Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.     

BH3 activation blocks Hdmx suppression of apoptosis and cooperates with Nutlin to induce cell death

The Hdmx protein restricts p53 activity in vivo and is overexpressed in a significant fraction of human tumors that retain the wild type p53 allele. An understanding of how Hdmx limits p53 activation and blocks apoptosis could therefore lead to development of novel therapeutic agents. We previously showed that Hdmx modulates tumor cell sensitivity to Nutlin-3a, a potent antagonist of the p53/Hdm2 interaction. In this report, we demonstrate that this also applies to MI-219, another Hdm2 antagonist. Thus, the inability to disrupt Hdmx/p53 complexes is a potential barrier to the efficacy of these compounds as single agents. We show that sensitivity to apoptosis in cells with high Hdmx levels is restored by combined treatment with Hdm2 and a Bcl-2 family member antagonist to activate Bax. The data are consistent with a model in which Hdmx attenuates p53-dependent activation of the intrinsic apoptotic pathway, and that this occurs upstream of Bax activation. Thus, selectively inhibiting Hdm2 and activating Bax is one effective strategy to induce apoptosis in tumors with high Hdmx levels. Our findings also indicate that preferential induction of apoptosis in tumor versus normal cells occurs using appropriate drug doses.     

Hdmx modulates the outcome of p53 activation in human tumor cells

Tumors that express wild-type P53 provide a target for therapies designed to reactivate P53 function. This is supported by the potent activation of P53 in tumor cells by Nutlin, a cis-imidazoline that inhibits the Hdm2-P53 interaction. The efficacy of Hdm2.P53 antagonists could be compromised if they do not antagonize Hdmx, an Hdm2 homolog that inhibits P53 transactivation. We evaluated the role of Hdmx expression in sensitivity to Nutlin in a range of cancer cell lines. Nutlin reduced Hdmx levels in normal cells and some cancer cell lines, whereas other cancer cells were refractory to such down-regulation. Strikingly, Nutlin did not disrupt Hdmx.P53 complexes, and in cell lines where no Hdmx degradation occurred, Nutlin failed to induce apoptosis. shRNA-mediated reduction of Hdmx sensitized cells to apoptosis, but caspase-3 was neither required nor sufficient for Hdmx degradation or apoptosis. Our data imply that Hdmx is an important determinant of the outcome of P53 activation. Thus, targeting Hdmx may be a therapeutic strategy that complements drugs such as Nutlin.     

Differential roles of ATM- and Chk2-mediated phosphorylations of Hdmx in response to DNA damage

The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.     

Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity

Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14(ARF) or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRas(V12). Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.     

Solution structure of the p53 regulatory domain of the p19Arf tumor suppressor protein

Arf is a tumor suppressor that regulates p53 function and is a frequent target for loss in human cancers. Through two novel mechanisms, Arf inhibits the oncoprotein Hdm2, a negative regulator of p53. (1) Arf inhibits the E3 ubiquitin ligase activity of Hdm2 that leads to p53 degradation, and (2) Arf sequesters Hdm2 within nucleoli. These activities of Arf promote p53-mediated cell cycle arrest and apoptosis. Fundamental to these processes are interactions between Arf and Hdm2. Here we show that a peptide containing the 37 N-terminal amino acids of mouse Arf (mArfN37) localizes to nucleoli, sequesters Hdm2 within nucleoli, and causes cell cycle arrest. Circular dichroism and NMR spectroscopy show that mArfN37 is largely unstructured under aqueous conditions; however, the peptide adopts two alpha-helices (helix 1, residues 4-14; and helix 2, residues 20-29) in 2,2,2-trifluoroethanol (TFE). Each helix contains an amino acid motif that is repeated twice in mArfN37, once in each helix. The two helices, however, do not interact but are connected by an apparently flexible linker. The repeated motif contains Arg residues spaced by a hydrophobic segment that may be involved in Hdm2 recognition and binding. The RRPR nucleolar localization signal, contained within residues 31-34, appears to be disordered under all conditions. The identification of two Arf structural modules suggests that short peptides containing the repeated motif may function as Arf mimics and may allow the design of small molecule Arf mimics in the future.     

The ubiquitin-protein ligase activity of Hdm2 is inhibited by nucleic acids

The proto-oncoprotein Hdm2 is a member of the RING finger-type family of ubiquitin-protein ligases E3. The RING finger domain is assumed to mediate the specific interaction of an E3 with its cognate ubiquitin-conjugating enzyme E2, which catalyzes the covalent attachment of ubiquitin to substrate proteins. In addition, the RING finger domain of Hdm2 is involved in Hdm2 homooligomer formation and has the capacity to bind to RNA in a sequence-specific manner. Here we report that interaction with nucleic acids interferes with both Hdm2/Hdm2 complex formation and auto-ubiquitination of Hdm2 in vitro. Furthermore, although binding of Hdm2 to the tumor suppressor p53 is not inhibited by nucleic acids, Hdm2-mediated ubiquitination of p53 is significantly decreased. Taken together, these results provide the first example of an E3 whose activity can be regulated by direct interaction with nucleic acids.     

