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P53 and disease: When the guardian angel fails
Abstract
The p53 tumor suppressor gene (TP53) is mutated more often in human cancers than any other gene yet reported. Of importance, it is mutated frequently in the common human malignancies of the breast and colorectum and also, but less frequently, in other significant human cancers such as glioblastomas. There is also one inherited cancer predisposing syndrome called Li-Fraumeni that is caused by TP53 mutations. In this review, we discuss the significance of p53 mutations in some of the above tumors with a view to outlining how p53 contributes to malignant progression. We also discuss the usefulness of TP53 status as a prognostic marker and its role as a predictor of response to therapy. Finally, we outline some evidence that abnormalities in p53 function contribute to the etiology of other non-neoplastic diseases.
... Alterations of many signalling pathways such as PI3-kinase/AKT, Ras/Raf/MEK/ERK, WNT, APC, and p53 alone or in combinations, associate with CRC (Bahrami et al., 2018). Mutation or deletion of the tumor suppressor p53 is associated with up to 60% of CRC (Royds and Iacopetta, 2006). Alteration in these pathways often activates mammalian target of rapamycin (mTOR) kinase, a critical catalytic component of two distinct multi-protein complexes, mTORC1 and mTORC2 (Doherty and Baehrecke, 2018;Xie et al., 2016). ...
... The findings unearthed by this study is summarized in Figure 7. AFE equally sensitized HT29 cells which carry a negative dominant mutation (R273H) in the DNA binding domain of p53 (p53 R273H ) (Brosh and Rotter, 2009;Kaeser et al., 2004). This is significant because p53 mutation plays a critical determinant in the development as well as aggressiveness of CRC (Royds and Iacopetta, 2006;Solomon et al., 2018). p53-mutant CRC are resistant to 5FU or cisplatin-based chemotherapy. ...
Ethnopharmacological relevance
Ervatamia coronaria, a popular garden plant in India and some other parts of the world is known traditionally for its anti-inflammatory and anti-cancer properties. The molecular bases of these functions remain poorly understood.
Aim of the study
Efficacies of the existing therapies for colorectal cancer (CRC) are limited by their life-threatening side effects and unaffordability. Therefore, identifying a safer, efficient, and affordable therapeutic is urgent. We studied the anti-CRC activity of an alkaloid-rich fraction of E. coronaria leaf extracts (AFE) and associated underlying mechanism.
Materials and methods
Activity guided solvant fractionation was adopted to identify the activity in AFE. Different cell lines, and tumor grown in syngeneic mice were used to understand the anti-CRC effect. Methodologies such as LCMS, MTT, RT-qPCR, immunoblot, immunohistochemistry were employed to understand the molecular basis of its activity.
Results
We showed that AFE, which carries about six major compounds, is highly toxic to colorectal cancer (CRC) cells. AFE induced cell cycle arrest at G1 phase and p21 and p27 genes, while those of CDK2, CDK-4, cyclin-D, and cyclin-E genes were downregulated in HCT116 cells. It predominantly induced apoptosis in HCT116p53+/+ cells while the HCT116p53-/- cells under the same treatment condition died by autophagy. Notably, AFE induced upregulation of AMPK phosphorylation, and inhibition of both of the mTOR complexes as indicated by inhibition of phosphorylation of S6K1, 4EBP1, and AKT. Furthermore, AFE inhibited mTOR-driven conversion of cells from reversible cell cycle arrest to senescence (geroconversion) as well as ERK activity. AFE activity was independent of ROS produced, and did not primarily target the cellular DNA or cytoskeleton. AFE also efficiently regressed CT26-derived solid tumor in Balb/c mice acting alone or in synergy with 5FU through inducing autophagy as a major mechanism of action as indicated by upregulation of Beclin 1 and phospho-AMPK, and inhibition of phosphor-S6K1 levels in the tumor tissue lysates.
Conclusion
AFE induced CRC death through activation of both apoptotic and autophagy pathways without affecting the normal cells. This study provided a logical basis for consideration of AFE in future therapy regimen to overcome the limitations associated with existing anti-CRC chemotherapy.
... More than 50% of all human cancers have been associated with missense [758] and/or synonymous mutations of the p53 gene [742,[759][760][761], where close to 95% of these mutations are located in the DNA-binding domain (DBD) [12,742,[762][763][764][765] which is regulated by interactions with the extensive intrinsically disordered regions of the C-and N-termini that flank the DBD [547,[766][767][768]. IDRs are believed to modulate LLPS [557]; therefore, it is not surprising that aggregates of both wild-type and mutant p53 have been observed in cancer cells and tissues and are regarded as a hallmark for p53 inactivation [769]. In addition, the formation of amyloid oligomers and fibrils in the DBD of p53 may produce prion-like characteristics [770][771][772] that can result in the gain or loss of functions [773]. ...
... Dysregulation of m 6 A modifiers can lead to changes in the regulation of gene expression, affecting cancer [1003,1004], neurodegenerative diseases [990,991,1005], aortic dissection [1006], blood pressure regulation [1007], and cardiac function [1008]. More than 50% of all human cancers have been associated with missense [758] and/or synonymous mutations of the p53 gene [759][760][761]. An analysis of datasets from the Cancer Genome Atlas Research Network (TCGA) acute myeloid leukemia (AML) study revealed that mutations and/or copy number variations in genes that write, read, or erase m 6 A methylations, such as METTL3, METTL14, YTHDF1, YTHDF2, FTO, and ALKBH5, are significantly correlated with p53 mutations in AML patients [1009]. ...
Biomolecular condensates are membraneless organelles (MLOs) that form dynamic, chemically distinct subcellular compartments organizing macromolecules such as proteins, RNA, and DNA in unicellular prokaryotic bacteria and complex eukaryotic cells. Separated from surrounding environments, MLOs in the nucleoplasm, cytoplasm, and mitochondria assemble by liquid–liquid phase separation (LLPS) into transient, non-static, liquid-like droplets that regulate essential molecular functions. LLPS is primarily controlled by post-translational modifications (PTMs) that fine-tune the balance between attractive and repulsive charge states and/or binding motifs of proteins. Aberrant phase separation due to dysregulated membrane lipid rafts and/or PTMs, as well as the absence of adequate hydrotropic small molecules such as ATP, or the presence of specific RNA proteins can cause pathological protein aggregation in neurodegenerative disorders. Melatonin may exert a dominant influence over phase separation in biomolecular condensates by optimizing membrane and MLO interdependent reactions through stabilizing lipid raft domains, reducing line tension, and maintaining negative membrane curvature and fluidity. As a potent antioxidant, melatonin protects cardiolipin and other membrane lipids from peroxidation cascades, supporting protein trafficking, signaling, ion channel activities, and ATPase functionality during condensate coacervation or dissolution. Melatonin may even control condensate LLPS through PTM and balance mRNA- and RNA-binding protein composition by regulating N6-methyladenosine (m6A) modifications. There is currently a lack of pharmaceuticals targeting neurodegenerative disorders via the regulation of phase separation. The potential of melatonin in the modulation of biomolecular condensate in the attenuation of aberrant condensate aggregation in neurodegenerative disorders is discussed in this review.
... Mutant p53 loss-of-function, dominant-negative and gain-offunction properties are all important for tumorigenesis in humans, with gain-of-function activities being particularly relevant. 24 Currently, 80% of LFS families that fulfıll the classical clinical criteria (Table 1) harbor TP53 germline mutations 25 -most of which reside in the DBD. Causal mutations in genes other than TP53 have not been reproducibly observed, although it is anticipated the new platforms for whole genome and whole exome sequencing may uncover candidate genes. ...
... Ultimately, determining the exact nature of the TP53 mutation may be of prognostic signifıcance and therefore important for the clinical management of these patients. 25 Most importantly, a high index of suspicion should be maintained for known LFS carriers who complain of persisting but unexplained symptoms. ...
An understanding of the genetic causes and molecular pathways of hereditary cancer syndromes has historically informed our knowledge and treatment of all types of cancers. For this review, we focus on three rare syndromes and their associated genetic mutations including BAP1, TP53, and SDHx (SDHA, SDHB, SDHC, SDHD, SDHAF2). BAP1 encodes an enzyme that catalyzes the removal of ubiquitin from protein substrates, and germline mutations of BAP1 cause a novel cancer syndrome characterized by high incidence of benign atypical melanocytic tumors, uveal melanomas, cutaneous melanomas, malignant mesotheliomas, and potentially other cancers. TP53 mutations cause Li-Fraumeni syndrome (LFS), a highly penetrant cancer syndrome associated with multiple tumors including but not limited to sarcomas, breast cancers, brain tumors, and adrenocortical carcinomas. Genomic modifiers for tumor risk and genotype-phenotype correlations in LFS are beginning to be identified. SDH is a mitochondrial enzyme complex involved in the tricarboxylic acid (TCA) cycle, and germline SDHx mutations lead to increased succinate with subsequent paragangliomas, pheochromocytomas, renal cell carcinomas (RCCs), gastrointestinal stromal tumors (GISTs), and other rarer cancers. In all of these syndromes, the molecular pathways have informed our understanding of tumor risk and successful early tumor surveillance and screening programs.
... In our case, the patient was previously diagnosed with Li-FRAUMENI syndrome, an inherited autosomal dominant disorder, with elevated cancer predisposition due to abnormalities in the p53 tumor suppressor gene (TP53) [15]. It is noteworthy that not all tumors positively stained for p53 on IHC are associated with mutations in this gene and the diagnosis of LFS should be based on genetic testing for clinically selected individuals [15,16]. ...
... In our case, the patient was previously diagnosed with Li-FRAUMENI syndrome, an inherited autosomal dominant disorder, with elevated cancer predisposition due to abnormalities in the p53 tumor suppressor gene (TP53) [15]. It is noteworthy that not all tumors positively stained for p53 on IHC are associated with mutations in this gene and the diagnosis of LFS should be based on genetic testing for clinically selected individuals [15,16]. In ALCL, p53 expression was found in the majority of cases, but this did not necessarily correlated with evidence of gene mutation [17,18]. ...
Background
Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare malignancy, recently recognized as a provisional entity by the World Health Organization. Although increasing data have been published on this entity in recent years, a great number of patients and health professionals remain unaware of this diagnosis. Case presentationWe herein report the case of a 56-year-old female with Li-FRAUMENI syndrome who presented with late right-sided recurrent breast swelling after prophylactic adenomastectomy with implant reconstruction. Imaging scans revealed an heterogeneous mass adjacent to the implant fibrous capsule. A biopsy of the lesion rendered the diagnosis of a BIA-ALCL. Conclusions
This case presents similarities with previous reports, but also some particularities, which should be stressed in order to make the diagnosis the earliest possible. The most distinct feature is that this is the second report of BIA-ALCL arising in the setting of Li-FRAUMENI syndrome.
... The tumor suppressor protein p53 is a transcription factor that can trigger cell cycle arrest, replicative senescence, or apoptosis (8). The induction of p53 expression by a wide variety of cellular stresses such as hypoxia, UV, and cytotoxic drugs is involved in accelerated cellular senescence as well as G1/S and G2/M cell cycle checkpoint activation (9)(10)(11). The TP53 gene encoding p53 is the most frequently inactivated gene in human malignancy, and somatic missense mutations of p53 are found in ~50% of human cancers (12). ...
... p53 is a tumor suppressor gene that is stimulated by cellular stress including hypoxia, ultraviolet radiation, and oxidative stress (9)(10)(11). In addition, p53 is a known transcription factor that induces the expression of various genes associated with apoptosis, cell cycle arrest, and DNA repair (19). ...
Aberrant fibronectin (FN) expression is associated with poor prognosis, cell adhesion, and cell motility in a variety of cancer cells. In this study, we investigated the relationship between p53 and FN expression in breast cancer cells. Basal FN expression was significantly decreased by treatment with the p53 activator III, RITA, in MCF7 breast cancer cells with wild-type p53. In addition, overexpression of wild-type p53 markedly decreased the level of FN expression in p53-mutant breast cancer cells. To examine the mechanism underlying the relationship between p53 and FN expression, we treated MCF7 breast cancer cells with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). Our results showed that basal FN expression was increased by TPA treatment in a time-dependent manner. In contrast, the level of p53 expression was decreased by TPA treatment. However, the expression of FN and p53 was not altered by TPA in p53-mutant breast cancer cells. Furthermore, the alterations in FN and p53 expression in response to TPA were prevented by a specific MEK inhibitor, UO126. Finally, we demonstrated that TPA triggers degradation of p53 through the proteasomal pathway in MCF7 cells. TPA-induced FN expression was decreased by the proteasome inhibitor MG132. Under the same condition, p53 protein expression, but not mRNA expression, was reversed by MG132. Taken together, our data demonstrate that the level of FN expression is associated with the status and expression of p53 in breast cancer cells.
... TP53 activation induces TP53-responsive genes at the transcriptional level, resulting in cell cycle arrest, senescence, or apoptosis [2]. Disruption of the TP53 pathway is considered to occur early during oncogenesis [3]. ...
... The failure to respond, despite wild-type TP53 may be due to defects either upstream (p14 ARF ) or downstream (p21 Cip1/WAF1 , Puma) in the TP53 pathway [3,13]. Of note, tumor lines with known MDM2 amplification were not responsive to RG7112 treatment. ...
Background:
MK-8242 is an inhibitor of MDM2 that stabilizes the tumor suppressor TP53 and induces growth arrest or apoptosis downstream of TP53 induction.
Procedures:
MK-8242 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10.0 μM and against the PPTP in vivo xenograft panels using oral gavage on Days 1-5 and Day 15-19 at a dose of 125 mg/kg (solid tumors) or 75 mg/kg (acute lymphoblastic leukemia [ALL] models).
Results:
The median IC50 for MK-8242 was 0.07 μM for TP53 wild-type cell lines versus >10 μM for TP53 mutant cell lines. MK-8242 induced a twofold or greater delay in time to event in 10 of 17 (59%) of TP53 wild-type solid tumor xenografts, excluding osteosarcoma xenografts that have very low TP53 expression. Objective responses were observed in seven solid tumor xenografts representing multiple histotypes. For the systemic-disease ALL panel, among eight xenografts there were two complete responses (CRs) and six partial responses (PRs). Two additional MLL-rearranged xenografts (MV4;11 and RS4;11) grown subcutaneously showed maintained CR and PR, respectively. The expected pharmacodynamic responses to TP53 activation were observed in TP53 wild-type models treated with MK-8242. Pharmacokinetic analysis showed that MK-8242 drug exposure in SCID mice appears to exceed that was observed in adult phase 1 trials.
