P53 Response Research Articles

Enhancement of anticancer activity by silibinin and paclitaxel combination on the ovarian cancer

Background: Ovarian carcinoma is the most lethal cancer among all gynaecological malignancies. One of the most chemotherapy drugs used for ovarian cancer is paclitaxel which induces apoptosis. Paclitaxel has been used for many years. Similar to the most cancers this responds to chemotherapy initially but in a long run, drug resistance happens which fails the treatment procedure. Combination of chemotherapy drugs has been suggested to deal with this issue. Silibinin, a plant extraction, has been used from ancient time in traditional medicine and identified to have powerful antioxidant activity.Aim: The aim of this study was to examine the effect of paclitaxel and silibinin combination on SKOV-3 cancer cell line.Materials and methods: The human epithelial ovarian cancer cell line, SKOV-3, was cultured and treated with paclitaxel, silibinin and paclitaxel plus silibinin for 48 hours. MTT assay was carried out to determine cell viability. For apoptotic process, we used real-time PCR to study P53 and P21 genes expression after drug treatment and network analysis was performed using Pathway Studio web tool (Elsevier).Results: Cell growth was inhibited considerably (p < .05) by combination of paclitaxel and silibinin after 48 hours of treatment. Also silibinin and paclitaxel combination induced apoptosis in SKOV-3 cells. Expression analysis by real-time PCR showed the significant up-regulation of two tumour suppressor genes, P53 and P21 in response to combination of silibinin and paclitaxel. In addition, computational network analysis demonstrated the crosstalk between paclitaxel, silibinin and ovarian cancer.Conclusions: Our results showed that combination of chemotherapy drugs of silibinin and paclitaxel can be more efficient in treatment of ovarian cancer cells.

Functional consequences of inducible genetic elements from the p53 SOS response in a mammalian organ system

In response to DNA damage from ultraviolet (UV) radiation, bacteria deploy the SOS response in order to limit cell death. This bacterial SOS response is characterized by an increase in the recA gene that transactivates expression of multiple DNA repair genes. The current series of experiments demonstrate that a mammalian organ system (the cochlea) that is not evolutionarily conditioned to UV radiation can elicit SOS responses that are reminiscent of that of bacteria. This mammalian SOS response is characterized by an increase in the p53 gene with activation of multiple DNA repair genes that harbor p53 response elements in their promoters. Furthermore, the experimental results provide support for the notion of a convergent trigger paradox, where independent SOS triggers facilitate disparate physiologic sequelae (loss vs. recovery of function). Therefore, it is proposed that the mammalian SOS response is multifunctional and manipulation of this endogenous response could be exploited in future biomedical interventions.

ZNF509S1 downregulates PUMA by inhibiting p53K382 acetylation and p53-DNA binding

Expression of the POK family protein ZNF509L, and -its S1 isoform, is induced by p53 upon exposure to genotoxic stress. Due to alternative splicing of the ZNF509 primary transcript, ZNF509S1 lacks the 6 zinc-fingers and C-terminus of ZNF509L, resulting in only one zinc-finger. ZNF509L and -S1 inhibit cell proliferation by activating p21/CDKN1A and RB transcription, respectively. When cells are exposed to severe DNA damage, p53 activates PUMA (p53-upregulated modulator of apoptosis) transcription. Interestingly, apoptosis due to transcriptional activation of PUMA by p53 is attenuated by ZNF509S1. Thus we investigated the molecular mechanism(s) underlying the transcriptional attenuation and anti-apoptotic effects of ZNF509S1. We show that ZNF509S1 modulation of p53 activity is important in PUMA gene transcription by modulating post-translational modification of p53 by p300. ZNF509S1 directly interacts with p53 and inhibits p300-mediated acetylation of p53 lysine K382, with deacetylation of p53 K382 leading to decreased DNA binding at the p53 response element 1 of the PUMA promoter. ZNF509S1 may play a role not only in cell cycle arrest, by activating RB expression, but also in rescuing cells from apoptotic death by repressing PUMA expression in cells exposed to severe DNA damage.

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.

Assessment of the DNA damaging potential of environmental chemicals using a quantitative high-throughput screening approach to measure p53 activation.

Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53-signaling pathway using a quantitative high-throughput screening assay employing HCT-116 cells (p53+/+ ) containing a stably integrated β-lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (-S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 μM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in-depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct-acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494-507, 2017. © 2017 Wiley Periodicals, Inc.

