Carcinoma-associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor-positive cancers, and explore the influences of one of these, miR-26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR-26b was the most highly deregulated microRNA. Using qPCR, miR-26b was confirmed as down-regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR-26b expression changed breast fibroblast behaviour. Reduced miR-26b expression caused fibroblast migration and invasion to increase by up to three-fold in scratch-closure and trans-well assays. Furthermore, in co-culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR-26b expression enhanced both MCF7 migration in trans-well assays and MCF7 invasion from three-dimensional spheroids by up to five-fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR-26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR-26b. In addition, three novel miR-26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR-26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor-positive cancers, and we have identified key genes and molecular pathways that act downstream of miR-26b in CAFs. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ER␣ phosphorylation correlates with patient outcome, ER␤ is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ER␤ that is phosphorylated at serine 105 (S105-ER␤) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ER␤ expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ER␤ regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ER␤ was observed in breast carcinoma and was associated with better survival (Allred score >3), even in tamoxifen-resistant cases, and additionally correlated with ER␤1 and ER␤2 expression. Distinct S105-ER␤ nuclear speckles were seen in some higher grade tumors. S105-ER␤ levels increased in MCF-7 cells in response to 17␤-estradiol, the ER␤-specific agonist diarylpropionitrile , and the partial ER␤-agonist genistein. S105-ER␤ nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17␤-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ER␤1 and ER␤2. Presence of S105-ER␤ in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ER␤ might be a useful additional prognostic marker in this disease.

BackgroundMicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells.Methodology/Principal FindingsWe used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR–92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells.ConclusionsmiR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer.

The presence of fibroblasts with nuclear β-catenin in tumours is a good prognostic indicator, although in the context of tissue culture models these cells can increase the growth and metastatic potential of cancer cells. These apparently paradoxical observations underline the complexity of epithelial-stromal signalling within tumours and highlight an area for further study.

Background: Despite many published studies on ERβ, progress towards understanding its role in breast cancer remains slow. This is largely due to discordant data between mRNA and protein studies as well as failure to take into account the biologically distinct ERβ isoforms and their heterogeneous expression profile.Methods: We compared expression of ERβ, -2 and -5 genes in HB2 and MCF-7 breast cell lines, primary breast fibroblasts (n=5) and whole tissue and laser microdissected epithelial and stromal cells obtained from 25 human breast tumours.Results: Our study shows that the level of gene expression of ERβ isoforms depends on the cell population within a given tumour and varies dramatically in different cellular compartments. This has implications for gene expression analyses and could explain some of the contradictory data published to date, rendering “grind and bind” analyses of ERβ uninformative.Conclusions: With the technology now available, we suggest a more refined approach be adopted to help resolve some of the controversy surrounding ERβ.