Direct P53 Target Gene Research Articles

P53 target ANKRA2 cooperates with RFX7 to regulate tumor suppressor genes

The transcription factor regulatory factor X 7 (RFX7) has been identified as a tumor suppressor that is recurrently mutated in lymphoid cancers and appears to be dysregulated in many other cancers. RFX7 is activated by the well-known tumor suppressor p53 and regulates several other known tumor suppressor genes. However, what other factors regulate RFX7 and its target genes remains unclear. Here, reporter gene assays were used to identify that RFX7 regulates the tumor suppressor gene PDCD4 through direct interaction with its X-box promoter motif. We utilized mass spectrometry to identify factors that bind to DNA together with RFX7. In addition to RFX7, we also identified RFX5, RFXAP, RFXANK, and ANKRA2 that bind to the X-box motif in the PDCD4 promoter. We demonstrate that ANKRA2 is a bona fide direct p53 target gene. We used transcriptome analyses in two cell systems to identify genes regulated by ANKRA2, its sibling RFXANK, and RFX7. These results revealed that ANKRA2 functions as a critical cofactor of RFX7, whereas RFXANK regulates largely distinct gene sets.

LINC01021 Attenuates Expression and Affects Alternative Splicing of a Subset of p53-Regulated Genes.

Loss of the p53-inducible LINC01021 in p53-proficient CRC cell lines results in increased sensitivity to DNA-damaging chemotherapeutics. Here, we comprehensively analyze how LINC01021 affects the p53-induced transcriptional program. Using a CRISPR/Cas9-approach, we deleted the p53 binding site in the LINC01021 promoter of SW480 colorectal cancer cells and subjected them to RNA-Seq analysis after the activation of ectopic p53. RNA affinity purification followed by mass spectrometry was used to identify proteins associated with LINC01021. Loss of the p53-inducibility of LINC01021 resulted in an ~1.8-fold increase in the number of significantly regulated mRNAs compared to LINC01021 wild-type cells after ectopic activation of p53. A subset of direct p53 target genes, such as NOXA and FAS, displayed significantly stronger induction when the p53-inducibility of LINC01021 was abrogated. Loss of the p53-inducibility of LINC01021 resulted in alternative splicing of a small number of mRNAs, such as ARHGAP12, HSF2, and LYN. Several RNA binding proteins involved in pre-mRNA splicing were identified as interaction partners of LINC01021 by mass spectrometry. Our results suggest that LINC01021 may restrict the extent and strength of p53-mediated transcriptional changes via context-dependent regulation of the expression and splicing of a subset of p53-regulated genes.

ID3 is a novel target gene of p53 and modulates lung cancer cell metastasis

The tumor suppressor p53 prevents cancer development by regulating dozens of target genes with diverse biological functions. Although numerous p53 target genes have been identified to date, the dynamics and function of the regulatory network centered on p53 have not yet been fully elucidated. We herein identified inhibitor of DNA-binding/differentiation-3 (ID3) as a direct p53 target gene. p53 bound the distal promoter of ID3 and positively regulated its transcription. ID3 expression was significantly decreased in clinical lung cancer tissues, and was closely associated with overall survival outcomes in these patients. Functionally, ID3 deficiency promoted the metastatic ability of lung cancer cells through its effects on the transcriptional regulation of CDH1. Furthermore, the ectopic expression of ID3 in p53-knockdown cells restored E-cadherin expression. Collectively, the present results demonstrate that ID3 plays a tumor-suppressive role as a downstream effector of p53 and impedes lung cancer cell metastasis by regulating E-cadherin expression.

TP53 CRISPR/Cas9 Reveals Strict BAX Dependence in the Response to BH3 Mimetic Targeting MCL1