HdmX overexpression inhibits oncogene induced cellular senescence

Cellular senescence is an irreversible state of terminal growth arrest that requires functional p53. Acting to block tumor formation, induction of senescence has also been demonstrated to contribute to tumor clearance via the immune system following p53 reactivation.^{1, 2} The Hdm2-antagonist, Nutlin-3a, has been shown to reactivate p53 and induce a quiescent state in various cancer cell lines,^{3, 4} similar to the G1 arrest observed upon RNAi targeting of Hdm2 in MCF7 breast cancer.⁵ In the present study we show that HdmX, a negative regulator of p53, impacts the senescence pathway. Specifically, overexpression of HdmX blocks Ras mediated senescence in primary human fibroblasts. The interaction of HdmX with p53 and the re-localization of HdmX to the nucleus through Hdm2 association appear to be required for this activity. Furthermore, inhibiting HdmX in prostate adenocarcinoma cells expressing wild-type p53, mutant Ras and high levels of HdmX induced cellular senescence as measured by an increase in irreversible b-galactosidase staining. Together these results suggest that HdmX overexpression may contribute to tumor formation by blocking senescence and that targeting HdmX may represent an attractive anti-cancer therapeutic approach.     

Abrogation of Wip1 expression by RITA-activated p53 potentiates apoptosis induction via activation of ATM and inhibition of HdmX

Inactivation of the p53 tumour suppressor, either by mutation or by overexpression of its inhibitors Hdm2 and HdmX is the most frequent event in cancer. Reactivation of p53 by targeting Hdm2 and HdmX is therefore a promising strategy for therapy. However, Hdm2 inhibitors do not prevent inhibition of p53 by HdmX, which impedes p53-mediated apoptosis. Here, we show that p53 reactivation by the small molecule RITA leads to efficient HdmX degradation in tumour cell lines of different origin and in xenograft tumours in vivo. Notably, HdmX degradation occurs selectively in cancer cells, but not in non-transformed cells. We identified the inhibition of the wild-type p53-induced phosphatase 1 (Wip1) as the major mechanism important for full engagement of p53 activity accomplished by restoration of the ataxia telangiectasia mutated (ATM) kinase-signalling cascade, which leads to HdmX degradation. In contrast to previously reported transactivation of Wip1 by p53, we observed p53-dependent repression of Wip1 expression, which disrupts the negative feedback loop conferred by Wip1. Our study reveals that the depletion of both HdmX and Wip1 potentiates cell death due to sustained activation of p53. Thus, RITA is an example of a p53-reactivating drug that not only blocks Hdm2, but also inhibits two important negative regulators of p53 - HdmX and Wip1, leading to efficient elimination of tumour cells.     

Differential recognition by the tumor suppressor protein p53 of DNA modified by the novel antitumor trinuclear platinum drug BBR3464 and cisplatin

The trinuclear platinum agent BBR3464, a representative of a new class of anticancer drugs, is more potent than conventional mononuclear cisplatin [cis-diamminedichloroplatinum(II)]. BBR3464 retains significant activity in human tumor cell lines and xenografts that are refractory or poorly responsive to cisplatin, and displays a high activity in human tumor cell lines that are characterized by both wild-type and mutant p53 gene. In contrast, on average, cells with mutant p53 are more resistant to the effect of cisplatin. It has been hypothesized that the sensitivity or resistance of tumor cells to cisplatin might be also associated with cell cycle control and repair processes that involve p53. DNA is a major pharmacological target of platinum compounds and DNA binding activity of the p53 protein is crucial for its tumor suppressor function. This study, using gel-mobility-shift assays, was undertaken to examine the interactions of active and latent p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by BBR3464 in a cell free medium and to compare these results with those describing the interactions of these proteins with DNA modified by cisplatin. The results indicate that structurally different DNA adducts of BBR3464 and cisplatin exhibit a different efficiency to affect the binding affinity of the modified DNA to p53 protein. It has been suggested that different structural perturbations induced in DNA by the adducts of BBR3464 and cisplatin produce a differential response to p53 protein activation and recognition and that a 'molecular approach' to control of downstream effects such as protein recognition and pathways of apoptosis induction may consist in design of structurally unique DNA adducts as cell signals.     

Intrinsically unstructured domains of Arf and Hdm2 form bimolecular oligomeric structures in vitro and in vivo

Arf, Hdm2, and p53 regulate the tumor-suppressor pathway that is most frequently disrupted in human cancer. In the absence of tumorigenic stress, Hdm2 actively attenuates p53-dependent cell cycle arrest and apoptosis by mediating ubiquitination-dependent degradation of p53. Mitogenic stress activates Arf, which indirectly activates p53 by binding to and nullifying the anti-p53 activities of Hdm2. Small conserved domains within Arf and Hdm2 mediate their direct interaction. Individually, these domains are intrinsically unstructured and, when combined in vitro, cofold into bimolecular oligomeric structures that resemble amyloid fibrils in some features. Detailed structural characterization of Hdm2/Arf complexes has previously been hampered by their heterogeneity and large size. Here, we report that a nine-residue fragment of the N-terminus of mouse Arf (termed "A1-mini") cofolds specifically with the Arf-binding domain of Hdm2 to form bimolecular oligomers. We characterized these unprecedented structures using analytical ultracentrifugation and NMR spectroscopy, providing insights into their structural organization. The A1-mini peptide not only binds specifically to Hdm2 in vitro but also recapitulates the nucleolar localization features of full-length Arf in cells. Furthermore, larger fragments of Arf that contain the A1-mini segment have previously been shown to activate p53 in mouse and human cells. Our studies provide the first insights into the molecular basis through which Arf nullifies the p53-inhibiting activity of Hdm2, indirectly activating the tumor-suppressor function of p53 in mammalian cells.