Wild-type P53 Function Research Articles

Abstract 1295: Evaluation of the efficacy of TRAIL plus quercetin as a potential breast carcinoma therapeutic

Abstract Breast cancer is the most commonly diagnosed cancer in women in the United States. There is a continued need for the development of selective and specific treatment options for all types of breast cancer, including hormone-dependent and triple-negative subtypes. Recombinant human Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (rhTRAIL), the optimized form of the endogenous death ligand, shows potential as an effective anti-cancer therapeutic due to its ability to induce apoptosis in cancer independent of wild-type p53 function, while displaying minimal toxicity to normal cells. However, a majority of breast cancer cell lines exhibit resistance to TRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors 4 and 5. To overcome TRAIL resistance, a cotreatment option has been explored utilizing the natural compound Quercetin (Q). Q is a flavonol found in certain fruits, vegetables, and teas. As a single agent, Q has been shown to have antiproliferative and pro-apoptotic effects on a variety of cancer cell lines. The aim of this study is to examine the capacity of Q to enhance TRAIL's pro-apoptotic and antiproliferative effects on breast cancer cells. Sulphorhodamine B (SRB) assays were performed on hormone dependent (MCF-7) and triple negative (BT-20) breast cancer cell lines to determine if the cotreatment of Q and TRAIL hinders cell growth. Growth for both MCF-7 and BT-20 cells was substantially inhibited by single agent Q treatments (12.5 μM; ∼20%, 25 μM; ∼40%, 50 μM; ∼60%) but not by single agent TRAIL treatment (100 ng/mL; <20%). Moreover, BT-20 and MCF-7 cell growth was further inhibited by cotreatment with both agents; for example, the cotreatment of 50 μM Q and 100 ng/mL TRAIL inhibited BT-20 and MCF-7 cell growth by 80% and 90%, respectively. As a single agent, Q was able to induce apoptosis in a dose-dependent manner in both breast cancer cells; thereafter, Q's ability to sensitize breast cancer cells to TRAIL-induced apoptosis was examined by western blot analysis. Compared to single agent treatments, the combination of Q and TRAIL enhanced the induction of apoptosis as indicated by increased PARP cleavage (a hallmark of apoptosis) and the activation of the executioner caspases 3 and 7. Furthermore, the cotreatment of breast cancer cells with Q and TRAIL enhanced the activation of the extrinsic and intrinsic apoptotic pathways, as assessed by the activation of caspase 8 and the release of cytochrome c from the mitochondria, respectively. Overall, these findings suggest that the cotreatment of Q and rhTRAIL possesses the potential to be an anti-breast cancer therapeutic by enhancing pro-apoptotic and anti-proliferative effects in hormone dependent and triple-negative breast cancer cells. Citation Format: Jasmine M. Manouchehri, Michael Kalafatis, Daniel Lindner. Evaluation of the efficacy of TRAIL plus quercetin as a potential breast carcinoma therapeutic. [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 1295.

Abstract 3816: Reactive oxygen species and ERK2 phosphorylation are required for NSC59984 to induce mutant p53 protein degradation and restore p53 signaling

Abstract Tumor suppressor p53 is mutated in over 50% of human cancers. p53 mutation abolishes wild-type p53 function and also endows mutant p53 with a gain-of-function (GOF) that drives tumor growth and drug resistance. Targeting mutant p53 is an attractive strategy for cancer therapy. We recently reported (Zhang et al., Cancer Research, 2015) a small-molecule NSC59984 with dual capabilities to restore p53 signaling and destabilize mutant p53 protein (depleting GOF). We now demonstrate the role of reactive oxygen species (ROS) and ERK2 in the mechanism of action of NSC59984. We observe a sustained-phosphorylation of ERK2 in cancer cells treated with NSC59984. Blockade of ERK2 rescues mutant p53 from NSC59984-mediated degradation, and inhibits restoration of p53 signaling in mutant p53-expressing cells. Thus, sustained ERK2 phosphorylation is required for NSC59984-induced degradation of mutant p53 protein, and depletion of mutant p53 contributes to the restoration of p53 pathway. NSC59984 induces MDM2 phosphorylation that correlates with ERK2 phosphorylation. The effect of NSC59984 on MDM2 phosphorylation is blocked by U0126 and knockdown of ERK2. NSC59984-mediated mutant p53 protein degradation is inhibited by MDM2 knockdown, and enhanced by MDM2 overexpression. Thus, ERK2-dependent MDM2 phosphorylation is a major determinant of NSC59984-mediated mutant p53 degradation. We investigated the role of ROS in the effect of NSC59984 on ERK2 phosphorylation. ROS is induced by NSC59984 and a decrease in ROS by NAC inhibits NSC59984-induced ERK2 phosphorylation, and mutant p53 protein degradation. Increased ROS by BSO treatment enhances the NSC59984 effect on ERK2 phosphorylation, mutant p53 protein degradation and restoration of p53 signaling. We conclude that ROS is required for NSC59984 to sustain ERK2 phosphorylation, which, in turn, is required for NSC59984-induced mutant p53 protein degradation via MDM2. The combination of NSC59984 and BSO synergistically induces cell death in colorectal cancer cells. ROS and ERK2 are two important factors required for NSC59984 to degrade mutant p53 protein, restore p53 signaling and induce cell death. These results provide a rationale for clinical testing of NSC59984 in tumors with high ROS. Citation Format: Shengliang Zhang, Lanlan Zhou, David Dicker, Wafik S. El-Deiry. Reactive oxygen species and ERK2 phosphorylation are required for NSC59984 to induce mutant p53 protein degradation and restore p53 signaling. [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 3816.