Conclusions:
MK-8242-induced tumor regressions across multiple solid tumor histotypes and induced CRs or PRs for most ALL xenografts. This activity was observed at MK-8242 drug exposures that appear to exceed those observed in human phase 1 trials.
... Losing the function of the p53 gene can cause genomic instability and thus disrupt the ability of the genome to replicate accurately. This has been observed in about half of human malignant cancers [63] and underscores the vital role of p53 in tumor suppression. Therefore, the use of these techniques cannot be considered a safe and risk-free option for cell immortalization. ...
Cells are very important to researchers due to their use in various biological studies in in vitro and in vivo settings. This importance stems from the short lifespan of most cells under laboratory conditions, which can pose significant challenges, such as the difficulties associated with extraction from the source tissue, ethical concerns about separating cells from human or animal models, limited cell passage ability, and variation in results due to differences in the source of the obtained cells, among other issues. In general, cells in laboratory conditions can divide into a limited number, known as the Hayflick limit, due to telomere erosion at the end of each cellular cycle. Given this problem, researchers require cell lines that do not enter the senescence phase after a limited number of divisions. This can allow for more stable studies over time, prevent the laborious work associated with cell separation and repeated cultivation, and save time and money in research projects. The aim of this review is to summarize the function and effect of immortalization techniques, various methods, their advantages and disadvantages, and ultimately the application of immortalization and cell line production in various research fields.
... In fact, mice with mutation of Ser15 in the p53 gene spontaneously develop late-onset lymphomas and exhibit accelerated aging 10 , suggesting that p53 activity reflected by p53 phosphorylation at Ser15 is indispensable for both antiaging and tumor suppression in mammals. These p53 functions decline with age 11 and result in accelerated cancer progression in mammals after middle age 12 . Indeed, p53 −/− mice die within 1 year 13,14 . ...
Understanding the biological effects of low-dose (<100 mGy) ionizing radiation (LDR) is technically challenging. We investigated age-dependent LDR effects using adaptive response experiments in young (7-to 12-week-old) and middle-aged (40-to 62-week-old) C57BL/6 mice. Compared with 3 Gy irradiation, 0.02 Gy preirradiation followed by 3 Gy irradiation prolonged life in young mice but not middle-aged mice. Preirradiation also suppressed irradiation-induced 53BP1 repair foci in the small intestines, splenic apoptosis, and p53 activity in young mice but not middle-aged mice. Young p53 +/− C57BL/6 mice did not show these adaptive responses, indicating that insufficient p53 function in young mice mitigated the adaptive responses. Interestingly, p53 activation in middle-aged mice spontaneously became approximately 4.5-fold greater than that in young mice, possibly masking LDR stresses. Furthermore, adaptive responses in young mice, but not in middle-aged mice, suppressed some senescence-associated secretory phenotype (SASP) factors ( IL-6, CCL2 , CCL5 , CXCL1 ). Thus, LDR-induced adaptive responses associated with specific SASP factors may be attenuated by a combination of reduced DNA damage sensor/transducer function and chronic p53 activation in middle-aged mice.
... In CRC, P53 mutation is found in approximately 50% of all cases, which could contribute to genome instability and regulate the occurrence and progression of tumours [9][10][11]. The importance of P53 mutation in chemoresistance has also been reported for different cancers, such as CRC and breast cancer [12,13]. Targeting the key factors of the P53 signalling pathway to rescue the chemoresistance caused by P53 mutation has become a research focus [14]. ...
P53 mutation is an important cause of chemoresistance in colorectal cancer (CRC). The investigation and identification of the downstream targets and underlying molecular mechanism of chemoresistance induced by P53 abnormalities are therefore of great clinical significance. In this study, we demonstrated and reported for the first time that leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is a key functional downstream factor and therapeutic target for P53 mutation-induced chemoresistance. Due to its RNA binding function, LRPPRC specifically bound to the mRNA of multidrug resistance 1 (MDR1), increasing MDR1 mRNA stability and protein expression. In normal cells, P53 induced by chemotherapy inhibited the expression of LRPPRC via miR-34a and in turn reduced the expression of MDR1. However, chemotherapy-induced P53/miR-34a/LRPPRC/MDR1 signalling pathway activation was lost when P53 was mutated. Additionally, the accumulated LRPPRC and MDR1 promoted drug resistance. Most importantly, gossypol-acetic acid (GAA) was recently reported by our team as the first specific inhibitor of LRPPRC. In CRC cells with P53 mutation, GAA effectively induced degradation of the LRPPRC protein and reduced chemoresistance. Both in vivo and in vitro experiments revealed that combination chemotherapy with GAA and 5-fluorouracil (5FU) yielded improved treatment outcomes. In this study, we reported a novel mechanism and target related to P53-induced drug resistance and provided corresponding interventional strategies for the precision treatment of CRC.
... In addition, significant biological heterogeneity is evident within each non-WNT subgroup, for instance, TP53 mutations associate with a poor outcome in SHH [5,38]. The loss of p53 function is thought to confer resistance to chemotherapy [39,40], and effective anti-tumoural treatments have yet to be established for this group, which represents approximately 10 patients in Europe per year. In contrast, Group 3 and Group 4 harbour few mutations but multiple DNA copy number alterations [34]. ...
Simple Summary
Patients with medulloblastoma receive treatment according to a risk stratification, which is a combination of clinical and biological factors. To date there have been a limited number of trials for high-risk disease in children older than 3 years, with a wide range of treatment philosophies that usually involve higher doses of radiotherapy delivered either conventionally or in hyper-fractionated/accelerated regimens. Similarly, both standard and high-dose chemotherapies were assessed. However, to date, trials in high-risk medulloblastoma have commonly been institutional or national, based on modest cohort sizes, and have not evaluated the relative performance of different strategies in a randomised fashion. We describe the concepts and design of the SIOP-E high-risk medulloblastoma clinical trial (SIOP-HR-MB), the first international, biomarker-driven, randomised clinical trial for high-risk medulloblastoma. SIOP-HR-MB is programmed to recruit >800 patients in 16 countries across Europe; its primary objectives are to assess the relative efficacies of the alternative established regimens.
Abstract
Medulloblastoma patients receive adapted therapies stratified according to their risk-profile. Favourable, standard, and high disease-risk groups are each defined by the status of clinical and pathological risk factors, alongside an evolving repertoire of diagnostic and prognostic biomarkers. Medulloblastoma clinical trials in Europe are coordinated by the International Society for Paediatric Oncology (SIOP-Europe) brain tumour group. Favourable and standard-risk patients are eligible for the SIOP-PNET5-MB clinical trial protocol. In contrast, therapies for high-risk disease worldwide have, to date, encompassed a range of different treatment philosophies, with no clear consensus on approach. Higher radiotherapy doses are typically deployed, delivered either conventionally or in hyper-fractionated/accelerated regimens. Similarly, both standard and high-dose chemotherapies were assessed. However, trials to date in high-risk medulloblastoma have commonly been institutional or national, based on modest cohort sizes, and have not evaluated the relative performance of different strategies in a randomised fashion. We describe the concepts and design of the SIOP-E high-risk medulloblastoma clinical trial (SIOP-HR-MB), the first international biomarker-driven, randomised, clinical trial for high-risk medulloblastoma. SIOP-HR-MB is programmed to recruit >800 patients in 16 countries across Europe; its primary objectives are to assess the relative efficacies of the alternative established regimens. The HR-MB patient population is molecularly and clinically defined, and upfront assessments incorporate a standardised central review of molecular pathology, radiology, and radiotherapy quality assurance. Secondary objectives include the assessment of (i) novel therapies within an upfront ‘window’ and (ii) therapy-associated neuropsychology, toxicity, and late effects, alongside (iii) the collection of materials for comprehensive integrated studies of biological determinants within the SIOP-HR-MB cohort.
... This evidence was supported by previous studies that reported that the treatment outcome of KRAS mutation-positive biliary tract cancer patients was significantly worse than that of KRAS wildtype patients [22], and point mutations of KRAS are usually associated with the onset of acquired resistance to anti-EGFR treatment [23]. In addition, TP53 mutation has been reported to be a negative factor for the outcome of patients with TKI therapy [24] and chemotherapeutic drugs including gemcitabine, cisplatin, and 5-FU [25,26]. Unfortunately, there is no data on mutation status of HuCCA-1 cell. ...
Purpose The potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells.
Materials and Methods ErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digital-droplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles in vivo cell environments.
Results CCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib.
Conclusion Afatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients.
Key words Cholangiocarcinoma, ErbB receptors, ErbB targeted drugs, Antineoplastic agents, Gemcitabine, Afatinib
... Also, the expression of p53 is considered as a prognostic marker that greatly determines the therapeutic outcome in neoplastic diseases. However, mutations in p53 are frequently observed in many human malignancies including breast, colorectal, and glioblastomas, which later paves the way for cancer progression 50,51 . Pro-EMT genes and transcription factors are also increasingly recognized as repressors of p53 functions; For example, Twist1, a mesenchymal marker and transcription factor, is involved in the degradation of p53 in sarcomas. ...
Epithelial-mesenchymal transition (EMT) is critical for the metastatic dissemination of cancer cells and contributes to drug resistance. In this study, we observed that epithelial colorectal cancer (CRC) cells transiently exposed to 5-fluorouracil (5-FU) (a chemotherapeutic drug for CRC) as well as 5-FU-resistant cells (5-FUR) develop EMT characters as evidenced by activation of Vimentin and augmented invasive properties. On the other hand, 4DPG (4′-demethyl-deoxypodophyllotoxin glucoside), a natural podophyllotoxin analog attenuates EMT and invadopodia formation abilities of HCT-116/5-FUR and SW-620/5-FUR cells. Treatment with 4DPG restrains Vimentin phosphorylation (Ser38) in 5-FUR cells, along with downregulation of mesenchymal markers Twist1 and MMP-2 while augmenting the expression of epithelial markers E-cadherin and TIMP-1. Moreover, 4DPG boosts the tumor-suppressor protein, checkpoint kinase 2 (Chk2) via phosphorylation at Thr68 in a dose-dependent manner in 5-FUR cells. Mechanistically, SiRNA-mediated silencing of Chk2, as well as treatment with Chk2-specific small-molecule inhibitor (PV1019), divulges that 4DPG represses Vimentin activation in a Chk2-dependent manner. Furthermore, immunoprecipitation analysis unveiled that 4DPG prevents complex formation between Vimentin and p53 resulting in the rescue of p53 and its nuclear localization in aggressive 5-FUR cells. In addition, 4DPG confers suitable pharmacokinetic properties and strongly abrogates tumor growth, polyps formation, and lung metastasis in an orthotopic rat colorectal carcinoma model. In conclusion, our findings demonstrate 4DPG as a targeted antitumor/anti-metastatic pharmacological lead compound to circumvent EMT-associated drug resistance and suggest its clinical benefits for the treatment of aggressive cancers.
... It is well known that p53 suppresses tumor formation and renders protection against DNA damage by inducing cell cycle arrest, DNA repair, or apoptosis [18]. Mutation of p53 is often observed in cancer, especially in late events in malignant progression [19,20]. ...
The tumor suppressor p53 is considered the “guardian of the genome” that can protect cells against cancer by inducing cell cycle arrest followed by cell death. However, STAT3 is constitutively activated in several human cancers and plays crucial roles in promoting cancer cell proliferation and survival. Hence, STAT3 and p53 have opposing roles in cellular pathway regulation, as activation of STAT3 upregulates the survival pathway, whereas p53 triggers the apoptotic pathway. Constitutive activation of STAT3 and gain or loss of p53 function due to mutations are the most frequent events in numerous cancer types. Several studies have reported the association of STAT3 and/or p53 mutations with drug resistance in cancer treatment. This review discusses the relationship between STAT3 and p53 status in cancer, the molecular mechanism underlying the negative regulation of p53 by STAT3, and vice versa. Moreover, it underlines prospective therapies targeting both STAT3 and p53 to enhance chemotherapeutic outcomes.
... It lacks additional clinical features and is only characterized by the high frequency of malignancies in multiple organs, making it a difficult syndrome to diagnose in the absence of a significant family history of multiple cancers (7). The involved gene encodes the TP53 transcription factor, tumor protein p53, also known as the "guardian of the genome" (8). TP53 is involved in cellular growth control by regulating the expression of several genes causing cell-cycle arrest and apoptosis in response to DNA damage. ...
Pediatric High-Grade Gliomas (pHGG) are among the deadliest childhood brain tumors and can be associated with an underlying cancer predisposing syndrome. The thorough understanding of these syndromes can aid the clinician in their prompt recognition, leading to an informed genetic counseling for families and to a wider understanding of a specific genetic landscape of the tumor for target therapies. In this review, we summarize the main pHGG-associated cancer predisposing conditions, providing a guide for suspecting these syndromes and referring for genetic counseling.
... This evidence was supported by previous studies that reported that the treatment outcome of KRAS mutation-positive biliary tract cancer patients was significantly worse than that of KRAS wildtype patients [22], and point mutations of KRAS are usually associated with the onset of acquired resistance to anti-EGFR treatment [23]. In addition, TP53 mutation has been reported to be a negative factor for the outcome of patients with TKI therapy [24] and chemotherapeutic drugs including gemcitabine, cisplatin, and 5-FU [25,26]. Unfortunately, there is no data on mutation status of HuCCA-1 cell. ...
Purpose:
The potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells.
Materials and methods:
ErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digital-droplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles in vivo cell environments.
Results:
CCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib.
Conclusion:
Afatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients.
... It is now well-established that about 50% of cancers are strongly associated with p53 mutation directly [112] or indirectly through overexpression of its negative regulators, like mouse double minute 2 homolog (Mdm)-2 or murine double minute (Mdm)-X [113]. Downregulation of negative regulators and subsequent increased expression of p53 and p21 were observed in malignant melanoma (A375) and breast cancer cells (MCF7) following ABZ treatment [105]. ...
