R249S Mutation Research Articles

Multiscale simulations reveal the driving forces of p53C phase separation accelerated by oncogenic mutations.

Liquid-Liquid phase separation (LLPS) of p53 to form liquid condensates has been implicated in cellular functions and dysfunctions. The p53 condensates may serve as amyloid fibril precursors to initiate p53 aggregation, which is associated with oncogenic gain-of-function and various human cancers. M237I and R249S mutations located in p53 core domain (p53C) have been detected respectively in glioblastomas and hepatocellular carcinoma. Interestingly, these p53C mutants can also undergo LLPS and liquid-to-solid phase transition, which are faster than wild type p53C. However, the underlying molecular basis governing the accelerated LLPS and liquid-to-solid transition of p53C remain poorly understood. Herein, we explore the M237I/R249S mutation-induced structural alterations and phase separation behavior of p53C by employing multiscale molecular dynamics simulations. All-atom simulations revealed conformational disruptions in the zinc-binding domain of the M237I mutant and in both loop3 and zinc-binding domain of the R249S mutant. The two mutations enhance hydrophobic exposure of those regions and attenuate intramolecular interactions, which may hasten the LLPS and aggregation of p53C. Martini 3 coarse-grained simulations demonstrated spontaneous phase separation of p53C and accelerated effects of M237I/R249S mutations on the phase separation of p53C. Importantly, we find that the regions with enhanced intermolecular interactions observed in coarse-grained simulations coincide with the disrupted regions with weakened intramolecular interactions observed in all-atom simulations, indicating that M237I/R249S mutation-induced local structural disruptions expedite the LLPS of p53C. This study unveils the molecular mechanisms underlying the two cancer-associated mutation-accelerated LLPS and aggregation of p53C, providing avenues for anticancer therapy by targeting the phase separation process.

TP53 Mutations R175H and R249S Are Rare in Indian Head and Neck Cancer Patients

Abstract Introduction Mutations in tumor suppressor gene TP53 are considered as one of the main causes for different types of cancer. Head and neck squamous cell carcinoma (HNSCC) is one of the common cancers found in India. Among the several mutations reported in the TP53 gene, R175H and R249S are linked to cause of several cancers. This work was carried out to study the prevalence of R175H and R249S mutations in HNSCC patients of Indian origin. Method Tumor samples were collected from 50 HNSCC patients and good quality genomic deoxyribonucleic acid (DNA) were obtained from 41 samples. Using this genomic DNA, polymerase chain reaction-linked restriction fragment length polymorphism technique was used to screen both the mutations in the TP53 gene of the patients. Result The results revealed that out of the 41 samples analyzed, all the samples were negative for the mutations both in homozygous and heterozygous condition. This experiment was repeated three times, and the representative image is shown. Conclusion This study suggests that mutations in codon 175 (R175H) and 249 (R249S) are rare in HNSCC patients of Indian origin.

Assessments of TP53 and CTNNB1 gene hotspot mutations in circulating tumour DNA of hepatitis B virus-induced hepatocellular carcinoma.