TP53 Mutations in Head and Neck Squamous Cell Carcinoma and Their Impact on Disease Progression and Treatment Response.

Recent studies describing the mutational landscape of head and neck squamous cell carcinoma (HNSCC) on a genomic scale by our group and others, including The Cancer Genome Atlas, have provided unprecedented perspective for understanding the molecular pathogenesis of HNSCC progression and response to treatment. These studies confirmed that mutations of the TP53 tumor suppressor gene were the most frequent of all somatic genomic alterations in HNSCC, alluding to the importance of the TP53 gene in suppressing the development and progression of this disease. Clinically, TP53 mutations are significantly associated with short survival time and tumor resistance to radiotherapy and chemotherapy in HNSCC patients, which makes the TP53 mutation status a potentially useful molecular factor for risk stratification and predictor of clinical response in these patients. In addition to loss of wild-type p53 function and the dominant-negative effect on the remaining wild-type p53, some p53 mutants often gain oncogenic functions to promote tumorigenesis and progression. Different p53 mutants may possess different gain-of-function properties. Herein, we review the most up-to-date information about TP53 mutations available via The Cancer Genome Atlas-based analysis of HNSCC and discuss our current understanding of the potential tumor-suppressive role of p53, focusing on gain-of-function activities of p53 mutations. We also summarize our knowledge regarding the use of the TP53 mutation status as a potential evaluation or stratification biomarker for prognosis and a predictor of clinical response to radiotherapy and chemotherapy in HNSCC patients. Finally, we discuss possible strategies for targeting HNSCCs bearing TP53 mutations. J. Cell. Biochem. 117: 2682-2692, 2016. © 2016 Wiley Periodicals, Inc.

Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing.

The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as ‘Low’, ‘Intermediate’ or ‘High’ for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.

Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides.

Cancer drugs often fail due to the emergence of clinical resistance. This can manifest through mutations in target proteins that selectively exclude drug binding whilst retaining aberrant function. A priori knowledge of resistance-inducing mutations is therefore important for both drug design and clinical surveillance. Stapled peptides represent a novel class of antagonists capable of inhibiting therapeutically relevant protein-protein interactions. Here, we address the important question of potential resistance to stapled peptide inhibitors. HDM2 is the critical negative regulator of p53, and is often overexpressed in cancers that retain wild-type p53 function. Interrogation of a large collection of randomly mutated HDM2 proteins failed to identify point mutations that could selectively abrogate binding by a stapled peptide inhibitor (PM2). In contrast, the same interrogation methodology has previously uncovered point mutations that selectively inhibit binding by Nutlin, the prototypical small molecule inhibitor of HDM2. Our results demonstrate both the high level of structural p53 mimicry employed by PM2 to engage HDM2, and the potential resilience of stapled peptide antagonists to mutations in target proteins. This inherent feature could reduce clinical resistance should this class of drugs enter the clinic.

