Induction Of P53 Target Genes Research Articles

Hepatic p53 is regulated by transcription factor FOXO1 and acutely controls glycogen homeostasis

The tumor suppressor p53 is involved in the adaptation of hepatic metabolism to nutrient availability. Acute deletion of p53 in the mouse liver affects hepatic glucose and triglyceride metabolism. However, long-term adaptations upon the loss of hepatic p53 and its transcriptional regulators are unknown. Here we show that short-term, but not chronic, liver-specific deletion of p53 in mice reduces liver glycogen levels, and we implicate the transcription factor forkhead box O1 protein (FOXO1) in the regulation of p53 and its target genes. We demonstrate that acute p53 deletion prevents glycogen accumulation upon refeeding, whereas a chronic loss of p53 associates with a compensational activation of the glycogen synthesis pathway. Moreover, we identify fasting-activated FOXO1 as a repressor of p53 transcription in hepatocytes. We show that this repression is relieved by inactivation of FOXO1 by insulin, which likely mediates the upregulation of p53 expression upon refeeding. Strikingly, we find that high-fat diet–induced insulin resistance with persistent FOXO1 activation not only blunted the regulation of p53 but also the induction of p53 target genes like p21 during fasting, indicating overlapping effects of both FOXO1 and p53 on target gene expression in a context-dependent manner. Thus, we conclude that p53 acutely controls glycogen storage in the liver and is linked to insulin signaling via FOXO1, which has important implications for our understanding of the hepatic adaptation to nutrient availability.

PARP1 Is Required for ATM-Mediated p53 Activation and p53-Mediated Gene Expression after Ionizing Radiation.

PARP1 and p53 are key players in maintaining genomic stability, but their interplay is still not fully understood. We investigated the impact of PARP1 knockout on the DNA damage response after ionizing radiation (IR) by comparing a U2OS-based PARP1-knockout cell line, established by using the genome-editing system CRISPR/Cas9, with its wild-type counterpart. We intended to gain more insight into the impact of PARP1 on the transcriptional level under basal conditions, after low dose (1 Gy) and high dose (10 Gy) DNA damage induced by IR, aiming to reveal the potential connections between the involved pathways. In the absence of additionally induced DNA damage, lacking PARP1 led to an increased up-regulation of CDKN1A (p21), which caused a G1 arrest and slightly diminished cell proliferation. While a small but comparable transcriptional DNA damage response was observed upon 1 Gy IR in both cell lines, a pronounced transcriptional induction of p53 target genes was evident after treatment with 10 Gy IR exclusively in PARP1-proficient cells, suggesting that PARP1 facilitates the p53 signaling response after IR. Additionally, PARP1 appeared to be required for the ATM-dependent activation of PLK3, which in turn activates p53, leading to its transcriptional damage response. Our results support the involvement of PARP1 activation among the first steps in IR-induced DNA damage response.

Insulin-like Growth Factor-1 Impacts p53 Target Gene Induction in UVB-irradiated Keratinocytes and Human Skin.

The tumor suppressor protein p53 limits mutagenesis in response to ultraviolet-B (UVB) light exposure by activating the transcription of genes that mitigate the damaging effects of UVB radiation on DNA. Because most nonmelanoma skin cancers (NMSCs) occur in older individuals, it is important to understand the process of mutagenesis in the geriatric skin microenvironment. Based on previous studies demonstrating that geriatric skin expresses lower levels of the growth factor insulin-like growth factor-1 (IGF-1) than young adult skin, a role for IGF-1 in the regulation of p53 target genes was investigated in both human keratinocytes in vitro and human skin explants ex vivo. The products of the p53 target genes p21 and DNA polymerase eta (pol η) were found to be increased by UVB exposure in both experimental systems, and this induction was observed to be partially abrogated by depriving keratinocytes of IGF-1 in vitro or by the treatment of keratinocytes in vitro and human skin explants with an IGF-1 receptor antagonist. Because p21 and pol η function to limit mutagenic DNA replication following UVB exposure, these results suggest that NMSC risk in geriatric populations may be due to age-dependent decreases in IGF-1 signaling that disrupt p53 function in the skin.

CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation

Casein kinase 1α (CK1α), a component of the β-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded by Csnk1a1) in skin physiology, we crossed mice harboring floxed Csnk1a1 with mice expressing K14-Cre-ERT2 to generate mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by β-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo after Csnk1a1 ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14-Cre-ERT2 CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte-stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.

Checkpoint kinase 2 is dispensable for regulation of the p53 response but is required for G2/M arrest and cell survival in cells with p53 defects under heat stress.

Hyperthermia induced by heat stress (HS) is known to inhibit proliferation and induce cell death in cancer. We previously demonstrated that checkpoint kinase 1 (Chk1) contributes to G2/M arrest and cell survival under HS; however, the role of Chk2, a functional analog of Chk1, in regulation of the cell cycle and cell death under HS is still unknown. Here, we addressed the role of Chk2 using Molt-4 cells with p53-targeted shRNA (Molt-4/shp53) and parental control cells (Molt-4/V). Chk2 inhibition suppressed C-terminal acetylation of p53 and delayed the induction of p53-target genes in Molt-4/V cells under HS; however, Chk2 inhibition failed to inhibit apoptosis induced by HS, indicating that Chk2 was dispensable for p53-dependent apoptosis under HS. In contrast, Chk2 inhibition abrogated G2/M arrest and promoted cell death induced by HS in HeLa cells and Molt-4/shp53 cells. Thus, we demonstrated for the first time that Chk2 was required for cell cycle arrest and cell survival, particularly in cells with p53 defects under HS. These findings indicated that Chk2 may be a selective target for p53-mutated or -deficient cancer treated with hyperthermia.

Abstract 3694: Selective killing of cancer cells by the styryl lactone (R)- goniothalamin is mediated through glutathione conjugation, oxidative stress and a marked reactivation of the R175H mutant p53 protein

Abstract Goniothalamin (GTN) is a secondary metabolite styryl lactone isolated from several species of the tropical medicinal tree Goniothalamus. GTN has been shown to be cytotoxic and induce apoptosis in many cancer cell lines. This study sought to define the molecular basis underlying the antiproliferative actions of GTN. We synthesized the R and S enantiomers of GTN and found the R form to be more cytotoxic against a panel of breast cancer and lung cancer cell lines. The IC50 of GTN against breast cancer cell lines was in the range of 10-20 μM and interestingly, the SKBR3 cells, which harbor a R175H mutation in p53 were more sensitive to the drug. In contrast, the normal breast epithelial MCF10A cells were not killed by GTN, and N-acetylcysteine prevented cell-killing, indicating the ROS involvement. Indeed, the flow cytometry and cell staining with DCF-DA showed high levels of ROS generation, and this was accompanied by significant S-glutathionylation of bulk proteins. Other studies showed that GTN forms conjugate with glutathione with ease and deplete GSH levels in cells. Because p53 is a redox-sensitive protein, we hypothesized that the redox imbalance induced by GTN may affect the structure of the R175H mutant p53 protein, and account for greater cytotoxicity. We also engineered the p53-null H1299 lung cancer cells to stably express the R175H mutant protein by lentiviral technology as an isogenic model. The conformation-specific antibodies for p53, namely Pab1620 that recognize the wt-p53 and pab420 that detects the mutant p53 were used for validating the p53 restoration. Immunoprecipitation and immunostaining using confocal microscopy showed that GTN treatment of R175H p53-containing cells results in a reciprocal loss of mutant protein and increase of wt-like protein. Further, the EMSA revealed a time-dependent restoration of DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes. The changes were associated with a G2/M arrest and significant apoptosis. Increased levels of apoptotic markers suggested a priming action of GTN on cell death pathways. GTN also suppressed the SKBR3 cell migration and invasion at 5-10 μM. In SKBR3 xenografts developed in nude mice, there was a marked tumor growth delay with either GTN alone or in combination with cisplatin. Our results shed light on the multiple mechanisms, including glutathione depletion, generation of redox imbalance, protein glutathionylation and p53 reactivation in the GTN cytotoxicity. We suggest that GTN-induced oxidative milieu facilitates a functional restoration of the mutant p53 through a thiolation of the redox-sensitive cysteines present in the DNA-binding domain. Our study will help to establish redox-perturbation as a paradigm for reactivation of the defective tumor suppressor (supported by a CPRIT grant [RP130266] to KSS). Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Debasish Basak, Kalkunte S. Srivenugopal. Selective killing of cancer cells by the styryl lactone (R)- goniothalamin is mediated through glutathione conjugation, oxidative stress and a marked reactivation of the R175H mutant p53 protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3694.

