Presence Of Mutant P53 Research Articles

Design, synthesis and evaluation of novel levoglucosenone derivatives as promising anticancer agents

A series of levoglucosenone-derived 1,2,3-triazoles and isoxazoles featuring a flexible spacer between the heteroaromatic and anhydropyranose cores have been designed and synthesized following an hetero Michael // 1,3-dipolar cycloaddition path. The use of a design of experiments approach allowed the optimization of the oxa-Michael reaction with propargyl alcohol as nucleophile, a key step for the synthesis of the target compounds. All of the compounds were tested for their anticancer activity on MDA-MB-231 cells, featuring mutant p53. The results highlighted the importance of the introduction of the flexible spacer as well as the higher activity of oxa-Michael isoxazole-derivatives. The most prominent compounds also showed anti-proliferative activities against lung and colon cancer cell lines. The compounds showed enhanced cytotoxic effects in the presence of mutant p53, determined both by endogenous mutant p53 knock down (R280K) and by reintroducing p53 R280K in cells lacking p53 expression.

Bicistronic transfer of CDKN2A and p53 culminates in collaborative killing of human lung cancer cells in vitro and in vivo.

Cancer therapies that target a single protein or pathway may be limited by their specificity, thus missing key players that control cellular proliferation and contributing to the failure of the treatment. We propose that approaches to cancer therapy that hit multiple targets would limit the chances of escape. To this end, we have developed a bicistronic adenoviral vector encoding both the CDKN2A and p53 tumor suppressor genes. The bicistronic vector, AdCDKN2A-I-p53, supports the translation of both gene products from a single transcript, assuring that all transduced cells will express both proteins. We show that combined, but not single, gene transfer results in markedly reduced proliferation and increased cell death correlated with reduced levels of phosphorylated pRB, induction of CDKN1A and caspase 3 activity, yet avoiding the induction of senescence. Using isogenic cell lines, we show that these effects were not impeded by the presence of mutant p53. In a mouse model of in situ gene therapy, a single intratumoral treatment with the bicistronic vector conferred markedly inhibited tumor progression while the treatment with either CDKN2A or p53 alone only partially controlled tumor growth. Histologic analysis revealed widespread transduction, yet reduced proliferation and increased cell death was associated only with the simultaneous transfer of CDKN2A and p53. We propose that restoration of two of the most frequently altered genes in human cancer, mediated by AdCDKN2A-I-p53, is beneficial since multiple targets are reached, thus increasing the efficacy of the treatment.

PO-091 Induction of anti-Fas regulates PUMA and MYC: P53-dependent apoptosis pathway in hepatocellular carcinoma cells

IntroductionIncreased oxidative stress is a hallmark of cancer cells, which makes them more vulnerable to induction of reactive oxygen species (ROS). P53 plays a crucial role in sensing and removing oxidative damage to DNA, and inactivating mutations in the TP53 gene attenuate this function. In addition, it was shown that mutant p53 is able to suppress the function of major antioxidant factors. Therefore, mutant p53 renders cancer cells even more susceptible to the induction of oxidative stress. Besides p53, the poly (ADP-ribose) polymerase 1 (PARP-1) protein plays an important role in the repair of ROS-induced DNA-damage. This led us to explore the potential of combining oxidative stress induction with the targeted inhibition of the PARP-1 protein to selectively target mutant p53 NSCLC cancer cells.Material and methodsAPR-246 and Auranofin (inhibition glutathione (GSH) and/or thioredoxin reductase 1 (TrxR1)) and Olaparib (PARP-1 inhibitor) were used. The cytotoxicity (SRB-assay) of these compounds was determined in a panel of NSCLC cell lines with different p53 status, including isogenic cell lines (p53 shRNA-knockdown, p53 knock-in). Total GSH content (GSH/GSSG-GloTM) and ROS content (CellROX) were determined. N-acetyl-l-cysteine (NAC) was used as a potent ROS-scavenger. Induction of apoptosis/cell death was determined by the AnnV/PI assay (FC) or the Cytotox Reagent (IncuCyte). DNA-damage was assessed by g-H2AX foci (IF) and the Comet Assay. Synergism was determined using the Additive model.Results and discussionsP53Mut knock-down reduces the cytotoxic effect of APR-246, Auranofin and Olaparib, while p53Mut knock-in sensitised cells for all three compounds. APR-246/Olaparib treatment reduced GSH levels and increased ROS content, resulting in a strong accumulation of DNA-damage and synergistic induction of cell death. Co-treatment with NAC or p53-knockdown significantly reduced this cytotoxic response. Similar synergistic effects were observed for Auranofin/Olaparib treatment in several cell lines with clinically relevant p53 mutations.ConclusionMutant p53 protein expression renders NSCLC cells more susceptible to APR-246, Auranofin and Olaparib treatment. In addition, the combination of oxidative stress induction (APR-246, Auranofin) with PARP-1 inhibition (Olaparib) results in remarkable synergistic effects in the presence of mutant p53. Therefore, this combination strategy could be a promising and selective treatment option for mutant p53 NSCLC patients in which resistance to standard therapies often occurs.