Origin of drug and radio-refractory clones, cancer stem-like cells, and rapid angiogenesis and metastasis are among the primary concerns that limit the efficacy of anticancer treatments, emphasizing the urgency of developing new therapeutics. Factors like high attrition rates, huge investments, patients' heterogeneity, and diverse molecular subtypes have challenged the rapid development of anticancer drugs. Treatment with repurposing pleiotropic benzimidazole antihelminthics, like mebendazole, albendazole, and flubendazole has recently opened a new window, owing to their easy access, low cost as a generic drug, and long track record of safe use in the human population. This review highlights the outcomes of preclinical and clinical studies of these drugs as a potent anticancer agent(s) conducted in the last two decades. Substantial preclinical studies, as well as limited clinical trials, suggest noteworthy anticancer potency of these pleiotropic benzimidazoles, particularly as potent microtubule disrupting, anti-angiogenic, and anti-metastatic agents, inhibitors of the immune checkpoint, hypoxia-inducible factor, epithelial-mesenchymal transition, cancer stemness, and multidrug resistance protein 1, and inducers of apoptosis and M1 polarization. These anticancer effects are attributed to multiple action points, including intrinsic apoptosis, canonical Wnt/β-catenin, JAK/STAT-3, JNK, MEK/ERK, and hedgehog signaling pathways. The effective anticancer properties of mebendazole, albendazole, and flubendazole either alone or synergistically with frontline drugs, warrant their validation through controlled clinical trials to use them as promising avenues to anticancer therapy.
... The tumor suppressor p53 protein plays an important role in the control of various aspects of health and disease [1][2][3]. ...
The onco-suppressor p53 protein plays also an important role in the control of various aspects of health and disease. p53 levels are low in normal cells and elevated under stress conditions. While low levels of p53 promote tumor formation, overactive p53 leads to premature aging and cell death. RNA degradation is a critical level of regulation contributing to the control of gene expression. p53, as an RNA-binding protein, exerts 3′ → 5′ exoribonuclease activity, mediating degradation of adenylate/uridylate-rich elements (ARE)–containing ssRNAs. The 3′-UTR of p53-mRNA, which is a target of p53 itself, harbors cis-acting AREs. Our results suggest that p53 controls its own expression through murine double-minute 2 (mdm2)–independent “RNA decay” function in cytoplasm. We demonstrate that p53 expresses an exoribonuclease activity through the binding to ARE sequences of p53-mRNA via translation-independent and translation-dependent polysome-associated pathways. Antagonistic interplay was detected between p53 levels and execution of its exoribonuclease function mirrored in low p53 levels in normal cells, due to the efficient exoribonuclease activity, and in the accumulation of p53 in cells exposed to p53-activating drugs in accordance with the reduced exoribonuclease activity. Apparently, p53, via control of its own mRNA stability and/or translation in cytoplasm, might act as a negative regulator of p53-mRNA levels. The observed connection between exoribonuclease activity and p53 abundance highlights the importance of this function affecting p53 expression, imperative for multiple functions, with implications for the steady-state levels of protein and for the p53 stress response. The modulation in expression of exoribonuclease activity would be translated into the alterations in p53 level.
Key messages
p53 controls its own expression through mdm2-independent “RNA decay” function in cytoplasm.
p53 expresses an exoribonuclease activity through the binding to ARE sequences of p53-mRNA.
Antagonistic interplay exists between stress-induced p53 and execution of its exoribonuclease function.
... The protein p53 is considered a tumor suppressor protein, it acts as a transcription factor and regulates the expression of genes involved in DNA repair, cell cycle arrest and apoptosis [12]. The association of p53 mutations and cancer was first established for a familial, early-onset, form of cancer susceptibility [13,14]. However, pathogenic somatic mutations in the p53 gene have been also identified in approximately 50% of epithelial cancers. ...
Diseases caused by protein misfolding are an emerging pathologic category that are thought to share some basic common mechanisms and display impressive heterogeneity in terms of tissue involvement, age of onset and clinical features. The growing recognition of the impact that protein misfolding has on human diseases is certainly related to the phenomenon of population aging and the expansion of the population in which these diseases are more frequent, but it is also based on a scientific revolution that looks at protein dynamics and relates these data to their potential pathologic implications. The multidisciplinary exchange of knowledge between experts in apparently unrelated diseases, such as sickle cell anemia and Alzheimer’s disease, has helped clarify the pathogenesis of these and many other diseases. The quick expansion of knowledge on the mechanisms of these diseases is priming pharmaceutical research that is now providing the first prototype drugs.
... These mutation frequencies of TP53 are lower in breast cancers than in other solid tumors (10). The level of TP53 protein expression is stabilized and activated in response to a wide variety of cellular stresses such as DNA damage, ultraviolet irradiation, hypoxia and nucleotide depletion (11)(12)(13). TP53 works as a transcriptional activator and regulates cell cycle arrest, senescence, apoptosis, DNA replication and repair through the expression of its downstream target genes such as p21, Bax, NOXA and p53R2 (14,15). ...
In a previous study, we reported that α‑smooth muscle actin (α‑SMA), one of the mesenchymal marker proteins, is highly expressed in tamoxifen‑resistant breast cancer (TamR) cells. However, the exact mechanism of α‑SMA expression in TamR cells is not fully understood. Here, we investigated the effect of TP53 on α‑SMA expression in breast cancer cells. The levels of α‑SMA mRNA and protein expression were analyzed by real‑time PCR and western blotting, respectively. In estrogen receptor‑positive [ER(+)] breast cancer patients, aberrant α‑SMA expression was found to be associated with a poor prognosis. The level of α‑SMA expression was significantly increased in established TamR cells compared to tamoxifen‑sensitive (TamS) cells. To verify the regulatory mechanism of α‑SMA expression, we analyzed diverse kinase activities between TamS and TamR cells. The activity of TP53 was markedly increased in the TamR cells. When TamS cells were treated with TP53 activator, Nutlin3 (Nut3), α‑SMA expression was increased in the TamS cells. In addition, α‑SMA expression was significantly increased by TP53 overexpression in breast cancer cells. On the contrary, the basal level of α‑SMA expression was decreased by the TP53 inhibitor, pifithrin‑α (PFT‑α). Taken together, we demonstrated that α‑SMA expression is regulated by TP53 activity in TamR cells.
... This result is in agreement with previous report showing that DHA inhibits growth of CRC cells independent of p53 mutational status. 16 As, this protein is frequently mutated in most hu- man malignancies, which is associated with poor prognosis and resistance to ionizing radiation, [32][33][34] this could demon- strate the advantage of using DHA as an attractive radio- sensitizer agent in mutant-p53 tumor cells compared with other compounds. ...
Mutant-p53 colorectal cancer (CRC) cells are often resistant to radiotherapy. Loss of suppressor activity of wt-p53 on survivin is responsible for the enhanced expression of survivin as a radioresistant factor in tumor cells. Yet, no survivin-modulating drug has been approved for clinical application in CRC. Thus, the search for safe compounds that modulate survivin expression and induce apoptosis irrespective of p53 status may potentiate the efficacy of radiotherapy in mutant-p53 CRC cells. Omega-3 docosahexaenoic acid (DHA) induces apoptosis in malignant cells without cytotoxicity in normal cells. However, little is known whether in vitro concentrations of DHA equal to the human plasma levels are able to modulate expression of survivin and sensitize mutant-p53 CRC cells to γ-irradiation. Radioresistant mutant-p53 HT-29 cells were pretreated with 50- and 100-μM DHA for 48-h before 2-, 4-, 6-, 8-, and 10-Gy of γ-irradiation. Thereafter, proliferation rates were measured after 6 d. HT-29 cells were also pretreated with 50- and 100-μM DHA for 4-h before 2- and 10-Gy of γ-irradiation after which, cell number, survivin expression, caspase-3 activation, apoptosis, and ED50 (γ-irradiation dose causing 50% growth inhibition) were evaluated. Pretreatment of HT-29 cells with 50- and 100-μM DHA for 48-h followed by 2- to 10-Gy of γ-irradiation induced a dose-dependent additive decrease in cell proliferation rate and ED50 values were decreased by 88%, 44%, 41%, and 27% for 500-, 1500-, 2500-, and 5000 cells per well pretreated with 100-μM DHA respectively. Pretreatment of 5 × 105 HT-29 cells per well with 100-μM DHA for 4-h followed by 2- or 10-Gy of irradiation resulted in 53% and 86% decreases in cell numbers, 2- and 5.1-fold activation in caspase-3 followed by 66% and 60% decreases in survivin mRNA levels respectively. DHA in combination with radiation increased total apoptotic rate 48-h post-treatment. DHA decreases survivin expression and induces caspase-3 activation irrespective of p53 status. Significant decreases in ED50 values at concentrations of DHA equal to human plasma levels, suggesting that DHA could be used as an attractive radiosensitizer agent in CRC patients with mutant-p53.
... Up to now, base editing 102 applications mainly focussed on correcting disease mutations, on genetic diversification using tiled 103 gRNA libraries [27][28][29], or on introducing premature stop codons as alternative to classic knock-out 104 strategies [30]. 105 p53 is arguably the most studied protein in health and disease [31,32]. Historically dubbed the 106 guardian of the genome [33], the encoding gene is mutated in 50% of all reported human cancer cases 107 [34], making it an attractive starting point for the development of anticancer strategies [35]. ...
The CRISPR/Cas9 revolution is profoundly changing the way life sciences technologies are used. Many assays now rely on engineered clonal cell lines to eliminate overexpression of bait proteins. Control cell lines are typically non-engineered cells or engineered clones implying a considerable risk for artefacts because of clonal variation. Genome engineering can also transform BioID, a proximity labelling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, resulting in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins wherein we introduce a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering, leading to bi-cistronic expression of both p53 and BirA* under control of the endogenous promoter. By targeting a Cas9-cytidine deaminase base editor to the T2A auto-cleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. Using quantitative proteomics we show significant benefits over classical ectopic expression of p53-BirA*, and we provide a first well-controlled view on the proximal proteins of endogenous p53 in colon carcinoma cells. This novel application for base editors expands the CRISPR/Cas9 toolbox and can be a valuable addition for synthetic biology.
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... Mutations in the TP53 gene are frequent genetic alterations in human GBM. They are associated not only with the loss of tumor suppressor function, but also with a gain of function by switching on genes involved in the glioblastoma pathway [35]. Deregulation of the p53 pathway may also contribute to the treatment-resistant phenotype of GBM. ...
Glioblastoma multiforme (GBM) is the most common and most aggressive subtype of malignant gliomas. The current standard of care for newly diagnosed GBM patients involves maximal surgical debulking, followed by radiation therapy and temozolomide chemotherapy. Despite the advances in GBM therapy, its outcome remains poor with a median survival of less than two years. This poor outcome is partly due to the ability of GBM tumors to acquire adaptive resistance to therapy and in particular to radiation. One of the mechanisms contributing to GBM tumor progression and resistance is an aberrant activation of NF-ĸB, a family of inducible transcription factors that play a pivotal role in regulation of many immune, inflammatory and carcinogenic responses. Acetyl-11-keto-β-boswellic acid (AKBA) is a pentacyclic terpenoid extracted from the gum Ayurvedic therapeutic plant Boswellia serrata. AKBA is anti-inflammatory agent that exhibits potent cytotoxic activities against various types of tumors including GBM. One of the mechanisms underlying AKBA anti-tumor activity is its ability to modulate the NF-ĸB signaling pathway. The present study investigated in vitro and in vivo the effect of combining AKBA with ionizing radiation in the treatment of GBM and assessed AKBA anti-tumor activity and radio-enhancing potential. The effect of AKBA and/or radiation on the survival of cultured glioblastoma cancer cells was evaluated by XTT assay. The mode of interaction of treatments tested was calculated using CalcuSyn software. Inducing of apoptosis following AKBA treatment was evaluated using flow cytometry. The effect of combined treatment on the expression of PARP protein was analysed by Western blot assay. Ectopic (subcutaneous) GBM model in nude mice was used for the evaluation of the effect of combined treatment on tumor growth. Immunohistochemical analysis of formalin-fixed paraffin-embedded tumor sections was used to assess treatment-related changes in Ki-67, CD31, p53, Bcl-2 and NF-ĸB-inhibitor IĸB-α. AKBA treatment was found to inhibit the survival of all four tested cell lines in a dose dependent manner. The combined treatment resulted in a more significant inhibitory effect compared to the effect of treatment with radiation alone. A synergistic effect was detected in some of the tested cell lines. Flow cytometric analysis with Annexin V-FITC/PI double staining of AKBA treated cells indicated induction of apoptosis. AKBA apoptotic activity was also confirmed by PARP cleavage detected by Western blot analysis. The combined treatment suppressed tumor growth in vivo compared to no treatment and each treatment alone. Immunohistochemical analysis showed anti-angiogenic and anti-proliferative activity of AKBA in vivo. It also demonstrated a decrease in p53 nuclear staining and in Bcl-2 staining and an increase in IĸB-α staining following AKBA treatment both alone and in combination with radiotherapy. In this study, we demonstrated that AKBA exerts potent anti-proliferative and apoptotic activity, and significantly inhibits both the survival of glioblastoma cells in vitro and the growth of tumors generated by these cells. Combination of AKBA with radiotherapy was found to inhibit factors which involved in cell death regulation, tumor progression and radioresistence, therefore it may serve as a novel approach for GBM patients.
... More specifically, p53 acts to suppress the inflammatory response and is also an inhibitor of NFkB. Adverse inhibition of p53 and intense NFkB activation can contribute to chronic inflammation via lack of apoptotic clearance and turnover of cells (Royds & Iacopetta 2006). On account that PKD causes chronic tissue inflammation as characterized by abnormal extra cellular matrix (ECM) dynamics and tissue fibrosis, we also measured FN. ...
Rainbow trout Oncorhynchus mykiss surviving pro-liferative kidney disease (PKD) are reported not to develop the disease upon re-exposure. However, the mechanisms involved in the immune response to re-exposure are unknown. We examined disease susceptibility and the immune response of naive 1+ rainbow trout when first exposed to Tetracap-suloides bryosalmonae in comparison with that of 1+ rainbow trout re-exposed to T. bryosalmonae. PKD pathogenesis, parasite burden and transcrip-tional signatures of the host immune response were assessed at 10, 25 and 40 d.p.e (days post-exposure). In addition, we evaluated the presence of IgM+ B cells in the blood and the posterior kidney. The exposure of 1+ rainbow trout to T. bryosalmonae for the first time resulted in 100% infection prevalence, high parasite burdens and severe clinical PKD, while re-exposed fish were either able to avoid reinfection completely or mount an earlier and more efficient adaptive-type immune response. This response was characterized by a greater amount of IgM+ B cells in the blood and elevated mRNA levels of secretory IgM in the posterior kidney which minimized pathogen burden and kidney inflammation. Our findings suggest that rainbow trout is able to develop immune protection against T. bryosalmonae.