Mutation R273H confers p53 a stimulating effect on the IGF-1R-AKT pathway via miR-30a suppression in breast cancer.

p53 is the most highly mutated tumor suppressor in human malignancies. A wide array of p53 mutations has been revealed to play pivotal roles during cancer progression, which abolish anti-tumor functions of wild type p53 but also elicit tumorigenic effects by activating a diverse subset of downstream molecules. R273H mutation of p53 has been closely implicated in human cancer. Here we report miR-30a as a novel downstream target of p53 R273H mutant, which binds to the promoter region to repress miR-30a expression. Consequently, p53 R273H mutant enhances the migratory capabilities of tumor cells that are compromised by exogenous miR-30a over-expression. Our further investigation indicates that p53 R273H mutation unleashes the inhibition effect of miR-30a on IGF-1R expression, thus leading to elevated activation of IGF-1R-AKT signaling cascade in tumor cells.

Targeting of Mutant p53 and the Cellular Redox Balance by APR-246 as a Strategy for Efficient Cancer Therapy.

TP53 is the most frequently mutated gene in cancer. The p53 protein activates transcription of genes that promote cell cycle arrest or apoptosis, or regulate cell metabolism, and other processes. Missense mutations in TP53 abolish specific DNA binding of p53 and allow evasion of apoptosis and accelerated tumor progression. Mutant p53 often accumulates at high levels in tumor cells. Pharmacological reactivation of mutant p53 has emerged as a promising strategy for improved cancer therapy. Small molecules that restore wild type activity of mutant p53 have been identified using various approaches. One of these molecules, APR-246, is a prodrug that is converted to the Michael acceptor methylene quinuclidinone (MQ) that binds covalently to cysteines in p53, leading to refolding and restoration of wild type p53 function. MQ also targets the cellular redox balance by inhibiting thioredoxin reductase (TrxR1) and depleting glutathione. This dual mechanism of action may account for the striking synergy between APR-246 and platinum compounds. APR-246 is the only mutant p53-targeting compound in clinical development. A phase I/IIa clinical trial in hematological malignancies and prostate cancer showed good safety profile and clinical effects in some patients. APR-246 is currently tested in a phase Ib/II trial in patients with high-grade serous ovarian cancer.

P53 Replacement Therapy for Cancer.

Tumor suppressor gene (TSG) replacement therapy that involves various delivery systems is emerging as a promising antitumor strategy because malignant tumors develop through genetic alterations in TSGs. The most potent therapeutic TSG for tumor suppression is the multifunctional transcription factor p53 gene that regulates diverse cellular phenomena such as cell cycle arrest, senescence, apoptosis, and autophagy. Since the p53 gene is frequently inactivated by aberrant genetic regulation in human cancers, p53 replacement therapy is widely and frequently used as a potent antitumor strategy to restore wild-type p53 function in the p53-inactivated tumors. This chapter focuses on four types of p53 transfer systems: cationic liposome-DNA plasmid complexes, a replication-deficient adenovirus vector, a replication-competent adenovirus vector, and a protein transduction system. Moreover, we discuss recent advances in our understanding of the molecular basis of the p53-mediated cell death signaling pathway and therapeutic methods for enhancing tumor cell death and induction of bystander effects within tumor tissues in p53 replacement therapy. Exploration of the molecular mechanism underlying the p53-mediated tumor-suppressive network system and development of an effective strategy for enhancing p53-mediated cell death signaling pathways would lead to an improvement in the clinical outcome of patients with p53-inactivated cancers.

Regulation of endoplasmic reticulum functions.

