Expression Of Mutant P53 Protein Research Articles

Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Abstract C029: The pattern of mutant p53 protein expression in cervical cancer-associated single and co-infection with human papillomavirus and Epstein-Barr virus

Abstract Introduction: There is mounting evidence indicating that the co-existence of Human papillomavirus (HPV), and Epstein-Barr virus (EBV) is associated with aggressive and poorly differentiated squamous cell carcinomas (SCC) phenotype. However, there is a paucity of data on the prevalence of EBV in cases of cervical cancer in Southern Nigeria or the effect of both viruses on p53 gene expression among patients diagnosed with cervical cancer. Therefore, this study investigated the prevalence of mutant p53 (mtp53) in single and co-presence of HPV and EBV-LMP1 in invasive and non-invasive cases of cervical cancer in order to determine the role of EBV in cervical carcinogenesis. Methods: This retrospective cohort study included 105 viable archived formalin-fixed paraffin-embedded tissues of cervical cancer, diagnosed between 2016 and 2019 in two tertiary health care facilities in Southern Nigeria. Tissue sections were stained for mtp53, EBV-LMP1, and HPV using the immunohistochemical technique and scored based on staining intensity and documented as 0, +1, +2, and +3. Tissues with scores of 0 and +1 were considered negative whereas those with scores of +2 and +3 were considered positive. Descriptive, Chi-square test and Pearson’s correlation were carried out and statistical significance was set at ≤0.05. Result: The mean age of participants with cervical cancer was 53.32 ± 12.54 years. The prevalence of invasive and non-invasive cases of cervical cancer were 62.9% and 37.1%, respectively. The prevalence of well, moderately, and poorly differentiated cervical cancer were 54.3%, 34.3%, and 11.4%, respectively. The prevalence of single EBV-LMP1, and HPV antigen were 21.0% (n= 21) and 14.2% (n= 15), respectively whereas the prevalence of EBV-LMP1/HPV co-infection was 42.9% (n= 45). The prevalence of HPV/EBV-LMP1 co=presence was high in invasive cervical cancer compared with non-invasive cervical cancer cases (45.5% versus 30.8%) at p= 0.154. The prevalence of mtp53 was higher in EBV-LMP1/HPV co-presence compared with HPV, and EBV-LMP1 mono-presence (80.0%, 60.0%, and 14.3%, respectively) at p= 0.001. Conclusion: This study suggests that EBV increases the risk of cervical carcinogenesis in HPV-infected individuals in Southern Nigeria. Thus, EBV DNA should equally be investigated during HPV testing of suspected cervical cancer cases to identify individuals with poorer prognoses. Citation Format: Jude Okoye, Anthony Ajuluchukwu Ngokere, Charles Chinedum Onyenekwe, Ukpai Eze, Okechi Obioma. The pattern of mutant p53 protein expression in cervical cancer-associated single and co-infection with human papillomavirus and Epstein-Barr virus [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C029.

Immunoexpression of p53 mutant-type in Iranian patients with primary and recurrence oral squamous cell carcinoma.

Mutations in tumor suppressor p53 protein can occur at different phases of malignant transformation and affect the patient's prognosis. This study aimed to evaluate the expression of mutant p53 protein in Iranian patients with the primary and recurrence oral squamous cell carcinoma (OSCC). This retrospective cross-sectional study conducted on a group of patients with the primary OSCC (n=122) and the control subjects with oral noncancerous reactive lesions (n=80). Immunohistochemistry was performed with the DO-7 monoclonal antibody against p53 protein, and samples with ≥10% immunostaining were considered positive. Statistical analyses were carried out using SPSS. Positive staining for p53 was observed in none of the control subjects and 57.4% (70 of 122) of the primary OSCC patients (p<0.0001, OR=107.69, 95%CI=6.49-179.0). The p53 immunopositivity had no significant differences between males and females (54.2% vs. 62%, p=0.390), but significantly different between those aged below and over 50 years (p<0.0001, OR=4.52, 95%CI=1.07-12.05). During follow-up, OSCC recurrence occurred in 104 patients, but the phenotype of the mutant p53 protein in patients who relapsed was the same as in matched primary tumors (p=0.763). Risk of recurrence had no significant differences between p53-positive and p53-negative cases (p=0.953), males and females (p=0.263), and age below and over 50 years (p=0.223). Despite its confirmed diagnostic value, the immunoexpression of the p53 mutant protein in OSCC in cancer recurrence was the same as in the primary tumor. However, further studies with a larger sample size and longer follow-up are needed to confirm or change our conclusions.

