that the 4-miRNAs can distinguish a  www.nature.com/scientificreports www.nature.com/scientificreports/ patient that will relapse to the brain from the one who will not, supporting the prognostic potential of the identified 4-miRNAs signature. Further to the ROC curve analysis, Kaplan-Meier survival plot showed that the low-risk group had a 60% brain metastasis-free survival rate and 67% OS, whereas the high-risk group had metastasis-free and overall survival rate lower than 10%.
Of interest, the oncogene MET, encoding the transmembrane receptor cMET, was common in all the IPA network analyses. In BC, cMET is associated with aggressive clinico-pathological features, shorter disease-free/ overall survival and brain metastasis [41][42][43] . Consistent with these data, we observed cMET overexpression in BCR and their paired BMs but not in the BCNR samples. Previously, we also demonstrated increased cMET copy number in paired primary BCR and their BMs 44 . The above, highlight an active cMET pathway in primary BC samples that recurred to the brain as well as in BCBM but not in primary BC cases that do not recur.
Microglial cells are known to express cMET and produce HGF, contributing to angiogenesis of brain tumours 42,45,46 . A paracrine cytokine loop present between tumour-associated astrocytes and cancer cells results in continuous activation of the cMET pathway leading to induction of angiogenesis 42 . Therefore, cMET appears able to promote brain colonisation and maintenance via promoting angiogenesis within the CNS. Silencing cMET in combination with radiotherapy in a brain metastatic mouse model significantly prolonged survival, demonstrating its therapeutic potential 47 . cMET activation is also know to modulate immune cell functions, therefore, inhibition of cMET signalling may not only suppress cancer cell growth but also stimulate the immune system by alleviating the immunosuppressive effects on macrophages and dendritic cells 45,46,48 . Given that there are available drugs targeting cMET, the potential benefit of targeting cMET-positive BCBM with these agents warrants investigation.
A limitation of our study is the challenge in collecting a larger number of matched primary BC and BM cases. Moreover, it had been previously observed that the miRNA levels gradually decreased over time in long-archived FFPE blocks 49 and this accounted for the limited number of BCNR cases with a long follow-up available in our study. Therefore, an analysis by subtypes was not feasible. In an attempt to reduce the possible effect of the TN cases (absent in our BCNR cohort) we further analysed the BCNR vs ER + /HER2 + BCR (Supplementary Table 3). Of the 4 miRNAs, miR-132-3p, miR-150-5p, miR-199a-5p and miR-155-5p, only miR-155-5p was absent in the combined BCNR vs ER + /HER2 + BCR analysis indicating that its expression may be more relevant to TNBC 23,28 . However, given the hormone loss in the metastatic setting and the previous report of miR-155-5p in brain metastasis, we believe that this miRNA is still important and its role requires further investigation. In addition, the majority of patients with metastatic breast cancer, might present metastases to other organs such as bones, liver, lungs prior to the development of BM. However, the presence of BM regardless of other metastatic sites is a critical clinical complication, conferring a dismal prognosis to the patients. The patients included in this study came from the biobank of a specialist tertiary referral neurosurgical centre (Walton Hospital) and the relevant detailed clinical information with regard to other involved anatomical sites was not collected by the biobank. Given the samples are anonymised we were unable to seek out and collect this information. Finally, the patients included in this study may have received some form of systemic therapy and/or radiotherapy prior to the biopsy of the metastatic lesion. Therefore, the detected miRNAs might be affected by the metastatic process, the therapy or a combination of both. Despite these limitations, this study provides a comprehensive analysis of differentially-expressed miRNAs in primary BCs that did not relapsed versus primary BCs that relapsed to the brain and their matched brain metastasis. It should be also noted, that the current study represents the largest set of unselected matched primary breast tumours and brain metastases so far.
In summary, this is the first study to report a panel of differentially-expressed miRNAs in BCBM that could potentially be used to identify BC patients at increased risk of BCBM as well as provide potential novel therapeutic targets. Since these miRNAs were also linked to cMET using network analysis, the data supports a novel therapeutic strategy for BCBM that is focused on targeting cMET.

Patients.
A total of 12 formalin-fixed paraffin-embedded (FFPE) primary BCNR and 40 primary BCR samples with their paired BMs were collected from the Liverpool Tissue Bank, Walton Research Tissue Bank (WRTB) Liverpool, UK and the Royal College of Surgeons Ireland (RCSI) National Breast Cancer Bioresource, Ireland. The specimens were reviewed by two pathologists (SR,KB) and defined as per local protocol. Briefly, ER, PgR positivity was defined using the Allred scoring system (3)(4)(5)(6)(7)(8), whereas HER2 status was classified as negative (staining 0-1), positive (staining 3 + ). Amplification of HER2 was confirmed by FISH (fluorescent in-situ hybridisation) if the staining was 2 + (equivocal). The cMET protein expression was assessed by immunohistochemistry (IHC). Intensity was scored according to a four-tier systems: 0, no staining; 1+, weak; 2+, moderate; and 3+, strong. The H-score was used as a semi-quantitative measure by multiplying the staining intensity (0-3) and the percentage of positive cells (0-100%) for a final IHC score ranging from 0 to 300. The study was performed in accordance with the Declaration of Helsinki and approved by the WRTB Ethics committee (WRTB15_06), the National Research Ethics Committee (NRES 11/WN003/2), the UK Health Research Authority (NRES 12/NW/0778) and the RCSI Institutional Review Board (#13/09; ICORG09/07). Appropriate approvals and written consent were in place before anonymised tissue and data were released.
RNA extraction and miRNA profiling. RNA was extracted using the miRNeasy FFPE kit (Qiagen, Manchester, UK) and quantified on the ND-Nanodrop1000 spectrometer (ThermoFisher Scientific, Wilmington, MA, USA). RNA integrity number (RIN) was determined using the 2100 Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). Profiling was performed using the NanoString ™ nCounter ™ miRNA Expression Assay (Human_v3 miRNA) according to the manufacturer's instructions. The raw data were quality control (QC) assessed and normalised by the NanoString ™ nSolver ™ analysis software. Fifteen cases (5 paired BCR-BM, 2 BCRs and 3 BMs) failed the NanoString ™ QC and normalisation and were excluded from downstream analysis Ethics approval and consent to participate. This project was peer reviewed by the Walton Research Tissue Bank (WRTB15_06), an approved repository under the Human Tissue Act (National Research Ethics Service: NRES 11/WN003/2), the UK Health Research Authority (NRES 12/NW/0778) and the RCSI Institutional Review Board (#13/09; ICORG09/07). Appropriate approvals and written consent were in place before anonymised tissue and data were released. The study was performed in accordance with the Declaration of Helsinki.

Data availability
All the data (raw counts and normalised counts) analysed in this study are available in the Additional File 1 (Additional file 1_Raw Counts and Normalised Data.xlsx) submitted with the manuscript.