Understanding cell cycle and cell death regulation provides novel weapons against human diseases.

Cell division, cell differentiation and cell death are the three principal physiological processes that regulate tissue homoeostasis in multicellular organisms. The growth and survival of cells as well as the integrity of the genome are regulated by a complex network of pathways, in which cell cycle checkpoints, DNA repair and programmed cell death have critical roles. Disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. Compromised cell death can also favour genomic instability. It is becoming increasingly clear that dysregulation of cell cycle and cell death processes plays an important role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation and neurodegenerative diseases. Research achievements in these fields have led to the development of novel approaches for treatment of various conditions associated with abnormalities in the regulation of cell cycle progression or cell death. A better understanding of how cellular life-and-death processes are regulated is essential for this development. To highlight these important advances, the Third Nobel Conference entitled 'The Cell Cycle and Cell Death in Disease' was organized at Karolinska Institutet in 2016. In this review we will summarize current understanding of cell cycle progression and cell death and discuss some of the recent advances in therapeutic applications in pathological conditions such as cancer, neurological disorders and inflammation.

PRIMA-1MET induces mitochondrial apoptosis through activation of caspase-2.

This corrects the article DOI: 10.1038/onc.2016.210.

APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells.

Two main causes of platinum resistance are mutation in the tumor suppressor gene TP53 and drug-induced increase in intracellular glutathione concentration. Mutations in TP53 occur in about 50% of human tumors. APR-246 (PRIMA-1(MET)) is the first clinical-stage compound that reactivates mutant p53 and induces apoptosis. APR-246 is a prodrug that is converted to the active compound methylene quinuclidinone (MQ), a Michael acceptor that binds to cysteine residues in mutant p53 and restores its wild-type conformation. Here, we show that MQ also binds to cysteine in glutathione, thus decreasing intracellular free glutathione concentration. We also show that treatment with APR-246 completely restores the cisplatin and doxorubicin sensitivity to p53-mutant drug-resistant ovarian cancer cells. We propose that this unique ability of APR-246/MQ to bind to cysteines in both mutant p53 and glutathione has a key role in the resensitization as well as in the outstanding synergistic effects observed with APR-246 in combination with platinum compounds in ovarian cancer cell lines and primary cancer cells. However, MQ binding to cysteines in other targets, for example, thioredoxin reductase, may contribute as well. Strong synergy was also observed with the DNA-damaging drugs doxorubicin and gemcitabine, while additive effects were found with the taxane docetaxel. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with platinum-based therapy in patients with p53-mutant recurrent high-grade serous (HGS) ovarian cancer. More than 96% of these patients carry TP53 mutations. Combined treatment with APR-246 and platinum or other DNA-damaging drugs could allow dramatically improved therapy of a wide range of therapy refractory p53 mutant tumors.

TP53: an oncogene in disguise.

The standard classification used to define the various cancer genes confines tumor protein p53 (TP53) to the role of a tumor suppressor gene. However, it is now an indisputable fact that many p53 mutants act as oncogenic proteins. This statement is based on multiple arguments including the mutation signature of the TP53 gene in human cancer, the various gains-of-function (GOFs) of the different p53 mutants and the heterogeneous phenotypes developed by knock-in mouse strains modeling several human TP53 mutations. In this review, we will shatter the classical and traditional image of tumor protein p53 (TP53) as a tumor suppressor gene by emphasizing its multiple oncogenic properties that make it a potential therapeutic target that should not be underestimated. Analysis of the data generated by the various cancer genome projects highlights the high frequency of TP53 mutations and reveals that several p53 hotspot mutants are the most common oncoprotein variants expressed in several types of tumors. The use of Muller's classical definition of mutations based on quantitative and qualitative consequences on the protein product, such as 'amorph', 'hypomorph', 'hypermorph' 'neomorph' or 'antimorph', allows a more meaningful assessment of the consequences of cancer gene modifications, their potential clinical significance, and clearly demonstrates that the TP53 gene is an atypical cancer gene.

Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ.

Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels.

APR-246/PRIMA-1MET inhibits thioredoxin reductase 1 and converts the enzyme to a dedicated NADPH oxidase.

The low-molecular-weight compound APR-246 (PRIMA-1(MET)) restores wild-type conformation and function to mutant p53, and triggers apoptosis in tumor cells. We show here that APR-246 also targets the selenoprotein thioredoxin reductase 1 (TrxR1), a key regulator of cellular redox balance. APR-246 inhibited both recombinant TrxR1 in vitro and TrxR1 in cells. A Sec-to-Cys mutant of TrxR1 was not inhibited by APR-246, suggesting targeting of the selenocysteine residue in wild-type TrxR1. Preheated APR-246 and its conversion product methylene quinuclidinone (MQ) were much more efficient TrxR1 inhibitors than APR-246 itself, indicating that MQ is the active compound responsible for TrxR1 enzyme inhibition. TrxR1 inhibited by MQ was still functional as a pro-oxidant NADPH oxidase. Knockdown of TrxR1 caused a partial and reproducible attenuation of APR-246-induced tumor cell death independently of p53 status. Cellular TrxR1 activity was also inhibited by APR-246 irrespective of p53 status. We show that APR-246 can directly affect cellular redox status via targeting of TrxR1. Our findings provide an explanation for the previously observed effects of APR-246 on tumor cells lacking mutant p53.