Hypoxia-Induced Cisplatin Resistance in Non-Small Cell Lung Cancer Cells Is Mediated by HIF-1α and Mutant p53 and Can Be Overcome by Induction of Oxidative Stress.

The compound APR-246 (PRIMA-1MET) is a known reactivator of (mutant) p53 and inducer of oxidative stress which can sensitize cancer cells to platinum-based chemotherapeutics. However, the effect of a hypoxic tumor environment has been largely overlooked in this interaction. This study focusses on the role of hypoxia-inducible factor-1α (HIF-1α) and the p53 tumor suppressor protein in hypoxia-induced cisplatin resistance in non-small cell lung cancer (NSCLC) cells and the potential of APR-246 to overcome this resistance. We observed that hypoxia-induced cisplatin resistance only occurred in the p53 mutant NCI-H2228Q331* cell line, and not in the wild type A549 and mutant NCI-H1975R273H cell lines. Cisplatin reduced HIF-1α protein levels in NCI-H2228Q331* cells, leading to a shift in expression from HIF-1α-dependent to p53-dependent transcription targets under hypoxia. APR-246 was able to overcome hypoxia-induced cisplatin resistance in NCI-H2228Q331* cells in a synergistic manner without affecting mutant p53Q331* transcriptional activity, but significantly depleting total glutathione levels more efficiently under hypoxic conditions. Synergism was dependent on the presence of mutant p53Q331* and the induction of reactive oxygen species, with depletion of one or the other leading to loss of synergism. Our data further support the rationale of combining APR-246 with cisplatin in NSCLC, since their synergistic interaction is retained or enforced under hypoxic conditions in the presence of mutant p53.

Abstract 4180: RS-41, a multi-targeted kinase inhibitor, induces cell cycle arrest and apoptosis in p53 mutant and wild type models of upper gastrointestinal cancers

Abstract Introduction: Upper Gastrointestinal Cancers (UGCs) are a leading cause of cancer-related mortality and account for approximately 1.1 million deaths worldwide. UGCs respond poorly to conventional chemotherapy due to constitutive over activity of multiple oncogenic signaling mechanisms, including the epidermal growth factor receptor (EGFR), ERBB2/HER-2, Aurora kinases, and JAK-STAT pathways. In addition, presence of mutant P53 further imparts resistance to conventional chemotherapeutic agents. Therefore, novel small molecule inhibitors that target multiple kinases associated with oncogenic progression could pave the way for improved chemotherapy and better therapeutic outcomes. In this study, we characterized the anticancer activity of RS-41, an investigational 4-phenylbenzamidopyrrolo[2,3-d]-pyrimidin-4-amine multi-kinase inhibitor, in P53 mutant and wild type models of UGC. Methods: Target kinase inhibition and selectivity screening assays were performed to determine potency and selectivity of kinase inhibition for RS-41. In addition, MTT-cell viability assay, clonogenic cell survival assay, cell cycle analyses, and western blot analyses were done to evaluated the effect of RS-41 treatment on cell viability, survival, cell cycle progression, and expression of apoptotic markers in P53 mutant (FLO-1) and P53 wild type (AGS) UGC cells, respectively. Results: The kinase selectivity screening assay characterized RS-41 for its selectivity against a panel of 90 human kinases. The kinase screening analyses showed that RS-41 selectively inhibits AURKA (IC50-0.96±0.03µM), JAK2 (IC50-1.21±0.17µM), and EGFR (IC50-5.92±0.75µM) kinases, respectively. The cell viability data indicate that treatment with RS-41 mediates significant (P≤0.05) inhibition of FLO-1 and AGS UGC cell viability. The clonogenic cell survival data showed that treatment with RS-41 for 24 hrs. suppresses subsequent formation of colonies in both FLO-1 and AGS UGC cells. The cell cycle data exhibited a marked increase (P≤0.05) in the percentage of FLO-1 and AGS cells in the sub-G1-phase (cell death) after treatment with RS-41 for 24 and 72 hrs., respectively. The western blotting data further confirmed induction of apoptosis in FLO-1 and AGS cells as evidenced by an increase in expression of various markers of apoptosis (P73/P53, cleaved PARP, and/or cleaved caspase 3) following treatment with RS-41 for 24 and 72 hrs. Conclusions: Our in vitro data indicate that inhibition of various oncogenic kinases with RS-41 is an effective therapeutic strategy for inducing apoptosis in both P53 mutant and wild type UGC cells. Our study suggests that RS-41 is a promising multi-kinase inhibitor with a potential to further enhance chemotherapeutic options for treatment of UGC. Citation Format: Tanvi Visal, Shraddha Patel, Priya Pancholi, Samhita Bapat, Amruta Samant, Dhvanir Kansara, Sonali Kurup, Vikas Sehdev. RS-41, a multi-targeted kinase inhibitor, induces cell cycle arrest and apoptosis in p53 mutant and wild type models of upper gastrointestinal cancers [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 4180. doi:10.1158/1538-7445.AM2017-4180

