Tumor Suppressor Protein, p53

Division Of Basic Sciences - Nci

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Abstract

The p53 tumor suppressor protein is a key component of the cellular response to stress. It is a homo-tetrameric, sequence-specific transcription factor activated by DNA damage, hypoxia, heat shock, and other types of stress and regulates DNA repair, cell cycle arrest, senescence, metabolism and apoptosis. It is maintained at low levels in unstressed cells but becomes stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of p53 PTMs to better understand how they modulate p53 functions. The tandem N-terminal transactivation domains (TADs) of p53 are crucial for p53 activity as a transcription factor. The two subdomains, TAD1 (residues 1-40) and TAD2 (residues 35-59), interact with several domains of the transcriptional coactivator p300. However, the two subdomains can function independently of one another, suggesting the participation of distinguishing transcriptional cofactors in transcriptional activation by TAD1 and TAD2 in which interactions may be differentially regulated by p53 phosphorylation. To identify distinct interacting partners for TAD1 and TAD2, peptides comprising TAD1 (residues 9-33) or TAD2 (residues 35-59), with and without phosphorylation at Thr 18 or Ser 46, respectively, were synthesized and covalently attached to biotin at the N-termini. We used these peptides as a bait for pulldown of interacting proteins from nuclear extracts prepared from MCF7 cells treated with etoposide; mass spectrometry analysis was used to identify and quantitatively compare the interactors to discriminate between those preferentially interacting with the TAD1 or TAD2 subdomains. Our experiments using biological triplicate pulldowns have identified a list of potential interactors that show a preference for either unmodified or modified p53 in untreated cells or following etoposide treatment. In addition to known binding partners of p53 TAD1 and TAD2, we identified several new interactors. We have validated a few of the new interactors identified and are performing functional experiments to investigate the consequences of these interactions on p53 activity and stability. We have also expanded the experiments to determine whether these interactions also occur under other stress conditions known to activate p53, including ER stress and UV damage. Understanding the effects of these new interactors on p53 regulation will open new methods to increase p53 activity in tumors. The C-terminus of p53 exhibits a diverse array of PTMs, including phosphorylation, methylation, acetylation, ubiquitination, sumoylation, neddylation and hydroxylation that are primarily localized to the terminal thirty residues of the protein. We have shown that p53 can be both mono- and dimethylated on Lys382, with the former modification repressing p53 transcriptional activity and the latter promoting DNA repair, in addition to demonstrated acetylation and ubiquitination of the same site. SETD8 monomethylates p53 on lysine 382, attenuating p53 pro-apoptotic and growth arrest functions. Using a high-content imaging siRNA screen and a chemical screen, in a collaboration with Drs. Veschi and Thiele, we identified SETD8 as a suppressor of p53 activity in neuroblastoma cell lines. Genetic or pharmacological inhibition of SETD8 activity resulted in activation of the p53 wild-type pathway by decreasing p53K382me1. We have initiated a collaboration with Drs. Veschi and Stassi and recently showed that SETD8 is highly expressed in colon cancer stem cells (CSCs), with commensurate increased levels of p53K382me1. These two effects may play a synergistic role in the tumorigenic and metastatic capabilities of CSCs. We are currently using a high-throughput assay to identify novel inhibitors of SETD8 with an improved activity and tolerability in vivo. p53 point mutations have been reported to occur in approximately half of all human tumors, with marked over-representation of specific "hot-spot" residues. These mutations abolish the ability of p53 to function as a transcription factor and tumor suppressor. Moreover, many mutant forms of p53 have acquired novel oncogenic activities through gain-of-function mechanisms. p53 mutations generally either affect DNA contact or cause structural instability with partial unfolding and aggregate formation, similar to that seen in amyloid diseases. We are examining a small molecule, NSC59984, in esophageal adenocarcinoma cells and investigating its mechanism of action and effects on the oncometabolic profile. The findings will help elucidate pathways critical for preventing tumor growth by inhibiting gain-of-function mutant p53 activities and restoring wild-type p53 activity. A second means to target mutant p53 was reported by Padmanabhan et al. (Nat Commun. 9(1):1270 (2018)), showing that an inhibitor targeting deubiquitinase USP15 specifically results in p53-R175H degradation by disrupting the USP15-p53-R175H interaction. This leads to increased p53-R175H ubiquitination and degradation of the mutant protein. Based on these findings, we have chosen to explore other members of the deubiquitinating enzyme (DUB) family as potential modulators of mutant p53 protein stability. Using CRISPR-interference and CRISPR-activation screens with DUB-targeting sgRNAs in ovarian cancer cell lines of varying p53 status, we are identifying DUBs that contribute to mutant p53 stability. These lines encompass several p53 expression models, p53-null, wild-type and mutant p53-R175H, commonly seen in ovarian cancer. The DUBs that are identified will be further analyzed as druggable targets. The ability to selectively target DUBs will be crucial, since they take on several roles in the ubiquitin pathway, particularly by focusing on the N-terminal domains adjacent to the catalytic domain, which vary significantly among USPs. The goal is to develop small molecules and/or peptidomimetic compounds that disrupt the accumulation of mutant p53 and/or rescue p53 wildtype tumor suppressive function.