Loss Of Wild-type P53 Function Research Articles

Targeting the Mevalonate Pathway in the Mitochondria-Dependent Chemotherapy Resistance of TP53 Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) bearing mutations in the TP53 tumor suppressor gene is the most fatal of AMLs with a two-year overall survival between 0-15%. Patients with TP53 mutant (MT) AML typically respond to initial therapy but quickly relapse and die of disease due to chemotherapy resistance. We have undertaken to understand the mechanisms of chemotherapy resistance in TP53 MT AML. Initially, we purified AML blasts to 99% purity using flow sorting and performed RNA sequencing on TP53 MT and wildtype samples. Gene set enrichment analysis demonstrated that TP53 MT AML patient samples were enriched in the cholesterol synthesis or mevalonate pathway. This was validated using the BEAT AML dataset. Interestingly, p53 loss and/or mutation in breast, liver, colon and pancreatic cancer models leads to a dependency on an upregulated mevalonate pathway. Despite these data, the role of the mevalonate pathway in TP53 MT AML is unknown. We have subsequently explored the role of the mevalonate pathway in TP53 MT AML chemotherapy resistance. Experiments were performed in CRISPR-engineered TP53 mutated human AML cells with isogenic controls. Treatment with DNA damaging agents and analysis for cell survival confirmed that loss of wildtype p53 function enhanced cell survival. Recent results have demonstrated that chemotherapy resistance in AML cells is associated with an increase in oxidative phosphorylation (OXPHOS), which is dynamically induced by DNA damage. We confirmed that TP53 MT AML cells increase OXPHOS after DNA damage (Figure 1). Treatment of cells with rosuvastatin, a potent inhibitor of the mevalonate pathway, blocked this increase in OXPHOS and induced chemosensitivity. Lipidomic analysis demonstrates that the increase in OXPHOS correlates with an increase in coenzyme Q10 (CoQ10), a byproduct of the mevalonate pathway. Importantly, co-treatment of cells with a soluble form of mevalonate or a downstream byproduct, geranylgeranyl pyrophosphate (GGPP), recovered OXPHOS and subsequent chemoresistance. This indicates rosuvastatin's activity is specific to the mevalonate pathway, independent of cholesterol, and dependent on the GGPP branch of the pathway. Analysis is ongoing to determine the role of GGPP in regulating the mitochondrial response to DNA damage in TP53 MT cells, including if GGPP is required for CoQ10 synthesis as this remains a controversy in the literature. Overall, this data supports a model where TP53 MT AML cells dynamically upregulate the mevalonate pathway to regulate OXPHOS and avoid DNA damage-induced cell death (Figure 2). To determine if this model is physiologically relevant, we studied a patient derived xenograft (PDX) model of TP53 MT AML cells. Engrafted mice were treated with standard AML chemotherapeutic agent, cytarabine (AraC), for five days alone or in combination with high dose rosuvastatin. Interestingly, mice sacrificed three days after completing AraC administration continue to demonstrate an enhanced OXPHOS response. This response is blunted by co-treatment with rosuvastatin. As expected, TP53 MT AML cells respond modestly to AraC alone and this response was increased with the combination therapy. Overall, these results demonstrate that TP53 MT AML requires the mevalonate pathway for chemotherapy resistance and targeting of the mevalonate pathway in combination with chemotherapy may improve clinical responses. Through detailed biochemical analysis of this mechanism, we will elucidate novel therapeutic approaches to TP53 MT AML. Figure Legend: (1) Oxygen consumption rate measured by seahorse technology in indicated cell lines and conditions. Statistics performed with unpaired T-test in GraphPad with stars indicating significance as per figure. (2) Proposed schema of mevalonate pathway dependencies in TP53 mutant (MT) AML. Bold arrows indicate increased byproduct dependency in TP53 MT AML cells treated with DNA damaging chemotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A p53 transcriptional signature in primary and metastatic cancers derived using machine learning.