P53 Dynamically Directs TFIID Assembly on Target Gene Promoters.

p53 is a central regulator that turns on vast gene networks to maintain cellular integrity in the presence of various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key issue, we have undertaken an integrated approach involving single-molecule fluorescence microscopy, single-particle cryo-electron microscopy, and biochemistry. Our real-time single-molecule imaging data demonstrate that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID's ability to bind DNA by stabilizing TFIID contacts with both the core promoter and a region within p53's response element. Analysis of single-molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID's conversion to a rearranged DNA binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID's interaction with DNA induces p53 to rapidly dissociate, which likely leads to additional rounds of p53-mediated recruitment of other basal factors. Collectively, these findings indicate that p53 dynamically escorts and loads TFIID onto its target promoters.

Abstract CT012: Novel effect of tamoxifen therapy: disruption of ER-p53 interaction leading to altered gene expression profile in human breast tumors

Abstract Introduction: We have reported that ERα binds to wild type (wt) p53 and inactivates its tumor suppressor activity. This interaction is augmented by estradiol and disrupted by tamoxifen (TAM). Moreover, retrospective clinical studies have shown that ER+ breast cancer (BC) expressing wt p53 is more responsive to TAM therapy compared to those expressing mutant p53. We conducted a window of opportunity trial in women with ER+ BC expressing wt p53 to test our hypothesis that TAM relieves the functional suppression of p53 by ERα. Methods: Women with ER+ invasive BC were randomized to 20mg TAM daily for 4 weeks prior to surgery or standard of care (SOC.) Wt p53 status in tumors was confirmed by massively parallel sequencing. Only ER+ wt p53 tumors were analyzed. ERα-p53 interaction was determined by Proximity Ligation Assay (PLA). Global transcriptome analysis in surgically resected tumors was conducted by RNA-seq. The multiplexed libraries were sequenced on HiSeq 2500 using 100 cycle paired end sequencing and an average 50 million paired end reads were generated for each sample. Immunohistochemistry was performed on FFPE tissue from diagnostic biopsies and surgically resected tumors to compare expression of ERα and p53 selected downstream targets.17β-estradiol and TAM metabolites were measured in the plasma, tumor and surrounding normal tissue using LC-MS/MS. Steady-state levels of TAM were confirmed. Polymorphisms affecting TAM metabolism enzymes were determined by genotyping. Findings: 53 women completed the study and 36 had adequate tissue for analysis (16 TAM and 20 SOC). PLA demonstrated that ERα-p53 interaction was disrupted in 75% of TAM treated patients. Differential gene expression (DEG) analysis using DESEq2 R package followed by GSEA pathway analysis showed that 300 genes were upregulated and 272 genes were downregulated (p&amp;lt;0.05) in response to TAM therapy. DEG included those involved in the regulation of cell cycle, apoptosis, immune response, energy metabolism, growth factors and receptors, and membrane transporters. Importantly, several p53-target genes responded strongly to TAM therapy (repression targets of p53 were downregulated and activation targets were upregulated) while pro-proliferative ERα targets were downregulated. Detailed results and analysis will be provided at the presentation. Conclusions: In human BC, we have shown that in addition to its conventional effects, TAM disrupts the ERα-p53 interaction leading to functional reactivation of p53. Our data 1) supports the role of ERα-p53 crosstalk in endocrine resistance of ER+ tumors, 2) provides insight into the mechanism underlying favorable response of wt p53 to TAM therapy, and 3) has implications toward stratifying ER+ BC patients to those who will or will not be responsive to TAM therapy. Citation Format: Swati A. Kulkarni, Chetan C. Oturkar, Stephen B. Edge, Jianmin Wang, John H. Walton, Wendy M. Swetzig, Araba Adjei, Robert Bies, Alan D. Hutson, Adrienne Groman, Morrison D. Carl, Jerry Fetterly, Schika Kumar, Gokul M. Das. Novel effect of tamoxifen therapy: disruption of ER-p53 interaction leading to altered gene expression profile in human breast tumors [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 CT012. doi:10.1158/1538-7445.AM2017-CT012

Abstract 119: Vitamin K2 targets castration-resistant prostate cancer VCaP cells by reactive oxygen species mediated apoptotic cell death

