High Incidence Of P53 Mutations Research Articles

Epithelial Mutant p53 Promotes Resistance to Anti-PD-1-Mediated Oral Cancer Immunoprevention in Carcinogen-Induced Mouse Models.

Simple SummaryImmune checkpoint blockade with anti-PD-1 antibodies blocks the development of oral squamous cell carcinomas (OSCCs) in preclinical models. Understanding whether the genetic alterations that accumulate during oral cancer development affect the response to PD-1 inhibitors is critical to identify patients who may benefit from immunoprevention interventions. Using genetically engineered mouse models that develop carcinogen-induced oral tumors that differ on the mutational status of the p53 gene, we demonstrated that expression of gain-of-function mutant p53 in the epithelial cells of the oral lesions promotes resistance to the immunopreventive effects of anti-PD-1. These novel findings could guide patient-specific strategies for oral cancer immunoprevention based on p53 profiling.Oral squamous cell carcinoma (OSCC) develops through the multistep malignant progression of squamous epithelium. This process can be prevented by PD-1 blockade in a mouse model for oral carcinogenesis. OSCCs exhibit a high incidence of p53 mutations that confer oncogenic gain-of-function (GOF) activities that promote resistance to standard therapies and poor clinical outcomes. To determine whether epithelial p53 mutations modulate anti-PD-1-mediated oral cancer immunoprevention, we generated mouse models for oral carcinogenesis by exposing mice carrying epithelial-specific p53 mutations to the carcinogen 4NQO. Consistent with the oncogenic functions of mutant p53, mice with OSCCs expressing the p53R172H GOF mutation developed higher metastasis rates than mice with loss-of-function (LOF) p53 deletion or with wild-type p53. Throughout oral cancer progression, pre-invasive and invasive lesions showed a gradual increase in T-cell infiltration, recruitment of immunosuppressive regulatory T-cells (Tregs), and induction of PD-1/PD-L1 immune checkpoint proteins. Notably, while PD-1 blockade prevented the development of OSCCs in mice with wild-type p53 or p53 deletion, GOF p53R172H abrogated the immunopreventive effects of anti-PD-1, associated with upregulation of IL17 signaling and depletion of exhausted CD8 cells in the microenvironment of the p53R172H tumors. These findings sustain a potential role for p53 profiling in personalized oral cancer immunoprevention.

Saikosaponin-d, a calcium mobilizing agent, sensitizes chemoresistant ovarian cancer cells to cisplatin-induced apoptosis by facilitating mitochondrial fission and G2/M arrest.

Cisplatin (CDDP) and its derivatives are first line anti-cancer drugs for ovarian cancer (OVCA). However, chemoresistance due to high incidence of p53 mutations leads to poor clinical prognosis. Saikosaponin-d (Ssd), a saponin from a herbal plant extract, has been shown to induce cell death and sensitize chemoresistant cells to chemotherapeutic agents. Here, we demonstrated that Ssd sensitized chemoresistant OVCA cells with either p53-wt, -mutant and -null to CDDP. The action of Ssd appears to be through induction of mitochondrial fragmentation and G2/M arrest. Ssd is mediated via calcium signaling, up-regulation of the mitochondrial fission proteins Dynamin-related protein 1 (Drp1) and optic atrophy 1 (Opa1), and loss in mitochondrial membrane potential (MMP). Moreover, in the presence of CDDP, Ssd also down-regulates protein phosphatase magnesium-dependent 1 D (PPM1D) and increases the phosphorylation of checkpoint protein kinases (Chk) 1, cell division cycle 25c (Cdc25c) and Cyclin dependent kinase 1 (Cdk1). Our findings suggest that Ssd could sensitize OVCA to CDDP independent of the p53 status through multiple signaling pathways. They support the notion that Ssd may be a novel adjuvant for the treatment of chemoresistant OVCA.