The tumor suppressor gene, TP53, has the highest rate of mutation among all genes in human cancer. This transcription factor plays an essential role in the regulation of many cellular processes. Mutations in TP53 result in loss of wild-type p53 function in a dominant negative manner. Although TP53 is a well-studied gene, the transcriptome modifications caused by the mutations in this gene have not yet been explored in a pan-cancer study using both primary and metastatic samples. In this work, we used a random forest model to stratify tumor samples based on TP53 mutational status and detected a p53 transcriptional signature. We hypothesize that the existence of this transcriptional signature is due to the loss of wild-type p53 function and is universal across primary and metastatic tumors as well as different tumor types. Additionally, we showed that the algorithm successfully detected this signature in samples with apparent silent mutations that affect correct mRNA splicing. Furthermore, we observed that most of the highly ranked genes contributing to the classification extracted from the random forest have known associations with p53 within the literature. We suggest that other genes found in this list including GPSM2, OR4N2, CTSL2, SPERT, and RPE65 protein coding genes have yet undiscovered linkages to p53 function. Our analysis of time on different therapies also revealed that this signature is more effective than the recorded TP53 status in detecting patients who can benefit from platinum therapies and taxanes. Our findings delineate a p53 transcriptional signature, expand the knowledge of p53 biology and further identify genes important in p53 related pathways.

TP53 mutations in head and neck cancer.

Head and neck squamous cell carcinomas (HNSCCs) arising in the mucosal linings of the upper aerodigestive tract are highly heterogeneous, aggressive, and multifactorial tumors affecting more than half a million patients worldwide each year. Classical etiological factors for HNSCC include alcohol, tobacco, and human papillomavirus (HPV) infection. Current treatment options for HNSCCs encompass surgery, radiotherapy, chemotherapy, or combinatorial remedies. Comprehensive integrative genomic analysis of HNSCC has identified mutations in TP53 gene as the most frequent of all somatic genomic alterations. TP53 mutations are associated with either loss of wild-type p53 function or gain of functions that promote invasion, metastasis, genomic instability, and cancer cell proliferation. Interestingly, disruptive TP53 mutations in tumor DNA are associated with aggressiveness and reduced survival after surgical treatment of HNSCC. This review summarizes the current evidence and impact of TP53 mutations in HNSCC.

Abstract A15: Elucidating the role of p53 in the cellular origins of pancreatic cancer development

Abstract The tumor suppressor gene TP53 encodes a transcriptional activator critical for suppressing pancreatic ductal adenocarcinoma (PDAC). TP53 mutations, which are most frequently missense mutations in the DNA binding domain, result in both loss of wild-type p53 function and gain of novel, oncogenic functions. Mouse models of PDAC relying on oncogenic Kras and p53 mutation have suggested that either pancreatic acinar or ductal cells, the two major epithelial cell types in the pancreas, may be the cell of origin for PDAC, although there is very limited understanding of the molecular pathways through which p53 mutations contribute to PDAC driven by oncogenic Kras. Moreover, the cellular functions of p53 involved in tumor suppression remain enigmatic. To set up a platform for deconstructing p53 loss-of-function (LOF) and gain-of-function (GOF) pathways, we have generated mouse models to study p53 function in adult mice, mimicking the context of human cancer, using tamoxifen-inducible Cre recombinases to activate Kras (KrasLSL-G12D) in adult mouse pancreatic acinar (Ptf1aCreER) and ductal (Sox9CreER) compartments, in the backdrop of different p53 alleles. To investigate p53 LOF, we generated mouse cohorts with wild-type p53 expression or conditional p53 knockout (p53fl/fl). To examine p53 GOF, we generated mouse cohorts with conditional expression of structural mutant p53R172H (p53LSL-R172H/fl), contact mutant p53R270H (p53LSL-R270H/fl) and conditional p53 knockout (p53null/fl) for comparison in a uniform p53 heterozygous genetic background of stromal tissues. Our tumor studies have demonstrated that oncogenic Kras expression and p53 knockout or mutation in acinar cells of adult mice leads to metastatic PDAC development with complete penetrance. This suggests a novel role for p53 in tumor suppression by blocking cellular reprogramming of acinar cells to ductal-like cells. In contrast, these mutations drive PDAC from ductal cells less efficiently. To understand the origins of human PDAC, we are investigating the molecular pathways altered in mouse acinar and ductal cell-derived tumors. Through comparison with human pancreatic cancer data, our studies will elucidate the cellular origins of different molecular subtypes of human PDAC. Elucidating the mechanisms of PDAC development is critical to improving detection and therapy in PDAC patients. Citation Format: Brittany M. Flowers, Hang Xu, Kathryn Hanson, Christina Curtis, Hannes Vogel, Laura D. Wood, Laura D. Attardi. Elucidating the role of p53 in the cellular origins of pancreatic cancer development [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A15.