The orphan nuclear receptor NR4A2 is part of a p53-microRNA-34 network.

Nuclear receptor subfamily 4 group A member 2 (NR4A2) is an orphan nuclear receptor that is over-expressed in cancer and promotes cell proliferation, migration, transformation, and chemoresistance. Increased expression and function of NR4A2 have been attributed to various signaling pathways, but little is known about microRNA (miRNA) regulation of NR4A2 in cancer. To investigate the posttranscriptional regulation of NR4A2, we used a 3′ untranslated region (UTR) reporter screen and identified miR-34 as a putative regulator of NR4A2. By using computer predictions, we identified and confirmed an miRNA recognition element in the 3′ UTR of NR4A2 that was responsible for miR-34–mediated suppression. We next demonstrated that overexpression of exogenous miR-34 or activation of the p53 pathway, which regulates endogenous miR-34 expression, decreased NR4A2 expression. Consistent with previous reports, overexpression of NR4A2 blocked the induction of p53 target genes, including mir-34a. This was a phenotypic effect, as NR4A2 overexpression could rescue cells from p53-induced inhibition of proliferation. In summary, our results are the first characterization of a cancer-related miRNA capable of regulating NR4A2 and suggest a network and possible feedback mechanism involving p53, miR-34, and NR4A2.

Piperlongumine exerts cytotoxic effects against cancer cells with mutant p53 proteins at least in part by restoring the biological functions of the tumor suppressor

Piperlongumine (PL), a small molecule alkaloid present in black pepper (Piper longum), has been reported to kill tumor cells irrespective of their p53 gene status, however, the mechanisms involved are unknown. Since p53 is a redox-sensitive protein, we hypothesized that the redox imbalance induced by PL may affect the structure and/or function of the mutant p53 protein and promote cell death. We used two human colon cancer cell lines, the HT29 and SW620 which harbor the R273H DNA contact abrogatory mutation in p53. PL treatment induced significant ROS production and protein glutathionylation with a concomitant increase in Nrf-2 expression in both cell lines. Surprisingly, immunoprecipitation with wt-p53 specific antibodies (PAb1620) or direct western blotting showed a progressive generation of wild-type-like p53 protein along with a loss of its mutant counterpart in PL-treated HT29 and SW620 cells. Moreover, the EMSA and DNA-affinity blotting revealed a time-dependent restoration of DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes, MDM2 and Bax. PL, while cytotoxic by itself, also increased the cell killing by many anticancer drugs. In nude mice bearing the HT29 tumors, PL alone (7.5 mg/kg daily) produced a 40% decrease in tumor volume, which was accompanied by diminished intratumoral mutant p53 protein levels. The antitumor efficacy of BCNU or doxorubicin in HT29 xenografts was highly potentiated by PL, followed by expression of apoptotic proteins. These clinically-relevant findings suggest that PL-induced oxidative milieu facilitates a weak functional restoration of mutant p53 through protein glutathionylation and contributes to the increased drug sensitivity.

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.

A new genotoxicity assay based on p53 target gene induction

The p53 tumor suppressor protein has emerged as a universal sensor of genotoxic stress that regulates the transcription of numerous genes required for appropriate cellular response to DNA damage. Therefore, transcriptional induction of p53 target genes can be considered as a global and early indicator of genotoxic stress. By performing expression microarrays and RNA-Seq analysis on wild-type and mutant TP53 human lymphocytes respectively derived from controls and Li-Fraumeni patients and exposed to different classes of genotoxic agents, we first determined a common p53-dependent transcriptional signature of DNA damage. We then derived a simple and fast assay based on the exposure of wild-type TP53 lymphocytes to physical or chemical agents and on the quantitative measurement of selected p53 target gene transcriptional induction. The specificity of the p53 genotoxicity assay can easily be demonstrated by performing the same experiment in control lymphocytes with heterozygous TP53 mutations, which compromise responses to DNA damage. This assay allowed us to show that most of the drugs commonly used in cancer treatment, except the microtubule poisons, are highly genotoxic. The p53 genotoxicity assay should facilitate the measurement of the genotoxic effects of chemical and physical agents and the identification of drugs that are not genotoxic and do not expose patients to the risk of secondary malignancies, especially those with a constitutional defect in response to DNA damage, such as patients with Li-Fraumeni syndrome.

