Regulator Of P53 In Response Research Articles

Crosstalk between tumor suppressors p53 and PKCδ: Execution of the intrinsic apoptotic pathways

p53 and PKCδ are tumor suppressors that execute apoptotic mechanisms in response to various cellular stresses. p53 is a transcription factor that is frequently mutated in human cancers; it regulates apoptosis in transcription-dependent and -independent ways in response to genotoxic stresses. PKCδ is a serine/threonine protein kinase and mutated in human cancers. Available evidence shows that PKCδ activates p53 by direct and/or indirect mechanisms. Moreover, PKCδ is also implicated in the transcriptional regulation of p53 in response to DNA damage. Recent findings demonstrated that p53, in turn, binds onto the PKCδ promoter and induces its expression upon DNA damage to facilitate apoptosis. Both p53 and PKCδ are associated with the apoptotic mechanisms in the mitochondria by regulating Bcl-2 family proteins to provide mitochondrial outer membrane permeabilization. This review discusses the crosstalk between p53 and PKCδ in the context of apoptotic cell death and cancer therapy.

Abstract 159: LncRNA RoR enhances the c-Myc mRNA stability in colon cancer

Abstract Background: Emerging evidence has implicated long non-coding RNAs (lncRNAs) as master gene regulators; and they are often aberrantly expressed in a variety of human diseases including cancer. One of the mechanisms of lncRNA-mediated gene expression involves modulation of translation or mRNA stability by interacting with RNA binding proteins. We have previously demonstrated that lncRNA-RoR is a strong negative regulator of p53 in response to DNA damage. The present study was to determine whether and how lncRNA-RoR regulates c-Myc in colon cancer. Methods: MTT assays were used to determine cell growth. Colon cancer tissue array was used to determine the level of lncRNA-RoR in colon cancer tissues by qRT-PCR. Interaction between lncRNA-RoR and RNA binding proteins was confirmed by RNA co-immunoprecipitation and RNA precipitation. LncRNA-RoR knockout in colon cancer cells was through CRISPR/Cas9 system. Dual fluorescent in situ hybridization (FISH) was used to determine the relative levels of lncRNA-RoR and c-Myc mRNA, and their co-localizations in colon cancer specimens. The effect of lncRNA-RoR on tumor growth was determined by a xenograft mouse model. Results: Ectopic expression of lncRNA-RoR upregulated, whereas RoR-siRNA inhibited c-Myc mRNA and protein level, which was further confirmed by lncRNA-RoR knockout. Mechanistically, we showed that lncRNA-RoR directly interacted with RNA binding proteins, hnRNP U and hnRNP D, both of which are implicated in stability of c-Myc mRNA. This interaction was further confirmed by RNA immunoprecipitation. In consistent with these findings, ectopic expression of lncRNA-RoR stimulated cell proliferation but knockout of lncRNA-RoR significantly decreased cell proliferation. Moreover, ectopic expression of lncRNA-RoR in HCT-116 cells significantly promoted tumor growth in a xenograft mouse model. Finally, qPCR array showed the significant upregulation of lncRNA-RoR in colon cancer tissues. Similarly dual FISH revealed a strong correlation between lncRNA-RoR and c-Myc mRNA in colon cancer tissues. Conclusion: It is well known that c-Myc mRNA has a very short half-life and regulation of c-Myc mRNA stability is complex. Our results suggest a novel mechanism of regulation of c-Myc mRNA stability involving lncRNA-RoR, hnRNP U and hnRNP D. Citation Format: Jianguo Huang. LncRNA RoR enhances the c-Myc mRNA stability in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 159. doi:10.1158/1538-7445.AM2015-159

