Donnarumma, Elvira (2015) Cancer-associated fibroblasts (CAFs) release exosomal microRNAs that dictate an aggressive phenotype in breast cancer cells. [Tesi di dottorato]

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Despite many novel therapeutic approaches, breast cancer remains one of the leading cause of cancer mortality among women. Recent findings indicate that cancer-associated fibroblasts (CAFs), the major components of the tumor microenvironment, play a crucial role in breast cancer progression, but how they promote tumorigenesis is poorly understood. Increasing evidence indicates that exosomes, membrane vesicles sized 30-100 nm in diameter, play an important role in cell-cell communication. Exosomes and their cargo, including microRNAs (miRs), may vehicolate between cells and affect the biological behavior of recipient cells. Therefore, one alternative mechanism of the promotion of breast cancer progression by CAFs may be through cancer-associated fibroblast-secreted exosomes, which would deliver oncogenic miRs to breast cancer cells. Firstly, to investigate the potential role of miRs in stroma-tumor communication, we compared miR expression profile in exosomes from 2 cancer-associated fibroblasts and 2 normal fibroblasts. We found that in CAF exosomes the levels of miR-21, miR-378e, and miR-143 were increased as compared to normal fibroblast exosomes, and we validated the array data by real-time PCR. By immunofluorescence experiments, we demonstrated that PKH26-labeled-exosomes could be transferred from fibroblasts to a breast cancer epithelial cell line, T47D. Furthermore, to elucidate whether the identified miRs were shuttled into T47D cells via exosomes, we transfected CAFs with cy3-labeled-miRs (cy3-miR-21, cy3-miR-143, cy3-miR-378e), and, then, we isolated the released exosomes. Interestingly, when these exosomes were added to T47D cells, the cy3-miRs were detected in the cytoplasm of T47D cells, and they co-localized with the signals of an exosomal marker, CD63. Then, we demonstrated that TGF-β, apart from its direct role in the activation of normal fibroblasts to CAFs, increased the levels of these miRs in normal fibroblast exosomes. Finally, for the first time, we provided evidence of the role of CAF exosomes and their miR contents in the induction of stemness phenotype in T47D cells. In fact, T47D cells exposed to CAF exosomes or transfected with the identified miRs exhibited a significantly increased capacity to form mammospheres, and increased stem cell and epithelial-mesenchymal transition markers, SOX2, Nanog, Oct3/4, Snail and Zeb. We conclude that CAFs regulate the stemness phenotype of breast cancer cells through exosome-mediated delivery of oncogenic miRs. Our data provide insight into the mechanisms underlying the stemness maintenance in breast cancer.

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