Abstract 3949: p53-restoring small molecule CB001 suppresses tumor growth and metastasis

Abstract The uncontrolled growth and metastasis of tumor cells leads to morbidity and mortality in patients with cancer. The tumor suppressor p53, as a guardian of genome integrity, plays an important role in suppressing tumor growth and metastasis. However, loss of p53 pathway signaling frequently occurs in advanced tumors. Dysfunction of the p53 pathway is due to either p53 gene mutation or inactivation of p53. Mutations not only result in loss of wild-type p53 function, but also endow mutant p53 with a gain-of function, which further confers tumor aggressiveness and metastasis. Restoring p53 pathway signaling is an attractive strategy for suppressing tumor growth and metastasis in cancer therapy. Small-molecule CB001 restores p53 pathway signaling in p53-deficient colorectal cancer cells. We examined p53 pathway signaling based on the p53-responsive bioluminescence and the protein level of p53 targets. CB001 restores p53 pathway signaling in a p53-dependent and -independent manner in cancer cells. CB001 restores p53 pathway signaling via p73 in mutant p53-expressing cancer cells, and activates the p53 pathway in wild-type p53-expressing cancer cells. Correlated with restoration of p53 pathway signaling, CB001 induces cell death in cancer cells, but not in normal cells. Neither phosphorylation of H2AX in cancer cells nor DNA intercalation was observed upon CB001 treatment at the tested doses, suggesting little or no genotoxicity of CB001 in human colorectal cancer cells. Furthermore, both cell scratch assay and Boyden chamber assay reveal that CB-001 inhibits cancer cell migration. We further demonstrate that CB001 induces cell death and inhibits cell migration via p73 in p53-mutant colorectal cancer cells. These results taken together suggest that CB001 is a promising lead for drug development with a strong capability to suppress tumor growth and metastasis through restoring p53 pathway in cancer cells. Citation Format: Shengliang Zhang, Lanlan Zhou, Jessica Wagner, David T. Dicker, Wafik S. El-Deiry. p53-restoring small molecule CB001 suppresses tumor growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3949.

Abstract A28: Understanding the role of the tumor suppressor p53 in pancreatic cancer development

Abstract In the next decade, pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer-related death. This high rate of mortality is attributed to late-stage diagnosis and lack of effective therapeutic strategies. The tumor-suppressor gene TP53 is mutated in ~75% of PDAC patients. The most frequently observed TP53 mutations in PDAC occur within the DNA binding domain at codons 175 and 273. TP53 mutations may induce loss of wild-type p53 function and gain of novel, oncogenic functions. We aim to use different mouse models of PDAC to identify the molecular pathways deregulated when wild-type p53 is lost and mutant p53 is expressed, to elucidate the molecular mechanisms driving PDAC development. While previous studies have suggested that point mutant p53 drives gain-of-function phenotypes, our studies of aging Pdx1-Cre;KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato mice with conditional p53 knockout (p53Flox/Flox or p53Flox/Null) or p53 mutation (p53LSL-R172H/Flox or p53LSL-R172H/Null) did not demonstrate a clear mutant p53 gain-of-function phenotype in tumor latency. We hypothesize that tumor latency is too rapid in these models to manifest a clear gain-of-function phenotype, requiring the establishment of different model systems. Indeed, our preliminary studies using tumor-derived p53R172H cell lines and isogenic p53 null cell lines suggest a role for mutant p53 gain of function in later-stage tumorigenesis, specifically in metastatic seeding. Furthermore, we are evaluating the role of the putative PDAC cells of origin, pancreatic acinar and ductal cells, in PDAC development. Using both tumor-derived mouse cell lines and pancreatic lineage-specific genetically engineered mouse models of PDAC, we can evaluate the consequences of loss of wild-type p53 expression and gain of mutant p53 expression in different pancreatic compartments. Ultimately, using these systems we aim to elucidate the target genes and molecular pathways controlled by wild-type p53 or by mutant p53 to regulate PDAC initiation. Understanding the molecular mechanisms driving PDAC initiation is critical for the development of new therapeutic strategies for early detection and effective intervention in PDAC patients. Citation Format: Brittany M. Flowers, Patty B. Garcia, Barbara M. Grüner, Monte M. Winslow, Laura D. Attardi. Understanding the role of the tumor suppressor p53 in pancreatic cancer development [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A28.

