P53 Hotspot Mutants Research Articles

P53-R273H upregulates neuropilin-2 to promote cell mobility and tumor metastasis

Mounting evidence indicates that hotspot p53 mutant proteins often possess gain-of-function property in promoting cell mobility and tumor metastasis. However, the molecular mechanisms are not totally understood. In this study, we demonstrate that the hotspot mutation, p53-R273H, promotes cell migration, invasion in vitro and tumor metastasis in vivo. p53-R273H significantly represses expression of DLX2, a homeobox protein involved in cell proliferation and pattern formation. We show that p53-R273H-mediated DLX2 repression leads to upregulation of Neuropilin-2 (NRP2), a multifunctional co-receptor involved in tumor initiation, growth, survival and metastasis. p53-R273H-induced cell mobility is effectively suppressed by DLX2 expression. Furthermore, knockdown of NRP2 significantly inhibits p53-R273H-induced tumor metastasis in xenograft mouse model. Together, these results reveal an important role for DLX2-NRP2 in p53-R273H-induced cell mobility and tumor metastasis.

Abstract LB-030: Specific missense mutations in TP53 elicit contrasting biochemical and biological outcomes affecting prostate cancer progression

Abstract Prostatic adenocarcinoma (PCa) remains the most frequently diagnosed cancer type and the third leading cause of cancer death in men in the United States. While organ-confined disease is manageable, treatment options for disseminated PCa remain limited. First-line therapy for metastatic PCa functions through targeting the androgen receptor (AR) signaling axis, as AR activity is required for tumor maintenance. However, while treatment is initially effective, these tumors invariably recur, displaying reactivation of AR signaling despite continued therapeutic targeting. Moreover, no durable treatment currently exists for this ultimately fatal disease stage, termed castrate-resistant PCa (CRPC). Significantly, recent high profile, genome-wide studies have revealed that missense mutations in the TP53 gene occur in nearly all cancer types, and these mutations frequently occur at specific TP53 “hotspots”. Interestingly, enrichment of certain hotspot p53 mutants specifically occurs in PCa. While the underlying causes of PCa-enriched mutations remain undefined, their presence elicits contrasting, pro-oncogenic biochemical and biological phenotypes. Unpublished data suggests that mutant p53 isoforms act in a mutation-specific fashion to drive PCa progression through alterations in the canonical activity of p53, identified through characterization of their related transcriptomes and cistromes. Biologically, these mutant p53 isoforms also confer differential proliferative and survival phenotypes. Moreover they act in a mutation-dependent fashion in response to castration therapy both in vitro and in vivo. Mechanistically, pro-oncogenic mutant p53 appears to manipulate both conventional and novel pathways leading to PCa progression. These findings both corroborate and expand the recent findings that mutations in p53 drive cancer progression, and further suggest a critical role for p53 mutations as regulators of disease progression in PCa. Citation Format: Jennifer J. McCann, Jeffry L. Dean, Renee De Leeuw, Matthew J. Schiewer, Christopher M. McNair, Karen E. Knudsen. Specific missense mutations in TP53 elicit contrasting biochemical and biological outcomes affecting prostate cancer progression [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-030. doi:10.1158/1538-7445.AM2017-LB-030

Abstract AP16: THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS

Abstract BACKGROUND: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism underlying metastasis is still not clear. Twist1 is a transcriptional factor that promotes epithelial-mesenchymal transition (EMT) and enhances the migration and tumor-initiation of ovarian cancer cells. It also has been associated with cancer stem cells and chemoresistance. The purpose of our study is to better understand the regulation of Twist1 and to identify molecular pathways that can be targeted in order to control metastatic diseases. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We hypothesize that through their interaction, p53 promotes the proteasomedependent degradation of Twist1 and consequently inhibits EMT in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p53's ability to inhibit Twist1, leading to enhanced EMT and metastasis. METHOD: In ubiquitin assay p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Coimmunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Plasmid constructs containing different Twist1 mutants were created to select the degradation resistant mutation. ΔC-Twist1 (C-terminal deletion) was used in in vivo study. RESULT: 1) In EOC cells, proteasome-dependent degradation suppressed Twist1 protein. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In ovarian cancer cells, wild type p53 overexpression reduced Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to enhance Twist1 degradation. E3 ligase, Pirh2, formed complex with p53 and Twist1 to induce Twist1 degradation. 2) ΔC-Twist1 was resistant to p53-regulated degradation. In xenograft mouse model, EOC cells overexpressing ΔC-Twist1 could form more aggressive tumors with increased numbers of metastatic lesions than wild type Twist1 group. 3) In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. Nine samples with high levels of wild type p53 all showed low or no Twist1 expression. CONCLUSION: Our data revealed a mechanism by which Twist1 is regulated through p53-promoted proteasome-dependent degradation. p53 facilitates the formation of a Twist1-p53-Pirh2 complex and the Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS [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 AP16.