... TP53 je nejčastěji postiženým genem v mutagenezi mnoha nádorových onemocnění. Mutace bývá nalézána až u 50 % všech nádorů [32]. Je známo celkem 2 314 mutací TP53, které jsou buď aktivační (cca 71 %, protein se váže na Mdm2 a dojde k jeho degradaci), nebo inaktivační (29 %, nemožnost regulace genové exprese) [33]. ...
Background:
Colorectal cancer (CRC) remains a major health burden with an incidence of 1.3 million new cases worldwide and a mortality of almost 8.5%. It is the 2nd most common cancer in women (1st breast carcinoma) and 3rd most common in men (1st lung carcinoma, 2nd prostate carcinoma). CRC alongside breast, lung, prostate and stomach cancer is in the top five most common cancers in men and women worldwide. There are still more than 50% of CRC patients diagnosed with advanced disease (stage III and IV) in the Czech Republic. Genetically, CRC is a very heterogeneous disease with many factors playing key roles in pathogenesis. There are two types of CRC, hereditary with an incidence of between 5% and 10% with APC (FAP, aFAP) or MMR (HNPCC) genes affected, and sporadic colorectal cancer with an incidence of 90-95% with a lot of mutations in variable genes that accumulate during pathogenesis (APC, KRAS, MMR, microRNA, CIMP etc.). Knowledge of the molecular pathogenesis of CRC (hereditary, sporadic) is crucial for treatment, assessment of risk, prognosis, and patient follow-up.
Conclusion:
This article summarizes the molecular pathogenesis of sporadic and hereditary CRC.
... 48 p53 and its isoforms are associated with development of neurodegenerative diseases such as Parkinson's and Alzheimer's because of its induction of cell death in response to stress and interaction with distinct cellular factors that have the ability to facilitate the development of these diseases. [49][50][51][52] Recent accumulating data suggest that p53 has a role in regulation of PNS regeneration. Di Giovanni et al. 53 have shown that TP53 null mice exhibited limited nerve regeneration and suggested that this is due to the transcriptional activity mediated by p53, which activates the axon regenerating genes Coronin 1b and Rab13. ...
Regeneration and tumorigenesis share common molecular pathways, nevertheless the outcome of regeneration is life, whereas tumorigenesis leads to death. Although the process of regeneration is strictly controlled, malignant transformation is unrestrained. In this review, we discuss the involvement of TP53, the major tumor-suppressor gene, in the regeneration process. We point to the role of p53 as coordinator assuring that regeneration will not shift to carcinogenesis. The fluctuation in p53 activity during the regeneration process permits a tight control. On one hand, its inhibition at the initial stages allows massive proliferation, on the other its induction at advanced steps of regeneration is essential for preservation of robustness and fidelity of the regeneration process. A better understanding of the role of p53 in regulation of regeneration may open new opportunities for implementation of TP53-based therapies, currently available for cancer patients, in regenerative medicine.Cell Death and Differentiation advance online publication, 21 October 2016; doi:10.1038/cdd.2016.117.
... The tumor protein p53 (TP53) is a frequently mutated gene in human cancers, once mutated gains oncogenic role [2][3][4]. Metaphorically speaking, when the guardian of the genome TP53 fails, it leads to the activation of the early carcinogenic events [5]. This protein have an important role, the determination of TP53 mutation is used as diagnostic and prognostic marker, but also is conserved as a molecular target in pharmacological investigation [6][7][8]. ...
Oral squamous cell carcinoma (OSCC) is a malignancy with elevated prevalence and somber prognosis due to the fact that most of the patients are diagnosed at an advanced stage. p53 has a crucial role in proliferation and apoptosis during the occurrence and development of numerous malignant tumors. The impact of mutated p53 on the development and progression of OSCC is unclear and might have therapeutic implications. Using an in vitro RNA interference experiment, we have evaluated the impact of p53 knockdown on cell viability, apoptosis, migration, and gene expression for key genes involved in apoptosis and angiogenesis. We observed that inhibiting the expression of p53 decreased the proliferation ability and induced apoptosis/autophagy in SSC-4 cells. Moreover, we observed that this has decreased migration and has blocked the expression of VEGF. In conclusion, our research provides a proof that a direct connection between p53 knockdown and OSCC cell death can be established, therefore opening new potential directions in OSCC molecular therapeutics and management.
... P53 protein is frequently mutated in most human cancers, which is related to poor prognosis [30]. As prodigiosininduced apoptosis in colorectal cancer cells and other malignant cells is p53 independent [13,31], this could mean an advantage over other chemotherapeutic drugs that need functional p53 to provoke its cytotoxic effect [32]. ...
Over-expression of the proto-oncogene survivin in colorectal cancer stem cells (CCSCs) is thought to be one the primary causes for therapy failure. It has also been reported that tumor suppressor miR-16-1 is down-regulated in colorectal cancer (CRC) cells. Therefore, the search for new anti-proliferative agents which target survivin or miR-16-1 in CCSCs is warranted. Several studies have shown that prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in different kinds of cancer cells. Here, we investigated the effects of prodigiosin on HCT-116 cells that serve as a model for CRC initiating cells with stem-like cells properties. HCT-116 cells were treated with 100, 200 and 400 nM prodigiosin after which cell number, viability, growth-rate, survivin and miRNA-16-1 expression, caspase-3 activation and apoptotic rate were evaluated. Prodigiosin decreased significantly growth-rate in a dose-and time-dependent manner. After a 48 h treatment with 100, 200 and 400 nM prodigiosin, growth-rates were measured to be 84.4 ± 9.2 %, 58 ± 6.5 % and 46.3 ± 5.2 %, respectively, compared to untreated cells. We also found that treatment for 48 h with indicated concentrations of prodigiosin resulted in 41 %, 54.5 % and 63 % decrease in survivin mRNA levels and induced 32 %, 48 % and 61 % decrease in survivin protein levels as well as resulted in 128.3 ± 10 %, 178.7 ± 6.1 % and 205 ± 7.6 % increase in caspase-3 activation respectively compared to untreated cells. Prodigiosin caused a significant increase in miRNA-16-1 expression at a concentration of 100 nM and treatment with different concentrations of prodigiosin resulted in 2.2- to 3-fold increase in miRNA-16-1/survivin ratios compared to untreated cells. An increase in number of apoptotic cells ranging from 28.2 % to 86.8 % was also observed with increasing prodigiosin concentrations. Our results provide the first evidence that survivin and miRNA-16-1 as potential biomarkers could be targeted in CRC initiating cells with stem-like cells properties by prodigiosin and this compound with high pro-apoptotic capacity represents the possibility of its therapeutic application directed against CCSCs.
... P53 protein is frequently mutated in most human cancers, which is related to poor prognosis [30]. As prodigiosininduced apoptosis in colorectal cancer cells and other malignant cells is p53 independent [13,31], this could mean an advantage over other chemotherapeutic drugs that need functional p53 to provoke its cytotoxic effect [32]. ...
Over-expression of the proto-oncogene survivin in colorectal cancer stem cells (CCSCs) is thought to be one the primary causes for therapy failure. It has also been reported that tumor suppressor miR-16-1 is down-regulated in colorectal cancer (CRC) cells. Therefore, the search for new anti-proliferative agents which target survivin or miR-16-1 in CCSCs is warranted. Several studies have shown that prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in different kinds of cancer cells. Here, we investigated the effects of prodigiosin on HCT-116 cells that serve as a model for CRC initiating cells with stem-like cells properties. HCT-116 cells were treated with 100, 200 and 400 nM prodigiosin after which cell number, viability, growth-rate, survivin and miRNA-16-1 expression, caspase-3 activation and apoptotic rate were evaluated. Prodigiosin decreased significantly growth-rate in a dose-and time-dependent manner. After a 48 h treatment with 100, 200 and 400 nM prodigiosin, growth-rates were measured to be 84.4 ± 9.2 %, 58 ± 6.5 % and 46.3 ± 5.2 %, respectively, compared to untreated cells. We also found that treatment for 48 h with indicated concentrations of prodigiosin resulted in 41 %, 54.5 % and 63 % decrease in survivin mRNA levels and induced 32 %, 48 % and 61 % decrease in survivin protein levels as well as resulted in 128.3 ± 10 %, 178.7 ± 6.1 % and 205 ± 7.6 % increase in caspase-3 activation respectively compared to untreated cells. Prodigiosin caused a significant increase in miRNA-16-1 expression at a concentration of 100 nM and treatment with different concentrations of prodigiosin resulted in 2.2- to 3-fold increase in miRNA-16-1/survivin ratios compared to untreated cells. An increase in number of apoptotic cells ranging from 28.2 % to 86.8 % was also observed with increasing prodigiosin concentrations. Our results provide the first evidence that survivin and miRNA-16-1 as potential biomarkers could be targeted in CRC initiating cells with stem-like cells properties by prodigiosin and this compound with high pro-apoptotic capacity represents the possibility of its therapeutic application directed against CCSCs.
... Depending on the type and amount of stress, and the type of tissue, the p53-dependent response leads to DNA repair, cell cycle arrest, metabolic reprogramming, apoptosis or senescence, thus preventing the development of cancer (5). Mutations in the TP53 gene are associated with more than a half of all forms of human malignancies (6)(7)(8). ...
p53 tumor suppressor is a transcription factor that controls cell cycle and genetic integrity. In response to genotoxic stress p53 activates DNA repair, cell cycle arrest, apoptosis or senescence, which are initiated via p53 binding to its specific DNA response elements (RE). The consensus p53 DNA RE consists of two decameric palindromic half-site sequences. Crystallographic studies have demonstrated that two isolated p53 DNA-binding core domains interact with one half-site of the p53 DNA REs suggesting that one p53 tetramer is bound to one RE. However, our recent 3D cryo-EM studies showed that the full-length p53 tetramer is bound to only one half-site of RE.
Here, we have used biochemical and electron microscopy (EM) methods to analyze DNA-binding of human and murine p53 tetramers to various p53 DNA REs. Our new results demonstrate that two p53 tetramers can interact sequence-specifically with one DNA RE at the same time. In particular, the EM structural analysis revealed that two p53 tetramers bind one DNA RE simultaneously with DNA positioned between them. These results demonstrate a mode different from that assumed previously for the p53-DNA interaction and suggest important biological implications on p53 activity as a transcriptional regulator of cellular response to stress.
... Previous studies in liver cancer reported that the upregulation of stemness and anti-apoptotic genes, the enhanced expression of dysadherin and the overproduction of chemokines o serve crucial roles in therapy failure, cancer metastases and invasion (2,3,11,12). In addition, point mutations in the p53 gene and the inactivation of Bcl-2 leads to the upregulation of anti-apoptotic pathways in CSCs (13). Furthermore, previous studies in breast, liver and colon cancer (5,6,14) have suggested that the Wnt/β-catenin pathway is involved in the regulation of CSC survival maintenance. ...
Wnt/β‑catenin is an important signaling pathways involved in the tumorgenesis, progression and maintenance of cancer stem cells (CSCs). In the present study, the role of Wnt/β‑catenin signaling in CSC‑mediated tumorigenesis and invasion in liver CSCs was investigated. A small population of cancer stem‑like side population (SP) cells (3.6%) from liver cancer samples were identified. The cells were highly resistant to drug treatment due to the enhanced expression of drug efflux pumps, such as ABC subfamily G member 2, multidrug resistance protein 1 and ATP‑binding cassette subfamily B member 5. Furthermore, using TOPflash and reverse transcription‑quantitative polymerase chain reaction analysis, Wnt/β‑catenin signaling and the transcriptional regulation of Wnt/β‑catenin target genes including dickkopf Wnt signaling pathway inhibitor 1, axis inhibition protein 2 and cyclin D1 were observed to be markedly upregulated in liver cancer SP cells. As a consequence, SP cells possessed infinite cell proliferation potential and the ability to generating tumor spheres. In addition, upon reducing Wnt/β‑catenin signaling, the rates of proliferation, tumor sphere formation and tumor invasion of SP cells were markedly reduced. Therefore, these data suggest that Wnt/β‑catenin signaling is a potential therapeutic target to reduce CSC‑mediated tumorigenicity and invasion in liver cancer.
... TP53, located on the short arm of chromosome 17, is one of the most important tumour suppressor genes. It plays a critical role in response to cellular stress including regulation of target genes, DNA repair, inducing cell-cycle arrest, apoptosis and senescence (Royds & Iacopetta, 2006). Its role has been studied extensively in various types of cancer including haematological malignancies. ...
Treatment success in patients with acute myeloid leukaemia (AML) is heterogeneous. Cytogenetic and molecular alterations are strong prognostic factors, which have been used to individualize treatment. Here, we studied the impact of TP53 mutations on the outcome of AML patients with adverse cytogenetic risk treated with allogeneic haematopoietic stem cell transplantation (HSCT). Samples of 97 patients with AML and adverse-risk cytogenetics who had received a HSCT within three randomized trials were analysed. Complete sequencing of the TP53 coding region was performed using next generation sequencing. The median age was 51 years. Overall, TP53 mutations were found in 40 patients (41%). With a median follow up of 67 months, the three-year probabilities of overall survival (OS) and event-free survival for patients with TP53 wild type were 33% [95% confidence interval (CI), 21% to 45%] and 24% (95% CI, 13% to 35%) compared to 10% (95% CI, 0% to 19%) and 8% (95% CI, 0% to 16%) (P = 0·002 and P = 0·007) for those with mutated TP53, respectively. In multivariate analysis, the TP53-mutation status had a negative impact on OS (Hazard Ratio = 1·7; P = 0·066). Mutational analysis of TP53 might be an important additional tool to predict outcome after HSCT in patients with adverse karyotype AML.
