The PI discovered the first metastasis suppressor gene, nm23. Basic and translational research has investigated the role of Nm23 in the regulation of tumor metastasis. Eleven 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 identified an interaction between Nm23-H1 and Kinase suppressor of ras (Ksr), a putative scaffold protein for the Erk Map kinase complex. Nm23-H1 bound and phosphorylated Ksr in vitro. Transfection of breast carcinoma cells with wild type Nm23-H1 reduced Map kinase activation as compared to vector transfectant controls. We suggested the hypothesis that Nm23-H1 interaction with Ksr resulted in altered scaffold function, reduced Map kinase signaling and altered metastatic potential. Recently we found that Nm23-H1 expression level altered the stoichiometry of client protein binding to the Ksr scaffold. In transient and stable Nm23-H1 overexpressing breast cells, greater Hsp90 was bound to Ksr than in control transfectants. The greater Hsp90 binding was accompanied by accelerated degradation of Ksr, and enhanced sensitivity to the geldanamycins in anchorage independent proliferation assays. The data indicate a potential mechanism of Nm23-H1 modulation of Ksr scaffold function, and suggest an interaction of metastatic potential and drug sensitivity.A second potential mechanism of action is under study, based on microarray analysis of control- and Nm23-H1 transfectants. We have identified several genes preferentially expressed in the control transfectants, including c-met, CTGF, EDG2, FZD1, L1CAM, NETO2, PTN, SMOH, etc. Expression of each gene was elevated in transfectants expressing Nm23 mutants which failed to suppress motility. Several of the genes, including c-met, L1CAM, PTN, GA17, RSK1, DDR1 and CP exhibited the same expression trend in a public database of human breast cancer microarray data when supervised by Nm23 expression level, confirming their potential relevance to human cancer metastasis. Transfections are underway to determine the functional impact of these genes on motility and soft agar colonization, aspects of metastasis. Translational research on Nm23 proposed 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. Analysis of the nm23-H1 promoter revealed a 400 bp region which controlled expression, and contained a cassette of transcription factors regulated by a glucocorticoid response element (GRE). Deletion studies showed that these sites were functional in regulating nm23-H1 transcription. Medroxyprogesterone acetate (MPA), an unusual agonist for GR, as well as the androgen receptor and progesterone receptor, elevated Nm23-H1 expression of breast carcinoma cell lines in vitro. MPA acted via a post-transcriptional mechanism using the GR, at pharmacologic doses. We have reported preclinical experiments to determine if MPA can halt metastatic colonization. Mice were injected iv with metastatic human MDA-MB-231 breast carcinoma cells, and permitted to develop micrometastases for one month. Mice were then randomized to vehicle or MPA, the latter given in a one month induction and subsequent bimonthly maintenance dose. Mice receiving MPA had significantly fewer gross metastases in the lung, a smaller proportion of mice with metastases and smaller metastases. Immunohistochemistry revealed that MPA treated mice had a greater proportion of pulmonary metastases with high Nm23 expression. Side effects included weight gain, but no effects on bone mineral density or mammary histology. The data indicate that agents elevating metastsis suppressor gene expression may be effective against metastatic colonization.
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