Abstract 1218: Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors

Abstract Introduction: TP53 is mutated in 30-40% of breast cancers and is associated with poor prognosis. Mutation of TP53 causes increased cellular proliferation, migration and invasion, and downstream activation of multiple pathways. HMG CoA reductase inhibitors, such as simvastatin, inhibit tumorigenic properties induced by TP53 mutation. Mechanism of this response is an important question in targeted cancer therapy. It is well known that HMG CoA reductase inhibitors block cholesterol production and prenylation. Therefore, we hypothesized that prenylation inhibitors would target p53 mutant cells primarily by inhibiting activation of the ras family induced by TP53 mutation. Method: We generated 5 MCF10A stably transduced cell lines over-expressing each of the common TP53 point mutations, R273H, G245S, R248Q, Y234C, or wild type p53. Total Ras, Ras-GTP, total RhoA and RhoA-GTP was measured by immunoprecipitation and immunoblot. Proliferation was measured using CellTiterGlo. The functional effects of a panel of prenylation inhibitors was measured using a caspase3 reporter assay, and migration was measured with a scratch assay. Illumina mRNA sequencing was performed to measure gene expression of mutant and wild type cells before and after simvastatin treatment (2.5µl, 48 h). The RNAseq was analyzed with the DAVID Enrichment tool to evaluate ras pathway activation induced by TP53 mutation. Results: Mutation in TP53 was associated with significant activation of both wild-type Ras and RhoA. Proliferation of cells expressing mutant TP53 was sensitive to a panel of prenylation inhibitors, including simvastatin. Growth inhibition by simvastatin correlated with induction of apoptosis, and could be fully rescued by addition of farnesylpyrophosphate or geranylgeranylpyrophosphate, suggesting that simvastatin functionally blocks both prenylation pathways which are activated in the presence of mutant p53 (IC50(wt/mut) = 4). RNAseq analysis confirms that Ras signaling pathways, including GAPs and GEFs are enriched in the presence of mutant TP53, and FOXO signaling is significantly targeted post statin treatment (P<0.01). Conclusion: p53 mutant breast cancer cells are highly sensitive to prenylation inhibition due to activation of both ras and rhoA. Simvastatin inhibits both farnesylation and geranylgeranylation, effectively blocking ras pathway activation and induction of proliferation in p53 mutant cell lines. A gene expression panel associated with ras pathway activation was identified and may be predictive biomarkers for sensitivity to statin therapy. Citation Format: Shay R. Ferdosi, Benjamin Katchman, Jia Loo, Harneet Grewal, Seron Eaton, Shanshan Yang, Jin Park, Joshua Labaer, Karen S. Anderson. Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors [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 1218. doi:10.1158/1538-7445.AM2017-1218

Identification, validation, and targeting of the mutant p53-PARP-MCM chromatin axis in triple negative breast cancer