Background: Hepatitis B virus (HBV) infection is one of the major causes of chronic liver disease, which progresses from chronic hepatitis B (CHB) to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Early detection and laboratory-based screening of hepatocellular carcinoma are still major challenges. This study was undertaken to determine whether the cancer hallmark gene signatures that are released into circulation as circulating tumour DNA (ctDNA) can be used as a liquid biopsy marker for screening, early detection, and prognosis of HCC. Methods: A total of 130 subjects, including HBV-HCC (n = 80), HBV-cirrhotic and non-cirrhotic (n = 35), and healthy (n = 15) controls, were evaluated for TP53 and beta-catenin (CTNNB1) gene hotspot mutations in ctDNA by Sanger-based cycle sequencing and droplet digital PCR (ddPCR) assays. Mutation detection frequency, percentage mutant fractions, and their association with tumour stage, mortality, and smoking habits were determined. Results: Sanger-based cycle sequencing was carried out for 32 HCC patients. Predict SNP Tools analysis indicated several pathogenic driver mutations in the ctDNA sequence, which include p.D228N, p.C229R, p.H233R, p.Y234D, p.S240T, p.G245S, and p.R249M for TP53 gene exon 7 and p.S33T for CTNNB1 gene exon 3. The TP53 c.746G>T (p.R249M) mutation was detected predominately (25% cases) by sequencing, but there was no dominant mutation at position c.747G>T (p.R249S) that was reported for HBV-HCC patients. A dual-probe ddPCR assay was developed to determine mutant and wild-type copy numbers of TP53 (p.R249M and p.R249S) and CTNNB1 (p.S45P) and their percentage mutant fraction in all 130 subjects. The TP53 R249M and CTNNB1 S45P mutations were detected in 31.25% and 26.25% of HCC patients, respectively, with a high mutant-to-wild-type fraction percentage (1.81% and 1.73%), which is significant as compared to cirrhotic and non-cirrhotic patients. Poor survival was observed in HCC patients with combined TP53 and CTNNB1 gene driver mutations. The TP53 R249M mutation was also significantly (p < 0.0001) associated with smoking habits (OR, 11.77; 95% CI, 3.219-36.20), but not the same for the TP53 R249S mutation. Conclusion: Screening of ctDNA TP53 and CTNNB1 gene mutations by ddPCR may be helpful for early detection and identifying the risk of HCC progression.

Abstract 2590: TP53 R249S mutation confers hepatic organoids with gain-of-function (GOF) tumorigenic features through transcriptional activation of ZMIZ2

Abstract Hepatocellular Carcinoma (HCC) represents the third leading cause of cancer-related mortality worldwide. TP53 mutations are pivotal genomic drivers for HCC development through frequent concurrent loss-of-function (LOF) aberrations and protein-altering missense mutations. Over 90% of the TP53 missense mutations are distributed within the core DNA binding domain (DBD), where R249S is the most common. Cumulative studies have demonstrated the cancer-promoting properties of TP53R249S mutation in HCC, but its biological impact on tumor initiation remains to be defined. To mimic the early stages of liver carcinogenesis, we generated normal hepatic organoids from human liver tissues of 3 individuals. CRISPR-Cas9 mediated knockout of TP53 in liver organoids resulted in pleomorphic malignant features, including dysplasia, hyperchromasia, atypical and frequent mitosis, loss of polarity, and increased nuclear to cytoplasm ratio. To recapitulate TP53R249S genotype in HCC, we ectopically expressed R249S mutant in liver organoids through lentiviral infection. TP53R249S liver organoids displayed tumorigenic properties as evidenced by increased lesion forming incidence (37.5%) in subcutaneous xenografts when compared with TP53KO (17%) and TP53 wild-type (WT) organoids (0%). Chromatin immunoprecipitation sequencing (ChIP-seq) analysis with HCC cell lines substantiated the specific gain-of-function (GOF) transcriptional activities of R249S mutant. A unique subset of transcription start site-proximal peaks was exclusively found in R249S mutant cells when compared with other TP53 missense mutants and WT. Integration of ChIP-seq and RNA-seq analysis identified transcription factor ZMIZ2 as a direct transcription target of R249S mutant. Our results showed that ZMIZ2 is preferentially overexpressed in HCC patients carrying TP53R249S mutation and exerts a vital role for proliferation of R249S mutant HCC cells. Knockdown of ZMIZ2 profoundly suppressed global H3K27 acetylation (H3K27ac) and H3K4 trimethylation (H3K4me3) in HCC cells. Transcriptome profiling of ZMIZ2 knockdown cells identified multiple downstream targets enriched in chromatin binding and interaction with histone deacetylase, further reinforcing its involvement in epigenetic regulation. In summary, our study revealed that TP53R249S mutation confers distinct advantages in increased tumorigenicity to human liver organoids through GOF transcriptional activities. ZMIZ2 serves as a direct downstream effector contributing to the oncogenic growth arising from R249S mutation plausibly through altering chromatin remodelling. Citation Format: Mingjing Xu, Yin Kau Lam, Jianqing Yu, Kelvin Kwok Chai Ng, Nathalie Wong. TP53 R249S mutation confers hepatic organoids with gain-of-function (GOF) tumorigenic features through transcriptional activation of ZMIZ2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2590.