Constant rate of p53 tetramerization in response to DNA damage controls the p53 response.

The dynamics of the tumor suppressor protein p53 have been previously investigated in single cells using fluorescently tagged p53. Such approach reports on the total abundance of p53 but does not provide a measure for functional p53. We used fluorescent protein-fragment complementation assay (PCA) to quantify in single cells the dynamics of p53 tetramers, the functional units of p53. We found that while total p53 increases proportionally to the input strength, p53 tetramers are formed in cells at a constant rate. This breaks the linear input–output relation and dampens the p53 response. Disruption of the p53-binding protein ARC led to a dose-dependent rate of tetramers formation, resulting in enhanced tetramerization and induction of p53 target genes. Our work suggests that constraining the p53 response in face of variable inputs may protect cells from committing to terminal outcomes and highlights the importance of quantifying the active form of signaling molecules in single cells.Quantification of the dynamics of p53 tetramers in single cells using a fluorescent protein-fragment complementation assay reveals that, while total p53 increases proportionally to the DNA damage strength, p53 tetramers are formed at a constant rate.

The p53 Reactivating Drug PRIMA-1Met Induces Apoptosis Of Myeloma Cells Via ROS Production

Patients with multiple myeloma (MM) carrying a del(17)(p13) (del17p) deletion and/or TP53 mutation at diagnosis have a shortened survival whatever the treatment received. By using a large and characterized collection of human myeloma cell lines (HMCLs), we have shown that TP53mutated/deleted cell lines are more resistant to melphalan and nutlin3a (MDM2 inhibitor) than TP53wild-type HMCLs (Surget S, Cancer Res 2012;72:4562). The absence of a functional p53 pathway prevents the induction of p53 target genes, including pro-apoptotic genes, and therefore impairs apoptosis. In cancer cells, p53 mutations are frequent and highly expressed mutant p53 proteins exert a dominant effect. Pharmacological drugs such as PRIMA-1Met/APR246, which has been recently evaluated in a phase I/II study (Lehmann S J Clin Oncol 2012;30:3633) offers an interesting opportunity to redirect the activity of mutant misfolded p53 proteins. With this aim, the efficiency of PRIMA-1Met was assessed in 22 HMCLs and 16 primary MM samples characterized for del17p. HMCLs were heterogeneously sensitive to PRIMA-1Met, with an LD50 median value of 35 µM (range 4->100 µM). We failed to observe any correlation with HMCL's TP53 status since the LD50 median values of TP53wt, TP53mut and TP53del HMCLs were not significantly different (p=0.1, Kruskal Wallis test) at respectively 21 µM (range 4-70, n=7), 45 µM (21->100, n=13) and 21 µM (8-33, n=2). Primary MM samples were also heterogeneously sensitive, with a median cell death value induced by 10 µM PRIMA-1Met of 51% (n=16, range 5-100%). The 5 del17p+ samples showed no significant difference in their sensitivity to PRIMA-1Met from the 11 del17p- (p=0.31 Mann Whitney). Moreover, the sensitivity of 2 TP53wt and 2 TP53mut HMCLs was unchanged upon p53 silencing, and PRIMA-1Met failed to increase the expression of such p53 target genes as p21, Puma or DR5 (TRAIL-R2) in both TP53wt and TP53mut/del HMCLs. By contrast, nutlin3a induced cell death and increased DR5 expression in all TP53wt but in none of the TP53mut/del HMCLs (p<0.001, n=22) and both cell death and DR5 expression increase was prevented upon p53 silencing. In primary samples, nutlin3a increased DR5 expression in del17p- (n=9, median fold-increase 1.29) but not in del17p+ samples (n=4, median fold-increase 0.90, p=0.05) while PRIMA-1Met increased DR5 expression in neither del17p- nor del17p+ samples (0.90 versus 0.96, p=0.94). Despite a lack of activation of p53 target genes, PRIMA-1Met induced apoptosis as shown by caspase 3 and PARP cleavages. Interestingly, PRIMA-1Met induced a burst of ROS (shown by DHE staining