Microenvironmental Changes in DCIS: Myoepithelial Cells Change from Tumour Suppressor to Tumour Promoter.

Abstract

Abstract Background: The mechanism of transition of ductal carcinoma in-situ (DCIS) to invasive cancer is poorly understood but recently changes in the microenvironment have been implicated. A unique feature of the DCIS microenvironment is the presence of a myoepithelial population. Normal myoepithelial cells (MECs) have a broad tumour suppressor function but these cells show changes in gene expression and phenotype in DCIS.Hypothesis: We hypothesised that during the evolution of DCIS breast myoepithelial cells undergo a switch in gene expression profile that compromises their suppressor function and may lead to promotion of tumour progression.Materials and methods: To investigate this, Immuno-Laser Capture Microdissection was used to isolate MECs from normal and DCIS breast tissues followed by whole genome expression profiling using Affymetrix HGU-133 plus2.0 arrays. The data were analysed using Bioconductor (www.bioconductor.org) packages within the open source R statistical environment (www.r-project.org). We set a double threshold for significant changes in gene expression with a False Discovery Rate (FDR) <= 0.05 and an absolute fold change ≥4.5.Results: Ingenuity Pathways software analysis showed clustering of most of the altered genes in cancer, cell death and cell-cell signalling networks, with the integrin pathway as the top canonical pathway. Validation using QRT-PCR and immunohistochemistry (IHC) revealed a 71.4% correlation rate with the array results. Most dramatic was up-regulation of Fibronectin-1 (FN1) in DCIS MEC. IHC analysis for FN1 on normal (n=88) and DCIS tissues with and without associated invasion (n=87) confirmed a strong correlation between FN1 protein expression by MEC and DCIS (p<0.0001). We set cut off point of 50% for percentage of ducts showing positive staining for FN1 by MECs in pure DCIS cases and DCIS cases that have progressed to IDC and found a statistically significant correlation between high expression level (>50%) and presence of invasion (p=0.006). Moreover, culture of normal and DCIS-modified MEC lines on a Fibronectin matrix revealed that FN1 modifies MEC function and abrogates their tumour suppressor activity.Other key alterations identified and validated include up-regulation of the recently characterised Nephronectin and down-regulation of PTHLH, FGFR2, ADAMTS5, TGFBR3, and CAV2, each of which has been implicated in suppressing tumour growth and invasion or inhibition of angiogenesis.Conclusion and significance: This is the first study to use this in-vivo technique to investigate molecular changes in MEC in DCIS and to reveal that DCIS associated MECs show up-regulation of the tumour-promoting molecule FN1. Our data support the hypothesis that there is switch in MEC function from suppressor to potential promoter activity that can influence the progression of in-situ to invasive disease and may lead to novel predictive and therapeutic targets. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4161.