Uncovering a novel pathway for p16 silencing: Therapeutic implications for lung cancer.

A key step during onset of most cases of non-small cell lung cancer (NSCLC) is the loss of the tumor suppressor p16INK4a (best known as p16), commonly due to promoter hypermethylation. We recently reported a novel regulatory pathway involving E6-associated protein and cell division control protein 6, which provides a methylation-independent mechanism for p16 silencing in patients with a particularly aggressive form of NSCLC.

Restoration of tumor suppression in prostate cancer by targeting the E3 ligase E6AP.

Restoration of tumor suppression is an attractive onco-therapeutic approach. It is particularly relevant when a tumor suppressor is excessively degraded by an overactive oncogenic E3 ligase. We previously discovered that the E6-associated protein (E6AP; as classified in the human papilloma virus context) is an E3 ligase that has an important role in the cellular stress response, and it directly targets the tumor-suppressor promyelocytic leukemia protein (PML) for proteasomal degradation. In this study, we have examined the role of the E6AP-PML axis in prostate cancer (PC). We show that knockdown (KD) of E6AP expression attenuates growth of PC cell lines in vitro. We validated this finding in vivo using cell line xenografts, patient-derived xenografts and mouse genetics. We found that KD of E6AP attenuates cancer cell growth by promoting cellular senescence in vivo, which correlates with restoration of tumor suppression by PML. In addition, we show that KD of E6AP sensitizes cells to radiation-induced death. Overall, our findings demonstrate a role for E6AP in the promotion of PC and support E6AP targeting as a novel approach for PC treatment, either alone or in combination with radiation.

Targeting Mdmx to treat breast cancers with wild-type p53.

The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53.

Expression of E6AP and PML predicts for prostate cancer progression and cancer-specific death.

BACKGROUND: The promyelocytic leukemia (PML) tumor suppressor plays an important role in the response to a variety of cellular stressors and its expression is downregulated or lost in a range of human tumors. We have previously shown that the E3 ligase E6-associated protein (E6AP) is an important regulator of PML protein stability but the relationship and clinical impact of PML and E6AP expression in prostatic carcinoma is unknown. METHODS: E6AP and PML expression was assessed in tissue microarrays from a phase I discovery cohort of 170 patients treated by radical prostatectomy for localized prostate cancer (PC). Correlation analysis was carried out between PML and E6AP expression and clinicopathological variates including PSA as a surrogate of disease recurrence. The results were confirmed in a phase II validation cohort of 318 patients with associated clinical recurrence and survival data. RESULTS: Survival analysis of the phase I cohort revealed that patients whose tumors showed reduced PML and high E6AP expression had reduced time to PSA relapse (P = 0.012). This was confirmed in the phase II validation cohort where the expression profile of high E6AP/low PML was significantly associated with reduced time to PSA relapse (P < 0.001), clinical relapse (P = 0.016) and PC-specific death (P = 0.014). In multivariate analysis, this expression profile was an independent prognostic indicator of PSA relapse and clinical relapse independent of clinicopathologic factors predicting recurrence. CONCLUSION: This study identifies E6AP and PML as potential prognostic markers in localized prostate carcinoma and supports a role for E6AP in driving the downregulation or loss of PML expression in prostate carcinomas.

The E6AP E3 ubiquitin ligase regulates the cellular response to oxidative stress.

The E6AP E3 ubiquitin ligase has been linked to the regulation of cell growth and to the cellular stress response. However, the specific stress conditions that are controlled by E6AP have not been defined. An important stress condition that controls cell growth is oxidative stress, where the levels of intracellular reactive oxygen species (ROS) regulate the appropriate cellular response. Here, we describe a novel role for E6AP in the control of oxidative stress response. Cells lacking E6AP expression have reduced capacity to accumulate ROS, and oxidative DNA damage, in response to 20% cell culture oxygen levels, treatment with hydrogen peroxide and expression of oncogenic RAS. This effect of E6AP is associated with the regulation of the anti-oxidant enzyme, Prx1, a previously identified target of E6AP, and can be corrected by downregulation of Prx1 or by reconstitution of E6AP expression. Consequently, cells with compromised E6AP have impaired senescent and apoptotic response to sub-lethal and lethal doses of oxidative stress, respectively. In a xenograft model, downregulation of E6AP renders transplanted tumours refractory to growth-suppressive effects of hydrogen peroxide. Our results provide the first demonstration that E6AP is an important regulator of ROS-mediated cellular senescence and cell death.

AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy.

The phosphatidylinositol 3-kinase (PI3K)/AKT and RAS oncogenic signalling modules are frequently mutated in sporadic human cancer. Although each of these pathways has been shown to play critical roles in driving tumour growth and proliferation, their activation in normal human cells can also promote cell senescence. Although the mechanisms mediating RAS-induced senescence have been well characterised, those controlling PI3K/AKT-induced senescence are poorly understood. Here we show that PI3K/AKT pathway activation in response to phosphatase and tensin homolog (PTEN) knockdown, mutant PI3K, catalytic, α polypeptide (PIK3CA) or activated AKT expression, promotes accumulation of p53 and p21, increases cell size and induces senescence-associated ß-galactosidase activity. We demonstrate that AKT-induced senescence is p53-dependent and is characterised by mTORC1-dependent regulation of p53 translation and stabilisation of p53 protein following nucleolar localisation and inactivation of MDM2. The underlying mechanisms of RAS and AKT-induced senescence appear to be distinct, demonstrating that different mediators of senescence may be deregulated during transformation by specific oncogenes. Unlike RAS, AKT promotes rapid proliferative arrest in the absence of a hyperproliferative phase or DNA damage, indicating that inactivation of the senescence response is critical at the early stages of PI3K/AKT-driven tumourigenesis. Furthermore, our data imply that chronic activation of AKT signalling provides selective pressure for the loss of p53 function, consistent with observations that PTEN or PIK3CA mutations are significantly associated with p53 mutation in a number of human tumour types. Importantly, the demonstration that mTORC1 is an essential mediator of AKT-induced senescence raises the possibility that targeting mTORC1 in tumours with activated PI3K/AKT signalling may exert unexpected detrimental effects due to inactivation of a senescence brake on potential cancer-initiating cells.

E6AP is required for replicative and oncogene-induced senescence in mouse embryo fibroblasts.

Cellular senescence is important for the maintenance of tissue homeostasis, and has recently been shown to pose a natural barrier to tumorigenesis. The E3 ubiquitin ligase, E6AP, has been linked to a number of protein regulators of the cell cycle as well as the cellular stress response. We therefore explored the role of E6AP in the cellular response to stress. We found that mouse embryo fibroblasts (MEFs) lacking E6AP escape replicative senescence, as well as Ras-induced senescence associated with impaired markers. E6AP-deficient MEFs exhibit a range of transformed phenotypes: these include the ability to grow under stress conditions (such as low serum and DNA damage), enhanced proliferation, anchorage independent growth and enhanced growth of xenografts in mice. The transformed phenotype of E6AP-deficient MEFs is associated with lower basal and stress-induced accumulation of p53. Overall, our study implicates E6AP as an important regulator of the cellular response to stress, in particular through the regulation of replicative and oncogene-induced senescence.

E6AP promotes the degradation of the PML tumor suppressor.

The promyelocytic leukemia (PML) tumor suppressor is essential for the formation of PML nuclear bodies (NBs). PML and PML-NBs have been implicated in the regulation of growth inhibition, senescence and apoptosis. PML is activated in response to stress signals and is downregulated in certain human cancers. However, the factors mediating PML stability are incompletely understood. Here we demonstrate that a catalytically active form of the mammalian E3 ligase E6AP (HPV E6-associated protein) acts to reduce the half-life of the PML protein by promoting its degradation in the proteasome. E6AP mediates the ubiquitination of PML in an in vitro ubiquitination assay. E6AP and PML interact at physiological levels and colocalize in PML-NBs. Importantly, PML protein expression is elevated in multiple organs and cell types from E6AP null mice and in lymphoid cells is associated with increased number and intensity of PML-NBs. This PML elevation is enhanced in response to DNA damage. Our results identify E6AP as an important regulator of PML and PML-NBs.

p53 controls hPar1 function and expression.

