Regulation of growth and tumorigenicity of breast cancer cells by the low molecular weight GTPase Rad and Nm23

Rad is the prototypic member of a family of novel Ras-related GTPases that is normally expressed in heart, skeletal muscle, and lung and that has been shown to exhibit a novel form of hi-directional interaction with the nm23 metastasis suppressor. In the present study, we hale investigated the expre ssion of Rad in normal and neoplastic breast tissues by Western blot and im munohistochemistry and the functional effect of altered Rad expression in b reast cancer cell lines. We found that, although Rad is frequently expresse d in normal breast tissue (23/30 Rad+ve), expression is usually lost in adj acent invasive carcinoma (8/30 Rad+ve: P < 0.0001). However, where Rad expr ession persists in a small proportion of tumors, it is associated with high er grade, larger size, and extensive axillar?; nodal involvement (n = 48; P = 0.035, P = 0.016, P = 0.022, respectively). Furthermore, Rad is also hig hly expressed in a breast cancer cell line with high tumorigenic and metast atic potential (MDA-MB231). To further examine the role of Rad in breast ca ncer, we stably transfected a Rad-ve breast cancer cell line (MDA-MB435). W e observed an increase in growth and marked increased colony formation in s oft agar in vitro (P < 0.05) and an increase in tumor growth rate in nude m ice (P < 0.05). Moreover. coexpression of nm23 with wild-type Rad inhibited the effect of Rad on growth of these cells in culture and markedly inhibit ed tumor growth in vivo. Additional transfection studies with mutated Rad c DNAs revealed that the growth-promoting effects of Rad appeared to be media ted through its NH2- and COOH-terminal regions, rather than its GTPase doma in, and might involve acceleration of cell cycle transition. These findings suggest that Rad may act as an oncogenic protein in breast tissues and dem onstrate a potential mechanism by which interaction between Rad and nm23 ma y regulate growth and tumorigenicity of breast cancer.