MAB19562X Sigma-AldrichAnti-Laminin-5 Antibody, clone D4B5, Alexa Fluor® 488 conjugated

The cell surface glycoprotein Trask/CDCP1 is phosphorylated during anchorage loss in epithelial cells in which it inhibits integrin clustering, outside-in signaling, and cell adhesion. Its role in cancer has been difficult to understand, because of the lack of a discernible pattern in its various alterations in cancer cells. To address this issue, we generated mice lacking Trask function. Mammary tumors driven by the PyMT oncogene and skin tumors driven by the SmoM2 oncogene arose with accelerated kinetics in Trask-deficient mice, establishing a tumor suppressing function for this gene. Mechanistic investigations in mammary tumor cell lines derived from wild-type or Trask-deficient mice revealed a derepression of integrin signaling and an enhancement of integrin-growth factor receptor cross-talk, specifically in unanchored cell states. A similar restrictive link between anchorage and growth in untransformed epithelial cells was observed and disrupted by elimination of Trask. Together our results establish a tumor-suppressing function in Trask that restricts epithelial cell growth to the anchored state.

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 pro-apoptotic 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.