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Signaling and Regulation

Immortalized Mammary Epithelial Cells Overexpressing Protein Kinase C Acquire a Malignant Phenotype and Become Tumorigenic in Vivo¹

¹ Department of Cell Biology, Research Area, Institute of Oncology "Angel H. Roffo," University of Buenos Aires, Argentina, and
² Rochelle Belfer Chemotherapy Foundation Laboratory, Division of Hematology/Oncology, Department of Medicine, Mount Sinai School of Medicine, New York University, New York, NY

Requests for reprints: Julio A. Aguirre-Ghiso, Division of Hematology/Oncology, Department of Medicine, Mount Sinai School of Medicine, New York University (Box 1178), One Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-3194; Fax: (212) 996-5787. E-mail: julio.aguirre-ghiso{at}mssm.edu

We have investigated the role of a classical isoform of protein kinase C (PKC) in promoting immortalized mammary cell tumorigenesis in vivo and the contribution of proteases and adhesion molecules to this process. We hypothesized that overexpression of PKC in immortalized mammary epithelial cells may initiate, by activating the mitogenic ERK pathway, early changes in proteases, adhesion molecules, and markers of an epithelium-to-mesenchyme transition that may contribute to in vivo tumorigenesis. Here we show that compared to vector-transfected cells, immortalized murine mammary epithelial cells (NMuMG) overexpressing PKC have stronger activation of (5-fold) ERK1/2 MAPKs, which results in a similar increase in cyclin D1. In addition, PKC-expressing cells showed increased levels of vimentin, fibronectin (FN), ß1-integrins, enhanced adhesion to fibronectin, and its organization into fibrils. Concomitantly, PKC induced a dramatic down-regulation of E-cadherin protein levels and its localization to cell-cell junctions. NMuMG cells expressing PKC became resistant to death by anoikis and formed colonies in soft agar. This effect was dependent on ERK activation, because Mek1/2 inhibition with PD98059 abrogated anchorage-independent growth. Most importantly, unlike control NMuMG cells, PKC-transfected cells inoculated s.c. into nude mice displayed tumorigenic and invasive capacity and were able to spontaneously metastasize. This behavior correlated with increased production of uPA and MMPs-9/-2 induced by PKC. These results suggest that PKC overexpression in immortalized mammary epithelial cells may generate, through an increase in ERK, signaling changes in the expression of genes associated with an epithelium-to-mesenchyme transition that may be sufficient to favor tumor growth in vivo.