INNV-06. DETECTION OF MUTANT P53 POSITIVE GLIOBLASTOMA CELLS IN CEREBROSPINAL FLUID (CSF) USING A MICROFLUIDIC BASED IMMUNOASSAY

Abstract BACKGROUND Metastatic involvement of the CSF by non-CNS neoplasms surpasses that of primary brain tumors, although conventional glioblastoma (GBM) can occasionally be identified in the CSF. Here, we apply Biocept’s CNSide test to examine a patient CSF sample with GBM, verified with an antibody against mutant p53. The p53 pathway is deregulated in 84% of glioblastoma patients and point mutations lead to expression of mutant p53 protein. METHODS A CSF sample from 65-year-old male patient, with GBM diagnosed by MRI &amp; CT, and prior identified p53 mutation on R273C by NGS was collected into Biocept’s CSF collection tubes at Providence Saint John’s Health Center. CSF-cells were incubated, 2 days post collection, with a proprietary antibody cocktail, including anti-CD9, followed with a biotinylated secondary, which enables enrichment of capture antibody labeled cells in a streptavidin coated microfluidic device. Cells were fixed and permeabilized immunofluorescence was performed against mutant p53, CD45, fluorescently labeled streptavidin, for capture antibody detection, and DAPI. Microfluidic chips were scanned and analyzed on a Bioview system. The specificity of the mutant p53 antibody was verified using the same test on tumor cells with either wildtype p53 vs various p53 mutations. RESULTS CSF analysis by the CNSide platform detected 3 tumor cells / mL. Immunofluorescence confirmed strong expression of mutant p53 protein on about 1/3 of cells identified as tumor cells. Besides the patient R273C mutation, we demonstrated detection of P223L/V274F, E285K and Q331R mutations on tumor cell lines. DISCUSSION Our work suggests that a microfluidic based GBM capture test paired with appropriate biomarkers can be used as sensitive means to detect GBM cells in CSF and may be useful for diagnostics and treatment monitoring. Further, we demonstrated the utility of a mutant p53 antibody to identify tumor cells. Technical and clinical studies are needed to substantiate this hypothesis.

Structural Basis of Mutation-Dependent p53 Tetramerization Deficiency

The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA-binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation present in the population in south Brazil. In this work, we combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. In addition to the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt-bridge interaction that stabilizes the p53 tetramerization domain were studied (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay, performed in yeast by tuning the expression of wild-type and mutant p53 proteins, revealed that p53 tetramerization mutations could decrease the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53’s conformational stability and function.

Suppression of the Proliferation of Huh7 Hepatoma Cells Involving the Downregulation of Mutant p53 Protein and Inactivation of the STAT 3 Pathway with Ailanthoidol.

Ailanthoidol (ATD) has been isolated from the barks of Zanthoxylum ailanthoides and displays anti-inflammatory, antioxidant, antiadipogenic, and antitumor promotion activities. Recently, we found that ATD suppressed TGF-β1-induced migration and invasion of HepG2 cells. In this report, we found that ATD exhibited more potent cytotoxicity in Huh7 hepatoma cells (mutant p53: Y220C) than in HepG2 cells (wild-type p53). A trypan blue dye exclusion assay and colony assay showed ATD inhibited the growth of Huh7 cells. ATD also induced G1 arrest and reduced the expression of cyclin D1 and CDK2. Flow cytometry analysis with Annexin-V/PI staining demonstrated that ATD induced significant apoptosis in Huh7 cells. Moreover, ATD increased the expression of cleaved PARP and Bax and decreased the expression of procaspase 3/8 and Bcl-xL/Bcl-2. In addition, ATD decreased the expression of mutant p53 protein (mutp53), which is associated with cell proliferation with the exploration of p53 siRNA transfection. Furthermore, ATD suppressed the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and the expression of mevalonate kinase (MVK). Consistent with ATD, the administration of S3I201 (STAT 3 inhibitor) reduced the expression of Bcl-2/Bcl-xL, cyclin D1, mutp53, and MVK. These results demonstrated ATD’s selectivity against mutp53 hepatoma cells involving the downregulation of mutp53 and inactivation of STAT3.