in flow cytometry), involved in cell death since the ROS scavengers NAC (5 mM), N-acetyl-L-cysteine, as well as 5 mM GSH-MEE (gluthatione reduced ethyl ester, data not shown), strongly inhibited cell death in both HMCLs and primary cells with respective median inhibitions of 98% (n=8, p=0.01) and 93% (n=11, p<0.001 Wilcoxon matched-pairs signed rank test). Reciprocally, 0.5 mM BSO (L-buthionine-sulfoximine), an irreversible inhibitor of gamma-glutamyl cysteine synthetase, which depletes GSH in situ and in turn increases ROS, strongly synergized with 10 µM PRIMA-1Met and overcame resistance to apoptosis in 91% of HMCLs (n=22) and in 100% of primary samples (n=6). The median value of HMCL cell death was respectively 92% for 10µM PRIMA-1Met plus 0.5 mM BSO versus 2% for 10 µM PRIMA-1Met (n=22, p<0.001) and 4% for 0.5 mM BSO (n=22, p<0.001), and that of primary samples was 88% for PRIMA-1Met /BSO versus 22% for PRIMA-1Met (n=6, p=0.03) or 1% for BSO (n=6, p=0.03, Wilcoxon matched-pairs signed rank test). Notably, cell death induced by nutlin3a was not inhibited by NAC nor did BSO synergize with nutlin3a, confirming that nutlin3a and PRIMA-1Met have distinct death inducing pathways. Interestingly, the sensitivity of HMCLs to PRIMA-1Met alone correlated to the expression of an enzyme involved in oxidative stress (p=0.04, Spearman test), the participation of which is currently under investigation. Finally, the 10 µM PRIMA-1Met plus 0.5 mM BSO combination was found to be effective in only 2 of 6 mantle cell lymphoma cell lines, suggesting that it could be cell-type specific. These data show that PRIMA-1Met in combination with BSO has a strong pro-apoptotic activity in MM, independently of the p53 pathway, and thus could be of interest for del17p+ patients refractory to current therapies. Disclosures:No relevant conflicts of interest to declare.

Corrigendum to “Chromium-mediated apoptosis: involvement of DNA-dependent protein kinase (DNA-PK) and differential induction of p53 target genes” [DNA Repair 7 (9) (2008) 1484–1499

Corrigendum to “Chromium-mediated apoptosis: involvement of DNA-dependent protein kinase (DNA-PK) and differential induction of p53 target genes” [DNA Repair 7 (9) (2008) 1484–1499

Long-Term Effects of Ionizing Radiation on Gene Expression in a Zebrafish Model

Understanding how initial radiation injury translates into long-term effects is an important problem in radiation biology. Here, we define a set of changes in the transcription profile that are associated with the long-term response to radiation exposure. The study was performed in vivo using zebrafish, an established radiobiological model organism. To study the long-term response, 24 hour post-fertilization embryos were exposed to 0.1 Gy (low dose) or 1.0 Gy (moderate dose) of whole-body gamma radiation and allowed to develop for 16 weeks. Liver mRNA profiles were then analyzed using the Affymetrix microarray platform, with validation by quantitative PCR. As a basis for comparison, 16-week old adults were exposed at the same doses and analyzed after 4 hours. Statistical analysis was performed in a way to minimize the effects of multiple comparisons. The responses to these two treatment regimes differed greatly: 360 probe sets were associated primarily with the long-term response, whereas a different 2062 probe sets were associated primarily with the response when adults of the same age were irradiated 4 hours before exposure. Surprisingly, a ten-fold difference in radiation dose (0.1 versus 1.0 Gy) had little effect. Analysis at the gene and pathway level indicated that the long-term response includes the induction of cytokine and inflammatory regulators and transcription and growth factors. The acute response includes the induction of p53 target genes and modulation of the hypoxia-induced transcription factor-C/EBP axis. Results help define genes and pathways affected in the long-term, low and moderate dose radiation response and differentiate them from those affected in an acute response in the same tissue.