Abstract 3537: Suppression of miR-145 by long noncoding RNA RoR in colon cancer

Abstract Background: The human genome makes a large number of non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs. Compared to well-characterized microRNAs, much less is known about lncRNAs. However, emerging evidence has implicated lncRNAs as master gene regulators; and they are often aberrantly expressed in a variety of human diseases including cancer. In particular, several studies suggest that lncRNAs can modulate the concentration and biological functions of microRNAs through a competing endogenous RNA (ceRNA) mechanism. We have previously demonstrated that lncRNA-RoR is a strong negative regulator of p53 in response to DNA damage and miR-145 is a direct transcription target for p53. Therefore, it is important to further characterize this p53 regulatory network involving lncRNA-RoR and miR-145, and to determine their role in tumorigenesis. Methods: To determine cell growth, we performed MTT assays. Protein levels from treated cells were analyzed by western blot. Real-time RT-PCR was used to determine relative expression of genes. Interaction between lncRNA-RoR and the key component (AGO2) of the RNA-induced silencing complex (RISC) was confirmed by RNA co-immunoprecipitation and RNA precipitation. Dual fluorescent in situ hybridization (FISH) was used to determine the relative levels of lncRNA-RoR and miR-145, and their co-localizations in colon cancer specimens. The effect of lncRNA-RoR on tumor growth was determined by a xenograft mouse model. Results: LncRNA-RoR suppressed miR-145 and upregulated c-Myc, one of major miR-145 target genes. In contrast, RoR-siRNA increased miR-145 expression and inhibited c-Myc. Of interest, miR-145 can also repress lncRNA-RoR expression. This reciprocal repression between lncRNA-RoR and miR-145 appeared to work through the CeRNA mechanism. Mechanistically, we showed that lncRNA-RoR directly interacted with AGO2, a key component of RISC complex, by RNA precipitation assays. This interaction was further confirmed by RNA immunoprecipitation with AGO2 antibody. In consistent with these finding, lncRNA-RoR stimulated proliferation in different cancer cells. Moreover, ectopic expression of lncRNA-RoR in HCT-116 cells significantly promoted tumor growth in a xenograft mouse model. Finally, experiments with dual FISH revealed an inverse correlation between lncRNA-RoR and miR-145 in colon cancer specimens. LncRNA-RoR was upregulated in colon cancer whereas miR-145 was downregulated in the same specimens. Conclusion: Taken together, these results suggest that lncRNA-RoR plays an oncogene role in colon cancer in part through suppression of the tumor suppressor miR-145. Thus, a better understanding of the p53-RoR-miR-145 regulatory system will provide new insight of colon cancer therapy. Citation Format: Jianguo Huang, Yin-Yuan Mo. Suppression of miR-145 by long noncoding RNA RoR in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3537. doi:10.1158/1538-7445.AM2014-3537

Programmed cell death 6, a novel p53‐responsive gene, targets to the nucleus in the apoptotic response to DNA damage

The cellular response to genotoxic stress is multifaceted in nature. Following DNA damage, the tumor suppressor gene p53 activates and plays critical roles in cell cycle arrest, activation of DNA repair and in the event of irreparable damage, induction of apoptosis. The breakdown of apoptosis causes the accumulation of mutant cells. The elucidation of the mechanism for the p53-dependent apoptosis will be crucial in applying the strategy for cancer patients. However, the mechanism of p53-dependent apoptosis remains largely unclear. Here, we carried out ChIP followed by massively parallel DNA sequencing assay (ChIP-seq) to uncover mechanisms of apoptosis. Using ChIP-seq, we identified PDCD6 as a novel p53-responsive gene. We determined putative p53-binding sites that are important for p53 regulation in response to DNA damage in the promoter region of PDCD6. Knockdown of PDCD6 suppressed p53-dependent apoptosis. We also observed that cytochrome c release and the cleavage of PARP by caspase-3 were suppressed by depletion of PDCD6. We further observed that PDCD6 localizes in the nucleus in response to DNA damage. We identified the nuclear localization signal of PDCD6 and, importantly, the nuclear accumulation of PDCD6 significantly induced apoptosis after genotoxic stress. Therefore, we conclude that a novel p53-responsive gene PDCD6 is accumulated in the nucleus and induces apoptosis in response to DNA damage.