TP53 copy number and protein expression inform mutation status across risk categories in acute myeloid leukemia

AbstractMutant TP53 is an adverse risk factor in acute myeloid leukemia (AML), but large-scale integrated genomic-proteomic analyses of TP53 alterations in patients with AML remain limited. We analyzed TP53 mutational status, copy number (CN), and protein expression data in AML (N = 528) and provide a compilation of mutation sites and types across disease subgroups among treated and untreated patients. Our analysis shows differential hotspots in subsets of AML and uncovers novel pathogenic variants involving TP53 splice sites. In addition, we identified TP53 CN loss in 70.2% of TP53-mutated AML cases, which have more deleterious TP53 mutations, as well as copy neutral loss of heterozygosity in 5/32 (15.6%) AML patients who had intact TP53 CN. Importantly, we demonstrate that mutant p53 protein expression patterns by immunohistochemistry evaluated using digital image-assisted analysis provide a robust readout that integrates TP53 mutation and allelic states in patients with AML. Expression of p53 by immunohistochemistry informed mutation status irrespective of TP53 CN status. Genomic analysis of comutations in TP53-mutant AML shows a muted landscape encompassing primarily mutations in genes involved in epigenetic regulation (DNMT3A and TET2), RAS/MAPK signaling (NF1, KRAS/NRAS, PTPN11), and RNA splicing (SRSF2). In summary, our data provide a rationale to refine risk stratification of patients with AML on the basis of integrated molecular and protein-level TP53 analyses.

An Immunohistochemical Assessment of Ki-67 and P53 Over-expression in Helicobacter pylori-positive Gastritis

AIM: Helicobacter pylori infection of the stomach is a common disease and the resulting changes from it are many and deserve to be in the focus of researchers’ attention, to assess the expression of mutant P53 protein and Ki-67 markers in patients with gastritis secondary to H. pylori. METHODS: Thirty samples with positive H. pylori gastritis were included in a retrospective study in Mosul/Iraq. The histological parameters were assessed using the Sydney system, then, the expression of Ki-67 and P53 expression was studied by immunohistochemical methods. The significance level was appointed at (0.05). RESULTS: Ki-67 and P53 expression were found in 83.3% of the total cases. The study results show that 92% of positive Ki-67 and P53 cases had chronic inflammatory cell infiltration, polymorph nuclear cells infiltration, and atrophy. Whereas 96% of positive Ki-67 cases had no metaplasia, 92% of the positive P53 cases had no metaplasia. The results also showed that only 16% of the positive Ki-67 cases had dysplastic changes, and 24% of the positive cases of P53 cases were showed dysplasia. whenever P53 was negative; there is neither metaplasia nor dysplasia in the tissue, this does not apply to Ki-67 negative cases. CONCLUSIONS: Ki-67 and P53 expressions increase with chronicity of Helicobacter pylori-positive gastritis. P53 expression is amplified when atrophy is present in these samples.