NuMA Is Required for the Selective Induction of p53 Target Genes

The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription.

Abstract 525: Induction of apoptosis in ovarian clear cell carcinomas with wild-type TP53 by inhibiting PI3K/mTOR signaling pathway.

Abstract Background:Epithelial ovarian cancer is a leading cause of death from gynecological malignancies.Ovarian clear cell adenocarcinoma (OCCA) is the second most common cause of death from ovarian cancer with a higher incidence in Asia, especially Japan (&amp;gt;25%). Due to low response rates to conventional platinum-based chemotherapy, the clinical outcome is generally poor. PIK3CA mutations are frequent (&amp;gt;40%) in OCCA compared with other histology types and overexpression of receptor tyrosine kinases (RTKs), including HER2 and cMET, is also frequently observed in OCCA. Thus, the RTK-PI3K (phosphatidylinositol 3-kinase)/mTOR (mammalian target of rapamycin) signaling axis is broadly activated in OCCA. This study evaluated the anti-tumor efficacy of DS-7423, a novel PI3K/mTOR dual inhibitor, in a panel of OCCA cell lines. Material and methods: We evaluated the phosphorylation status of RTK-PI3K/mTOR signaling pathway in 9 OCCA cell lines. Four of those cell lines possessed PIK3CA mutations; 3 of the 5 remaining cell lines overexpressed HER2, HER3 and/or cMET. We determined the effect of DS-7423 by MTT assay, FACS analysis, and Western blotting. Apoptosis induction was detected by annexin-V and propidium iodide (PI) staining. Induction of p53 target genes, including p53AIP1, was evaluated by RT-PCR. Results: IC50 values for DS-7423 were &amp;lt;100 nM for all 9 OCCA cell lines. Efficacy did not significantly differ between PIK3CA mutant and wild type OCCA cells. AKT and its downstream targets (GSK3β, FOXO1/3a, MDM2, 4EBP1, and S6) were phosphorylated in OCCA cell lines regardless of PIK3CA mutational status. DS-7423 suppressed the phosphorylation levels of AKT and its target proteins in all these OCCA cell lines. FACS analysis revealed an increase in sub-G1 cell populations by treatment at ≥156 nM DS-7423 in 6 out of 9 OCCA cell lines. In these 6 cell lines, the number of apoptotic cells, as determined by annexin-V and PI staining, increased in a dose-dependent manner and DS-7423 induced apoptosis more effectively in TP53 wild type OCCA cell lines than in TP53 mutant OCCA cell lines. In accordance with decreased phospho-MDM2 levels, phosphorylation levels of p53 (Ser46) were increased and expression of p53AIP1 and PUMA, genes regulating p53-dependent apoptosis, were induced by DS-7423 in these p53 wild type cells. We also evaluated the expression status of bcl-2 family proteins in the OCCA cell line OVISE, and observed that DS-7423 treatment induced up-regulation of proapoptotic Bim and down-regulation of anti-apoptotic Mcl-1. Conclusions: Dual PI3K/mTOR inhibitors such as DS-7423 may constitute a promising molecular targeted therapy for OCCA, regardless of PIK3CA status. DS-7423 may induce apoptosis via activation of p53 and bcl-2 family members. As p53 mutations ar generally rare in OCCA, DS-7423 might induce p53-dependent apoptosis in this cancer type. Citation Format: Tomoko Kashiyama, Katsutoshi Oda, Yuji Ikeda, Yoshinobu Shiose, Yasuhide Hirora, Aki Miyasaka, Takahiro Koso, Kanako Inaba, Tomohiko Fukuda, Keiko Shoji, Michihiro Tanikawa, Kazunori Nagasaka, Osamu Hiraike-Wada, Kei Kawana, Hiroyuki Aburatani, Shunsuke Nakagawa, Tomoyuki Fujii, Tetsu Yano, Shiro Kozuma. Induction of apoptosis in ovarian clear cell carcinomas with wild-type TP53 by inhibiting PI3K/mTOR signaling pathway. [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 525. doi:10.1158/1538-7445.AM2013-525

Abstract 2171: A new small molecule compound restoring p53 pathway induces cell death via active p73 and degradation of mutant p53 in colorectal cancer cells.