First Molecular Characterization of Chronic Hepatitis B Carriers in Timbuktu, Mali.

In Mali, hepatocellular carcinoma (HCC) is the third and sixth most common cancer in men and women, respectively. Mali comprises several distinct climato-ecological zones. Most studies to date have been conducted in the sub-Sahelian zone of southern Mali, including the capital city Bamako. In this part of the country, the main risk factors for HCC are chronic hepatitis B virus (HBV) carriage and dietary exposure to aflatoxins, a well-known hepatocarcinogen. Data are scarce for other ecological zones, but our preliminary data from 721 blood donors in the area of Timbuktu, presented in this study, suggest that chronic HBV carriage is also endemic in the northern Saharan zone of Mali. For further study, 29 healthy HBV chronic carrier volunteers were recruited from the blood transfusion center in Timbuktu. Successful viral genotyping in 20 volunteers revealed HBV genotype E in 13 cases and D in 7 cases, suggesting that this geographical and anthropological transition zone may also represent a transition zone between HBV genotypes that dominate sub-Saharan and northern Africa, respectively. Sequencing of circulating cell-free plasma DNA (cfDNA) from donors did not reveal the presence of the TP53 R249S mutation in these donors, a marker of dietary exposure to aflatoxins in sub-Saharan Africa. These results suggest that the geo-epidemiological distribution of the risk factors for HCC is not uniform across Mali, but is dependent upon climatic, socioeconomic and anthropological factors that might have an impact on patterns of chronic liver disease and cancer.

TP53 R249S mutation in hepatic organoids captures the predisposing cancer risk.

Major genomic drivers of hepatocellular carcinoma (HCC) are nowadays well recognized, although models to establish their roles in human HCC initiation remain scarce. Here, we used human liver organoids in experimental systems to mimic the early stages of human liver carcinogenesis from the genetic lesions of TP53 loss and L3 loop R249S mutation. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) of HCC cell lines shed important functional insights into the initiation of HCC consequential to the loss of tumor-suppressive function from TP53 deficiency and gain-of-function activities from mutant p53. Human liver organoids were generated from surgical nontumor liver tissues. CRISPR knockout of TP53 in liver organoids consistently demonstrated tumor-like morphological changes, increased in stemness and unrestricted in vitro propagation. To recapitulate TP53 status in human HCC, we overexpressed mutant R249S in TP53 knockout organoids. A spontaneous increase in tumorigenic potentials and bona fide HCC histology in xenotransplantations were observed. ChIP-seq analysis of HCC cell lines underscored gain-of-function properties from L3 loop p53 mutants in chromatin remodeling and overcoming extrinsic stress. More importantly, direct transcriptional activation of PSMF1 by mutant R249S could increase organoid resistance to endoplasmic reticulum stress, which was readily abrogated by PSMF1 knockdown in rescue experiments. In a patient cohort of primary HCC tumors and genome-edited liver organoids, quantitative polymerase chain reaction corroborated ChIP-seq findings and verified preferential genes modulated by L3 mutants, especially those enriched by R249S. We showed differential tumorigenic effects from TP53 loss and L3 mutations, which together confer normal hepatocytes with early clonal advantages and prosurvival functions.

Metabolic Syndrome Instead of Aflatoxin-Related TP53 R249S Mutation as a Hepatocellular Carcinoma Risk Factor

Metabolic Syndrome Instead of Aflatoxin-Related TP53 R249S Mutation as a Hepatocellular Carcinoma Risk Factor

Genome-wide association study of the TP53 R249S mutation in hepatocellular carcinoma with aflatoxin B1 exposure and infection with hepatitis B virus.