... Его биологическая роль заключается в обеспечении стабильности генома и генетической однородности клеток в целостном организме. Не случайно ген р53 часто характеризуют метафорически, как «страж генома» [1], «ангелхранитель» [2], «ген совести клетки» [3], а мутантный ген -как «падший ангел» [4]. Эти эпитеты хорошо отражают роль р53 в предотвращении болезней и драматизм событий, связанных с утратой его функций. ...
... As a tumor suppressor and regulator of hundreds of target genes, TP53 can regulate numerous cellular processes, including cell cycle progression, apoptosis, cellular senescence, and DNA repair [80]. More recently, TP53 was implicated as a regulator of aging and thus could contribute to many aspects of aging and age-related diseases, such as cardiovascular and metabolic disorders [81,82]. The actions of aging proteins like TP53 on CVD have been well studied [83] and suggest that TP53 and its cellular pathways contribute to disease pathogenesis. ...
The objective of this study was to perform a systematic review of published literature on differentially expressed genes (DEGs) in human epicardial adipose tissue (EAT) to identify molecules associated with CVDs. A systematic literature search was conducted in PubMed, SCOPUS, and ISI Web of Science literature databases for papers published before October 2014 that addressed EAT genes and cardiovascular diseases (CVDs). We included original papers that had performed gene expressions in EAT of patients undergoing open-heart surgery. The Reporting Recommendations for Tumor Marker Prognostic Studies (PRIMARK) assessment tool was also used for methodological quality assessment. From the 180 papers identified by our initial search strategy, 40 studies met the inclusion criteria and presented DEGs in EAT samples from patients with and without CVDs. The included studies reported 42 DEGs identified through comparison of EAT-specific gene expression in patients with and without CVDs. Among the 42 DEGs, genes involved in regulating apoptosis had higher enrichment scores. Notably, interleukin-6 (IL-6) and tumor protein p53 (TP53) were the main hub genes in the network. The results suggest that regulation of apoptosis in EAT is critical for CVD development. Moreover, IL-6 and TP53 as hub genes could serve as biomarkers and therapeutic targets for CVDs.
Molecular glues are typically small chemical molecules that act on the interface between the target protein and the degradation machinery to trigger ternary complex formation. Identification of molecular glues is challenging, and there has been a lack of target-upregulating molecular glues, which are desired for many targets such as tumor suppressor proteins (TSPs). TSPs are usually degraded by the proteasome through polyubiquitination (poly-ub) by specific E3 ligases, whereas deubiquitinases (DUBs) are capable of removing poly-ub conjugates to counteract these E3 ligases. Thus, small molecular glues that enhance the anchoring of TSPs to DUBs may stabilize them through deubiquitination. Here, through small-molecule microarray-based technology and unbiased screening, we identified three potential molecular glues that may tether P53 to the DUB USP7 and elevate the P53 level. Among them, bromocriptine (BC) is an FDA-approved drug showing the most robust effects. We further demonstrated that BC increased P53 stability via the predicted molecular glue mechanism engaging USP7. To confirm the generality of the screening platform, we identified another USP7-engaging molecular glue that upregulates PTEN, which is another well-known TSP. Taken together, we established a potential screening platform that may facilitate the discovery of novel molecular glues stabilizing TSPs via engaging the DUB USP7. Similar strategies could be applied to the identification of other types of molecular glues that may benefit drug discovery and chemical biology studies.
The dualistic subtyping propagated by Bokhman et al. provided the foundation for the prediction of endometrial cancer for several decades; however, over the years it has exhibited uncertainties in terms of tumor cell graduation and overall survival of patients. Molecular analyses identified the new subgroups POLE ultramutated, mismatch repair deficient (MMRd), p53 mutated, and endometrial cancer with an unspecific molecular profile. The predictive evidence of the molecular subgroups was confirmed by several studies and is significantly superior to the model published by Bokhman et al. In the present review article, the typing of endometrioid adenocarcinomas and the associated molecular pathological background are described based on five histopathological case examples from routine diagnostics. Furthermore, the last case report describes a subtype not yet included in the World Health Organization (WHO) classification of female genital tumors.
Cancer pathologies are associated with the unfolding and aggregation of most recurring mutations in the DNA Binding Domain (DBD) of p53 that coordinate the destabilization of protein. Substitution at the 175th codon with arginine to histidine (R175H, a mutation of large to small side-chain amino acid) destabilizes the DBD by 3 kcal/mol and triggers breasts, lung cancer, etc. Stabilizing the p53 mutant by small molecules offers an attractive drug-targeted anti-cancer therapy. The thiosemicarbazone (TSC) molecules NPC and DPT are known to act as zinc-metallochaperones to reactivate p53R175H. Here, a combination of LESMD simulations for 10 TSC conformations with a p53R175H receptor, single ligand-protein conformation MD, and ensemble docking with multiple p53R175H conformations observed during simulations is suggested to identify the potential binding site of the target protein in light of their importance for the direct TSC - p53R175H binding. NPC binds mutant R175H in the loop region L2-L3, forming pivotal hydrogen bonds with HIS175, pi‑sulfur bonds with TYR163, and pi-alkyl linkages with ARG174 and PRO190, all of which are contiguous to the zinc-binding native site on p53DBD. DPT, on the other hand, was primarily targeting alternative binding sites such as the loop-helix L1/H2 region and the S8 strand. The similar structural characteristics of TSC-bound p53R175H complexes with wild-type p53DBD are thought to be attributable to involved interactions that favour binding free energy contributions of TSC ligands. Our findings may be useful in the identification of novel pockets with druggable properties.
CNS tumors are a diverse group of neoplasms that emerge from a variety of different CNS cell types. These tumors may be benign, malignant, or borderline in nature. The majority of high grade glial tumors are fatal, with the exception of pilocytic astrocytoma. Primary malignant CNS tumors occur at a global annual rate of 2.1 to 5.8 per 100,000 persons. Males are more likely to develop malignant brain tumors than females, whereas benign meningiomas are more common in adult females. Additionally, gender inequalities in non-malignant tumors peak between the ages of 25 and 29 years. Only a small number of genetic variants have been associated with survival and prognosis. Notably, central nervous system (CNS) tumors exhibit significant age, gender, and race variation. Race is another factor that affects the incidence of brain and spinal cord tumors. Different races exhibit variation in terms of the prevalence of brain and CNS malignancies. This chapter discusses ongoing research on brain and spinal cord tumor epidemiology, as well as the associated risks and accompanied disorders.KeywordsEpidemiologyBrain tumorsGliomaGlioblastomaNeurological Surgery
Mitochondria, which serve as the energy factories of cells, are involved in cell differentiation, calcium homeostasis, amino acid and fatty acid metabolism and apoptosis. In response to environmental stresses, mitochondrial homeostasis is regulated at both the organelle and molecular levels to effectively maintain the number and function of mitochondria. The mitochondrial unfolded protein response (UPRmt) is an adaptive intracellular stress mechanism that responds to stress signals by promoting the transcription of genes encoding mitochondrial chaperones and proteases. The mechanism of the UPRmt in Caenorhabditis elegans (C. elegans) has been clarified over time, and the main regulatory factors include ATFS-1, UBL-5 and DVE-1. In mammals, the activation of the UPRmt involves eIF2α phosphorylation and the uORF-regulated expression of CHOP, ATF4 and ATF5. Several additional factors, such as SIRT3 and HSF1, are also involved in regulating the UPRmt. A deep and comprehensive exploration of the UPRmt can provide new directions and strategies for the treatment of human diseases, including aging, neurodegenerative diseases, cardiovascular diseases and diabetes. In this review, we mainly discuss the function of the UPRmt, describe the regulatory mechanisms of the UPRmt in C. elegans and mammals, and summarize the relationship between the UPRmt and various human diseases.
Cockle Cerastoderma edule is a species of great commercial interest in Galicia. It is one of the bivalve species with more annual catches and, therefore, has a high economic and ecological importance in this region. Pathological studies associated with cockle mortality events in Galicia detected high prevalence of a pathological condition known as disseminated neoplasia. Knowledge of cancer in molluscs is scarce. This study aims to deep in the knowledge about the epidemiology of this disease, its etiology and the biochemical and physiological changes it causes. A thorough knowledge of this disease is required to develop strategies to fight it.
The clearance of damaged or unwanted mitochondria by autophagy (also known as mitophagy) is a mitochondrial quality control mechanism postulated to play an essential role in cellular homeostasis, metabolism, and development and confers protection against a wide range of diseases. Proper removal of damaged or unwanted mitochondria is essential for organismal health. Defects in mitophagy are associated with Parkinson's, Alzheimer's disease, cancer, and other degenerative disorders. Mitochondria regulate organismal fitness and longevity via multiple pathways, including cellular senescence, stem cell function, inflammation, mitochondrial unfolded protein response (mtUPR), and bioenergetics. Thus, mitophagy is postulated to be pivotal for maintaining organismal healthspan and lifespan and the protection against aged‐related degeneration. In this review, we will summarize recent understanding of the mechanism of mitophagy and aspects of mitochondrial functions. We will focus on mitochondria‐related cellular processes that are linked to aging and examine current genetic evidence that supports the hypothesis that mitophagy is a pro‐longevity mechanism.
The process of phase separation allows for the establishment and formation of subcompartmentalized structures, thus enabling cells to perform simultaneous processes with precise organization and low energy requirements. Chemical modifications of proteins, RNA, and lipids alter the molecular environment facilitating enzymatic reactions at higher concentrations in particular regions of the cell. In this review, we discuss the nucleolus as an example of the establishment, dynamics, and maintenance of a membraneless organelle with a high level of organization.
Background
Lung cancer is a leading fatal malignancy due to the high incidence of treatment failure. Dysfunction of the tumor suppressor p53 contributes to cancer initiation, progression, and therapeutic resistance. Targeting MDM2, a negative regulator of p53, has recently attracted interest in cancer drug research as it may restore tumor suppressive function.
Purpose
The present study aimed to investigate the effect of 3,4-dihydroxy-5,4′-dimethoxybibenzyl (DS-1) on targeting MDM2 and restoring p53 function in lung cancer cells.
Methods
The efficacy of DS-1 alone or in combination with cisplatin in lung cancer cells was determined by MTT, nuclear staining, and annexin V/PI assay. The expression of apoptosis-related proteins was determined by western blot analysis. To evaluate the role of DS-1 on the stabilization and degradation of p53, cycloheximide chasing assay and immunoprecipitation were conducted, and the active form of p53 was investigated by immunofluorescent staining assay. To confirm and demonstrate the site interaction between DS-1 and the MDM2 protein, in silico computational analysis was performed.
Results
DS-1 exhibited a cytotoxic effect and sensitized lung cancer cells to cisplatin-induced apoptosis. DS-1 caused a significant increase in the cellular level of p53 protein, while the active form of p53 (phosphorylation at Ser15) was unaltered. DS-1 treatment in combination with cisplatin could enhance activated p-p53 (Ser15) and p53 downstream signaling (Bax, Bcl-2, and Akt), leading to a higher level of apoptosis. Immunoprecipitation analysis revealed that DS-1 decreased the p53-ubiquitin complex, a prerequisite step in p53 proteasomal degradation. Molecular docking simulation further evidenced that DS-1 interacts with MDM2 within the p53-binding domain by carbon–hydrogen bond interaction at Lys27, π–alkyl interactions at Ile37 and Leu30, and van der Waals interactions at Ile75, Val51, Val69, Phe67, Met38, Tyr43, Gly34, Phe31, and Lys27. Treatment by DS-1 and cisplatin in patient-derivated primary lung cancer cells showed consistent effects by increasing cisplatin sensitivity.
Conclusions
Our findings provide evidence that DS-1 is an MDM2 inhibitor and its underlying mechanism involves MDM2 binding and p53 induction, which may benefit the development of this compound for lung cancer treatment.
The immortalized cell is an essential research tool that uses robust growth properties for the functional investigation of gene products. Immortalized mammalian cells have mainly been established using three methods: expression of simian vacuolating virus 40 T antigen (the SV40 method); human papilloma virus-derived oncoprotein E6/E7 (the E6/E7 method); or combinatorial expression of R24C mutant cyclin dependent kinase 4, cyclin D1, and telomerase reverse transcriptase (the K4DT method). However, it is unclear as to which method is optimal for an in vitro model. Here, we compared the biological characteristics and genome-wide expression profiles of immortalized human dermal papilla cells generated by the SV40, E6/E7, or K4DT method. To our knowledge, this is the first study to comprehensively compare expression profiles to determine the optimal immortalization method for maintaining the original nature of the wild type cells. This data would be valuable to scientists aiming to establish new immortalized cell lines.
An oncoprotein MDM2 binds to the extreme N-terminal peptide region of a tumor suppressor protein p53 (p53NTD) and inhibits its anticancer activity. We recently discovered a peptide named MIP which exhibits much higher binding affinity for MDM2 than p53NTD. Experiments showed that the binding free energy (BFE) of MDM2-MIP is lower than that of MDM2-p53NTD by ~−4 kcal/mol. Here we develop a theoretical method which is successful in reproducing this quantitative difference and elucidating its physical origins. It enables us to decompose the BFE into a variety of energetic and entropic components, evaluate their relative magnitudes, and identify the physical factors driving or opposing the binding. It should be applicable also to the assessment of differences among ligands in the binding affinity for a particular receptor, which is a central issue in modern chemistry. In the MDM2 case, the higher affinity of MIP is ascribed to a larger gain of translational, configurational entropy of water upon binding. This result is useful to the design of a peptide possessing even higher affinity for MDM2 as a reliable drug against a cancer.
Breast cancer is the leading cause of cancer-related deaths in women and earlier detection can substantially reduce deaths from breast cancer. Polymers with targeted ligands are widely used in the field of molecular ultrasound imaging and targeted tumor therapy. In our study, the nanotheranostic agent was fabricated through filling perfluoropropane (C3F8) into poly(D,L-lactic-co-glycolic acid) nanoparticles (PLGA NPs), followed by the formation of gold nanoshell on the surface, then conjugated with anti p53 antibody which has high specificity with the p53 protein overexpressing in breast cancer. The average diameter of the gold nanoshelled PLGA NPs carrying anti p53 antibody (p53-PLGA@Au NPs) was 247±108.2 nm. The p53-PLGA@Au NPs had well-defined spherical morphology and hollow interiors observed by electron microscope, and had a good photothermal effect under the irradiation of an 808nm laser. The results of laser scanning confocal microscope (LSCM) and flow cytometer (FCM) indicated the specific targeting of p53-PLGA@Au NPs conjugating with breast cancer MCF-7 cells overexpressing p53 protein in vitro. Also the ultrasound imaging experiments in vitro showed that p53-PLGA@Au NPs were suitable for ultrasound contrast imaging. In conclusion, the p53-PLGA@Au NPs are demonstrated to be novel targeted UCAs and may have potential applications in the early diagnosis and targeted near-infrared (NIR) photothermal therapy of breast cancer in the future.
