Ian M. Love

CBP and p300 are cytoplasmic E4 polyubiquitin ligases for p53

p300 and CREB-binding protein (CBP) act as multifunctional regulators of p53 via acetylase and polyubiquitin ligase (E4) activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. Consistent with the cytoplasmic localization of its E3/E4 activity, CBP deficiency specifically stabilized cytoplasmic, but not nuclear p53. The N-terminal 616 aa of CBP, which includes the conserved Zn2+-binding C/H1-TAZ1 domain, was the minimal domain sufficient to destabilize p53 in vivo, and it included within an intrinsic E3 autoubiquitination activity and, in a two-step E4 assay, exhibited robust E4 activity for p53. Cytoplasmic compartmentalization of p300/CBPs ubiquitination function reconciles seemingly opposed functions and explains how a futile cycle is avoided—cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while physically separate nuclear p300/CBP activities, such as p53 acetylation, activate p53.

The SWI/SNF chromatin remodeling subunit BRG1 is a critical regulator of p53 necessary for proliferation of malignant cells

The tumor suppressor p53 preserves genome integrity by inducing transcription of genes controlling growth arrest or apoptosis. Transcriptional activation involves nucleosomal perturbation by chromatin remodeling enzymes. Mammalian SWI/SNF remodeling complexes incorporate either the Brahma-related gene 1 (BRG1) or Brahma (Brm) as the ATPase subunit. The observation that tumor cell lines harboring wild-type p53 specifically maintain expression of BRG1 and that BRG1 complexes with p53 prompted us to examine the role of BRG1 in regulation of p53. Remarkably, RNAi depletion of BRG1, but not Brm, led to the activation of endogenous wild-type p53 and cell senescence. We found a proline-rich region unique to BRG1 was required for binding to the histone acetyl transferase protein, CBP, as well as to p53. Ectopic expression of a proline-rich region deletion mutant BRG1 that is defective for CBP binding inhibited p53 destabilization. Importantly, RNAi knockdown of BRG1 and CBP reduced p53 poly-ubiquitination in vivo. In support of p53 inactivation by the combined activities of BRG1 and CBP, we show that DNA damage signals promoted disassociation of BRG1 from CBP, thereby allowing p53 accumulation. Our data demonstrate a novel function of the evolutionarily conserved chromatin remodeling subunit BRG1, which cooperates with CBP to constrain p53 activity and permit cancer cell proliferation.

The histone acetyltransferase PCAF regulates p21 transcription through stress-induced acetylation of histone H3

The activity of p53 as a tumor suppressor primarily depends on its ability to transactivate specific target genes in response to genotoxic and other potentially mutagenic stresses. Several histone acetyl transferases (HATs), including p300, CBP, PCAF and GCN5 have been implicated in the activation of p53-dependent transcription of the cyclin-dependent kinase (cdk) inhibitor p21 as well as other target genes. Here we show that PCAF, but not CBP or p300, is a critical regulator of p53-dependent p21 expression in response to multiple p53-activating stresses. PCAF was required for the transcriptional activation of p21 in response to exogenous p53 in p53-null cells, nutlin-3, DNA damaging agents and p14ARF expression, suggesting a broad requirement for PCAF in p53 signaling to p21 after stress. Importantly, cells lacking PCAF failed to undergo cell cycle arrest in response to nutlin-3 treatment or p14ARF expression, consistent with a physiologically important role for PCAF in this p53 function. Surprisingly, the role for PCAF in induction of p21 was independent of p53 lysine 320 acetylation, a previously suggested target of PCAF-mediated acetylation. Though p21 promoter occupancy by p53 was not altered by PCAF knockdown, activation of p21 transcription required an intact PCAF HAT domain, and induction of chromatin marks acetyl-H3K9 and acetyl-H3K14 at the p21 promoter by p53 was dependent upon physiologic levels of PCAF. Together, our experiments indicate that PCAF is required for stress-responsive histone 3 acetylation at the p21 promoter, p53-directed transcription of p21 and the resultant growth arrest.

It Takes 15 to Tango: Making Sense of the Many Ubiquitin Ligases of p53

The transcription factor p53 regulates numerous cellular processes to guard against tumorigenesis. Cell-cycle inhibition, apoptosis, and autophagy are all regulated by p53 in a cell- and context-specific manner, underscoring the need for p53 activity to be kept low in most circumstances. p53 is kept in check primarily through its regulated ubiquitination and degradation by a number of different factors, whose contributions may reflect complex context-specific needs to restrain p53 activity. Chief among these E3 ubiquitin ligases in p53 homeostasis is the ubiquitously expressed proto-oncogene MDM2, whose loss renders vertebrates unable to limit p53 activity, resulting in early embryonic lethality. MDM2 has been validated as a critical, universal E3 ubiquitin ligase for p53 in numerous tissues and organisms to date, but additional E3 ligases have also been identified for p53 whose contribution to p53 activity is unclear. In this review, we summarize the recent advances in our knowledge regarding how p53 activity is apparently controlled by a multitude of ubiquitin ligases beyond MDM2.