Human protease-activated receptor 1 (hPar1) is a bona fide receptor of the hemostatic protease thrombin, and has a central function in tumor progression. Inactivation of the tumor suppressor gene p53 is one of the most common genomic alterations occurring in cancer. Here, we address the interrelations between p53 and hPar1 in cancer. We demonstrate an inverse correlation between hPar1 gene expression and wild-type (wt) p53 levels, and a direct correlation with levels of the mutant (mt) p53. Bioinformatic search revealed the presence of at least two p53 motifs in the hPar1 promoter. Indeed, temperature-sensitive (ts) p53 forms reduced hPar1 promoter activity on wt p53 expression. Ectopic introduction of the p53R175H mutant into cells lacking p53 caused a moderate two-fold induction of hPar1 promoter activity. Chromatin immunoprecipitation (ChIP) analyses confirmed a physical association between the p53 protein and hPar1 chromatin fragments. In parallel, PAR1 function is attenuated by p53, as shown by inhibition of pFAK levels and a Matrigel invasion assay. Ectopic reinforcement of hPar1 rescued the inhibition conferred by p53, confirming that p53 directly affects hPar1 expression and function. Altogether, we provide evidence for a direct binding between p53 and hPar1 chromatin, and assign hPar1 as a target of p53.

PML enhances the regulation of p53 by CK1 in response to DNA damage.

In response to stress, p53 is accumulated and activated to induce appropriate growth inhibitory responses. This requires the release of p53 from the constraints of its negative regulators Mdm2 and Mdm4. A key event in this dissociation is the phosphorylation of p53 at threonine residue (Thr18) within the Mdm2/4-binding domain. Casein kinase 1 (CK1) plays a major role in this phosphorylation. The promyelocytic leukemia protein (PML) regulates certain modifications of p53 in response to DNA damage. Here, we investigated the role of PML in the regulation of Thr18 phosphorylation. We found that PML enhances Thr18 phosphorylation of endogenous p53 in response to stress. On DNA damage, CK1 accumulates in the cell, with a proportion concentrated in the nucleus together with p53 and PML. Furthermore, CK1 interacts with endogenous p53 and PML, and this interaction is enhanced by genotoxic stress. Inhibition of CK1 impairs the protection of p53 by PML from Mdm2-mediated degradation. Our findings support a role for PML in the regulation of p53 by CK1. We propose that following DNA damage, PML facilitates Thr18 phosphorylation by recruiting p53 and CK1 into PML nuclear bodies, thereby protecting p53 from inhibition by Mdm2, leading to p53 activation.

Tumor suppressor p53 regulates heparanase gene expression.

Mammalian heparanase degrades heparan sulfate, the most prominent polysaccharide of the extracellular matrix. Causal involvement of heparanase in tumor progression is well documented. Little is known, however, about mechanisms that regulate heparanase gene expression. Mutational inactivation of tumor suppressor p53 is the most frequent genetic alteration in human tumors. p53 is a transcription factor that regulates a wide variety of cellular promoters. In this study, we demonstrate that wild-type (wt) p53 binds to heparanase promoter and inhibits its activity, whereas mutant p53 variants failed to exert an inhibitory effect. Moreover, p53-H175R mutant even activated heparanase promoter activity. Elimination or inhibition of p53 in several cell types resulted in a significant increase in heparanase gene expression and enzymatic activity. Trichostatin A abolished the inhibitory effect of wt p53, suggesting the involvement of histone deacetylation in negative regulation of the heparanase promoter. Altogether, our results indicate that the heparanase gene is regulated by p53 under normal conditions, while mutational inactivation of p53 during cancer development leads to induction of heparanase expression, providing a possible explanation for the frequent increase of heparanase levels observed in the course of tumorigenesis.

c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination.

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.

Transactivation-deficient p73alpha (p73Deltaexon2) inhibits apoptosis and competes with p53.

p73 has recently been identified as a structural and functional homolog of the tumor suppressor protein p53. Overexpression of p53 activates transcription of p53 effector genes, causes growth inhibition and induced apoptosis. We describe here the effects of a tumor-derived truncated transcript of p73alpha (p73Deltaexon2) on p53 function and on cell death. This transcript, which lacks the acidic N-terminus corresponding to the transactivation domain of p53, was initially detected in a neuroblastoma cell line. Overexpression of p73Deltaexon2 partially protects lymphoblastoid cells against apoptosis induced by anti-Fas antibody or cisplatin. By cotransfecting p73Deltaexon2 with wild-type p53 in the p53 null line Saos 2, we found that this truncated transcript reduces the ability of wild-type p53 to promote apoptosis. This anti-apoptotic effect was also observed when p73Deltaexon2 was co-transfected with full-length p73 (p73alpha). This was further substantiated by suppression of p53 transactivation of the effector gene p21/Waf1 in p73Deltaexon2 transfected cells and by inhibition of expression of a reporter gene under the control of the p53 promoter. Thus, this truncated form of p73 can act as a dominant-negative agent towards transactivation by p53 and p73alpha, highlighting the potential implications of these findings for p53 signaling pathway. Furthermore, we demonstrate the existence of a p73Deltaexon2 transcript in a very significant proportion (46%) of breast cancer cell lines. However, a large spectrum of normal and malignant tissues need to be surveyed to determine whether this transdominant p73 variant occurs in a tumor-specific manner.

