Abstract A75: Retinoblastoma protein promotes Wnt signaling pathway activation in MC3T3 osteoblasts
Authors: Viviana Váazquez-Rivera, Bernadette Sosa-García, Nismaliry Martínez-García, Pedro Santiago-Cardona
Journal: Cancer Research (2009)

Abstract Disruption of the retinoblastoma (pRb) pathway is observed in most cancers. Mutational inactivation of pRb itself is observed at high frequencies in retinoblastomas, osteosarcomas, and small cell lung carcinoma. pRb blocks cell cycle progression by repressing E2F-dependent transcription of S-phase-related genes inducing cellular differentiation by serving as co-activator of tissue-specific transcription factors. Another pathway frequently targeted in human cancers is the Wnt pathway, which regulates a variety of processes such as chondrocyte and osteoblast differentiation. Wnt activation hinders β-catenin degradation by proteosomes with consequent β-catenin nuclear accumulation and transcription of Wnt target genes. In contrast, the absence of the Wnt signal allows the recruitment of CK1, APC, GSK3-β, and β-catenin by Axin. This complex phosphorylates β-catenin at specific residues serving as a tag for recognition and degradation. Given that inactivation of both pRb and Wnt pathways is commonly observed in osteosarcomas, we sought to determine if these pathways are functionally linked in such a manner that inactivation of one of them leads to deregulation of the other. To test this, we performed qRT-PCR of pRb-expressing and pRb-deficient osteoblasts (MC3T3 Rb+/+ and MC3T3 Rb−/−, respectively) in order to compare the mRNA steady state levels of the Wnt pathway components. We found a 1.8 fold increase (p&amp;lt;0.05) in the levels of PP2, an inducer of β-catenin degradation, in MC3T3 Rb-deficient relative to MC3T3 Rb-expressing. Whereas, we observed a 0.4 fold decrease (p&amp;lt;0.005) in Dvl, an inhibitor of β-catenin degradation, in MC3T3 Rb-deficient relative to MC3T3 Rb-expressing. Taken together these results indicate that pRb promotes Wnt signaling by inhibiting β-catenin's degradation. Wnt activation requires β-catenin's nuclear relocalization, a process blocked in part by phosphorylation of β-catenin at Serine 33 (pSer33) by GSK3-β. To test if pRb regulates β-catenin's relocalization we performed immunocytochemistry assay. Our immunocytochemistry studies showed an increase in nuclear localization of β-catenin and β-catenin pSer33 in the pRb-expressing compared to the pRb-deficient osteoblasts. Upon nuclear relocalization, β-catenin must bind to the Tcf/Lef transcriptional activators forming a complex capable of transcribingWnt targets. To determine if the nuclear β-catenin observed in the pRb-expressing cell line can induce expression of Wnt target genes, we transfected osteoblasts with a TopFlash construct consisting of a luciferase reporter under the control of a β-catenin/Tcf/Lef responsive promoter. Using Dual-Luciferase Assay we demonstrated a 2.8 increase in reporter activity of the pRb-expressing over the pRb-deficient cell line. This increase in luciferase activity of the pRb-expressing indicates an increase in β-catenin Tcf/lef dependent transcription. Our data show that, in the presence of a functional pRb, β-catenin degradation is blocked, proposing a novel role for pRb as a promoter of the Wnt signaling pathway in osteoblasts. This project was supported by PSM Institutional Funds, American Cancer Society's Institutional Research Grant #93-032-13. Citation Information: Cancer Res 2009;69(23 Suppl):A75.