Molecular Biology of the Metastatic Phenotype

Division Of Clinical Sciences - Nci

Investigators

Linked publications & trials

Abstract

The PI discovered the first metastasis suppressor gene, Nm23 (NME). Metastasis suppressor genes are genes that, when re-introduced into metastatic tumor cells at physiologic levels of expression, significantly inhibit metastasis in vivo without affecting primary tumor size. These genes provide a window on the metastatic process independent of events in the primary tumor. Many metastasis suppressor genes have now been described and our goal is to translate this knowledge to the clinic. In my lab, basic and translational research has investigated the role of NME1 in the regulation of tumor metastasis. Eleven transfection studies have documented that overexpression of NME1 or NME2 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 complex. In the past year we have conducted two fundamental mechanistic studies: (1) The lab investigated the role of dynamin 2 (DNM2) in NME function. The role of dynamin (DNM2), a GTPase regulating membrane scission of vesicles in endocytosis, was examined in NME1 and NME2 regulation of tumor cell motility and metastasis. Overexpression of NMEs in two cancer cell lines increased endocytosis of transferrin receptor (TfR) and EGF receptor (EGFR) concurrent with motility and migration suppression; internalized vesicles costained with Rab5, depleted AP2 from cell surface, and exhibited increased Rab5-GTP levels, consistent with endocytosis. The dynamin inhibitors Iminodyne-22 and Dynole-34-2, or shRNA-mediated downregulation of DNM2, impaired NME ability to augment endocytosis or suppress tumor cell motility. In a lung metastasis assay NME1 overexpression failed to significantly suppress metastasis in the DNM2 knockdown cells. Using the EGF-EGFR signaling axis as a model in MDA-MB-231T cells, NME1 decreased pEGFR and pAkt expression in a DNM2-dependent manner, indicating the relevance of this interaction to downstream signaling. NME/DNM2 interaction was confirmed in two-way coimmunoprecipitations. Using transfection of a NME1 site directed mutant lacking a histidine protein kinase activity but retaining a nucleoside diphosphate kinase (NDPK) activity, the NDPK activity of NME was insufficient to promote endocytosis or inhibit EGFR signaling. We report a novel mechanism of action: Addition of NME1 or NME2 to DNM2 in vitro facilitated DNM2 oligomerization and increased GTPase activity, both required for vesicle scission. A NMEDNM2 interaction may contribute to metastasis suppression by altering the tumor endocytic and motility phenotypes. (2) We investigated a potential role for NME1 as a component of tumor exosomes. NME1 is encapsulated into exosomes from tumor cells at levels equal to or higher than in the cytoplasm. Exosomal NME1 inhibits motility and migration in vitro and showed a trend for metastasis prevention in vivo. To enhance NME delivery, liposomes containing NME were prepared which showed significant metastasis preventive activity. This research points to a potential new clinical approach to preventing metastasis (Clinical and Experimental Metastasis 39: 815-831, 2022).