Apoptotic Function Of P53 Research Articles

LincRNA-RoR regulates the apoptotic function of p53 via targeting miR-204/MDM2 in human esophageal squamous cell carcinoma

LincRNA-RoR regulates the apoptotic function of p53 via targeting miR-204/MDM2 in human esophageal squamous cell carcinoma

Abstract 4830: Selective DNA-PK inhibitor, M3814, boosts p53 apoptotic response to DNA double strand breaks and effectively kills acute leukemia cells: Implications for AML therapy

Abstract M3814 is a selective inhibitor of DNA-PK in clinical development. It effectively blocks non-homologous end joining repair of DNA double strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By inhibiting DNA-PK catalytic activity in the presence of DSB damage, M3814 disrupts a previously unrecognized negative regulatory loop between DNA-PK and ATM, leading to increased activation of the ATM pathway, including its downstream targets, CHK2 and p53. As a result, in cancer cells expressing wild-type p53, DNA-PK inhibitor strongly activates the p53 response to DSB DNA damage and its two main functions- cell cycle arrest and apoptosis. While p53 activation in p53 wild-type epithelial cancer cells causes effective cell cycle arrest, the majority of the epithelial tumors lose their ability to undergo p53 dependent apoptosis. In contrast, tumor cells from patients with acute leukemia express wild-type p53 in with preserved p53 apoptotic function, making them amenable to p53 activation approaches. Here, we investigate the therapeutic potential of M3814 in combination with DSB-inducing agents in acute leukemia cells. M3814 strongly potentiated the activity of IR in three p53 wild-type leukemia cell lines (MOLM-13, MOLT-4, MV4-11) but had minimal effect on the p53-null HL-60 and THP-1 lines. This potentiation was due to enhanced activation of the p53 pathway as demonstrated by substantially increased expression of p53 transcriptional targets, p21 and PUMA, and induction of apoptosis only in p53 wild-type cells. M3814 showed synergism in p53-dependent cell killing with the topoisomerase inhibitors, daunorubicin and idarubicin, and 2-3 fold increase in the potency of daunorubicin/cytarabine combination in p53 wild-type acute myeloid leukemia (AML) cells. Daunorubicin/cytarabine/M3814 combinations were better tolerated by CD34+ bone marrow cells harvested from healthy volunteers and tested in vitro. These results support further exploration of M3814 as a novel combination partner in the treatment of p53 wild-type AML. Citation Format: Eric Haines, Astrid Zimmermann, Frank Zenke, Andree Blaukat, Lyubomir T. Vassilev. Selective DNA-PK inhibitor, M3814, boosts p53 apoptotic response to DNA double strand breaks and effectively kills acute leukemia cells: Implications for AML therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4830.

P53-Dependent PUMA to DRAM antagonistic interplay as a key molecular switch in cell-fate decision in normal/high glucose conditions

BackgroundAs an important cellular stress sensor phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The p53 activity mainly depends on its transactivating function, however, how p53 can select one or another biological outcome is still a matter of profound studies. Our previous findings indicate that switching cancer cells in high glucose (HG) impairs p53 apoptotic function and the transcription of target gene PUMA.Methods and resultsHere we report that, in response to drug adriamycin (ADR) in HG, p53 efficiently induced the expression of DRAM (damage-regulated autophagy modulator), a p53 target gene and a stress-induced regulator of autophagy. We found that ADR treatment of cancer cells in HG increased autophagy, as displayed by greater LC3II accumulation and p62 degradation compared to ADR-treated cells in low glucose. The increased autophagy in HG was in part dependent on p53-induced DRAM; indeed DRAM knockdown with specific siRNA reversed the expression of the autophagic markers in HG. A similar outcome was achieved by inhibiting p53 transcriptional activity with pifithrin-α. DRAM knockdown restored the ADR-induced cell death in HG to the levels obtained in low glucose. A similar outcome was achieved by inhibition of autophagy with cloroquine (CQ) or with silencing of autophagy gene ATG5. DRAM knockdown or inhibition of autophagy were both able to re-induce PUMA transcription in response to ADR, underlining a reciprocal interplay between PUMA to DRAM to unbalance p53 apoptotic activity in HG. Xenograft tumors transplanted in normoglycemic mice displayed growth delay after ADR treatment compared to those transplanted in diabetics mice and such different in vivo response correlated with PUMA to DRAM gene expression.ConclusionsAltogether, these findings suggest that in normal/high glucose condition a mutual unbalance between p53-dependent apoptosis (PUMA) and autophagy (DRAM) gene occurred, modifying the ADR-induced cancer cell death in HG both in vitro and in vivo.