Wig-1, a novel regulator of N-Myc mRNA and N-Myc-driven tumor growth.

Wig-1 is a transcriptional target of the p53 tumor suppressor and encodes an mRNA stability-regulating protein. We show here that Wig-1 knockdown causes a dramatic inhibition of N-Myc expression and triggers differentiation in neuroblastoma cells carrying amplified N-Myc. Transient Wig-1 knockdown significantly delays development of N-Myc-driven tumors in mice. We also show that N-Myc expression is induced upon moderate p53-activating stress, suggesting a role of the p53-Wig-1-N-Myc axis in promoting cell cycle re-entry upon p53-induced cell cycle arrest and DNA repair. Moreover, our findings raise possibilities for the improved treatment of poor prognosis neuroblastomas that carry amplified N-Myc.

The p53 target Wig-1: a regulator of mRNA stability and stem cell fate?

Wig-1 is a transcriptional target of the tumor suppressor p53 and encodes an unusual zinc-finger protein involved in post-transcriptional gene regulation. Wig-1 is expressed in all cell types investigated so far, with the highest levels in the brain, and is enriched in stem cells as compared with more differentiated cells of the same lineage. Wig-1 binds to both long double-stranded (ds) RNA and short microRNA-like dsRNA. We have shown that Wig-1 acts in a positive feedback loop that stabilizes p53 mRNA through an AU-rich element (ARE) in the p53 3'untranslated region. Our preliminary data indicate a more general effect of Wig-1 on ARE-containing mRNA. Here we shall summarize current knowledge about Wig-1 and discuss possible implications on p53 function and other cellular processes.

PRIMA-1(MET)/APR-246 targets mutant forms of p53 family members p63 and p73.

The low molecular weight compound PRIMA-1 and the structural analog PRIMA-1(MET), also named APR-246, reactivate mutant p53 through covalent binding to the core domain and induce apoptosis in tumor cells. Here, we asked whether PRIMA-1(MET)/APR-246 can rescue mutant forms of the p53 family members p63 and p73 that share high sequence homology with p53. We found that PRIMA-1(MET)/APR-246 can restore the pro-apoptotic function to mutant TAp63γ and TAp73ß in tumor cells but has less effect on TAp73α. Moreover, PRIMA-1(MET)/APR-246-stimulated DNA binding of mutant TAp63γ and induced expression of the p53/p63/p73 downstream targets p21 and Noxa. The reactivation of mutant p53, p63 and p73 by PRIMA-1(MET)/APR-246 indicates a common mechanism, presumably involving homologous structural elements in the p53 family proteins. Our findings may open avenues for therapeutic intervention in human developmental disorders with mutations in p63.

Pharmacological reactivation of mutant p53: from protein structure to the cancer patient.

The p53 tumor suppressor pathway blocks tumor development by triggering apoptosis or cellular senescence in response to oncogenic stress. A large fraction of human tumors carry p53 mutations that disrupt DNA binding of p53 and transcriptional regulation of target genes. Reconstitution of wild-type p53 in vivo triggers rapid elimination of tumors. Therefore, pharmacological reactivation of mutant p53 is a promising strategy for novel cancer therapy. Several approaches for identification of small molecules that target mutant p53 have been applied, including rational design and screening of chemical libraries. The compound PhiKan083 binds with high affinity to a crevice created by the Y220C mutation in p53 and stabilizes the mutant protein. The compound PRIMA-1 (p53 reactivation and induction of massive apoptosis) restores wild-type conformation to mutant p53 by binding to the core and induces apoptosis in human tumor cells. The PRIMA-1 analog APR-246 is currently tested in a clinical trial. Successful development of mutant p53-reactivating anticancer drugs should have a major impact on the treatment of cancer.

Mutant p53 reactivation by PRIMA-1MET induces multiple signaling pathways converging on apoptosis.

The low molecular weight compound PRIMA-1(MET) reactivates mutant p53 and triggers mutant p53-dependent apoptosis in human tumor cells. We investigated the effect of PRIMA-1(MET) on global gene expression using microarray analysis of Saos-2 cells expressing His273 mutant p53 and parental p53 null Saos-2 cells. PRIMA-1(MET) affected transcription of a significantly larger number of genes in the mutant p53-expressing cells compared to the p53 null cells. Genes affected by PRIMA-1(MET) in a mutant p53-dependent manner include the cell-cycle regulators GADD45B and 14-3-3gamma and the pro-apoptotic Noxa. Several of the affected genes are known p53 target genes and/or contain p53 DNA-binding motifs. We also found mutant p53-dependent disruption of the cytoskeleton, as well as transcriptional activation of the XBP1 gene and cleavage of its mRNA, a marker for endoplasmic reticulum stress. Our data show that PRIMA-1(MET) induces apoptosis through multiple transcription-dependent and -independent pathways. Such integral engagement of multiple pathways leading to apoptosis is consistent with restoration of wild-type properties to mutant p53 and is likely to reduce the risk of drug resistance development in clinical applications of PRIMA-1(MET).

