PH domain-only protein PHLDA3 is a p53-regulated repressor of Akt.

p53 And Akt are critical players regulating tumorigenesis with opposite effects: whereas p53 transactivates target genes to exert its function as a tumor suppressor, Akt phosphorylates its substrates and transduces downstream survival signals. In addition, p53 and Akt negatively regulate each other to balance survival and death signals within a cell. We now identify PHLDA3 as a p53 target gene that encodes a PH domain-only protein. We find that PHLDA3 competes with the PH domain of Akt for binding of membrane lipids, thereby inhibiting Akt translocation to the cellular membrane and activation. Ablation of endogenous PHLDA3 results in enhanced Akt activity and decrease of p53-dependent apoptosis. We also demonstrate the suppression of anchorage-independent cell growth by PHLDA3. Loss of the PHLDA3 genomic locus was frequently observed in primary lung cancers, suggesting a role of PHLDA3 in tumor suppression. Our results reveal a new mode of coordination between the p53 and Akt pathways.

TSPAN12 is a critical factor for cancer-fibroblast cell contact-mediated cancer invasion.

Communication between cancer cells and their microenvironment controls cancer progression. Although the tumor suppressor p53 functions in a cell-autonomous manner, it has also recently been shown to function in a non-cell-autonomous fashion. Although functional defects have been reported in p53 in stromal cells surrounding cancer, including mutations in the p53 gene and decreased p53 expression, the role of p53 in stromal cells during cancer progression remains unclear. We herein show that the expression of α-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts (CAFs), was increased by the ablation of p53 in lung fibroblasts. CAFs enhanced the invasion and proliferation of lung cancer cells when cocultured with p53-depleted fibroblasts and required contact between cancer and stromal cells. A comprehensive analysis using a DNA chip revealed that tetraspanin 12 (TSPAN12), which belongs to the tetraspanin protein family, was derepressed by p53 knockdown. TSPAN12 knockdown in p53-depleted fibroblasts inhibited cancer cell proliferation and invasion elicited by coculturing with p53-depleted fibroblasts in vitro, and inhibited tumor growth in vivo. It also decreased CXC chemokine ligand 6 (CXCL6) secretion through the ß-catenin signaling pathway, suggesting that cancer cell contact with TSPAN12 in fibroblasts transduced ß-catenin signaling into fibroblasts, leading to the secretion of CXCL6 to efficiently promote invasion. These results suggest that stroma-derived p53 plays a pivotal role in epithelial cancer progression and that TSPAN12 and CXCL6 are potential targets for lung cancer therapy.

PHLDA3 is a novel tumor suppressor of pancreatic neuroendocrine tumors.

The molecular mechanisms underlying the development of pancreatic neuroendocrine tumors (PanNETs) have not been well defined. We report here that the genomic region of the PHLDA3 gene undergoes loss of heterozygosity (LOH) at a remarkably high frequency in human PanNETs, and this genetic change is correlated with disease progression and poor prognosis. We also show that the PHLDA3 locus undergoes methylation in addition to LOH, suggesting that a two-hit inactivation of the PHLDA3 gene is required for PanNET development. We demonstrate that PHLDA3 represses Akt activity and Akt-regulated biological processes in pancreatic endocrine tissues, and that PHLDA3-deficient mice develop islet hyperplasia. In addition, we show that the tumor-suppressing pathway mediated by MEN1, a well-known tumor suppressor of PanNETs, is dependent on the pathway mediated by PHLDA3, and inactivation of PHLDA3 and MEN1 cooperatively contribute to PanNET development. Collectively, these results indicate the existence of a novel PHLDA3-mediated pathway of tumor suppression that is important in the development of PanNETs.

Novel p53 target gene FUCA1 encodes a fucosidase and regulates growth and survival of cancer cells.

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α-l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.

MOZ increases p53 acetylation and premature senescence through its complex formation with PML.

Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)- and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML-MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML.

Interaction between RB protein and NuMA is required for proper alignment of spindle microtubules.