Chemoprevention of spontaneous ovarian cancer in the domestic hen

The hen is an attractive animal model for in vivo testing of agents that thwart ovarian carcinogenesis because ovarian cancer in the domestic hen features clinical and molecular alterations that are similar to ovarian cancer in humans, including a high incidence of p53 mutations. The objective of the study was to test the potential ovarian cancer chemopreventive effect of the p53 stabilizing compound CP-31398 on hens that spontaneously present the ovarian cancer phenotype. Beginning at 79 wk of age, 576 egg-laying hens (Gallus domesticus) were randomized to diets containing different amounts of CP-31398 for 94 wk, 5 d, comprising a control group (C) (n = 144), which was fed a diet containing 0ppm (mg/kg) of CP-31398; a low-dose treatment (LDT) group (n = 144), which was fed a diet containing 100ppm of CP-31398; a moderate-dose treatment (MDT) group (n = 144) which was fed a diet containing 200ppm of CP-31398; and a high-dose treatment (HDT) group (n = 144), which was fed a diet containing 300ppm of CP-31398. Hens were killed at 174 wk of age to determine the incidence of ovarian and oviductal adenocarcinomas. Whereas the incidence of localized and metastatic ovarian cancers in the MDT and HDT groups was significantly lower (up to 77%) compared to levels in the C and LDT groups (P < 0.05), the incidence of oviductal cancer was unaffected by CP-31398. CP-31398 appears to be an effective tool for chemoprevention against ovarian malignancies, but does not appear to affect oviductal malignancies.

Effect of amplification of xenobiotic detoxification pathways genes on survival in lung adenocarcinoma versus squamous cell carcinoma bases on the Cancer Genome Atlas provisional database.

e14093 Background: Ral interacting protein (RLIP76) is a stress-responsive anti-apoptotic efflux transporter of the mercapturic acid pathway (MPy). The MPy enzymes are an essential component of the broader xenobiotic detoxification pathways (XDP) that are regulated by p53 (TP53) in response to xenobiotic/oxidative stress. Homozygous Rlip knockout (Rlip-/-) mice are resistant to benzopyrene induced adenocarcinoma and have constitutive activation of p53. The cancer-resistant phenotype of Rlip-/- mice is the diametric opposite of p53 homozygous knockout mice (p53-/-) mice, which die before the age of 24 weeks of spontaneous malignancy. Methods: Our preliminary studies show that creating Rlip deficiency through pharmacological or genetic methods caused all p53-/- mice to remain cancer-free at an unprecedented age of 32 weeks. This degree of cancer prevention has never been observed with any previous pharmacological or genetic intervention in p53-/- mice. Transcriptomic and epigenomic profiling of the cancer-free p53-/- mice revealed that the direction of change in expression of a network of Rlip-linked XDP enzymes and signaling proteins was identical to that in the cancer-resistant Rlip-/- mice. Results: We hypothesized that the activity of this Rlip-linked subset of the XDP enzymes/signaling proteins (designated XMN1) is a determinant of carcinogenic susceptibility caused by p53 loss. To examine this possibility, we queried the TCGA (The Cancer Genome Atlas) database to determine whether components of XMN1 were differentially expressed in human malignancy, and whether this was associated with overall survival. We focused on non-small cell lung cancer because of the high incidence of p53 mutations in these cancers. To our surprise, we found amplification or upregulation of XMN1 components conferred a reduced survival in lung adenocarcinoma but prolonged survival in squamous cell carcinoma. Conclusions: The mechanisms underlying this remarkable diametrically opposite effect on prognosis of are unknown and suggest opposed functions of XMN1 in Squamous verses Adenocarcinoma.

P53 mutations cooperate with oncogenic Kras to promote adenocarcinoma from pancreatic ductal cells.