The endoplasmic reticulum (ER) is a multifunctional organelle that is specialized for lipid synthesis, Ca2+ homeostasis, and protein synthesis. Protein synthesis is a multistep process involving folding and manipulation of proteins as well as the exportation of proteins to an appropriate location. Several stress responses, such as metabolic and anaerobic stress, induce ER dysfunction and protein unfolding, which accelerates the disruption of ER homeostasis. To prevent the production of unfolded proteins, cells induce the unfolded protein response (UPR), which is a highly regulated protein quality surveillance system. UPR maintains and restores ER homeostasis to increase the secretory function of the ER via the induction of the ER chaperone protein. This stimulates re-folding or degradation of unfolded proteins. However, irreparable ER stress induces a cell death response to eliminate these damaged cells [1]. Three types of ER transmembrane proteins, a protein-kinase and site-specific endoribonuclease, inositol-requiring enzyme 1 α (IRE1α), protein kinase R-like ER kinase/pancreatic eukaryotic initiation factor 2 kinase, and activating transcription factor 6 are essential for the mammalian UPR system. In particular, IRE1α is an essential signal transducer involved in the maintenance of ER function. The IRE1α pathway induces X-box binding protein 1(S) [XBP1(S)], which is the active form of XBP1 generated by IRE1α-dependent splicing of the XBP1 mRNA. XBP1(S) is a transcription factor that increases the expression of the ER chaperone, thus enhancing the secretory function of the ER and suppressing ER stress-mediated cell death [1]. Cancer cells alternate between glycolysis and protein synthesis pathways to survive and proliferate during stress associated with tumor growth and prolonged metabolic and anaerobic stress [2]. Cancer cells produce a lot of new proteins to satisfy their rapid growth using an energy source, such as the cancer-specific aerobic glycolysis pathway. Thus, the augmented function of the ER and resistance of ER stress-mediated cell death is essential for tumor proliferation and survival. Many reports have shown the enhancement of ER function in a variety of human cancers, but the mechanism for the cancer cell-specific regulation of ER function remains unresolved. Our recent study reveals that the tumor suppression gene, p53, is a key regulator of ER function [3]. To the best of our knowledge, this is the first report to connect the cancer-related gene to ER function. p53 is mutated in at least half of human cancers, and defects in the p53 response lead to tumor development. The functions of p53 influence many types of cell signaling transduction pathways, but the role of p53 in the ER is largely unknown [4]. We found that the loss of p53 function increased IRE1α, XBP1(S), and ER chaperone expression in non-stressed and ER-stressed conditions and also increased ER function. Interestingly, IRE1α was abundantly expressed in mutant p53 cell lines compared with wild-type p53 cell lines. The upregulation of IRE1α expression triggered the activation of the IRE1α/XBP1 pathway. Further, our experiments identified the mechanism for increased IRE1 expression with p53 deficiency, and our results revealed that suppressed p53-dependent IRE1α-synoviolin-1 (SYVN1) association induced IRE1α proteasomal degradation. Therefore, wild-type p53 suppresses ER function and adapts to ER stress by keeping IRE1α expression levels low, which highlights the anti-cancer effects of p53. However, loss of p53 function activates the IRE1α/XBP1 pathway and stimulates resistance to ER stress-mediated cell death. Thus, up-regulation of IRE1α expression via the loss of p53 function is one of the mechanisms by which a cancer-specific increase in ER function and activation of the IRE1α/XBP1 pathway is related to some parts of the aggressive cancer phenotype found in p53-mutant cancers. Another important finding of our current study is that an inhibitor of IRE1 selectively prevents the growth of p53-null tumors in vivo compared with that of wild type p53 tumors [3]. Several reports suggested that the inhibition of the IRE1α/XBP1 pathway is a new anti-cancer target. Cubillos-Ruiz et al. reported that the inhibition of activated XBP1 stimulates tumor-infiltrating dendritic cell-induced anti-tumor immunity [5]. The IRE1α inhibitor has an anti-cancer effect on human multiple myeloma and pancreatic cancers [6, 7]. Furthermore, DNA-damaging agents are widely used as treatments for various forms of cancer in the clinic, and its anti-cancer effects depend on the appropriate function of wild-type p53. Thus, mutant p53 cancer cells have been reported to show chemoresistance to many anti-cancer agents, and there is a need for novel therapies against mutant p53 cancers. Therefore, preventing the activation of the IRE1α/XBP1 pathway is a promising target for anti-tumor agents of mutant p53 cancers. In summary, cancer cells need to increase ER function to survive stressful environments. Loss of p53 function augments ER function via the activation of the IRE1α/XBP1 pathway by the disruption of the p53-SYVN1-IRE1α complex. Thus, targeting the IRE1α/XBP1 pathway has potential as a novel therapeutic strategy for malignancy and chemo-resistance of mutant p53 cancers.

Losing p53 loosens up ER-stress.