Abstract 4913: Invasive melanoma cells commandeer p53 activity to promote the survival of a therapy resistant subpopulation

Abstract Metastatic melanoma is highly aggressively and often therapy resistant. Signaling pathways which promote invasion of metastatic melanoma, also promote therapy resistance. Therapy resistant melanomas are characterized by high levels of the pro-invasive non-canonical Wnt molecule, Wnt5A. Previously, we described an adaptive stress response in highly invasive melanoma cells, which is characterized by a growth arrest and an increase in senescence markers, yet these cells retain the ability to invade and form colonies. These highly invasive cells do not undergo apoptosis following treatment with DNA damaging agents such as doxorubicin and are resistant to BRAFV600E targeted therapy. Recently, Lukin et al. have shown that p53 expression promotes survival of colorectal cancer cells via p21 expression and a reversible cell cycle arrest, allowing for repair of damaged DNA. Here, we show that highly invasive and therapy resistant melanoma cells express Wnt5A, p53 and p21, which regulate proliferation and slow cycling in invasive melanoma cells. The expression of p53 is promoted by Wnt5A, knock down of Wnt5A decreases p53 and p21 expression in these cells and decreases the number of cells arrested in G2/M following DNA damage. The cell cycle and apoptotic functions of p53 are highly regulated. iASPP, MDMX, and MDM2 have been shown to regulate the function of wild type p53 in melanoma cells. MDM2, an E4 ubiquitin ligase, regulates p53 by shuttling it out of the nucleus and targeting it for proteasomal degradation. We found that MDM2 expression increases in invasive melanoma cells following DNA damage, however, in these cells it is phosphorylated at serine 395. Phosphorylation of MDM2 at ser395 blocks its ability to export p53 from the nucleus, leading to increased p53 expression. Even with increased p53 expression, invasive melanoma cells undergo a cell cycle arrest following stress instead of apoptosis. The apoptotic function of p53 has been shown to be inhibited by nuclear iASPP, which is enriched in metastatic melanoma. We found that knocking down Wnt5A in invasive melanoma cells decreases the expression of iASPP. These data suggest that Wnt5A promotes MDM2 Ser395 phosphorylation and iASPP expression, blocking down regulation of p53 and its apoptotic function, while promoting cell cycle arrest and survival following stress. These data may reveal a mechanism by which highly invasive melanoma cells evade therapy to form therapy resistant sub-colonies at distant sites. Citation Format: Marie R. Webster, Amanpreet Kaur, Abibatou Ndoye, Curtis Kugel, Subhasree Basu, Alexander Valiga, Jessica Appleton, Ying-Jie Wang, Maureen Murphy, Ashani T. Weeraratna. Invasive melanoma cells commandeer p53 activity to promote the survival of a therapy resistant subpopulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2017-4913

P16INK4A enhances the transcriptional and the apoptotic functions of p53 through DNA-dependent interaction.