Over 80% of triple negative breast cancers express mutant p53. Mutant p53 often gains oncogenic function suggesting that triple negative breast cancers may be driven by p53 protein type. To determine the chromatin targets of this gain-of-function mutant p53 we used inducible knockdown of endogenous gain-of-function mtp53 in MDA-MB-468 cells in conjunction with stable isotope labeling with amino acids in cell culture and subcellular fractionation. We sequenced over 70,000 total peptides for each corresponding reciprocal data set and were able to identify 3010 unique cytoplasmic fraction proteins and 3403 unique chromatin fraction proteins. The present proteomics experiment corroborated our previous experiment-based results that poly ADP-ribose polymerase has a positive association with mutant p53 on the chromatin. Here, for the first time we report that the heterohexomeric minichromosome maintenance complex that participates in DNA replication initiation ranked as a high mutant p53-chromatin associated pathway. Enrichment analysis identified the minichromosome maintenance members 2–7. To validate this mutant p53- poly ADP-ribose polymerase-minichromosome maintenance functional axis, we experimentally depleted R273H mutant p53 and found a large reduction of the amount of minichromosome maintenance complex proteins on the chromatin. Furthermore a mutant p53-minichromosome maintenance 2 direct interaction was detected. Overexpressed mutant p53, but not wild type p53, showed a protein-protein interaction with minichromosome maintenance 2 and minichromosome maintenance 4. To target the mutant p53- poly ADP-ribose polymerase-minichromosome maintenance axis we treated cells with the poly ADP-ribose polymerase inhibitor talazoparib and the alkylating agent temozolomide and detected synergistic activation of apoptosis only in the presence of mutant p53. Furthermore when minichromosome maintenance 2–7 activity was inhibited the synergistic activation of apoptosis was blocked. This mutant p53- poly ADP-ribose polymerase -minichromosome maintenance axis may be useful for theranostics.

Improvement of gemcitabine sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells by RUNX2 depletion-mediated augmentation of TAp73-dependent cell death.

Pancreatic cancer exhibits the worst prognostic outcome among human cancers. Recently, we have described that depletion of RUNX2 enhances gemcitabine (GEM) sensitivity of p53-deficient pancreatic cancer AsPC-1 cells through the activation of TAp63-mediated cell death pathway. These findings raised a question whether RUNX2 silencing could also improve GEM efficacy on pancreatic cancer cells bearing p53 mutation. In the present study, we have extended our study to p53-mutated pancreatic cancer MiaPaCa-2 cells. Based on our current results, MiaPaCa-2 cells were much more resistant to GEM as compared with p53-proficient pancreatic cancer SW1990 cells, and there existed a clear inverse relationship between the expression levels of TAp73 and RUNX2 in response to GEM. Forced expression of TAp73α in MiaPaCa-2 cells significantly promoted cell cycle arrest and/or cell death, indicating that a large amount of TAp73 might induce cell death even in the presence of mutant p53. Consistent with this notion, overexpression of TAp73α stimulated luciferase activity driven by p53/TAp73-target gene promoters in MiaPaCa-2 cells. Similar to AsPC-1 cells, small interfering RNA-mediated knockdown of RUNX2 remarkably enhanced GEM sensitivity of MiPaCa-2 cells. Under our experimental conditions, TAp73 further accumulated in RUNX2-depleted MiaPaCa-2 cells exposed to GEM relative to GEM-treated non-silencing control cells. As expected, silencing of p73 reduced GEM sensitivity of MiPaCa-2 cells. Moreover, GEM-mediated Tyr phosphorylation level of TAp73 was much more elevated in RUNX2-depleted MiaPaCa-2 cells. Collectively, our present findings strongly suggest that knockdown of RUNX2 contributes to a prominent enhancement of GEM sensitivity of p53-mutated pancreatic cancer cells through the activation of TAp73-mediated cell death pathway, and also provides a promising strategy for the treatment of patients with pancreatic cancer bearing p53 mutation.