The endoplasmic reticulum (ER) is the central intracellular organelle responsible for proper folding, localization, and post-translational modifications of proteins in eukaryotic cells. During conditions of cellular stress, incorrectly folded proteins can accumulate in the ER and trigger the unfolded protein response (UPR), which involves the activation of signal transduction pathways designed to restore correct protein folding and recovery from ER stress [1]. In rapidly dividing tumor cells, UPR can become constitutively activated, presumably because higher levels of proliferation require increased protein synthesis [1, 2]. In addition, the tumor micro-environment is often host to additional adverse conditions including nutrient and oxygen deprivation, leading to further perturbations of ER function. Therefore, it may come as no surprise that UPR has been shown to play a crucial cytoprotective role in tumor cells [2]. We have recently unveiled an unexpected role for the tumor suppressor p53 in regulating ER function via the IRE1α/XBP1 pathway [3]. IRE1α is an ER stress sensor that activates the UPR to restore ER function and maintain cellular homeostasis. It was observed that wild-type p53 suppresses IRE1α expression and activation, while the loss of active p53 potentiates the IRE1α/XBP1 pathway, enhancing ER function [3]. This finding suggests a survival benefit for cancer cells that can inactivate p53. Because cancer cells with high protein load on the ER would need enhanced ER function to cope with constant ER stress, losing p53 function would assist tumors to grow in adverse conditions by increasing IRE1α/XBP1 pathway activation. In addition, we found that a p53 target, CDIP1, acts as a proapototic signal mediator in conditions of ER stress [4]. The CDIP1/BAP31 complex was shown to transduce ER stress-mediated apoptotic signaling to the mitochondria and activate the intrinsic apoptosis pathway, suggesting that loss of p53 function may contribute to the survival of cancer cells by enhancing ER function as well as hindering ER stress-mediated apoptosis signaling. The loss of p53 function in relation to ER stress is likely to have important consequences for the sustenance of cancer cell proliferation. Previous studies have demonstrated that the activation of p53 suppresses mTOR activity and consequently inhibits protein translation [5, 6]. The elimination of wild-type p53 function may therefore provide an opportunity for cancer cells to further promote unregulated proliferation, via elevated protein synthesis and a subsequent increase in ER function. We have revealed that wild-type p53 directly interacts with synoviolin (SYVN1) and can thereby stimulate SYVN1-dependent degradation of IRE1α. The loss of p53 disrupts SYVN1-mediated IRE1α downregulation, producing an ER stress-resistant phenotype by increased IRE1α/XBP1 pathway activity [3]. However, although the loss of p53 function facilitates enhanced ER function to manage high ER stress, this also generates an overreliance on IRE1α in cancer cells with p53 mutations. Indeed, we have found that pharmacological inhibition of IRE1α strongly suppresses protein secretion and potently induces cell death in such p53-deficient cancer cells [3]. Importantly, the IRE1α inhibitor exhibited significant inhibitory effects against p53-deficient human tumors in vivo compared to those with wild-type p53. These results illuminate an important proof-of-concept for targeting tumors with mutant p53 via inhibition of the IRE1α/XBP1 pathway. Malignant transformation occurs through a combination of oncogenic pathway activation and the loss of tumor suppression. Understanding the consequences and identifying vulnerabilities caused by such changes can provide promising opportunities to selectively target cancer cells. Mutation in the p53 gene is the most frequent genetic alteration found in human cancer and inactivation of p53 is known as one of the strongest driving factors in cancer development and chemoresistance [7]. The new finding that loss of p53 activates the IRE1α/XBP1 pathway leading to enhanced ER function broadens our understanding of how cancer cells with mutant p53 survive, and also highlights a possibility for the development of novel therapeutic strategies.

Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) via a direct protein—protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.

CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.

Abstract 2640: Small molecule compound NCI-8 induces ERK2-dependent mutant-p53 protein degradation