D4S234E, a novel p53-responsive gene, induces apoptosis in response to DNA damage

Understanding intracellular transduction pathways on apoptosis is indispensable for clinical application to develop effective cancer therapies. Transcription factor p53 is activated in response to genotoxic damage and plays crucial roles in apoptosis. p53 regulates the expression of numerous apoptosis-related genes to induce cell death. Here, through genome-wide comprehensive gene expression profile, we identified D4S234E as a novel p53-responsive gene. We determined the p53-binding region in the D4S234E promoter, which is important for p53 regulation in response to DNA damage. Inhibition of D4S234E expression by RNA interference suppressed apoptosis. Furthermore, we observed that D4S234E partially localizes in the endoplasmic reticulum (ER). We found a functional ER retention signal in D4S234E and, more importantly, ER targeting is essential for D4S234E-mediated apoptosis. Finally, depletion of D4S234E diminished genotoxic stress-induced reduction of Bcl-2 and augmentation of CHOP. We thus concluded that a novel p53-responsive gene D4S234E is accumulated in the ER and induces apoptosis in response to DNA damage.

Artemis is a negative regulator of p53 in response to oxidative stress

Artemis is a multifunctional phospho-protein with roles in V(D)J recombination, repair of double-strand breaks by nonhomologous end-joining, and regulation of cell cycle checkpoints after DNA damage. Here, we describe a novel function of Artemis as a negative regulator of p53 in response to oxidative stress in both primary cells and cancer cell lines. We show that depletion of Artemis under typical culture conditions (21% oxygen) leads to a spontaneous phosphorylation and stabilization of p53, and resulting cellular G1 arrest and apoptosis. These effects are suppressed by co-depletion of DNA-PKcs, but not ATM, indicating that Artemis is an inhibitor of DNA-PKcs-mediated stabilization of p53. Culturing of cells at 3% oxygen or treatment with an antioxidant abrogated p53 stabilization indicating that oxidative stress is the responsible cellular stimulus. Treatment with IR or hydrogen peroxide did not cause activation of this signaling pathway, while inhibitors of mitochondrial electron transport were effective in reducing its activation. In addition, we show that p53-inducible genes involved in reducing reactive oxygen species (ROS) are upregulated by Artemis depletion. These findings indicate that Artemis and DNA-PKcs participate in a novel, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.

AIMP2/p38, the scaffold for the multi-tRNA synthetase complex, responds to genotoxic stresses via p53

AIMP2/p38 is a scaffolding protein required for the assembly of the macromolecular tRNA synthetase complex. Here, we describe a previously unknown function for AIMP2 as a positive regulator of p53 in response to genotoxic stresses. Depletion of AIMP2 increased resistance to DNA damage-induced apoptosis, and introduction of AIMP2 into AIMP2-deficient cells restored the susceptibility to apoptosis. Upon DNA damage, AIMP2 was phosphorylated, dissociated from the multi-tRNA synthetase complex, and translocated into the nuclei of cells. AIMP2 directly interacts with p53, thereby preventing MDM2-mediated ubiquitination and degradation of p53. Mutations in AIMP2, affecting its interaction with p53, hampered its ability to activate p53. Nutlin-3 recovered the level of p53 and the susceptibility to UV-induced cell death in AIMP2-deficient cells. This work demonstrates that AIMP2, a component of the translational machinery, functions as proapoptotic factor via p53 in response to DNA damage.

Cooperative Roles of c-Abl and Cdk5 in Regulation of p53 in Response to Oxidative Stress

