The pseudo-caspase FLIP(L) regulates cell fate following p53 activation

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

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FLIP(L) determines p53 induced life or death

10.1101/858688 ◽

2019 ◽

Author(s):

Andrea Lees ◽

Alexander J. McIntyre ◽

Fiammetta Falcone ◽

Nyree T. Crawford ◽

Christopher McCann ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Fate ◽

Target Genes ◽

Hdac Inhibitors ◽

Caspase 8 ◽

Cycle Arrest ◽

P53 Activation ◽

P53 Target Genes ◽

Mdm2 Inhibitor

AbstractHow p53 differentially activates cell cycle arrest versus cell death remains poorly understood. Here, we demonstrate that upregulation of canonical pro-apoptotic p53 target genes in colon cancer cells imposes a critical dependence on the long splice form of the caspase-8 regulator FLIP (FLIP(L)), which we identify as a direct p53 transcriptional target. Inhibiting FLIP(L) expression with siRNA or Class-I HDAC inhibitors promotes apoptosis in response to p53 activation by the MDM2 inhibitor Nutlin-3A, which otherwise predominantly induces cell-cycle arrest. When FLIP(L) upregulation is inhibited, apoptosis is induced in response to p53 activation via a novel ligand-independent TRAIL-R2/caspase-8 complex, which, by activating BID, induces mitochondrial-mediated apoptosis. Notably, FLIP(L) depletion inhibits p53-induced expression of the cell cycle regulator p21 and enhances p53-mediated upregulation of PUMA, with the latter activating mitochondrial-mediated apoptosis in FLIP(L)-depleted, Nutlin-3A-treated cells lacking TRAIL-R2/caspase-8. Thus, we report two previously undescribed, novel FLIP(L)-dependent mechanisms that determine cell fate following p53 activation.

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Growth Suppression by Acute PromyelocyticLeukemia-Associated Protein PLZF Is Mediated by Repression ofc-mycExpression

Molecular and Cellular Biology ◽

10.1128/mcb.23.24.9375-9388.2003 ◽

2003 ◽

Vol 23 (24) ◽

pp. 9375-9388 ◽

Cited By ~ 99

Author(s):

Melanie J. McConnell ◽

Nathalie Chevallier ◽

Windy Berkofsky-Fessler ◽

Jena M. Giltnane ◽

Rupal B. Malani ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Cell Cycle Arrest ◽

Target Genes ◽

Ectopic Expression ◽

Growth Suppression ◽

Quiescent State ◽

Cycle Arrest

ABSTRACT The transcriptional repressor PLZF was identified by its translocation with retinoic acid receptor alpha in t(11;17) acute promyelocytic leukemia (APL). Ectopic expression of PLZF leads to cell cycle arrest and growth suppression, while disruption of normal PLZF function is implicated in the development of APL. To clarify the function of PLZF in cell growth and survival, we used an inducible PLZF cell line in a microarray analysis to identify the target genes repressed by PLZF. One prominent gene identified was c-myc. The array analysis demonstrated that repression of c-myc by PLZF led to a reduction in c-myc-activated transcripts and an increase in c-myc-repressed transcripts. Regulation of c-myc by PLZF was shown to be both direct and reversible. An interaction between PLZF and the c-myc promoter could be detected both in vitro and in vivo. PLZF repressed the wild-type c-myc promoter in a reporter assay, dependent on the integrity of the binding site identified in vitro. PLZF binding in vivo was coincident with a decrease in RNA polymerase occupation of the c-myc promoter, indicating that repression occurred via a reduction in the initiation of transcription. Finally, expression of c-myc reversed the cell cycle arrest induced by PLZF. These data suggest that PLZF expression maintains a cell in a quiescent state by repressing c-myc expression and preventing cell cycle progression. Loss of this repression through the translocation that occurs in t(11;17) would have serious consequences for cell growth control.

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Distinct mechanisms control genome recognition by p53 at its target genes linked to different cell fates

Nature Communications ◽

10.1038/s41467-020-20783-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Marina Farkas ◽

Hideharu Hashimoto ◽

Yingtao Bi ◽

Ramana V. Davuluri ◽

Lois Resnick-Silverman ◽

...

