Abstract
Abnormally suppressed levels of cyclin-dependent kinase inhibitors (CKIs) are associated with aggressive androgen independent (AI) prostate cancer and are thought to contribute to its uncontrolled proliferation. Consistent with such a role, AI human prostate cancer cell lines, LNCaP-104R1, ALVA31 and PC-3, express low levels of the CKI, p21WAF1,CIP1, compared to the less malignant, androgen dependent (AD) LNCaP cells. Rho GTPases, including RhoA, Rac1 and Cdc42, are signaling proteins that regulate dynamic cytoskeletal rearrangements and play an important role in other cellular processes including cell cycle progression. Signaling via RhoA has been associated with suppression of p21, especially in the context of oncogenic Ras mutations. We examined effects of Rho GTPases on p21 in models of highly malignant, AI prostate cancer (LNCaP-104R1, ALVA31 and PC-3) and in the less aggressive, AD LNCaP cells. Inhibition of Rho GTPases with Clostridium dificile toxin A results in upregulation of p21 in AI lines but has no effect on p21 levels in LNCaP cells. Interestingly, GTPase activity demonstrates significantly higher Rac1 activity in all three AI cell lines compared to LNCaP cells. Specific inhibition of Rac1 by dominant negative (DN) mutant or introduction of the Rac/Cdc42 binding domain from the effector protein, PAK, results in upregulation of p21 protein and mRNA exclusively in the AI cells. Thus, in three models of aggressive human prostate cancer, increased levels of GTP-bound Rac1 correspond with suppressed expression of p21. Additionally, p21 protein and mRNA expression is increased upon specific inhibition of RhoA by DN mutant or introduction of Clotstridium botulinum C3 exoenzyme. Upregulation of p21 via Rac1 or RhoA inhibition was functionally significant as evidenced by decreased cyclin-dependent kinase 2 activity and decreased cell proliferation. Rho-kinase, a RhoA effector, has been shown to play a role in conferring invasive properties in some cells. We tested whether RhoA's signaling to p21 in AI cells was mediated by Rho-kinase. Using DN Rho-kinase mutant, we show that p21 expression is upregulated in ALVA31 and PC-3 cells. Taken together, we implicate a role for both Rac1 and RhoA signaling in driving rapid cellular proliferation, suggesting possible therapeutic targets for advanced prostate disease.