P53 Activity Research Articles

Celecoxib enhances the response of tumor cells to cisplatin through upregulating PUMA in non–small cell lung cancer carrying wild-type p53

Abstract Celecoxib, a cyclooxygenase-2 inhibitor, can enhance the efficacy of chemotherapy; however, its effect seems inconsistent. In this study, we investigated whether celecoxib would increase the antiproliferative effects of cisplatin in human lung cancer cells. Our data demonstrated the synergistic effects of celecoxib with cisplatin in wild-type p53 cells and their antagonistic effects in mutated or deleted p53 cells. Combination indices of 0.82 to 0.93 reflected a synergistic effect between celecoxib and cisplatin in lung cancer cells with wild-type p53. Combination indices of 1.63 to 3.00 reflected antagonism between celecoxib and cisplatin in lung cancer cells with mutated or deleted p53. Compared with that in cells with mutated or deleted p53, apoptosis significantly increased with the addition of celecoxib and cisplatin in wild-type p53 cells (P < 0.05). Moreover, the results in vivo were similar to those in vitro: celecoxib combined with cisplatin slowed tumor growth in wild-type p53 groups and not in mutated or deleted p53 groups. In addition, celecoxib promoted p53 translocation into the nucleus and upregulated active p53 expression in wild-type p53 cells. Celecoxib combined with cisplatin upregulated PUMA (PUMA is a downstream gene of p53) after active p53 increased in wild-type p53 cells. In summary, the combination of celecoxib and cisplatin demonstrates clear synergistic effects in wild-type p53 cells and antagonistic effects in mutated or deleted p53 cells. The synergistic effect was achieved by apoptosis, induced by upregulating PUMA. Our results will provide a new treatment strategy for patients carrying wild-type p53, insensitive to cisplatin.

Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease – with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Efficacy and safety of brigimadlin (BI 907828), an MDM2–p53 antagonist, in patients (pts) with advanced biliary tract cancer: Data from two phase Ia/Ib dose-escalation/expansion trials.

487 Background: Outcomes remain poor for pts treated with standard-of-care chemotherapy for advanced biliary tract cancer (BTC). Hence, there is a clinical need for novel therapeutic strategies. One such emerging strategy is antagonism of mouse double minute 2 (MDM2), which is amplified in 5–8% of cases of BTC. Brigimadlin (BI 907828) is a MDM2–p53 antagonist that blocks the MDM2–p53 interaction, thereby restoring p53 activity and leading to cell-cycle arrest and apoptosis in TP53 wild-type tumors. Brigimadlin is currently being assessed in two Phase Ia/Ib dose-escalation/expansion trials in pts with advanced/metastatic solid tumors as monotherapy (NCT03449381) and in combination with an anti-PD-1 antibody, ezabenlimab (NCT03964233). Here, we present data in pts with advanced BTCs treated in these trials. Methods: Pts in the monotherapy trial received escalating doses of brigimadlin on day 1 of 21-day cycles (Q3W). Pts in the combination trial received escalating doses of brigimadlin and 240 mg ezabenlimab on day 1 Q3W (doublet); one pt also received the anti-LAG-3 antibody BI 754111 (which has since been discontinued; triplet). Results: At data cut-off (June 2023), a total of 16 pts with BTC have been enrolled (10 in the monotherapy and 6 in the combination trial). In the monotherapy trial, 9 pts received brigimadlin 45 mg Q3W and 1 received 80 mg Q3W. In the combination trial, 5 pts received 30 mg/45 mg brigimadlin doublet and 1 received 45 mg triplet. Across both trials, 7 pts with MDM2 -amplified tumors achieved partial response (PR), 3 in the monotherapy trial and 4 in the combination trial. In the monotherapy trial, the responding pts had intrahepatic cholangiocarcinoma (iCC; 80 mg, 73% tumor shrinkage, progression-free survival [PFS] 13.3 months), ampullary adenocarcinoma (45 mg, 72% tumor shrinkage, PFS censored at 666 days), and extrahepatic cholangiocarcinoma (45 mg, 68% tumor shrinkage, PFS censored at 164 days). In the combination trial, 3 responding pts had iCC (pt 1, 30 mg doublet, 54% tumor shrinkage, PFS 9.6 months; pt 2, 45 mg doublet, 49% tumor shrinkage, PFS 7.5 months; pt 3, 45 mg doublet, 39% tumor shrinkage, PFS censored at 5.5 months) and 1 had gallbladder cancer (45 mg doublet, 50% tumor shrinkage, PFS 7.9 months). A further 7 pts (5 in the monotherapy trial and 2 in the combination trial) achieved stable disease (SD). In both trials, the most common any-grade treatment-related AE (TRAE) was nausea; the most common grade ≥3 TRAEs were thrombocytopenia and neutropenia. Conclusions: Brigimadlin showed a manageable safety profile and encouraging preliminary efficacy in pts with BTC, with 7 PRs and 7 SDs in 16 pts. A Phase IIa/IIb trial of brigimadlin in patients with MDM2 -amplified, TP53 wild-type BTC and other solid tumors is ongoing (Brightline-2). Clinical trial information: NCT03449381 and NCT03964233 .

