The genetic regulation of tumor metastasis was investigated. Nm23 was identified on the basis of its reduced steady state RNA levels in highly metastatic murine melanoma cell lines. Reduced nm23 steady state RNA levels were observed in a subset of human breast carcinoma patients; in two reports these patients exhibited greater numbers of lymph node metastases, and significantly reduced disease free and overall survival. Two human nm23 genes were identified, nm23-H1 and nm23-H2, both of which encode 17 kDa proteins. Nm23 has been demonstrated to have two characteristics of a cancer suppressor gene: Transfection of murine nm23-1 into highly metastatic murine K-1735 TK melanoma cells resulted in a reduced incidence of primary tumor formation, significant reductions in tumor metastatic potential, and altered in vitro responsiveness to the cytokine TGF-B. Nm23-H1 was mapped to 17q21 in the C.E.P.H. database, and its somatic allelic deletion was noted in human breast, lung, renal and colorectal carcinomas. A homozygous deletion of nm23-H1 was also observed. Multiple regulatory mechanisms for nm23 in human cancer have been discovered, in addition to its reduced expression. In colorectal carcinoma nm23-H1 allelic deletion, and not overall protein/RNA expression, was significantly correlated with the development of distant metastases. In childhood neuroblastoma, preliminary data have correlated increased nm23 RNA expression and gene amplication with high metastatic potential. The human nm23-H1 and nm23-H2 cDNAs have been subcloned into an expression construct, and human breast and ovarian carcinoma cell lines are in the process of being transfected. The murine nm23-1, and human nm23-H1 and nm23-H2 cDNAs have been expressed in bacteria, and demonstrated to encode a nucleoside diphosphate kinase (NDPK). Comparison of tumor cell NDPK activity and nm23 expression suggests that variations in m23 expression and the metastatic phenotype cause only minor changes in total NDPK activity. In order to determine the relationship between NDPK function and biological suppression of tumor incidence and metastasis, eleven site directed mutant nm23 constructs were prepared. The NDPK activity of the mutated nm23-1 cDNAs will be determined by bacterial expression, and metastasis regulatory activity by transfection into K-1735 TK melanoma cells.
