Cepeda Mario A, Evered Caitlin L, Pelling Jacob J H, Damjanovski Sashko
Department of Biology, Faculty of Science, University of Western Ontario, BGS room 3053, 1151 Richmond St N, London, ON, N6A 5B7, Canada.
Lawson Health Research Institute, London, ON, Canada.
J Cell Commun Signal. 2017 Jun;11(2):167-179. doi: 10.1007/s12079-016-0373-3. Epub 2017 Jan 9.
Membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14) is a unique protease that cleaves extracellular proteins, activates proMMPs, and initiates intracellular signalling. MCF-7 cells are non-invasive and deficient in MT1-MMP, MMP-2, and MMP-9 expression. We created an MCF-7 cell line (C2) that stably produces active MT1-MMP and demonstrated increased ERK1/2 phosphorylation. MAPK inhibition in this cell line showed an inverse relationship in MMP-2 and MMP-9 transcripts where levels of these genes increased and decreased, respectively. Using invasive MDA-MB 231 cells that endogenously produce MT1-MMP and have naturally high pERK levels, we demonstrated the identical inverse relationship between MMP-2 and -9 transcript and protein levels, suggesting that this novel relationship is conserved amongst MT1-MMP positive breast cancer cells. To further analyze the relationship between MMP-2 and -9 levels, we chemically inhibited activation and catalytic activity of MT1-MMP using a furin and MMP inhibitor, respectively, to show that interference with the functions of MT1-MMP induced changes in MMP-2 and 9 transcript levels that were always inverse of each other, and likely mediated by differential transcriptional activity of the NF-κB transcription factor. Furthermore, we analyzed the functional consequences of these expression changes to show MMP, and in particular ERK, inhibition decreased migration and invasion using 2D culture, and inhibits the formation of an invasive phenotype in Matrigel 3D culture. This study demonstrated a novel inverse transcriptional relationship between MMP-2 and -9 levels and MT1-MMP activity that have functional consequences, and also showed that increases in the levels of MMPs does not necessarily correlate with an invasive phenotype.
膜型-1基质金属蛋白酶（MT1-MMP，MMP-14）是一种独特的蛋白酶，可切割细胞外蛋白质、激活前MMP，并启动细胞内信号传导。MCF-7细胞无侵袭性，且MT1-MMP、MMP-2和MMP-9表达缺失。我们构建了一个稳定产生活性MT1-MMP的MCF-7细胞系（C2），并证明其ERK1/2磷酸化增加。对该细胞系进行MAPK抑制后，MMP-2和MMP-9转录本呈现相反的关系，即这些基因的水平分别升高和降低。使用内源性产生MT1-MMP且天然具有高pERK水平的侵袭性MDA-MB 231细胞，我们证明了MMP-2和-9转录本与蛋白水平之间存在相同的反向关系，表明这种新关系在MT1-MMP阳性乳腺癌细胞中是保守的。为了进一步分析MMP-2和-9水平之间的关系，我们分别使用弗林蛋白酶抑制剂和MMP抑制剂化学抑制MT1-MMP的激活和催化活性，结果表明干扰MT1-MMP的功能会诱导MMP-2和9转录本水平发生相互反向的变化，这可能是由NF-κB转录因子的差异转录活性介导的。此外，我们分析了这些表达变化的功能后果，结果表明抑制MMP，特别是ERK，使用二维培养可降低迁移和侵袭，并抑制基质胶三维培养中侵袭性表型的形成。本研究证明了MMP-2和-9水平与MT1-MMP活性之间存在一种新的反向转录关系，且具有功能后果，同时还表明MMP水平的升高不一定与侵袭性表型相关。
