P53 Nuclear Localization Research Articles

Forkhead Box O3A (FOXO3) and the Mitochondrial Disulfide Relay Carrier (CHCHD4) Regulate p53 Protein Nuclear Activity in Response to Exercise

Although exercise is linked with improved health, the specific molecular mechanisms underlying its various benefits require further clarification. Here we report that exercise increases the nuclear localization and activity of p53 by acutely down-regulating coiled-coil-helix-coiled-coil-helix domain 4 (CHCHD4), a carrier protein that mediates p53 import into the mitochondria. This response to exercise is lost in transgenic mice with constitutive expression of CHCHD4. Mechanistically, exercise-induced nuclear transcription factor FOXO3 binds to the CHCHD4 promoter and represses its expression, preventing the translocation of p53 to the mitochondria and thereby increasing p53 nuclear localization. The synergistic increase in nuclear p53 and FOXO3 by exercise can facilitate their known interaction in transactivating Sirtuin 1 (SIRT1), a NAD+-dependent histone deacetylase that mediates adaptation to various stresses. Thus, our results reveal one mechanism by which exercise could be involved in preventing cancer and potentially other diseases associated with aging.

Differential expression of multidrug resistance‑related proteins in adriamycin‑resistant (pumc‑91/ADM) and parental (pumc‑91) human bladder cancer cell lines.

Multidrug resistance (MDR) is the major obstacle to bladder cancer chemotherapy. Several mechanisms have been implicated in the development of MDR, including extrusion of the drug by cell membrane pumps, associated with P‑glycoprotein (P‑gp) and multidrug resistance‑associated protein (MRP); increased DNA damage repair, associated with topoisomeraseII (TopoII); suppression of drug‑induced apoptosis, associated with p53; and regulation of cancer cell growth, associated with vascular endothelial growth factor (VEGF). In the present study, the expression levels of these five markers were detected in an adriamycin (ADM)‑resistant human bladder cancer cell line (pumc‑91/ADM) and its parental cell line (pumc‑91), in order to determine which marker is more important, or whether all of them participate in drug resistance. The expression levels of P‑gp, MRP, TopoII, VEGF and p53 were measured in the two cell lines by reverse transcription‑quantitative polymerase chain reaction, western blotting and immunohistochemistry. A significant increase in P‑gp, MRP and VEGF, and a decrease in TopoII mRNA expression were detected in the pumc‑91/ADM drug‑resistant cell line compared with the pumc‑91 cell line; however, no difference in p53 mRNA expression was detected between the cells. In pumc‑91/ADM cells, the protein expression levels of P‑gp and MRP were upregulated, whereas TopoII was significantly decreased. However, no marked differences in p53 or VEGF expression were detected between the two cell lines at the protein level. The cytoplasmic and cell membrane localization of P‑gp and MRP, the cytoplasmic localization of VEGF, and the nuclear localization of p53 and TopoII were confirmed in the two cell lines. The present study detected increased P‑gp and MRP, and reduced TopoII expression in pumc‑91/ADM cells compared with pumc‑91 cells; however, no difference was detected in p53 and VEGF expression between the cell lines. In conclusion, a significant upregulation of MRP and downregulation of TopoII were detected in the ADM‑resistant human bladder cancer cell line (pumc‑91/ADM) compared with in the parental cell line (pumc‑91).

A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53.

p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways.

O‐GlcNAc Regulates p53 in Ovarian Cancer

O‐GlcNAcylation is a dynamic post‐translational modification (PTM) that consists of an addition of a single N‐acetylglucosamine sugar to serine and threonine residues in nuclear, cytoplasmic and mitochondrial proteins. The enzyme O‐linked β‐N‐acetylglucosamine transferase (OGT) adds the modification, while enzyme O‐GlcNAcase (OGA) removes the modification. O‐GlcNAc regulates enzymatic activity, localization, stability and protein‐protein interactions on a wide range of proteins such as transcription factors, structural proteins and kinases. In cancer, when comparing O‐GlcNAc levels in normal and tumor cells, tumor samples present higher O‐GlcNAcylation in vitro and in vivo. This aberrant O‐GlcNAc pattern contributes to changes in important signaling pathways and could contribute to the progression of the disease. Interestingly, the tumor suppressor p53 is modified by O‐GlcNAc. The transcription factor p53 regulates diverse cellular processes like cell survival and the cell cycle. Most solid tumors contain mutations in p53 leading to increased degradation or loss of p53 function, which promotes malignant tumor growth. Ovarian cancer has one of the highest frequencies of p53 mutation rates. The goal of this work is to understand the relationship between O‐GlcNAcylation and p53 function in ovarian cancer. First, O‐GlcNAcylation, OGT and OGA protein and mRNA levels were measured in a series of human ovarian cancer cell lines and no correlation was found between p53 genetic status and O‐GlcNAcylation. Overexpression or silencing of p53 in a p53 wild‐type cell line (A2780) had little effect on O‐GlcNAcylation. On the other hand, pharmacological inhibition of OGA with the inhibitor Thiamet‐G (TMG) leads to an increase in nuclear localization of p53 and increased mRNA levels of p53‐target genes (p21 and Bax). Overexpression of OGA or OGT leads to increased p53 protein but not mRNA levels and a concurrent increase in p53‐targets (Mdm‐2, p21 and Bax) expression. Also, p53 acetylation on lysine 382, a transcriptional activating modification, is higher in OGA/OGT overexpressing cells. In conclusion, O‐GlcNAcylation regulates p53 function through stabilization and activation.Support or Funding InformationSupport and funding: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Science Without Borders Program, NIH NIDDK R01 DK 100595.

Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types.

Chelators such as 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) target tumor cell iron pools and inhibit proliferation. These agents also modulate multiple targets, one of which is the cyclin-dependent kinase inhibitor, p21. Hence, this investigation examined the mechanism of action of these compounds in targeting p21. All the chelators up-regulated p21 mRNA in the five tumor cell-types assessed. In contrast, examining their effect on total p21 protein levels, these agents induced either: (1) down-regulation in MCF-7 cells; (2) up-regulation in SK-MEL-28 and CFPAC-1 cells; or (3) had no effect in LNCaP and SK-N-MC cells. The nuclear localization of p21 was also differentially affected by the ligands depending upon the cell-type, with it being decreased in MCF-7 cells, but increased in SK-MEL-28 and CFPAC-1 cells. Further studies assessing the mechanisms responsible for these effects demonstrated that p21 expression was not correlated with p53 status, suggesting a p53-independent mechanism. Considering this, we examined proteins that modulate p21 independently of p53, namely NDRG1, MDM2 and ΔNp63. These studies demonstrated that a dominant negative MDM2 isoform (p75(MDM2)) closely resembled p21 expression in response to chelation in three cell lines. These data suggest MDM2 may be involved in the regulation of p21 by chelators.

Functional variant analyses (FVAs) predict pathogenicity in the BRCA1 DNA double-strand break repair pathway.

Heritable mutations in the BRCA1 and BRCA2 and other genes in the DNA double-strand break (DSB) repair pathway disrupt binding of the encoded proteins, transport into the nucleus and initiation of homologous recombination, thereby increasing cancer risk [Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. and Livingston, D.M. (1997) Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell, 88, 265-275, Chen, J., Silver, D.P., Walpita, D., Cantor, S.B., Gazdar, A.F., Tomlinson, G., Couch, F.J., Weber, B.L., Ashley, T., Livingston, D.M. et al. (1998) Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol. Cell, 2, 317-328]. To meet the challenge of correct classification, flow cytometry-based functional variant analyses (FVAs) were developed to determine whether variants in DSB repair genes disrupted the binding of BRCA1 to BARD1, PALB2, BRCA2 and FANCD2, phosphorylation of p53 or BRCA1 nuclear localization in response to DNA damage caused by diepoxybutane, mitomycin C and bleomycin. Lymphoblastoid cells from individuals with BRCA1 pathogenic mutations, benign variants, and variants of uncertain significance or with known BRCA2, FANCC or NBN mutations were tested. Mutations in BRCA1 decreased nuclear localization of BRCA1 in response to individual or combination drug treatment. Mutations in BRCA1 reduced binding to co-factors, PALB2 and FANCD2 and decreased phosphorylation of p53. Mutations in BRCA2, FANCC and NBN decreased nuclear localization of BRCA1 in response to drug treatment, cofactors binding and p53 phosphorylation. Unsupervised cluster analysis of all and as few as two assays demonstrated two apparent clusters, high-risk BRCA1 mutations and phenocopies and low-risk, fully sequenced controls and variants of uncertain significance (VUS). Thus, two FVA assays distinguish BRCA1 mutations and phenocopies from benign variants and categorize most VUS as benign. Mutations in other DSB repair pathway genes produce molecular phenocopies. FVA assays may represent an adjunct to sequencing for categorizing VUS or may represent a stand-alone measure for assessing breast cancer risk.

Transcriptional Activation of p53 during Cold Induced Torpor in the 13-Lined Ground Squirrel Ictidomys tridecemlineatus.