Abstract 1220: MDM2 promotes p53-independent breast cancer metastatic phenotypes

Abstract Many human cancers overexpress the E3 ubiquitin ligase MDM2. The cancer genome atlas (TCGA) found that increased MDM2 expression is one of four gene pathways that correlate with all breast cancer subtypes. An increasing body of literature strongly suggests that overexpression of MDM2 can drive breast oncogenesis in the presence of mutant p53 (mtp53), as well as through molecular pathways that do not require wild-type p53 degradation. Therefore, the canonical wild-type p53 degradation pathway by MDM2 is clearly not the only important pathway. MDM2 expression has been shown to influence breast cancer cell invasion and metastasis. One potential mechanism for this is through the activation of SLUG (a transcription repressor of E-Cadherin) and the process of E-cadherin degradation. To investigate the p53-independent influence of MDM2 we engineered human breast cancer clones with inducible shRNA to knockdown MDM2 in MDA-MB-231 (mtp53 R280K), T47D (mtp53 L194F) and MCF-7 (wild-type p53) cells. Wound healing assays showed that MDM2 knockdown in all three cell lines reduced cell migratory capacity without altering p53 protein levels. In T47D.shmdm2 cells, a decrease in SLUG expression was observed with MDM2 knockdown indicating SLUG is a p53-independent target of MDM2. The knockdown of MDM2 also decreased slug and mmp9 mRNA detected by quantitative RT-PCR. To determine the biological significance of MDM2 in a system to recapitulate breast cancer architecture we examined T47D.shmdm2 cells in a 3-dimensional (3D) cell culture model. Importantly, the depletion of MDM2 in T47D.shmdm2 cells grown in 3D culture promoted luminal clearance. Our findings suggest that MDM2 can influence SLUG and MMP9 levels associated with breast cancer metastasis through a mechanism that does not require degradation of wild-type p53. Moreover our work suggests that MDM2 may promote changes to breast cancer architecture that drive metastasis. Acknowledgements. This work was supported by a grant to JB from the Breast Cancer Research Foundation. Citation Format: Chong Gao, Nandini Kundu, Jill Bargonetti. MDM2 promotes p53-independent breast cancer metastatic phenotypes. [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 1220. doi:10.1158/1538-7445.AM2015-1220

Diclofenac Induces Apoptosis and Suppresses Diffuse Large B-Cell Lymphoma Proliferation Independent of P53 Status

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL).Despite long-term remission achieved with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), relapse occurs in almost one third of the patients. Therefore, there is a need for novel therapeutic targets that are relevant to DLBCL pathogenesis. TP73 gene is a member of the p53 tumor suppressor gene family, which is critical in the regulation of cell cycle and apoptosis. TP73 is located in distal 1p36 chromosomal region that is commonly disrupted in DLBCL. Our previous studies had shown that the differential expression of p73 isoforms correlates with proliferation and apoptosis in DLBCL patient specimens. Furthermore, the experimental modulation of p73 isoforms using expression vectors or siRNA modulates the behavior and regulate the chemotherapeutic response of DLBCL cell line models. Diclofenac is NSAID that has been shown to increase p73 activity, substitute p53 activity and suppress the growth of neuroblastoma. In the present study, we investigated whether diclofenac modulates DLBCL apoptosis and cell cycle progression independent of p53 status. We used cell line models of the GCB-DLBCL (DHL-16 and OCI-Ly7) and the ABC-DLBCL (OCI-Ly3 and Pfeiffer). Because OCI-Ly7 and Pfeiffer have a mutant p53, these cells can model the activity of diclofenac in the presence of mutant p53. We used MTT assay to study the response of the DLBCL cells to various concentrations of diclofenac (25, 50, 100, 150, 200, 250 µM) and at different time points (24, 48, and 72 hours). To decipher the biological effects of diclofenac treatment on DLBCL cells Hema-3 staining was done to visualize morphologic evidence of cell death; propidium iodide-based flow cytometric analysis for cell cycle progression; BrdU incorporation for proliferation; and Annexin-V-Flous flow cytometric analysis for apoptosis. Molecularly, Caspase-GLO assay was used for evaluation of Caspase-3, 7, 8 activity and qRT-PCR was used to estimate the effect of diclofenac treatment on p73 and the p53 family transcriptional target regulating cell cycle (p21) and apoptosis (PUMA, NOXA, BIM, and CD95). Mann-Whitney (for two groups) or ANOVA (for more than two groups) analyses were used to determine the statistical significance for comparisons between different treatment groups. Diclofenac treatment displayed a concentration and duration-dependent suppressive cell proliferative activity against a panel of DLBCL cells independent of p53 status including experimental therapy-resistant models. Diclofenac treatment resulted in cell cycle arrest mainly at the G2/M phase, decreased proliferation, and caused profound cell death (mainly apoptosis and possibly necroptosis). Molecularly, diclofenac treatment was associated with increased activity of caspases- 3, -7 and -8. Increased p53 pathway activity as suggested by induction of expression of a panel of p53 transcriptional targets including the cell cycle regulatory molecule p21 and the pro-apoptotic molecules, PUMA, NOXA, BIM, and CD95, was detected in diclofenac treated DLBCL cells. More importantly diclofenac treatment was associated with enhanced expression of the pro-apoptotic isoforms of the p53 homologue, TAp73. Together, our data demonstrate that clinically non-toxic doses of diclofenac treatment, induces apoptosis and cell cycle arrest of both GCB and ABC-DLBCL cells independent of p53 status and is associated with increased expression of the p73 homologue TAp73. These data highlight the potential of diclofenac as a novel adjuvant therapy in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Mutant p53 and the response to chemotherapy and radiation.

