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Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.

Recurrent respiratory papillomas are premalignant tumors of the airway caused by human papillomaviruses (HPVs), primarily Types 6 and 11. We had reported that respiratory papillomas overexpress the epidermal growth factor receptor (EGFR), the small GTPase Rac1 and cyclooxygenase-2 (COX-2), and have enhanced nuclear factor-kappaB (NFkappaB) activation with decreased levels of IkappaB-beta but not IkappaB-alpha. We also showed that EGFR-activated Rac1 mediates expression of COX-2 through activation of p38 mitogen-activated protein kinase. We have now asked whether the p21-activated kinases Pak1 or Pak2 mediate activation of p38 by Rac1 in papilloma cells. Pak1 and Pak2 were constitutively activated in vivo in papilloma tissue compared with normal epithelium, and Rac1 siRNA reduced the level of both phospho-Pak1 and phospho-Pak2 in cultured papilloma cells. Reduction in Pak1 and Pak2 with siRNA decreased the COX-2 expression in papilloma cells, increased the levels of IkappaB-beta and reduced the nuclear localization of NF-kappaB, but had no effect on p38 phosphorylation. Our studies suggest that Rac1 --> Pak1/Pak2 --> NFkappaB is a separate pathway that contributes to the expression of COX-2 in HPV-induced papillomas, independently of the previously described Rac1 --> p38 --> COX-2 pathway.

Recurrent respiratory papillomas are epithelial tumors of the airway caused by human papillomaviruses. We previously reported that the epidermal growth factor receptor (EGFR) is overexpressed in papilloma cells, that cyclooxygenase-2 (COX-2) is induced, and that COX-2 expression in primary papilloma cells requires activation of the EGFR but not Erk. Rac1, a member of the Rho family of GTPases, is a key signaling element that is known to control multiple pathways downstream of the EGFR. Here we report that Rac1 is overexpressed in papilloma cells compared with normal laryngeal epithelial cells and that the increased levels of Rac1 are mediated by EGFR activation. Transfecting cells with Rac1-specific siRNA suppressed COX-2 expression. Surprisingly, Rac1 mediated phosphorylation of p38 mitogen-activated kinase in papilloma cells but not normal cells, and inhibition of p38 with the specific inhibitor SB202190 suppressed COX-2 expression in papilloma cells but had no effect on low-level COX-2 expression in normal cells. Thus, the signaling cascades that regulate COX-2 expression are different in HPV-infected papilloma cells, with a significant contribution by the EGFR-- Rac1-->p38 pathway.