Abstract Tumor suppressor p53 is critical for protection against cancer. Over 50% of human cancers harbor mutant p53 which is hyper-stabilized and blocks p53 family member p73/p63 activity. Mutations not only abrogate the p53 tumor suppressor function, but also endow mutant p53 with gain-of-function (GOF) as pro-oncogene which contributes to tumor progression and chemotherapy resistance. Targeting mutant p53 to restore the p53 pathway is a promising strategy for cancer therapy. Small molecule restoring p53 pathway is mostly based on conformational change in mutant p53 or upregulation of wild-type p53. It is barely reported about small molecule compound with ability to both restore p53 pathway and deplete mutant p53 GOF. In this study, we describe a new small molecular compound, NCI-8 that specifically depletes mutant p53 protein and activates p73 to induce p53 responsive transcriptional activity and cell death in mutant p53 colorectal cancer cells. NCI-8 treatment increased p53 responsive bioluminescence and p53 target gene expression such as P21, Puma and DR5 specifically in mutant p53 colorectal cancer cells. Accompanied with p53 pathway restoration, NCI-8 down-regulated mutant p53 in cancer cells with no evident wild-type conformational change in mutant p53. NCI-8- mediated down-regulation of mutant p53 was rescued by MG132, a proteasome inhibitor and nutlin-3, an MDM2 inhibitor. NCI-8 induced ubiquitination of mutant p53. Taken together these results indicate that NCI-8 induces mutant p53 protein degradation via an MDM2-mediated ubiquitin-proteasome pathway. Further, we examined the role of p73 in NCI-8 action to p53 pathway restoration. NCI-8 enhanced p53-responsive bioluminescence with10-fold increase in cells overexpressing p73, but only 2-fold increase in p53-overexpressing cells. NCI-8- induced p53-responsive bioluminescence was significantly reduced by knock-down of p73, but not by knock-down of mutant p53. Knock-down of p73 significantly reduced NCI-8 induction of p53 target genes in mutant p53 cancer cells. These results indicate that p73 is required for NCI-8 to restore the p53 pathway in mutant p53-expressing cancer cells. Importantly, NCI-8 induced cell death in cancer cells, but not in normal cells. No genotoxicity of NCI-8 was detected in cancer and normal cells. Knock-down of p73 reduced cell apoptosis in mutant p53-expressing cancer cells treated with NCI-8, while, overexpression of p73 sensitized these cancer cells to NCI-8 induced cell death. Combination treatments demonstrate that NCI-8 synergizes with CPT11chemotherapy to induce cell death in mutant p53 colorectal cancer cells. These results suggest that p53 pathway restoration and cell death induced by NCI-8 occur via activation of p73 and depletion of mutant p53 GOF. NCI-8 is a promising lead compound specifically targeting mutant p53 for the development of new anti-cancer drugs. Citation Format: Shengliang Zhang, Lanlan Zhou, Bo Hong, Noel Warfel, Antonius Van den heuvel, David Dicker, Levy Kopelovich, Wafik El-Deiry. A new small molecule compound restoring p53 pathway induces cell death via active p73 and degradation of mutant p53 in colorectal 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 2171. doi:10.1158/1538-7445.AM2013-2171

Abstract 1130: Antitumor activity of a pyrrole-imidazole polyamide in three tumor xenograft models.

