title: Characterization of downstream Ras signals that induce alternative protease-dependent invasive phenotypes.
abstract: Invasive and metastatic cells require protease expression for migration through the extracellular matrix. Metastatic NIH 3T3 fibroblasts transformed by different activated ras genes showed two different protease phenotypes, rasuPA+/CL- and rasCL+/uPA- (Zhang, J-Y., and Schultz, R. M. (1992) Cancer Research 52, 6682-6689). Phenotype rasuPA+/CL- is dependent on expression of the serine-type protease urokinase plasminogen activator (uPA) and the phenotype rasCL+/uPA- on the cystine-type protease cathepsin L (CL) for lung colonization in experimental metastasis. The existence of multiple invasive phenotypes on ras-isoform transformation implied the activation of alternative pathways downstream from Ras. We now show that c-Raf-1, extracellular signal-regulated protein kinase (ERK)-1, and ERK-2 are hyperphosphorylated, and the ERK activity is high in both the uPA- and CL-dependent ras-transformed invasive phenotypes. Levels of c-Jun and c-Jun NH2-terminal kinase (JNK) activity are also high in the uPA-dependent phenotype, but they are almost undetectable in the CL-dependent phenotype. The uPA Ras-response element is a PEA3/URTF element, and mobility shift assays show a strong PEA3/URTF protein band in the uPA-dependent phenotype. This band is competed by a consensus AP-1 DNA sequence and by antibodies to PEA3 and c-Jun. Thus, the uPA-invasive phenotype appears to require the activation of Ets/PEA3 and c-Jun transcription factors activated by the ERK and JNK pathways, while the CL-invasive phenotype appears to require ERK activity with suppression of JNK and c-Jun activities. These postulates are supported by the introduction of a dominant negative c-Jun, TAM67, into cells of phenotype rasuPA+/CL-, which down-regulated the high uPA mRNA levels characteristic of this phenotype to basal levels and up-regulated basal levels of CL mRNA to levels similar to those observed in cells of phenotype rasCL+/uPA-. We conclude that the JNK pathway acts as a switch between two distinct protease phenotypes that are redundant in their abilities to grow tumors and metastasize.
authors.affiliation: Department of Pathology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.
authors.forename: S
authors.initials: S
authors.lastname: Silberman
authors.forename: M
authors.initials: M
authors.lastname: Janulis
authors.forename: R M
authors.initials: RM
authors.lastname: Schultz
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D011905:Genes, ras; D006801:Humans; D051379:Mice; D008954:Models, Biological; D010641:Phenotype; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010450:Endopeptidases