Understanding the molecular basis of tumor metastasis should provide important insights into new strategies for the treatment of cancer. In contrast to the greater progress achieved in elucidating molecular genetic mechanisms of neoplastic transformation, the nature of the genes responsible for metastasis is far less well understood. Only a few genes directly or indirectly associated with metastatic behavior have been identified so far. This may be related to the possible recessive nature of many of the genetic alterations that lead to tumor cell survival and proliferation in a heterologous tissue environment. While dominant genes can be detected and characterized by gene transfer, identification of recessive mechanisms of metastasis may be achieved by the application of the recently developed general procedure for functional selection of recessive genes using genetic suppressor elements (GSEs) which induce dominant phenotypic changes by interfering with the function of genes from which they are derived. The methodology for GSE isolation has been developed on the model of human topoisomerase II (topo II). In that study expression selection of GSEs was carried out from a mixture of recombinant retroviruses containing a library of random fragment of topo II cDNA. A set of topo II-cDNA-derived fragments, encoding either antisense RNA sequences or short truncated proteins were selected for their ability to induce cellular resistance to topo II-interactive drugs by suppressing topo II expression or function. The GSE selection approach was then expanded to include all the expressed genes by preparing a large (3X10 independent clones) library from normalized (uniform abundance) cDNA of NIH 3T3 cells which should contain GSEs for most of the genes expressed in these cells. By using different selection strategies, we have isolated from this library several biologically active GSEs representing short fragments of known and unknown genes and inducing different selectable phenotypes (drug resistance or neoplastic transformation of NIH 3T3 cells). In the present project, we are planning to isolate from the same library GSEs capable of conferring the ability for spontaneous or experimental metastasis on non-metastasizing mouse cells, by in vivo selection of cells carrying such GSEs. The cloned GSEs will be used as probes for the isolation of the full-length cDNAs of their corresponding genes, whose function would be associated with preventing metastatic conversion. Cell phenotypes associated with suppression or overexpression of these genes in different rodent tumor cell lines will be characterized. The GSE approach should allow the identification and functional analysis of new metastasis suppressor genes.