The transcription factor p53 is located at the centre of multiple pathways relating the cellular response to stress. Commonly known as a tumor suppressor, it is responsible for initiating diverse actions to protect the integrity of the genome, ranging from cell cycle arrest to apoptosis. This study investigated the regulation of p53 protein in hibernating 13-lined ground squirrel Ictidomys tridecemlineatus during multiple stages of the torpor-arousal cycle. Transcript and protein levels of p53 were both elevated in the skeletal muscle during early and late torpor stages of the hibernation cycle. Nuclear localization of p53 was also increased during late torpor, and this is associated with an increase in its DNA binding activity and expression of p53 transcriptional targets p21CIP, gadd45α, and 14-3-3σ. The increase in p53 transcriptional activity appears to be independent of its phosphorylation at Ser-15, Ser-46, and Ser-392, consistent with an absence of checkpoint kinase activation during torpor. Sequence analysis revealed unique amino acid substitutions in the ground squirrel p53 protein, which may contribute to an increase in protein stability compared to nonhibernators. Overall, the study results provided evidences for a potential role of p53 in the protection of the skeletal muscle during torpor.

Histone demethylase Jumonji D3 (JMJD3/KDM6B) at the nexus of epigenetic regulation of inflammation and the aging process.

Histone methylation is involved in the epigenetic control of immune responses and cellular senescence. Jumonji domain-containing protein 3 (JMJD3), also called lysine-specific demethylase 6B (KDM6b), is an inducible histone demethylase which enhances immune responses and can trigger cellular senescence. JMJD3 potentiates gene expression by demethylating repressive H3K27me3 epigenetic marks in promoters and gene bodies. Moreover, JMJD3 also stimulates transcription in a demethylase-independent manner by mediating interactions between chromatin modifiers. JMJD3 can enhance both pro-inflammatory and anti-inflammatory responses by targeting distinct transcription factors in a context-dependent manner in gene promoters. For instance, JMJD3 can induce macrophage M2 polarization via STAT6 signaling. JMJD3 also interacts with T-bet factor and induces Th1 differentiation of CD4(+) T cells. Moreover, JMJD3 can activate TGF-β signaling through the SMAD3 pathway. Conversely, JMJD3 displaces polycomb complexes from the INK4 box, which induces the expression of INK4a and triggers cellular senescence. JMJD3 can also enhance the nuclear localization of p53 and thus regulate its function. The control of INK4 box and p53 is closely related to the regulation of the aging process. We will briefly review the inducible properties of JMJD3 expression and then focus on the role of JMJD3 in the regulation of inflammation and senescence through different signaling pathways. We emphasize that an inflammatory milieu and cellular stress can enhance immune responses and provoke cellular senescence via epigenetic regulation through JMJD3 activation.

The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

BackgroundDesmoplastic small round cell tumor (DSRCT) is characterized by the presence of a fusion protein EWS/WT1, arising from the t (11;22) (p13;q12) translocation. Here we examine the oncogenic properties of two splice variants of EWS/WT1, EWS/WT1-KTS and EWS/WT1 + KTS.MethodsWe over-expressed both EWS/WT1 variants in murine embryonic fibroblasts (MEFs) of wild-type, p53+/- and p53-/- backgrounds and measured effects on cell-proliferation, anchorage-independent growth, clonogenicity after serum withdrawal, and sensitivity to cytotoxic drugs and gamma irradiation in comparison to control cells. We examined gene expression profiles in cells expressing EWS/WT1. Finally we validated our key findings in a small series of DSRCT.ResultsNeither isoform of EWS/WT1 was sufficient to transform wild-type MEFs however the oncogenic potential of both was unmasked by p53 loss. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 enhanced cell-proliferation, clonogenic survival and anchorage-independent growth. EWS/WT1 expression in wild-type MEFs conferred resistance to cell-cycle arrest after irradiation and daunorubicin induced apoptosis. We show DSRCT commonly have nuclear localization of p53, and copy-number amplification of MDM2/MDMX. Expression of either isoform of EWS/WT1 induced characteristic mRNA expression profiles. Gene-set enrichment analysis demonstrated enrichment of WNT pathway signatures in MEFs expressing EWS/WT1 + KTS. Wnt-activation was validated in cell lines with over-expression of EWS/WT1 and in DSRCT.ConclusionIn conclusion, we show both isoforms of EWS/WT1 have oncogenic potential in MEFs with loss of p53. In addition we provide the first link between EWS/WT1 and Wnt-pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterize DSRCT.

Abstract 2163: Increased overall survival in platinum-resistant ovarian cancer: paradigmatic use of novel SINE (selective inhibitor of nuclear export), which restores p53 nuclear localization and activation.