In addition to playing roles in the genesis and progression of cancer, mutant p53 also appears to play a significant role in the response to cancer therapy. In response to chemotherapy and radiation, two mainstays of cancer treatment, most cancer cells harboring p53 mutations show a reduced sensitivity compared to cells lacking p53 or those with wild type p53. However, there are also many instances where mutant p53 has shown no effect or enhances cellular sensitivity to chemotherapy and radiation. Similar to the in vitro cellular studies, the majority of clinical studies show a correlation between the presence of mutant p53 in patient tumors and adverse outcomes following treatment with chemotherapy agents or radiation in comparison to tumors with wild-type p53. However, it still remains unclear whether the presence of mutant p53 in tumors can serve as a reliable prognostic factor and aid in treatment planning. Thus, as genomic analysis of patient tumors becomes more cost effective, the role of mutant p53 in tumor responses from cancer therapy ultimately needs to be addressed. This chapter will discuss current mechanisms of how p53 mutations affect cellular responses to chemotherapy and radiation and discuss patient outcomes based on p53 status.

Abstract 2188: A screen to identify novel p53 family member activity-restoring compounds with a focus on p73.

Abstract Tumor suppressor p53 family regulates gene transcription, and thereby cell cycle, apoptosis, metabolism and other processes. The p53 gene is found mutated in over 50% of all human cancers, and it has been of great interest to identify drugs that can restore p53 function. While p53 can display a wide variety of mutations, which potentially could complicate function restoration therapy, family member p73 gene mutation is rare. Therefore, we sought to identify novel compounds that are able to restore the activity of the p53 family, with a focus on p73 dependency in the presence of mutant p53. To this end, a functional cell-based screening assay was set up for cancer cell death-inducing small molecules that activate p53 pathway-dependent gene transcription. SW480 colon carcinoma cells, which carry R273H/R309S mutations in p53, and stably express a p53 family-responsive luciferase construct, were utilized, with parental cells and cells displaying stable knockdown of p73 being subjected to compounds from NCI Diversity Set 3. This set contains about 1600 compounds that are relatively well characterized and structurally rigid. After various treatment periods, p53 activity was assessed, as well as cell viability. Subsequently, a total of 26 compounds were selected for further investigation and validation. Follow up studies will include dose and time courses for p53 family activation and viability in a variety of cell lines that are wild-type, mutant or null for p53. Also mechanistic studies and cellular assays will be performed, with the hopes to identify novel drugs that are able to restore activity of the p53 family, and subsequent cancer cell death, and thus providing novel candidates for the treatment of human cancer. Citation Format: Antonius P. van den Heuvel, Wenge Wang, Levy Kopelovich, Wafik S. El-Deiry. A screen to identify novel p53 family member activity-restoring compounds with a focus on p73. [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 2188. doi:10.1158/1538-7445.AM2013-2188

P53 mutants induce transcription of NF-κB2 in H1299 cells through CBP and STAT binding on the NF-κB2 promoter and gain of function activity