AFB1 hepatocarcinogenesis is via lipid peroxidation that inhibits DNA repair, sensitizes mutation susceptibility and induces aldehyde-DNA adducts at p53 mutational hotspot codon 249.

Aflatoxin B1 (AFB1) contamination in the food chain is a major cause of hepatocellular carcinoma (HCC). More than 60% of AFB1 related HCC carry p53 codon 249 mutations but the causal mechanism remains unclear. We found that 1) AFB1 induces two types of DNA adducts in human hepatocytes, AFB1-8,9-epoxide-deoxyguanosine (AFB1-E-dG) induced by AFB1-E and cyclic α-methyl-γ-hydroxy-1,N2-propano-dG (meth-OH-PdG) induced by lipid peroxidation generated acetaldehyde (Acet) and crotonaldehyde (Cro); 2) the level of meth-OH-PdG is >30 fold higher than the level of AFB1-E-dG; 3) AFB1, Acet, and Cro, but not AFB1-E, preferentially induce DNA damage at codon 249; 4) methylation at –CpG- sites enhances meth-OH-PdG formation at codon 249; and 5) repair of meth-OH-PdG at codon 249 is poor. AFB1, Acet, and Cro can also inhibit DNA repair and enhance hepatocyte mutational sensitivity. We propose that AFB1-induced lipid peroxidation generated aldehydes contribute greatly to hepatocarcinogenesis and that sequence specificity of meth-OH-PdG formation and repair shape the codon 249 mutational hotspot.

The dominant-negative interplay between p53, p63 and p73: A family affair.

The tumor suppression activity of p53 is frequently impaired in cancers even when a wild-type copy of the gene is still present, suggesting that a dominant-negative effect is exerted by some of p53 mutants and isoforms. p63 and p73, which are related to p53, have also been reported to be subjected to a similar loss of function, suggesting that a dominant-negative interplay might happen between p53, p63 and p73. However, to which extent p53 hotspot mutants and isoforms of p53, p63 and p73 are able to interfere with the tumor suppressive activity of their siblings as well as the underlying mechanisms remain undeciphered. Using yeast, we showed that a dominant-negative effect is widely spread within the p53/p63/p73 family as all p53 loss-of-function hotspot mutants and several of the isoforms of p53 and p73 tested exhibit a dominant-negative potential. In addition, we found that this dominant-negative effect over p53 wild-type is based on tetramer poisoning through the formation of inactive hetero-tetramers and does not rely on a prion-like mechanism contrary to what has been previously suggested. We also showed that mutant p53-R175H gains the ability to inhibit p63 and p73 activity by a mechanism that is only partially based on tetramerization.

Abstract SY04-03: Improving survival by exploiting tumor dependence on stabilized mutant p53 in mouse models