Retinoblastoma protein (pRB) controls cell cycle progression and cell cycle exit through interactions with cellular proteins. Many pRB-binding proteins, which function in gene transcription or modulation of chromatin structure, harbor LXCXE motifs in their binding domain for pRB. In this study, we found that nuclear mitotic apparatus protein (NuMA), a mitotic spindle organizer, interacts with pRB through LSCEE sequences located in its C-terminal region. siRNA-mediated down-regulation of pRB caused aberrant distribution of NuMA and alignment of spindle microtubules in mitotic cells. Abnormal organization of spindle microtubules was also accompanied by misalignment of an over-expressed NuMA mutant (mut-NuMA) with a defect in pRB binding caused by an LSGEK mutation. The mut-NuMA-over-expressing cells showed lower potency for survival than wild-type NuMA (wt-NuMA)-over-expressing cells during 2 weeks of culture. Interestingly, knockdown of pRB reduced the population of wt-NuMA-over-expressing cells to the same level as mut-NuMA cells after 2 weeks. Taken together, pRB may have a novel function in regulating the mitotic function of NuMA and spindle organization, which are required for proper cell cycle progression.

Induction of ZEB proteins by inactivation of RB protein is key determinant of mesenchymal phenotype of breast cancer.

We previously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition. To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation and induced epithelial marker expression. These results implicate ZEB in induction of the epithelial-mesenchymal transition, as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway.

The cellular DNA-damage response is a signaling network that is vigorously activated by cytotoxic DNA lesions, such as double-strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which modulates this process by phosphorylating key players in these pathways. A long-standing question in this field is whether DSB formation affects chromatin condensation. Here, we show that DSB formation is followed by ATM-dependent chromatin relaxation. ATM's effector in this pathway is the protein KRAB-associated protein (KAP-1, also known as TIF1beta, KRIP-1 or TRIM28), previously known as a corepressor of gene transcription. In response to DSB induction, KAP-1 is phosphorylated in an ATM-dependent manner on Ser 824. KAP-1 is phosphorylated exclusively at the damage sites, from which phosphorylated KAP-1 spreads rapidly throughout the chromatin. Ablation of the phosphorylation site of KAP-1 leads to loss of DSB-induced chromatin decondensation and renders the cells hypersensitive to DSB-inducing agents. Knocking down KAP-1, or mimicking a constitutive phosphorylation of this protein, leads to constitutive chromatin relaxation. These results suggest that chromatin relaxation is a fundamental pathway in the DNA-damage response and identify its primary mediators.

Complex regulation of p73 isoforms after alteration of amyloid precursor polypeptide (APP) function and DNA damage in neurons.

Genetic ablations of p73 have shown its implication in the development of the nervous system. However, the relative contribution of ΔNp73 and TAp73 isoforms in neuronal functions is still unclear. In this study, we have analyzed the expression of these isoforms during neuronal death induced by alteration of the amyloid-ß precursor protein function or cisplatin. We observed a concomitant up-regulation of a TAp73 isoform and a down-regulation of a ΔNp73 isoform. The shift in favor of the pro-apoptotic isoform correlated with an induction of the p53/p73 target genes such as Noxa. At a functional level, we showed that TAp73 induced neuronal death and that ΔNp73 has a neuroprotective role toward amyloid-ß precursor protein alteration or cisplatin. We investigated the mechanisms of p73 expression and found that the TAp73 expression was regulated at the promoter level. In contrast, regulation of ΔNp73 protein levels was regulated by phosphorylation at residue 86 and multiple proteases. Thus, this study indicates that tight transcriptional and post-translational mechanisms regulate the p73 isoform ratios that play an important role in neuronal survival.

Cancer susceptibility polymorphism of p53 at codon 72 affects phosphorylation and degradation of p53 protein.

The common polymorphism of p53 at codon 72, either encoding proline or arginine, has drawn attention as a genetic factor associated with clinical outcome or cancer risk for the last 2 decades. We now show that these two polymorphic variants differ in protein structure, especially within the N-terminal region and, as a consequence, differ in post-translational modification at the N terminus. The arginine form (p53-72R) shows significantly enhanced phosphorylation at Ser-6 and Ser-20 compared with the proline form (p53-72P). We also show diminished Mdm2-mediated degradation of p53-72R compared with p53-72P, which is at least partly brought about by higher levels of phosphorylation at Ser-20 in p53-72R. Furthermore, enhanced p21 expression in p53-72R-expressing cells, which is dependent on phosphorylation at Ser-6, was demonstrated. Differential p21 expression between the variants was also observed upon activation of TGF-ß signaling. Collectively, we demonstrate a novel molecular difference and simultaneously suggest a difference in the tumor-suppressing function of the variants.

Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2.

Transcriptional activity of p53, a central regulatory switch in a network controlling cell proliferation and apoptosis, is modulated by protein stability and post-translational modifications including phosphorylation and acetylation. Here we demonstrate that the human serine/threonine kinase homeodomain-interacting protein kinase-2 (HIPK2) colocalizes and interacts with p53 and CREB-binding protein (CBP) within promyelocytic leukaemia (PML) nuclear bodies. HIPK2 is activated by ultraviolet (UV) radiation and selectively phosphorylates p53 at Ser 46, thus facilitating the CBP-mediated acetylation of p53 at Lys 382, and promoting p53-dependent gene expression. Accordingly, the kinase function of HIPK2 mediates the increased expression of p53 target genes, which results in growth arrest and the enhancement of UV-induced apoptosis. Interference with HIPK2 expression by antisense oligonucleotides impairs UV-induced apoptosis. Our results imply that HIPK2 is a novel regulator of p53 effector functions involved in cell growth, proliferation and apoptosis.

Hedgehog signaling overrides p53-mediated tumor suppression by activating Mdm2.

The hedgehog (Hh) signaling pathway regulates the development of many organs in mammals, and activation of this pathway is widely observed in human cancers. Although it is known that Hh signaling activates the expression of genes involved in cell growth, the precise role of the Hh pathway in cancer development is still unclear. Here, we show that constitutively activated mutants of Smoothened (Smo), a transducer of the Hh signaling pathway, inhibit the accumulation of the tumor suppressor protein p53. This inhibition was also observed in the presence of Hh ligand or with the overexpression of the transcription factors Gli1 and Gli2, downstream effectors of Smo, indicating that this inhibition is specific for the Hh pathway. We also report that Smo mutants augment p53 binding to the E3 ubiquitin-protein ligase Mdm2 and promote p53 ubiquitination. Furthermore, Hh signaling induced the phosphorylation of human Mdm2 protein on serines 166 and 186, which are activating phosphorylation sites of Mdm2. Smo mutants enhanced the proliferation of mouse embryonic fibroblasts (MEFs) while inducing a DNA-damage response. Moreover, Smo partially inhibited p53-dependent apoptosis and cell growth inhibition in oncogene-expressing MEFs. We also found that accumulation of p53 is inhibited by Hh signaling in several human cancer cell lines. Therefore, the Hh pathway may be a powerful accelerator of oncogenesis by activating cell proliferation and inhibiting the p53-mediated anti-cancer barrier induced by oncogenic stress.

p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease.

We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington's disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific, as it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-alpha, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. Our findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD.

Cytoplasmic tethering is involved in synergistic inhibition of p53 by Mdmx and Mdm2.

The mdm2 and mdmx oncogenes play essential yet nonredundant roles in synergistic inactivatiosn of p53. However, the biochemical mechanism by which Mdmx synergizes with Mdm2 to inhibit p53 function remains obscure. Here we demonstrate that, using nonphosphorylatable mutants of Mdmx, the cooperative inhibition of p53 by Mdmx and Mdm2 was associated with cytoplasmic localization of p53, and with an increase of the interaction of Mdmx to p53 and Mdm2 in the cytoplasm. In addition, the Mdmx mutant cooperates with Mdm2 to induce ubiquitination of p53 at C-terminal lysine residues, and the integrity of the C-terminal lysines was partly required for the cooperative inhibition. The expression of subcellular localization mutants of Mdmx revealed that subcellular localization of Mdmx dictated p53 localization, and that cytoplasmic Mdmx tethered p53 in the cytoplasm and efficiently inhibited p53 activity. RNAi-mediated inhibition of Mdmx or introduction of the nuclear localization mutant of Mdmx reduced cytoplasmic retention of p53 in neuroblastoma cells, in which cytoplasmic sequestration of p53 is involved in its inactivation. Our data indicate that cytoplasmic tethering of p53 mediated by Mdmx contributes to p53 inactivation in some types of cancer cells.

Regulation of clathrin-mediated endocytosis by p53.