Frequency of Somatic TP53 Mutations in Combination with Known Pathogenic Mutations in Colon Adenocarcinoma, Non–Small Cell Lung Carcinoma, and Gliomas as Identified by Next-Generation Sequencing

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer. It encodes p53, a DNA-binding transcription factor that regulates multiple genes involved in DNA repair, metabolism, cell cycle arrest, apoptosis, and senescence. TP53 is associated with human cancer by mutations that lead to a loss of wild-type p53 function as well as mutations that confer alternate oncogenic functions that enable them to promote invasion, metastasis, proliferation, and cell survival. Identifying the discrete TP53 mutations in tumor cells may help direct therapies that are more effective. In this study, we identified the frequency of individual TP53 mutations in patients with colon adenocarcinoma (48%), non–small cell lung carcinoma (NSCLC) (36%), and glioma/glioblastoma (28%) at our institution using next-generation sequencing. We also identified the occurrence of somatic mutations in numerous actionable genes including BRAF, EGFR, KRAS, IDH1, and PIK3CA that occurred concurrently with these TP53 mutations. Of the 480 tumors examined that contained one or more mutations in the TP53 gene, 219 were colon adenocarcinomas, 215 were NSCLCs, and 46 were gliomas/glioblastomas. Among the patients positive for TP53 mutations diagnosed with colon adenocarcinoma, 50% also showed at least one mutation in pathogenic genes of which 14% were BRAF, 33% were KRAS, and 3% were NRAS. Forty-seven percent of NSCLC patients harboring TP53 mutations also had a mutation in at least one actionable pathogenic variant with the following frequencies: BRAF: 4%, EGFR: 10%, KRAS: 28%, and PIK3CA: 4%. Fifty-two percent of patients diagnosed with glioma/glioblastoma with a positive TP53 mutation had at least one concurrent mutation in a known pathogenic gene of which 9% were CDKN2A, 41% were IDH1, and 11% were PIK3CA.

Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21)-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy) results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation) retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and the majority (>60%) exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate) triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT) as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays.

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.

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

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.

Evidence for Mutant p53 Gain-of-Function Effects in Normal Haemopoietic Cells and Myc-Driven Lymphoma

Deregulated c-MYC expression and mutations in p53 are among the most common changes detected in human cancer. It is now established that mutant p53 proteins confer a poor prognosis in human cancer through both loss of wild-type p53 activity as well as various proposed gain-of-function properties. The specific role of mutant p53 in MYC-driven tumorigenesis is not known. The Eμ-Myc mouse model carries a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma (BL). These mice develop aggressive pre-B or B cell lymphomas and ~20% of those tumours exhibit p53 mutations. We have shown that MYC-driven lymphomas are exquisitely dependent on the pro-survival BCL-2 family member MCL-1 such that loss of a single allele of Mcl-1 leads to dramatic tumour regression and prolonged animal survival. Interestingly, we found that this dependency on MCL-1 is reduced, but not completely ablated, by the presence of a p53 mutation. This suggests an important role for mutant p53 in the sustained survival of MYC-driven lymphomas. We are investigating the effects of five different mutant mouse p53 proteins (V170M, I192S, G280, R246Q, R270H) on tumour initiation, sustained growth and chemoresistance in the Eμ-Myc mouse model.We are further examining the effect of p53 mutations on MCL-1 dependence by using a floxed Mcl-1 gene and a tamoxifen-inducible Cre-recombinase in established Eμ-Myc lymphomas. Preliminary data suggest that both loss of wild-type p53 function as well as retroviral over-expression of mutant p53 can compensate for reduced levels of MCL-1 (loss of one Mcl-1 allele). The underlying mechanisms for this are under investigation.The role of mutant p53 in lymphoma cell survival has been further examined in Eμ-Myc lymphoma-derived cell lines. Enforced over-expression of mutant p53 in cell lines containing wild-type p53 impaired induction of apoptosis by Nutlin3A, an inhibitor of Mdm-2 (the major negative regulator of p53). Remarkably, Nutlin-3a-induced apoptosis was impaired although it caused substantial transcriptional induction of the p53 apoptosis effectors, Puma and Noxa. Importantly, different mutant p53 proteins conferred different levels of protection against cell death. The observed protection against cell death may be partly due to dominant-negative effects of mutant p53, however, it does not appear to be robust enough to account for the extent of cell survival. Furthermore, mutant p53 conferred resistance to docetaxol, which is thought to induce cell death through predominantly p53-independent mechanisms. These data suggest that mutant p53 can protect against both p53-dependent and p53-independent cell death processes. Conversely, transcriptional induction of Noxa and Puma implies that “p53-restoration therapy” may remain a feasible treatment strategy even in tumours that bear mutations in p53 and that the role of a dominant-negative effect for some mutant p53 proteins may be less important than previously considered, at least in lymphoma cells.We are also examining the effect of mutant p53 on lymphoma development utilizing a hematopoietic reconstitution model and retroviral over-expression of mutant p53 proteins. The different mutant p53 proteins investigated exhibited distinct effects during tumorigenesis. The R246Q mutant p53 protein markedly accelerated lymphoma development in the context of MYC over-expression. The R246Q mutant p53 protein demonstrated strong selection in p53-deficient (p53-/-) hematopoietic cells during reconstitution indicative of an advantageous activity in emergency hematopoiesis.Overall, these findings provide evidence for a positive oncogenic role of mutant p53 in hematopoietic cells that provides a particularly potent selective advantage in the context of MYC driven lymphoma development. Importantly, different p53 mutations exhibit different functional properties such that different p53 mutations are likely to be associated with distinct risk in human malignant disease. DisclosuresNo relevant conflicts of interest to declare.

