Abstract 1176: Bypassing the dominant-negative effect of mutant p53 in cancer cells

Abstract

Abstract The tumor suppressor p53 is mutated in more than 50% of all cancers. The transcriptional activity of p53 depends on localization to the nucleus and formation of p53 tetramers. Use of wild-type (wt) p53 for gene therapy is limited due to the dominant-negative effect of mutant (mut) p53 over wt p53. Mut p53 will hetero-oligomerize with wt p53 via the tetramerization domain (TD) found in p53, deactivating the tumor suppressor function of wt p53. In order to use p53 for gene therapy, an alternative oligomerization domain for p53 was investigated. The coiled-coil (CC) domain from Bcr (breakpoint cluster region), similar to the native TD of p53, can form an antiparallel dimer of dimers. The TD from p53 was then substituted with this CC, and the activity of this new p53 (p53-CC) was tested. We evaluated the transcriptional activity of p53-CC compared to wt p53 in a dual luciferase reporter gene assay. To demonstrate the ability of our p53-CC to bypass the dominant-negative effect prompted by hetero-oligomers, the reporter gene assay was carried in human breast adenocarcinoma (MCF-7) and human breast ductal carcinoma (T47D) cell lines. MCF-7 cells contain wt p53 population that is mislocalized to the cytoplasm, whereas, T47D cells contain mutant p53. Indeed, data from the reporter gene experiment shows a reduction in wt p53 activity introduced to T47D cells compared to MCF-7 cells, while the activity of p53-CC remains consistent regardless of the presence of an endogenous mut p53 population. We also tested the biological activity of p53-CC and its ability to induce apoptosis using 7-AAD and Annexin-V assays (late stage apoptosis). In addition, Caspase-3/7 assay (mid-stage apoptosis) was performed to further validate a p53-dependent apoptotic pathway. Data obtained from the aforementioned experiments suggest that p53-CC has a very similar biological activity to wt p53. Furthermore, we examined the gene expression profile of p53-CC using wt p53 as a reference. The assay was performed using real-time PCR array technology to analyze the expression of 84 genes related to p53-mediated signal transduction. Two negative controls were used; the first is a p53 construct that lacks its tetramerization domain (p53ΔTDC) and should not possess any transcriptional activity, and the second is the CC by itself. Indeed, p53-CC shows comparable gene expression profile to wt p53 and has the ability to transactivate the various p53 target genes. In summary, p53-CC retains p53 activity and excludes hetero-oligomer formation, overcoming a major barrier in using p53 for cancer gene therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1176. doi:1538-7445.AM2012-1176