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Abstract

Metastatic disease at distant sites is the main cause of death for patients of breast cancer. Among them, patients diagnosed with the subtype termed Triple Negative (TNBC) are affected the most by both residual disease after treatment and distant metastases. It is therefore of utmost importance to investigate more in depth the role of pathways related to TNBC development and metastasization, possibly identifying novel therapeutic or diagnostic targets. In this PhD project, the effect of the miRNome (i.e. known miRNAs encoded in the genome) was analysed on three pathways relevant for TNBC metastatic processes. A high-throughput proteomic approach termed Reverse Phase Protein Arrays (RPPA) was exploited to quantify the effect of miRNAs on the expression of 62 proteins belonging to c-Met, Integrin, and WNT/β-catenin signalling pathways. Then, a novel network analysis method was developed, taking into consideration the role of the proteins within the pathway of interest, and therefore contextualising the miRNA:target interactions. miRNAs identified as modulators of WNT/β-catenin signalling were tested functionally by means of a reporter gene assay, characterising miR-193b, -409, -494-, and -92b as strong repressors of the pathway. Their ability to regulate proliferation was assessed in a context of pathway overactivation, revealing that miR-193b, -409, and -494 reduce it. Analysis of stem-associated surface markers CD44 and CD24 showed that miR-193b is able to decrease the CD44+ population. Additionally, direct molecular interactions with predicted target sequences in 3’UTRs of mRNAs corresponding to the proteins probed were validated for miR-193b and miR-494.

In summary, the findings of this dissertation describe the role of miRNAs as putative repressors or activators of metastatic pathways in TNBC, validating miR-193b, miR-409, and miR-494 as highly integrated within the network of WNT/β-catenin signalling. Furthermore, the RPPA results provide a useful resource available to the scientific community for further studies related to miRNA function in TNBC.