Redox signalling regulates breast cancer metastasis via phenotypic and metabolic reprogramming due to p63 activation by HIF1α.

Redox signaling caused by knockdown (KD) of Glutathione Peroxidase 2 (GPx2) in the PyMT mammary tumour model promotes metastasis via phenotypic and metabolic reprogramming. However, the tumour cell subpopulations and transcriptional regulators governing these processes remained unknown. We used single-cell transcriptomics to decipher the tumour cell subpopulations stimulated by GPx2 KD in the PyMT mammary tumour and paired pulmonary metastases. We analyzed the EMT spectrum across the various tumour cell clusters using pseudotime trajectory analysis and elucidated the transcriptional and metabolic regulation of the hybrid EMT state. Integration of single-cell transcriptomics between the PyMT/GPx2 KD primary tumour and paired lung metastases unraveled a basal/mesenchymal-like cluster and several luminal-like clusters spanning an EMT spectrum. Interestingly, the luminal clusters at the primary tumour gained mesenchymal gene expression, resulting in epithelial/mesenchymal subpopulations fueled by oxidative phosphorylation (OXPHOS) and glycolysis. By contrast, at distant metastasis, the basal/mesenchymal-like cluster gained luminal and mesenchymal gene expression, resulting in a hybrid subpopulation using OXPHOS, supporting adaptive plasticity. Furthermore, p63 was dramatically upregulated in all hybrid clusters, implying a role in regulating partial EMT and MET at primary and distant sites, respectively. Importantly, these effects were reversed by HIF1α loss or GPx2 gain of function, resulting in metastasis suppression. Collectively, these results underscored a dramatic effect of redox signaling on p63 activation by HIF1α, underlying phenotypic and metabolic plasticity leading to mammary tumour metastasis.

Exploring the Role of Globular Domain Locations on an Intrinsically Disordered Region of p53: A Molecular Dynamics Investigation.

The pre-tetramerization loop (PTL) of the human tumor suppressor protein p53 is an intrinsically disordered region (IDR) necessary for the tetramerization process, and its flexibility contributes to the essential conformational changes needed. Although the IDR can be accurately simulated in the traditional manner of molecular dynamics (MD) with the end-to-end distance (EEdist) unhindered, we sought to explore the effects of restraining the EEdist to the values predicted by electron microscopy (EM) and other distances. Simulating the PTL trajectory with a restrained EEdist , we found an increased agreement of nuclear magnetic resonance (NMR) chemical shifts with experiments. Additionally, we observed a plethora of secondary structures and contacts that only appear when the trajectory is restrained. Our findings expand the understanding of the tetramerization of p53 and provide insight into how mutations could make the protein impotent. In particular, our findings demonstrate the importance of restraining the EEdist in studying IDRs and how their conformations change under different conditions. Our results provide a better understanding of the PTL and the conformational dynamics of IDRs in general, which are useful for further studies regarding mutations and their effects on the activity of p53.

