Tumor Suppressor Protein, p53

Division Of Basic Sciences - Nci

Investigators

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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 one of the new interactors, BRAT1 (BRCA1 Associated ATM Activator 1) as a direct binding partner of p53 and have shown its levels change following induction of stress with etoposide. Current work aims to knockdown BRAT1 isoforms to identify which isoform is directly regulating p53 and affects mitochondrial function and cell proliferation. 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. Interestingly, p53K382me1 expression is undetectable in the healthy mucosa, while it is highly expressed in the tumor leading edge region together with the CD44v6+ marker which is required for the CSC migration and generation of metastasis. It is known that chronic inflammatory stimuli may promote tumor progression and metastasis. In inflammatory conditions of the lower gastrointestinal tract, p53 inactivation represents an early step of tumorigenesis. We detected high levels of p53K382me1 in immune cell infiltrates in CRCs which were significantly associated with worse prognosis. 50% of macrophages (CD163+, CD6 8+) expressed p53K382me1 whereas T lymphocytes (CD4+or CD8+), neutrophils (CD15+) and B cells (CD20+) expressed low levels. Cross talk between cancer cells and macrophages in the tumor microenvironment may lead to functional inactivation of p53; therefore, p53K382me1 could represent the first known mechanism of p53 inactivation mediated by an epigenetic regulator in the M2 macrophage population. Preliminary data showed that treatment with conditioned media collected upon direct interaction between CR-CSCs and macrophages, high levels of IL-6 and monocyte-chemoattractant-protein (MCP-1) were detected accompanied with down-regulation of p53 target genes, suggesting that this may be an early mechanism of p53 functional inactivation during CRC tumorigenesis. Currently, there are few available substrate-competitive cell-permeable inhibitors targeting SETD8. The identification of a compound specific for the SETD8-p53 interaction is urgently needed. We are 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 onco-metabolic 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. NSC59984, first identified from a high-throughput screen, induces wildtype p53 signaling and anti-proliferative effects while inhibiting mutant p53 gain-of-function activities. Recently, we have investigated its specific mechanism of action against p53. We found that NSC59984 reacts with thiols via an unusual Michael addition at the alpha-carbon. Covalent modification of p53 Cys124 and Cys229 was observed both following in vitro reaction and upon treatment of cells. We also used a biotinylated form of NSC59984 and, separately, thermal proteome profiling to examine off-target effects, identifying several proteins involved in cellular metabolism as potential targets. These results demonstrate that covalent modification of p53 by NSC59984 leads to increased wildtype activity and suggest that potential reaction with metabolic enzymes may contribute to antiproliferative function.