Abstract 2489: A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis

Abstract Two of the most common events in human tumors are mutation of the tumor suppressor gene TP53 and development of aneuploidy. In addition to losing their wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mechanistic understanding of mutant p53 GOF activities is complicated by the diversity and context-specific nature of reported GOF phenotypes. The study of mutant p53 GOF activities is especially challenging because mutations in p53 are positively correlated with the development of aneuploidy, which can increase heterogeneity through diverse chromosomal alterations and itself contributes to tumorigenesis. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic epithelial cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Additionally, the use of clonally-derived cell lines originating from the same near diploid parental genetic background allows for assessment of the genomic alterations and resulting molecular heterogeneity following mutation of TP53. Through genomic, transcriptomic, and cellular based assays we have validated our cell line models and found that missense mutant and p53 null cells display loss of p53 function. Through functional genomics analyses comparing isogenic epithelial cells, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and aneuploidy and assessed whether our clonal cell lines display several previously reported mutant p53 GOF phenotypes such as altered gene expression, proliferation, metabolism, drug sensitivity, and migration. Further, using lentiviral mediated knockdown of p53 protein we evaluated the dependency of these phenotypes on expression of mutant p53 protein. Finally, data from The Cancer Genome Atlas (TCGA) was used for the analysis of manifestations of clinical mutant p53 GOF phenotypes. The dissection of mutant p53 GOF phenotypes will improve the current understanding of the role of mutant p53 in tumorigenesis. The results generated from these studies have the potential for clinical translation in major types of human cancer that have high-frequency p53 mutation. Citation Format: Lindsay N. Redman-Rivera, Timothy M. Shaver, Hailing Jin, Johanna M. Schafer, Quanhu Sheng, Rachel A. Hongo, Kathryn E. Beckermann, Brian D. Lehmann, Ferrin C. Wheeler, Jennifer A. Pietenpol. A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2489.

The Correlation Between Mutant p53 Protein Expression and Cell Atypia in Early Differentiated Gastric Adenocarcinoma.

ObjectiveThis study aims to explore the correlation between the expression of mutant p53 protein and cellular atypia in early differentiated gastric adenocarcinoma (DGA).MethodsA total of 107 cases of early DGA samples resected by endoscopic submucosal dissection (ESD) were collected from the Pathology Department of Beijing Friendship Hospital from January 2018 to December 2019. The EnVision two-step immunohistochemical method was used to detect the expression of mutant p53 protein in these cancer tissues, and the correlation with cell atypia was analyzed.ResultsIn early DGA tissues, the expression rate of mutant p53 protein was significantly higher than in normal gastric mucosa (P < 0.01). However, the expression of mutant p53 protein was not correlated to age or gender (P > 0.05) but to the location of the tumor, depth of invasion, and degree of differentiation (P < 0.01). The expression of mutant p53 protein was closely correlated to cell atypia. Furthermore, this was weakly positive in low-grade atypical adenocarcinoma but strongly positive or negative in high-grade atypical adenocarcinoma, and there was a significant difference between these two (P < 0.01).ConclusionMutant p53 protein is highly expressed in early DGA, which can be used as an auxiliary index for the diagnosis of early gastric cancer. The different expression patterns of mutant p53 protein in high-grade and low-grade atypical gastric cancers suggest that these may have different genetic changes.

A mutant p53/Hif1α/miR-30d axis reprograms the secretory pathway promoting the release of a prometastatic secretome.

TP53 missense mutations are frequent driver events during tumorigenesis. The majority of TP53 mutations are missense and occur within the DNA binding domain of p53, leading to expression of mutant p53 (mut-p53) proteins that not only lose the tumor suppressive functions of the wild-type (wt-p53) form, but can also acquire novel oncogenic features fostering tumor growth, metastasis and chemoresistance. Mut-p53 affects fundamental cellular pathways and functions through different mechanisms, a major one being the alteration of gene expression. In our recent work (Capaci et al., 2020, Nat Commun) we found that mut-p53, via miR-30d, modifies structure and function of the Golgi apparatus (GA) and induces increased rate of trafficking. This culminates in the release of a pro-malignant secretome, which is capable of remodeling the tumor microenvironment (TME), to increase stiffness of the extracellular matrix (ECM), favouring metastatic colonization, as shown by cell-based assays and experiments of metastatic niche preconditioning in mouse xenograft models. This study provides new insights into the mechanisms by which mut-p53, through induction of non-coding RNAs, can exert pro-tumorigenic functions in a non-cell-autonomous fashion, and highlights potential non-invasive biomarkers and therapeutic targets to treat tumors harboring mut-p53 (Figure 1).