Tumor suppressor p53 and its gain-of-function mutants in cancer

Tumor suppressor p53 plays a pivotal role in tumor suppression. p53 is the most frequently mutated gene in cancer. As a transcription factor, p53 mainly exerts its role in tumor suppression through transcriptional regulation of its downstream target genes. Thus, p53 and its target genes form a complex p53 signaling pathway to regulate a wide variety of biological processes to prevent tumorigenesis. Recent studies have revealed that in addition to apoptosis, cell cycle arrest and senescence, p53's functions in the regulation of energy metabolism and anti-oxidant defense contribute significantly to its role in tumor suppression. Studies further show that many tumor-associated mutant p53 proteins not only lose tumor suppressive functions of wild-type p53, but also gain new oncogenic activities that are independent of wild-type p53, including promoting tumor cell proliferation, survival, metabolic changes, angiogenesis, and metastasis, which are defined as mutant p53 gain-of-function. The frequent loss of wild-type p53 function and the gain-of-function of mutant p53 in human tumors make p53 an extremely attractive target for cancer therapy. Different strategies and many small-molecule drugs are being developed for the p53-based tumor therapy. Here, we review the mechanisms of p53 in tumor suppression and gain-of-function mutant p53 in tumor development, as well as the recent advances in the development of the p53-based tumor therapy.

Increased Oxidative Damage in Carriers of the Germline TP53 p.R337H Mutation

Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early onset cancers. In Brazil, a variant form of LFS/LFL is commonly detected because of the high prevalence of a founder mutation at codon 337 in TP53 (p.R337H). The p53 protein exerts multiple roles in the regulation of oxidative metabolism and cellular anti-oxidant defense systems. Herein, we analyzed the redox parameters in blood samples from p.R337H mutation carriers (C, n = 17) and non-carriers (NC, n = 17). We identified a significant increase in erythrocyte GPx activity and in plasma carbonyl content,an indicator of protein oxidative damage, in mutation carriers compared to non-carriers (P = 0.048 and P = 0.035, respectively). Mutation carriers also showed a four-fold increase in plasma malondialdehyde levels, indicating increased lipid peroxidation (NC = 40.20±0.71, C = 160.5±0.88, P<0.0001). Finally, carriers showed increased total antioxidant status but a decrease in plasma ascorbic acid content. The observed imbalance could be associated with deregulated cell bioenergetics and/or with increased inflammatory stress, two effects that may result from loss of wild-type p53 function. These findings provide the first evidence that oxidative damage occurs in carriers of a germline TP53 mutation, and these may have important implications regarding our understanding of the mechanisms responsible for germline TP53 p.R337H mutation-associated carcinogenesis.