Abstract Ovarian cancer (OvCa) is the most lethal female reproductive tract malignancy worldwide with >190,000 new cases diagnosed yearly. Overall, ∼60% of patients will relapse after primary therapy and for the majority of women with advanced disease, their platinum-resistant cancers are currently incurable. During OvCa progression, multiple tumor suppressor proteins (TSPs), most notably p53 (>95% of late-stage cases), are inactivated. Chromosomal region maintenance 1 (CRM1) is solely responsible for the nuclear export of the major TSPs, and increased CRM1 expression has been linked to advanced OvCa stage and poor overall survival. To address the therapeutic role of CRM1 inhibition in OvCa, we tested two novel SINEs in vitro and in vivo. For in vitro studies, we used KPT-185 as a single agent and in combination with cisplatinum in the isogenic cell lines A2780 and CP70. KPT-185 cytotoxicity was highly specific for tumor cell lines. The IC50 for the normal ovarian surface epithelial cell line IOSE527 (4000nM) was 40-90 times higher than A2780 (46.5nM) and CP70 (111.7nM) lines. Combination treatment demonstrated an additive effect on cell death, overcoming cisplatinum resistance. A2780 displayed G1/G2 arrest whereas CP70, only G2 arrest. p53 nuclear accumulation, and downstream activation of targets, was demonstrated following SINEs treatment. Two complementary in vivo models were used. First, the orally bioavailable SINE KPT-330, currently in Phase 1 clinical studies, was given as a single agent in a Rag1 KO mouse model with 4 unrelated patient-derived chemonaive cell lines. Oral KPT-330 inhibited subcutaneous tumor growth and markedly increased survival: all chronically treated mice continued to survive longer (>78d) than controls (all died at 23d). Cross-over of control mice to KPT-330 resulted in tumor growth arrest. In the second model, highly aggressive, luciferase positive, cisplatin resistant CP70 cells were injected i.p into nu/nu mice (n=34) and tumor growth followed in real-time using WBLI. SINE treatment led to marked reductions in tumor burden as well as survival improvements: control mice were all dead by 25 days whereas cisplatin (2.5mg/kg, 2x wk; 31d), KPT-330 (15mg/kg, twice weekly; 41d) and KPT-330+cisplatin (same as single treatments, respectively; 48d) treated mice have significantly improved survival outcomes (p<0.0005 for each treatment versus control). Together, these studies demonstrate the first successful use of highly selective, potent and irreversible CRM1 inhibitors in overcoming cisplatin resistance and prolonging survival in OvCa models. Restoration of p53 nuclear localization and functional activity and RNASeq identified pathways at serially increasing doses will be discussed. First-in-man trials have begun and trials in ovarian cancer are planned for 2013. Citation Format: Ying Chen, Eva Kalir, Catalina Camacho-Vanegas, Fei Huang, Elena Pereira, Yosef Landesman, Dilara McCauley, Mansoor Raza Mirza, Michael Kauffman, Sharon Shacham, Peter R. Dottino, John A. Martignetti. Increased overall survival in platinum-resistant ovarian cancer: paradigmatic use of novel SINE (selective inhibitor of nuclear export), which restores p53 nuclear localization and activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2163. doi:10.1158/1538-7445.AM2013-2163

Epigallocatechin gallate promotes p53 accumulation and activity via the inhibition of MDM2-mediated p53 ubiquitination in human lung cancer cells

Epigallocatechin gallate (EGCG), which is derived from green tea, is well known for its chemopreventive activity. Several studies have shown that p53 plays an important role in the activity of EGCG; however, the mechanism by which EGCG regulates p53 requires further investigation. In the present study, we showed that EGCG inhibits anchorage-independent growth of human lung cancer cells by upregulating p53 expression. EGCG treatment can substantially increase p53 stability, promote nuclear localization of p53 and decrease nuclear accumulation of MDM2. We also found that EGCG increases the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity. Although EGCG promotes MDM2 expression in a p53-dependent manner, the interaction between MDM2 and p53 was significantly inhibited following EGCG treatment, which resulted in the inhibition of MDM2-mediated p53 ubiquitination. Thus, our results suggest that the stabilization and activation of p53 may partly contribute to the anticancer activity of EGCG.