Pancreatic cancer is one of the most lethal malignancies, with virtually all patients eventually succumbing to their disease. Mutations in p53 have been documented in >50% of pancreatic cancers. Owing to the high incidence of p53 mutations in PanIN 3 lesions and pancreatic tumors, we interrogated the comparative ability of adult pancreatic acinar and ductal cells to respond to oncogenic Kras and mutant Tp53(R172H) using Hnf1b:CreER(T2) and Mist1:CreER(T2) mice. These studies involved co-activation of a membrane-tethered GFP lineage label, allowing for direct visualization and isolation of cells undergoing Kras and mutant p53 activation. Kras activation in Mist1(+) adult acinar cells resulted in brisk PanIN formation, whereas no evidence of pancreatic neoplasia was observed for up to 6 months following Kras activation in Hnf1beta(+) adult ductal cells. In contrast to the lack of response to oncogenic Kras alone, simultaneous activation of Kras and mutant p53 in adult ductal epithelium generated invasive PDAC in 75% of mice as early as 2.5 months after tamoxifen administration. These data demonstrate that pancreatic ductal cells, whereas exhibiting relative resistance to oncogenic Kras alone, can serve as an effective cell of origin for pancreatic ductal adenocarcinoma in the setting of gain-of-function mutations in p53.

Effects of prostaglandin E2on p53 mRNA transcription and p53 mutagenesis during T‐cell‐independent human B‐cell clonal expansion

Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM(+)IgD(+)CD27(-) B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID(+) cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5 × 10(-4)) greater than the baseline error rate (<0.8 × 10(-4)). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.

Characterization of p53 expression in rainbow trout

The tumour suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. Given the high incidence of p53 mutations in human cancers, it has been extensively studied, though only a small fraction of these investigations have been in non-mammalian systems. For the present study, an anti-rainbow trout p53 polyclonal antibody was generated. A variety of rainbow trout (Oncorhynchus mykiss) tissues and cell lines were examined through western blot analysis of cellular protein extracts, which revealed relatively high p53 levels in brain and gills. To evaluate the checkpoint response of rainbow trout p53, RTbrain-W1 and RTgill-W1 cell lines were exposed to varying concentrations of the DNA damaging agent bleomycin and ribonucleotide reductase inhibitor hydroxyurea. In contrast to mammals, these checkpoint-inducing agents provoked no apparent increase in rainbow trout p53 levels. These results infer the presence of alternate DNA damage checkpoint mechanisms in rainbow trout cells.

The dl1520 Virus Is Found Preferentially in Tumor Tissue after Direct Intratumoral Injection in Oral Carcinoma

The dl1520 Virus Is Found Preferentially in Tumor Tissue after Direct Intratumoral Injection in Oral Carcinoma

The cancer connection: BRCA1 and BRCA2 tumor suppression in mice and humans.

BRCA1 and BRCA2 mutations are estimated to be responsible for the great majority of familial breast and ovarian cancers. Much progress has been made toward the understanding of the function of these proteins through genetic, biochemical, and structural studies. The embryonic lethality encountered in the knockout mouse initially hindered the development of mouse models aimed at studying tumor suppression. However, mice that harbor hypomorphic Brca1 and Brca2 alleles and cre-mediated tissue-specific deletions for Brca1 and Brca2 have been generated. Mice deficient for either Brca1 or Brca2 sustain a wide range of carcinoma and mammary epithelium deleted for Brca1 or Brca2 is highly susceptible to mammary tumorigenesis. Mammary (and other) tumors occur at long latency as compared to oncogene-induced mouse tumors. p53 deficiency is highly cooperative with both Brca1 and Brca2 in promoting tumorigenesis. Analysis of Brca1-associated mammary tumors reveals significant similarities to BRCA1-associated breast cancer in regard to high tumor grade, hormone receptor negativity, a high incidence of p53 mutations and genetic instability.