Exposure to dietary aflatoxin B1 (AFB1) induces DNA damage and mutation in the TP53 gene at codon 249, known as the TP53 R249S mutation, and is a major risk factor for hepatocellular carcinoma (HCC). AFB1 and the hepatitis B virus (HBV) together exert synergistic effects that promote carcinogenesis and TP53 R249S mutation in HCC. A genome-wide association study (GWAS) of whole genome exons was conducted using 485 HCC patients with chronic HBV infection. This was followed by an independent replication study conducted using 270 patients with chronic HBV infection. Immunohistochemistry was used to evaluate TP53 expression in all samples. This showed a correlation between codon 249 mutations and TP53 expression. Susceptibility variants for the TP53 R249S mutation in HCC were identified based on both the GWAS and replication study. The associations between identified variants and the expression levels of their located genes were analyzed in 20 paired independent samples. The likelihood of positive TP53 expression was found to be higher in HCC patients with the R249S mutation both in the GWAS (P<0.001) and the replication study (P=0.006). The combined analyses showed that the TP53 R249S mutation was significantly associated with three single nucleotide polymorphisms (SNPs): ADAMTS18 rs9930984 (adjusted P=4.84×10-6), WDR49 rs75218075 (adjusted P=7.36×10-5), and SLC8A3 rs8022091 (adjusted P=0.042). The TP53 R249S mutation was found to be highly associated with the TT genotypes of rs9930984 (additive model, P=0.01; dominant model, P=6.43×10-5) and rs75218075 (additive model, P=0.002; dominant model, P=2.16×10-4). Additionally, ADAMTS18 mRNA expression was significantly higher in HCC tissue compared with its expression in paired non-tumor tissue (P=0.041), and patients carrying the TT genotype at rs9930984 showed lower ADAMTS18 expression in non-tumor tissue compared with patients carrying the GT genotype (P=0.0028). WDR49 expression was markedly lower in HCC tissue compared with paired non-tumor tissue (P=0.0011). TP53 expression is significantly associated with the R249S mutation in HCC. Our collective results suggest that rs9930984, rs75218075, and rs8022091 are associated with R249S mutation susceptibility in HCC patients exposed to AFB1 and HBV infection.

TP53 R249S mutation detected in circulating tumour DNA is associated with Prognosis of hepatocellular carcinoma patients with or without hepatectomy.

Somatic mutation R249S in TP53 is highly common in hepatocellular carcinoma (HCC). We aim to investigate the effects of R249S in ctDNA on the prognosis of HCC. We analysed three cohorts including 895 HCC patients. TP53 mutation spectrum was examined by direct sequencing of genomic DNA from tissue specimens in HCC patients with hepatectomy (Cohort 1, N=260). R249S and other recurrent missense mutations were assessed for their biological functions and associations with overall survival (OS) and progression-free survival (PFS) of HCC patients in Cohort 1. R249S within circulating tumour DNA (ctDNA) was detected through droplet digital polymerase chain reaction (ddPCR) and its association with OS and PRS was analysed in HCC patients with (Cohort 2, N=275) or without (Cohort 3, N=360) hepatectomy. In Cohort 1, R249S occupied 60.28% of all TP53 mutations. Overexpression of R249S induced more serious malignant phenotypes than those of the other three identified TP53 recurrent missense mutations. Additionally, R249S, but not other missense mutations, was significantly associated with worse OS (P=.006) and PFS (P=.01) of HCC patients. Consistent with the results in Cohort 1, HCC patients in Cohorts 2 and 3 with R249S had worse OS (P=8.291×10-7 and 2.608×10-7 in Cohorts 2 and 3, respectively) and PFS (P=5.115×10-7 and 5.900×10-13 in Cohorts 2 and 3, respectively) compared to those without this mutation. TP53 R249S mutation in ctDNA may serve as a promising prognosis biomarker for HCC patients with or without hepatectomy.

Analysis of TP53 aflatoxin signature mutation in hepatocellular carcinomas from Guatemala: A cross-sectional study (2016-2017).

