Slits are a group of secreted proteins that regulate the migration of neuronal cells and axon guidance and play important roles in the development of nerves system. Some recent studies implicate that Slit gene transcription is inactivated in tumor cells and Slits may function as tumor suppressors, while in different contexts, Slits have been demonstrated to be up-regulated in tumors and enhance tumor development. We are exploring these paradoxical observations by knockdown of Slit2 gene in tumor cells by using retroviral vector driven RNAi and examining the effect on tumors in vivo and in vitro. We report here that many human and mouse tumor cell lines as well as clinical tumor samples express Slits and their transmembrane receptor Robos. Knockdown of Slit2 gene in a mouse breast cancer cell line, 4T1, significantly suppressed tumor growth and reduced metastasis in lungs in animals. This phenomenon was also observed in immune incompetent nude mice, suggesting that Slit2 may have oncogenic effects on tumor development. In vitro cultures showed that silencing of Slit2 gene did not significantly affect tumor growth but resulted in growth arrest when cells reached confluence. In contrast to control tumor cells that overgrew and died after confluence, the Slit2 gene knockdown tumor cells remained living up to 2 weeks in confluent cultures with or without replacement of mediums, implicating that the cells became quiescent. These cells could, however, grow normally after subculture at low densities, suggesting that contact inhibition mediated by high cell density might result in the growth arrest. Further analysis indicated that knockdown of Slit2 lead to cell cycle arrest at G0/G1 phase at confluence and impaired directional motility of the tumor cells in a monolayer wound healing assay. Addition of exogenous recombinant Slit2 proteins in cultures prior to confluence could restore the cell cycle of Slit2 silenced tumor cells at confluence to the control level, suggesting that Slit2 may act through interaction with its receptors Robos. We suggest that Slits may have oncogenic effects on tumor cells in an autonomous manner and may regulate tumorigenesis and metastasis through a mechanism related to contact inhibition. Our on-going experiments will further determine molecular mechanisms for Slit2/Robo mediated effects on tumor cells.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]