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 Ezrin has been documented to bind Nm23 through its FERM domain. Nm23 inhibited Ezrin induction of motility in vitro and metastasis in vivo. Nm23 binding to Ezrin outcompeted for Ezrin-Fak association, which may represent a new mechanism of inhibition of metastatic signaling. 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. The interaction of Nm23 and Gelsolin has been demonstrated to influence actin dynamics, a potential mechanistic explanation for Nm23's anti-metastatic effects. One of the potential avenues to drug a suppressor gene is to identify inverse correlates of its expression. 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. Other 4T1 experiments demonstrated that shRNA down-regulation of LPA1 also prevented metastasis, and is the mechanism of action of 0719. Treatment with 0719 only after mammary fat pad removal, akin to adjuvant therapy, was also metastasis suppressive.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. When animals injected with MDA-MB-231T cells were treated with 0719 and the drug was removed, death ensued. 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, but0719 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.These data, to our knowledge, represent the first drug candidate to induce metastatic dormancy. A manuscript describing this project is in press in JNCI.In a high risk, high impact effort, I have collaborated with Dr. George Sledge to identify inverse expression correlates for multiple metastasis suppressor genes, not just Nm23. Using a gene expression analysis of siRNA knockdown of 19 metastasis suppressors, fifteen genes have been identified that are each inversely correlated to at least five, and up to eighteen metastasis suppressors. This effort is expected to lead to the identification of new genes mediating metastatic colonization, and new preventives and therapeutics.
NIH ExPorter
NIH ExPorter