Abstract The tumor suppressor p53 is mutated in over 50% of human cancers leading not only to loss of wild-type p53 function, but also to gain-of-function (GOF) mutant p53 which acts as an oncogene. Mutant p53 is expressed at very high levels and can inhibit residual wild-type p53 activity as well as the function of p53 family member proteins such as p73 or p63. Therefore, targeting mutant p53 by either restoring the p53 pathway or depleting its GOF is an attractive strategy for cancer therapy. NCI-8 is a small molecule compound with dual abilities to induce p53 signal pathway and destabilize mutant p53 protein (deplete GOF) in mutant p53 expressing colorectal cancer cells. It remains unclear how NCI-8 regulates mutant p53 protein degradation. ERK2 is a member of the MAP kinase family which plays a critical role in regulating cell growth and differentiation by phosphorylating substrates including wild-type p53. We demonstrate that NCI-8-induces mutant p53 protein degradation via activation of ERK2 signaling. We observed a sustained phosphorylation of ERK2 upon NCI-8 treatment of cancer cells, but not in normal cells. MEK inhibitor U0126 treatment completely blocked NCI-8-mediated phosphorylation of ERK2 in cancer cells. These results, taken together, suggest that NCI-8 activates the ERK2 signaling pathway in cancer cells. There was a correlation between mutant p53 degradation and phosphorylation of ERK2 in cancer cells treated with NCI-8. We further examined the role of ERK2 phosphorylation in NCI-8- mediated mutant p53 protein degradation. Cancer cells were treated with U0126 to block ERK2 phosphorylation or transiently transfected with siRNA to knockdown ERK2. U0126 treatment or knockdown of ERK2 rescued mutant p53 from NCI-8-mediated protein degradation, suggesting that NCI-8-mediated phosphorylation of ERK2 is required for mutant p53 degradation. We further found that U0126 treatment inhibited NCI-8 induction of p21, puma and Noxa expressions in cancer cells. Consistently, transient over-expression of exogenous mutant p53 blocked NCI-8-mediated restoration of p53 pathway signaling in mutant-p53 expressing cancer cells. These results suggest that NCI-8 restores p53 signal pathway via ERK2-dependent mutant p53 protein degradation. Correlated with the U0126-rescue of mutant p53 protein, the percentage of cells with sub-G1 content induced by NCI-8 is decreased in response to U0126 treatment in cancer cells, suggesting that cell death induced by NCI-8 depends on ERK2-mediated mutant p53 degradation. Furthermore, combinational indices showed that U0126 antagonized NCI-8- induced cell death, but no antagonism was found in combinational treatment of NCI-8 and EGFR inhibitors in cancer cells. Our data indicate an important role of NCI-8-phosphorylated ERK2 in regulation of mutant p53 protein degradation and cell death in cancer cells, and provide a rationale for clinical testing of NCI-8 and ERK2 pathway-related factors in cancer therapy. Citation Format: Shengliang Zhang, Lanlan Zhou, David T. Dicker, Wafik S. EL-Deiry. Small molecule compound NCI-8 induces ERK2-dependent mutant-p53 protein degradation. [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 2640. doi:10.1158/1538-7445.AM2015-2640

Reactivating p53 functions by suppressing its novel inhibitor iASPP: a potential therapeutic opportunity in p53 wild-type tumors.

Although mutational inactivation of p53 is found in 50% of all human tumors, a subset of tumors display defective p53 function, but retain wild-type (WT) p53. Here, direct and indirect mechanisms leading to the loss of WT p53 activities are discussed. We summarize the oncogenic roles of iASPP, an inhibitor of WT p53, in promoting proliferation, invasion, drug or radiation-resistance and metastasis. From the therapeutic view, we highlight promising perspectives of microRNA-124, peptide and small molecules that reduce or block iASPP for the treatment of cancer. High iASPP expression enhances proliferation, aggressive behavior, the resistance to radiation/chemotherapy and correlates with poor prognosis in a range of human tumors. Overexpression of iASPP accelerates tumorigenesis and invasion through p53-dependent and p53-independent mechanisms. MicroRNA-124 directly targets iASPP and represses the growth and invasiveness of cancer cells. The disruption of iASPP-p53 interaction by a p53-derived peptide A34 restores p53 function in cancer cells. The inhibition of iASPP phosphorylation with small molecules induces p53-dependent apoptosis and growth suppression. The mechanisms underlying aberrant expression of iASPP in human tumors should be further investigated. Reactivating WT p53 functions by targeting its novel inhibitor iASPP holds promise for potential therapeutic interventions in the treatment of WT p53-containing tumors.

Genotype Versus Phenotype: The Yin and Yang of Germline TP53 Mutations in Li-Fraumeni Syndrome

Genotype Versus Phenotype: The Yin and Yang of Germline <i>TP53</i> Mutations in Li-Fraumeni Syndrome

Can we be SMaRT-er in our approach to cancer therapy?

Can we be SMaRT-er in our approach to cancer therapy?

Trans-splicing repair of mutant p53 suppresses the growth of hepatocellular carcinoma cells in vitro and in vivo.