Genome-wide association study in diffusely infiltrating type cholangiocarcinoma (CC) can be limited due to the difficulty of obtaining tumor tissue. We aimed to evaluate the genomic alterations of diffusely infiltrating type CC using next-generation sequencing (NGS) of bile and to compare the variations with those of mass-forming type CC. A total of 24 bile samples obtained during endoscopic retrograde cholangiopancreatography (ERCP) and 17 surgically obtained tumor tissue samples were evaluated. Buffy coat and normal tissue samples were used as controls for a somatic mutation analysis. After extraction of genomic DNA, NGS analysis was performed for 48 cancer related genes. There were 27 men and 14 women with a mean age of 65.0±11.8years. The amount of extracted genomic DNA from 3cm(3) of bile was 66.0±84.7μg and revealed a high depth of sequencing coverage. All of the patients had genomic variations, with an average number of 19.4±2.8 and 22.3±3.3 alterations per patient from the bile and tumor tissue, respectively. After filtering process, damaging SNPs (8 sites for each type of CC) were predicted by analyzing tools, and their target genes showed relevant differences between the diffusely infiltrating and mass-forming type CC. Finally, in somatic mutation analysis, tumor-normal paired 14 tissue and 6 bile samples were analyzed, genomic alterations of EGFR, FGFR1, ABL1, PIK3CA, and CDKN2A gene were seen in the diffusely infiltrating type CC, and TP53, KRAS, APC, GNA11, ERBB4, ATM, SMAD4, BRAF, and IDH1 were altered in the mass-forming type CC group. STK11, GNAQ, RB1, KDR, and SMO genes were revealed in both groups. The NGS analysis was feasible with bile sample and diffusely infiltrating type CC revealed genetic differences compared with mass-forming type CC. Genome-wide association study could be performed using bile sample in the patients with CC undergoing ERCP and a different genetic approach for accurate diagnosis, pathogenesis study, and targeted therapy will be needed in diffusely infiltrating type CC.
Rare Diseases and Orphan Drugs shows that much of what we now know about common diseases has been achieved by studying rare diseases. It proposes that future advances in the prevention, diagnosis, and treatment of common diseases will come as a consequence of our accelerating progress in the field of rare diseases. Understanding the complex steps in the development of common diseases, such as cancer, cardiovascular disease, and metabolic diseases, has proven a difficult problem. Rare diseases, however, are often caused by aberrations of a single gene. In rare diseases, we may study how specific genetic defects can trigger a series of events that lead to the expression of a particular disease. Often, the disease process manifested in a certain rare disease is strikingly similar to the disease process observed in a common disease. This work ties the lessons learned about rare diseases to our understanding of common ones. Chapters covering the number of common diseases are minimized, while rare diseases are introduced as single diseases or as members of diseases classes. After reading this book, readers will appreciate how further research into the rare diseases may lead to new methods for preventing, diagnosing, and treating all diseases, rare or common. • Makes rare diseases relevant to clinicians and researchers by tying lessons learned about the rare diseases to our understanding of the common diseases • Stresses basic pathologic mechanisms that account for human disease (e.g., disorders of cell development, replication, maintenance, function and structure), that can be understood without prior training in pathology • Discusses advanced concepts in molecular biology and genetics in a simple, functional context appropriate for medical trainees and new researchers • Offers insights into how further research into rare diseases may lead to new methods for preventing, diagnosing, and treating all diseases.
This chapter brings together all of the scientific concepts introduced in earlier chapters to synthesize a coherent explanation of the biological relationship between the rare diseases and the common diseases. The rare diseases share genes and pathways with the common diseases. In some cases, populations of individuals with a common disease contain subsets of individuals whose disease has a monogenic cause (i.e., rare disease inside a common disease). New data produced by genome wide association studies suggest that disease pathways have genetic correlates, and that classes of genes may be involved in disease processes that happen to employ the same pathways.
It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.
The mechanisms causing resistance to chemotherapeutic drugs in cancer patients are poorly understood. Recent evidence suggests that different forms of chemotherapy may exert their cytotoxic effects by inducing apoptosis. The tumor suppressor gene P53 has a pivotal role inducing apoptosis in response to cellular damage. In vitro investigations have shown intact p53 to play a critical role executing cell death in response to treatment with cytotoxic drugs like 5-fluorouracil, etoposide and doxorubicin. Recently, mutations in the P53 gene were found to confer resistance to anthracyclines in a mouse sarcoma tumor model, and overexpression of the p53 protein (which, in most cases, is due to a mutated gene) was found to be associated with lack of response to cisplatin-based chemotherapy in non-small cell lung cancer. Previous studies have shown mutations in the P53 gene or overexpression of the p53 protein to predict a poor prognosis, but also a beneficial effect of adjuvant radiotherapy or chemotherapy in breast cancer. In this study we present data linking specific mutations in the P53 gene to primary resistance to doxorubicin therapy and early relapse in breast cancer patients.
Chemotherapeutic management of ovarian cancers is a difficult task as these neoplasms show significant differences in chemosensitivity, even if they share identical clinicopathological features. The present study was undertaken to investigate the prognostic and predictive role of p53 alterations in ovarian cancer. To this end, using different technical approaches, i.e. genetic and immunohistochemical analyses, we analysed a series of 68 ovarian neoplasms including 15 low malignant potential (LMP) tumours and 53 invasive carcinomas. We never observed p53 abnormalities in LMP tumours. p53 alterations were present only in invasive ovarian carcinomas, and they were detected much more frequently in tumours characterized by high histological grade (P = 0.01) and advanced-stage disease (P = 0.006 and P = 0.05 for gene mutations and protein expression respectively). For 33 patients with invasive ovarian cancer, information was available concerning response to cisplatin-based chemotherapy. A strong correlation (P = 0.001) has emerged between p53 alterations and response to chemotherapy; only one (14%) of seven patients who had a pathological complete response to antiblastic drugs showed p53 aberrations, whereas 18 (82%) of 22 cases with partial response and all of the four non-responsive patients scored positive for p53 abnormalities. We also observed that patients with p53 mutations had a significantly shorter progression-free survival than patients with p53-negative tumours (P = 0.05). Taken together, our results strongly suggest that in epithelial ovarian malignancies tumours showing p53 aberrations are significantly less sensitive to chemotherapy and more aggressive than those with functional p53. Thus, a routine analysis of this gene could have profound implications for the treatment of ovarian cancer.
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The tumour-suppressor gene TP53 is frequently mutated in breast tumours, and the majority of the mutations are clustered within the core domain, the region involved in DNA binding. We searched for alterations in this central domain of the TP53gene in 222 human breast cancer specimens using polymerase chain reaction-single-strand conformation analysis (PCR-SSCA) followed by sequencing. TP53 gene mutations were observed in 66 tumours (31%), including three tumours that contain two mutations. Fifty-four (78%) of these mutations were missense point mutations, one was a nonsense mutation and four were deletions and/or insertions causing disruption of the protein reading frame, whereas four mutations were either silent or a polymorphism (at codon 213; n = 6). Interestingly, the majority of missense mutations were observed at codon 248. The outcome has been related with patient and tumour characteristics, and with prognosis in 177 patients who were eligible for analysis of both relapse-free and overall survival (median survival for patients alive was 115 months). There was no significant association between the frequency of TP53 mutations and menopausal or nodal status, or tumour size. In a Cox univariate analysis, TP53 gene mutation was significantly associated with poor relapse-free survival (RFS: P = 0.02) but not with overall survival (OS: P = 0.07). In a Cox multivariate analysis, including classical prognostic factors, TP53 gene mutation independently predicted poor RFS and OS (RHR = 1.8 and 1.6 respectively). Unexpectedly, the median relapse-free survival of patients with a polymorphism at codon 213 or with a silent mutation was shorter (median 11 months) than the median relapse-free survival of patients with or without a TP53 gene mutation (median 34 or 48 months respectively). In an exploratory subset analysis, mutations in codons that directly contact DNA were related with the poorest relapse-free (P < 0.05) and overall survival (P < 0.02). These data imply that in the analysis of the prognostic value of TP53, the type of mutation and its biological function should be considered.
We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.
Many studies have investigated the association between alterations in the p53 gene and clinical outcome of breast cancer, and most investigators have reported poorer overall and disease-free survival (as indicated by a relative hazard (RH) greater than one) in breast cancer cases with somatic mutations in p53. However, different studies have produced widely differing RH estimates, ranging from no risk (RH = 1) to a relative hazard of 23, and not all of these results have been statistically significant. We have therefore reviewed all the published studies that have investigated the association between somatic mutations in the p53 gene and breast cancer prognosis and used standard techniques of meta-analysis to combine the results of these studies to produce a more precise estimate of the prognostic significance of p53 mutations. Eleven studies investigated overall survival in a total of 2319 unselected cases. The RH estimates from these ranged from 1 to 23.4 with a combined RH estimate of 2.0 (confidence interval 1.7-2.5). Three studies investigated the role of p53 in node-negative patients and in these, the combined estimate of RH was 1.7 (1.2-2.3). For three studies of node-positive breast cancer the combined risk estimate was 2.6 (1.7-3.9). The inclusion of p53 mutation screening in large breast cancer clinical trials seems warranted in the light of these results. Analysis of large numbers of cases matched for stage and therapy will allow definitive clarification of the value of p53 mutational status in prognostication, and possibly choice of therapy.
There is evidence from experimental work that hypoxia induces apoptosis in apoptosis-sensitive neoplastic cells and that this apoptotic sensitivity is lost during malignant progression. Oxygenation profiles and apoptotic indices in human squamous cell cancer of the uterine cervix have been determined, and a subgroup of tumors has been identified with low apoptotic index despite pronounced hypoxia representing carcinomas that consist of neoplastic cells with diminished apoptotic potential. These hypoxic low-apoptotic tumors show a high probability for lymphatic spread and for recurrence despite adjuvant treatment with radiation or chemotherapy in addition to radical surgery. The clinical results presented strongly support the hypothesis derived from experimental studies that the selection of apoptosis-insensitive neoplastic cell phenotypes in a hypoxic microenvironment is an important mechanism for malignant progression in solid tumors.
The family history of cancer in children treated for a solid malignant tumour in the Paediatric Oncology Department at Institute Gustave-Roussy, has been investigated. In order to determine the role of germline p53 mutations in genetic predisposition to childhood cancer, germline p53 mutations were sought in individuals with at least one relative (first- or second-degree relative or first cousin) affected by any cancer before 46 years of age, or affected by multiple cancers. Screening for germline p53 mutation was possible in 268 index cases among individuals fulfilling selection criteria. Seventeen (6.3%) mutations were identified, of which 13 were inherited and four were de novo. Using maximum likelihood methods that incorporate retrospective family data and correct for ascertainment bias, the lifetime risk of cancer for mutation carriers was estimated to be 73% for males and nearly 100% for females with a high risk of breast cancer accounting for the difference. The risk of cancer associated with such mutations is very high and no evidence of low penetrance mutation was found. These mutations are frequently inherited but de novo mutations are not rare.
TP53 status [mutations, immunostaining, and loss of heterozygosity (LOH)], expression of c-erbB-2, bcl-2, and histological grading were correlated to the response to doxorubicin monotherapy (14 mg/m2) administered weekly to 90 patients with locally advanced breast cancer. Mutations in the TP53 gene, in particular those affecting or disrupting the loop domains L2 or L3 of the p53 protein, were associated with lack of response to chemotherapy (P = 0.063 for all mutations and P = 0.008 for mutations affecting L2/L3, respectively). Similarly, expression of c-erbB-2 (P = 0.041), a high histological grade (P = 0.023), and lack of expression of bcl-2 (P = 0.018) all predicted chemoresistance. No statistically significant association between either p53 immunostaining or TP53 LOH and response to therapy was recorded, despite the finding that both were associated with TP53 mutation status (p53 immunostaining, P < 0.001; LOH, P = 0.021). Lack of immunostaining for p53 despite mutation of the TP53 gene was particularly seen in tumors harboring nonsense mutations or deletions/splices (7 of 10 negative for staining compared with 4 of 16 with missense mutations). TP53 mutations (total/affecting L2/L3 domains) were associated with expression of c-erbB-2 (P < 0.001 for both), high histological grade (P = 0.001 and P = 0.025), and bcl-2 negativity (P = 0.003 and P = 0.002). TP53 mutations, histological grade, and expression of bcl-2 (but not LOH or c-erbB-2 expression) all predicted for relapse-free as well as breast cancer-specific survival in univariate analysis (Ps between <0.0001 and 0.0155), but only tumor grade was found to be predictive in multivariate analysis (P = 0.01 and P = 0.0007, respectively). Our data are consistent with the hypothesis that certain TP53 mutations predict for resistance to doxorubicin in breast cancer patients. However, the observation that the majority of patients with TP53 mutations affecting or disrupting the L2/L3 domains with LOH in addition (n = 12) obtained a partial response (n = 4) or stabilization of disease (n = 5) during chemotherapy suggests redundant mechanisms to compensate for loss of p53 function. Our findings are consistent with the hypothesis that other defects may act in concert with loss of p53 function, causing resistance to doxorubicin in breast cancers.
Survivin is a member of the inhibitor of apoptosis family. This apoptosis inhibitor also has an evolutionarily conserved role as a mitotic spindle checkpoint protein. Previous studies on p53-repressed genes have implicated several genes involved in the G(2)/M transition of the cell cycle as targets of negative regulation by p53. However, few targets of p53 repression that are anti-apoptotic have been identified. This study identifies the anti-apoptotic survivin gene as a p53-repressed gene. Notably, Survivin repression by p53 is shown to be distinct from p53-dependent growth arrest. Chromatin immunoprecipitations indicate that p53 binds the survivin promoter in vivo; immunobinding studies indicate that this site overlaps with a binding site for E2F transcription factors and is subtly distinct from a canonical p53-transactivating element. The survivin-binding site contains a 3-nucleotide spacer between the two decamer "half-sites" of the p53 consensus element; deletion of this spacer is sufficient to convert the survivin site into a transactivating element. Finally, we show that overexpression of Survivin in cells sensitive to p53-dependent cell death markedly inhibits apoptosis induced by ultraviolet light. The identification of survivin as a p53 repressed gene should aid in the elucidation of the contribution of transcriptional repression to p53-dependent apoptosis.