RAC1b Collaborates with TAp73α-SMAD4 Signaling to Induce Biglycan Expression and Inhibit Basal and TGF-β-Driven Cell Motility in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by a marked desmoplastic tumor stroma that is formed under the influence of transforming growth factor (TGF)-β. Data from mouse models of pancreatic cancer have revealed that transcriptionally active p73 (TAp73) impacts the TGF-β pathway through activation of Smad4 and secretion of biglycan (Bgn). However, whether this pathway also functions in human PDAC cells has not yet been studied. Here, we show that RNA interference-mediated silencing of TAp73 in PANC-1 cells strongly reduced the stimulatory effect of TGF-β1 on BGN. TAp73-mediated regulation of BGN, and inhibition of TGF-β signaling through a (Smad-independent) ERK pathway, are reminiscent of what we previously observed for the small GTPase, RAC1b, prompting us to hypothesize that in human PDAC cells TAp73 and RAC1b are part of the same tumor-suppressive pathway. Like TAp73, RAC1b induced SMAD4 protein and mRNA expression. Moreover, siRNA-mediated knockdown of RAC1b reduced TAp73 mRNA levels, while ectopic expression of RAC1b increased them. Inhibition of BGN synthesis or depletion of secreted BGN from the culture medium reproduced the promigratory effect of RAC1b or TAp73 silencing and was associated with increased basal and TGF-β1-dependent ERK activation. BGN also phenocopied the effects of RAC1b or TAp73 on the expression of downstream effectors, like the EMT markers E-cadherin, Vimentin and SNAIL, as well as on negative regulation of the ALK2-SMAD1/5 arm of TGF-β signaling. Collectively, we showed that tumor-suppressive TAp73-Smad4-Bgn signaling also operates in human cells and that RAC1b likely acts as an upstream activator of this pathway.

Targeting p53 misfolding conundrum by stabilizing agents and their analogs in breast cancer therapy: a comprehensive computational analysis.

Cancer continues to be a major global public health concern and one of the foremost causes of death. Delays in the diagnosis and cure may cause an increase in advanced stage disease and mortality. The most common cancer found in women currently is breast carcinoma. Breast carcinoma has surpassed lung carcinoma and currently represents the chief type of cancer diagnosed (2.3 million new cases, which amount to 11.7% of all cancer cases). In addition, by 2040, the incidence will increase by more than 46% as per the estimates of GLOBOCAN. Triple-negative breast cancer (TNBC) represents a highly aggressive and invasive subtype of breast cancer, characterized by rapid progression, short response time to the available treatment, and poor clinical results. Thus, it is very crucial to develop novel diagnostic tools and therapeutics with good efficacy. A majority of cancers display malfunction along the p53 pathway. Moreover, p53 not only loses its function but is also prone to misfolding and aggregation, leading to formation of amyloid aggregates as well. Research is being carried out to find ways to restore the normal action and expression of p53. Here, we have explored PhiKan-083 for its possible stabilizing effect on p53 in order to address the problem with its misfolding. Thus, examining the analogs of PhiKan-083 that have a role in p53 stability will help update our understanding of cancer progression and may expedite the progress of new anticancer treatments. We anticipate that the drug molecules and their analogs targeting p53 aggregation may be used in combination with other anticancer compounds to solve the problem with p53 aggregation. In this study, by employing ADMET analysis, the compounds were screened, and we further examined the chosen compounds with the help of molecular docking. By using databases like UALCAN, TIMER, GEPIA, and PredictProtein, we investigated TP53's expression pattern and prognostic relevance in various cancer settings.