p16INK4A and p53 are two important tumor suppressor proteins that play essential roles during cell proliferation and aging through regulating the expression of several genes. Here, we report that p16INK4A and p53 co-regulate a plethora of transcripts. Furthermore, both proteins colocalize in the nucleus of human primary skin fibroblasts and breast luminal cells, and form a heteromer whose level increases in response to genotoxic stress as well as aging of human fibroblasts and various mouse organs. CDK4 is also present in this heteromeric complex, which is formed only in the presence of DNA both in vitro using pure recombinant proteins and in vivo. We have also shown that p16INK4A enhances the binding efficiency of p53 to its cognate sequence presents in the CDKN1A promoter in vitro, and both proteins are present at the promoters of CDKN1A and BAX in vivo. Importantly, the fourth ankyrin repeat of p16INK4A and the C-terminal domain of p53 were necessary for the physical association between these two proteins. The physiologic importance of this association was revealed by the inability of cancer-associated p16INK4A mutants to interact with p53 and to transactivate the expression of its major targets CDKN1A and BAX in the p16-defective U2OS cells expressing either wild-type or mutated p16INK4A . Furthermore, the association between p16INK4A and p53 was capital for their nuclear colocalization, the X-ray-dependent induction of p21 and Bax proteins as well as the induction of apoptosis in various types of cells. Together, these results show DNA-dependent physical interaction between p16INK4A and p53.

Hyperglycemia triggers HIPK2 protein degradation.

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies.

The Machado-Joseph Disease Deubiquitinase Ataxin-3 Regulates the Stability and Apoptotic Function of p53.

As a deubiquitinating enzyme (DUB), the physiological substrates of ataxin-3 (ATX-3) remain elusive, which limits our understanding of its normal cellular function and that of pathogenic mechanism of spinocerebellar ataxia type 3 (SCA3). Here, we identify p53 to be a novel substrate of ATX-3. ATX-3 binds to native and polyubiquitinated p53 and deubiquitinates and stabilizes p53 by repressing its degradation through the ubiquitin (Ub)-proteasome pathway. ATX-3 deletion destabilizes p53, resulting in deficiency of p53 activity and functions, whereas ectopic expression of ATX-3 induces selective transcription/expression of p53 target genes and promotes p53-dependent apoptosis in both mammalian cells and the central nervous system of zebrafish. Furthermore, the polyglutamine (polyQ)-expanded ATX-3 retains enhanced interaction and deubiquitination catalytic activity to p53 and causes more severe p53-dependent neurodegeneration in zebrafish brains and in the substantia nigra pars compacta (SNpc) or striatum of a transgenic SCA3 mouse model. Our findings identify a novel molecular link between ATX-3 and p53-mediated cell death and provide an explanation for the direct involvement of p53 in SCA3 disease pathogenesis.

The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression.

P53 is a transcription factor highly inducible by many stress signals such as DNA damage, oncogene activation, and nutrient deprivation. Cell-cycle arrest and apoptosis are the most prominent outcomes of p53 activation. Many studies showed that p53 cell-cycle and apoptosis functions are important for preventing tumor development. p53 also regulates many cellular processes including metabolism, antioxidant response, and DNA repair. Emerging evidence suggests that these noncanonical p53 activities may also have potent antitumor effects within certain context. This review focuses on the cell-cycle arrest and apoptosis functions of p53, their roles in tumor suppression, and the regulation of cell fate decision after p53 activation.

Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma.

BackgroundAdrenocortical carcinoma (ACC) is an aggressive cancer with a 5 year survival rate of 20–30 %. Various factors have been implicated in the pathogenesis of ACC including dysregulation of the G2/M transition and aberrant activity of p53 and MDM2. Polo-like kinase 1 (PLK-1) negatively modulates p53 functioning, promotes MDM2 activity through its phosphorylation, and is involved in the G2/M transition. Gene expression profiling of 44 ACC samples showed that increased expression of PLK-1 in 29 % of ACC. Consequently, we examined PLK-1’s role in the modulation of the p53 signaling pathway in adrenocortical cancer.MethodsWe used siRNA knock down PLK-1 and pharmacological inhibition of PLK-1 and MDM2 ACC cell lines SW-13 and H295R. We examined viability, protein expression, p53 transactivation, and induction of apoptosis.ResultsKnocking down expression of PLK-1 with siRNA or inhibition of PLK-1 by a small molecule inhibitor, BI-2536, resulted in a loss of viability of up to 70 % in the ACC cell lines H295R and SW-13. In xenograft models, BI-2536 demonstrated marked inhibition of growth of SW-13 with less inhibition of H295R. BI-2536 treatment resulted in a decrease in mutant p53 protein in SW-13 cells but had no effect on wild-type p53 protein levels in H295R cells. Additionally, inhibition of PLK-1 restored wild-type p53’s transactivation and apoptotic functions in H295R cells, while these functions of mutant p53 were restored only to a smaller extent. Furthermore, inhibition of MDM2 with nutlin-3 reduced the viability of both the ACC cells and also reactivated wild-type p53′s apoptotic function. Inhibition of PLK-1 sensitized the ACC cell lines to MDM2 inhibition and this dual inhibition resulted in an additive apoptotic response in H295R cells with wild-type p53.ConclusionsThese preclinical studies suggest that targeting p53 through PLK-1 is an attractive chemotherapy strategy warranting further investigation in adrenocortical cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-015-0080-3) contains supplementary material, which is available to authorized users.