Background In multiple myeloma (MM), 1q gain/amplification (1q+) and deletion of the short arm of chromosome 17 (del17p), encompassing MCL1 and TP53, respectively, as well as the t(4;14) translocation are adverse events that reduce both event-free survival and overall survival of patients. The genome gatekeeper p53 is well known for its multiple roles in the regulation of mitochondrial apoptosis: p53 induces early direct transactivation of the BH3-only protein PUMA (BBC3) and the death effector BAX (BAX) and late direct-to-indirect transactivation of NOXA (PMAIP1) and BAK (BAK1). To investigate the direct impact of loss of p53 function in myeloma cells, we generated TP53 CRISPR/Cas9 isogenic cells from cell lines harboring t(4;14) and 1q+. Materials and Methods To knockout the expression of all p53 isoforms in two TP53wt t(4;14)+ 1q+ myeloma cell lines, NCI-H929 and XG7 cells were sequentially transduced with 2 lentiviruses expressing Cas9 and a doxycycline-inducible TP53-specific sgRNA targeting exon 5. TP53-/-clones (n=3) and control clones (n=3) were derived for each cell line by limiting dilution. Results RNAseq analysis was performed in triplicate in the 12 clones of the two MM cell lines: analysis of the 6 TP53-/-clones versus the 6 control clones showed significant changes (log2FC>0.4, p>0.05) in the expression of 92 genes under steady-state conditions (and of more than 3000 upon nutlin3a-induced p53 accumulation) : 27% of the genes were known to be direct p53 target genes, 39% were known to be indirectly regulated, and 35% of the genes were not identified as p53 targets in solid cancer cell lines (Andrysik Z Genome Res 2017). Among the 51 downregulated genes, only 2 belonged to apoptosis pathways, namely BAX and TNFRSF10B, both direct early targets of p53. Of note, the expression of BAK1, PMAIP1 or BBC3 was not significantly modified. To directly assess the impact of BAX decrease in the 6 TP53-/- clones, we used 3 BH3 mimetics specific for BCL2, Venetoclax, MCL1, S63845, and BCLXL, A1155463. The XG7 and NCI-H929 cell lines were predominantly resistant to Venetoclax and to A1155463, and TP53 knockout did not alter responses. In contrast, as compared to control clones, the response to S63845 was decreased 5.7 to 7.4-fold in NCI-H929 TP53-/- (LD50 median values increased from 7 to 40nM, p<0.0001) and XG7 TP53-/- clones (LD50 median values increased from 25 to 186nM, p=0.0007), respectively. This result suggested a major role of BAX in the response to S63845. Indeed, BAX silencing significantly inhibited S63845-induced cell death while BAK silencing had no protective effect, demonstrating the BAX dependence of the S63845 response. Of note, this strong dependence on p53-dependent BAX expression was not limited to XG7 or NCI-H929 : in 21 TP53abnormal myeloma cell lines, BAX expression (GEP) was 1.7-fold lower than in 11 TP53wt cell lines (LD50 median values 34 vs. 59, p=0.0004), and sensitivity to S63845 was also significantly lower in TP53abn MM cell lines compared to MM TP53wt (LD50 median values 75 nM vs. 12 nM, p=0.0296). The resistance of TP53-/- cells to S63845 was overcome by the addition of Venetoclax, although the death remained lower in TP53-/- clones compared to control clones (p=0.001). BAX and BAK activation and MCL1 co-immunoprecipitation assays further demonstrated the central role of MCL1/BAX expression and complexes in the response to S63845. In 63 consecutive independent samples from patients with MM (23 at diagnosis, 40 at relapse), 33 1q+ and 30 1q-, we show that 1q gain/amplification favored response to 25 nM S63845 alone (death median 16% in 29 1q- vs 30% in 34 1q+, p=0.0132) and had a trend in combination with 300 nM Venetoclax (80% vs 89%, p=0.152). Finally, we show that 1q was sufficient to induce responses to S63845 plus Venetoclax regardless of del17p status (death median 30% in 26 1q+ del17p- vs. 40% in 6 1q+ del17p+ samples). To analyze the response to the combination of S63845 and Venetoclax at the single cell level, we performed scRNASeq analysis in t(4;14)+ 1q+ del17p+/- patients and determined the transcriptomic profile of cells surviving the combination: we show that BH3 mimetics combination heterogeneously induced death in and between patient's samples and we identified a resistance signature that is under evaluation. All these results support the S63845 / Venetoclax combination for patients with double (1q+, del17p) to triple (t(4;14), 1q+, del17p) hit. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Global Analyses to Identify Direct Transcriptional Targets of p53.

The transcription factor p53 controls a gene expression program with pleiotropic effects on cell biology including cell cycle arrest and apoptosis. Identifying direct p53 target genes within this network and determining how they influence cell fate decisions downstream of p53 activation is a prerequisite for designing therapeutic approaches that target p53 to effectively kill cancer cells. Here we describe a comprehensive multi-omics approach for identifying genes that are direct transcriptional targets of p53. We provide detailed procedures for measuring global RNA polymerase activity, defining p53 binding sites across the genome, and quantifying changes in steady-state mRNA in response to p53 activation.

