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 mechanism of Nm23 suppression of metastasis is likely complex. Previous work has demonstrated a histidine protein kinase activity for Nm23 and identified Ksr and disruption of the Erk signaling pathway as substrates. Another potential mechanism of action of Nm23 is through binding to other proteins. This project asked, in an unbiased manner, which proteins bind to a Flag tagged Nm23-H1 and -M1 in vitro and in vivo. Murine 4T1 cells were implanted in the mammary fat pad (mfp), a primary tumor was removed 10 days later, and metastases were evident in spleen, liver, lung and lymph nodes eight weeks post-injections. Transfection of 4T1 cells with Nm23-H1 and -M1 inhibited metastasis to the liver by 69% and 75%, respectively. In collaboration with Dr. Tim Veenstra Nm23-H1 co-immunoprecipitating proteins have been identified from from 4T1 cells in vitro, primary in vivo tumors and metastases. A previously undisclosed interaction of Nm23 and Ezrin has been validated. Ezrin links the cytoskeleton with signaling complexes and could exert multiple effects on the metastatic process. Co-immunoprecipiations have been validated, and the Ezrin binding site for Nm23 is under investigation using site directed mutagenesis. Overexpression of the N- and C-terminal pieces of Ezrin have shown varying effects on Nm23 binding and in vitro motility. Gelsolin is another new validated Nm23 binding protein. The interaction of Nm23 and Gelsolin only occurs in cell lines with relatively high levels of Nm23 expression, a trend not previously observed. We hypothesize that the interation of Nm23 and Gelsolin may mediate the nonmetastatic phenotype of Nm23. Site directed mutagenesis of Gelsolin is underway to identify the Nm23 binding site. A third potential contributor to Nm23 suppression of metastasis is downstream changes in gene expression, based on microarray analysis of control- and Nm23-H1 transfectants. The lysophosphatidic acid receptor (LPA1), a G-protein coupled receptor for the serum lysophosphatidic acid (LPA) was found to be inversely related to Nm23 expression in cell lines, human breast tumors, and knock-out animal tissues. Forced re-expression of LPA1 overcame Nm23 suppression of tumor cell motility in vitro and metastasis in vivo. We hypothesized that an inhibitor of LPA1 would act as a metastasis suppressor and have preclinically validated Debio 0719. Administration of 0719 to animals injected into the mammary fat pad with murine 4T1 cells had no effect on primary tumor size. After surgical removal of the primary tumor, metastasis in the lymph nodes, lungs and liver was quantified 10 weeks post-injection. 0719 induced a significant suppression of metastasis in all sites. Confirmatory experiments showed a non-significant effect of 0719 on MDA-MB-231T human breast tumor growth in the mammary fat pad, but a significant inhibition of lung colonization after tail vein injection. Analysis of tissue from vehicle and 0719 treated animals showed that: (1) proliferation measured by Ki67 staining was high and equivalent in primary tumors treated with vehicle or 0719, but was reduced in lung and liver metastases treated with 0719;(2) similar trends were observed in pErk staining;(3) the opposite trend was osberved for p-p38 staining- no difference was observed in primary tumors, but 0719 treated lung and liver metastases exhibited higher levels of expression than vehicle controls. The data indicate that 0719 induced site specific dormancy in metastasis, a novel indication for a drug to date. The mechanism of metastatic dormancy is under investigation.Using culture supernatants from primary mammary fat pad fibroblasts, lung fibroblasts and liver parenchymal cells, different patterns of cytokines were expressed in primary versus metastatic tissue microenvironments. Debio 0719 is hypothesized to influence the pattern of cytokine expression in specific microenvironments, and the pattern of cytokine receptors on tumor cells, to favor a dormancy respons. A third project is exploring the contribution of Nm23 binding proteins to its regulation of tumor 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. A Phase II trial of MPA opened at Indiana University, (PI Kathy Miller) in 2007, funded by an Avon-NCI grant to Dr. Miller. Patients will be post-menopausal and have tumors that are hormone receptor negative, thus promoting utilization of MPA through the GR. Patients will be randomized to MPA or, in a second cohort, MPA plus metronomic IdoCM as an anti-angiogenenic agent. The primary objective is clinical benefit rate;secondary objectives include toxicity, PK, and surrogates of MPA effect in skin and tissue biopsies and the primary tumor. Our laboratory and that of Dr. Merino will participate in the tissue analysis.
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