Abstract 24: The phosphorylation of p53 at serine 46 is essential to induce cell death through palmdelphin in response to DNA damage

Abstract Tumor suppressor p53 plays a pivotal role in cell cycle arrest, DNA repair, and apoptosis in response to DNA damage. Promoter selectivity of p53 depends mainly on post-translational modification. Notably, the apoptotic function of p53 is related to its phosphorylation at serine-46 (ser46) to promote pro-apoptotic genes. However, little is known about the pro-apoptotic genes induced by Ser46 phosphorylation. Our research achieved to investigate the pro-apoptotic genes induced by p53 in a phospho-ser46-specific manner using microarray and ChIP sequencing in human cancer cell lines. As a result, palmdelphin (PALMD), an isoform of paralemmin protein, was strongly elicited from the phosphorylation of ser46. The mRNA and protein expression of PALMD increased only in wild type p53 transfected cells, but not in ser46-mutated cells. Importantly, PALMD moved to the nucleus in response to DNA damage and the apoptotic function of PALMD was tightly exerted with localization into nucleus. Interestingly, down-regulation of PALMD by siRNA resulted in necroptosis-like cell death through ATP depletion. Moreover, we found vimentin as a PALMD interacting protein and the depletion of vimentin increased PALMD level to accelerate apoptosis. These results demonstrate that p53 regulates cell death fate (apoptosis or necroptosis-like cell death) through promoting PALMD expression in a phospho-ser46-specific manner in response to DNA damage. Citation Format: Nurmaa Khund Dashzeveg, Kiyotsugu Yoshida. The phosphorylation of p53 at serine 46 is essential to induce cell death through palmdelphin in response to DNA damage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 24. doi:10.1158/1538-7445.AM2015-24

Abstract 2964: The codon 47 Pro47Ser polymorphism influences p53 phosphorylation and apoptotic function

Abstract The p53 tumor suppressor protein is a central signaling protein in response to oncogenic, genotoxic and metabolic stress. Single nucleotide polymorphisms (SNPs) in p53 can have a marked impact on p53 tumor suppressor function, and have been associated with increased cancer risk. Amino acid 47 of p53 is a conserved residue, located within the second transactivation domain of this protein. In wild type p53, this residue is proline (P47, or WT). Importantly, this proline is critical for the ability of proline-directed kinases to phosphorylate p53 on the adjacent residue, serine 46. Phosphorylation of serine 46 of p53 greatly enhances the ability of p53 to transactivate certain target genes, and to induce apoptosis. In some Africans and African Americans, amino acid 47 of p53 encodes serine (S47) instead of proline. We have shown that the S47 polymorphism eliminates phosphorylation on serine 46, and reduces p53's ability to induce apoptosis by at least 3-fold. We have created mice that contain the humanized p53 gene (Hupki) for either the S47 or WT alleles of p53. In new data, we show that tissues from these mice have significantly decreased apoptosis after gamma radiation. We also show that apoptosis induced by cisplatin is markedly defective in S47 mice and cell lines. Further, MEFs from S47 mice have a 15-fold increase in IC50 for cisplatin, indicating that this polymorphism will impact the efficacy of cisplatin therapy in Africans and African Americans. Finally, we have identified two critical p53 target genes, GLS2 (metabolism) and NOXA (apoptosis), which show significantly impaired transactivation by S47. The long-term objective of this research is to further characterize the function of the S47 variant, and to identify the contribution of this variant to cancer risk and progression in Africans and African Americans. Citation Format: Matthew Jennis, Monica Hollstein, Maureen E. Murphy. The codon 47 Pro47Ser polymorphism influences p53 phosphorylation and apoptotic function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2964. doi:10.1158/1538-7445.AM2014-2964