Conservation and divergence of the p53 gene regulatory network between mice and humans

Understanding the p53 tumor suppressor pathway remains crucial for the design of anticancer strategies. Studies in human tumors and mouse models help to unravel the molecular mechanisms that underlie the p53 signaling pathway. Yet, the p53 gene regulatory network (GRN) is not the same in mice and humans. The comparison of the regulatory networks of p53 in mice and humans reveals that gene up- and down-regulation by p53 are distinctly affected during evolution. Importantly, gene up-regulation by p53 underwent more rapid evolution and gene down-regulation has been evolutionarily constrained. This difference stems from the two major mechanisms employed by p53 to regulate gene expression: up-regulation through direct p53 target gene binding and indirect down-regulation through the p53-p21-DREAM pathway. More than 1000 genes have been identified to differ in their p53-dependent expression between mice and humans. Analysis of p53 gene expression profiles and p53 binding data reveal that turnover of p53 binding sites is the major mechanism underlying extensive variation in p53-dependent gene up-regulation. Only a core set of high-confidence genes appears to be directly regulated by p53 in both species. In contrast to up-regulation, p53-induced down-regulation is well conserved between mice and humans and controls cell cycle genes. Here a curated data set is provided that extends the previously established web-atlas at www.targetgenereg.org to assess the p53 response of any human gene of interest and its mouse ortholog. Taken together, the analysis reveals a limited translation potential from mouse models to humans for the p53 GRN.

DNA repair processes are critical mediators of p53-dependent tumor suppression

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence1,2. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function3-5. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.

CRP Stimulates GDF15 Expression in Endothelial Cells through p53.

Growth differentiation factor 15 (GDF15) is a multifunctional, secreted protein that is a direct target gene of p53. GDF15 is a prospective biomarker of cardiovascular disease (CVD). C-reactive protein (CRP), like GDF15, is implicated in inflammation and an independent biomarker of CVD. However, the molecular interactions between GDF15 and CRP remain unexplored. In women, we found a significant relationship between hsCRP and GDF15 serum and mRNA levels. In vitro treatment of cultured human aortic endothelial cells (HAECs) with purified CRP or transfection of a CRP plasmid into HAECs induced GDF15 expression. Dual-luciferase reporter assays confirmed that CRP significantly increased the levels of GDF15 promoter luciferase activity, indicating that CRP induces GDF15 transcription. Chromatin immunoprecipitation (ChIP) assays confirmed that p53 was recruited to both p53 binding sites 1 and 2 in the GDF15 promoter in response to CRP. We have uncovered a linkage between CRP and GDF15, a new clue that could be important in the pathogenesis of endothelial inflammation.

The Alzheimer's disease-associated TREM2 gene is regulated by p53 tumor suppressor protein

TREM2 mutations evoke neurodegenerative disorders, and recently genetic variants of this gene were correlated to increased risk of Alzheimer's disease. The signaling cascade originating from the TREM2 membrane receptor includes its binding partner TYROBP, BLNK adapter protein, and SYK kinase, which can be activated by p53. Moreover, in silico identification of a putative p53 response element (RE) at the TREM2 promoter led us to hypothesize that TREM2 and other pathway elements may be regulated in p53-dependent manner. To stimulate p53 in synergistic fashion, we exposed A549 lung cancer cells to actinomycin D and nutlin-3a (A + N). In these cells, exposure to A + N triggered expression of TREM2, TYROBP, SYK and BLNK in p53-dependent manner. TREM2 was also activated by A + N in U-2 OS osteosarcoma and A375 melanoma cell lines. Interestingly, nutlin-3a, a specific activator of p53, acting alone stimulated TREM2 in U-2 OS cells. Using in vitro mutagenesis, chromatin immunoprecipitation, and luciferase reporter assays, we confirmed the presence of the p53 RE in TREM2 promoter. Furthermore, activation of TREM2 and TYROBP by p53 was strongly inhibited by CHIR-98014, a potent and specific inhibitor of glycogen synthase kinase-3 (GSK-3). We conclude that TREM2 is a direct p53-target gene, and that activation of TREM2 by A + N or nutlin-3a may be critically dependent on GSK-3 function.