Delivery to, and Reactivation of, the p53 Pathway in Cancer Cells Using a Grafted Cyclotide Conjugated with a Cell-Penetrating Peptide.

Peptides are promising drug modalities that can modulate protein-protein interactions, but their application is hampered by their limited ability to reach intracellular targets. Here, we improved the cytosolic delivery of a peptide blocking p53:MDM2/X interactions using a cyclotide as a stabilizing scaffold. We applied several design strategies to improve intracellular delivery and found that the conjugation of the lead cyclotide to the cyclic cell-penetrating peptide cR10 was the most effective. Conjugation allowed cell internalization at micromolar concentration and led to elevated intracellular p53 levels in A549, MCF7, and MCF10A cells, as well as inducing apoptosis in A549 cells without causing membrane disruption. The lead peptide had >35-fold improvement in inhibitory activity and increased cellular uptake compared to a previously reported cyclotide p53 activator. In summary, we demonstrated the delivery of a large polar cyclic peptide in the cytosol and confirmed its ability to modulate intracellular protein-protein interactions involved in cancer.

SIRT7 and p53 interaction in embryonic development and tumorigenesis.

p53 is a hallmark tumor suppressor due in part to its role in cell cycle progression, DNA damage repair, and cellular apoptosis; its protein activity interrelates with the Sirtuin family of proteins, major regulators of the cellular response to metabolic, oxidative, and genotoxic stress. In the recent years, mammalian Sirtuin 7 (SIRT7) has emerged as a pivotal regulator of p53, fine-tuning its activity in a context dependent manner. SIRT7 is frequently overexpressed in human cancer, yet its precise role in tumorigenesis and whether it involves p53 regulation is insufficiently understood. Depletion of SIRT7 in mice results in impaired embryo development and premature aging. While p53 activity has been suggested to contribute to tissue specific dysfunction in adult Sirt7 -/- mice, whether this also applies during development is currently unknown. By generating SIRT7 and p53 double-knockout mice, here we show that the demise of SIRT7-deficient embryos is not the result of p53 activity. Notably, although SIRT7 is commonly considered an oncogene, SIRT7 haploinsufficiency increases tumorigenesis in p53 knockout mice. Remarkably, in specific human tumors harboring p53 mutation, we identified that SIRT7 low expression correlates with poor patient prognosis. Transcriptomic analysis unveils a previously unrecognized interplay between SIRT7 and p53 in epithelial-to-mesenchymal transition (EMT) and extracellular matrix regulation with major implications for our understanding of embryonic development and tumor progression.

Cyclin B2 impairs the p53 signaling in nasopharyngeal carcinoma

BackgroundCyclin B2 (CCNB2), a member of the cyclin family, is an oncogene in multiple cancers, including nasopharyngeal carcinoma (NPC). However, the epigenetics mechanism for CCNB2 overexpression in NPC remains unclear. This study dissects the regulatory role of CCNB2 in NPC and the molecular mechanism.MethodsDifferentially methylated genes (DMG) and differentially expressed genes (DEG) were screened out in GSE52068 and GSE13597 databases, respectively, and candidate targets were identified by the Venn diagram. GO annotation and pathway enrichment analyses were performed on selected DMG and DEG, and a PPI network was constructed to pinpoint hub genes. PCR and qMSP were conducted to detect the expression and methylation of CCNB2 in cells. The siRNA targeting CCNB2 was transfected into NPC cells, and the migration, proliferation, cell cycle, epithelial-mesenchymal transition (EMT), tumorigenesis, and metastasis were examined. The upstream factor responsible for CCNB2 overexpression in NPC was explored. The p53 activity in NPC cells was assessed using western blot analysis.ResultsCCNB2 showed hypomethylation and overexpression in NPC. CCNB2 silencing inhibited cell migration, proliferation, cell cycle entry, and EMT. JMJD6 was overexpressed in NPC and upregulated CCNB2 through demethylation. JMJD6 reversed the effects of CCNB2 downregulation, resulting in elevated cellular activity in vitro and tumorigenic and metastatic activities in vivo. CCNB2 blocked the p53 pathway, while the p53 pathway inhibitor reversed the effect of CCNB2 silencing to increase the activity of NPC cells.ConclusionsJMJD6 enhanced CCNB2 transcription by demethylating CCNB2, thereby repressing the p53 pathway and promoting NPC progression.

