Abstract B127: Loss of miR-92 expression in breast epithelial cells is associated with cancer progression
Authors: Smith Laura , Baxter Euan , Hanby Andrew , Hughes Thomas , Millican-Slater Rebecca , Verghese Eldo , Speirs Valerie 
Published: May 2015

Abstract
MicroRNAs (miR) are a class of short non-coding RNAs that regulate gene expression and are commonly dysregulated in cancers, including those of the breast. MiR-92 is thought to function as an oncogene, promoting cell proliferation and reducing apoptosis. We have previously shown that expression of ERβ1 is negatively regulated by miR-92 in unselected non-microdissected breast cancers, providing a mechanism for down-regulation of this putative tumour suppressor gene. Here, we aimed to confirm the function of ERβ1 in breast cancer cells and examined expression of miR-92, specifically in the breast epithelium, during breast cancer progression. We also investigated the role of miR-92 in fibroblasts and examined whether differential expression in these cells might modify the behaviour of breast cancer epithelial cells.
Stable ERβ1 overexpression was achieved using a MoMLV-based vector (pFBneo) or silenced by siRNA and effects were examined using various functional assays. We used laser microdissection (LMD) to isolate matched normal, co-incident DCIS and invasive tissue from 10 breast cancer patients. RNA was extracted, cDNA synthesised and preamplified using MegaPlex PreAmp Primers. MiR-92 expression was analysed by real-time PCR. Expression of ERβ1 protein was concurrently analysed for each patient by immunohistochemistry. LMD was also used to isolate matched normal and cancer-associated fibroblasts from 21 breast cancer patients and miR-92 expression was examined as above. Loss and gain of miR-92 in fibroblasts was achieved using anti-miR-92 miRNA and miRNASelect pEP-hsa-mir-92a-1 expression vector, respectively. The invasive potential of these fibroblasts was examined using a 2D modified Boyden chamber assay.
ERβ1 overexpression decreased growth and induced cell death in T47D and BT-20 breast cancer cells. Surprisingly, we found that whilst absolute miR-92 expression levels varied across patients the expression pattern of miR-92 was consistent in all cases; miR-92 levels decreased during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p&lt;0.01) and lowest in invasive breast tissue (p&lt;0.01). Expression of ERβ1 and miR-92 were not inversely correlated as expected. Further examination of the staining patterns of ERβ1 showed nuclear expression in normal breast epithelium with increased cytoplasmic immunoreactivity during progression to DCIS and invasive breast cancer. ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues, however miR-92 expression levels remained unchanged between matched microdissected adjacent normal and cancer-associated fibroblasts. Despite this, we demonstrated that fibroblasts can contribute to breast tumour progression as manipulation of miR-92 levels influenced the invasive potential of breast cancer epithelial cells.
In summary, whilst confirming a potential tumour suppressor role for ERβ1 we have shown that expression is not inversely correlated with its negative regulator miR-92 in breast epithelium, suggesting other methods of regulation. Expression of miR-92 was lost, rather than gained, in breast epithelial cells during breast cancer progression correlating with a shift in ERβ1 staining from nuclei to the cytoplasm. While miR-92 expression levels remained unchanged in stromal fibroblasts, our data support a functional role in fibroblasts and show that differential miR-92 expression in these cells can influence the invasive capacity of breast cancer epithelial cells. Finally, our study highlights the importance of isolating specific cell types prior to expression analyses since changes in the epithelium may be masked by expression from the stroma and vice versa.
Citation Format: Laura Smith, Euan Baxter, Andrew Hanby, Thomas Hughes, Rebecca Millican-Slater, Eldo Verghese, Valerie Speirs. Loss of miR-92 expression in breast epithelial cells is associated with cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B127.