p53 Ubiquitination and Proteasomal Degradation

p53 levels and activity are controlled in large part through regulated ubiquitination and subsequent destruction by the 26S proteasome. Monoubiquitination of p53 is mediated primarily by the RING-finger E3 ubiquitin ligase MDM2 and impacts p53 activity through modulation of p53 localization and transcription activities. Recently, several E4 ubiquitin ligases (E4s) have been identified which serve to extend these monoubiquitin chains. The ubiquitin ligase activity of these factors toward p53, and their contribution to p53 degradation, can be studied using a variety of in vitro and in vivo methods and reagents which will be described in this chapter. These methods include in vivo ubiquitination of p53 using HA-ubiquitin or his-ubiquitin; the in vitro E3 ubiquitin ligase assay, in which ubiquitin reaction components (URC) are incubated with a purified E3 or E4 ligase; a one-step E4 assay, in which URC are incubated with a substrate, E3, and E4; and a two-step E4 assay in which p53 is monoubiquitinated in an E3 reaction, and subsequently purified and incubated with an E4. Finally, we will describe an in vitro degradation assay in which ubiquitinated p53 is incubated with purified 26S proteasomes. Together, these assays can be used to provide insight into the biochemical nature of p53 ubiquitination and degradation.

Constitutive Activation of DNA Damage Checkpoint Signaling Contributes to Mutant p53 Accumulation via Modulation of p53 Ubiquitination

Many mutant p53 proteins exhibit an abnormally long half-life and overall increased abundance compared with wild-type p53 in tumors, contributing to mutant p53s gain-of-function oncogenic properties. Here, a novel mechanism is revealed for the maintenance of mutant p53 abundance in cancer that is dependent on DNA damage checkpoint activation. High-level mutant p53 expression in lung cancer cells was associated with preferential p53 monoubiquitination versus polyubiquitination, suggesting a role for the ubiquitin/proteasome system in regulation of mutant p53 abundance in cancer cells. Interestingly, mutant p53 ubiquitination status was regulated by ataxia–telangectasia mutated (ATM) activation and downstream phosphorylation of mutant p53 (serine 15), both in resting and in genotoxin-treated lung cancer cells. Specifically, either inhibition of ATM with caffeine or mutation of p53 (serine 15 to alanine) restored MDM2-dependent polyubiquitination of otherwise monoubiquitinated mutant p53. Caffeine treatment rescued MDM2-dependent proteasome degradation of mutant p53 in cells exhibiting active DNA damage signaling, and ATM knockdown phenocopied the caffeine effect. Importantly, in cells analyzed individually by flow cytometry, p53 levels were highest in cells exhibiting the greatest levels of DNA damage response, and interference with DNA damage signaling preferentially decreased the relative percentage of cells in a population with the highest levels of mutant p53. These data demonstrate that active DNA damage signaling contributes to high levels of mutant p53 via modulation of ubiquitin/proteasome activity toward p53. Implication: The ability of DNA damage checkpoint signaling to mediate accumulation of mutant p53 suggests that targeting this signaling pathway may provide therapeutic gain. Mol Cancer Res; 14(5); 423–36. ©2016 AACR.

Inhibition of C-terminal binding protein attenuates transcription factor 4 signaling to selectively target colon cancer stem cells

Selective targeting of cancer stem cells (CSCs), implicated in tumor relapse, holds great promise in the treatment of colorectal cancer. Overexpression of C-terminal binding protein (CtBP), an NADH dependent transcriptional regulator, is often observed in colon cancer. Of note, TCF-4 signaling is also up-regulated in colonic CSCs. We hypothesized that CtBP, whose dehydrogenase activity is amenable to pharmacological inhibition by 4-methylthio-2-oxobutyric acid (MTOB), positively regulates TCF-4 signaling, leading to CSC growth and self-renewal. CSCs demonstrated significant upregulation of CtBP1 and CtBP2 levels (mRNA and protein) and activity partly due to increased NADH/NAD ratio, as well as increased TCF/LEF transcriptional activity, compared to respective controls. Depletion of CtBP2 inhibited, while its overexpression enhanced, CSC growth (1° spheroids) and self-renewal (2°/3° spheroids). Similarly, MTOB caused a robust inhibition of spheroid growth and self-renewal in a dose dependent manner. MTOB displayed significantly greater selectivity for growth inhibition in the spheroids, at least in part through induction of apoptosis, compared to monolayer controls. Moreover, MTOB inhibited basal as well as induced (by GSK-3β inhibitor) TCF/LEF activity while suppressing mRNA and protein levels of several β-catenin target genes (CD44, Snail, C-MYC and LGR5). Lastly, CtBP physically interacted with TCF-4, and this interaction was significantly inhibited in the presence of MTOB. The above findings point to a novel role of CtBPs in the promotion of CSC growth and self-renewal through direct regulation of TCF/LEF transcription. Moreover, small molecular inhibition of its function can selectively target CSCs, presenting a novel approach for treatment of colorectal cancer focused on targeting of CSCs.

Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.

NIAM's tangled web of growth control.

Aside from its canonical activity activating p53 in response to oncogene activation, the tumor suppressor (alternate reading frame) ARF exhibits p53-independent antiproliferative and tumor-suppressive activities. In search of the mechanism underpinning these activities, a number of ARF-interacting proteins have been identified in the years since the ARF/MDM2 interaction was characterized as the means by which ARF activates p53. These novel ARF interactors include ARF-binding protein1 (ARF-BP1), nucleophosmin, C-terminal binding protein (CtBP)-family co-repressors, and the nuclear interactor of ARF and MDM2 (NIAM).1 Of these known ARF interactors, NIAM is unique in its action as a putative tumor suppressor and suppressor of cell growth, rather than an oncogene or growth/survival factor that is antagonized by ARF. In the April 15, 2014 issue of Cell Cycle, Reed et al.2 investigated the pathways downstream of NIAM required for its growth-suppressive properties. What little was previously known about NIAM strongly suggested its ability to induce cell cycle arrest channels through multiple parallel pathways.3 NIAM promotes both ARF-dependent and -independent activation of p53 transcriptional activity and is downregulated in a number of pancreatic adenocarcinoma cell lines and human tumors. Mysteriously, however, NIAM also promotes cell cycle arrest through a p53-independent pathway. These observations suggest an unusually complex interplay among NIAM, ARF, p53, and the cell cycle-regulatory machinery. Reed et al. attempt to elucidate the mechanisms underlying NIAM’s control of the p53-dependent arm of its cell cycle-inhibitory activities, discovering that physiologic levels of NIAM are necessary for full p53 transactivation of the p21 promoter, and that NIAM promotes acetylation of lysine 120 of p53, a modification regarded as critical for activation of the apoptotic arm of the p53 transcription program.4,5 The authors further demonstrate that p53 activation by NIAM occurs through 2 distinct pathways: disruption of the p53–MDM2 complex, and association of NIAM with the Tip60 acetyltransferase. In a surprising twist, the authors show that depletion of Tip60 only partially reverses NIAM-mediated K120 acetylation, and that an N-terminal NIAM truncation mutant, competent for MDM2 binding, Tip60 binding, chromatin association, and cell cycle arrest, is unable to drive K120 acetylation of p53. While it is possible that residual Tip60 or the presence of hMOF is sufficient for acetylation of a limited pool of p53, the inability of the truncation mutant to promote p53 acetylation despite retaining robust Tip60 interaction is surprising and may suggest that the NIAM C terminus recruits additional functions necessary for in vivo acetylation of p53 at sites of p53-dependent transcription. The data are also a clear indication that the K120 acetylation driven by NIAM is dispensable for its broader functions in inhibiting MDM2–p53 interaction and promoting cell cycle arrest. Together, Reed et al.’s data support a model whereby one pool of NIAM sequesters MDM2 from p53 and allows p53 activation, while another pool promotes activation and association with the p53 acetylase and coactivator Tip60. However, advances in our understanding often raise as many questions as they provide answers, and regulation of proliferation by NIAM is no exception. For example, if K120 acetylation by NIAM is separable from Tip60 binding and p53 activation, how is NIAM regulating Tip60 function? It is possible that Tip60 in this model functions as a canonical histone acetyltransferase, acetylating histones in the vicinity of p53 target promoters, and that NIAM facilitates this activity through enhancing DNA binding of Tip60, or modulating its interaction with negative regulators. Interestingly, Tip60 is known to interact with MDM2 and negatively regulate MDM2-mediated NEDDylation,6 while NIAM interacts with both of these factors but, notably, not p53. It is also interesting to note that the NIAM N terminus encodes all functions necessary for p53-dependent growth arrest, including binding of Tip60 and MDM2, while the C terminus, upon which K120 acetylation is dependent, seems to provide some function necessary to direct the acetylase activity of Tip60 toward p53. Future work will be instrumental in determining whether NIAM regulation of p53 activities occurs through regulation of a Tip60–MDM2 interaction. Of broader interest will be the exploration of NIAM’s role in tumorigenesis—its downregulation suggests that NIAM activity may be selected against during tumorigenesis, so it will be critical to develop animal models to probe NIAM function and to carefully analyze the wealth of available high-throughput sequence and microarray data to determine whether NIAM functions as a tumor suppressor in certain tumors, perhaps in a mutually exclusive fashion with Tip60 and p53.