Basic and translational research has investigated the role of nm23 in the regulation of tumor metastasis. Nine transfection studies have documented that overexpression of nm23 in various tumor cell lines resulted in a 50-90% decrease in tumor metastatic potential in vivo. The biochemical mechanism whereby Nm23 suppresses metastatic potential is under investigation. We have previously reported that a histidine dependent protein phosphotransferase activity correlated with Nm23-H1 suppression of tumor cell motility, an aspect of metastasis. Using a computer homology search to lower eukaryotic proteins that interact with histidine kinases, we identified the Kinase suppressor of ras (Ksr) as a binding partner and substrate of Nm23-H1 in vitro. Nm23-H1 phosphorylates at least one serine in the CA4 domain of KSR in the manner of a histidine protein kinase. Ksr is thought to be a scaffold molecule in the Map kinase pathway. The data suggests the hypothesis that Nm23-H1 may function through modulation of signal transduction in this pathway. Translational research on nm23 proposes that elevation of Nm23 expression in micrometastatic or overtly metastatic breast or other carcinomas may limit colonization, motility and de-differentiation, with a clinical benefit. In order to determine how to elevate breast carcinoma Nm23 expression a model system consisting of four well characterized human cell lines differing in Nm23 expression and in vivo metastatic potential was used. The nm23-H1 promoter was cloned and a 2.1 kb fragment shown to confer differential expression when transfected into each cell line tethered to a reporter gene. Deletion analysis revealed a 450 bp promoter fragment which contributed to the regulation of Nm23-H1 expression. Within this fragment a striking pattern of transcription factor binding sites emerged: Sites known to determine mammary specific gene expression in the MMTV-LTR and milk genes (MAF, NF1, F11) were all present as well as the glucocorticoid response elements needed to activate them. Deletion of the mammary specific sites decreased reporter gene expression. Based on these observations we found that dexamethasone, prednisolone and other glucocorticoid agonists elevated Nm23-H1 expression in vitro, but at """"""""physiologic"""""""" rather than """"""""pharmacologic"""""""" levels. A related compound which binds to the glucocorticoid receptor, medroxyprogesterone acetate (MPA)elevated Nm23-H1 expression in vitro at pharmacologic levels and was chosen for futher study. At low doses MPA is used as a contraceptive, but at high doses it has shown efficacy in advanced breast and endometrial cancers. An optimal dose and schedule has not been established clinically. We find that MPA uses the glucocorticoid receptor (GR), and not the androgen or progesterone receptors to elevate Nm23-H1, and that it results in GR translocation to the nucleus. In vivo work is commencing to determine whether MPA elevates Nm23-H1, and reduces metastasis in vivo.
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