P63α and ΔNp63α can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes

The p53 tumor suppressor protein plays a critical role in the regulation of the cell cycle and apoptosis. The importance of p53's functions is underscored by the high incidence of p53 mutations in human cancers. Recently, two p53-related proteins, p73 and p63, were identified as members of the p53 gene family. Multiple isoforms of p73 have been found, including DeltaN variants in which the N-termini are truncated. p63 is expressed as three major forms, p63alpha, p63beta and p63gamma, each of which differ in their C-termini. All three forms can be alternatively transcribed from a cryptic promoter located within intron 3, producing DeltaNp63alpha, DeltaNp63beta and DeltaNp63gamma. The high degree of similarity of p73 and p63 to evolutionarily conserved regions of p53 suggests that these proteins play an important and potentially redundant role in regulating cell cycle arrest and apoptosis. Here we describe the characterization of cell lines generated to inducibly express p63alpha and DeltaNp63alpha. We have found that p63alpha and DeltaNp63alpha can differentially regulate endogenous p53 target genes and induce cell cycle arrest and apoptosis. Deletion of the N-terminal 26 amino acids of DeltaNp63alpha abolished its ability to transactivate p53 target genes and induce cell cycle arrest and apoptosis. This indicates that a putative transactivation domain exists within the N-terminus of the DeltaN variants of p63. Furthermore, the differential regulation of p53 target genes by p63alpha and DeltaNp63alpha suggests that p63 and p53 utilize both similar and different signaling pathways to execute their cellular functions.

Restoration of p53 function in anaplastic Wilms' tumor

Recent studies have reported a high incidence of p53 mutations in anaplastic Wilms' tumors (WT). Restoration of the normal p53 state by current gene therapy techniques is thus an attractive potential mode of therapy for this tumor, which is poorly responsive to standard therapy. The purpose of this study is to determine whether gene delivery of normal p53 is possible and to characterize the subsequent effect of restoring the wild-type p53 state. Anaplastic WT RM1 cells (mutant p53) were transduced with replication-deficient adenoviral vectors containing either the wild-type p53 gene (rAd-p53) or the gene encoding a green fluorescent protein (rAd-GFP). The transduction efficiency of adenovirus for RM1 cells was determined by flow cytometric analysis of rAd-GFP-transduced cells. The effect of p53 transduction on cell viability was evaluated using a colorimetric proliferation assay. Apoptosis was evaluated by labeling DNA breaks using a TUNEL assay (Apo-Direct kit). Cells treated with increasing concentrations of viral particles relative to tumor cells (multiplicity of infection-MOI) showed a dose-dependent increase in the number of cells transduced. Twenty-four hours after viral treatment, the percentage of cells transduced for MOIs of 10, 50, 100, and 500 was 29.5, 60.9, 74.6, and 92.4, respectively; at 48 hours the percentage of cells transduced increased to 70.8, 90.7, 93. 7, and 96.3, respectively. Viral treatment at an MOI of 50 reduced cell proliferation by 10% at 17 hours and 97% at 5 days; at an MOI of 100, the relative reduction in proliferation was 15% and 99.8%, respectively. When assayed, 30% of cells became apoptotic at an MOI of 50, and 48% at an MOI of 100. Highly efficient delivery of the p53 tumor suppressor gene by adenoviral vector to anaplastic WT is possible. Subsequent restoration of the normal p53 state results in reduced viability and increased apoptosis. Gene replacement of p53 may represent a novel therapeutic agent for anaplastic Wilms' tumors.

Genetic background determines the response to adenovirus-mediated wild-type p53 expression in pancreatic tumor cells.

The development of new therapies is particularly urgent with regard to pancreatic tumors. Gene therapy approaches involving p53 replacement are promising due to the central role of p53 in the cellular response to DNA damage and the high incidence of p53 mutations in pancreatic tumors. Adenoviruses containing wild-type (wt) p53 cDNA (Ad5CMV-p53) were introduced into four human pancreatic cell lines to examine the impact caused by exogenous wt p53 on these cells. Introduction of wt p53 in mutant p53 cells (NP-9, NP-18, and NP-31) caused marked falls in cell proliferation and rises in the level of apoptosis. In contrast, overexpression of p53 did not induce apoptosis in NP-29 (wt p53). The presence of p16 contributes to the induction of apoptosis, as demonstrated by introduction of the wt p16 gene (Ad5RSV-p16). Analysis of cell cycle and apoptosis in etoposide-treated cells corroborated the inability of NP-29 to die by apoptosis, suggesting that this wt p53 cell line lacks p53 downstream functions in the apoptosis pathway. Taken together, our results indicate that the effects elicited by exogenous p53 protein depend upon the molecular alterations related to p53 actions on cell cycle and apoptosis. Therefore, knowledge of the genetic background of tumor cells is crucial to the development of efficient therapies based on the introduction of tumor suppressor genes.

Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas.

Glioblastoma multiforme, the most malignant human brain tumor, may develop de novo (primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). We present further evidence that primary and secondary glioblastomas constitute distinct disease entities which develop through the acquisition of different genetic alterations. We analyzed p53 mutations, p53 protein accumulation and epidermal growth factor receptor (EGFR) overexpression in 49 biopsies classified as primary or secondary glioblastoma according to clinical and histopathologic criteria. Patients with primary glioblastoma were selected on the basis of a clinical history of less than 3 months and histopathologic features of glioblastoma at the first biopsy (19 cases; mean age, 55 years). The diagnosis of secondary glioblastomas required at least two biopsies and clinical as well as histologic evidence of progression from low grade or anaplastic astrocytoma (30 cases; mean age, 39 years). DNA sequence analysis showed that p53 mutations were rare in primary glioblastomas (11%) while secondary glioblastomas had a high incidence of p53 mutations (67%), of which 90% were already present in the first biopsy. The incidence of p53 protein accumulation (nuclear immunoreactivity to PAb 1801) was also lower in primary (37%) than in secondary glioblastomas (97%). In contrast, immunoreactivity for the EGF receptor prevailed in primary glioblastomas (63%) but was rare in secondary glioblastomas (10%). Only one out of 49 glioblastomas showed EGFR overexpression and a p53 mutation. These data indicate that overexpression of the EGF receptor and mutations of the p53 tumor suppressor gene are mutually exclusive events defining two different genetic pathways in the evolution of glioblastoma as the common phenotypic endpoint.

Relationship between p53 mutation incidence in oral cavity squamous cell carcinomas and patient tobacco use.

It is well-established that a high incidence of p53 mutations exist in oral cavity squamous cell carcinomas (OCSCCs). To determine whether p53 mutations are etiologically associated with OCSCC development or are associated with exposure to specific carcinogens, we have analyzed the conserved regions of the p53 gene (exons 5-9) in 48 OCSCCs obtained from patients with varied tobacco and alcohol use histories by polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP) and DNA sequencing analysis. Thirty-eight percent (18/48) of the OCSCCs exhibited a mutation in exons 5-9 of the p53 gene. There was a significantly higher incidence of p53 mutations in OCSCCs from tobacco users (predominantly cigarette smokers) compared to those who had never used tobacco. No increase in the incidence of p53 mutation was observed in tobacco users who drank alcohol. G to A transitions and deletions were the predominant mutations observed in OCSCCs from tobacco users. No specific pattern of mutation was observed in OCSCCs from those subjects who had never used tobacco. These data suggest that a history of tobacco use was associated with a high incidence of p53 mutations in patients with OCSCC and that tobacco carcinogens include a specific pattern of mutations in oral cavity tissue in vivo.

P53 mutations and hepatitis B virus: cofactors in hepatocellular carcinoma.

We studied 80 hepatocellular carcinomas from three continents for p53 gene (TP53) mutations and hepatitis B virus (HBV) sequences. p53 mutations were frequent in tumors from Mozambique but not in tumors from South Africa, China, and Germany. Independent of geographic origin, most tumors were positive for HBV sequences. X gene coding sequences of HBV were detected in 78% of tumors, whereas viral sequences in the surface antigen- and core antigen-encoding regions were present in less than 45% of tumors. These observations indicate that hepatocellular carcinomas are genetically heterogeneous. Mozambican-type of hepatocellular carcinomas are characterized by a high incidence of p53 mutations related to aflatoxins. In other tumors, the rarity of p53 mutations combined with the frequent presence of viral X gene coding sequences suggests a possible interference of HBV with the wild-type p53 function.

MDS and AML with 17p deletion are associated to specific dysgranulopoiesis and a high incidence of p53 mutations

MDS and AML with 17p deletion are associated to specific dysgranulopoiesis and a high incidence of p53 mutations