Delin Chen

Negative Control of p53 by Sir2α Promotes Cell Survival under Stress

The NAD-dependent histone deacetylation of Sir2 connects cellular metabolism with gene silencing as well as aging in yeast. Here, we show that mammalian Sir2alpha physically interacts with p53 and attenuates p53-mediated functions. Nicotinamide (Vitamin B3) inhibits an NAD-dependent p53 deacetylation induced by Sir2alpha, and also enhances the p53 acetylation levels in vivo. Furthermore, Sir2alpha represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sir2alpha point mutant increases the sensitivity of cells in the stress response. Thus, our findings implicate a p53 regulatory pathway mediated by mammalian Sir2alpha. These results have significant implications regarding an important role for Sir2alpha in modulating the sensitivity of cells in p53-dependent apoptotic response and the possible effect in cancer therapy.

Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization

The p53 tumour suppressor is a short-lived protein that is maintained at low levels in normal cells by Mdm2-mediated ubiquitination and subsequent proteolysis. Stabilization of p53 is crucial for its tumour suppressor function. However, the precise mechanism by which ubiquitinated p53 levels are regulated in vivo is not completely understood. By mass spectrometry of affinity-purified p53-associated factors, we have identified herpesvirus-associated ubiquitin-specific protease (HAUSP) as a novel p53-interacting protein. HAUSP strongly stabilizes p53 even in the presence of excess Mdm2, and also induces p53-dependent cell growth repression and apoptosis. Significantly, HAUSP has an intrinsic enzymatic activity that specifically deubiquitinates p53 both in vitro and in vivo. In contrast, expression of a catalytically inactive point mutant of HAUSP in cells increases the levels of p53 ubiquitination and destabilizes p53. These findings reveal an important mechanism by which p53 can be stabilized by direct deubiquitination and also imply that HAUSP might function as a tumour suppressor in vivo through the stabilization of p53.

Deacetylation of p53 modulates its effect on cell growth and apoptosis

The p53 tumour suppressor is a transcriptional factor whose activity is modulated by protein stability and post-translational modifications including acetylation. The mechanism by which acetylated p53 is maintained in vivo remains unclear. Here we show that the deacetylation of p53 is mediated by an histone deacetylase-1 (HDAC1)-containing complex. We have also purified a p53 target protein in the deacetylase complexes (designated PID; but identical to metastasis-associated protein 2 (MTA2)), which has been identified as a component of the NuRD complex. PID specifically interacts with p53 both in vitro and in vivo, and its expression reduces significantly the steady-state levels of acetylated p53. PID expression strongly represses p53-dependent transcriptional activation, and, notably, it modulates p53-mediated cell growth arrest and apoptosis. These results show that deacetylation and functional interactions by the PID/MTA2-associated NuRD complex may represent an important pathway to regulate p53 function.

ARF-BP1/mule is a critical mediator of the ARF tumor suppressor

Although the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.

Transcription-independent ARF regulation in oncogenic stress-mediated p53 responses

The tumour suppressor ARF is specifically required for p53 activation under oncogenic stress. Recent studies showed that p53 activation mediated by ARF, but not that induced by DNA damage, acts as a major protection against tumorigenesis in vivo under certain biological settings, suggesting that the ARF–p53 axis has more fundamental functions in tumour suppression than originally thought. Because ARF is a very stable protein in most human cell lines, it has been widely assumed that ARF induction is mediated mainly at the transcriptional level and that activation of the ARF–p53 pathway by oncogenes is a much slower and largely irreversible process by comparison with p53 activation after DNA damage. Here we report that ARF is very unstable in normal human cells but that its degradation is inhibited in cancerous cells. Through biochemical purification, we identified a specific ubiquitin ligase for ARF and named it ULF. ULF interacts with ARF both in vitro and in vivo and promotes the lysine-independent ubiquitylation and degradation of ARF. ULF knockdown stabilizes ARF in normal human cells, triggering ARF-dependent, p53-mediated growth arrest. Moreover, nucleophosmin (NPM) and c-Myc, both of which are commonly overexpressed in cancer cells, are capable of abrogating ULF-mediated ARF ubiquitylation through distinct mechanisms, and thereby promote ARF stabilization in cancer cells. These findings reveal the dynamic feature of the ARF–p53 pathway and suggest that transcription-independent mechanisms are critically involved in ARF regulation during responses to oncogenic stress.

Differential Effects on ARF Stability by Normal versus Oncogenic Levels of c-Myc Expression

ARF suppresses aberrant cell growth upon c-Myc overexpression by activating p53 responses. Nevertheless, the precise mechanism by which ARF specifically restrains the oncogenic potential of c-Myc without affecting its normal physiological function is not well understood. Here, we show that low levels of c-Myc expression stimulate cell proliferation, whereas high levels inhibit by activating the ARF/p53 response. Although the mRNA levels of ARF are induced in both scenarios, the accumulation of ARF protein occurs only when ULF-mediated degradation of ARF is inhibited by c-Myc overexpression. Moreover, the levels of ARF are reduced through ULF-mediated ubiquitination upon DNA damage. Blocking ARF degradation by c-Myc overexpression dramatically stimulates the apoptotic responses. Our study reveals that ARF stability control is crucial for differentiating normal (low) versus oncogenic (high) levels of c-Myc expression and suggests that differential effects on ULF- mediated ARF ubiquitination by c-Myc levels act as a barrier in oncogene-induced stress responses.

