Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let-7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let-7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let-7-regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.

Mitogen-activated protein (MAP) kinases play distinct roles in a variety of cellular signaling pathways and are regulated through multiple mechanisms. In this study, a novel 61-kDa member of the MAP kinase family, termed extracellular signal-regulated kinase 7 (ERK7), has been cloned and characterized. Although it has the signature TEY activation motif of ERK1 and ERK2, ERK7 is not activated by extracellular stimuli that typically activate ERK1 and ERK2 or by common activators of c-Jun N-terminal kinase (JNK) and p38 kinase. Instead, ERK7 has appreciable constitutive activity in serum-starved cells that is dependent on the presence of its C-terminal domain. Interestingly, the C-terminal tail, not the kinase domain, of ERK7 regulates its nuclear localization and inhibition of growth. Taken together, these results elucidate a novel type of MAP kinase whereby interactions via its C-terminal tail, rather than extracellular signal-mediated activation cascades, regulate its activity, localization, and function.

To elucidate signal transduction pathways leading to neuronal differentiation, we have investigated a conditionally immortalized cell line from rat hippocampal neurons (H19-7) that expresses a temperaturesensitive simian virus 40 large T antigen. Treatment of H19-7 cells with the differentiating agent basic fibroblast growth factor at 39؇C, the nonpermissive temperature for T function, resulted in the activation of c-Raf-1, MEK, and mitogen-activated protein (MAP) kinases (ERK1 and -2). To evaluate the role of Raf-1 in neuronal cell differentiation, we stably transfected H19-7 cells with v-raf or an oncogenic human Raf-1-estrogen receptor fusion gene (⌬Raf-1:ER). ⌬Raf-1:ER transfectants in the presence of estradiol for 1 to 2 days expressed a differentiation phenotype only at the nonpermissive temperature. However, extended exposure of the ⌬Raf-1:ER transfectants to estradiol or stable expression of the v-raf construct yielded cells that extended processes at the permissive as well as the nonpermissive temperature, suggesting that cells expressing the large T antigen are capable of responding to the Raf differentiation signal. ⌬Raf-1:ER, MEK, and MAP kinase activities in the ⌬Raf-1:ER cells were elevated constitutively for up to 36 h of estradiol treatment at the permissive temperature. At the nonpermissive temperature, MEK and ERKs were activated to a significantly lesser extent, suggesting that prolonged MAP kinase activation may not be sufficient for differentiation. To test this possibility, H19-7 cells were transfected or microinjected with constitutively activated MEK. The results indicate that prolonged activation of MEK or MAP kinases (ERK1 and -2) is not sufficient for differentiation of H19-7 neuronal cells and raise the possibility that an alternative signaling pathway is required for differentiation of H19-7 cells by Raf.Biochemical and genetic studies of cells from a wide variety of species have demonstrated that the signals leading to differentiation are highly conserved and that in many cases, the same compounds are involved in both cell growth and transformation. Two general hypotheses have been suggested to explain why common signaling pathways can lead to different endpoints. First, it has been suggested that both the amplitude and the duration of a particular signal can influence the final biological endpoint (reviewed in reference 18). Alternatively, differences in phenotypes have been attributed to differences in specific cellular environments (both internal and external). While it is likely that both factors contribute to the final biological endpoint, it is important to investigate a number of differentiation systems in order to identify their relative contributions.One major problem, particularly with respect to neuronal cell differentiation, has been the limited availability of appropriate model systems. The best-characterized system to date is the pheochromocytoma cell line PC12, which is derived from a transplantable rat adrenal pheochromocytoma (13). One problem that potentially compl...