Background and aimsGuatemala has the highest incidence of hepatocellular carcinoma (HCC) in the Western hemisphere. The major risk factors in Guatemala are not well characterized, but the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) appears to be low, while the prevalence of aflatoxin (AFB1) exposure appears to be high. To examine whether AFB1 may contribute to the elevated incidence of HCC in Guatemala, this study examined the frequency of the AFB1‐signature mutation in the TP53 gene (R249S) as well as other somatic mutations. In addition, we assessed whether the frequency of the TP53 mutation differed by sex.MethodsFormalin‐fixed, paraffin‐embedded (FFPE) HCC tissues were obtained from three hospitals in Guatemala City between 2016 and 2017. In addition, tumor tissues preserved in RNAlater were also obtained. Sociodemographic and clinical information including HBV and HCV status were collected. Targeted sequencing of TP53 was performed in the FFPE samples, and a panel of 253 cancer‐related genes was sequenced in the RNAlater samples.ResultsNinety‐one FFPE tissues were examined, from 52 men and 39 women. Median (IQR) age at diagnosis was 62 (51‐70). Among those with known HBV and HCV status, two were HBV+ and three were HCV+. Overall, 47% of the HCCs had a TP53 mutation. The AFB1‐signature R249S mutation was present in 24%. No overlap between the R249S mutation and HBV+ was observed in this cohort. Among 18 RNAlater samples examined, 44% had any TP53 mutation and 33% had the R249S mutation. Other somatic mutations were identified in known HCC driver genes.ConclusionsThe presence of the TP53 R249S mutation in the samples studied suggests that AFB1 may contribute to the high incidence of HCC in Guatemala. The proportion of HBV+ tumors was low, suggesting that AFB1 may be associated with HCC in the absence of concomitant HBV infection. Further investigation of AFB1 and other risk factors for HCC in Guatemala is warranted.

Lack of hotspot mutations other than TP53 R249S in aflatoxin B1 associated hepatocellular carcinoma

Abstract Objective Despite the recent advances in diagnosis and treatment of hepatocellular carcinoma (HCC), it is still a major health problem. Therefore, understanding the molecular mechanism is very important. Our aim is to investigate the molecular basis of aflatoxin B1 (AFB1) induced HCC other than the hotspot TP53 p.Arg249Ser (c.747G&gt;T) (R249S) mutation. Methods 525 genes previously reported to be involved in carcinogenesis with mutations in different cancer types were analyzed by next generation sequencing for 525 cancer-gene panel (Roche/NimbleGen) in one tumor sample (T29) and one cell line (MAHLAVU) carrying TP53 R249S mutation. Additionally, ARID2 and BCORL1 genes were analyzed by Sanger sequencing for MAHLAVU and Primary Liver Carcinoma/Poliomyelitis Research Foundation/5 (PLC/PRF/5) cell lines. Results No other common gene mutations were found in the analyzed T29 and MAHLAVU samples and also no genetic variation possibly associated with AFB1 was detected in PLC/PRF/5 cell line and 68 COSMIC HCC samples. Likewise, no pathogenic mutation was detected in ARID2 and BCORL1 genes of MAHLAVU and PLC/PRF/5 cell lines. Conclusion No fingerprint mutations were detected in the analyzed genes. To the best of our knowledge, other hotspot mutations appear to be absent if not at a very low frequency in HCC carrying TP53 R249S mutation.

Microsecond molecular dynamics simulations reveal the allosteric regulatory mechanism of p53 R249S mutation in p53-associated liver cancer