The mutational patterns of the p53 gene for exons 4-9 were analyzed in 30 recurring tumors compared with the p53 status of the corresponding 30 primary breast cancers. The prevalence of p53 mutations was higher, although not statistically significant (P = 0.07), in the evaluable recurring tumors compared with the corresponding primaries, 12 of 29 (41%) versus 7 of 30 (23%). Twenty-one of the patients had unchanged p53 mutation status in the recurring compared with the primary tumors, whereas 8 had an altered mutational status or pattern in the sequential tumor. These findings indicate that p53 mutations may be an important factor for tumor progression in human breast cancer.
We investigated the prognostic significance of mutation to the TP53 tumor suppressor gene in a series of 908 breast cancer patients treated with or without adjuvant therapies. The frequency of TP53 mutation detected by single strand conformation polymorphism (SSCP) was 19.4% (176/908) in the overall tumor series. In multivariate analysis, TP53 mutation was independently associated with worse survival in the overall (HR = 2.1, 95% CI [1.5-3.1], P<0.0001), non-adjuvant treated (HR=2.2, 95% CI [1.2-4.2], P=0.017) and adjuvant treated (HR= 2.0, 95% CI [1.3-3.1], P = 0.0009) patients.
Purpose and Experimental Design: Telomeres of tumor cells may be maintained by telomerase or by alternative lengthening of telomeres (ALT). The standard ALT assay requires Southern analysis of high molecular weight genomic DNA. We aimed to establish and validate an ALT assay suitable for archived paraffin-embedded tumors and to use it to examine the prevalence and clinical significance of ALT in various types of tumors that are often telomerase negative.
Results: To assay for ALT, we detected ALT-associated promyelocytic leukemia (PML) bodies (APBs) by combined PML immunofluorescence and telomere fluorescence in situ hybridization. APBs are PML nuclear domains containing telomeric DNA and are a known hallmark of ALT in cell lines. The APB assay concurred with the standard ALT assay in 62 of 62 tumors and showed that 35% of 101 soft tissue sarcomas (STS), 47% of 58 osteosarcomas (especially younger patients), 34% of 50 astrocytomas, and 0% of 17 papillary thyroid carcinomas were ALT positive (ALT+). The prevalence of ALT varied greatly among different STS subtypes: malignant fibrous histiocytomas, 77%; leiomyosarcomas, 62%; liposarcomas, 33%; synovial sarcomas, 9%; and rhabdomyosarcomas, 6%. ALT correlated with survival in glioblastoma multiforme and occurred more often in lower-grade astrocytomas, but ALT+ and ALT− sarcomas were equally aggressive in terms of grade and clinical outcome.
Conclusion: The APB assay for ALT is suitable for paraffin-embedded tumors. It showed that a substantial proportion of STS, osteosarcomas, and astrocytomas, but not papillary thyroid carcinomas use ALT. APB positivity correlated strongly with survival of patients with astrocytomas.
We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non‐isotopic single‐strand conformation polymorphism (SSCP) method we screened for mutations in exons 4–10 of the p53 gene in 375 primary breast cancers from patients with a median follow‐up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S‐phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node‐negative and hormone receptor‐positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S‐phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction‐SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information. Int. J. Cancer 74:642–647, 1997.© 1997 Wiley‐Liss, Inc.
Purpose: Although the genetic alterations in glioblastoma have been well characterized, reports regarding their prognostic effects have been inconsistent.
Experimental Design: In this series of 140 consecutive cases of glioblastoma treated at a single center, we analyzed the frequency, age dependency and prognostic effects of TP53 mutation, CDKN2A/p16 deletion, EGFR amplification, as well as loss of chromosome 1p, chromosome 10q, and chromosome 19q. The complete set of genetic alterations was available on 60 of 140 patients.
Results: In this cohort of glioblastoma cases, TP53 mutation was significantly associated with patient age. The prognostic effects of TP53 mutation, EGFR amplification, CDKN2A/p16 alterations, and loss of chromosome 1p were dependent on the age of the patient.
Conclusions: This is the first observation that the prognostic effects of TP53, 1p, and CDKN2A/p16 alterations are dependent on patient age. These observations concerning the interactions of age and genetic changes in glioblastoma suggest that tumorigenic pathways to glioblastoma vary with the age of the patient and that future molecular marker studies should carefully evaluate the potential age-dependent prognostic effects of these biological variables. The inconsistent or negative prognostic effects of molecular markers reported in prior studies of glioblastoma may be because different effects at different ages may have resulted in a cancellation of an overall effect in the entire cohort.
Epidemiological studies on intracranial tumors have suggested that the observed familial aggregation of a proportion of gliomas may be due to inherited predisposition to their development. In the Li-Fraumeni syndrome (LFS) associated with germ-line mutations of the p53 gene, nervous-system tumors are observed with increased frequency. However, the contribution of germ-line p53 mutation to the incidence of brain tumors has not been investigated. In order to address this point, we have performed 2 independent investigations. First, we have examined an unselected series of brain tumors. Whenever the presence of a p53 mutation in the tumor was observed, the possible germ-line origin of the mutation was investigated. Germ-line p53 mutations were also analyzed in constitutional DNA of patients with gliomas that had been selected for an unusual personal or familial history of cancer. Germ-line p53 mutations were detected in 1 out of 80 unselected cases and in 3 out of 15 selected cases (20%). We conclude that germ-line p53 mutation may contribute to a small fraction of gliomas that develop in the general population. The presence of a personal or familial history of cancer in a patient with glioma should prompt the search for a germ-line p53 mutation. However, the low frequency of p53 germ-line mutation suggests that alterations of this gene may not account for most familial cases of gliomas. © 1995 Wiley-Liss, Inc.
We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non-isotopic single-strand conformation polymorphism (SSCP) method we screened for mutations in exons 4–10 of the p53 gene in 375 primary breast cancers from patients with a median follow-up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S-phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node-negative and hormone receptor-positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S-phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction-SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information. Int. J. Cancer 74:642–647, 1997.© 1997 Wiley-Liss, Inc.
There are now reports of nearly 250 independent germline TP53 (p53) mutations in over 100 publications. Such mutations are typically associated with Li-Fraumeni or Li-Fraumeni-like syndrome, although many have been identified in cohorts of patients with tumors considered to be typical of LFS. In general, the spectrum of mutations that has been detected in the germline reflects that found in tumors, although there are some notable exceptions in certain tumor types. Detailed knowledge of the pedigrees allows a comprehensive analysis of genotype–phenotype correlations and an understanding of the tumors that are associated with germline TP53 mutations. This review will discuss the spectrum of mutations and the methods for mutation detection, the tumors associated with inheritance of a germline mutation, and some of the ethical and clinical problems in patients with a germline TP53 mutation. Hum Mutat 21:313–320, 2003. © 2003 Wiley-Liss, Inc.
The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Here, it is shown that
heterozygous germ line mutations in hCHK2occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in
theTP53 gene. These observations suggest that hCHK2is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a
link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast.
The entire coding sequence of the p53 gene was analysed for the presence of mutations in 12 families conforming to a restricted definition of Li-Fraumeni syndrome (classic LFS) and nine families with features of LFS conforming to a broader definition. Mutations were detected in seven families. Six were point mutations with one each affecting codons 175, 180, and 220 and three affecting codon 248. The seventh was a deletion/insertion mutation in exon 4. Germline mutations in p53 were a feature of families which included children with rhabdomyosarcoma and/or adrenal cortical carcinoma. Germline p53 mutations were detected in six of the nine families with such tumors. An analysis of these 7 mutations, together with 34 published examples, showed that more than one-half were transitions at CpG dinucleotides, suggesting that the majority of germline p53 mutations may arise as a result of spontaneous events. The most common cancers occurring in the 41 families with germline p53 mutations, in common with classic LFS, were bone and soft tissue sarcoma, breast cancer, brain tumors, leukemia, and adrenocortical carcinoma, although less than one-half of the probands with germline p53 mutations came from classic LFS families. More than one-half of the cancers overall and nearly one-third of the breast cancers were diagnosed before 30 years of age. These observations have important implications for asymptomatic carriers of germline p53 mutations, and there is a need for international collaboration in the development of protocols for the management of such families.
Epidemiological studies on intracranial tumors have suggested that the observed familial aggregation of a proportion of gliomas may be due to inherited predisposition to their development. In the Li-Fraumeni syndrome (LFS) associated with germ-line mutations of the p53 gene, nervous-system tumors are observed with increased frequency. However, the contribution of germ-line p53 mutation to the incidence of brain tumors has not been investigated. In order to address this point, we have performed 2 independent investigations. First, we have examined an unselected series of brain tumors. Whenever the presence of a p53 mutation in the tumor was observed, the possible germ-line origin of the mutation was investigated. Germ-line p53 mutations were also analyzed in constitutional DNA of patients with gliomas that had been selected for an unusual personal or familial history of cancer. Germ-line p53 mutations were detected in 1 out of 80 unselected cases and in 3 out of 15 selected cases (20%). We conclude that germ-line p53 mutation may contribute to a small fraction of gliomas that develop in the general population. The presence of a personal or familial history of cancer in a patient with glioma should prompt the search for a germ-line p53 mutation. However, the low frequency of p53 germ-line mutation suggests that alterations of this gene may not account for most familial cases of gliomas.
Since the majority of germline mutations in the TP53 gene seem to occur in LFS or LFL families, and these are rare, research is best conducted in a collaborative setting (Li and Fraumeni, in press). In a report from a meeting at Bethesda in 1993, the following areas were outlined for collaborative study: the correlation (if any) of phenotypes with specific mutation; age specific penetrance; cumulative cancer incidence; gender differences in tumour development in carriers; the effects of DNA damaging agents on individuals with a TP53 mutation; the frequency of TP53 germline mutations in cohorts of patients with rare childhood tumours (eg adrenocortical carcinoma); and the psychosocial aspects of predictive TP53 testing. In addition, if, as seems likely from recent data, X irradiation in these individuals induces DNA damage that is tolerated, urgent collaborative studies are needed to investigate new methods of screening, such as magnetic resonance imaging. Treatment modalities should be carefully chosen, and for this reason alone, predictive testing may be desirable in all LFS and LFL families. Individuals carrying TP53 mutations could be offered chemoprevention within trials in an effort to reduce their mortality from cancer.
Immunohistochemical (IHC) detection of p53 protein was compared with the presence of p53 gene mutation in many colorectal (n = 100), breast (n = 92), endometrial (n = 122), and gastric (n = 116) carcinomas. Two commercially available antibodies, DO7 and CM1, were used for IHC analysis of paraffin-embedded tissue sections. Screening for gene mutations in frozen and paraffin-embedded tumor samples was carried out using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). The frequency of nuclear staining with DO7 or CM1 for each tumor type, respectively, was colorectal (36%, 23%); breast (15%, 19%); endometrial (21%, 33%); and gastric (23%,-). Overall correlation between the two antibodies for nuclear staining was 90% for the 314 tumors analyzed. Cytoplasmic staining was observed with DO7 in 7% of breast and 5% of gastric carcinomas and with CM1 in 17% of breast and 54% of endometrial carcinomas. p53 gene mutation was found in 39% of colorectal, 28% of breast, 13% of endometrial, and 25% of gastric cancers. The concordance between p53 nuclear overexpression and gene mutation (both positive or both negative) was 68% for colorectal, 79% for breast, 76% for endometrial, and 73% for gastric carcinomas. This study provides further evidence that IHC detection of p53 protein accumulation does not always indicate the presence of a gene mutation and vice versa. Discordant results were observed in approximately 20% to 30% of the tumors studied, highlighting the need for careful characterization of both p53 gene and protein alterations when assessing the relationship between p53 status and tumor behavior.
The gradual loss of DNA from the ends of telomeres has been implicated in the control of cellular proliferative potential. Telomerase is an enzyme that restores telomeric DNA sequences, and expression of its activity was thought to be essential for the immortalization of human cells, both in vitro and in tumor progression in vivo. Telomerase activity has been detected in 50-100% of tumors of different types, but not in most normal adult somatic tissues. It has also been detected in about 70% of human cell lines immortalized in vitro and in all tumor-derived cell lines examined to date. It has previously been shown that in vitro immortalized telomerase-negative cell lines acquire very long and heterogeneous telomeres in association with immortalization presumably via one or more novel telomere-lengthening mechanisms that we refer to as ALT (alternative lengthening of telomeres). Here we report evidence for the presence of ALT in a subset of tumor-derived cell lines and tumors. The maintenance of telomeres by a mechanism other than telomerase, even in a minority of cancers, has major implications for therapeutic uses of telomerase inhibitors.
We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non-isotopic single-strand conformation polymorphism (SSCP) method we screened for mutations in exons 4-10 of the p53 gene in 375 primary breast cancers from patients with a median follow-up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S-phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node-negative and hormone receptor-positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S-phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction-SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information.
The tumor suppressor protein p53 is an essential molecule in cell proliferation and programmed cell death (apoptosis), and has been postulated to play a principal part in the development of atherosclerosis. We have examined the effect of p53 inactivation on atherogenesis in apoE-knockout mice, an animal model for atherosclerosis. We found that, compared with p53+/+/apoE-/- mice, p53-/-/apoE-/- mice developed considerably accelerated aortic atherosclerosis in the presence of a similar serum cholesterol in response to a high-fat diet. Furthermore, the atherosclerotic lesions in p53-/-/apoE-/- mice had a significant (approximately 280%) increase in cell proliferation rate and an insignificant (approximately 180%) increase in apoptosis compared with those in p53+/+/apoE-/- mice. Our observations indicate that the role of p53 in atherosclerotic lesion development might be associated with its function in cell replication control, and that p53-independent mechanisms can mediate the apoptotic response in atherosclerosis.