CBIO-22. p53 GAIN-OF-FUNCTION MUTATIONS DRIVE SEX SPECIFIC EFFECTS ON GLIOMA TUMORIGENESIS

Abstract Sex differences in malignant brain tumors are well-established: Males exhibit greater incidence and poorer survival. Understanding the biology behind these sex differences requires investigation of the pathways known to drive gliomagenesis. The transcription factor TP53 (p53) is one of the most commonly mutated genes in glioblastoma. Most p53 mutations are missense mutations in the DNA-binding domain that lead to the expression of a full length mutant p53 protein. These mutations can endow p53 with oncogenic gains-of-function through aberrant DNA binding and regulation of noncanonical cancer-promoting target genes. Previously, we analyzed patient mutation data and identified six p53 mutations with sex differences in prevalence. In this study, we developed an in vitro mutant p53 glioma model to investigate the sex specific effects of three p53 point mutations: R175H, Y205C, and Y220C (Mm R172H, Y202C, and Y217C respectively). Male and female astrocytes isolated from p53flox/- mouse pups were transduced with a retrovirus expressing mutant p53, followed by a lentivirus expressing CRE recombinase to remove the endogenous WTp53. We then assessed cell proliferation, clonogenicity, and in vivo tumorigenesis in these cells. All three mutations assayed displayed sex differences in proliferation, with male cells overexpressing p53:Y202C and p53:Y217C growing faster than female cells, and female cells overexpressing p53:R172H growing faster than male cells. Male Y202C and Y217C expressing astrocytes also exhibited a trend toward greater clonogenicity compared to female astrocytes. This observation is supported by higher expression of the stem cell markers SOX2 and NESTIN in the male cells. We performed parallel flank injections of male and female astrocytes expressing each mutation or p53 KO. Only male astrocytes expressing p53:Y202C or p53:Y217C and female astrocytes expressing p53:R172H mutation were able to form tumors in vivo. Together, these data support a sex specific gain-of-function phenotype for three different p53 mutations observed in glioma.

Pterostilbene as a Phytochemical Compound Induces Signaling Pathways Involved in the Apoptosis and Death of Mutant P53-Breast Cancer Cell Lines

Pterostilbene is a natural nonflavonoid polyphenolic compound. It shows a remarkable range of biological activities, including antiproliferative, antiinflammatory, and antioxidant activity. However, the mechanism of action of PT in breast cancer cells containing mutant p53 protein has not been fully elucidated. Therefore, the present study was aimed at investigating the influence of PT on signaling pathways involved in the apoptosis of mutant p53-breast cancer cell lines. Immunocytochemistry and Western Immunoblotting techniques were used in this study. The present data showed that the viabilities and the proliferations of MDA-MB-231 and T-47D decreased significantly (P < 0.001) after treatment with different concentrations of PT. In addition, the morphological characteristics of both cell lines were changed after treatment with PT. Decreased protein expression of mutant p53 (R280 K, L194F) in MDA-MB-231 and T-47D breast cancer cell lines has also been achieved. In addition, overexpression of pro-apoptotic (Bax) protein, caspase-3 activity and histone release were increased after treatment of both cell lines with different PT concentrations. Furthermore, the protein expressions of cyclin D1, mTOR, and oncogenic β-catenin were significantly downregulated after treatment of both cell lines with PT. In conclusion, downregulations of protein expression of mutant p53, cyclin D1, mTOR, and β-catenin were increased after both cell lines had been treated with pterostilbene. PT could point to a promising use against the development and the progression of breast cancer as a natural therapeutic agent.

Potensi Ekstrak Etanol 96% Daun Salam (Syzigium polyanthum Wight. (Walp.)) terhadap Ekspresi p53 pada Sel Kanker HeLa Cell Lines