Abstract IA07: Deconstructing p53 pathways in tumor suppression

Abstract The p53 transcription factor is a critical tumor suppressor in humans and mice. Despite this essential function, the molecular pathways through which p53 acts in tumor suppression remain enigmatic. To define the transcriptional programs through which p53 suppresses carcinogenesis, we have taken combined mouse genetic and genomic approaches. We previously generated a panel of p53 knock-in mouse strains expressing mutants in the first (p5325,26), second (p5353,54), or both (p5325,26,53,54) of two amino-terminal transcriptional activation domains (TADs). In terms of transcriptional activity, we have discovered that p5325,26 is severely impaired for transactivation of the majority of canonical p53 target genes (e.g. p21, Puma, Noxa), but retains the ability to activate a set of primarily novel p53 target genes, while p5325,26,53,54 lacks transactivation activity altogether. The p5353,54 mutant, in contrast, is uncompromised for transactivation of p53 target genes. The p5325,26,53,54 mutant is completely defective in tumor suppression, underscoring the importance of transcriptional activation for p53-mediated tumor suppression. Intriguingly, the p5325,26 mutant retains full activity in suppressing a variety of cancers, indicating that efficient transactivation of most canonical p53 target genes is dispensable for tumor suppression. As p5325,26 activates only a subset of p53-inducible genes, yet retains tumor-suppressor activity, it has helped pinpoint a small set of novel, direct p53-inducible tumor suppression-associated target genes (TSAGs) whose functions we are currently interrogating through genetic screens. Additionally, we have made the surprising discovery that the p5353,54 mutant suppresses pancreatic cancer more efficiently than wild-type p53, and that this capacity correlates with the ability of p5353,54 to hyperactivate a subset of p53 target genes. Indeed, our analysis of these genes has uncovered Ptpn14, a direct p53 target gene critical for mediating p53 function in suppressing pancreatic cancer cell growth. Ptpn14 encodes a protein tyrosine phosphatase that negatively regulates the oncoprotein Yap in both human and mouse cells. These studies thus reveal a p53-Ptpn14-Yap axis in pancreatic cancer. Together, these strategies will help to elaborate the transcriptional networks fundamental for p53 function in tumor suppression. Citation Format: Stephano Spano Mello, Kathryn Bieging-Rolett, Alyssa Kaiser, Elizabeth Joy Valente, Nitin Raj, Jacob McClendon, Brittany Maria Flowers, David Warren Morgens, Michael Cory Bassik, Laura Donatella Attardi. Deconstructing p53 pathways in tumor suppression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr IA07.

P53-mediated regulation of bile acid disposition attenuates cholic acid-induced cholestasis in mice.

The tumour suppressor p53 is traditionally recognized as a surveillance molecule to preserve genome integrity. Recent studies have demonstrated that it contributes to metabolic diseases. Here, we investigated the role of p53 in the regulation of bile acid disposition and cholestasis. The bile acid disposition-related gene expression profile affected by p53 activation was assessed in mouse primary hepatocytes with p53 depletion and in Trp53-null mice. Dual luciferase reporter assay was used to detect the transcriptional activities of target genes. Anticholestatic effects of p53 activator doxorubicin were investigated in a 0.5% cholic acid-fed mouse model of cholestasis. Changes in bile acids were evaluated using metabolomics analysis. Doxorubicin-mediated p53 activation induced Cyp2b10, Sult2a1 and Abcc2/3/4 expression in mice in vitro and in vivo. ABCC3 and CYP2B6 (human orthologue of Cyp2b10) were identified as direct p53 target genes. Doxorubicin attenuated cholic acid-induced cholestasis in mice, as demonstrated by shrunken gall bladder, decreased serum total bile acid and total bilirubin levels and alkaline phosphatase activity. Targeted metabolomics analysis revealed that doxorubicin enhanced the excretion of bile acid metabolites from serum and liver to intestine and faeces. Up-regulation of Cyp2b10, Sult2a1 and Abcc2/3/4 expression was further confirmed in cholestatic mice. Cholic acid-induced cholestatic injury was aggravated in p53-deficient mice and levels of bile acid in intestine and faeces were decreased. Our findings suggest a novel role of p53 in promoting bile acid disposition and alleviating cholestatic syndrome, which provides a potential therapeutic target for cholestasis.