Abstract Missense mutations in p53 generate aberrant proteins with abrogated tumour suppressor functions that can also acquire oncogenic gain-of-function activities that promote malignant progression, invasion, metastasis and chemoresistance (1-5). Mutant p53 (mutp53) proteins undergo massive constitutive stabilization specifically in tumours, which is the key requisite for the acquisition of gain-of- functions activities (6-8). Although currently 11 million patients worldwide live with tumours expressing highly stabilized mutp53, it is unknown whether mutp53 is a therapeutic target in vivo. Here we use a novel mutp53 knockin mouse model expressing a conditional inactivatable R248Q hotspot mutation (called floxQ; present in human cancers) to show that tumours depend on sustained mutp53 protein expression. Upon Tamoxifen-induced mutp53 ablation, allotransplanted and autochthonous tumours curb their growth, thus extending animal survival by 37%, and advanced tumours undergo apoptosis and tumour regression or stagnation. The HSP90/HDAC6 chaperone machinery, which is significantly upregulated in cancer compared with normal tissues, is a major determinant of mutp53 stabilization (9-12). We show that long-term HSP90 inhibition significantly extends the survival of hotspot mutp53 Q/- (R248Q allele; Ref 2) and H/H (R172H allele; Ref 3) mice by 59% and 48%, respectively, but not their corresponding p53 -/- (also known as Trp53 -/-) littermates. This mutp53-dependent drug effect occurs in tumor-bearing H/H mice treated with 17DMAG/SAHA, and in H/H and Q/- mice treated with the potent Hsp90 inhibitor ganetespib (the latter given as a once-weekly monotherapy). Notably, drug activity correlates with induction of mutp53 degradation, tumour cell apoptosis and strong prevention of T-cell lymphomagenesis. Together, these findings support the notion that tumors expressing mutp53 depend on it for tumor survival and maintenance, and fundamentally differ in their oncogenic wiring from p53-null tumors. Moreover, and surprisingly, the mechanism of action of these pleiotropic Hsp90 inhibitors in this specific setting is largely via targeting mutp53. Missense mutp53 is highly expressed in ∼50% of all human tumors. One therapeutic strategy, considered for nearly two decades, is to develop small compounds capable of restoring the lost wildtype function of mutp53 proteins (13). In the face of this daunting and still elusive goal, the results presented here show that eliminating stabilized mutp53 protein has positive therapeutic effects in vivo, even in the absence of a wildtype allele, since such tumors show exploitable dependence on its gain-of-function. Moreover, targeting Hsp90 might represent the first and currently only viable clinical strategy to achieve this goal. Overall, these proof-of-principle data identify mutp53 as a potentially actionable cancer-specific drug target.

Abstract LB-352: p53-promoted Twist1 degradation inhibits EMT in ovarian cancer cells

Abstract Background: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism behind ovarian cancer metastasis is still not clear. Twist1 is associated with the increased ability of migration and tumor-initiation of ovarian cancer cells; therefore, understanding the regulation of Twist is critical for the prevention of metastatic disease. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We propose that p53 functions as a normal inhibitor of Twist1. We hypothesize that through their interaction, p53 promotes the proteasome-dependent degradation of Twist1 and consequently inhibits epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p53's ability to inhibit Twist1, leading to enhanced EMT and metastasis. Method: p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Co-immunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Result: Wild type p53 overexpression reduces Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to regulate Twist1 degradation. E3 ligase, Pirh2, forms complex with p53 and Twist1 to induce Twist1 degradation. In EOC cells, Twist1 is suppressed by proteasome-dependent degradation. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. The 9 samples with high levels of wild type p53 all showed low or no Twist1 expression. Conclusion: Our data revealed a mechanism by which a crucial EMT inducer, Twist1, is regulated by wild type p53 through enhancing its proteasome-dependent degradation. p53 promotes the formation of a Twist1-p53-Pirh2 complex and facilitates Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. p53-promoted Twist1 degradation inhibits EMT in ovarian cancer cells. [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 LB-352.

Abstract 3688: The C-terminal domain of tumor-derived mutant p53 is required for its oncogenic gain-of-function activity