Administration of grape seed extract alleviates age-associated decline in ubiquitin-proteasome system and cardiomyocyte apoptosis in rats

Effective clearance of oxidized, damaged, and/or misfolded proteins in the cell by the ubiquitin-proteasome system (UPS) is critical for cell homeostasis, survival and function. We hypothesized that in the aging heart, generation of free radicals could impair UPS where the associated build-up of polyubiquitinated proteins could trigger programmed cell death. To test this, we used young (4 months old) and aged (24 months old) rats to analyze polyubiquitinated proteins, proteasome activity and programmed cell death in the ventricular tissue samples. Our studies reveal excessive deposition of polyubiquitinated proteins in the ventricular tissue extracts of old rats when compared to younger rats. The increased ubiquitination was accompanied by a significant decrease in 20S proteasome activity. Since the loss of proteasome-mediated clearance of ubiquitinated proteins is linked to programmed cell death, we measured TUNEL activity in aged rat heart and compared with younger animals. Aged animal hearts showed a substantial increase in programmed cell death as evidenced by TUNEL positive nuclei and DNA fragmentation. Analyses of cell death/survival pathways support our findings in terms of age-associated increase in the nuclear localization of p53, Bax/Bcl2 ratio and cleaved (active) caspase-3 and decreased expression of cellular inhibitor of apoptosis (cIAP1). Administration of grape seed extract (GSE) as a source of antioxidants significantly reduced these age-associated deleterious changes suggesting that free radicals primarily contribute to impaired UPS function and increased programmed cell death and that administration of antioxidants during aging could protect cardiac muscle cells and preserve ventricular function.

Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency by Modulating p53 Activity in Models of Diamond Blackfan Anemia

Abstract 512Ribosomal protein mutations are common in patients with Diamond Blackfan anemia (DBA), who have red cell aplasia and craniofacial abnormalities, and rps14 deficiency has been linked to 5q- myelodysplastic syndrome. We have characterized a zebrafish mutant in rps29, a ribosomal protein in the small subunit. Rps29−/− embryos have hematopoietic and endothelial defects, including decreased hematopoietic stem cells, defects in hemoglobinization, and decreased staining of vessel markers. Heads of mutant embryos are also morphologically affected, with increased apoptosis. Consistent with other models of DBA, knockdown of p53 completely rescues the rps29 hematopoietic and apoptotic phenotypes. We wanted to identify chemicals that could rescue the rps29 mutant phenotype. Using 600 compounds from known bioactive libraries, we performed an in vivo chemical screen for rescue of the rps29 apoptotic and endothelial defects. Treatment with one compound, A-3, improved the morphology of the apoptotic embryo head. A-3 is an inhibitor of calmodulin, which interacts with a host of calmodulin-dependent enzymes and can bind to other proteins in the cell, including p21. W-7, another calmodulin inhibitor related to A-3, can rescue the endothelial defect in the rps29 mutant. Calmodulin inhibition in rps29 mutant embryos rescues the hemoglobin defect. Furthermore, structurally unrelated chemicals that inhibit calmodulin rescue the endothelial defect. We hypothesized that calmodulin inhibition was affecting some part of the p53 pathway. We tested whether A-3 had an effect on the phenotype in vitro in a cancer cell line where shRNA knockdown of RPS19 has been shown to stabilize p53 and induce p21. When rps19 is knocked down in A549 cells, treatment with calmodulin inhibitors resulted in a decrease of p21 protein, as measured by flow cytometry, although p53 protein levels were stable. This effect was also observed in primary human cord blood-derived CD34+ hematopoietic stem and progenitor cells. In situ immunofluorescence in A549s further revealed that nuclear localization of p53 and p21 upon RPS19 knockdown was disrupted by chemical treatment. Calmodulin inhibitors also rescue ribosomal protein deficiency-mediated erythroid defects both in vitro and in vivo. When RPS19 is knocked down in CD34+ cells, there is a defect in erythroid differentiation. Treatment with calmodulin inhibitors rescued production of CD71+ cells upon differentiation. In conclusion, we have demonstrated the use of calmodulin inhibitors as a novel approach for mediating p53 activation upon ribosomal protein knockdown, thereby rescuing the erythroid defects of ribosomal deficiency. Our data suggests that small molecules that inhibit calmodulin may be effective therapies for patients with ribosomal deficiency. Disclosures:Ebert:Celgene: Consultancy; Genoptix: Consultancy. Zon:Fate Therapeutics: Founder Other.

Endoplasmic reticulum stress stimulates p53 expression through NF-κB activation.

BackgroundInduction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood.Principal FindingsIn this report, we demonstrate that induction of p53 expression is mediated through NF-κB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-κB and activity of NF-κB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-κB inhibitor, Bay 11-7082 and downregulation of NF-κB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells.SignificanceTaken together, NF-κB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress.