Bay leaves (Syzigium polyanthum Wight. (Walp.) have been utilized as one of traditional medicines in Indonesian. However, the potency of the leaves for cancer treatment has not been well-explored. The aim of this study is to determine the expression of p53 tumor suppressor gene after administration of 96% ethanol extract of Bay leaves on HeLa cell lines. This study is begin by making extract using ethanol 96% as a solvent followed by phytochemical screening and extract evaluation of HeLa cells by immunohistochemistry. The observation of p53 protein expression is conducted using the Image J Profiler Plugin software 1.52a version. The H-score data is processed using the One-Way ANNOVA IBM SPSS 20 version program and continued with Post Hoc analysis using the Duncan test. The identification of 96% ethanol extract result showed that bay leaves contain flavonoids, alkaloids, saponins, phenols, terpenoids, and steroids. Based on the Post Hoc test results, the administration of 96% ethanol extract bay leaves with three dose variations (156 µg/ml, 234 µg/ml, 312 µg/ml) and negative control had significant differences, while the high dose (312 µg/ml) had not significant differences to positive control. The result showed that 96% ethanol extract of bay leaves by the dose of 312 ug/ml can inhibit the expression of mutant p53 protein in cervical cancer cells HeLa cell lines.

Abstract 2962: Molecular mechanisms underlying the sensitizing effect of mutant p53 protein expression for Auranofin treatment of NSCLC and PDAC cells

Abstract Introduction: Auranofin (AF) is an orally available, organogold compound that is FDA-approved for its use in rheumatoid arthritis. AF gained interest as an anti-cancer agent since it acts as a thioredoxin reductase (TrxR) inhibitor. Data extracted from The Cancer Genome Atlas show that TXNRD1 is abundantly expressed in lung adenocarcinoma patients and negatively affects their outcome, making TrxR1 an attractable target in non-small cell lung cancer (NSCLC) patients. The goal of this study was to determine if mutant p53 expression can be used as a predictive biomarker for the treatment of AF in NSCLC. In addition, we aimed to obtain a profound understanding of the molecular mechanisms that govern the sensitizing effect of mutant p53 towards AF-treated NSCLC cells by investigating the type of cell death. Material and methods: We determined IC50-values using the sulforhodamine-B assay for AF in a panel of NSCLC and pancreatic ductal adenocarcinoma (PDAC) cell lines with different p53 mutations and expression levels. In addition, we correlated these IC50-values to baseline expression levels of different antioxidant related proteins. Results were confirmed in the NSCLC cell line NCI-H1299 (p53 deletion, null) and its two isogenic derivates (mutant p53 R175H or R273H knock-in). mRNA-Seq of the isogenic cell line panel was performed after exposure to a medium dose of AF. Finally, the IncuCyte ZOOM system was used to determine the percentage of AF-induced cell death by the means of the Cytotox Green reagent after pretreatment of the tumor cells with a panel of apoptosis, necroptosis and ferroptosis inhibitors. Results: A strong inverse correlation was observed between mutant p53 protein expression and AF IC50-values and baseline Trx levels in the NSCLC and PDAC cells, indicating that mutant p53 can sensitize cancer cells for AF. This sensitizing effect was confirmed in the p53Mutant isogenic NSCLC cells. Transcriptome analysis revealed the up- and downregulation of specific genes involved in ferroptosis. More specifically, AF induced cell death through various mechanisms, including apoptosis and ferroptosis, which were dependent on the expression levels of p53 rather than the type of p53 mutation. In cells with high expression levels of mutant p53, AF induced ferroptosis and lipid peroxidation. At lower expression levels of p53, the apoptosis pathway was triggered by AF with an increase in caspase-3/7 positive cells and inhibition of cell death by the caspase inhibitor Z-VAD-FMK. Conclusion: Mutant p53 protein expression renders NSCLC and PDAC cells more susceptible to AF. In an isogenic setting, medium levels of mutant p53 sensitize NSCLC cells for apoptosis while cells with higher levels are effectively killed through ferroptosis. Altogether, AF presents a potential novel therapeutic strategy to efficiently kill p53 mutated NSCLC tumor cells by various types of cell death. Citation Format: Laurie Freire Boullosa, Filip Lardon, Evelien Smits, Christophe Deben. Molecular mechanisms underlying the sensitizing effect of mutant p53 protein expression for Auranofin treatment of NSCLC and PDAC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2962.