The tumor suppressor p53, a transcription factor, plays a critical role in many cellular processes, including DNA repair and apoptosis and cell cycle arrest. Missense mutations in the p53 have closely related to human cancer. R249S mutation at the p53 core DNA binding domain (DBD) is frequently observed in hepatocellular carcinoma. This mutation is away from the p53 DBD-DNA binding interface. However, how the R249S mutation causes the structural changes of p53 DBD that lead to weak the binding of p53 mutant to DNA has not been clearly understood. Here, microsecond-scale molecular dynamics (MD) simulations of p53 DBD in the wild type (WT) and R249S mutated states in the absence of DNA binding were performed to explore the effect of the R249S mutation on the conformational dynamics of p53 DBD. The R249S mutation does not cause the global conformational changes, and it only affects the local domains at the mutation site and the DNA binding interface, particularly at the S1-S2 turn. The allosteric effects of the S1-S2 turn induced by the R249S mutation lead to the extension of the S1-S2 turn into the β-strands, which in turn interferes with the binding of DNA at the major groove. The results can help decipher the allosteric regulatory mechanism by which the R249S mutation of p53 DBD affects the p53 DBD-DNA interactions.

Targeted genomic profiling identifies frequent deleterious mutations in FAT4 and TP53 genes in HBV-associated hepatocellular carcinoma

BackgroundHepatitis B virus (HBV) is the major risk factor for hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV-associated HCC pathogenesis is still unclear. Genetic alterations in cancer-related genes have been linked to many human cancers. Here, we aimed to explore genetic alterations in selected cancer-related genes in patients with HBV-associated HCC.MethodsTargeted sequencing was used to analyze six cancer-related genes (PIK3CA, TP53, FAT4, IRF2, HNF4α and ARID1A) in eight pairs of HBV-associated HCC tumors and their adjacent non-tumor tissues. Sanger sequencing, quantitative PCR, Western-blotting and RNAi-mediated gene knockdown were used to further validate findings.ResultsTargeted sequencing revealed thirteen non-synonymous mutations, of which 9 (69%) were found in FAT4 and 4 (31%) were found in TP53 genes. Non-synonymous mutations were not found in PIK3CA, IRF2, HNF4α and ARID1A. Among these 13 non-synonymous mutations, 12 (8 in FAT4 and 4 in TP53) were predicted to have deleterious effect on protein function by in silico analysis. For TP53, Y220S, R249S and P250R non-synonymous mutations were solely identified in tumor tissues. Further expression profiling of FAT4 and TP53 on twenty-eight pairs of HCC tumor and non-tumor tissues confirmed significant downregulation of both genes in HCC tumors compared with their non-tumor counterparts (P < 0.001 and P < 0.01, respectively). Functional analysis using RNAi-mediated knockdown of FAT4 revealed an increased cancer cell growth and proliferation, suggesting the putative tumor suppressor role of FAT4 in HCC.ConclusionsThis study highlights the importance of FAT4 and TP53 in HCC pathogenesis and identifies new genetic variants that may have potentials for development of precise therapy for HCC.

Epidemiological and Serological Profile of Hepatitis B Virus in an Urban Area in Mali

The objective of our study was to evaluate hepatitis B virus (HBV) infection in an urban population. This longitudinal study was conducted in Bamako District and Kati Commune. After a preparatory phase, the persons who accepted the protocol were assessed for HBsAg. HBsAg carriers had blood collection for HBeAg assay, viral load assessment, genotyping, DNA mutation testing, and severity of hepatic fibrosis and necrosis. At the end of this study, 1475 persons were included, of which 195 had HBsAg positive confirmed, that is to say 13.97%. The mean age of HBsAg positive patients was 35.11 ± 11.12 years with a sex ratio of 2.68. HBeAg was found in 8.9% of the patients tested for this antigen. The viral load was undetectable in 10.52% of patients and greater than 2000 IU/mL in 32.24% of cases. Fibrosis ≥ F2 and necrosis ≥ A2 were found in respectively 19.72% and 6.80% of cases. Genotype E was found in 91.6 patients and an R249S mutation observed in 39.04% of cases. Conclusion: HBV infection has a serious impact on socio-economic development in Mali because it affects mainly the young male population, hence the need to organize preventive measures effectively.

