Role of Rac1 GTPase hyperactivity in suppressing p21Cip1 levels and stimulating prostate cancer cell proliferation and tumor formation

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Abnormally suppressed levels of cyclin-dependent kinase inhibitors (CKIs) are associated with aggressive, androgen-independent prostate cancer and contribute to deregulated cell proliferation. The androgen-independent human prostate cancer cell lines, LNCaP-104R1, ALVA31, and PC-3, express low levels of the CKI, p21Cip1, compared to the less malignant, androgen-dependent LNCaP cells. We investigated the mechanism underlying p21 suppression by examining the role of Rho GTPases, signaling proteins that play important roles in cell cycle progression, at least in part through regulation of CKIs. In particular, we tested the hypothesis that increased activity of Rho GTPases in highly malignant, androgen-independent prostate cancer cells represses p21, thereby permitting uncontrolled cell cycle progression. Inhibition of the Rho family member, Rac1, using a dominant negative Rac1 mutant or by introduction of exogenous binding domain from the Rac1 effector, PAK-1, induced p21 expression in androgen-independent lines. Rac1 inhibition had no effect on the higher p21 levels characteristic of LNCaP cells; however, overexpression of constitutively active Rac1 suppressed p21 levels in LNCaP. Thus, Rac1 activity is both necessary and sufficient for suppression of p21 in prostate cancer cells. The induction of p21 in the androgen-independent cell lines was functionally significant as demonstrated by inhibition of cyclin-dependent kinase 2 activity and decreased cell proliferation. Furthermore, Rac1 activity, but not Rac1 protein levels, was significantly higher in all three androgen-independent cell lines compared to LNCaP cells. To assess the role of Rac1 in tumorigenesis, we stably expressed constitutively active Rac1 in LNCaP cells (LNCaP/CA-Rac1) and assessed tumor formation in nude mice. LNCaP/CA-Rac1 xenografts exhibited a higher tumor take rate and grew more rapidly than LNCaP controls expressing the vector alone (LNCaP/neo). These results are unique in describing a role for Rac1 in p21 regulation and implicate the Rac1 signaling pathway as a potential therapeutic target for controlling prostate cancer cell growth following progression to androgen independence. Supported by U.S. Department of Defense DAMD17-02-1-0094.