NRF2 Is a Major Target of ARF in p53-Independent Tumor Suppression

Although ARF can suppress tumor growth by activating p53 function, the mechanisms by which it suppresses tumor growth independently of p53 are not well understood. Here, we identified ARF as a key regulator of nuclear factor E2-related factor 2 (NRF2) through complex purification. ARF inhibits the ability of NRF2 to transcriptionally activate its target genes, including SLC7A11, a component of the cystine/glutamate antiporter that regulates reactive oxygen species (ROS)-induced ferroptosis. As a consequence, ARF expression sensitizes cells to ferroptosis in a p53-independent manner while ARF depletion induces NRF2 activation and promotes cancer cell survival in response to oxidative stress. Moreover, the ability of ARF to induce p53-independent tumor growth suppression in mouse xenograft models is significantly abrogated upon NRF2 overexpression. These results demonstrate that NRF2 is a major target of p53-independent tumor suppression by ARF and also suggest that the ARF-NRF2 interaction acts as a new checkpoint for oxidative stress responses.

Controlling ARF stability: New players added to the team

Oncogenic stress induces the transcription of the tumor suppressor ARF, which regulates cellular responses predominantly through p53 activation. Specifically, ARF inhibits the degradation of p53 by blocking either MDM2 or ARF-BP1, known E3 ubiquitin ligases for p53 (reviewed in ref. 1). Once activated, p53 initiates cell cycle arrest or apoptosis functioning as effective barriers against tumorigenesis. As the transcriptional activation of ARF is well studied upon various cellular stress, recent focus has turned to post-translational regulation of ARF. Interestingly, ARF is a very stable protein in cancer cells yet short-lived in many normal human cell lines. ARF predominantly localizes in the nucleolus and complexes with nucleophosmin (NPM), which helps preserve ARF stability and heighten its function. Accordingly, our group previously aimed to identify potential interacting proteins with ARF through biochemical purification of NPM followed by mass spectrometry. We identified a novel E3 ubiquintin ligase for ARF, coining it ubiquitin ligase for ARF or ULF. Inhibition of ULF increases ARF levels, thereby activating p53 and decreasing cellular proliferation. Further, we show that NPM prevents ULF-mediated ubiquitination of ARF by sequestering ARF in the nucleolus and not the nucleoplasm where ULF localizes. Lastly, as c-MYC is a potent activator of ARF transcription, we analyzed the interaction between ULF and ARF. Indeed, expression of c-MYC neutralizes ULF-mediated ARF ubiquitination and stabilizes ARF protein levels. Aberrant expression of c-MYC is a common driver to many cancers; high levels of c-MYC activate the ARF/p53 axis, which inhibits cell proliferation, while low levels seem to circumvent ARF activation and promote tumorigenesis. Interestingly, basal levels of c-MYC are required for cellular homeostasis and development. Therefore, we aimed to mechanistically dissect the paradoxical roles of the cellular response difference between high and low c-MYC levels in terms of ARF regulation. Infecting cells with c-MYC at 2 different multiplicities of infection (moi), designated as either high or low c-MYC, Chen et al. show that cells infected under high c-MYC levels grew significantly slower than cells infected under low c-MYC conditions. Both conditions activated ARF transcription, yet only high c-MYC infection resulted in ARF protein elevation and subsequent p53-mediated cellular responses. High c-MYC infection disrupted the ULF-mediated ARF ubiquitination, allowing for ARF stabilization and p53-dependent apoptosis, which was exacerbated when combined with genotoxic stress. Interestingly, ARF expression gradually decreased after DNA damage, while its mRNA remained stable. Combining DNA damage with ULF knockdown rescued both the ARF depletion and starkly increased p53-dependent apoptosis. Taken in aggregate, disrupting ULF-mediated ubiquitination of ARF in combination with either oncogenic and/ or genotoxic stress stabilizes ARF and primes cells for p53-mediated apoptosis or cell cycle arrest (Fig. 1). These results, in part, help understand how cells with basal levels of c-MYC do not activate the ARF/p53 axis. The interplay between DNA damage and ARF stability warrants more study, as ubiquitin-mediated ARF degradation may not be the sole regulator of ARF protein levels. Recently, Vassilis Gorgoulis’ group defined new players in the pathway regulating ARF protein stability. Previously reported, yet through unknown mechanisms, mouse embryonic fibroblasts null for ATM, a protein kinase that regulates the DNA damage response cascade, have elevated ARF proteins levels. Velimezi et al. extended this observation to human cancer cells through ATM ablation. ARF transcription is unaffected upon ATM depletion, yet ARF protein levels are stabilized. Conversely, activation of ATM through DNA damage decreased ARF protein levels, which was reversed by ATM abrogation. Interestingly, concomitant depletion of NPM and ATM did not result in ARF stabilization, suggesting that ATM regulates NPM function. Surprisingly, by inhibiting ATM, NPM phosphorylation increased (showing stronger binding/sequestration ability for ARF), while under DNA damage, NPM phosphorylation decreased (having weaker binding to ARF). This suggested that ATM activates a phosphatase to target NPM. PP1 is a reported ATM-activated phosphatase that also dephosphorylates NPM. Indeed, after PP1 depletion, ARF levels increased and remained high with genotoxic stress, as phosphorylated NPM blocks ARF degradation. Next, seeking out the kinase involved in phosphorylating NPM upon genotoxic stress, researchers intuitively chose NEK2, a kinase previously reported to interact with NPM. Dual inhibition of NEK2 and ATM prevented ARF upregulation. Collectively, these experiments show novel opposing roles of PP1 and NEK2 in the ATM–ARF pathway (Fig. 1). Lastly, Velimezi et al.

ARF–NRF2: A new checkpoint for oxidative stress responses?

ABSTRACT NRF2 (nuclear factor erythroid 2-related factor 2) is a transcription factor which plays a major role in oxidative stress responses by regulating antioxidant gene expression. We have recently identified the ARF tumor suppressor as a key regulator of NRF2. ARF can significantly inhibit NRF2 transcriptional activities, and the ARF-NRF2 interaction may function as a novel checkpoint for oxidative stress responses.