Abstract 493: A novel mechanism of colorectal tumorigenesis based on the functional inactivation of p53 by the pituitary tumor transforming gene binding factor (PBF)
Authors: Robert I. Seed, Martin L. Read, Jim Cw Fong, Gregory D. Lewy, Vicki E. Smith, Rachel Rj Watkins, Perkin Pk Kwan, Neil Sharma, Gavin A. Ryan, Jayne A. Franklyn, Kristien Boelaert, Chistopher J. McCabe
Journal: Cancer Research (2010)

Abstract PBF is the poorly characterized binding partner of the pituitary tumor transforming gene (PTTG). PTTG is a multifunctional proto-oncogene overexpressed in colorectal cancer, which regulates the function of the tumor suppressor protein p53. Recent data indicates that PBF can transform cells independently of PTTG and that subcutaneous expression of PBF elicits large tumors in nude mice. Given that PBF is both transforming in vitro and tumorigenic in vivo, our present studies aim to investigate whether PBF has a role in colorectal tumorigenesis. Initially we demonstrated that PBF represses p53-mediated gene regulation through Hdm2 promoter assays in p53-null H1299 cells. Transfection of p53 elicited a 30.7 ± 2.6-fold stimulation of promoter activity. However, co-transfection of PBF significantly repressed p53 transcriptional activity (16.3 ± 1.1-fold; p&amp;lt;0.0003). Exposure of HCT116 cells to gamma-irradiation resulted in significantly increased endogenous PBF protein expression from 4 hours after treatment and reached maximal levels after 24 hours, in keeping with the observed increase in p53 protein stability. Furthermore, co-immunoprecipitation assays revealed a direct interaction between PBF and p53 in vivo, with a significant increase in binding following gamma-irradiation. In immunofluorescence studies, HCT116 and COS-7 cells treated with gamma-irradiation displayed increased nuclear localisation of PBF compared to untreated controls, resulting in an enhanced degree of colocalisation of both PBF and p53 within the nucleus. Transient overexpression of PBF in HCT116 cells resulted in substantially elevated nuclear p53 protein expression from as early as 2 hours after treatment, with increased p53 stabilization persisting for 24 hours compared to mock-transfected controls. Finally, in a cohort of matched cancer versus normal colorectal tissue samples we observed a significant increase in PBF expression at both the mRNA (2.4-fold, n=24, p=0.009) and protein level in tumors, which correlated with a significant increase in p53 protein expression. Taken together, these results highlight a potential role for PBF within the DNA damage response, whereby PBF stabilizes in response to genotoxic stress, binds directly to p53 and inhibits p53 mediated responses. From these findings we propose that aberrant regulation of PBF leads to the functional inactivation of p53, and thus represents a potential novel mechanism for colorectal tumorigenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 493.