Abstract Prostate Cancer (PCa) is the second most common cancer in western countries especially in US population, in which castration-resistant prostate cancer (CRPC) is the major cause for patient mortality. Current treatment options available for CRPC are not efficient and have undesirable side effects. Hence there is an urgent need to develop non-toxic and effective treatment strategies for CRPC. Vitamin K2 (VK2), a natural menaquinone has several medicinal values including anti-cancer activity and anti-osteoporosis effect. The aim of this study is to evaluate the therapeutic effects of Vitamin K2 (VK2) and its anti-cancer mechanism against CRPC. In this study, we have used VCaP cell line (ATCC) which is established from a patient with hormone refractory prostate cancer. VCaP cells were treated with various concentrations of VK2 to evaluate its effects on cell viability by MTT assay, anchorage independent growth by soft agar assay, cellular senescence by beta-galactosidase staining assay and cancer cell migration by wound healing assay. We have also assessed the VK2-induced production of intracellular reactive oxygen species (ROS) using DCF (2′,7′-dichlorofluorescein) probe based fluorescence assay. VK2 induced apoptosis was detected by Annexin-V FITC and TUNEL assays. Western blot analysis was utilized to uncover the anti-proliferative and anti-metastatic mechanisms of VK2 against CRPC. Our results showed that VK2 significantly inhibits the proliferation of VCaP cells in a dose dependent manner at 48 hrs treatment in vitro. MTT data also showed that anti-proliferative effects of VK2 were significantly abrogated in the presence of anti-oxidant N-acetyl cysteine (NAC) and caspase inhibitor Z-VAD-FMK suggesting that ROS and caspase activation as the underlying anti-cancer mechanisms of VK2 in CRPC cells. In addition, VK2 reduced the migration potential of VCaP cells in wound healing assay and inhibited anchorage independent growth of these cells when compared to untreated cells. Annexin-V and TUNEL assays confirmed that VK2 induces apoptosis in VCaP cells. Our results also suggested that the VK2 has the ability to enforce growth arrest in CRPC cells by activating cellular senescence. Western blot analysis revealed that VK2 downregulated the expression of BiP, survivin, MMP-2, and PCNA while activating PARP-1, p21 and DNA damage response marker, phospho-H2AX in VCaP cells. Furthermore, VCaP cells treated with VK2 resulted in the activation of Caspase-3 and-7 apoptotic mediators. These results correlated with translocation of Bax and Cytochrome C to cytoplasm following VK2 treatment in VCaP cells as determined by confocal immunofluorescence analysis. In conclusion our study suggests that VK2 might be an effective anti-proliferative and anti-metastatic agent for CRPC by specifically targeting key anti-apoptotic, cell cycle progression and metastasis promoting signaling molecules. Citation Format: Subramanyam Dasari, Maarten C. Bosland, Andre Kajdacsy- Balla, Gnanasekar Munirathinam. Vitamin K2 targets castration-resistant prostate cancer VCaP cells by reactive oxygen species mediated apoptotic cell death [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 119. doi:10.1158/1538-7445.AM2017-119

