Basic and translational research has investigated the role of nm23 in the regulation of tumor metastasis. Seven transfection studies have documented that overexpression of nm23 cDNA in either breast or prostate carcinoma or melanoma cell lines results in a 50-90% decrease in tumor metastatic potential in vivo. The biochemical mechanism whereby Nm23 suppresses metastatic potential is under investigation. We have transfected site directed mutants of nm23-H1 into breast carcinoma cell lines and examined their in vitro motility to correlate Nm23 structure and biological function. Two mutations abrogated the tumor cell motility suppressive capacity of Nm23: proline 96, the killer of prune mutation in the Drosophila nm23 homolog, which can cause aberrant differentiation; serine 120, a site of mutation in human Stage IV neuroblastomas, and phosphorylation. In vitro assays of purified wild- type and mutant Nm23-H1 proteins found that the proline 96 and serine 120 mutant proteins were uniquely deficient in aspects of histidine- dependent protein phosphotransferase pathways. Histidine protein kinases are poorly described in mammalian cells, but form the two- component or histidyl-aspartyl phosphorelay signal transduction system in prokaryotes. Using a computer homology search, we have identified three mammalian proteins which share amino acid sequences with eukaryotic two-component response regulators. We hypothesize that Nm23 may interact with these proteins in an analogous method, and are currently conducting two-hybrid, kinase and developmental studies. 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. Two observations have been made in this model system: (1) Footprinting of the minimal region of the promoter determining differential expression has revealed usage of 21 transcription factor binding sites comparably by all four cell lines. Seven sites were used to different extents by the four cell lines and selected for futher analysis among 11 breast carcinoma cell lines. Of these, 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, CTF/NF1, F11, Glucocorticoid receptor, metal response element) were all differentially used. Transfection studies, currently underway for individual sites, have confirmed that the F11 site acts as a repressor for Nm23 expression, and interference with this site may constitute a strategy for elevation of Nm23 expression. Future studies will extend this research to combinations of sites and characterization of functional transcription factors using these sites. (2) One metastatically competent breast carcinoma cell line, MDA-MB-231, exhibited altered DNA methylation of the nm23-H1 promoter on southern blots. Studies using 5-aza-2-deoxycytidine (5azadC), a DNA methylation inhibitor, increased Nm23 expression in this cell line, and three others. In collaboration with Baylor University, we are asking whether differences in the DNA methylation status of the nm23-H1 promoter are found in human breast carcinomas. In vivo investigation of 5azadC is planned. - breast cancer, metastasis,
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