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. For example, the two subdomains, TAD1 (residues 1-40) and TAD2 (residues 35-59), interact with several domains of the transcriptional coactivator p300. However, they 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 Thr18 or Ser46, 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 are currently validating some of these results as direct binding partners of p53 via immunoprecipitation, measuring levels before and after induction of cellular stress. We are also exploring the functional effects of these phosphorylation-specific interactors. 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 attenuating p53 pro-apoptotic and growth arrest functions and the latter promoting DNA repair. In collaboration with Drs. Giorgio Stassi and Veronica Veschi (University of Palermo and Rome, Italy), we have used a collection of primary colorectal cancer (CRC) cell cultures established in their laboratory to show that Cancer Stem Cells (CSC) at the front of CRC invasion express high levels of SETD8 and p53-K382Me1. 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. Further, 50% of macrophages (CD163+, CD6 8+) expressed p53K382me1. Cross talk between cancer cells and macrophages in the tumor microenvironment may lead to functional inactivation of p53; therefore, SETD8-mediated p53K382me1 could represent the first mechanism of p53 inactivation mediated by an epigenetic regulator in the M2 macrophage population. These findings were recently published in Mol Cancer. Currently, there are few available substrate-competitive cell-permeable inhibitors targeting SETD8. In a pre-clinical xenograft colon tumor animal model, inhibition by UNC0379, a selective, substrate-competitive inhibitor of SETD8, produced a significant survival advantage, suggesting that SETD8 may be an effective therapeutic target in colon cancer. Because SETD8 has a wide range of substrates with different functions in the cell, the identification of a compound specific for the SETD8-p53 interaction is urgently needed. We are using a high-throughput assay that will allow us to identify novel inhibitors of SETD8 with 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. In 2022, we published on the effects of a small molecule, NSC59984, in esophageal adenocarcinoma cells, investigated its mechanism of action and effects on the onco-metabolic profile. More recently, we have published a follow-up on the mechanism of action of this small molecule in ACS Pharmacol and Transl Sci. We used biophysical and mass spectrometry approaches to investigate the specific reaction of NSC59984 with p53. Similar to other reactivators of mutant p53, we show that NSC59984 covalently modifies p53 on specific cysteine residues in the DNA-binding domain. Molecular simulations predict that these modifications affect the conformation of the domain and stabilize conformations that mimic wild-type activity. Further, we examined potential off-target effects of NSC59984 and identified potential targets among metabolic enzymes.