Abstract LB-169: Targeted tumor prevention in Li-Fraumeni syndrome

Abstract Li-Fraumeni syndrome (LFS) is a rare, dominant cancer predisposition disorder. The majority of LFS cases are brought about by germline p53 mutations. Individuals affected develop a spectrum of cancers occurring since childhood or early adulthood (Mai et al, 2012). Tumors associated with LFS include but are not limited to sarcomas, lymphomas and leukemias, breast cancers, brain tumors, etc (Olivier et al, 2010; Mai et al, 2012). p53 is a tumor suppressor gene that regulates many cellular processes and prevents cancer formation, growth and dissemination. When inactivated by mutation, it not only loses its tumor suppressive function, but also acquires oncogenic properties. Mutations of p53 promote destabilization of its three-dimensional structure, which can cause p53 to partially unfold, aggregate and accumulate in cells (Xu et al, 2011). We developed a peptide, ReACp53, to arrest p53 clumping and accumulation, chaperone p53 to a functional state and promote its degradation, hence arresting tumor proliferation (Soragni et al, 2016). ReACp53 can also target p63 and p73 (Zheng et al, 2016). It has shown good tolerability and a favorable safety profile in vivo. In LFS patients and LFS animal models, p53 is undetectable in normal tissues. However, the protein starts to accumulate in tumors and pre-cancerous lesions (Lang et al, 2004; Soragni et al, unpublished data). As p53 accumulation is correlated with aggregation (Xu et al, 2011; Soragni et al, 2016), we postulated that, by periodically administering ReACp53, we could target this initial event early on, and prevent p53 accumulation and/or push the p53 aggregates-ridden cells into apoptosis (Soragni et al, 2016). Here, we tested ReACp53 as a tumor preventive agent, by administering it twice a week from weaning to LFS mice carrying the aggregation-prone R172H mutation (R175H in humans). ReACp53 delayed cancer onset resulting in a significant increase in overall survival (OS). The homozygous cohort (n=16/group) showed a 50% increase in median survival in ReACp53-treated mice (median OS 15.79 vs 23.93 weeks for vehicle vs ReACp53, p=0.005; HR=2.6). Heterozygous mice (n=8/group) showed a 36% increase in OS with a median of 58.86 vs 80.50 weeks for vehicle vs ReACp53 (p=0.03, HR=2.6). No long-term side effect was observed upon ReACp53 administration. The tumor spectrum reported differed between the two groups. Activation of a functional p53 response in mouse cells and LFS patient cells treated with ReACp53 is undergoing. Overall, our study supports ReACp53 therapy as a potential approach to tumor prevention in LFS. Citation Format: Alice Soragni, Nhan Phan, David Eisenberg. Targeted tumor prevention in Li-Fraumeni syndrome [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 LB-169. doi:10.1158/1538-7445.AM2017-LB-169

Abstract 5508: Histone modifications affect differential regulation of TGF-beta-induced Nox4 by p53

Abstract Recent studies suggest p53 plays an important role in TGF-β-mediated cell signaling and migration. Previously, we showed wild-type (WT) and mutant (mt) forms of p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (NOX4). We found that WT-p53 suppresses TGF-β-induced NOX4, ROS production, and cell migration, whereas tumor-associated p53-mt proteins enhance NOX4 expression and cell migration by TGF-β/SMAD3-dependent mechanisms. In this study, we utilized The Cancer Genome Atlas (TCGA) to perform statistical analysis on gene expression data from primary tumor samples and found a correlation between tumors with p53 “hotspot” mutations and increased NOX4 mRNA expression. Furthermore, we examined the basis of human NOX4 promoter regulation by p53 and SMAD3. By deletion analysis of the NOX4 promoter, we found two critical SMAD3 binding elements (SBE) are also required for p53-dependent promoter activity. Conversely, promoter activity was abolished by dose-dependent heterologous expression of p53-WT. Moreover, expression of active SMAD3 resulted in robust NOX4 promoter activity, which was abolished when co-expressed with p53-WT. Chromatin immunoprecipitation (ChIP) analysis revealed SMAD3 and p53-WT or p53-mt were associated with specific SBEs and p53 response elements (p53-RE) in a TGF-β-dependent manner. Interestingly, the repressive effect by p53- WT on NOX4 was relieved upon treatment with histone deacetylase (HDAC) inhibitors or mutation of the transactivation domain. Overexpression of p300, a known p53-mt-binding transcriptional co-regulator and histone acetyltranserfase (HAT), enhanced p53-mt-mediated NOX4 promoter activity, whereas the HAT-inactive p300-mt reduced promoter activity. Furthermore, overexpression of p53-mt augmented TGF-β-mediated acetylation of histones associated with the NOX4 promoter. Finally, scratch wound assays demonstrated NOX4 and p300 promote TGF-β/mutant p53-mediated cell migration. Collectively, these data and provide new insight on TGF-β/SMAD3 and mutant p53-mediated NOX4 induction involving epigenetic control of NOX4 in tumor cell migration. Citation Format: Howard E. Boudreau, Wei Feng MA, Agnieszka Korzeniowska, Jonathan J. Park, Thomas L. Leto. Histone modifications affect differential regulation of TGF-beta-induced Nox4 by p53 [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 5508. doi:10.1158/1538-7445.AM2017-5508

A Kinase-Independent Role for Cyclin-Dependent Kinase 19 in p53 Response.

