CHARACTERIZATION OF DOWNSTREAM RAS SIGNALS THAT INDUCE ALTERNATIVE PROTEASE-DEPENDENT INVASIVE PHENOTYPES

Invasive and metastatic cells require protease expression for migratio n through the extracellular matrix. Metastatic NIH 3T3 fibroblasts tra nsformed by different activated ras genes showed two different proteas e phenotypes, ras(UPA+/CL-) and ras(CL+/uPA-) (Zhang, J-Y,, and Schult z, R. M. (1992) Cancer Research 52, 6682-6689). Phenotype ras(uPA+/CL- ) is dependent ras the serine-type protease urokinase plasminogen acti vator (uPA) and the phenotype ras(CL+/uPA-) on the cystine-type protea se cathepsin L (CL) for lung colonization in experimental metastasis, The existence of multiple invasive phenotypes on ras-isoform transform ation implied the activation of alternative pathways downstream from R as. We now show that c-Raf-1, extracellular signal-regulated protein k inase (ERK)-1, and ERK-2 are hyperphosphorylated, and the ERK activity is high in both the uPA- and CL-dependent ras-transformed invasive ph enotypes. Levels of c-Jun and c-Jun NH2-terminal kinase (JNK) activity are also high in the uPA-dependent phenotype, but they are almost und etectable in the CL-dependent phenotype, The uPA Ras-response element is a PEA3/URTF element, and mobility shift assays show a strong PEA3/U RTF 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 acti vity with suppression of JNK and c-Jun activities. These postulates ar e supported by the introduction of a dominant negative c-Jun, TAM67, i nto cells of phenotype ras(uPA+/CL-), which down-regulated the high uP A 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 ras(CL+/uPA-). We conclude that the JNK pathway acts as ras a switch between two distinct protease phenotypes that are redundant in their abilities to grow tumors and metastasize.