Identification of a p53 target, CD137L, that mediates growth suppression and immune response of osteosarcoma cells

p53 encodes a transcription factor that transactivates downstream target genes involved in tumour suppression. Although osteosarcoma frequently has p53 mutations, the role of p53 in osteosarcomagenesis is not fully understood. To explore p53-target genes comprehensively in calvarial bone and find out novel druggable p53 target genes for osteosarcoma, we performed RNA sequencing using the calvarial bone and 23 other tissues from p53+/+ and p53−/− mice after radiation exposure. Of 23,813 genes, 69 genes were induced more than two-fold in irradiated p53+/+ calvarial bone, and 127 genes were repressed. Pathway analysis of the p53-induced genes showed that genes associated with cytokine-cytokine receptor interactions were enriched. Three genes, CD137L, CDC42 binding protein kinase gamma and Follistatin, were identified as novel direct p53 target genes that exhibited growth-suppressive effects on osteosarcoma cell lines. Of the three genes, costimulatory molecule Cd137l was induced only in calvarial bone among the 24 tissues tested. CD137L-expressing cells exhibited growth-suppressive effects in vivo. In addition, recombinant Fc-fusion Cd137l protein activated the immune response in vitro and suppressed osteosarcoma cell growth in vivo. We clarified the role of CD137L in osteosarcomagenesis and its potential therapeutic application. Our transcriptome analysis also indicated the regulation of the immune response through p53.

P53 target miR-29c-3p suppresses colon cancer cell invasion and migration through inhibition of PHLDB2

miR-29c-3p is a potential tumor suppressor microRNA that is reportedly downregulated in several types of human cancers, but its role in colon cancer remains to be elucidated. Meanwhile, TP53, one of the most important tumor suppressors, is highly mutated in colon cancer. In the attempt to connect p53 and miR-29c-3p, we found that the upstream of miR-29c-3p gene contains a functional p53 consensus responsive element that is driven by p53 transcriptional factor activity, suggesting miR-29c-3p as a direct p53 target gene. Through online software prediction and in vivo validation, we demonstrated that Pleckstrin Homology Like Domain Family Member 2 (PHLDB2) is a valid miR-29c-3p target gene. Analysis of human cancer databases available from PROGgeneV2 showed that higher expression of PHLDB2 is associated with shorter overall survival and metastasis-free survival of colon cancer patients. Further, suppression of colon cancer cell invasion and migration by miR-29c-3p was significantly attenuated in the presence of ectopic PHLDB2, indicating PHLDB2 is a critical downstream target of miR-29c-3p. Collectively, our findings present the first to elucidate that miR-29c is a direct p53 target gene, and also identify PHLDB2 as an important miR-29c target gene involved in colon cancer metastasis.

Abstract PR07: Deconstructing p53 transcriptional networks in pancreatic cancer suppression

Abstract The p53 transcription factor is mutated in the majority of human pancreatic cancers, underscoring its critical role in suppressing pancreatic cancer development. Although p53 can restrain neoplastic cell expansion by inducing cell-cycle arrest or apoptosis in response to diverse stress signals, the molecular pathways through which p53 acts in tumor suppression remain largely elusive. To define the transcriptional networks underlying p53 tumor suppressor function, we have used mouse genetic and genomic approaches. We generated a set of p53 knock-in mouse strains expressing mutants in the first (p53 25,26), second (p5353,54), or both (p5325,26,53,54) of two transcriptional activation domains (TADs). Using a combination of ChIP-sequencing and expression profiling on cells expressing these mutants, we have been able to define a limited list of direct p53 target genes whose expression is tightly correlated with tumor suppression. Specifically, we found that p5325,26 is severely compromised for transactivation of most classical p53 target genes (e.g. p21, Puma), but retains the ability to activate a subset of p53 target genes, while p5325,26,53,54 lacks transactivation activity completely. The p5325,26,53,54 mutant is completely inactive in tumor suppression, highlighting the importance of transactivation for p53-mediated tumor suppression. Interestingly, however, p5325,26 retains full activity in suppressing various cancers, including pancreatic cancer, indicating that efficient transactivation of most canonical p53 targets is dispensable for tumor suppression. As p5325,26 activates only a subset of p53-dependent genes, yet retains tumor suppressor activity, it has helped pinpoint a small set of novel, direct p53-inducible tumor suppression-associated genes (TSAGs) whose functions we are currently interrogating through genetic screens. In addition, we have made the surprising discovery that the p5353,54 mutant suppresses pancreatic cancer more effectively than wild-type p53, and that this capacity correlates with the ability of p5353,54 to hyperactivate a subset of p53 target genes. Indeed, our analysis of these genes has unveiled p53 targets critical for mediating p53 function in suppressing mouse and human pancreatic cancer cell growth, including a protein tyrosine phosphatase that negatively regulates Yap signaling. Collectively, these approaches will delineate the transcriptional networks fundamental for p53 function in tumor suppression, an approach that will ultimately facilitate the design of novel therapies for pancreatic cancers with p53 mutation. Citation Format: Laura D. Attardi, Stephano Spano Mello, Elizabeth J. Valente, Pawel K. Mazur, Nitin Raj, Kathryn Bieging-Rolett, Hannes Vogel.{Authors}. Deconstructing p53 transcriptional networks in pancreatic cancer suppression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr PR07.