ABSTRACTThe human Mediator complex regulates RNA polymerase II transcription genome-wide. A general factor that regulates Mediator function is the four-subunit kinase module, which contains either cyclin-dependent kinase 8 (CDK8) or CDK19. Whereas CDK8 is linked to specific signaling cascades and oncogenesis, the cellular roles of its paralog, CDK19, are poorly studied. We discovered that osteosarcoma cells (SJSA) are naturally depleted of CDK8 protein. Whereas stable CDK19 knockdown was tolerated in SJSA cells, proliferation was reduced. Notably, proliferation defects were rescued upon the reexpression of wild-type or kinase-dead CDK19. Comparative RNA sequencing analyses showed reduced expression of mitotic genes and activation of genes associated with cholesterol metabolism and the p53 pathway in CDK19 knockdown cells. SJSA cells treated with 5-fluorouracil, which induces metabolic and genotoxic stress and activates p53, further implicated CDK19 in p53 target gene expression. To better probe the p53 response, SJSA cells (shCDK19 versus shCTRL) were treated with the p53 activator nutlin-3. Remarkably, CDK19 was required for SJSA cells to return to a proliferative state after nutlin-3 treatment, and this effect was kinase independent. These results implicate CDK19 as a regulator of p53 stress responses and suggest a role for CDK19 in cellular resistance to nutlin-3.

Human cancer-associated fibroblasts enhance glutathione levels and antagonize drug-induced prostate cancer cell death

Drug resistance is a major problem in cancer therapy. A growing body of evidence demonstrates that the tumor microenvironment, including cancer-associated fibroblasts (CAFs), can modulate drug sensitivity in tumor cells. We examined the effect of primary human CAFs on p53 induction and cell viability in prostate cancer cells on treatment with chemotherapeutic drugs. Co-culture with prostate CAFs or CAF-conditioned medium attenuated DNA damage and the p53 response to chemotherapeutic drugs and enhanced prostate cancer cell survival. CAF-conditioned medium inhibited the accumulation of doxorubicin, but not taxol, in prostate cancer cells in a manner that was associated with increased cancer cell glutathione levels. A low molecular weight fraction (<3 kDa) of CAF-conditioned medium had the same effect. CAF-conditioned medium also inhibited induction of reactive oxygen species (ROS) in both doxorubicin- and taxol-treated cancer cells. Our findings suggest that CAFs can enhance drug resistance in cancer cells by inhibiting drug accumulation and counteracting drug-induced oxidative stress. This protective mechanism may represent a novel therapeutic target in cancer.

Abstract NTOC-108: ACTIVITY OF DIANHYDROGALACTITOL (VAL–083) IN OVARIAN TUMOR MODELS, SENSITIVE OR RESISTANT TO CISPLATIN

Abstract PURPOSE: Platinum drug resistance is a major clinical impediment that is normally ascribed to several mechanisms, with dysfunctional p53 playing a critical role, particularly in the highly lethal high-grade ovarian serous carcinoma, where p53 mutations can be &amp;gt;80%. Since restoration of p53 function could restore drug-dependent cytotoxicity, the goal of this study was to examine dianhydrogalactitol (VAL-083) for its potential in an ovarian tumor panel representing different p53 statuses and cisplatin-resistance. VAL-083 has demonstrated clinical activity against a range of tumor types, including ovarian cancer, in historical NCI-sponsored clinical studies, but its activity in a cisplatin-resistance setting is not known. VAL-083 is a bi-functional alkylating agent with a distinct mechanism of action, forming DNA interstrand cross links at the N7 position of guanine, whereas cisplatin and carboplatin predominantly form intrastrand DNA cross-links. METHODS: The tumor panel was composed of cisplatin-sensitive wild type (wt) p53 ovarian cancer cell line A2780, cisplatin-resistant A2780-derived heterozygous p53-V172F mutant 2780CP/Cl-16 cells, and three other cisplatin-resistant models harboring mutant (OVCAR-10) or wt p53 (Hey and OVCA-433). Cells were exposed to cisplatin or VAL-083 and the IC50 cytotoxic parameter was determined by fitting the 5-day MTT cell survival data to a 4-parameter sigmoidal curve. MTT assay was used in conjunction with Bliss Independence Model and combination index approaches to assess VAL-083/cisplatin combinations. Immunoblot analysis assessed p53 response. RESULTS: The IC50 of cisplatin in the cisplatin-sensitive A2780 model was 0.2-0.3 µM. In contrast, the IC50 in the four cisplatin-resistant models (2780CP/Cl-16, OVCAR-10, Hey and OVCA-433) was 3-8 µM; a 10- to 27-fold increase. The corresponding IC50 for VAL-083 was about 0.5 µM in A2780 cells and 2-4 µM in the four cisplatin-resistant models; a 4- to 7-fold increase, suggesting a distinct mode of action of VAL-083 and the ability to circumvent cisplatin resistance. In addition, these results further indicate that VAL-083 activity is less dependent on p53 status. However, immunoblots of 2780CP/Cl-16 cells suggest partial dependency on p53 by demonstrating that VAL-083, unlike cisplatin, can activate mutant p53-V172F through Ser20 phosphorylation, thereby preventing binding to p53 of inhibitory MDM4. VAL-083 thus restores p53 function, which is consistent with its greater potency in p53 mutated cisplatin-resistant ovarian cancer. These results suggest that VAL-083 have two mechanisms: one dependent on p53 and another on a different pathway. The apparent differences in the mechanism between cisplatin and VAL-083 prompted a combination study with the two agents, and consistent synergy was demonstrated. CONCLUSIONS: VAL-083 can circumvent cisplatin-resistance in ovarian tumor models, and its activity is less dependent on p53 status. Together with overt synergy between VAL-083 and cisplatin, our results demonstrate the effectiveness of VAL-083 against cisplatin-resistant ovarian cancer as a single agent or in combination with cisplatin. Citation Format: Anne Steino, Michelle Martinez-Rivera, Guanghan He, Xiaolei Xie, Jeffrey A. Bacha, Dennis M. Brown and Zahid H. Siddik. ACTIVITY OF DIANHYDROGALACTITOL (VAL–083) IN OVARIAN TUMOR MODELS, SENSITIVE OR RESISTANT TO CISPLATIN [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-108.

Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity.

MDM2-MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2-MDMX-E2(UbcH5B)-ubiquitin complex, we designed MDM2 mutants that prevent E2-ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53's transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors.

Histone modifications affect differential regulation of TGFβ- induced NADPH oxidase 4 (NOX4) by wild-type and mutant p53.

Previously, we showed wild-type (WT) and mutant (mut) p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (NOX4): p53-WT suppresses TGFβ-induced NOX4, ROS and cell migration, whereas tumor-associated mut-p53 proteins enhance NOX4 expression and cell migration. Here, we extended our findings on the effects of p53 on NOX4 in several tumors and examined the basis of NOX4 transcriptional regulation by p53 and SMAD3. Statistical analysis of expression data from primary tumors available from The Cancer Genome Atlas (TCGA) detected correlations between mut-p53 and increased NOX4 expression. Furthermore, by altering p53 levels in cell culture models we showed several common tumor-associated mutant forms support TGFβ/SMAD3-dependent NOX4 expression. Deletion analysis revealed two critical SMAD3 binding elements (SBE) required for mut-p53-dependent NOX4 induction, whereas p53-WT caused dose-dependent suppression of NOX4 transcription. ChIP analysis revealed SMAD3 and p53-WT or mut-p53 associate with SBEs and p53 response elements in a TGFβ-dependent manner. Interestingly, the repressive effects of p53-WT on NOX4 were relieved by mutation of its transactivation domain or histone deacetylase (HDAC) inhibitor treatment. Overexpression of p300, a transcriptional co-regulator and histone acetyltransferase (HAT), enhanced p53-mediated NOX4 induction, whereas HAT-inactive p300 reduced NOX4 expression. Mut-p53 augmented TGFβ-stimulated histone acetylation within the NOX4 promoter. Finally, wound assays demonstrated NOX4 and p300 promote TGFβ/mut-p53-mediated cell migration. Our studies provide new insight into TGFβ/SMAD3 and mut-p53-mediated NOX4 induction involving epigenetic control of NOX4 in tumor cell migration, suggesting NOX4 is a potential therapeutic target to combat tumor progression and metastasis.

Hepatitis C Virus Indirectly Disrupts DNA Damage-Induced p53 Responses by Activating Protein Kinase R.