The p53 gene encodes a multi-functional protein to prevent tumorigenesis. Although there have been many reports of the nuclear functions of p53, little is known about the cytosolic functions of p53. Here, we found that p53 is present in cytosol as well as nuclei under unstressed conditions and binds to clathrin heavy chain (CHC). CHC is known to play a role in receptor-mediated endocytosis. Based on our findings, we examined the effect of p53 on clathrin-mediated endocytosis of epidermal growth factor receptor (EGFR). Surprisingly, p53 co-localized with CHC at the plasma membrane in response to EGF stimulation. In cells with ablated p53 expression by RNAi, EGFR internalization was delayed and intracellular signaling from EGFR was altered. Thus, our findings provide evidence that cytosolic p53 may participate in the regulation of clathrin-mediated endocytosis to control the correct signaling from EGFR.

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.

DNA damage-induced phosphorylation of MdmX at serine 367 activates p53 by targeting MdmX for Mdm2-dependent degradation.

Understanding how p53 activity is regulated is crucial in elucidating mechanisms of cellular defense against cancer. Genetic data indicate that Mdmx as well as Mdm2 plays a major role in maintaining p53 activity at low levels in nonstressed cells. However, biochemical mechanisms of how Mdmx regulates p53 activity are not well understood. Through identification of Mdmx-binding proteins, we found that 14-3-3 proteins are associated with Mdmx. Mdmx harbors a consensus sequence for binding of 14-3-3. Serine 367 (S367) is located within the putative binding sequence for 14-3-3, and its substitution with alanine (S367A) abolishes binding of Mdmx to 14-3-3. Transfection assays indicated that the S367A mutation, in cooperation with Mdm2, enhances the ability of Mdmx to repress the transcriptional activity of p53. The S367A mutant is more resistant to Mdm2-dependent ubiquitination and degradation than wild-type Mdmx, and Mdmx phosphorylated at S367 is preferentially degraded by Mdm2. Several types of DNA damage markedly enhance S367 phosphorylation, coinciding with increased binding of Mdmx to 14-3-3 and accelerated Mdmx degradation. Furthermore, promotion of growth of normal human fibroblasts after introduction of Mdmx is enhanced by the S367 mutation. We propose that Mdmx phosphorylation at S367 plays an important role in p53 activation after DNA damage by triggering Mdm2-dependent degradation of Mdmx.

p53AIP1 regulates the mitochondrial apoptotic pathway.

We identified recently the p53AIP1 gene, a novel p53 target that mediates p53-dependent apoptosis. In the experiments reported here, ectopic expression of p53AIP1 induced down-regulation of mitochondrial DeltaPsim and release of cytochrome c from mitochondria in human cells. Immunoprecipitation and immunostaining experiments indicated interaction between p53AIP1 and bcl-2 proteins at mitochondria. Overexpression of bcl-2 blocked the down-regulation of mitochondrial DeltaPsim and the proapoptotic activity of p53AIP1. Our results implicate p53AIP1 as a pivotal mediator of the p53-dependent mitochondrial apoptotic pathway.

IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis.

The transcription factors HSF1 and p53 both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both have the potential to promote tumor development. Here we show that a p53 target gene, IER5, encodes an activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. IER5 is also transcriptionally upregulated in various cancers, although this upregulation is not always p53-dependent. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation of these cells under stressed conditions. These results reveal the existence of a novel IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 observed in various cancers.

Functional role of Mdm2 phosphorylation by ATR in attenuation of p53 nuclear export.

Mdm2 oncoprotein plays a major role in inhibiting the p53 tumor suppressor protein. Here, we investigate phosphorylation of Mdm2 at serine 407 (S407). S407 is phosphorylated in cells after treatment with camptothecin (CPT) or hydroxyurea, inhibitors of DNA replication. S407 phosphorylation after CPT treatment is induced upon cell cycle arrest during S phase and prevented if entry into S phase of cell cycle is blocked. We found that a major kinase responsible for S407 phosphorylation is ATR, a DNA damage checkpoint protein that induces cell cycle arrest and promotes DNA repair in response to impaired DNA replication; induction of S407 phosphorylation is enhanced after expression of wild-type ATR, while it is inhibited by a dominant-negative form of ATR. Further, S407 is specifically phosphorylated by ATR in vitro. Substitution of S407 with aspartate (S407D), but not with alanine (S407A), promotes nuclear localization of p53. Taken together, our data indicate that S407 phosphorylation of Mdm2 by ATR reduces Mdm2-dependent export of p53 from nuclei to cytoplasm.