Abstract p53 is the most commonly mutated gene in human cancers. Unlike other tumor suppressors in which loss of protein expression is common, the vast majority of changes in p53 arise as point mutations. Tumor associated mutation hotspots are localized to the DNA binding domain, either in residues involved in direct DNA contact or those that regulate its conformation. In either case, such mutations have been shown to confer gain of function activity. This was first shown in soft agar assays in which over-expression of mutant p53 increased transformation of cells on a p53 null background. Furthermore, knock-in mouse models of mutant p53 showed alterations in tumor spectrum with the development of carcinomas not seen in p53 null mice. However, the mechanism by which mutant p53 actively aids in tumorigenesis remains unclear. Although mutant p53 causes loss of sequence specific DNA binding via the core domain, it retains the ability to interact with multiple proteins including transcriptional cofactors shown to have tumor suppressor activity such as p300 and CBP. These interactions occur through multiple protein binding domains including the C-terminal domain of p53. Our laboratory and others have shown that C-terminal truncation of wildtype p53 results in multiple deficiencies. Binding of mutant p53 to p300/CBP via the C-terminus is therefore an attractive hypothesis to explain mutant p53 gain of function. To determine the role of the C-terminus in mutant p53 gain of function activity, we have expressed constructs containing a C-terminal truncation in combination with varying hotspot mutations. Transfection of mutant p53 into a p53 null cell line was required for the formation of colonies in a soft agar assay, indicating an increase in tumorigenicity. Furthermore, C-terminal truncation of the mutant p53 abrogated this phenotype suggesting that this domain is required for mutant p53 gain of function. This was independent of expression levels as shown by immunoblot. We have also extended the findings of mutant p53 interaction with p300 by IP analysis of a panel of cell lines with varying p53 hotspot mutations. To confirm the role of the C-terminus in mutant p53 gain of function, we are developing a xenograft model to detect changes in tumorigenicity in vivo. These results will therefore explore the mechanism by which mutant p53 acts as an oncogene. Citation Format: Caleb C. Lee, James Manfredi. The C-terminal domain of tumor-derived mutant p53 is required for its oncogenic gain-of-function activity. [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 3688.

Mutant p53 and mTOR/PKM2 regulation in cancer cells

Mutations of TP53 gene are the most common feature in aggressive malignant cells. In addition to the loss of the tumor suppressive role of wild-type p53, hotspot mutant p53 isoforms display oncogenic proprieties notoriously referred as gain of functions (GOFs) which result in chemoresistance to therapies, genomic instability, aberrant deregulation of cell cycle progression, invasiveness and enhanced metastatic potential, and finally, in patient poor survival rate. The identification of novel functional oncogenic pathways regulated by mutant p53 represent and intriguing topic for emerging therapies against a broad spectrum of cancer types bearing mutant TP53 gene. Mammalian target of rapamycin (mTOR), as well as pyruvate kinase isoform M2 (PKM2) are master regulators of cancer growth, metabolism, and cell proliferation. Herein, we report that GOF mutant R175H and R273H p53 proteins trigger PKM2 phosphorylation on Tyr 105 through the involvement of mTOR signaling. Our data, together with the newly discovered connection between mutant p53 and mTOR stimulation, raise important implications for the potential therapeutic use of synthetic drugs inhibiting mTOR/PKM2 axis in cancer cells bearing mutant TP53 gene. We further hypothesize that mTOR/PKM2 pathway stimulation serves to sustain the oncogenic activity of mutant p53 through both the enhancement of chemoresistance and of aerobic glycolysis of cancer cells. © 2016 IUBMB Life, 68(9):722-726, 2016.

P53 Deletion or Hotspot Mutations Enhance mTORC1 Activity by Altering Lysosomal Dynamics of TSC2 and Rheb.

The activity of mammalian target of rapamycin complex 1 (mTORC1) is frequently enhanced in carcinomas, an effect thought to contribute to the malignant phenotype. Here, it is demonstrated that either deletion or mutation of TP53 in colon or lung carcinoma cells substantially enhances mTORC1 kinase activity by an effect downstream of and independent of AMPK. Mechanistically, it was determined that loss or mutation of p53 decreased expression of TSC2 and Sestrin2 (SESN2). Complementation of p53 null cells with TSC2 or Sestrin2 reduced mTORC1 activity to levels found in p53 wild-type (wt) cells, whereas their genetic depletion enhanced mTORC1 activity in p53 wt cells. However, the primary causal event in enhanced mTORC1 activity upon loss of p53 appeared to be a diminished distribution of TSC2 to lysosomal membranes containing mTOR. Subsequently, there was increased Rheb in the lysosomal compartment, and a higher mTOR association with Raptor. Transfection of TSC2 into p53 null cells replaced TSC2 and diminished Rheb at the lysosome, recapitulating cells with wt p53. In contrast, transfection of Sestrin2 decreased mTOR in lysosomes, but the lower levels of Sestrin2 in p53 null cells did not change lysosomal mTOR. In summary, loss of the transcriptional activity of p53, either by deletion or by key mutations in the DNA-binding domain, diminishes expression of TSC2 and Sestrin2, thus, shifting membrane-bound TSC2 out of lysosomal membranes, increasing lysosomal Rheb and increasing the kinase activity of mTORC1. This study establishes that loss of p53 function decreases lysosomal TSC2 and increases lysosomal Rheb resulting in hyperactive mTORC1, findings that are consistent with a more malignant phenotype.

Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities.

Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.

Abstract 2523: Strong synergistic effects with APR-246 and cisplatin in p53-mutant lung cancer cells

Abstract Background: Platinum compounds have been used as first-line treatment for many solid tumors including non small cell (NSCLC) and small cell (SCLC) lung cancer. However, patients with lung cancer often develop resistance to platinum compounds and eventually die of chemotherapy refractory disease. Mutation in the tumor suppressor protein p53 is common in lung cancer, ranging from 33% in adenocarcinomas to 70% in SCLC (The p53 website, http://p53.free.fr), and is one of the main causes for resistance to chemotherapy. APR-246 (PRIMA-1MET) is the first compound in clinical development that reactivates mutant p53 by inducing its wild type conformation thus triggering apoptosis (Lambert et al. Cancer Cell 15, 2009). APR-246 has yielded promising results in a first-in-human clinical trial in patients with hematological malignancies and prostate cancer (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing. Previously we have shown strong synergy with APR-246 and platinum compounds in p53-mutant drug-resistant ovarian cancer cells (AACR abstract # 3448, 2013). Moreover, APR-246 completely restored the sensitivity of cisplatin to resistant p53-mutant ovarian cancer cells (AACR abstract #1801, 2014). The aim of the current study was to investigate whether strong synergistic effect can also be observed in p53-mutant lung cancer cells. Methods: Cell viability was determined with FMCA or Cell Titer-Glo assay, p53 gene status by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Combination Index (CI) was calculated according to Additive model. Results: We observed strong synergistic effect (CI<0.5) with APR-246 and cisplatin in lung cancer cell lines carrying homozygous p53 hotspot mutations; NCI-H1770 (R248W), NCI-H1975 (R273H), NCI-H596 (G245C), PC-14 (R248W) and PC-14/CDDP (R248W). All these cell lines expressed a high level of p53. Synergistic (CI<0.8) or strong synergistic effect was found in lung cancer cell lines NCI-H378 (Y163C) and NCI-2087 (V157F) with homozygous mutations that occur frequently but are not hotspot mutations. NCI-2087 cells with the smoking-related V157F mutation express a high level of p53, while NCI-H378 cells express lower level. Mixed antagonist/additive/synergistic effects were observed in the p53 null cell line HOP-62, which does not express p53. Conclusions: Treatment with APR-246 in combination with cisplatin resulted in strong synergistic effect in both NSCL and SCLC cancer cells. Strongest synergies were observed in lung cancer cells with p53 hotspot mutations expressing a high level of p53. Studies to investigate the molecular mechanisms underlying the synergistic effects are ongoing. Our results suggest that combination treatment with APR-246 and platinum drugs may allow an improved therapy of p53-mutant lung cancer. Citation Format: Nina Mohell, Åsa Fransson, Jessica Alfredsson, Mikael von Euler, Ulf Björklund, Lars Abrahmsen. Strong synergistic effects with APR-246 and cisplatin in p53-mutant lung cancer cells. [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 2523. doi:10.1158/1538-7445.AM2015-2523

TP53: an oncogene in disguise.