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Cell Cycle Arrest ◽

Cell Fate ◽

Molecular Mechanisms ◽

Target Genes ◽

Binding Modes ◽

Cell Fate Decisions ◽

Cycle Arrest

AbstractThe tumor suppressor p53 integrates stress response pathways by selectively engaging one of several potential transcriptomes, thereby triggering cell fate decisions (e.g., cell cycle arrest, apoptosis). Foundational to this process is the binding of tetrameric p53 to 20-bp response elements (REs) in the genome (RRRCWWGYYYN0-13RRRCWWGYYY). In general, REs at cell cycle arrest targets (e.g. p21) are of higher affinity than those at apoptosis targets (e.g., BAX). However, the RE sequence code underlying selectivity remains undeciphered. Here, we identify molecular mechanisms mediating p53 binding to high- and low-affinity REs by showing that key determinants of the code are embedded in the DNA shape. We further demonstrate that differences in minor/major groove widths, encoded by G/C or A/T bp content at positions 3, 8, 13, and 18 in the RE, determine distinct p53 DNA-binding modes by inducing different Arg248 and Lys120 conformations and interactions. The predictive capacity of this code was confirmed in vivo using genome editing at the BAX RE to interconvert the DNA-binding modes, transcription pattern, and cell fate outcome.

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W436, a novel SMART derivative, exhibits anti‐hepatocarcinoma activity by inducing apoptosis and G2/M cell cycle arrest in vitro and in vivo and induces protective autophagy

Journal of Biochemical and Molecular Toxicology ◽

10.1002/jbt.22831 ◽

2021 ◽

Author(s):

Shuai Man ◽

Zhuzhu Wu ◽

Rui Sun ◽

Qi Guan ◽

Zengqiang Li ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

M Cell ◽

Cycle Arrest

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Menadione reduces CDC25B expression and promotes tumor shrinkage in gastric cancer

Therapeutic Advances in Gastroenterology ◽

10.1177/1756284819895435 ◽

2020 ◽

Vol 13 ◽

pp. 175628481989543

Author(s):

Amanda Braga Bona ◽

Danielle Queiroz Calcagno ◽

Helem Ferreira Ribeiro ◽

José Augusto Pereira Carneiro Muniz ◽

Giovanny Rebouças Pinto ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Specific Inhibitor ◽

Gastric Carcinogenesis ◽

In Vivo Experiments ◽

Cycle Arrest

Background: Gastric cancer is one of the most incident types of cancer worldwide and presents high mortality rates and poor prognosis. MYC oncogene overexpression is a key event in gastric carcinogenesis and it is known that its protein positively regulates CDC25B expression which, in turn, plays an essential role in the cell division cycle progression. Menadione is a synthetic form of vitamin K that acts as a specific inhibitor of the CDC25 family of phosphatases. Methods: To better understand the menadione mechanism of action in gastric cancer, we evaluated its molecular and cellular effects in cell lines and in Sapajus apella, nonhuman primates from the new world which had gastric carcinogenesis induced by N-Methyl-N-nitrosourea. We tested CDC25B expression by western blot and RT-qPCR. In-vitro assays include proliferation, migration, invasion and flow cytometry to analyze cell cycle arrest. In in-vivo experiments, in addition to the expression analyses, we followed the preneoplastic lesions and the tumor progression by ultrasonography, endoscopy, biopsies, histopathology and immunohistochemistry. Results: Our tests demonstrated menadione reducing CDC25B expression in vivo and in vitro. It was able to reduce migration, invasion and proliferation rates, and induce cell cycle arrest in gastric cancer cell lines. Moreover, our in-vivo experiments demonstrated menadione inhibiting tumor development and progression. Conclusions: We suggest this compound may be an important ally of chemotherapeutics in the treatment of gastric cancer. In addition, CDC25B has proven to be an effective target for investigation and development of new therapeutic strategies for this malignancy.

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