Abstract 1831: Selective inhibitors of nuclear export (SINE) induce multiple tumor suppressor proteins (TSP) activity and show single agent antitumor effect and synergy with Bcl-2 antagonist in non-small cell lung cancer

Abstract Neoplastic cells must inactivate their tumor suppressor proteins (TSP) in order to perpetuate their growth. Oncogene and growth factor driven nuclear export of TSPs is an increasingly recognized mechanism for TSP inactivation. We have developed orally active, small molecule SINE that irreversibly block the major nuclear export protein CRM1 (exportin 1, XPO1) and selectively induce the death of cancer cells. Forced nuclear accumulation of TSPs is believed to initiate a “genome survey” leading to the death of cancer cells, whereas normal cells undergo transient, reversible cell cycle arrest. We show that the small molecule SINE CRM1 inhibitor KPT-185 potently kills many types of tumor cells in vitro (IC50 20-500nM, IC80 ≤ 1µM at 72 hours) including ∼75% of NSCLC lines with diverse genetic signatures (EGFR wt & mut, p53 wt & mut, K-ras wt & mut). In contrast, ∼25% of NSCLC lines with similar genotypes show cytotoxicity IC50s ≤ 1.5µM and IC80 is not reached. Treatment of the SINE-resistant A549 NSCLC (adenocarcinoma, EGFR wt, p53 wt, K-ras mut, KPT-185 IC50 = 1.75µM) leads to nuclear localization of p53, p21, FOXO, E2F4, IkB, underphosphorylated pRb and other CRM1 cargoes at concentrations similar to that of sensitive NSCLC cell lines, but apoptosis is not readily induced. To test whether the resistance to CRM1 inhibition-mediated cytotoxicity is due to activation of anti-apoptotic (or inactivation of pro-apoptotic) BCL-2 family proteins, we combined SINE with ABT-737, a small-molecule BCL-2 inhibitor currently in Phase 2 clinical trials, and examined the molecular mechanisms leading to tumor cell death. We show that combination of low doses (500nM, the IC20) of KPT-185 with non-cytotoxic doses of the Bcl-2 inhibitor (≥ 100µM) induces synergistic cytotoxicity. Moreover, the antitumor effects of the combination therapy are manifested within 48 hours, whereas significant cytotoxicity by higher doses of SINE alone requires at least 72 hours. Similar results have been obtained with another SINE resistant cell line, HCC-2935 (EGFR mut, p53 mut, K-ras mut). We noted that in the resistant A549 line, levels of the pro-apoptotic BCL2 member, Bax, were reduced by ∼50% with SINE treatment, but that addition of the BCL2 inhibitor reversed this effect and induced significant synergistic cytotoxicity. The sensitive NSCLC cell line H-226 (EGFR wt, p53wt, K-ras wt; IC50 12nM) showed nuclear localization of CRM1 cargoes when treated with KPT-185 (100nM), but only modest additional killing by the addition of the Bcl-2 inhibitor. We conclude that mechanism of resistance to SINE mediated cytotoxicity can be overcome by antagonism of Bcl-2. In vivo studies using A-549 xenografts comparing the effects of KPT-SINE and ABT-737 alone or in combination are ongoing and will be reported at the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1831. doi:1538-7445.AM2012-1831

Abstract 1627: Chloroquine-induced inhibition of Arginase-1 promotes the nuclear localization of p53 in colon cancer cell lines

Abstract Emerging studies have identified the overexpression of Arginase-1 (ARG-1) in a variety of human malignancies, including cancer of the colon, leading to the promotion of tumor cell growth and invasion. Inactivation of the p53 tumor suppressor protein is known to involve somatic mutation or binding of viral oncoproteins to the p53 protein. However, several types of malignant and premalignant tissues harbor a genetically wild-type, but transcriptionally inactive, form of p53, often localized in the cytoplasm. The transactivation of the inactive p53 gene is thought to lead to its tumor suppressor function. The present report showed that the treatment of cultured human colon cancer cell lines (HT-29 and HCT-116) with 10 or 20 µM chloroquine (CQ), a known anti-rheumatoid/antimalarial drug, inhibited arginase activity with a concomitant nuclear accumulation of the p53 protein. By using specific antibodies it was demonstrated that the decreased in arginase activity correlated with the reduction of arg-1 levels in the cells. Upon treatment of cells with 20 µM CQ, the levels of arginase activity or arg-1 decreased by 43.5 % or 38.3 % in HT-29 cells and 40.9 or 39.4 % in HCT-116 respectively. Further the treatment of cells with 20 µM CQ for 48 h led to an increased level of p53 in the nucleus in comparison with the control samples (p <0.01). The nuclear:cytosol ratios of p53 levels were 5.3 (control) and 83 (20 µM CQ) in HCT-116 (wild type) cells and 2.6 (control) and 40.9 (20 µM CQ) in the HT-29 (mutant type) cells. These results indicate that the CQ-induced inhibition of arg-1 led to the nuclear accumulation of p53 in colon cancer cells. Thus the inhibition of arg-1 by CQ appears to protect the p53 tumor suppressor function.In an effort to expand our understanding of the molecular link between β-catenin levels, increased nuclear accumulation of p53 and CQ-mediated inhibition of arginase activity, HT-29 cells were co-incubated with CQ (0-50 µM) for 48 h and the level of β-catenin were determined by western blot assay. The results of this study indicate that CQ inhibited the β-catenin protein in a dose dependent manner. This decrease in the β-catenin level corresponded to an increase in the nuclear accumulation of p53 and corresponding diminution of intracellular Arg-1. In conclusion, the data presented herein represent a new and important improvement in understanding the pleiotropic effects of CQ on arg-1, with specific reference to its participation in the enhancement of the nuclear accumulation of p53 with the resultant down regulation of β-catenin protein. Thus this observation that CQ may inhibit the initial step of the arginine-polyamine pathway that results in the enhanced nuclear accumulation of p53 and a concomitant down regulation of β-catenin protein may provide a paradigm for targeted therapy in colorectal cancer chemoprevention and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1627. doi:1538-7445.AM2012-1627