Endogenous S100P-mediated autophagy regulates the chemosensitivity of leukemia cells through the p53/AMPK/mTOR pathway.

Autophagy, a highly regulated lysosome-dependent catabolic pathway, has garnered increasing attention because of its role in leukemia resistance. Among the S100 family of small calcium-binding proteins, S100P is differentially expressed in various tumor cell lines, thereby influencing tumor occurrence, invasion, metastasis, and drug resistance. However, the relationship between S100P and autophagy in determining chemosensitivity in leukemia cells remains unexplored. Our investigation revealed a negative correlation between S100P expression and the clinical status in childhood leukemia, with its presence observed in HL-60 and Jurkat cell lines. Suppression of S100P expression resulted in increased cell proliferation and decreased chemosensitivity in leukemia cells, whereas enhancement of S100P expression inhibited cell proliferation and increased chemosensitivity. Additionally, S100P knockdown drastically promoted autophagy, which was subsequently suppressed by S100P upregulation. Moreover, the p53/AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway was found to be functionally associated with S100P-mediated autophagy. Knockdown of S100P expression led to a decrease in p53 and p-mTOR levels and an increase in p-AMPK expression, ultimately promoting autophagy. This effect was reversed by administration of Tenovin-6 (a p53 activator) and Compound C (an AMPK inhibitor). The findings of our in vivo experiments provide additional evidence supporting the aforementioned data. Specifically, S100P inhibition significantly enhanced the growth of HL-60 tumor xenografts and increased the expression of microtubule-associated protein 1 light chain 3 and p-AMPK in nude mice. Consequently, it can be concluded that S100P plays a regulatory role in the chemosensitivity of leukemia cells by modulating the p53/AMPK/mTOR pathway, which controls autophagy in leukemia cells.

Progress in deciphering the role of p53 in diffuse large B-cell lymphoma: mechanisms and therapeutic targets.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, accounting for 30%-40% of non-Hodgkin lymphoma in adults. The mechanisms underlying DLBCL occurrence are extremely complex, and involve the B-cell receptor (BCR) and Toll-like receptor (TLR) signaling pathways, as well as genetic abnormalities and other factors. With the development of high-throughput sequencing, an increasing number of abnormal genes have been identified in DLBCL. Among them, the tumor protein p53 (TP53/p53) gene is important in DLBCL occurrence. Patients with DLBCL carrying TP53 gene abnormalities generally have poor prognosis and studies of p53 have potential to provide a better basis for their treatment. Normally, p53 is maintained at low levels through its interaction with murine double minute 2 (MDM2), and prevents tumorigenesis by mediating cell cycle arrest, apoptosis, and repair of damaged cells, among other processes. Therefore, the prognosis of patients with DLBCL harboring TP53 gene abnormalities (mutations, deletions, etc.) is poor, and targeting p53 for tumor therapy has become a research hotspot, following developments in molecular biology technologies. Current treatments targeting p53 mainly act by restoring the function or promoting degradation of mutant p53, and enhancing wild-type p53 protein stability and activity. Based on the current status of p53 research, exploration of existing therapeutic methods to improve the prognosis of patients with DLBCL with TP53 abnormalities is warranted.

Genes of Inflammation and Placental Function GWAS Associated with Idiopathic Recurrent Miscarriage in the Kazakh Population.