Phosphorylation status of ASPP2 modulates p53 apoptotic function in oxaliplatin-induced apoptosis of colorectal cancer HCT116 cells

To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2) phosphorylation status in the regulation of ASPP2-p53 apoptotic pathway activity. Cells were individually transfected with green fluorescent protein (GFP)-encoding vector, constitutively non-phosphorylatable ASPP2 mutant-ASPP2 (Am)-encoding vector, and wild type ASPP2 (Aw)-encoding vector) plasmids, respectively, to make them overexpressing phosphorylated and non-phosphorylated ASPP2 proteins, respectively. Cell apoptosis was induced by oxaliplatin. The apoptosis rate of cells was determined by flow cytometry after staining with FITC-conjugated annexin V and PI. ASPP2 protein level and its phosphorylation status were observed by Western blot. The interaction between ASPP2 and p53 was observed by immunoprecipitation assay. Oxaliplatin induced cell apoptosis and caused phosphorylation of ASPP2 at ser92/ser361 in the HCT116 cells. The apoptosis rate of Aw and Am plasmids-transfected cells were (3.8 ± 1.0)% and (3.9 ± 1.2)% respectively, statistically with a non-significant difference (P > 0.05) in comparison with that of the GFP plasmid-transfected cells [(4.0 ± 0.8)%]. After oxaliplatin treatment, the apoptosis rate of Aw plasmid-transfected cells was (46.7 ± 3.9)%, significantly higher than that of the Am and GFP plasmid-transfected cells [(40.1 ± 10.2)% and (37.1 ± 6.9)%, respectively, P < 0.05], however, there was no statistically significant difference (P > 0.05) between Am and GFP plasmid-transfected cells. These results indicate that phosphorylated ASPP2 promoted the oxaliplatin-induced apoptosis of HCT116 cells through a p53-dependent pathway. Phosphorylation status of ASPP2 influenced its binding activity to p53. Phosphorylation status of ASPP2 modulates p53 apoptotic function in oxaliplatin-induced apoptosis of colorectal cancer HCT116 cells.

Ubiquitin-like (UBX)-domain-containing protein, UBXN2A, promotes cell death by interfering with the p53-Mortalin interactions in colon cancer cells.

Mortalin (mot-2) induces inactivation of the tumor suppressor p53's transcriptional and apoptotic functions by cytoplasmic sequestration of p53 in select cancers. The mot-2-dependent cytoprotective function enables cancer cells to support malignant transformation. Abrogating the p53-mot-2 interaction can control or slow down the growth of cancer cells. In this study, we report the discovery of a ubiquitin-like (UBX)-domain-containing protein, UBXN2A, which binds to mot-2 and consequently inhibits the binding between mot-2 and p53. Genetic analysis showed that UBXN2A binds to mot-2's substrate binding domain, and it partly overlaps p53's binding site indicating UBXN2A and p53 likely bind to mot-2 competitively. By binding to mot-2, UBXN2A releases p53 from cytosolic sequestration, rescuing the tumor suppressor functions of p53. Biochemical analysis and functional assays showed that the overexpression of UBXN2A and the functional consequences of unsequestered p53 trigger p53-dependent apoptosis. Cells expressing shRNA against UBXN2A showed the opposite effect of that seen with UBXN2A overexpression. The expression of UBXN2A and its apoptotic effects were not observed in normal colonic epithelial cells and p53−/− colon cancer cells. Finally, significant reduction in tumor volume in a xenograft mouse model in response to UBXN2A expression was verified in vivo. Our results introduce UBXN2A as a home defense response protein, which can reconstitute inactive p53-dependent apoptotic pathways. Inhibition of mot-2-p53 interaction by UBXN2A is an attractive therapeutic strategy in mot-2-elevated tumors.

