The relevance of exosomal microRNAs in the re-education of stromal fibroblasts: a framework for the comprehension of triple negative breast cancer

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by elevated metastatic potential. TNBC progression is strongly sustained by the recruitment of the tumor microenvironment (TME), mainly composed of Cancer-Associated Fibroblasts (CAFs) able to endorse tumor hallmarks. Increasing evidence demonstrated that exosomes mediate the crosstalk between cancer cells and TME through delivering their molecular cargo. In this thesis, we examined the role of TNBC cell-derived exosomes and their microRNAs (miRNAs) cargo in the activation of stromal fibroblasts towards CAFs. We demonstrated that TNBC cell-derived exosomes increased fibroblast-mediated collagen contraction ability and migration potential alongside CAF-related molecular markers. Furthermore, fibroblasts activated by exosomes promoted the invasion potential of normal breast epithelial cells as assessed by the three-dimensional organotypic co-culture assay which resembled the in vivo context. We further investigated the role of TNBC cell-derived exosomes cargo in activating normal fibroblasts, by performing a small RNA-sequencing on fibroblasts incubated with exosomes. The findings revealed multiple upregulated miRNAs in fibroblasts upon exosome incubation. Among these, miRNAs-185-5p, -652-5p, and -1246 (combo miRs) were found to synergistically boost fibroblast migration, invasion, and contraction abilities together with the related molecular markers, thus promoting a specific CAF sub-specialization towards a pro-migratory functional state rather than a proliferative phenotype. Furthermore, conditioned medium from stromal fibroblasts transfected with combo miRs (CM_combo miRs) induced the migration of non-tumorigenic epithelial breast MCF10A cells, as assessed by transwell assay and increased the proliferation of breast cancer MCF7 cells, and MCF10A cells, as tested by MTS assay. Moreover, we investigated the invasion ability of patient-derived breast cancer organoids when incubated with CM_combo miRs through the in vitro collagen invasion assay. We found wider invasive protrusions in organoids cultured with CM_combo miRs compared to control (CM_Scra) indicating an increased invading trend of cancer cells mediated by fibroblasts activated by exosomal miRs. All together these data highlighted the role of exosomes and their miRNA cargo in the re-education of fibroblasts within the TME, shedding light on processes related to triple negative breast cancer evolution

The Role of Exo-miRNAs in Cancer: A Focus on Therapeutic and Diagnostic Applications

Exosomes are extracellular vesicles released into biological fluids where they act as carriers of various molecules, including proteins, lipids, and RNAs, between cells, modulating or perturbing specific physiological processes. Recently, it has been suggested that tumoral cells release excessive amounts of exosomes that, through their cargo, promote tumor progression, stimulating growth, angiogenesis, metastasis, insensitivity to chemotherapy, and immune evasion. Increasing evidence highlights exosomal microRNAs (exo-miRNAs) as important players in tumorigenesis. MicroRNA (miRNA) are a class of small non-coding RNA able to regulate gene expression, targeting multiple mRNAs and inducing translational repression and/or mRNA degradation. Exo-miRNAs are highly stable and easily detectable in biological fluids, and for these reasons, miRNAs are potential cancer biomarkers useful diagnostically and prognostically. Furthermore, since exosomes are natural delivery systems between cells, they can be appropriately modified to carry therapeutic miRNAs to specific recipient cells. Here we summarize the main functions of exo-miRNAs and their possible role for diagnostic and therapeutic applications

Global Impairment of Immediate-Early Genes Expression in Rett Syndrome Models and Patients Linked to Myelination Defects

Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient's peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs

miR-34c-3p targets CDK1 a synthetic lethality partner of KRAS in non-small cell lung cancer

Lung cancer is still the leading cause of death by cancer worldwide despite advances both in its detection and therapy. Multiple oncogenic driver alterations have been discovered, opening the prospective for new potential therapeutic targets. Among them, KRAS mutations represent the most frequent oncogene aberrations in non-small cell lung cancer (NSCLC) patients with a negative prognostic impact, but effective therapies targeting KRAS are not well characterized yet. Here, we demonstrate that the microRNA miR-34c-3p is a positive prognostic factor in KRAS-mutated NSCLC patients. Firstly, looking at the TGCA dataset, we found that high miR-34c-3p expression correlated with longer survival of KRAS-mutated NSCLC patients. In vitro assays on immortalized and patient-derived primary NSCLC cells revealed that miR-34c-3p overexpression increased apoptosis and lowered proliferation rate in KRASmut cells. Computational analysis and in vitro assays identified CDK1, one of the most promising lethal targets for KRAS-mutant cancer, as a target of miR-34c-3p. Moreover, the combination of CDK1 inhibition (mediated by RO3306) and miR-34c-3p overexpression resulted in an additive effect on the viability of KRASmut-expressing cells. Altogether, our findings demonstrate that miR-34c-3p is a novel biomarker that may allow tailored treatment for KRAS-mutated NSCLC patients