ABSTRACTMany DNA tumor viruses promote cellular transformation by inactivating the critically important tumor suppressor protein p53. In contrast, it is not known whether p53 function is disrupted by hepatitis C virus (HCV), a unique, oncogenic RNA virus that is the leading infectious cause of liver cancer in many regions of the world. Here we show that HCV-permissive, liver-derived HepG2 cells engineered to constitutively express microRNA-122 (HepG2/miR-122 cells) have normal p53-mediated responses to DNA damage and that HCV replication in these cells potently suppresses p53 responses to etoposide, an inducer of DNA damage, or nutlin-3, an inhibitor of p53 degradation pathways. Upregulation of p53-dependent targets is consequently repressed within HCV-infected cells, with potential consequences for cell survival. Despite this, p53 function is not disrupted by overexpression of the complete HCV polyprotein, suggesting that altered p53 function may result from the host response to viral RNA replication intermediates. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated ablation of double-stranded RNA (dsRNA)-activated protein kinase R (PKR) restored p53 responses while boosting HCV replication, showing that p53 inhibition results directly from viral activation of PKR. The hepatocellular abundance of phosphorylated PKR is elevated in HCV-infected chimpanzees, suggesting that PKR activation and consequent p53 inhibition accompany HCV infection in vivo. These findings reveal a feature of the host response to HCV infection that may contribute to hepatocellular carcinogenesis.

P53 dynamics in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM.

Cellular systems show a wide range of signaling dynamics. Many of these dynamics are highly stereotyped, such as oscillations at a fixed frequency. However, most studies looking at the role of signaling dynamics focus on one or a few cell lines, leaving the diversity of dynamics across tissues or cell lines a largely unexplored question. We focused on the dynamics of the tumor suppressor protein p53, which regulates cell cycle arrest and apoptosis in response to DNA damage. We established live-cell reporters for 12 cancer cell lines expressing wild-type p53 and quantified p53 dynamics in response to double-strand break-inducing DNA damage. In many of the tested cell lines, we found that p53 abundance oscillated in response to ionizing radiation or the DNA-damaging chemotherapeutic neocarzinostatin and that the periodicity of the oscillations was fixed. In other cell lines, p53 abundance dynamically changed in different ways, such as a single broad pulse or a continuous induction. By combining single-cell assays of p53 signaling dynamics, small-molecule screening in live cells, and mathematical modeling, we identified molecules that perturbed p53 dynamics and determined that cell-specific variation in the efficiency of DNA repair and the activity of the kinase ATM (ataxia-telangiectasia mutated) controlled the signaling landscape of p53 dynamics. Because the dynamics of wild-type p53 varied substantially between cell lines, our study highlights the limitation of using one line as a model system and emphasizes the importance of studying the dynamics of other signaling pathways across different cell lines and genetic backgrounds.

Excitability in the p53 network mediates robust signaling with tunable activation thresholds in single cells

Cellular signaling systems precisely transmit information in the presence of molecular noise while retaining flexibility to accommodate the needs of individual cells. To understand design principles underlying such versatile signaling, we analyzed the response of the tumor suppressor p53 to varying levels of DNA damage in hundreds of individual cells and observed a switch between distinct signaling modes characterized by isolated pulses and sustained oscillations of p53 accumulation. Guided by dynamic systems theory we show that this requires an excitable network structure comprising positive feedback and provide experimental evidence for its molecular identity. The resulting data-driven model reproduced all features of measured signaling responses and is sufficient to explain their heterogeneity in individual cells. We present evidence that heterogeneity in the levels of the feedback regulator Wip1 sets cell-specific thresholds for p53 activation, providing means to modulate its response through interacting signaling pathways. Our results demonstrate how excitable signaling networks can provide high specificity, sensitivity and robustness while retaining unique possibilities to adjust their function to the physiology of individual cells.