The loss of two or more pregnancies is considered recurrent miscarriage (RM). One of the causes of this pathology is the occurrence of mutations both in pleiotropic and pathway-specific regulators and in structural genes. The simplest type of such mutations is single nucleotide polymorphisms. The aim of the study is to study the relationship between gene polymorphisms of anti- and pro-inflammatory cytokines - interferon-gamma (T874A), interleukin (IL1B) (C3954T), IL6 (G572C) and IL10 (G1082A); placental function, apoptosis and angiogenesis - apolipoprotein C-III (APOC3) (G5163C), kinase insert domain receptor (A1719T, G1192A), P53 (Arg72Pro) and signal transducer and activator of transcription 3 (STAT3) (C1697G) with the development of idiopathic RM (iRM) in the Kazakh population. This was a case-control study. Molecular genetic studies were performed by TaqMan using a single site-specific amplification and real-time genotyping method in 302 women with iRM and 300 with normal reproduction. DNA isolation from the biomaterial was carried out using kits containing binding magnetic particles. Both samples were analysed for alleles and genotypes for the studied polymorphisms. For statistical data processing, Pearson's criterion, confidence interval (CI) and probability value were taken into account. It was found that the carriage of unfavourable genotypes (G/C, C/C) for the G5163C polymorphism of the APOC3 gene increases the risk of developing iRM by three times (odds ratio = 3.0; 95% CI = 2.24-4.07). Other studied polymorphisms in the genes of ILs, interferon, P53 proapoptotic protein, kinase domain receptor and STAT3 transcription activator were not associated with RM. Significant associations of APOC3 gene genotypes with the development of iRM in the Kazakh population indicate the involvement of the placental system, which is realised by vascularisation defects and defective embryo implantation and leads to early pregnancy termination.

Dysregulated Ribosome Biogenesis Is a Targetable Vulnerability in Triple-Negative Breast Cancer: MRPS27 as a Key Mediator of the Stemness-inhibitory Effect of Lovastatin.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited effective therapeutic options readily available. We have previously demonstrated that lovastatin, an FDA-approved lipid-lowering drug, selectively inhibits the stemness properties of TNBC. However, the intracellular targets of lovastatin in TNBC remain largely unknown. Here, we unexpectedly uncovered ribosome biogenesis as the predominant pathway targeted by lovastatin in TNBC. Lovastatin induced the translocation of ribosome biogenesis-related proteins including nucleophosmin (NPM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and the ribosomal protein RPL3. Lovastatin also suppressed the transcript levels of rRNAs and increased the nuclear protein level and transcriptional activity of p53, a master mediator of nucleolar stress. A prognostic model generated from 10 ribosome biogenesis-related genes showed outstanding performance in predicting the survival of TNBC patients. Mitochondrial ribosomal protein S27 (MRPS27), the top-ranked risky model gene, was highly expressed and correlated with tumor stage and lymph node involvement in TNBC. Mechanistically, MRPS27 knockdown inhibited the stemness properties and the malignant phenotypes of TNBC. Overexpression of MRPS27 attenuated the stemness-inhibitory effect of lovastatin in TNBC cells. Our findings reveal that dysregulated ribosome biogenesis is a targetable vulnerability and targeting MRPS27 could be a novel therapeutic strategy for TNBC patients.

Intracystic Malignant Adenomyoepithelioma of the Breast: A Case Report and Review of Literature

Adenomyoepithelioma (AME) of the breast is a rare neoplasm with a biphasic proliferation of epithelial and myoepithelial cells. The authors report an unusual case of AME of 51-year-old woman. The AME was located in a cystic structure 6 cm in diameter as a solid nodular lesion with 2cm size. Immunohistochemically, the nodule was comprised with 2 cell population of tubular structures. First population of the glandular epithelial cells with dark cytoplasm showed the activity of CK7(+), CAM5.2(+), CK5/6(partly +), p63(-), CD10(-), S-100(-), αSMA(-). Second population of outer myoepithelial cells with clear cytoplasm displayed the activity of p63(+), CD10(+), CK14(+) CK5/6 (partly +), CK7(weakly +), CAM5.2(weakly +), S-100(+), αSMA(+). Beside them, cellular cluster of the inner epithelial cells with prominent nuclear as well as cellular atypia, and atypical spindle cell proliferation emanating from the myoepithelial component were also present. Thus, the nodule was diagnosed as malignant AME. One year after the lumpectomy, pulmonal metastasis of the breast tumor was also found. So far as we are concerned, such case of intracystic malignant AME of the breast has never been described. Regarding histogenesis of the neoplasm growing under the unusual environment, a consideration with review of literature was done.