Cancer cells with p53 mutations, in general, grow more aggressively than those with wild-type p53 and show “gain of function” (GOF) phenotypes such as increased growth rate, enhanced resistance to chemotherapeutic drugs, increased cell motility and tumorigenicity; although the mechanism for this function remains unknown. In this communication we report that p53-mediated NF-κB2 up-regulation significantly contributes to the aggressive oncogenic behavior of cancer cells. Lowering the level of mutant p53 in a number of cancer cell lines resulted in a loss of GOF phenotypes directly implicating p53 mutants in the process. RNAi against NF-κB2 in naturally occurring cancer cell lines also lowers GOF activities. In H1299 cells expressing mutant p53, chromatin immunoprecipitation (ChIP) assays indicate that mutant p53 induces histone acetylation at specific sites on the regulatory regions of its target genes. ChIP assays using antibodies against transcription factors putatively capable of interacting with the NF-κB2 promoter show increased interaction of CBP and STAT2 in the presence of mutant p53. Thus, we propose that in H1299 cells, mutant p53 elevates expression of genes capable of enhancing cell proliferation, motility, and tumorigenicity by inducing acetylation of histones via recruitment of CBP and STAT2 on the promoters causing CBP-mediated histone acetylation.

Upregulation of the mitochondrial transport protein, Tim50, by mutant p53 contributes to cell growth and chemoresistance

The p53 gene is one of the most frequently mutated genes in human cancer. Some p53 mutations impart additional functions that promote oncogenesis. To investigate how these p53 mutants function, a proteomic analysis was performed. The protein, translocator of the inner mitochondrial membrane 50 (Tim50), was upregulated in a non-small cell lung carcinoma cell line (H1299) that expressed the p53 mutants R175H and R273H compared to cells lacking p53. Tim50 was also elevated in the breast cancer cell lines MDA-MB-468 and SK-BR-3, that endogenously express the p53 mutants R175H and R273H, respectively, compared to MCF-10A. The p53 mutants R175H and R273H, but not WT p53, upregulated the expression of a Tim50 promoter construct and chromatin immunoprecipitation (ChIP) analysis indicated increased histone acetylation and increased interaction of the transcription factors Ets-1, CREB and CREB-binding protein (CBP) with the Tim50 promoter in the presence of mutant p53. Finally, reduction of Tim50 expression reduced the growth rate and chemoresistance of cells harboring mutant p53 but had no effect upon cells lacking p53. Taken together, these findings identify the Tim50 gene as a transcriptional target of mutant p53 and suggest a novel mechanism by which p53 mutants enhance cell growth and chemoresistance.

Abstract 1091: ShcA-mediated ERK signaling contributes to the tumor-promoting activity of TGF-beta in the presence of mutant p53

Abstract Transforming growth factor-beta (TGF-β) signaling has been shown to inhibit tumorigenesis at the early stage and promote metastasis at the late stage. However, biomarkers for identifying the complicated molecular alterations mediating the switch of TGF-β from a tumor suppressor to a tumor promoter still remain obscure. Published studies have shown that the presence of mutated tumor p53 can switch TGF-β to becoming tumor-promoting. However, we found that in the p53-null PC-3 prostate cancer cell line, the ectopic expression of the mutant p53 R175H (mp53) enhanced the Smad-dependent signaling, but inhibited TGF-β-induced cell migration by downregulating the active ERK. Since TGF-β is known to activate MAPK/ERK pathway to induce cell migration, EMT and survival through direct phosphorylation of adaptor protein ShcA isoform p52, we investigated how the expression of the mp53 inhibited ERK activation. We found that the expression of mp53 led to downregulation of phospho-p52ShcA and upregulation of ERK antagonist p66ShcA isoform. Interestingly, we found partial depletion of p52ShcA by using siRNA enhanced the TGF-β/Smad signaling and inhibited the TGF-β/ERK signaling in PC-3 cells and spontaneously immortalized and non-tumorigenic human mammary epithelial MCF-10A cell line, suggesting ShcA may act as an additional switcher of the TGF-β. Furthermore, reactivation of ERK by the expression of its activator, MEK1, restored active ERK level and TGF-β-induced cell migration in PC-3/mp53 cell. Those findings show that mp53 expression alone is not sufficient to promote the malignant phenotype of TGF-β in certain cells and the cooperation of mp53 and ShcA-mediated ERK signaling may switch TGF-β to be a tumor promoter. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1091. doi:10.1158/1538-7445.AM2011-1091

PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein

The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 null cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET.

A systematic review of the role of mutant p53 in predicting outcome or response to treatment in head and neck squamous cell carcinoma (HNSCC).

