MicroRNAs are small non-coding RNA molecules that control gene expression post-transcriptionally, and are known to be altered in many diseases including breast cancer. The aim of this study was to determine the relevance of miR-379 in breast cancer. miR-379 expression was quantified in clinical samples including tissues from breast cancer patients (n=103), healthy controls (n=30) and patients with benign breast disease (n=35). The level of miR-379 and its putative target Cyclin B1 were investigated on all breast tissue specimens by RQ-PCR. Potential relationships with gene expression and patient clinicopathological details were also determined. The effect of miR-379 on Cyclin B1 protein expression and function was investigated using western blot, immunohistochemistry and proliferation assays respectively. Finally, the levels of circulating miR-379 were determined in whole blood from patients with breast cancer (n=40) and healthy controls (n=34). The level of miR-379 expression was significantly decreased in breast cancer (Mean(SEM) 1.9 (0.09) Log10 Relative Quantity (RQ)) compared to normal breast tissues (2.6 (0.16) Log10 RQ, p<0.01). miR-379 was also found to decrease significantly with increasing tumour stage. A significant negative correlation was determined between miR-379 and Cyclin B1 (r=-0.31, p<0.001). Functional assays revealed reduced proliferation (p<0.05) and decreased Cyclin B1 protein levels following transfection of breast cancer cells with miR-379. Circulating miR-379 was not significantly dysregulated in patients with breast cancer compared to healthy controls (p=0.42). This data presents miR-379 as a novel regulator of Cyclin B1 expression, with significant loss of the miRNA observed in breast tumours.
While a range of miRNAs have been shown to be dysregulated in the circulation of patients with breast cancer, little is known about the relationship between circulating levels and tumour characteristics. The aim of this study was to analyse alterations in circulating miRNA expression during tumour progression in a murine model of breast cancer, and to detemine the clinical relevance of identified miRNAs at both tissue and circulating level in patient samples. Athymic nude mice received a subcutaneous or mammary fat pad injection of MDA-MB-231 cells. Blood sampling was performed at weeks 1, 3 and 6 following tumour induction, and microRNA extracted. MicroRNA microArray analysis was performed comparing samples harvested at week 1 to those collected at week 6 from the same animals. Significantly altered miRNAs were validated across all murine samples by RQ-PCR (n = 45). Three miRNAs of interest were then quantified in the circulation(n = 166) and tissue (n = 100) of breast cancer patients and healthy control individuals. MicroArray-based analysis of murine blood samples revealed levels of 77 circulating microRNAs to be changed during disease progression, with 44 demonstrating changes >2-fold. Validation across all samples revealed miR-138 to be significantly elevated in the circulation of animals during disease development, with miR-191 and miR-106a levels significantly decreased. Analysis of patient tissue and blood samples revealed miR-138 to be significantly up-regulated in the circulation of patients with breast cancer, with no change observed in the tissue setting. While not significantly changed overall in breast cancer patients compared to controls, circulating miR-106a and miR-191 were significantly decreased in patients with basal breast cancer. In tissue, both miRNAs were significantly elevated in breast cancer compared to normal breast tissue. The data demonstrates an impact of tumour epithelial subtype on circulating levels of miRNAs, and highlights divergent miRNA profiles between tissue and blood samples from breast cancer patients.
Intercellular communication between breast tumour cells and stromal cells plays a fundamental role in cancer initiation and progression. Mesenchymal Stem Cells(MSCs) are multipotent cells with the proven ability to home to the site of breast tumours and integrate into the tumour architecture. The mechanisms of MSC interaction with breast cancer cells are poorly defined. Exosomes are microvesicles secreted by cells that have the capacity to transport genetic material including microRNAs(miRNAs), which are important regulators of gene expression. However, the miRNAs selectively packaged into exosomes, and their true role in the tumour microenvironment, is poorly understood. Aim: Investigate the presence of MSCs in primary breast tumours, and identify exosome-encapsulated miRNAs secreted by breast cancer cells and MSCs in vitro. Methods: Stromal cells were isolated from primary breast tumours and characterized based on MSC-associated cell surface antigens. Tumour stromal cells were cultured in appropriate conditions to investigate potential for differentiation into osteoblasts or adipocytes, followed by Von Kossa staining of calcium deposition, and Oil Red O staining of fat droplets respectively. MSCs isolated from healthy volunteers and breast cancer cell lines were cultured in exosome-depleted media for 48 hrs, and secreted exosomes isolated using ultracentrifugation. Transmission Electron Microscopy(TEM) and Western Blot were performed to confirm the presence of exosomes in the purified fraction. Extracted miRNA was subjected to global miRNA expression analysis using the Exiqon miRCURY™ LNA Array. Results: A subpopulation of tumour stromal cells expressed the full panel of surface markers associated with MSCs. This subpopulation also had the capacity to differentiate into osteoblasts and adipocytes, demonstrated by calcium deposition and formation of fat droplets. Exosomes were successfully isolated from conditioned media of breast cancer cell lines and MSCs. TEM revealed microvesicles with the appropriate size of 40-100nm. Western Blot confirmed the presence of the exosome-associated protein CD63. miRNA array analysis identified 413 miRNAs encapsulated in MSC-secreted exosomes from a panel of 2089 analysed, while each of the breast cancer cell lines secreted from 381 to 394 miRNAs in exosomes. Of these, 287 were common to all four breast cancer cell lines. Interestingly, a subset of these miRNAs are known to target genes associated with cell cycle regulation and apoptosis (e.g. miR-15a, miR-204, miR-221, miR-223). A panel of the miRNAs also appeared to have differential expression with reference to epithelial subtype. Conclusion: MSCs are present in primary breast tumours, and elucidating their interaction with breast cancer cells will be fundamental to understanding their role in the tumour microenvironment. Bidirectional transfer of exosome-encapsulated miRNAs may play an important role in this intercellular communication. Citation Format: Claire Glynn, Sonja Khan, Cathy Brougham, Cillian Clancy, Doireann Joyce, Peter Dockery, Michael J. Kerin, Roisin M. Dwyer. Investigation of exosome-encapsulated microRNA secretion in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3590. doi:10.1158/1538-7445.AM2014-3590
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