Markers of fibrosis and epithelial to mesenchymal transition demonstrate field cancerization in histologically normal tissue adjacent to breast tumors

Abstract

Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT-PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient-matched, tumor-adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN-1 and TAHN-5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra-lobular fibroblasts expressing MMP2, SPARC, TGF-β3, which are inducers of EMT, were both prevalent in TAHN-1 tissues, sparse in TAHN-5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α-smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN-1 tissues. These results identify cellular processes that are differentially activated between TAHN-1 and TAHN-5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors.

Figure 1

Differential gene expression in TAHN-1 tissues. (a) Listing of genes over-expressed in TAHN-1 tissues. (b) Transcript levels measured by microarray in RNA from individual TAHN-1, TAHN-5 and RM samples measured by microarray (m) or qRT-PCR (p). Asterisk denotes statistically significant (p < 0.05) differences between TAHN-1 and TAHN-5 tissues. (c) Transcript levels of additional up-regulated genes identified by PCR array. Asterisk denotes statistically significant (p < 0.05) differences between TAHN-1 and TAHN-5 tissues. All Y axes denote fold expression over control RNA from disease-free breast tissues.

Figure 2

Immunohistochemical staining of fibroblast populations demonstrates the presence of myofibroblasts within TAHN-1 tissue. (a) Immunohistochemical (IHC) staining for vimentin, α-smooth muscle actin (α-SMA), TGF-β3, MMP2, SPARC and Masson’s Trichrome Stain for collagen in TAHN-1, TAHN-5 and RM tissues. Areas within boxes in α-SMA, TGF-β3 and MMP2 figures are shown at a higher magnification in (b). IHC staining was performed using a diaminobenzadine stain (brown) and nuclei were counterstained with hematoxylin (blue). Since α-SMA stains both myoepithelial cells and myofibroblasts, these populations are identified by morphology and indicated by arrows in the α-SMA panels. “Fib” indicates fibroblasts and “Myo-epi” indicates myoepithelial cells. In Masson’s Trichrome stained tissues, nuclei appear maroon/black and collagen appears blue.

Figure 3

Immunohistochemical staining demonstrates abnormal myoepithelial cells within TAHN-1 tissues. (a) Staining for the myoepithelial marker, α-smooth muscle actin (α-SMA) in TAHN-1 and TAHN-5 issues. Since α-SMA stains both myoepithelial cells and myofibroblasts, myoepithelial populations are identified by morphology and indicated by arrows. (b) Staining for MMP2, SPARC in TAHN-1 and TAHN-5 tissues. (c) Staining for the proliferation marker, Ki67, in TAHN-1 and RM tissues. Immunohistochemical staining was performed as in Figure 2. Arrows marked “Myo-epi” indicate myoepithelial cells.

Figure 4

Double immunofluorescence demonstrates increased expression of S100A4 and α-SMA, common markers of EMT, in both luminal and myoepithelial populations. (a) Double immunofluorescence for S100A4 (green) and α-SMA (red) in RM, TAHN-5 and TAHN-1 tissues. Nuclei are stained with DAPI (blue). (b) Quantitation of α-SMA in luminal epithelial population and S100A4 in luminal and myoepithelial populations. See methods for additional details.

Figure 5

Double immunofluorescence demonstrates loss of cytokeratin in luminal epithelial cells highly expressing S100A4. (a) Double immunofluorescence for S100A4 (green) and pan-cytokeratin (red) in RM TAHN-5 and TAHN-1. Nuclei are stained with DAPI (blue). (b) Quantitation of cytokeratin in luminal epithelial population and S100A4 in luminal and myoepithelial populations. See methods for additional details.

Figure 6

Double immunofluorescence demonstrates additional markers of EMT in TAHN-1 tissues. (a) Double immunofluorescence for TGFβ (green) and α-SMA (red), (b) vimentin (red) and (c) vimentin (red) and SNAIL (green). Nuclei are stained with DAPI (blue). Quantitation of each marker is shown for luminal and myoepithelial populations on the right of each panel.