Myrtenal averts apoptotic evasion of cancer cells in 7,12-dimethylbenz(a)anthracene induced experimental oral carcinogenesis

Apoptotic avoidance is one of the foremost characteristic features of tumour cells. Apoptotic induction in cancer cells could thus help to identify new anticancer agents from the natural products. The present study has taken an effort to investigate the apoptotic efficacy of myrtenal, a monoterpene, in 7,12-dimethylbenz(a)anthracene (DMBA) induced oral carcinoma in male golden Syrian hamsters. The present study used the potent carcinogen, 7,12- dimethylbenz(a)anthracene, to develop oral tumours in the buccal pouches of the golden Syrian hamsters. Topical application of the above said carcinogen on the buccal mucosa (3 times a week for 14 weeks) resulted in well differentiated oral squamous cell carcinoma. The apoptotic potential of myrtenal was investigated using a spectrum of apoptotic markers, including pro-apoptotic and anti-apoptotic markers. Oral tumours developed in the buccal mucosa of hamsters showed higher expression of mutant p53 protein, Bcl-2 and lowered expression of Bax, Bad, Bid, caspase 3 and caspase 9. Myrtenal treatment at a dose of 230mg/kg bw orally to the hamsters treated with DMBA modulated the above said apoptotic markers towards tumour suppression or inhibition. The present findings thus suggests that the tumour preventive effect of myrtenal could partly be ascribed to its apoptotic induction potential during DMBA induced oral carcinogenesis.

Investigation on the Genomic Characterization of Uterine Sarcoma for rAd-p53 Combined with Chemotherapy Treatment.

The aim is to investigate the genomic characterization of uterine sarcoma for rAd-p53 (Gendicine®) combined with chemotherapy treatment. We recently published an article on 12 cases of uterine sarcomas, which were treated with rAd-p53 combined with chemotherapy. We found that rAd-p53 combined with chemotherapy is effective for various uterine sarcomas. Pretreatment pathological specimens of four uterine sarcoma patients were collected from the above recent clinical research and numbered 1-4A/B. Tumor samples were subjected to targeted sequencing by using a 416 genes panel. We profiled the mutation spectrum and tumor mutation burden in the tumors, identified mutated genes, and explored their gene function. We also verified the p53 protein expression using immunohistochemistry. We identified a total of 30 mutated genes that were found from the next-generation sequencing test results. The average number of mutated genes was up to seven in the five samples. TP53 gene was mutated in two of the four patients, No. 1 and No. 4B. They are c.C833G (p.P278R) missense mutation and a point mutation (C141*) that result in a premature stop codon. We did not find a mutated TP53 gene in the other two cases, but we identified mutated genes, including CREBBP, LYN, CDKN2A, and JAK2, which were located upstream of the TP53 gene; they may have an impact on TP53. We also identified 11 additional genes which are involved in p53-related signaling pathways or have interaction with p53. Compared to solid tumor mutational burden (TMB) distribution, none of their TMB was ranking in the top 25%. Mutant p53 protein expression was positive in two specimens. Our results demonstrated that the TP53 signaling pathway plays an important role in uterine sarcoma tumorigenesis. TP53 and the upstream genes such as CREBBP, LYN, CDKN2A, and JAK2 may be involved in the genomic characterization for rAd-p53 (Gendicine) combined with chemotherapy in uterine sarcoma. Besides, the average amount of mutated genes from every patient is large.

Assessment of TP53 lesions for p53 system functionality and drug resistance in multiple myeloma using an isogenic cell line model

Recent advances in molecular diagnostics have shown that lesions affecting both copies of the gene for tumor suppressor protein 53 (TP53) count among the most powerful predictors for high-risk disease in multiple myeloma (MM). However, the functional relevance and potential therapeutic implications of single hits to TP53 remain less well understood. Here, we have for the first time approximated the different constellations of mono- and bi-allelic TP53 lesions observed in MM patients within the frame of a single MM cell line model and assessed their potential to disrupt p53 system functionality and to impart drug resistance. Both types of common first hit: point mutation with expression of mutant p53 protein or complete loss of contribution from one of two wildtype alleles strongly impaired p53 system functionality and increased resistance to melphalan. Second hits abolished remaining p53 activity and increased resistance to genotoxic drugs even further. These results fit well with the clinical drive to TP53 single- and double-hit disease in MM patients, provide a rationale for the most commonly observed double-hit constellation (del17p+ TP53 point mutation), and underscore the potential increases in MM cell malignancy associated with any type of initial TP53 lesion.

