Disruption of the Rb-Raf-1 Interaction Inhibits Tumor Growth and Angiogenesis

Abstract

The retinoblastoma tumor suppressor protein (Rb) plays a vital role in regulating mammalian cell cycle progression and inactivation of Rb is necessary for entry into S phase. Rb is inactivated by phosphorylation upon growth factor stimulation of quiescent cells, facilitating the transition from G 1 phase to S phase. Although the signaling events after growth factor stimulation have been well characterized, it is not yet clear how these signals contact the cell cycle machinery. We had found previously that growth factor stimulation of quiescent cells lead to the direct binding of Raf-1 kinase to Rb, leading to its inactivation. Here we show that the Rb-Raf-1 interaction occurs prior to the activation of cyclin and/or cyclin-dependent kinases and facilitates normal cell cycle progression. Raf-1-mediated inactivation of Rb is independent of the mitogen-activated protein kinase cascade, as well as cyclin-dependent kinases. Binding of Raf-1 seemed to correlate with the dissociation of the chromatin remodeling protein Brg1 from Rb. Disruption of the Rb-Raf-1 interaction by a nine-amino-acid peptide inhibits Rb phosphorylation, cell proliferation, and vascular endothelial growth factor-mediated capillary tubule formation. Delivery of this peptide by a carrier molecule led to a 79% reduction in tumor volume and a 57% reduction in microvessel formation in nude mice. It appears that Raf-1 links mitogenic signaling to Rb and that disruption of this interaction could aid in controlling proliferative disorders.The retinoblastoma tumor suppressor protein, Rb, plays a vital role in regulating the mammalian cell proliferation and its inactivation facilitates S-phase entry (i.e., entry into S phase) (64). Rb is inactivated during normal cell cycle progression by a cascade of phosphorylation events mediated mainly by kinases associated with D and E type cyclins (45,55). Previous studies have shown that inhibition of Rb phosphorylation can lead to G 1 arrest and that phosphorylation site mutants of Rb have enhanced growth suppressive properties (2, 17, 31). The growth-inhibitory properties of Rb are primarily mediated by its interaction with the E2F family of transcription factors (10, 18); Rb binds to E2Fs 1, 2, and 3 and suppresses their transcriptional activity (4, 33). Inactivation of Rb by phosphorylation leads to the dissociation and activation of E2F, allowing the expression of many genes required for cell cycle progression and S-phase entry (5, 7, 48).In addition to its role in regulating cell proliferation, Rb affects chromatin structure and function as well (14,25,49). It has been shown recently that Rb induces heterochromatin formation and inhibition of E2F-regulated genes during cellular senescence (46). Further, Rb has been shown to localize to the chromatin and suppress abnormal endoreduplication that might occur after DNA damage (3). Rb has also been shown to possess antiapoptotic activity by repression of E2F1-regulated proapoptotic genes such as p73, Apaf-1, and caspase-3 (43, 51). These observations indicate that...

Involvement of RB gene family in tumor angiogenesis

“…Resulting from evidence that Raf-1 can bind to and inactivate the pRb protein (Wang et al, 1998), a peptide corresponding to the Raf-1 amino-acid residue involved in binding to pRb was synthesized. This Raf-1 peptide is able to disrupt the physical interaction between Raf-1 and pRb, both in vitro and in vivo, and demonstrates a potent antiangiogenic activity (Dasgupta et al, 2004). In in vitro angiogenesis system, the Raf-1 peptide inhibits VEGF-induced capillary tube formation modulating endothelial cell adhesion, invasion and migration and determining a significant inhibition of VEGF-induced pRb phosphorylation.…”

Regulation of miR106b cluster through the RB pathway

“…[40][41][42][43] The mRNA signals were normalized to GAPDH mRNA internal control and the primers used are presented in the Table S1. Mir 25, miR93 and mir106b were analyzed by QRT-PCR by using specific RT-PCR primers from Applied Biosystem, Inc.…”

Reprogramming of the SWI/SNF complex for co-activation or co-repression in prohibitin-mediated estrogen receptor regulation

“…Alteration of nucleosome structure by ATP-dependent remodeling complexes is a critical step in transcriptional regulation (Muchardt and Yaniv, 1993;Dunaief et al, 1994;Peterson and Tamkun, 1995;Goodwin, 1997;Muchardt et al, 1998;DeCristofaro et al, 1999;Murphy et al, 1999;Yaniv, 1999, 2001;Dahiya et al, 2000;de la Serna et al, 2001;Betz et al, 2002;Reisman et al, 2002;Roy et al, 2002;Wang et al, 2002bWang et al, , 2004Dasgupta et al, 2004;Roche et al, 2004;Inayoshi et al, 2006). The molecular mechanism responsible for these divergent functions of the SWI/ SNF complex has not been fully defined.…”