A role for the polyproline domain of p53 in its regulation by Mdm2.

The p53 protein plays a key role in the cellular response to stress by inducing cell growth arrest or apoptosis. The polyproline region of p53 has been shown to be important for its growth suppression activity. p53 protein lacking the polyproline region has impaired apoptotic activity and altered specificity for certain apoptotic target genes. Here we describe the role of this region in the regulation of p53 by its inhibitor Mdm2. p53 lacking the polyproline region was identified to be more susceptible to inhibition by Mdm2. Furthermore, the absence of this region renders p53 more accessible to ubiquitination, nuclear export, and Mdm2-mediated degradation. This increased sensitivity to Mdm2 results from an enhanced affinity of Mdm2 toward p53 lacking the polyproline region. Our results provide a new explanation for the impaired growth suppression activity of p53 lacking this region. The polyproline region is proposed to be important in the modulation of the inhibitory effects of Mdm2 on p53 activities and stability.

c-Jun and p53 activity is modulated by SUMO-1 modification.

The ubiquitin-related SUMO-1 molecule has been shown recently to modify covalently a number of cellular proteins including IkappaBalpha. SUMO-1 modification was found to antagonize IkappaBalpha ubiquitination and protect it from degradation. Here we identify the transcription factors c-Jun and p53, two well known targets of ubiquitin, as new substrates for SUMO-1 both in vitro and in vivo. In contrast to ubiquitin, SUMO-1 preferentially targets a single lysine residue in c-Jun (Lys-229), and the abrogation of SUMO-1 modification does not compromise its ubiquitination. Activation of Jun NH(2)-terminal kinases, which induces a reduction in c-Jun ubiquitination, similarly decreases SUMO-1 modification. Accordingly, loss of the two major Jun NH(2)-terminal kinase phosphorylation sites in c-Jun, Ser-63 and Ser-73, greatly enhances conjugation by SUMO-1. A SUMO-1- deficient c-JunK229R mutant shows an increased transactivation potential on an AP-1-containing promoter compared with wild-type c-Jun, suggesting that SUMO-1 negatively regulates c-Jun activity. As with c-Jun, SUMO-1 modification of p53 is abrogated by phosphorylation but remains unaltered upon chemical damage to DNA or Mdm2-mediated ubiquitination. The SUMO-1 attachment site in p53 (Lys-386) resides within a region known to regulate the DNA binding activity of the protein. A p53 mutant, defective for SUMO-1 conjugation, shows unaltered ubiquitination but has a slightly impaired apoptotic activity, indicating that modification by SUMO-1 might be important for the full biological activity of p53. Taken together, these data provide a first link between the SUMO-1 conjugation pathway and the regulation of transcription factors.

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent.

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.

The cellular response to p53: the decision between life and death.

The p53 tumor suppressor protein plays a crucial role in regulating cell growth following exposure to various stress stimuli. p53 induces either growth arrest, which prevents the replication of damaged DNA, or programmed cell death (apoptosis), which is important for eliminating defective cells. Whether the cell enters growth arrest or undergoes apoptosis, depends on the final integration of incoming signals with antagonistic effects on cell growth. Many factors affect the cellular response to activated p53. These include the cell type, the oncogenic status of the cell with emphasis on the Rb/E2F balance, the extracellular growth and survival stimuli, the intensity of the stress signals, the level of p53 expression and the interaction of p53 with specific proteins. p53 is regulated both at the levels of protein stability and biochemical activities. This complex regulation is mediated by a range of viral and cellular proteins. This review discusses this intriguing complexity which affects the cell response to p53 activation.