HDM2 Regulation by AURKA Promotes Cell Survival in Gastric Cancer

Suppression of P53 (tumor protein 53) transcriptional function mediates poor therapeutic response in patients with cancer. Aurora kinase A (AURKA) and human double minute 2 (HDM2) are negative regulators of P53. Herein, we examined the role of AURKA in regulating HDM2 and its subsequent effects on P53 apoptotic function in gastric cancer. Primary tumors and in vitro gastric cancer cell models with overexpression or knockdown of AURKA were used. The role of AURKA in regulating HDM2 and cell survival coupled with P53 expression and activity were investigated. Overexpression of AURKA enhanced the HDM2 protein level; conversely, knockdown of endogenous AURKA decreased expression of HDM2 in AGS and SNU-1 cells. Dual co-immunoprecipitation assay data indicated that AURKA was associated with HDM2 in a protein complex. The in vitro kinase assay using recombinant AURKA and HDM2 proteins followed by co-immunoprecipitation revealed that AURKA directly interacts and phosphorylates HDM2 protein in vitro. The activation of HDM2 by AURKA led to induction of P53 ubiquitination and attenuation of cisplatin-induced activation of P53 in gastric cancer cells. Inhibition of AURKA using an investigational small-molecule specific inhibitor, alisertib, decreased the HDM2 protein level and induced P53 transcriptional activity. These effects markedly decreased cell survival in vitro and xenograft tumor growth in vivo. Notably, analysis of immunohistochemistry on tissue microarrays revealed significant overexpression of AURKA and HDM2 in human gastric cancer samples (P < 0.05). Collectively, our novel findings indicate that AURKA promotes tumor growth and cell survival through regulation of HDM2-induced ubiquitination and inhibition of P53. Clin Cancer Res; 20(1); 76-86. ©2013 AACR.

Phosphorylation of ASPP2 by RAS/MAPK Pathway Is Critical for Its Full Pro-Apoptotic Function

We reported recently that apoptosis-stimulating protein of p53 (ASPP) 2, an activator of p53, co-operates with oncogenic RAS to enhance the transcription and apoptotic function of p53. However, the detailed mechanism remains unknown. Here we show that ASPP2 is a novel substrate of mitogen-activated protein kinase (MAPK). Phosphorylation of ASPP2 by MAPK is required for RAS-induced increased binding to p53 and increased transactivation of pro-apoptotic genes. In contrast, an ASPP2 phosphorylation mutant exhibits reduced p53 binding and fails to enhance transactivation and apoptosis. Thus phosphorylation of ASPP2 by RAS/MAPK pathway provides a novel link between RAS and p53 in regulating apoptosis.

Abstract 2472: Small-molecule-induced reactivation of mutant p53 in cancer cells.

Abstract The tumour suppressor p53 inhibits tumour growth by induction of cell cycle arrest, apoptosis or senescence. However, p53 mutations occur frequently in human tumours. About one-third of the mutations lower the melting temperature of the protein and cause rapidly unfolds at body temperature. The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface crevice that can be targeted by small-molecule stabilizers. Through biophysical and cell-based screening, we identified a compound, PK7088, which binds to the Y220C mutant with a dissociation constant of 140 μM. The binding mode of this compound class was determined by X-ray crystallography. In cancer cells carrying the Y220C mutant, PK7088 increased the amount of folded mutant protein with wild-type like conformation, as monitored by immunofluorescence, and restored its transcriptional functions. It induced p53-Y220C-dependent growth inhibition, cell-cycle arrest and apoptosis. This was confirmed by p53 silencing. Most notably, PK7088 increased the expression levels of p21 and the proapoptotic NOXA protein. PK7088 worked synergistically with Nutlin-3 on up-regulating p21 and NOXA expression, whereas Nutlin-3 on its own had no effect, consistent with its mechanism of action. PK7088 also restored non-transcriptional apoptotic functions of p53 by triggering nuclear export of BAX to the mitochondria. These findings provide novel insights into p53 mutant reactivation and may facilitate the development of more potent and selective small-molecule drugs for specific mutant p53 rescue. Citation Format: Xiangrui Liu, Rainer Wilcken, Andreas Joerger, Irina Chuckowree, John Spencer, Alan Fersht. Small-molecule-induced reactivation of mutant p53 in cancer cells. [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 2472. doi:10.1158/1538-7445.AM2013-2472

