Poster 053: Rap1 Enhances Beta-Catenin-Dependent Transcription and Cell Invasion in Oral and Oropharyngeal Squamous Cell Carcinoma

Abstract

Statement of the Problem: In the U.S., oral and oropharyngeal squamous cell carcinoma (OSCC) accounts for more deaths annually than cervical cancer, melanoma, or lymphoma. Despite the aggressive surgery and radiation therapy for this disease, the prognosis has not improved in four decades. Elucidation of signaling pathways that regulate proliferation in OSCC will facilitate the identification of novel treatment targets. Previously, we reported that active, GTP-bound Rap1 is strongly expressed in nuclei of OSCC. Importantly, because it has a canonical nuclear localization sequence that binds armadillo proteins, Rap1 is a candidate protein for the nuclear transport of armadillo proteins. One such protein is beta-catenin (beta-cat), which is a central molecule in the Wnt signal pathway. Beta-cat translocates from the cytoplasm to the nucleus, where as a co-factor with T-cell factor/ lymphoid enhancer factor (TCF/LEF), it triggers the expression of genes that control proliferation, migration, and invasion, and has been implicated in various cancers. However, there are conflicting reports on the nuclear translocation of beta-cat in SCC. Hence, the objective of this study was to investigate whether; a) beta-cat localizes in the nucleus of OSCC cells; b) Rap1 interacts with beta-cat; c) Rap1 induces nuclear translocation of beta-cat and enhances TCF-dependent transcription of beta-cat target genes; d) Rap1 promotes invasion of OSCC cells. Materials and Methods: Western blot analysis: Wholecell lysates were electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose membranes and blotted with mouse anti-Rap1 monoclonal antibody or mouse anti-beta-cat monoclonal antibody. Rap1 activation assay: Active, GTP-bound Rap1 was detected by the ralGDS pull down assay followed by immunoblot analysis with the Rap1 antibody. Transfection and luciferase assay: To determine if Rap1 induces nuclear translocation and transcription of beta-cat target genes, cells were co transfected with TOPFlash or FOPFlash, renilla luciferase (internal control), beta-cat construct, and Rap1AG12 and luminescence was measured 24 h later. TOPFlash contains the TCF binding site for beta-cat, upstream of the luciferase gene. FOPFlash, which has a scrambled binding site, was used as a negative control. Immunostaining: Immunohistochemistry was performed on human OSCC tissue sections or human OSCC cells with mouse anti-beta-cat monoclonal antibody or rabbit anti-beta-cat polyclonal antibody, respectively. Double-labeling immunofluorescence on transfected cells was also performed to identify the nuclear localization of beta-cat. Cell invasion assay: Cell invasion was determined by Matrigel invasion chamber (BD Biosciences) according to the manufacturer’s instructions. Method of Data Analysis: Statistical analysis was performed using Student’s t-test. P value of 0.05 or less was considered statistically significant. Results: a) Immunohistochemical studies showed nuclear beta-cat expression in OSCC cell lines and human tissue. b) The ralGDS pull down assay indicated that beta-cat binds to active, GTP-bound Rap1. c) Immunofluorescence studies showed that Rap1A enhanced nuclear translocation of beta-cat. d) Consistent with these findings, Rap1A upregulates beta-cat induced transcription and enhances beta-cat-mediated invasion of OSCC cells. Conclusion: Rap1 binds to beta-cat and upregulates beta-cat-dependent transcription and invasion, consistent with the notion that Rap1 facilitates nuclear transport of beta-cat. Since free beta-cat translocates to the nucleus and induces the expression of genes that regulate proliferation and invasion, elucidating the nuclear transport mechanism in OSCC could identify an important therapeutic target.