PRIMA-1MET induces mitochondrial apoptosis through activation of caspase-2.

p53 mutations occur frequently in human tumors. The low-molecular-weight compound PRIMA-1(MET) reactivates mutant p53, induces apoptosis in human tumor cells and inhibits tumor xenograft growth in vivo. Here, we show that PRIMA-1(MET) induces mutant p53-dependent mitochondria-mediated apoptosis through activation of caspase-2 with subsequent cytochrome c release and further activation of downstream caspase-9 and caspase-3. Inhibition of caspase-2 by a selective inhibitor and/or siRNA prevents cytochrome c release on PRIMA-1(MET) treatment and causes a significant reduction in PRIMA-1(MET)-induced cell death. Our findings highlight a chain of cellular events triggered by PRIMA-1(MET) that lead to apoptotic cell death. This should facilitate further development and optimization of efficient PRIMA-1(MET)-based anticancer drugs.

Proteomic identification of p53-dependent protein phosphorylation.

The p53 tumor suppressor regulates transcription of target genes. We have previously analysed the p53-dependent proteome and identified novel protein targets. Here we have examined p53-dependent phosphorylation using two-dimensional gel electrophoresis and staining with the fluorescent phosphoprotein dye Pro-Q Diamond. We report that p53 induces phosphorylation of a subset of proteins including Nm23, DJ-1, ANXA1 and PrxII. Our identification of p53-dependent phosphorylation of specific target proteins reveals new aspects of the p53-dependent cellular response and suggests that such posttranslational modifications may contribute to p53-mediated tumor suppression.

Mutated and non-mutated TP53 as targets in the treatment of leukaemia.

TP53 is mutated in 10-20% of cases of chronic lymphocytic leukaemia (CLL) and 3-8% of cases of acute myeloid leukaemia (AML). Recently, two classes of compounds that restore the function of p53 in tumours have been described. PRIMA-1 (p53-dependent reactivation and induction of massive apoptosis) restores the wild-type conformation of mutant TP53, whereas RITA (reactivation of p53 and induction of tumour cell apoptosis) increases intracellular levels of p53. We evaluated the effects of RITA alone and in combination with PRIMA-1 or conventional cytostatics on leukaemic cells isolated from AML and CLL patients. AML samples with -17, which are more resistant to daunorubicin and cytarabine compared with samples without -17, were effectively killed by PRIMA-1. RITA, which stabilizes the function of wild-type p53, induced apoptosis in AML cells. In contrast to that seen with PRIMA-1, AML patient samples without -17 were significantly more sensitive to RITA. Similarly, RITA exerted dose-dependent apoptosis and cytotoxicity in CLL cells, which was significantly more pronounced in samples without hemizygous TP53 deletion. Notably, a synergistic effect was observed in all CLL samples with RITA and fludarabine in combination. In both AML and CLL cells exposure to RITA resulted in induction of intracellular p53. We conclude that small molecules targeting p53 might be of clinical importance in the future for treating drug-resistant leukaemia.

Reactivation of mutant p53: molecular mechanisms and therapeutic potential.

The p53 tumor suppressor gene is the most frequently mutated gene in cancer. Most p53 mutations are missense point mutations that cluster in the DNA-binding core domain. This results in distortion of core domain folding and disruption of DNA binding and transcriptional transactivation of p53 target genes. Structural studies have demonstrated that mutant p53 core domain unfolding is not irreversible. Mutant p53 is expressed at high levels in many tumors. Therefore, mutant p53 is a promising target for novel cancer therapy. Mutant p53 reactivation will restore p53-dependent apoptosis, resulting in efficient removal of tumor cells. A number of strategies for targeting mutant p53 have been designed, including peptides and small molecules that restore the active conformation and DNA binding to mutant p53 and induce p53-dependent suppression of tumor cell growth in vitro and in vivo. This opens possibilities for the clinical application of mutant p53 reactivation in the treatment of cancer.

Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis.

p53 triggers apoptosis in response to cellular stress. We analyzed p53-dependent gene and protein expression in response to hypoxia using wild-type p53-carrying or p53 null HCT116 colon carcinoma cells. Hypoxia induced p53 protein levels and p53-dependent apoptosis in these cells. cDNA microarray analysis revealed that only a limited number of genes were regulated by p53 upon hypoxia. Most classical p53 target genes were not upregulated. However, we found that Fas/CD95 was significantly induced in response to hypoxia in a p53-dependent manner, along with several novel p53 target genes including ANXA1, DDIT3/GADD153 (CHOP), SEL1L and SMURF1. Disruption of Fas/CD95 signalling using anti-Fas-blocking antibody or a caspase 8 inhibitor abrogated p53-induced apoptosis in response to hypoxia. We conclude that hypoxia triggers a p53-dependent gene expression pattern distinct from that induced by other stress agents and that Fas/CD95 is a critical regulator of p53-dependent apoptosis upon hypoxia.