The p53 tumor suppressor protein, a critical modulator of cellular stress responses, is activated through diverse mechanisms that result in its stabilization and transcriptional activation. p53 activity is controlled by transcriptional, translational, and post-translational regulation. The major mechanisms of p53 regulation occur primarily through interactions with HDM2, an E3 ubiquitin ligase that leads to p53 nuclear export and degradation. Here, we demonstrate that hydrogen peroxide-induced oxidative stress elicits down-regulation of HDM2. c-Abl mediates down-regulation of HDM2, leading to an increase of p53 level. Moreover, Cdk5 (cyclin-dependent kinase 5), a proline-directed Ser/Thr kinase, additionally increases p53 stability via post-translational modification of p53 in response to hydrogen peroxide. The p53 protein stabilized by c-Abl and Cdk5 is transcriptionally active; however, transcription of its target gene is differentially regulated with selective binding of p53 on promoter regions of its target genes by c-Abl. In addition, c-Abl modulates Cdk5 activity via phosphorylation of tyrosine 15 in cooperation with cleavage of p35 to p25. Our results show that c-Abl and Cdk5 cooperatively regulate maximal activation of p53, resulting in neuronal death in response to oxidative stress by hydrogen peroxide. These findings aid in clarifying the mechanism underlying the occurrence of neuronal apoptosis as a result of c-Abl and Cdk5-mediated p53 stabilization and transcriptional activation.

P53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK

The p53 protein is one of the major tumor suppressor proteins. In response to DNA damage, p53 is prevented from degradation and accumulates to high levels. Ionizing radiation leads to hypophosphorylation of the p53 ubiquitin ligase Mdm2 at sites where phosphorylation is critical for p53 degradation and to the phosphorylation and activation of Akt/PKB, a kinase that phosphorylates and inhibits GSK-3. GSK-3, which normally phosphorylates Mdm2, is inactivated in response to ionizing radiation. We show that p53 accumulates in lymphoblasts from patients with the hereditary disorder ataxia telangiectasia in response to ionizing radiation despite the absence of a functional ATM kinase. Also, knockdown of ATR did not prevent p53 accumulation in response to ionizing radiation. Instead, p53 stabilization in response to ionizing radiation depended on the inactivation of GSK-3 and the presence of Akt/PKB. Akt/PKB is a target of DNA-PK, a kinase that is activated after ionizing radiation. Correspondingly, down-regulation of DNA-PK prevented phosphorylation of Akt/PKB and GSK-3 after ionizing radiation and strongly reduced the accumulation of p53. We therefore propose a signaling cascade for the regulation of p53 in response to ionizing radiation that involves activation of DNA-PK and Akt/PKB and inactivation of GSK-3 and Mdm2.

Cancer development induced by graded expression of Snail in mice

The zinc-finger transcription factor Snail is believed to trigger epithelial-mesenchymal transitions (EMTs) during cancer progression. This idea is supported by analysis of Snail knockout mice, which uncovered crucial role of Snail in gastrulation, and of individuals with cancer, in whom Snail expression is frequently upregulated. However, these results have not shown a direct link between Snail and the pathogenesis of cancer. Here we show that mice carrying hypomorphic tetracycline-repressible Snail transgenes, that increase Snail expression to 20% above normal levels, exhibit no morphological alterations and develop both epithelial and mesenchymal tumours (leukaemias). Suppression of the Snail transgene did not rescue the malignant phenotype, indicating that alterations induced by Snail are irreversible. CombitTA-Snail murine embryonic fibroblasts show similar migratory ability to that of control mouse embryonic fibroblasts (MEFs). However, CombitTA-Snail-MEFs induce tumour formation in nude mice. CombitTA-Snail expression results in increased radioprotection in vivo, although it does not affect p53 regulation in response to DNA damage. In concert with these results, Snail expression is repressed following DNA damage. This regulation of Snail by DNA damage is p53-independent. Our results connect DNA damage with the requirement of a critical level of an EMT regulator and provide genetic evidence that Snail plays essential roles in cancer development in mammals and thereby influences cell fate in the genotoxic stress response.