Abstract LB-176: Chemoprevention of NNK-induced lung adenoma and adenocarcinoma in A/J mice by p53 modulating agents, CP-31398 and Prima-1

Abstract Lung cancer is a leading cause of cancer deaths worldwide. p53 Mutations including wild-type p53 nuclear localization due to excessive endogenous electrophilic molecules are highly prevalent in tobacco associated lung cancers. Here, we report the chemopreventive effects of direct-acting p53 modulating agents, CP-31398 and Prima-1 on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung adenoma and adenocarcinoma in female A/J mice. Seven weeks-old mice on a regular AIN-76A diet, received one dose of 10 μmol NNK /mouse by i.p. injection. Three weeks after NNK treatment, groups of mice (25 mice/group) were fed control AIN-76A or experimental diets containing 50 and 100 ppm CP-31398, or 150 and 300 ppm Prima-1. Mice were sacrificed after 20 weeks (10 mice/group) and/or 36 weeks (15 mice/group) following NNK-treatment and evaluated for lung adenomas and adenocarcinomas by histopathological methods. Administration of 50 and 100 ppm CP-31398 significantly suppressed lung tumor formation by 29% and 39.5% (P<0.01-0.0001), respectively after 20 weeks and 32.2% and 40.1% (P<0.01-0.0001), respectively after 36 weeks. Similarly, 150 and 300 ppm Prima-1 significantly suppressed lung tumor formation by 22.2% and 34% (P<0.04-0.0001) after 20 weeks and 28.8% and 47.4% (P<0.01-0.0001) after 36 weeks, respectively. Inhibition of NNK-induced lung adenocarcinoma by either agent was greater than 55-72% (p<0.0001) after 20 weeks and about 40-57% after 36 weeks. These results suggest that restoring/activating wild-type p53 function by small molecules significantly inhibits NNK-induced lung tumor in A/J mice, most notably delaying progression of adenomas to adenocarcinoma. IHC analysis of lung tumors from NNK-treated mice exposed to either CP-31398 or Prima-1 showed a significantly reduced proliferating cell nuclear antigen (PCNA)-positive index, increased accumulation of nuclear wt-p53 including increased p21 and TUNNEL positive cells compared to lung tumors from NNK-treated mice fed with control diet. Incidentally, neither COX-1 nor iNOS were significantly affected by CP-31398 or Prima -1 in the NNK derived lung tumors. These results demonstrate, for the first time, the chemopreventive role of p53 modulating agents on tobacco specific carcinogen-induced lung adenocarcinomas. {Supported by NIH, NCI grants NO1-CN-53300}. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-176. doi:1538-7445.AM2012-LB-176

Dmp1 Physically Interacts with p53 and Positively Regulates p53′s Stability, Nuclear Localization, and Function

The transcription factor Dmp1 is a Ras/HER2-activated haplo-insufficient tumor suppressor that activates the Arf/p53 pathway of cell-cycle arrest. Recent evidence suggests that Dmp1 may activate p53 independently of Arf in certain cell types. Here, we report findings supporting this concept with the definition of an Arf-independent function for Dmp1 in tumor suppression. We found that Dmp1 and p53 can interact directly in mammalian cells via the carboxyl-terminus of p53 and the DNA-binding domain of Dmp1. Expression of Dmp1 antagonized ubiquitination of p53 by Mdm2 and promoted nuclear localization of p53. Dmp1-p53 binding significantly increased the level of p53, independent of the DNA-binding activity of Dmp1. Mechanistically, p53 target genes were activated synergistically by the coexpression of Dmp1 and p53 in p53(-/-);Arf(-/-) cells, and genotoxic responses of these genes were hampered more dramatically in Dmp1(-/-) and p53(-/-) cells than in Arf(-/-) cells. Together, our findings identify a robust new mechanism of p53 activation mediated by direct physical interaction between Dmp1 and p53.