Methylation of the Selected TP53 Introns in Advanced-Stage Ovarian Carcinomas.

Objective: Advanced-stage ovarian cancer (OC) is among the most fatal female genital tract neoplasms worldwide. Although different genetic mechanisms have been shown to be involved in ovarian carcinogenesis, the role of TP53 introns methylation is still unresolved. We performed methylation analysis of introns 1, 3, and 4 of the TP53 to identify patterns in primary stage III OCs, corresponding metastases, and healthy tissues. Methods: The study involved samples of paraffin-embedded tissues obtained from 80 patients with stage III OCs, who underwent surgery at the Department of Gynecology and Gynecologic Oncology of the Military Institute of Medicine in Warsaw, Poland. Altogether, 40 serous-type G2/3 OCs and 40 endometrioid-type G2/3 OCs were included. From the same patient, metastatic and normal tissues were simultaneously analyzed. As a control group, 80 tissue samples were collected from patients after bariatric operations. Human ovarian cancer A2780 cell line was also investigated. Total genomic DNA was isolated from paraffin-embedded tissue blocks and the methylation analysis was performed by bisulfite DNA conversion, DNA amplification with specific primers, cloning, and DNA sequencing. Results: All of the samples of intron 1 of TP53 were un-methylated in OCs, metastatic tissues, and in healthy tissues from the same patient. Also, no methylation of TP53 intron 1 was detected in cells from the human A2780 ovarian cancer cell line and in all samples from control group. In all samples, introns 3 and 4 of the TP53 were methylated in primary tumors, metastatic tissue, and in healthy tissue from the same patient, in human A2780 ovarian cell line, and in DNA samples from healthy patients. None of the clinicopatholocal features was related to the TP53 introns methylation status. Conclusions: Our data on TP53 introns methylation sheds new light on the mechanism of p53 activity for a better understanding of cancer biology. The study suggests the existence of an additional regulation rule of TP53 activity that involves demethylation-methylation mechanisms. Methylation at introns 3 and 4 may also overall help in protecting TP53 against damage by viral restrictases or viral DNA integration.

Comparative genomic analysis of Fischer F344 rat livers exposed for 90 days to 3-methylfuran or its parental compound furan

Furan is a naturally forming compound found in heat-processed foods such as coffee, canned meats, and jarred baby food. It is concurrently found with analogues including 2-methylfuran (2-MF) and 3-methylfuran (3-MF), and toxicity studies demonstrate all are potent liver toxins. Toxicity studies found 3-MF is more toxic than either furan, or 2-MF. The present analysis assesses the transcriptional response in liver samples taken from male Fischer (F344) rats exposed to furan or 3-MF from 0 to 2.0 and 0–1.0 mg/kg bw/day, respectively, for 90 days. Transcriptional analyses found decreased liver function and fatty acid metabolism are common responses to both furan and 3-MF exposure. Furan liver injury promotes a ductular reaction through Hippo and TGFB signalling, which combined with increased immune response results in ameliorating perturbed bile acid homeostasis in treated rats. Failure to activate these pathways in 3-MF exposed rats and decreased p53 activity leads to cholestasis, and increased toxicity. Finally, BMD analysis indicate many of the most sensitive pathways affected by furan and 3-MF exposure relate to metabolism - malate dehydrogenase and glucose metabolism with BMDLs of 0.03 and 0.01 mg/kg bw/day for furan and 3-MF exposure, respectively, which agrees with BMDLs previously reported for apical and microarray data.