Acridine derivatives activate p53 and induce tumor cell death through bax

CP-31398 activates wild-type p53 by a novel mechanism that does not involve phosphorylation of the amino-terminus of p53 and disassociation of MDM2. To identify more potent CP-31398-like p53 activators, we synthesized 4 acridine derivatives with a similar structure to CP-31398. These four compounds induced strong p53 transcription in cells with wild-type p53. We also found that several randomly chosen acridine derivatives, including 9- aminoacridine, amsacrine, quinacrine and acridine orange, induced p53 transcriptional activity. All these acridine derivatives stabilized p53 protein by blocking its ubiquitination, without phosphorylation of ser15 or ser20 on p53. Furthermore, acridine derivatives induced p53-dependent cell death. Knockout of Bax, a p53 target and a key cell death inducer in both intrinsic and extrinsic apoptotic pathways, blocked acridine derivatives from inducing cell death. In addition, in vivo delivery of quinacrine and amsacrine induced p53 transcriptional activity in tumor xenografts. Our results reveal that DNA-intercalating acridine derivatives can induce p53 stabilization by a manner similar to CP-31398. These findings provide insights into p53 regulation in response to DNA intercalating drugs and may assist new anticancer drug design.

Inhibition of Human p53 Basal Transcription by Down-regulation of Protein Kinase Cδ

In response to DNA damage, signal transduction pathways are activated that result in the increase of p53 protein levels, leading to either growth arrest or apoptosis. Protein kinase C (PKC) delta has been implicated as a tumor suppressor that is down-regulated by tumor-promoting phorbol esters in both mouse skin and cell culture models. We report here that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate prevents DNA damage-induced up-regulation of p53 by down-regulating PKC delta. Regulation of p53 in response to stress most commonly occurs by preventing ubiquitination and degradation of the p53 protein. Surprisingly, suppression of p53 expression by inhibition of PKC delta was caused by the inhibition of p53 synthesis, not increased degradation of p53 protein. Inhibiting PKC delta blocked both basal transcription of the human p53 gene and initiation of transcription from the human p53 promoter. Therefore, the tumor-suppressing effects of PKC delta are mediated at least in part through activating p53 transcription.

Regulation of p53 in response to DNA damage.

Activation of p53 can occur in response to a number of cellular stresses, including DNA damage, hypoxia and nucleotide deprivation. Several forms of DNA damage have been shown to activate p53, including those generated by ionising radiation (IR), radio-mimetic drugs, ultraviolet light (UV) and chemicals such as methyl methane sulfonate (MMS). Under normal conditions, p53 levels are maintained at a low state by virtue of the extremely short-half life of the polypeptide. In addition to this, p53 normally exists in an largely inactive state that is relatively inefficient at binding to DNA and activating transcription. Activation of p53 in response to DNA damage is associated with a rapid increase in its levels and with an increased ability of p53 to bind DNA and mediate transcriptional activation. This then leads to the activation of a number of genes whose products trigger cell-cycle arrest, apoptosis, or DNA repair. Recent work has suggested that this regulation is brought about largely through DNA damage triggering a series of phosphorylation, de-phosphorylation and acetylation events on the p53 polypeptide. Here, we discuss the nature of these modifications, the enzymes that bring them about, and how changes in p53 modification lead to p53 activation.

Regulation of p53 in response to ionizing radiation in ataxia telangiectasia fibroblasts

Purpose : An analysis of the structure and expression of p53 in fibroblasts from patients with ataxia telangiectasia (AT) is presented. Methods and Materials : p53 status in primary and SV40 T antigen-transformed AT cell lines was analyzed using immunocytochemistry and by sequencing with the dideoxynucleotide termination method. The expression of p53 transcript was measured by Northern analysis. The kinetics of p53 protein expression and DNA-binding activity were measured at various intervals following irradiation. Results : No mutation of p53 sequences was found in AT cells. Decreased levels of p53 mRNA and protein were observed in AT5BIVA cells compared to other SV40 T antigen-immortalized fibroblasts. Furthermore, DNA-protein binding analysis shows that a fraction of p53 in the nuclear extracts from AT5BIVA is regulated and binds to specific DNA sequence following irradiation. Conclusion : These data provide evidence for a heterogeneity of the p53 function in SV40-transformed AT cells. It also supports the hypothesis that a regulatory mechanism of p53 activity remains in T antigen-expressing cells in response to ionizing radiation damage.