Droplet digital PCR detects high rate of TP53 R249S mutants in cell-free DNA of middle African patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is still a major killing malignancy in sub-Saharan Africa. Lifelong intoxication with aflatoxin B1 is considered as one of the primary causes of this situation. The role of aflatoxin in HCC from a given population is commonly estimated through the prevalence of R249S mutation of TP53, a hallmark for previous exposure to the mycotoxin. However, the role of AFB1 is barely known in large part of Africa. We conducted a survey on circulating cell-free DNA from 149 patients with HCC and 213 control subjects with and without liver diseases from Cameroon and Central African Republic using droplet digital PCR technique. We observed a mutation prevalence of 24.8% (n = 37/149) in patients with tumor and 5.6% (n = 12/213) in controls (P = 2.2E−07). Patients with mutations usually displayed significantly increased circulating alpha-fetoprotein (AFP) values, high hepatitis B virus (HBV) DNA loads as well as worsened values of blood cells count. Interestingly, the fraction of droplets positive for R249S was significantly larger in patients with liver cancer (15.3 ± 3.7%) than in controls (0.5 ± 0.3%, P = 7.1E−04). Our survey indicates that AFB1 is instrumental for HCC development in Middle Africa and that droplet digital PCR might be used in the region both to diagnose HCC and to conduct public health surveys on populations at risk of chronic aflatoxin intoxication.

Abstract LB-229: Mutation spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellualr carcinoma

Abstract Responding to a need to identify the mutational processes that are responsible for malignant transformation, highly accurate duplex sequencing (DS) was employed to monitor acquisition of context-dependent high-resolution mutational spectra (MS) during the process of hepatocarcinogenesis. Aflatoxin B1 (AFB1) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB1-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C&amp;gt;T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Four-day old male mice were treated with AFB1 using a regimen that induced hepatocellular carcinoma within 72 weeks. The DS technology revealed a distinctive MS of AFB1 at 10 weeks after dosing, well before evidence of neoplasia. Importantly, 25% of all mutations were G&amp;gt;T in one trinucleotide context (5’-CGC-3’), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB1 exposure. At 72 weeks, livers were separated into tumor tissue and normal surrounding cellular fractions (primarily hepatocytes). The MS in normal cells surrounding the tumors revealed clear evidence of previous AFB1 exposure with predominantly G:C&amp;gt;T:A mutations and strong representation of the CGC&amp;gt;CTC hotspot. Analysis of tumor tissue revealed a more complex pattern than that observed in surrounding hepatocyte fraction; tumor MS were a composite of the 10-week spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. The 10-week AFB1-exposed mouse MS was then used as bait to computationally identify human HCC sequences that have similarity to it. This analysis revealed that the mouse spectrum has striking similarity to human HCCs from hepatitis B infected people who have the R249S mutation in P53. The data also provide insight into the molecular basis for the synergism between hepatitis B exposure and AFB1. The MS provided by DS technology provides a method to identify mutational processes in normal and tumor tissues enabling reconstruction of the molecular etiology of human hepatocarcinogenesis and other genetic diseases. Citation Format: Supawadee Chawanthayatham, Charles C. Valentine III, Bogdan I. Fedeles, Stephen L. Slocum, Gerald N. Wogan, Robert G. Croy, John M. Essigmann. Mutation spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellualr carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-229. doi:10.1158/1538-7445.AM2017-LB-229