The standard classification used to define the various cancer genes confines tumor protein p53 (TP53) to the role of a tumor suppressor gene. However, it is now an indisputable fact that many p53 mutants act as oncogenic proteins. This statement is based on multiple arguments including the mutation signature of the TP53 gene in human cancer, the various gains-of-function (GOFs) of the different p53 mutants and the heterogeneous phenotypes developed by knock-in mouse strains modeling several human TP53 mutations. In this review, we will shatter the classical and traditional image of tumor protein p53 (TP53) as a tumor suppressor gene by emphasizing its multiple oncogenic properties that make it a potential therapeutic target that should not be underestimated. Analysis of the data generated by the various cancer genome projects highlights the high frequency of TP53 mutations and reveals that several p53 hotspot mutants are the most common oncoprotein variants expressed in several types of tumors. The use of Muller's classical definition of mutations based on quantitative and qualitative consequences on the protein product, such as ‘amorph', ‘hypomorph', ‘hypermorph' ‘neomorph' or ‘antimorph', allows a more meaningful assessment of the consequences of cancer gene modifications, their potential clinical significance, and clearly demonstrates that the TP53 gene is an atypical cancer gene.

Insufficient sex description of cells supplied by commercial vendors.

evidence supporting sex as a biologically important variable in human and animal studies has been accumulating over recent decades ([1][1], [4][2], [6][3]–[8][4], [10][5]). As a result, the necessity of including female as well as male subjects in clinical studies has been widely recognized ([1][1

Gain-of-Function Mutant p53 Enhances Hematopoietic Stem Cell Self-Renewal

The tumor suppressor p53 is a critical regulator of hematopoietic stem cell (HSC) behavior and we demonstrated that p53 maintains HSC quiescence and regulates HSC response to irradiation (Liu et al., Cell Stem Cell, 2009).While TP53 mutations are less common in acute myeloid leukemia (5 to 8%) than in solid tumors (50%), they are associated with poor prognosis and abnormal cytogenetics, especially abnormalities in chromosomes 5 and 7. These mutations may abolish some but not necessarily all of the functions of p53 in regulating stem cell behavior. Therefore, this aspect of p53 function needs further investigation.To define the role of mutant p53 in the pathogenesis of AML, we introduced 9 hot-spot p53 mutants identified in AML patients, including p53R248W, p53R273H and p53Y220C, into wild type hematopoietic cells using retrovirus-mediated transduction and investigated the role of these p53 mutants in regulating HSC self-renewal. We found that hematopoietic cells expressing p53R248W, p53Y220C or p53R273H show enhanced repopulating potential 16 weeks following transplantation. As codon 248 of the p53 protein is most frequently mutated in AML, we decided to investigate the role of p53R248W mutant in HSCs by using the humanized knock-in mice of p53R248W. In p53 knockout mice, there is a dramatic increase of HSCs (CD48-CD150+Lin-Sca1+c-Kit+ cells); however,we found thatboth wild type andp53R248W mice have similar number of HSCs. While wild type p53 maintains HSC quiescence, expression of p53R248W in HSCs (CD48-CD150+LSKs) does not affect their quiescent state. Asp53R248W does not appear to affect HSC frequency and quiescence, it is not a loss-of-function mutant. We also used bone marrow cells isolated from both wild type and p53R248W mice to perform the serial replating assays and found that expressing p53R248W from the endogenous Trp53 promoter enhances the replating potential of hematopoietic cells. Moreover, we performed serial bone marrow repopulation (BMT) assays and found that the repopulating ability of p53R248W cells was significantly higher than that of the wild type cells in both primary and secondary BMT assays, demonstrating that the p53R248W mutant enhances HSC self-renewal in vivo. Furthermore, we observed that HSCs expressing p53R248W are resistant to genotoxic stress induced by irradiation and the p53R248W mice show extended survival following sub-lethal dose of total body irradiation. Ample data indicate that mutant p53 proteins not only lose their tumor suppressive functions, but also gain new abilities that promote tumorigenesis. To understand how mutant p53 enhances HSC self-renewal, we performed gene expression profiling assays by using HSCs isolated from wild type and p53R248W mice. We also utilized Ingenuity Pathway analysis software to group putative mutant p53 target genes into different pathways. While we did not observe change in the expression of p53 target genes in p53R248W HSCs, several pathways that are important for leukemogenesis, including epigenetic and DNA damage repair pathways, are altered in HSCs expressing p53R248W, demonstrating that p53R248W is a gain-of-function mutant. Given that TP53 mutations are correlated with poor prognosis, pharmacological inhibition of mutant p53 may be a promising therapeutic strategy for AML patients with TP53 mutations. Small molecule PRIMA-1 has been shown to restore wild-type conformation to some mutant p53 proteins and induce apoptosis in human tumor cells. We found that hematopoietic cells expressing mutant p53 are sensitive to PRIMA-1 treatment and undergo p53-dependent apoptosis. Furthermore, we observed that PRIMA-1 inhibits the growth of primary human AML cells with TP53 mutation in a dosage-dependent manner.Taken together, we demonstrated that gain-of-function mutant p53 enhances hematopoietic stem cell self-renewal through regulating epigenetic and DNA damage repair pathways. Our data also suggest that pharmacological inhibition of mutant p53 may sensitize the drug-resistant leukemia stem cells (LSCs) to chemotherapy and improves leukemia treatment. DisclosuresNo relevant conflicts of interest to declare.

