creased proliferation, growth, and survival by means other than Ras V12 are not sufficient to cause the metastatic progression of scrib -/cells. Thus, the metastasis-promoting effect of scrib inactivation is highly dependent on its specific cooperation with the Ras V12 allele. Moreover, aside from its known effects on proliferation, growth, and survival, Ras V12 may function through an as yet undefined cellular mechanism to elicit metastatic progression in scrib -/cells. It has proven difficult

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... to systematically study the genetic basis of metastasis with the currently available techniques. The Drosophila system described here circumvents the complication of acquired background mutations, which can occur through repeated passaging of cell lines or during the typically long latent period of mammalian tumor progression. In our initial screen, we found that mutations in different genes affecting the same physiological process-epithelial cell polarity maintenance-are sufficient in combination with Ras V12 to promote metastatic behavior in vivo. Interestingly, later stage human cancers typically lose cell polarity markers and epithelial structure during epithelial to mesenchymal transition (31). Also, E-cadherin loss, basement membrane degradation, and induction of cell migration and invasion relate well to observations made in human metastasis (15, 31, 32), which suggests that the ongoing screen will uncover genes and general mechanisms relevant to malignancy in humans. It has been proposed that oncogenes such as Ras may play a dual role in tumorigenesis and metastasis (33); however, this has not yet been rigorously proven in mammalian systems, as the effects of Ras in cell culture depend greatly on the particular cell line used. We provide experimental evidence that genetic alterations promoting noninvasive tumor growth can indeed make additional contributions to the development of metastatic behavior, as Ras V12 expression is a crucial factor in making cell polaritydeficient cells metastasize. Furthermore, we show that oncogenic Ras specifically cooperates with inactivation of cell polarity genes to promote metastatic behavior. This may provide an explanation for the different metastatic potential observed in tumors of distinctive origins. The Drosophila genetics techniques described here should make it easier to analyze the specific targets of Ras V12 in metastatic cells, to identify other genes that cooperate with Ras V12 or other oncogenic alterations in promoting metastasis, and to elucidate the cellular processes that go awry during metastatic progression. . 12. Three phenotypic classes were observed when Ras V12 -expressing cells in mosaic flies were also made homozygous for additional second-site mutations (862 mutant lines): reduced tumor growth (76 lines), enhanced tumor growth (9 lines), and tumors with metastatic behavior (2 lines).