Objectives. To systematically review whether the presence of mutant p53 alters patients’ prognosis in HNSCC. Method. Systematic searches of Medline, the Cochrane database of randomised controlled trials, Embase, review of conference abstracts and bibliographies of retrieved articles were performed. Studies were included if they; (1) considered biopsy proven primary HNSCC from the larynx, oropharynx, hypopharynx, or oral cavity. (2) Reported on the outcome following treatment that included radiotherapy, chemotherapy, surgery or a combination of these. (3) Attempted to assess p53 status and compared this with survival outcomes. (4) Measured survival outcomes at a minimum of 2 years post primary treatment. Primary outcome measures were overall survival (OS) and disease free survival (DFS). Hazard ratio (HR) with 95% confidence intervals (CI), was calculated from available data comparing outcome in mutant versus wild type p53 groups; initially for each study prior to aggregation. Results. 205 papers were fully scrutinised, of these 36 were suitable for meta-analysis. Larynx: OS HR = 1.00 (95% CI 0.82–1.22); DFS HR = 1.27 (95% CI 0.99–1.63). Oral Cavity: OS HR = 1.50 (95% CI 1.15–1.95), DFS HR = 1.46 (95% CI 1.12–1.91). Oropharynx: OS HR = 1.26 (95% CI 0.92–1.73), DFS HR = 0.45 (95% CI 0.28–0.73). Hypopharynx: OS HR = 1.59 (95% CI 1.07–2.35), no papers had sufficient information from which DFS results could be calculated. Conclusions. The results suggest a survival benefit for the p53 negative group, however there is marked heterogeneity between these studies, so any final conclusion should remain guarded.

Sequential Transcription Factor Targeting for Diffuse Large B-Cell Lymphomas

Transcription factors play a central role in malignant transformation by activating or repressing waves of downstream target genes. Therapeutic targeting of transcription factors can reprogram cancer cells to lose their advantages in growth and survival. The BCL6 transcriptional repressor plays a central role in the pathogenesis of diffuse large B-cell lymphomas (DLBCL) and controls downstream checkpoints, including the p53 tumor suppressor gene. We report that a specific inhibitor of BCL6 called BPI can trigger a p53 response in DLBCL cells. This was partially due to induction of p53 activity and partially due to relief of direct repression by BCL6 of p53 target genes. BPI could thus induce a p53-like response even in the presence of mutant p53. Moreover, sequential BCL6 peptide inhibitors followed by p53 peptide or small-molecule activators provided a more powerful antilymphoma effect than either treatment alone by maximally restoring p53 target gene expression. Therefore, tandem targeting of the overlapping BCL6 and p53 transcriptional programs can correct aberrant survival pathways in DLBCL and might provide an effective therapeutic approach to lymphoma therapy.

Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function?

Although still controversial, the presence of mutant p53 in cancer cells may result in more aggressive tumors and correspondingly worse outcomes. The means by which mutant p53 exerts such pro-oncogenic activity are currently under extensive investigation and different models have been proposed. We focus here on a proposed mechanism by which a subset of tumor-derived p53 mutants physically interact with p53 family members, p63 and p73, and negatively regulate their proapoptotic function. Both cell-based assays and knock-in mice expressing mutant forms of p53 support this model. As more than half of human tumors harbor mutant forms of p53 protein, approaches aimed at disrupting the pathological interactions among p53 family members might be of clinical value.

Silencing of stathmin induces tumor-suppressor function in breast cancer cell lines harboring mutant p53

Cancers harboring dominant-negative p53 mutations are often aggressive and difficult to treat. Direct attempts to restore wild-type p53 function have produced little clinical benefit. We investigated whether targeting a p53-target gene could induce certain tumor-suppressor characteristics. We found that inhibition of stathmin, a microtubule regulator that can be transcriptionally repressed by wild-type p53, restored certain wild-type functions to cancer cells with mutant p53. Silencing of stathmin by small interfering RNA (siRNA) in mutant p53 cell lines lowered expression to that observed following activation of wild-type p53 by DNA damage in wild-type p53 cell lines. siRNA-induced repression of stathmin decreased cell proliferation, viability and clonogenicity in mutant p53 cell lines. Furthermore, knockdown of stathmin partially restored cell-cycle regulation and activation of apoptosis. Therefore, targeting stathmin, a gene product that is overexpressed in the presence of mutant p53, may represent a novel approach to treating cancers with aberrant p53 function.