Slow repair of lipid peroxidation-induced DNA damage at p53 mutation hotspots in human cells caused by low turnover of a DNA glycosylase.

Repair of oxidative stress- and inflammation-induced DNA lesions by the base excision repair (BER) pathway prevents mutation, a form of genomic instability which is often observed in cancer as ‘mutation hotspots’. This suggests that some sequences have inherent mutability, possibly due to sequence-related differences in repair. This study has explored intrinsic mutability as a consequence of sequence-specific repair of lipid peroxidation-induced DNA adduct, 1, N6-ethenoadenine (εA). For the first time, we observed significant delay in repair of ϵA at mutation hotspots in the tumor suppressor gene p53 compared to non-hotspots in live human hepatocytes and endothelial cells using an in-cell real time PCR-based method. In-cell and in vitro mechanism studies revealed that this delay in repair was due to inefficient turnover of N-methylpurine-DNA glycosylase (MPG), which initiates BER of εA. We determined that the product dissociation rate of MPG at the hotspot codons was ≈5–12-fold lower than the non-hotspots, suggesting a previously unknown mechanism for slower repair at mutation hotspots and implicating sequence-related variability of DNA repair efficiency to be responsible for mutation hotspot signatures.

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

Background:Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development.Methods:To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53lox/lox/FAK+/+/WapCre, p53lox/lox/FAKflox/+/WapCre and p53lox/lox/FAKflox/−/WapCre mice, and mice with WapCre-mediated conditional expression of p53R270H, the mouse equivalent of human p53R273H hot spot mutation, together with conditional deletion of FAK, P53R270H/+/FAKlox/+/WapCre and p53R270H/+/FAKflox/−/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours.Results:Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53R270H-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures.Conclusions:Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53R270H mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53R270H-induced mammary tumour development.

Distinct Rayleigh Scattering from Hot Spot Mutant p53 Proteins Reveals Cancer Cells

The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor.

A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis.

Unequal prognostic potentials of p53 gain-of-function mutations in human cancers associate with drug-metabolizing activity

Mutation of p53 is the most common genetic change in human cancer, causing complex effects including not only loss of wild-type function but also gain of novel oncogenic functions (GOF). It is increasingly likely that p53-hotspot mutations may confer different types and magnitudes of GOF, but the evidences are mainly supported by cellular and transgenic animal models. Here we combine large-scale cancer genomic data to characterize the prognostic significance of different p53 mutations in human cancers. Unexpectedly, only mutations on the Arg248 and Arg282 positions displayed significant association with shorter patient survival, but such association was not evident for other hotspot GOF mutations. Gene set enrichment analysis on these mutations revealed higher activity of drug-metabolizing enzymes, including the CYP3A4 cytochrome P450. Ectopic expression of p53 mutant R282W in H1299 and SaOS2 cells significantly upregulated CYP3A4 mRNA and protein levels, and cancer cell lines bearing mortality-associated p53 mutations display higher CYP3A4 expression and resistance to several CYP3A4-metabolized chemotherapeutic drugs. Our results suggest that p53 mutations have unequal GOF activities in human cancers, and future evaluation of p53 as a cancer biomarker should consider which mutation is present in the tumor, rather than having comparison between wild-type and mutant genotypes.