Human Mammary Epithelial Cell Transformation by Rho GTPase through a Novel Mechanism
Final rept. 30 Jan 2005-28 Jul 2008
NEBRASKA UNIV MEDICAL CENTER OMAHA
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Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably utilized immortal or tumorigenic cell lines to study Rho GTPase functions and there are no studies on the potential of Rho small GTPase to overcome senescence checkpoints and induce preneoplastic transformation of human epithelial cells hMECs. We demonstrate here, that ectopic expression of wild-type RhoA as well as a constitutively-active RhoA mutant G14V in two independent primary hMEC strains led to their immortalization and preneoplastic transformation. These cells have continued to grow over 300 population doublings with no signs of senescence, whereas cells expressing the vector or dominant-negative RhoA mutant T19N senesced after 20 population doublings. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well known Rho-effectors including Rho-kinase, PKN and mDia 1 and 2, was also capable of immortalizing hMECs. Notably, similar to parental normal cells, Rho-immortalized cells have wild-type p53 and intact G1 cell cycle arrest upon adriamycin treatment. Rho-immortalized cells were anchorage-dependent. Lastly, microarray expression profiling of Rho-immortalized vs. parental cells showed altered expression of several genes previously implicated in immortalization and breast cancer progression. Taken together, these results demonstrate that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked cellular transformation and breast cancer.
- Medicine and Medical Research