Covering the Cover

Covering the Cover

Association of Aflatoxin and Gallbladder Cancer

Aflatoxin, which causes hepatocellular carcinoma, may also cause gallbladder cancer. We investigated whether patients with gallbladder cancer have higher exposure to aflatoxin than patients with gallstones. We measured aflatoxin B1 (AFB1)-lysine adducts in plasma samples from the Shanghai Biliary Tract Cancer case-control study, conducted from 1997 through 2001. We calculated age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) and the population-attributable fraction for 209patients with gallbladder cancer and gallstones vs 250 patients with gallstones without cancer (controls). In 54 patients with gallbladder cancer, tumor tissue was examined for the R249S mutation in TP53, associated with aflatoxin exposure, through targeted sequencing. The AFB1-lysine adduct was detected in 67 (32%) of 209 patients with gallbladder cancer and 37 (15%) of the 250 controls (χ2P< .0001), almost threefold more patients with gallbladder cancer than controls (OR, 2.71; 95% CI, 1.70-4.33). Among participants with detectable levels of AFB1-lysine, the median level of AFB1-lysine was 5.4 pg/mg in those with gallbladder cancer, compared with 1.2 pg/mg in controls. For patients in the fourth quartile of AFB1-lysine level vs the first quartile, the OR for gallbladder cancer was 7.61 (95% CI, 2.01-28.84). None of the 54 gallbladder tumors sequenced were found to have the R249S mutation in TP53. The population-attributable fraction for cancer related to aflatoxin was 20% (95% CI, 15%-25%). In a case-control study of patients with gallbladder cancer and gallstones vs patients with gallstones without cancer, we associated exposure to aflatoxin (based on plasma level of AFB1-lysine) with gallbladder cancer. Gallbladder cancer does not appear associate with the R249S mutation in TP53. If aflatoxin is a cause of gallbladder cancer, it may have accounted for up to 20% of the gallbladder cancers in Shanghai, China, during the study period, and could account for an even higher proportion in high-risk areas. If our findings are verified, reducing aflatoxin exposure might reduce the incidence of gallbladder cancer.

Whole genome sequencing of matched tumor, adjacent non-tumor tissues and corresponding normal blood samples of hepatocellular carcinoma patients revealed dynamic changes of the mutations profiles during hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) has become the third most deadly disease worldwide and HBV is the major factor in Asia and Africa. We conducted 9 WGS (whole genome sequencing) analyses for matched samples of tumor, adjacent non-tumor tissues and normal blood samples of HCC patients from three HBV positive patients. We then validated the mutations identified in a larger cohort of 177 HCC patients. We found that the number of the unique somatic mutations (average of 59,136) in tumor samples is significantly less than that in adjacent non-tumor tissues (average 83, 633). We discovered that the TP53 R249S mutation occurred in 7.7% of the HCC patients, and it was significantly associated with poor diagnosis. In addition, we found that the L104P mutation in the VCX gene (Variable charge, X-linked) was absent in white blood cell samples, but present at 11.1% frequency in the adjacent tissues and increased to 14.6% in HCC tissues, suggesting that this mutation might be a tumor driver gene driving HCC carcinogenesis. Finally, we identified a TK1-RNU7 fusion, which would result in a deletion of 103 amino acids from its C-terminal. The frequencies of this fusion event decreased from the adjacent tissues (29.2%) to the tumors (16.7%), suggesting that a truncated thymidine Kinase1 (TK1) caused by the fusion event might be deleterious and be selected against during tumor progression. The three-way comparisons allow the identification of potential driver mutations of carcinogenesis. Furthermore, our dataset provides the research community a valuable dataset for identifying dynamic changes of mutation profiles and driver mutations for HCC.

Histone methyltransferase SETDB1 regulates liver cancer cell growth through methylation of p53.

SETDB1 is a histone H3K9 methyltransferase that has a critical role in early development. It is located within a melanoma susceptibility locus and facilitates melanoma formation. However, the mechanism by which SETDB1 regulates tumorigenesis remains unknown. Here we report the molecular interplay between SETDB1 and the well-known hotspot gain-of-function (GOF) TP53 R249S mutation. We show that in hepatocellular carcinoma (HCC) SETDB1 is overexpressed with moderate copy number gain, and GOF TP53 mutations including R249S associate with this overexpression. Inactivation of SETDB1 in HCC cell lines bearing the R249S mutation suppresses cell growth. The TP53 mutation status renders cancer cells dependent on SETDB1. Moreover, SETDB1 forms a complex with p53 and catalyses p53K370 di-methylation. SETDB1 attenuation reduces the p53K370me2 level, which subsequently leads to increased recognition and degradation of p53 by MDM2. Together, we provide both genetic and biochemical evidence for a mechanism by which SETDB1 regulates cancer cell growth via methylation of p53.