Abstract Background: Pyrrole-imidazole polyamides are synthetic minor groove binders with modular sequence recognition. We developed a polyamide that targets the DNA sequence 5’-WGWWCW-3’ (W=A/T). This molecule is cytotoxic in a number of cancer cell lines including LNCaP (prostate cancer), DU145 (prostate cancer), and A549 (lung cancer). Data in cell culture suggests that this molecule interferes with RNA polymerase 2 activity resulting in proteasome-mediated degradation of the RNA Polymerase 2 large subunit, activation of p53 and induction of p53 target genes, and apoptosis. This polyamide demonstrates no detectable DNA damage by alkaline comet assay nor does it induce detectable phosphorylation of γH2A.X, ATM, DNA-PKcs, p53, or Chk2. This polyamide circulates in mice after intravenous and subcutaneous injection in saline vehicle. Methods: Male immune compromised mice were implanted with LNCaP, DU145, or A549 tumors. Tumors were allowed to grow until approximately 100 mm3 and then stratified into vehicle (5% DMSO/PBS) and treatment arms (1 mg/kg polyamide, in vehicle). Treatment was administered via subcutaneous injection every three days, for a total of three cycles (LNCaP), eight cycles (DU145), or six cycles (A549). At the experimental endpoint, tumors were resected and weighed immediately. For LNCaP xenografts, serum PSA pre- and post-treatment were also measured. In parallel, a group of immune compromised mice without xenografts were treated with an identical regimen for eight cycles and subsequently observed for three months. Results: Tumors resected from polyamide-treated mice were statistically smaller than those of vehicle-treated mice. The effect was greatest on LNCaP tumors (n=12, Treated (T)/Control (C): 32%, p=1.6E-5). DU145 tumors (n=6, T/C: 34%, p=0.013), and A549 tumors (n=8, T/C: 62%, p=0.047) were also affected. Post-treatment serum PSA in the polyamide-treated mice with LNCaP xenografts were lower than for the vehicle treated mice (p = 0.024). Statistical differences between treated and control groups were determined using two-tailed t-tests. Polyamide treatment resulted in no significant weight loss or observable distress in tumor-free mice and these mice appeared healthy for at least three months after treatment was complete. Conclusions: The polyamide demonstrated in vivo antitumor activity in this panel of tumor xenografts with limited host toxicity. Citation Format: Nicholas G. Nickols, Fei Yang, Benjamin C. Li, Georgi K. Marinov, Jonathan W. Said, Peter B. Dervan. Antitumor activity of a pyrrole-imidazole polyamide in three tumor xenograft models. [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 1130. doi:10.1158/1538-7445.AM2013-1130

P53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G(1) phase, although recent studies have identified a novel ER stress G(2) checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G(2) delay involving CHK1 and a later induction of G(1) arrest associated both with the induction of p53 target genes and loss of cyclin D(1). We show that substitution of p53/47 for p53 impairs the ER stress G(1) checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G(2) progression prior to ultimate G(1) arrest.

Runt-related Transcription Factor 1 (RUNX1) Stimulates Tumor Suppressor p53 Protein in Response to DNA Damage through Complex Formation and Acetylation

Representative tumor suppressor p53 plays a critical role in the regulation of proper DNA damage response. In this study, we have found for the first time that Runt-related transcription factor 1 (RUNX1) contributes to p53-dependent DNA damage response. Upon adriamycin (ADR) exposure, p53 as well as RUNX1 were strongly induced in p53-proficient HCT116 and U2OS cells, which were closely associated with significant transactivation of p53 target genes, such as p21(WAF)(1), BAX, NOXA, and PUMA. RUNX1 was exclusively expressed in the cell nucleus and formed a complex with p53 in response to ADR. Chromatin immunoprecipitation assay demonstrated that p53 together with RUNX1 are efficiently recruited onto p53 target gene promoters following ADR exposure, indicating that RUNX1 is involved in p53-mediated transcriptional regulation. Indeed, forced expression of RUNX1 stimulated the transcriptional activity of p53 in response to ADR. Consistent with these observations, knockdown of RUNX1 attenuated ADR-mediated induction of p53 target genes and suppressed ADR-dependent apoptosis. Furthermore, RUNX1 was associated with p300 histone acetyltransferase, and ADR-dependent acetylation of p53 at Lys-373/382 was markedly inhibited in RUNX1 knockdown cells. In addition, knockdown of RUNX1 resulted in a significant decrease in the amount of p53-p300 complex following ADR exposure. Taken together, our present results strongly suggest that RUNX1 is required for the stimulation of p53 in response to DNA damage and also provide novel insight into understanding the molecular mechanisms behind p53-dependent DNA damage response.