Citation: Cancer Research. 2019, 79(4)
Author: Giannoudis A.; Clarke K.; Zakaria R.; Vareslija D.; Farahani M.; Rainbow L.; Platt-Higgins A.; Ruthven S.; Brougham K.; Rudland P.S.; Jenkinson M.D.; Young L.; Falciani F.; Palmieri C.
Abstract: Background: Despite sequential improvements in the adjuvant treatment of breast cancer (BC), recurrence and metastasis remains a major clinical problem and in particular, brain metastasis (BCBM). A number of microRNAs (miRNAs) have been linked to the metastatic process in BC, but to date there is limited work on the microRNAs involved in BCBM. The current study aim to identify differentially expressed miRNAs within primary breast cancer who did not recur (BCNR) versus primary BC cases which did recur (BCR) and their matched BCBM cases.
Method(s): Formalin-fixed paraffin-embedded (FFPE) material was collected of 12 primary BCNRs from the Liverpool tissue bank and of 40 paired primary BCR samples and their matched BCBM from the Walton Research Tissue Bank and RCSI National Breast Cancer Bioresource. miRNA was extracted (Qiagen miRNeasy FFPE kit) and profiled using the NanoStringTM nCounterTM miRNA Expression Assay (Human v3 miRNA). The differentially expressed miRNAs between BCNR versus BCR and BCR versus their matched BCBM were identified by significance of microarray analysis (SAM) on the MeV4.9 software. Pathway analysis was performed using the DIANA-mirPath v3.0 software and the Ingenuity Pathway Analysis (IPA) to identify a network of genes/pathways regulated by the differentially expressed miRNAs.
Result(s): 12 BCNR and 30 matched pairs of BCR and BCBM passed the quality control and normalisation processes. Principal component analysis (PCA) performed on 166 miRNAs after QC/normalisation clearly distinguishes the BCNR and the primary BCR from the matched BCBM cases, whereas SAM revealed 58 differentially expressed miRNAs with a 10% FDR (false discovery rate) and an absolute log2 fold-change (FC) >1 between BCNR and BCR and 11 between the matched BCs and BCBMs. Pathway clustering revealed that these differentially expressed miRNAs (10% FDR, log2FC>1) within both BCNR vs BCR and BCR vs BCBM cohorts are highly enriched for genes involved in extracellular matrix (ECM)-receptor interactions, proteoglycans, adherens junctions, TGF-beta, P53 and Hippo signalling. IPA identified a network of genes, implicated in the processes of breast cancer invasion and metastasis, regulated by the identified miRNAs, such as, TWIST, MET, TP53, MYC, EZH2, ZEB1, TAGLN and BIRC5. Four of the significantly differentially expressed miRNAs, hsa-miR-132-3p, hsa-miR-199a-5p, hsa-miR-150-5p and hsa-miR-155-5p were present within both cohorts (BCNR vs BCR and BCR vs BCBM) and regulate genes involved in Hippo and TGF-beta signalling (DIANA-mirPath v3.0 analysis: p=5.23x10<sup>-08</sup> and p=2.67x10<sup>-07</sup> respectively).
Conclusion(s): The current study, utilising a large cohort of paired BCR and BCBM cases, provides novel insight into the molecular mechanisms and role of miRNAs in BCBM. Four miRNAs (hsa-miR-132-3p, hsa-miR-199a-5p, hsa-miR-150-5p and hsa-miR-155-5p) in particular could be potentially used to identify patients with increased risk of developing brain metastasis and help facilitate the development of specific treatments for BCBM, which to date have proved elusive. The miRNAs identified require further exploration as potential biomarkers as well as novel therapeutic targets.