Activation of the miR-34a/SIRT1/p53 Signaling Pathway Contributes to the Progress of Liver Fibrosis via Inducing Apoptosis in Hepatocytes but Not in HSCs.

Liver fibrosis results from a sustained wound healing response to chronic liver injury, and the activation of nonparenchymal hepatic stellate cells (HSCs) is the pivotal process. MicroRNA-34a (miR-34a) is the direct target gene of p53 and activates p53 through sirtuin 1 (SIRT1) simultaneously. The miR-34a/SIRT1/p53 signaling pathway thus forms a positive feedback loop wherein p53 induces miR-34a and miR-34a activates p53 by inhibiting SIRT1, playing an important role in cell proliferation and apoptosis. miR-34a expression has been found to be increased in animal models or in human patients with different liver diseases, including liver fibrosis. However, the exact role of this classical miR-34a/SIRT1/p53 signaling pathway in liver fibrosis remains unclear. In the present study, using a CCl4-induced rat liver fibrosis model, we found that the miR-34a/SIRT1/p53 signaling pathway was activated and could be inhibited by SIRT1 activator SRT1720. Further studies showed that the miR-34a/SIRT1/p53 signaling pathway was activated in hepatocytes but not in HSCs. The activation of this pathway in hepatocytes resulted in the apoptosis of hepatocytes and thus activated HSCs. Our data indicate that the miR-34a/SIRT1/p53 signaling pathway might be a promising therapeutic target for liver fibrosis.

Feedback modulation of neural network synchrony and seizure susceptibility by Mdm2-p53-Nedd4-2 signaling.

BackgroundNeural network synchrony is a critical factor in regulating information transmission through the nervous system. Improperly regulated neural network synchrony is implicated in pathophysiological conditions such as epilepsy. Despite the awareness of its importance, the molecular signaling underlying the regulation of neural network synchrony, especially after stimulation, remains largely unknown.ResultsIn this study, we show that elevation of neuronal activity by the GABA(A) receptor antagonist, Picrotoxin, increases neural network synchrony in primary mouse cortical neuron cultures. The elevation of neuronal activity triggers Mdm2-dependent degradation of the tumor suppressor p53. We show here that blocking the degradation of p53 further enhances Picrotoxin-induced neural network synchrony, while promoting the inhibition of p53 with a p53 inhibitor reduces Picrotoxin-induced neural network synchrony. These data suggest that Mdm2-p53 signaling mediates a feedback mechanism to fine-tune neural network synchrony after activity stimulation. Furthermore, genetically reducing the expression of a direct target gene of p53, Nedd4-2, elevates neural network synchrony basally and occludes the effect of Picrotoxin. Finally, using a kainic acid-induced seizure model in mice, we show that alterations of Mdm2-p53-Nedd4-2 signaling affect seizure susceptibility.ConclusionTogether, our findings elucidate a critical role of Mdm2-p53-Nedd4-2 signaling underlying the regulation of neural network synchrony and seizure susceptibility and reveal potential therapeutic targets for hyperexcitability-associated neurological disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0214-6) contains supplementary material, which is available to authorized users.

P53-Regulated Networks of Protein, mRNA, miRNA, and lncRNA Expression Revealed by Integrated Pulsed Stable Isotope Labeling With Amino Acids in Cell Culture (pSILAC) and Next Generation Sequencing (NGS) Analyses

We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p < 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3'-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the mechanisms of p53-mediated tumor suppression.

P53 directly activates cystatin D/CST5 to mediate mesenchymal-epithelial transition: a possible link to tumor suppression by vitamin D3.