Abstract 1719: Copper levels and the p53 protein: A delicate balance

Abstract The tumor suppressor p53 is mutated in 50% of all cancers and in about 90% of small lung cancers. Mutations lead to loss of p53 expression or the expression of mutant p53 proteins. These mutants often lose WT function, but they have also been shown to acquire invasive and metastatic functions. p53 requires a zinc molecule in its core domain to fold properly and to function as a transcription factor and tumor suppressor. Loss of this zinc through competition with copper causes p53 to unfold and to lose its transcription function. Copper is an essential heavy metal required for a large variety of biological processes as it is required for the function of many enzymes. In cancer patients, increased serum levels of copper have been found and people with a predisposition for copper accumulation in the liver (Wilson’s disease, mutations in ATP7B) are prone to develop liver cancers. Our preliminary results have indicated that an unfolded WT p53 molecule does not only lose its transcription function, but also gains mutant p53-like functions in promoting invasion. Copper-induced unfolded p53 interacted with the mutant p53 specific interacting proteins Dicer, Ago, p63 and p73 and impaired p63 function. These results raise the question as to why a WT molecule would lose its function and whether it perhaps could act as a redox sensor. Experiments are ongoing to explore p53 mediated transcription in response to copper exposure. In addition, we are setting up mouse models to investigate p53 folding in vivo in response to mild increases in copper serum levels and we are developing methods to better determine p53 folding in patients and in mice. These results will help us assess p53 status in relation to serum metal levels in vivo and determine whether p53 is important in the development of cancers and metastasis. Citation Format: Patricia Muller, Yannick von Grabowiecki, Callum Hall. Copper levels and the p53 protein: A delicate balance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1719.

Mutant p53 protein is prevalent in Aflatoxin B1 than in Hepatitis B Core antigen associated chronic liver diseases in Southern Nigeria

Background: Aflatoxin B1 (AFB1), hepatitis B virus (HBV) and p53 mutation are major risk factors for liver cancer. However, the presence of AFB1 and expression of mutant p53 protein during active HBV replication (nuclear expression of Hepatitis B Core Antigen; HBcAg) and HBV clearance (nuclear expression of HBcAg) is still under-investigated in Africa. Objectives: This study assessed the prevalence, extent of expression, interaction, and risk associated with single and binary expression of AFB1, HBcAg and mutant p53 (mtp53) in liver diseases in relation to age and sex. Materials and Methods: This retrospective study included 14 and 74 cases of liver cancer (LC) and chronic hepatitis, respectively. Tissues were histochemically and immunohistochemically stained, scored and documented as positive (+) or negative (-) for antigen. Result: In this study, the prevalence of AFB1, HBcAg, and mtp53 were 76.1%, 40.9%, and 61.4%, respectively. Higher prevalence and expression of AFB1 was observed in chronic hepatitis than in LC (p= 0.035 and 0.028, respectively). Higher prevalence of AFB1 and mtp53 and lower prevalence of HBcAg were observed in females than in males (p= 0.186, 0.0003 and 0.062, respectively). Cytoplasmic expression of HBcAg was higher in males (58 than in females were 58.6% (17/29) and 14.3% (1/7), respectively (p= 0.088). AFB1+ and HBcAg+ females were more and less likely to develop LC than males (OR: 3.00 and 0.39, 95%CI: 0.10-4.18 and 0.44-12.20, p= 0.012 and 0.003), respectively. AFB1+mtp53- (23.9%), AFB1+mtp53+ (52.3%), HBcAg+mtp53- (13.6%), HBcAg+mtp53+ (27.3%) and AFB1+HBcAg+ (34.1%) increased liver cancer risk (OR: 6.45, 21.50, 2.80, 0.78, and 5.11; 95%CI: 0.89-29.29, 0.95-43.86, 2.96-156.13, 0.60-13.01, and 0.14-4.25; p= 0.057, 0.002, 0.189, 0.772, and 0.067, respectively). Conclusion: This study suggests that women are at a higher risk of AFB1 exposure and p53 mutation, but are at a lower risk of viral replication than men.