Exposure to fluoride exacerbates the cognitive deficit of diabetic patients living in areas with endemic fluorosis, as well as of rats with type 2 diabetes induced by streptozotocin via a mechanism that may involve excessive activation of the poly(ADP ribose) polymerase-1/P53 pathway

Epidemiology has shown that fluoride exposure is associated with the occurrence of diabetes. However, whether fluoride affects diabetic encephalopathy is unclear. Elderly diabetic patients in areas with endemic (n = 169) or no fluorosis (108) and controls (85) underwent Montreal Cognitive Assessment. Sprague-Dawley rats receiving streptozotocin and/or different fluoride doses were examined for spatial learning and memory, brain morphology, blood-brain barrier, fasting blood glucose and insulin. Cultured SH-SY5Y cells were treated with 50 mM glucose and/or low- or high-dose fluoride, and P53-knockdown or poly-ADP-ribose polymerase-1 (PARP-1) inhibition. The levels of PARP-1, P53, poly-ADP-ribose (PAR), apoptosis-inducing factor (AIF), and phosphorylated-histone H2A.X (ser139) were measured by Western blotting. Reactive oxygen species (ROS), 8-hydroxydeguanosine (8-OHdG), PARP-1 activity, acetyl-P53, nicotinamide adenine dinucleotide (NAD+), activities of mitochondrial hexokinase1 (HK1) and citrate synthase (CS), mitochondrial membrane potential and apoptosis were assessed biochemically. Cognition of diabetic patients in endemic fluorosis areas was poorer than in other regions. In diabetic rats, fasting blood glucose, insulin resistance and blood-brain barrier permeability were elevated, while spatial learning and memory and Nissl body numbers in neurons declined. In these animals, expression and activity of P53 and PARP-1 and levels of NAD+, PAR, ROS, 8-OHdG, p-histone H2A.X (ser139), AIF and apoptosis content increased; whereas mitochondrial HK1 and CS activities and membrane potential decreased. SH-SY5Y cells exposed to glucose exhibited changes identical to diabetic rats. The changes in diabetic rats and cells treated with glucose were aggravated by fluoride. P53-knockout or PARP-1 inhibition mitigated the effects of glucose with/without low-dose fluoride. Elevation of diabetic encephalopathy was induced by exposure to fluoride and the underlying mechanism may involve overactivation of the PARP-1/P53 pathway.

Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer.

The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

The p53 protein is a suppressor of Atox1 copper chaperon in tumor cells under genotoxic effects.

The p53 protein is crucial for regulating cell survival and apoptosis in response to DNA damage. However, its influence on therapy effectiveness is controversial: when DNA damage is high p53 directs cells toward apoptosis, while under moderate genotoxic stress it saves the cells from death and promote DNA repair. Furthermore, these processes are influenced by the metabolism of transition metals, particularly copper since they serve as cofactors for critical enzymes. The metallochaperone Atox1 is under intensive study in this context because it serves as transcription factor allegedly mediating described effects of copper. Investigating the interaction between p53 and Atox1 could provide insights into tumor cell survival and potential therapeutic applications in oncology. This study explores the relationship between p53 and Atox1 in HCT116 and A549 cell lines with wild type and knockout TP53. The study found an inverse correlation between Atox1 and p53 at the transcriptional and translational levels in response to genotoxic stress. Atox1 expression decreased with increased p53 activity, while cells with inactive p53 had significantly higher levels of Atox1. Suppression of both genes increased apoptosis, while suppression of the ATOX1 gene prevented apoptosis even under the treatment with chemotherapeutic drugs. The findings suggest that Atox1 may act as one of key elements in promotion of cell cycle under DNA-damaging conditions, while p53 works as an antagonist by inhibiting Atox1. Understanding of this relationship could help identify potential targets in cell signaling pathways to enhance the effectiveness of combined antitumor therapy, especially in tumors with mutant or inactive p53.