MP39-13 IDENTIFICATION OF ISYNA1 AS A POTENTIAL PROGNOSTIC BIOMARKER FOR RENAL CANCER

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2017MP39-13 IDENTIFICATION OF ISYNA1 AS A POTENTIAL PROGNOSTIC BIOMARKER FOR RENAL CANCER Tomoyuki Koguchi, Kanako Matsuoka, Junya Hata, Yuichi Sato, Hidenori Akaihata, Masao Kataoka, Soichiro Ogawa, Nobuhiro Haga, Nobuhiro Kushida, Kei Ishibashi, Ken Ikawa, and Yoshiyuki Kojima Tomoyuki KoguchiTomoyuki Koguchi More articles by this author , Kanako MatsuokaKanako Matsuoka More articles by this author , Junya HataJunya Hata More articles by this author , Yuichi SatoYuichi Sato More articles by this author , Hidenori AkaihataHidenori Akaihata More articles by this author , Masao KataokaMasao Kataoka More articles by this author , Soichiro OgawaSoichiro Ogawa More articles by this author , Nobuhiro HagaNobuhiro Haga More articles by this author , Nobuhiro KushidaNobuhiro Kushida More articles by this author , Kei IshibashiKei Ishibashi More articles by this author , Ken IkawaKen Ikawa More articles by this author , and Yoshiyuki KojimaYoshiyuki Kojima More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1187AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In response to cellular stress, p53 exerts tumor-suppressive effects, such as apoptosis, cell cycle arrest, and senescence, through the induction of its target genes. Recently, p53 has been shown to control cellular homeostasis by regulating energy metabolism, glycolysis, antioxidant effects, and autophagy. A previous study has reported an association between the p53 expression pattern in immunohistochemical analyses of tumor and the prognosis of renal cell carcinoma (RCC). Although various tumors have p53 mutations, RCC has few p53 mutations. As such, we examined whether a p53 target gene might affect the prognosis of RCC patients. We identified a novel p53 target and examined whether it was useful as a prognostic factor for renal cancer. METHODS To screen for novel p53 target genes, we conducted a cDNA microarray analysis using mRNAs isolated from HCT116 p53+/+ and HCT116 p53-/- cells that were treated with 2 μg/ml of Adriamycin. To investigate whether mRNA transcription is regulated by p53, we performed a reporter assay and a chromatin immunoprecipitation (ChIP) assay using U373MG cells. To evaluate the effect of the novel p53 target on the biosynthesis of myo-inositol (MI), we performed a MI assay using HEK293T cells that were transfected with a plasmid expressing the novel p53 target, or HCT116 p53+/+ and HCT116 p53-/- cells that were treated with Adriamycin. To examine whether the novel p53 target would be useful as a prognosis factor for renal cancer, we performed a prognostic analysis using the mRNA expression ratio (tumor / normal kidney tissues) of the novel p53 target in RCC. RESULTS The results of the cDNA microarray analysis revealed inositol 3-phosphate synthase 1 ( ISYNA1 ) as a novel candidate gene. From the results of the reporter assay and the ChIP assay, we found p53 response elements in the promoter region and the seventh exon. As such, ISYNA1 was identified as a novel p53 target. The results of MI assay showed that ISYNA1 expression increased myo-inositol levels in the cells. In addition, DNA damage significantly increased the intracellular MI content in HCT116 p53+/+ cells, but did not affect the MI content in HCT116 p53-/- cells. The ISYNA1 mRNA expression ratios in renal cancer and renal clear cell carcinoma were significantly correlated with overall survival (p < 0.01). CONCLUSIONS We identified ISYNA1 as a novel p53 target gene and found that it regulated the intracellular MI content via a p53-ISYNA1 pathway. Our findings show that the ISYNA1 mRNA expression ratio is correlated to overall survival in RCC. Therefore, ISYNA1 might be useful as a novel prognostic factor for renal cancer. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e498 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Tomoyuki Koguchi More articles by this author Kanako Matsuoka More articles by this author Junya Hata More articles by this author Yuichi Sato More articles by this author Hidenori Akaihata More articles by this author Masao Kataoka More articles by this author Soichiro Ogawa More articles by this author Nobuhiro Haga More articles by this author Nobuhiro Kushida More articles by this author Kei Ishibashi More articles by this author Ken Ikawa More articles by this author Yoshiyuki Kojima More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Modeling amplified p53 responses under DNA-PK inhibition in DNA damage response.

During DNA double strand breaks (DSBs) repair, coordinated activation of phosphatidylinositol 3-kinase (PI3K)-like kinases can activate p53 signaling pathway. Recent findings have identified novel interplays among these kinases demonstrating amplified first p53 pulses under DNA-PK inhibition. However, no theoretical model has been developed to characterize such dynamics. In current work, we modeled the prolonged p53 pulses with DNA-PK inhibitor. We could identify a dose-dependent increase in the first pulse amplitude and width. Meanwhile, weakened DNA-PK mediated ATM inhibition was insufficient to reproduce such dynamic behavior. Moreover, the information flow was shifted predominantly to the first pulse under DNA-PK inhibition. Furthermore, the amplified p53 responses were relatively robust. Taken together, our model can faithfully replicate amplified p53 responses under DNA-PK inhibition and provide insights into cell fate decision by manipulating p53 dynamics.