N-methyl-N'-nitro-N-nitrosoguanidine에 의한 인체백혈병세포의 G2/M arrest 유발에서 Cdk inhibitor p21(WIP1/CIP1)의 관련성

본 연구에서는 monofunctional alkylating agent인 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 에 의한 인체 백혈병 U937 세포의 증식억제에 관한 기전 확인하였다. MNNG에 의한 U937 세포의 증식억제는 세포주기 G2/M arrest 및 apoptosis 유방과 연관이 있었으며, MNNG 는 G2/M기 조절에 관여하는 주요 cyclin 및 Cdk들의 발현 수준에는 큰 영향이 없었으나 cyclin B1 및 Cdk2-associated kinase의 활성을 매우 저하시켰다. MNNG 처리로 Cdk inhibit p2l(WAF1/CIP1)이 전사 및 번역 수준에서 발연이 증가되었으며, p21 promoter 의 활성도 증가되었다. p21 promoter deletion constructs을 이용한 연구에서 MNNG의 responsive element 부위는 전사 개시 부위 113-61 부근임을 확인하였다. 이 결과들은 MNNG에 의한 cyclin/Cdk 복합체의 kinase 활성 저하가 p53 비의존적인 p21의 활성 증가에 기인한 것임을 보여주는 것이며, 이는 MNNG의 암세포에서의 항암기전을 이해하는 귀중한 자료로서 제공될 것으로 기대된다. In this paper, to elucidate the further mechanisms of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced growth arrest, we investigated the effect of MNNG on cell cycle and proliferation in U937 cells, a p53-null human myeloid leukemia cell line. It was found that MNNG causes an arrest at the G2/M phase of the cell cycle and induces apoptosis, which is closely correlated to inhibition of cyclin B1 and cyelin-dependent kinase (Cdk) 2-associated kinase activities. MNNG treatment in. creased protein and mRNA levels of the Cdk inhibitor p21(WAF1/CIP1), and activated the reporter construct of a p21 promoter. By using p21 promoter deletion constructs, the MNNG-responsive element was mapped to a region between 113 and 61 relative to the transcription start site. These data indicate that in U937 cells MNNG can circumvent the loss of wild-type p53 function and induce critical downstream regulatory events leading to transcriptional activation of p21. Present results indicate that the p53-independent up-regulation of p21 by MNNG is likely responsible for the inhibition of cyclin/Cdk complex kinase activity rather than the down-regulation of cyclins and Cdks expression. These novel phenomena have not been previously described and provide important new insights into the possible biological effects of MNNG.

Mutant p53R270H gain of function phenotype in a mouse model for oncogene‐induced mammary carcinogenesis

In human breast cancer, mutations in the p53 gene are associated with poor prognosis. However, analysis of patient data so far did not clarify, whether missense point mutations in the p53 gene, in addition to causing loss of wild-type p53 function, also confer a gain of function phenotype to the encoded mutant p53. As heterogeneity of patient material and data might obscure a clear answer, we studied the effects of a coexpressed mutant p53(R270H) in transgenic mice in which SV40 early proteins initiate the development of mammary adenocarcinoma (WAP-T mice). In such tumors the endogenous wild-type p53 is functionally compromised by complex formation with SV40 T-antigen, thereby constituting a loss of wild-type p53 function situation that allowed analysis of the postulated gain of function effects of mutant p53(R270H). We found that mutant p53(R270H) in bi-transgenic mice enhanced the transition from intraepithelial neoplasia to invasive carcinoma, resulting in a higher frequency of invasive carcinoma per gland and per mouse, a more severe tumor phenotype, and more frequent pulmonary metastasis. Surprisingly, mutant p53(R270H) in this system does not increase genomic instability. Therefore, other postulated gain of function activities of mutant p53 must be responsible for the effects described here.

Frequent Overexpression of STK15/Aurora-A/BTAK and Chromosomal Instability in Tumorigenic Cell Cultures Derived From Human Ovarian Cancer

