Rac1 in human breast cancer: overexpression, mutation analysis, and characterization of a new isoform, Rac1b

Rac1 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangem ents and cell growth. Analogous to Ras, constitutively activating point mut ations of Rad cause tumorigenic transformation of cell lines. However, ther e is no information about whether Rad is also mutated in vivo, After RT-PCR of Rad, several clones of seven benign and 10 malignant breast cancer tiss ues as well as eight breast cancer cell lines were sequenced. Only single-n ucleotide polymorphisms of Rad could be detected, and none of these corresp onded to constitutively activating point mutations that have been used in c ell lines for transformation. While sequencing Rad in breast tissues, a new Rad isoform with an insertion of 19 codons within the reading frame of Rad close to switch region II was identified and named Rac1b, The Rac1b protei n acts like a fast cycling GTPase in GTP binding and hydrolysis assays, In Northern and Western blot experiments both Rad RNA and Rad protein had a si gnificantly higher expression in breast cancer tissues compared to normal b reast tissue samples. Immunohistochemical staining of Rad showed weak Rad e xpression in benign breast disease but high expression level in ductal carc inoma-in-situ, primary breast cancer, and lymph node metastases. In additio n, breast tumor cells from patients with recurrent disease had Rad expressi on at the plasma membrane, suggesting activation of Rad, in patients with a ggressive breast cancer.