ASPP1 and ASPP2 bind active RAS, potentiate RAS signalling and enhance p53 activity in cancer cells

RAS mutations occur frequently in human cancer and activated RAS signalling contributes to tumour development and progression. Apart from its oncogenic effects on cell growth, active RAS has tumour-suppressive functions via its ability to induce cellular senescence and apoptosis. RAS is known to induce p53-dependent cell cycle arrest, yet its effect on p53-dependent apoptosis remains unclear. We report here that apoptosis-stimulating protein of p53 (ASPP) 1 and 2, two activators of p53, preferentially bind active RAS via their N-terminal RAS-association domains (RAD). Additionally, ASPP2 colocalises with and contributes to RAS cellular membrane localisation and potentiates RAS signalling. In cancer cells, ASPP1 and ASPP2 cooperate with oncogenic RAS to enhance the transcription and apoptotic function of p53. Thus, loss of ASPP1 and ASPP2 in human cancer cells may contribute to the full transforming property of RAS oncogene.

Actinomycin-D treatment of high-risk ependymomas re-establishes the apoptotic function of p53

Actinomycin-D treatment of high-risk ependymomas re-establishes the apoptotic function of p53

High-mobility group A1 protein inhibits p53-mediated intrinsic apoptosis by interacting with Bcl-2 at mitochondria

The high-mobility group A (HMGA) proteins are a family of non-histone chromatin factors, encoded by the HMGA1 and HMGA2 genes. Several studies demonstrate that HMGA proteins have a critical role in neoplastic transformation, and their overexpression is mainly associated with a highly malignant phenotype, also representing a poor prognostic index. Even though a cytoplasmic localization of these proteins has been previously reported in some highly malignant neoplasias, a clear role for this localization has not been defined. Here, we first confirm the localization of the HMGA1 proteins in the cytoplasm of cancer cells, and then we report a novel mechanism through which HMGA1 inhibits p53-mitochondrial apoptosis by counteracting the binding of p53 to the anti-apoptotic factor Bcl-2. Indeed, we demonstrate a physical and functional interaction between HMGA1 and Bcl-2 proteins. This interaction occurs at mitochondria interfering with the ability of p53 protein to bind Bcl-2, thus counteracting p53-mediated mitochondrial apoptosis. This effect is associated with the inhibition of cytochrome c release and activation of caspases. Consistent with this mechanism, a strong correlation between HMGA1 cytoplasmic localization and a more aggressive histotype of thyroid, breast and colon carcinomas has been observed. Therefore, cytoplasmic localization of HMGA1 proteins in malignant tissues is a novel mechanism of inactivation of p53 apoptotic function.

DBC1 phosphorylation by ATM/ATR inhibits SIRT1 deacetylase in response to DNA damage

Human DBC1 (deleted in breast cancer-1; KIAA1967) is a nuclear protein that, in response to DNA damage, competitively inhibits the NAD(+)-dependent deacetylase SIRT1, a regulator of p53 apoptotic functions in response to genotoxic stress. DBC1 depletion in human cells increases SIRT1 activity, resulting in the deacetylation of p53 and protection from apoptosis. However, the mechanisms regulating this process have not yet been determined. Here, we report that, in human cell lines, DNA damage triggered the phosphorylation of DBC1 on Thr454 by ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia and Rad3-related) kinases. Phosphorylated DBC1 bound to and inhibited SIRT1, resulting in the dissociation of the SIRT1-p53 complex and stimulating p53 acetylation and p53-dependent cell death. Indeed, DBC1-mediated genotoxicity, which was shown in knockdown experiments to be dependent on SIRT1 and p53 expression, was defective in cells expressing the phospho-mutant DBC1(T454A). This study describes the first post-translational modification of DBC1 and provides new mechanistic insight linking ATM/ATR to the DBC1-SIRT1-p53 apoptotic axis triggered by DNA damage.