Reactivation of wild-type p53 (wt-p53) function is an attractive therapeutic approach to p53-defective cancers. An ideal p53-based gene therapy should restore wt-p53 production and reduces mutant p53 transcripts simultaneously. In this study, we described an alternative strategy named as trans-splicing that repaired mutant p53 transcripts in hepatocellular carcinoma (HCC) cells. The plasmids which encoded a pre-trans-splicing molecule (PTM) targeting intron 6 of p53 were constructed and then transfected into HCC cells carrying p53 mutation. Phenotypic changes of HCC cells induced by p53-PTM were analyzed through cell cycle, cell apoptosis and the expression of p53 downstream target genes. Spliceosome mediated RNA trans-splicing (SMaRT) reduced mutant p53 transcripts and produced functional wt-p53 protein after the delivery of p53-PTM plasmids, which resulted in phenotype correction of HCC cells. In tumor xenografts established by p53-mutated HCC cells, adenovirus encoding p53-PTM induced cell cycle arrest and apoptosis and then blocked the growth of tumors in mice. Collectively, our results demonstrated for the first time that mutant p53 transcripts were functionally corrected in p53-defective HCC cells and xenografts using trans-splicing, which indicated the feasibility of using trans-splicing to repair p53 mutation in p53-defective cancers.

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.

Comparison of effects of p53 null and gain-of-function mutations on salivary tumors in MMTV-Hras transgenic mice.

p53 is an important tumor suppressor gene which is mutated in ~50% of all human cancers. Some of these mutants appear to have acquired novel functions beyond merely losing wild-type functions. To investigate these gain-of-function effects in vivo, we generated mice of three different genotypes: MMTV-Hras/p53+/+, MMTV-Hras/p53-/-, and MMTV-Hras/p53R172H/R172H. Salivary tumors from these mice were characterized with regard to age of tumor onset, tumor growth rates, cell cycle distribution, apoptotic levels, tumor histopathology, as well as response to doxorubicin treatment. Microarray analysis was also performed to profile gene expression. The MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H mice displayed similar properties with regard to age of tumor onset, tumor growth rates, tumor histopathology, and response to doxorubicin, while both groups were clearly distinct from the MMTV-Hras/p53+/+ mice by these measurements. In addition, the gene expression profiles of the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors were tightly clustered, and clearly distinct from the profiles of the MMTV-Hras/p53+/+ tumors. Only a small group of genes showing differential expression between the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors, that did not appear to be regulated by wild-type p53, were identified. Taken together, these results indicate that in this MMTV-Hras-driven salivary tumor model, the major effect of the p53 R172H mutant is due to the loss of wild-type p53 function, with little or no gain-of-function effect on tumorigenesis, which may be explained by the tissue- and tumor type-specific properties of this gain-of-function mutant of p53.

Adenovirus-Mediated E2F-1 Gene Transfer Augments Gemcitabine-Induced Apoptosis in Human Colon Cancer Cells.

E2F-1 is a transcription factor that stimulates cellular proliferation and cell cycle progression. E2F-1 alone is sufficient to stimulate cells to initiate DNA synthesis, and this unscheduled entry into S phase is a potent trigger of apoptosis. Gemcitabine, a novel pyrimidine analogue with structural and metabolic similarities to cytarabine, also can efficiently induce apoptosis, especially for cancer cells that are already in S phase. Gemcitabine has established antitumor activity against solid tumors, including head and neck, ovarian, and non-small cell lung cancers. Therefore, we hypothesized that exogenous E2F-1 expression could accumulate cells in the S phase and thus sensitize them to gemcitabine. We constructed an adenoviral vector (AdCMVE2F-1) to transduce the exogenous E2F-1 gene into human cancer cells. Infection of human colon cancer cells with AdCMVE2F-1 resulted in the overexpression of E2F-1 mRNA and protein in a dose-dependent manner and consequently induced accumulation in S phase as measured by FACS analysis. To assess the synergistic antitumor effect of AdCMVE2F-1 and gemcitabine, the human colon cancer cel lines SW620, DLD-1, and LoVo were infected with AdCMVE2F-1 at various multiplicities of infection and then exposed to various concentrations of gemcitabine 24 hours after infection. Isobologram analysis showed that E2F-1-transduced cancer cells exhibited higher sensitivity to gemcitabine treatment compared to control virus-infected cells. Treatment with AdCMVE2F-1 plus gemcitabine enhanced endogenous p53 expression in LoVo cells, which contain wild-type p53; however, the finding that the synergistic effect can also be observed in mutant p53-expressing SW620 and DLD-1 cells suggests that wild-type p53 function may not be necessary for the therapeutic effects of this drug combination. Conclusions: Our data demonstrate that overexpression of ectopic E2F-1 protein may render cels more sensitive to gemcitabine-mediated apoptosis, an outcome that has important general implications for the treatment of human cancer.