The STK15 (also known as Aurora-A/BTAK) gene localized on chromosome 20q13 and encoding a centrosome-associated serine/threonine kinase is amplified and overexpressed in multiple human tumor cell types. Overexpression of this gene is involved in tumorigenic transformation, induction of centrosome duplication-distribution abnormalities, and aneuploidy in mammalian cells. To examine the potential role of STK15 in ovarian tumorigenesis, its mRNA and protein expression status were examined in cells grown in culture from 15 ovarian cancer specimens using semiquantitative RT-PCR and Western blot analysis. Normal ovarian surface tissues and the near diploid nontumorigenic breast epithelial cell line MCF10 were used as controls. The status of STK15 correlated with transformation-associated cellular phenotypes including tumorigenicity in nude mice, p53 expression level, and chromosomal ploidy. For chromosome ploidy analyses, FISH was carried out with direct fluorescence-labeled a-satellite probes for chromosome 3 and 17. STK15 mRNA was found overexpressed in 10 of the 15 ovarian cancer cell cultures. Five of these cell cultures revealed a truncated form of the STK15 protein with a molecular mass of 36 kDa. When tested for tumorigenicity in nude mice, 9 of the 10 cell cultures that overexpressed STK15 mRNA formed tumors in nude mice, while only one of the five cell cultures with no overexpression did. Cells overexpressing STK15 mRNA showed significant correlation with chromosome 3 polysomy. Six of the 13 (46%) cell cultures analyzed for p53 expression revealed overexpression of p53 and five of these six (83%) also overexpressed STK15. Four of the remaining seven cultures (57%) with overexpression of STK15 revealed minimal or no expression of p53. These results demonstrate that overexpression of STK15 significantly correlates with nude mice tumorigenicity and chromosomal aneuploidy in human ovarian cancer cells grown in vitro. Additionally, cells overexpressing STK15 also revealed frequent coordinate loss of wild-type p53 function manifested either as highly expressed intense staining reflective of a mutant form of p53 or almost complete absence of p53 staining. Overexpression of STK15 with coordinate loss of wild-type p53 function thus appears to play an important role in ovarian tumorigenesis and offers a novel molecular target in designing effective therapy of human ovarian cancer.

P53 accumulation in favorable-histology Wilms tumor is associated with angiogenesis and clinically aggressive disease

Background/Purpose: Unfavorable histology (UH) in Wilms tumor has been linked to malfunction of the p53 tumor suppressor gene, which regulates (1) the endogenous angiogenesis suppressor thrombospondin-1 (TSP-1) and (2) vascular endothelial growth factor (VEGF). The authors hypothesized that clinically aggressive favorable histology Wilms tumor (FH), like UH, but distinct from standard-risk FH disease, would display altered p53/TSP-1 function and upregulated angiogenesis. Methods: Three Wilms tumor specimens manifesting different histology and clinical behavior were obtained: clinically aggressive UH, clinically aggressive FH, and standard-risk FH disease. Xenografts were induced intrarenally in athymic mice. P53, TSP-1, and VEGF status and neovascularity were assessed in tumor tissues. Lungs were evaluated for metastasis. Results: Clinically aggressive FH Wilms tumor displayed progressive alteration in p53/TSP-1 status and upregulation of VEGF. Such alteration was observed in the UH tumor, but was absent from the standard-risk FH tumor. Xenografts from clinically aggressive tumors displayed brisk neoangiogenesis and yielded lung metastases. Conclusions: This is the first report of altered p53/TSP-1 function in association with clinically aggressive behavior in FH Wilms tumor. These characteristics were not observed in parallel studies of a nonaggressive FH tumor. Loss of wild-type p53 function may contribute to disease progression in FH Wilms tumor, in part by upregulation of VEGF.

Multiparameter flow cytometry for simultaneous assessment of p53 protein expression and cellular DNA content in oral squamous cell carcinomas: evidence for the development of aneuploid clones from p53-deficient diploid progenitor cells.

Diploid tumour cells regularly continue to progress after the development of aneuploid cell populations in head and neck squamous cell carcinomas. The coexistence of aneuploid clones with their diploid progenitor cells provides a unique opportunity to study the order of appearance of p53 mutation and aneuploidy in the same tumour. Multiparameter flow cytometry was therefore applied to 22 oral squamous cell carcinomas to simultaneously assess cellular DNA content and p53 protein expression on a single-cell basis. Concurrent measurements of cytokeratin expression served to identify tumour cells of epithelial origin. One of 5 diploid and 2 of 17 aneuploid carcinomas were p53-negative. For 15 p53-positive aneuploid tumours, overexpression of p53 protein was identified for the aneuploid clones as well as for coexisting diploid tumour cell populations in 14 cases. On the understanding that coexisting diploid and aneuploid tumour cell populations have a common clonal origin, these results provide evidence that aneuploid tumour clones typically develop from p53-deficient diploid progenitor cells. Loss of wild-type p53 function may therefore contribute to the development of aneuploidy in head and neck cancer.