Ataxia-telangiectasia mutated (ATM) is the product of the gene mutated in the human genetic disorder ataxia-telangeictasia (A-T). It is a 370 kDa protein that is a member of the phosphatidyl inositol 3-kinases superfamily. A-T cells and those derived from Atm-/- mice are characterized by hypersensitivity to ionizing radiation and defective cell cycle checkpoints. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation including the G1/S interface where ATM plays an important role in the activation of the tumour suppressor gene product p53. Activation leads to the induction of p21/WAF1, inhibition of cyclin-dependent kinase activity, failure to phosphorylate key substrates such as the retinoblastoma protein and consequently G1 arrest. ATM also plays an important role in the regulation and surveillance of meiotic progression. Absence of ATM gives rise to a spectrum of defects including immunodeficiency, neurodegeneration, radiosensitivity and cancer predisposition. It is clear that a better definition of the role of ATM in DNA damage recognition, cell cycle control and cell signalling may assist in the treatment of the progressive neurodegeneration in this syndrome.
Cathepsin B expression is increased at both the mRNA and protein levels in a wide variety of tumors. The mechanisms responsible for this regulation are not well elucidated. We have isolated a 2.2-kb cathepsin B genomic fragment that contains the 5'-flanking region of the cathepsin B gene. Using reporter gene analysis in human glioblastoma U87MG cells, we have mapped a 228-bp fragment (-172 to +56) having high promoter activity. This promoter region has a high G+C content; contains potential Spl, Ets, and USF binding motifs; and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site. Cotransfection experiments demonstrated that Spl and Ets1 could trans-activate cathepsin B transcription, whereas Ets2 could not. Electrophoretic mobility shift assays and supershift assays revealed that three of the four putative Sp1 sites in this promoter region form a specific complex containing the Sp1 transcription factor. Mutating all four of the Spl binding sites individually markedly reduced the promoter activity of transfected reporter genes in U87 cells. Cotransfection of this cathepsin B promoter construct with Spl family expression vectors in Schneider's Drosophila line 2 (SL2) cells demonstrated that Spl and Sp3, but not Sp4, activated cathepsin B transcription. Taken together, these results suggest that Sp1, Sp3, and Ets1 are important factors in cathepsin B transcription. The regulation of cathepsin B transcription by Sp1- and Sp1-related factors is mediated through multiple GC boxes.
The World Health Organization announces a new series entitled World Health Organization Classification of Tumors. This continuation of the International Histological Classification of Tumors project will include information on molecular genetics and will cover all tumor sites within the next 5 years.
The p53 tumour-suppressor gene integrates numerous signals that
control cell life and death. As when a highly connected node in the Internet
breaks down, the disruption of p53 has severe consequences.
Evidence suggests that p53 induces cell death by a dual mode of action involving activation of target genes and transcriptionally independent direct signaling. Mitochondria are major signal transducers in apoptosis. We recently discovered that a fraction of induced p53 protein rapidly translocates to mitochondria during p53-dependent apoptosis, but not during p53-independent apoptosis or p53-mediated cell cycle arrest. Importantly, specific targeting of p53 to mitochondria was sufficient to induce apoptosis in p53-deficient tumor cells. This led us to propose a model where p53 exerts a direct apoptogenic role at the mitochondria, thereby enhancing the transcription-dependent apoptosis of p53. Here we show for the first time that mitochondrial localization of endogenous p53 can be visualized by immunofluorescence of whole cells when stressed by hypoxic conditions. Suborganellar localization by limited trypsin digestion of isolated mitochondria from stressed cells suggests that a significant amount of mitochondrial p53 is located at the surface of the organelle. This mitochondrial association can be reproduced in vitro with purified p53. Together, our data provide further evidence for an apoptogenic signaling role of p53 protein in vivo at the level of the mitochondria.
In contrast to p53-mediated cell cycle arrest, the mechanisms of p53-mediated apoptosis in response to cellular stresses such as DNA damage, hypoxia and oncogenic signals still remain poorly understood. Elucidating these pathways is all the more pressing since there is good evidence that the activation of apoptosis rather than cell cycle arrest is crucial in p53 tumor suppression. Moreover, the therapeutic interest in p53 as the molecular target of anticancer intervention rests mainly on its powerful apoptotic capability. This puzzling elusiveness suggests that p53 not only engages a plethora of downstream pathways but itself might possess a biochemical flexibility that goes beyond its role as a mere transcription factor. Recent evidence of a direct pro-apoptotic role of p53 protein at mitochondria suggests a synergistic effect with its transcriptional activation function and brings an unexpected new level of complexity into p53 apoptotic pathways.
We recently presented evidence for tumor site and gender-specificity in the survival benefit from adjuvant chemotherapy in Stage III colorectal cancer (CRC). In the current study, we examined whether p53 alteration or the microsatellite instability (MSI) phenotype provide additional predictive information in CRC patients.
A retrospective series of 891 Stage III CRC patients with negative surgical margins was investigated. Thirty percent (270 of 891) received postoperative adjuvant chemotherapy with curative intent and comprising of 5-fluorouracil/levamisole. Adjuvant treatment and nontreatment patient groups were well matched for tumor site, grade, p53 alterations, and MSI. Surgical tumor specimens were investigated for p53 overexpression using immunohistochemistry and for p53 mutation and MSI using single-strand conformation polymorphism analysis. The predictive value of these markers was evaluated by comparing the survival of adjuvant-treated and nonadjuvant treated patients.
A strong inverse correlation was observed between p53 alteration and MSI (P < 0.0001). In univariate analysis, the factors of sex, site, p53 alteration, and MSI were each strong predictors of a survival benefit from chemotherapy. Multivariate analysis revealed that chemotherapy provided maximal survival benefit for female patients (P = 0.005) and for patients whose tumors contained normal p53 (P = 0.041). Males whose tumors contained a p53 alteration and were negative for MSI appeared not to benefit from chemotherapy.
Our findings suggest that p53 alteration and MSI could be clinically useful molecular predictive markers for the identification of CRC patients who might benefit from 5-fluorouracil-based chemotherapy.
While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14(ARF), Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage.
It has been shown that scatter photocoagulation induces regression of retinal neovascularization, but the mechanism for this effect is not completely understood. The main focus of our research is to determine the mechanism for the beneficial effects of photocoagulation. In the present study, we quantified the expression of growth factors and transcription factors that inhibit or induce angiogenesis in photocoagulated human retinal pigment epithelial (RPE) cells in vitro.
RPE cells were grown to confluence, and RNA was isolated from the RPE cells with or without photocoagulation. The following growth factors, their receptors and transcription factors were examined by reverse transcription polymerase chain reaction (RT-PCR): transforming growth factor (TGF)-beta 1, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor (KDR/flk-1), hypoxia-inducible factor (HIF)-1, ETS-1, nuclear factor kappa B (NF-kappa B), interleukin-8 (IL-8).
Laser photocoagulation increased the expression of TGF-beta 1. Expression of angiogenic factors bFGF, VEGF, IL-8 and their transcription factor, ETS1, was also increased. However, the up-regulation of these factors was observed early (6 h) after photocoagulation. Seventy-two hours after photocoagulation, when RPE cells were repaired, the expression of VEGF, IL-8, ETS-1, and NF-kappa B was decreased to the levels before photocoagulation.
These results suggest that TGF-beta produced by photocoagulated RPE cells and the down-regulation of angiogenic factors in repaired RPE cells, in all likelihood, play an important role in the processes that occur after laser photocoagulation.
Injury of endothelial cells has been assumed to be an initial trigger of the development of atherosclerosis. In this study, we investigated the molecular mechanisms of endothelial cell death induced by hypoxia, which leads to oxidative stress. To study the relation between hypoxia-induced cell death and activation of nuclear factor-kappaB (NF-kappaB) in a hypoxic state, we evaluated the effect of 2 antioxidant drugs, probucol and pyrrolidine dithiocarbamate (PDTC), on human endothelial apoptosis. Although hypoxic treatment of human aortic endothelial cells resulted in a significant decrease in cell number and a significant increase in apoptotic cells compared with that of cells under normoxia (P<0.01), treatment with probucol (50 micromol/L) or PDTC (100 micromol/L) significantly attenuated the decrease in cell number (P<0.01) and was accompanied by inhibition of NF-kappaB activation. Furthermore, downregulation of bcl-2 caused by hypoxia was inhibited by these drugs. We further investigated the translocation of bax protein from the cytoplasm to the mitochondrial heavy fraction membrane, as translocation of bax protein is considered to be a determinant of apoptosis. Interestingly, we found that antioxidant treatment inhibited the translocation of bax protein caused by hypoxia. Moreover, upregulation of p53, a proapoptotic molecule, was observed in hypoxia, whereas treatment with probucol attenuated the expression of p53 accompanied by suppression of NF-kappaB activation. These data suggest functional links between p53 and endothelial apoptosis through the activation of NF-kappaB. Overall, the current study demonstrated that oxidative stress induced apoptosis in human aortic endothelial cells through the downregulation of bcl-2, translocation of bax, and upregulation of p53, probably through NF-kappaB activation. Oxidative stress may play an important role in endothelial apoptosis mediated by hypoxia, through the activation of NF-kappaB.
A woman with a family history of brain tumors in her daughter and sister presented with a breast cancer. She subsequently developed two metachronous primary tumors: a small-cell lung cancer and a colon carcinoma. These tumors arose within the internal mammary radiotherapy field and within the field irradiated for ovariolysis. The p53 gene was analyzed in whole blood lymphocytes using a functional assay developed in yeast Saccharomyces cerevisiae, which tests the transcriptional competence of p53. DNA from the colon cancer cells was analyzed by polymerase chain reaction and sequencing. The patient had a germline-inactivating p53 mutation, confirming the diagnosis of Li-Fraumeni syndrome (LFS). The colon tumor and the lung tumor both conserved the mutant p53 allele but had lost the wild-type allele. This observation and the experimental data suggest an abnormal sensitivity of LFS patients to radiogenic carcinogenesis. The indications and extent of radiotherapy in patients with a clinical or molecular diagnosis of LFS should be discussed individually and should take into account the risk of secondary neoplasms arising in the radiation fields.
Many polymorphisms have been reported in the TP53 gene. Some of these are within the coding region, and may affect the function of the p53 protein, others are within introns or non-coding regions, and their significance is unclear. Recently, a number of publications have claimed that polymorphisms within intron 6 are responsible for inherited predisposition to childhood malignancies, familial breast cancer, and Li-Fraumeni syndrome (LFS). We find no evidence for intron 6 sequence variants predisposing to LFS in our cohort of families and, furthermore, we show that some of the conclusions of other groups cannot be supported by data from our analysis.
Li-Fraumeni syndrome (LFS) has been the most common terminology used for the syndrome. It is a rare familial dominantly inherited cancer syndrome characterized by a wide spectrum of neoplasms occurring in children and young adults. The canonical definition of LFS includes a proband diagnosed with sarcoma before 45 years of age, a first-degree relative with cancer before this same age and another first- or second-degree relative in the lineage with any cancer before this age or sarcoma at any age. Multiple studies have reported p53 germline mutations in LFS families in various parts of the world. As in sporadic tumors, loss of heterozygosity leading to the inactivation of the wild-type allele by deletion or mutation is observed in LFS tumors. Cancer-risk in mutation carriers has been estimated to be 73% in males and nearly 100% in females, the difference almost entirely explained by breast cancer. The identification of germline p53 mutations in rare cancer-prone families has given rise to the medical, counseling, psychological and ethical problems.
Mutations in the tumor suppressor gene TP53 are frequent in most human cancers. Comparison of the mutation patterns in different cancers may reveal clues on the natural history of the disease. Over the past 10 years, several databases of TP53 mutations have been developed. The most extensive of these databases is maintained and developed at the International Agency for Research on Cancer. The database compiles all mutations (somatic and inherited), as well as polymorphisms, that have been reported in the published literature since 1989. The IARC TP53 mutation dataset is the largest dataset available on the variations of any human gene. The database is available at www.iarc.fr/P53/. In this paper, we describe recent developments of the database. These developments include restructuring of the database, which is now patient-centered, with more detailed annotations on the patient (carcinogen exposure, virus infection, genetic background). In addition, a new on-line application to retrieve somatic mutation data and analyze mutation patterns is now available. We also discuss limitations on the use of the database and provide recommendations to users.
Abnormal vascular smooth muscle (VSM) cell proliferation contributes to the development of atherosclerosis and its associated disorders, including angioplasty restenosis. The tumor-suppressor protein p53 has been linked to the development of atherosclerotic lesions, and its homolog, p73, is proving to have contrasting functions in a variety of tissues. As an outgrowth of our previous finding that p73 is increased in serum-stimulated VSM cells and human atherosclerotic tissue, we examined p73 overexpression in VSM cells to elucidate causality of p73 expression with growth response. Overexpression of p73 results in decreased cell cycle transit and is accompanied by apoptosis. The apoptotic changes in p73 overexpressing VSM cells are independent of p53 and are associated with a decrease in levels of p21(waf1/cip1). In conjunction with our previous data finding that p73 is increased in serum-stimulated VSM cells, this work suggests a role for p73 in vascular proliferative diseases.
Drugs currently used for patients with Parkinson's disease provide temporary relief of symptoms but do not halt or slow the underlying neurodegenerative disease process. Increasing evidence suggests that neurons die in Parkinson's disease by a process called apoptosis, which may be triggered by mitochondrial impairment and oxidative stress. We report that two novel synthetic inhibitors of the tumor suppressor protein p53, pifithrin-alpha (PFT-alpha) and Z-1-117, are highly effective in protecting midbrain dopaminergic neurons and improving behavioral outcome in a mouse model of Parkinson's disease. Mice given intraperitoneal injections of PFT-alpha or Z-1-117 exhibited improved motor function, reduced damage to nigrostriatal dopaminergic neurons and reduced depletion of dopamine and its metabolites after exposure to the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP caused an increase in the level of the proapoptotic protein Bax, which was prevented by giving mice PFT-alpha and Z-1-117. PFT-alpha and Z-1-117 also suppressed Bax production and apoptosis in cultured dopaminergic cells exposed to MPP(+). Our findings demonstrate a pivotal role for p53 in experimental parkinsonism and identify a novel class of synthetic p53 inhibitors with clinical potential.
