Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells

Abstract

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the proapoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

HOXB7 Overexpression Leads Triple-Negative Breast Cancer Cells to a Less Aggressive Phenotype

“…Moreover, the search for potential downstream targets showed that the HOXB7overexpressing cells, along with decreased CTNNB1 expression, presented enriched direct interaction of HOXB7 with the promoter regions of EGFR, DNMT3B, COMMD7, and CDH1 (Figure 7). The maintenance of breast cells on a reconstituted basement membrane results in a 3D-growth that recapitulates several aspects of glandular architecture in vivo that are lost in 2D culture, as demonstrated with MCF10A cells [21,34]. Interestingly, MDA231 HOXB7overexpressing cells (D3) formed smaller and more compact spheroids compared to the spread and branched growth shown by the control cells.…”

“…The maintenance of breast cells on a reconstituted basement membrane results in a 3D-growth that recapitulates several aspects of glandular architecture in vivo that are lost in 2D culture, as demonstrated with MCF10A cells [ 21 , 34 ]. Interestingly, MDA231 HOXB7-overexpressing cells (D3) formed smaller and more compact spheroids compared to the spread and branched growth shown by the control cells.…”

Role of the integrin-linked kinase (ILK)/Rictor complex in TGFβ-1-induced epithelial–mesenchymal transition (EMT)

“…Normal human epithelial MCF10A and 184hTERT cells were cultured as described previously. 46,47 To examine Rictor phosphorylation, cells were serum starved for 24 h, then treated with or without 10% fetal bovine serum for 30 min. Cells were incubated with QLT0267 at concentrations specified in the text (QLT, Vancouver, BC, Canada), LY294002 (Sigma-Aldrich, Oakville, ON, Canada) or rapamycin (LC Laboratories, Woburn, MA, USA), 5 h, 1 h and 30 min.…”

Met Receptor Acts Uniquely for Survival and Morphogenesis of EGFR-Dependent Normal Mammary Epithelial and Cancer Cells

“…NMuMG and MCF-10A cell lines possess morphogenic capabilities when resuspended and cultured in a 3-D matrix like collagen [16] , [19] . AG1478 had a dramatic effect on 3D-morphogenesis of both cell lines, inducing death of all cells ( Fig.…”