Silibinin prevents ultraviolet B radiation-induced epidermal damages in JB6 cells and mouse skin in a p53-GADD45α-dependent manner

Better preventive strategies are required to reduce ultraviolet (UV)-caused photodamage, the primary etiological factor for non-melanoma skin cancer (NMSC). Accordingly, here we examined the preventive efficacy of silibinin against UVB-induced photodamage using mouse epidermal JB6 cells and SKH1 hairless mouse epidermis. In JB6 cells, silibinin pretreatment protected against apoptosis and accelerated the repair of cyclobutane pyrimidine dimers (CPD) induced by moderate dose of UVB (50 mJ/cm(2)), which we are at risk of daily exposure. Silibinin also reversed UVB-induced S phase arrest, reducing both active DNA synthesizing and inactive S phase populations. In mechanistic studies, UVB-irradiated cells showed a transient upregulation of both phosphorylated (Ser-15 and Ser-392) and total p53, whereas silibinin pretreatment led to a more sustained upregulation and stronger nuclear localization of p53. Silibinin also caused a marked upregulation of GADD45α, a downstream target of p53, implicated in DNA repair and cell cycle regulation. Importantly, under p53 and GADD45α knockdown conditions, cells were more susceptible to UVB-induced apoptosis without any significant S phase arrest, and protective effects of silibinin were compromised. Similar to the in vitro results, topical application of silibinin prior to or immediately after UVB irradiation resulted in sustained increase in p53 and GADD45α levels and accelerated CPD removal in the epidermis of SKH1 hairless mice. Together, our results show for the first time that p53-mediated GADD45α upregulation is the key mechanism by which silibinin protects against UVB-induced photodamage and provides a strong rationale to investigate silibinin in reducing the risk and/or preventing early onset of NMSC.

Abstract 9559: The Role of Myopalladin in the Interactions of ANKRD1/CARP with the Co-Activator p53 in Hypertrophic Cardiomyopathy

Background: Myopalladin (MYPN), a member of the Z-disc that interacts with other Z-disc proteins, including the cardiac ankyrin repeat protein ANKRD1 (CARP). Mutations in MYPN and CARP genes cause human dilated (DCM) and hypertrophic cardiomyopathies (HCM). Both MYPN and CARP have dual distributions in the cytoplasm and nucleus. In the nucleus, CARP is a transcriptional co-activator of p53, repressing expression of the cardiac proteins in an in vitro system. A MYPN-dependent cytoplasmic anchoring for CARP is described although the molecular basis of the MYPN modulation of CARP gene transcription is unclear. Previously, our laboratory identified a novel MYPN variant (MYPN Y20C ) in DCM and HCM patients. A transgenic MYPN Y20C cardiac-restricted overexpression mouse model recapitulates the HCM phenotype.. Methods and Results: In vitro and in vivo studies demonstrated that the MYPN Y20C mutation resulted in perinuclear localization in myoblasts while CARP retained its cytoplasmic/nuclear dual distribution; as did wild-type MYPN. Co-immunoprecipitation revealed decreased association between CARP and MYPN Y20C suggesting a dissociation of the cellular distributions of CARP and MYPN Y20C . In transgenic MYPN Y20C hearts, perinuclear MYPN Y20C had increased association with p53, a co-activator for negative gene regulation of CARP. Nuclear localization of p53 is a critical element in the activation of its transcription. Its sequestration in the cytoplasm renders the protein non-functional. This resulted in a loss-of-function of CARP gene transcription as demonstrated by increased expression of the CARP-specific sarcomeric protein myosin heavy chain (MHC), and atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) in vivo . CARP was serine phosphorylated and coexpression with MYPN led to suppression of CARP phosphorylation. Conclusion: Our study demonstrates a novel role for myopalladin in the development of HCM. The perinuclear/cytoplasmic sequestration of p53 by MYPN Y20C resulted in the inactivation of p53, which leads to loss-of-function of CARP gene regulation. Our data also suggests that myopalladin plays a pivotal role in relaying cardiac Z-disc signaling to those proteins involved in CARP phosphorylation/activation.