Cystatin D (CST5) encodes an inhibitor of cysteine proteases of the cathepsin family and is directly induced by the vitamin D receptor (VDR). Interestingly, vitamin D3 exerts tumor suppressive effects in a variety of tumor types. In colorectal cancer (CRC) cells CST5 was shown to mediate mesenchymal-epithelial transition (MET). Interestingly, vitamin D3 was shown to exert tumor suppressive effects in a variety of tumor types, including colorectal cancer (CRC). We recently performed an integrated genomic and proteomic screen to identify targets of the p53 tumor suppressor in CRC cells. Thereby, we identified CST5 as a putative p53 target gene. Here, we validated and characterized CST5 as a direct p53 target gene. After activation of a conditional p53 allele, CST5 was upregulated on mRNA and protein levels. Treatment with nutlin-3a or etoposide induced CST5 in a p53-dependent manner. These regulations were direct, since ectopic and endogenous p53 occupied a conserved binding site in the CST5 promoter region. In addition, treatment with calcitriol, the active vitamin D3 metabolite, and simultaneous activation of p53 resulted in enhanced CST5 induction and increased repression of SNAIL, an epithelial-mesenchymal transition (EMT) inducing transcription factor. Furthermore, CST5 inactivation decreased p53-induced mesenchymal-epithelial transition (MET) as evidenced by decreased inhibition of SNAIL and of migration by p53. Furthermore, CST5 expression was directly repressed by SNAIL. In summary, these results imply CST5 as an important mediator of tumor suppression by p53 in colorectal cancer. In addition, they suggest that a combined treatment activating p53 and the vitamin D3 pathway may function via induction of CST5.

5-Lipoxygenase is a direct p53 target gene in humans

The p53 tumor suppressor plays a critical role in cancer, and more than 50% of human tumors contain mutations or deletions of the TP53 gene. p53 can transactivate or repress target genes in response to diverse stress signals, such as transient growth arrest, DNA repair, cellular differentiation, senescence and apoptosis. Through an unbiased genome-wide ChIP-seq analysis, we have found that 5-lipoxygenase (ALOX5, 5-LO) which is a key enzyme of leukotriene (LT) biosynthesis, is a direct target gene of p53 and its expression is induced by genotoxic stress via actinomycin D (Act.D) or etoposide (Eto) treatment. 5-LO and LTs play a role in immunological diseases as well as in tumorigenesis and tumor growth. p53 binds to a specific binding site consisting of a complete p53 consensus-binding motif in ALOX5 intron G which is located about 64kbp downstream of the transcriptional start site. We confirmed the strong binding of p53 to the 5-LO target site in ChIP-qPCR experiments. Expression analyses by qRT-PCR and immunoblot further revealed that genotoxic stress induces the ALOX5 mRNA and protein expression in a p53-dependent manner. Knockdown of p53 in U2OS cells leads to a downregulation of 5-LO mRNA and protein expression. In addition, immunofluorescence and immunoprecipitation assays indicate the direct binding of 5-LO to p53 protein. Furthermore, we found that 5-LO can inhibit the transcriptional activity of p53 suggesting that 5-LO acts in a negative feedback loop to limit induction of p53 target genes.

Blm-s, a BH3-Only Protein Enriched in Postmitotic Immature Neurons, Is Transcriptionally Upregulated by p53 during DNA Damage

Programmed cell death is a pivotal process that regulates neuronal number during development. Key regulators of this process are members of the BCL-2 family. Using mRNA differential display, we identified a Bcl-2 family gene, Blm-s (Bcl-2-like molecule, short form), enriched in postmitotic neurons of the developing cerebral cortex. BLM-s functions as a BH3-only apoptosis sensitizer/derepressor and causes BAX-dependent mitochondria-mediated apoptosis by selectively binding to prosurvival BCL-2 or MCL-1. When challenged with γ-irradiation that produces DNA double-strand breaks (DSBs), Blm-s is transcriptionally upregulated in postmitotic immature neurons with concurrently increased apoptosis. RNAi-mediated depletion of Blm-s protects immature neurons from irradiation-induced apoptosis. Furthermore, Blm-s is a direct target gene of p53 and AP1 via the ataxia telangiectasia mutated (ATM)- and c-Jun N-terminal kinase (JNK)-signaling pathways activated by DSBs. Thus, BLM-s is likely an apoptosis sensor activated by DSBs accumulating in postmitotic immature neurons.