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Overexpression of RhoA Induces Preneoplastic Transformation of Primary Mammary Epithelial Cells

¹ Department of Genetics, Cell Biology and Anatomy and ² Eppley Institute for Cancer and Allied Diseases and UNMC-Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska; ³ New England Medical Center, Tufts University, Boston, Massachusetts; and ⁴ Evanston Northwestern Healthcare Research Institute, Department of Medicine, ⁵ Department of Bioinformatics, and ⁶ Genomic Core, Feinberg School of Medicine, Northwestern University, Chicago, Illinois

Requests for reprints: Vimla Band, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, 985805 Nebraska Medical Center, Omaha, NE 68198. Phone: 402-559-8565; Fax: 402-559-7328; E-mail: vband{at}unmc.edu.

Key Words: immortalization • Mammary cells • Rho GTPase

Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions, including actin cytoskeleton remodeling, cell migration, gene transcription, and cell proliferation. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably used 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 mammary epithelial cells (hMEC). We show here that ectopic expression of wild-type (WT) 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 (PD) with no signs of senescence, whereas cells expressing the vector or dominant-negative RhoA mutant (T19N) senesced after 20 PDs. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well-known Rho effectors including Rho kinase, PKN, mDia1, and mDia2, was also capable of immortalizing hMECs. Notably, similar to parental normal cells, Rho-immortalized cells have WT p53 and intact G₁ cell cycle arrest on Adriamycin treatment. Rho-immortalized cells were anchorage dependent and were unable to form tumors when implanted in nude mice. Lastly, microarray expression profiling of Rho-immortalized versus parental cells showed altered expression of several genes previously implicated in immortalization and breast cancer progression. Taken together, these results show that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked to cellular transformation and breast cancer. [Cancer Res 2009;69(2):483–91]