Myoepithelial Molecular Markers in Human Breast Carcinoma Pmc42-La Cells Are Induced by Extracellular Matrix and Stromal Cells

Abstract

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins alpha-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, alpha-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Contribution of Fibroblast and Mast Cell (Afferent) and Tumor (Efferent) IL-6 Effects within the Tumor Microenvironment

“…Primary human normal mammary fibroblasts (NMF) and breast cancer-associated fibroblasts (CAF) were previously described [15,16] Quantitative Real-time PCR All RNA preparations were extracted and purified via column chromatography (RNeasy kit, Qiagen, Doncaster, Victoria, Australia) as recommended by the manufacturer. The concentration and purity of RNAs was determined using spectrophotometry (Nanodrop ND-1000 spectrophotometer, Thermo Fisher Scientific, Scoresby, Victoria, Australia).…”

“…We found that CAF-conditioned medium induced myoepithelial molecular markers and increased the migratory ability of the epithelial breast cancer cell line PMC42-LA in a 3D organoid model [15,16]. We used the chemiarray system to examine specific soluble factor(s) in CAF-conditioned media that may be induce this EMT.…”

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation

“…We have previously shown the epithelial-like human breast carcinoma PMC42-LA subline undergoes an EMT-like change in response to EGF treatment [27], and in response to factors secreted by human breast carcinoma-derived stromal cells [28,36,37]. It also undergoes EMT at the monolayer wound edge in "scratch" assays which is synergistic with EGF-induced EMT (M. Leigh Ackland, unpublished observation).…”