Mazzoni Esteban, Adam Alejandro, Bal de Kier Joffe Elisa, Aguirre-Ghiso Julio A
Department of Cell Biology, Research Area, Institute of Oncology Angel H. Roffo, University of Buenos Aires, Argentina.
Mol Cancer Res. 2003 Aug;1(10):776-87.
We have investigated the role of a classical isoform of protein kinase C (PKCgamma) in promoting immortalized mammary cell tumorigenesis in vivo and the contribution of proteases and adhesion molecules to this process. We hypothesized that overexpression of PKCgamma 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 PKCgamma have stronger activation of (approximately 5-fold) ERK1/2 MAPKs, which results in a similar increase in cyclin D1. In addition, PKCgamma-expressing cells showed increased levels of vimentin, fibronectin (FN), beta1-integrins, enhanced adhesion to fibronectin, and its organization into fibrils. Concomitantly, PKCgamma induced a dramatic down-regulation of E-cadherin protein levels and its localization to cell-cell junctions. NMuMG cells expressing PKCgamma 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, PKCgamma-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 PKCgamma. These results suggest that PKCgamma 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.
我们研究了蛋白激酶C的一种经典同工型（PKCγ）在促进永生化乳腺细胞体内肿瘤发生中的作用，以及蛋白酶和黏附分子在此过程中的作用。我们假设，永生化乳腺上皮细胞中PKCγ的过表达可能通过激活促有丝分裂的ERK途径，引发蛋白酶、黏附分子以及上皮-间质转化标志物的早期变化，这些变化可能有助于体内肿瘤发生。在此我们表明，与载体转染细胞相比，过表达PKCγ的永生化小鼠乳腺上皮细胞（NMuMG）对ERK1/2丝裂原活化蛋白激酶的激活更强（约5倍），这导致细胞周期蛋白D1有类似程度的增加。此外，表达PKCγ的细胞中波形蛋白、纤连蛋白（FN）、β1整合素水平升高，对纤连蛋白的黏附增强，且纤连蛋白组织形成纤维。同时，PKCγ诱导E-钙黏蛋白蛋白水平显著下调及其在细胞间连接处的定位减少。表达PKCγ的NMuMG细胞对失巢凋亡诱导的死亡产生抗性，并在软琼脂中形成集落。这种效应依赖于ERK激活，因为用PD98059抑制Mek1/2可消除不依赖贴壁的生长。最重要的是，与对照NMuMG细胞不同，皮下接种到裸鼠体内的PKCγ转染细胞具有致瘤和侵袭能力，并且能够自发转移。这种行为与PKCγ诱导的尿激酶型纤溶酶原激活物（uPA）和基质金属蛋白酶-9/-2的产生增加相关。这些结果表明，永生化乳腺上皮细胞中PKCγ的过表达可能通过ERK增加，引发与上皮-间质转化相关基因表达的信号变化，这可能足以促进体内肿瘤生长。
