Synergy between histone deacetylase inhibitors and DNA- damaging agents is mediated by histone deacetylase 2 in colorectal cancer

Previous studies have associated the overexpression of histone deacetylase 2 (HDAC2) and the presence of TP53 mutations with the progression to advanced stage drug resistant colorectal cancer (CRC). However, the mechanistic link between HDAC2 expression and the TP53 mutational status has remained unexplored. Here, we investigated the function of HDAC2 in drug resistance by assessing the synergistic effects of DNA-targeted chemotherapeutic agents and HDAC inhibitors (HDACis) on two TP53-mutated colorectal adenocarcinoma CRC cell lines (SW480 and HT-29) and on the TP53-wild type carcinoma cell line (HCT116 p53+/+) and its TP53 deficient sub-line (HCT116 p53-/-). We showed that in the untreated SW480 and HT-29 cells the steady-state level of HDAC2 was low compared to a TP53-wild type carcinoma cell line (HCT116 p53+/+). Increased expression of HDAC2 correlated with drug resistance, and depletion by shRNA sensitised the multi-drug resistance cell line HT-29 to CRC chemotherapeutic drugs such as 5-fluorouracil (5-FU) and oxaliplatin (Oxa). Combined treatment with the HDACi suberoylanilide hydroxamic acid plus 5-FU or Oxa reduced the level of HDAC2 expression, modified chromatin structure and induced mitotic cell death in HT-29 cells. Non-invasive bioluminescence imaging revealed significant reductions in xenograft tumour growth with HDAC2 expression level reduced to <50% in treated animals. Elevated levels of histone acetylation on residues H3K9, H4K12 and H4K16 were also found to be associated with resistance to VPA/Dox or SAHA/Dox treatment. Our results suggest that HDAC2 expression rather than the p53 mutation status influences the outcome of combined treatment with a HDACi and DNA-damaging agents in CRC.

Genetics and Prognostication in Splenic Marginal Zone Lymphoma: Revelations from Deep Sequencing

Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies.

Experimental Design: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL.

Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02–4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05–4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08–5.2; P = 0.03).

Conclusions: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.

Indicatori patologici di rilevanza diagnostica nel carcinoma squamocellulare orale felino

Il carcinoma squamocellulare è il tumore maligno orale più frequente nel gatto e si caratterizza per diagnosi spesso tardiva e prognosi infausta. Il progetto riguarda la ricerca di marker di rilevanza dia-gnostica nel carcinoma squamocellulare orale felino (FOSCC), al fine di sviluppare un test di scree-ning non invasivo. È stata condotta un’analisi retrospettiva delle disregolazioni del gene oncosoppres-sore TP53 in campioni istologici di FOSCC e di una popolazione di controllo (lesioni infiammatorie croniche orali e mucose orali normali feline). Tramite next-generation sequencing (NGS) sono state rilevate mutazioni di TP53 nel 69% dei FOSCC, ed anche l’espressione immunoistochimica della pro-teina p53 era presente nel 69% dei tumori, con una concordanza discreta (77%) fra le due alterazioni. Nella popolazione di controllo erano presenti disregolazioni di p53 solo in due lesioni infiammatorie (3%). Successivamente è stata effettuata un’analisi prospettica con NGS della metilazione del DNA di 17 geni, noti per essere disregolati nel carcinoma squamocellulare orale umano o felino, insieme all’analisi mutazionale di TP53, in campioni istologici di FOSCC e in un gruppo di controllo. Le stesse indagini molecolari sono state svolte in parallelo su campioni di cellule prelevate mediante brushing orale. Utilizzando 6 dei geni indagati differenzialmente metilati nei FOSCC (FLI1, MiR124-1, KIF1A, MAGEC2, ZAP70, MiR363) e lo stato mutazionale diTP53, è stato impostato un algoritmo diagnostico per differenziare i FOSCC dalla mucosa orale non neoplastica. Applicato ai brushing, l’algoritmo è risultato positivo (indicativo di carcinoma) in 24/35 (69%) gatti con FOSCC, contro 2/60 (3%) controlli (sensibilità: 69%; specifici-tà: 97%). La quota di FOSCC identificati era significativamente maggiore nei gatti sottoposti a prelievo in anestesia generale rispetto ai gatti svegli. Questi risultati sono incoraggianti per il riconoscimento precoce del FOSCC tramite brushing orale. Saranno necessari ulteriori studi su casistiche più ampie per validare questa metodica e migliorarne la sensibilità.

Identification of molecular biomarkers in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) is a severe cancer that has been on the rise in Western nations over the past few decades. It has a high mortality rate and the 5-year survival rate is only 35%–45%. EAC has been included in a group of tumors with one of the highest rates of copy number alterations (CNAs), somatic structural rearrangements, high mutation frequency, with different mutational signatures, and with epigenetic mechanisms. The vast heterogeneity of EAC mutations makes it challenging to comprehend the biology that underlies tumor onset and development, identify prognostic biomarkers, and define a molecular classification to stratify patients. The only way to resolve the current disagreements is through an exhaustive molecular analysis of EAC. We examined the genetic profile of 164 patients' esophageal adenocarcinoma samples (without chemo-radiotherapy). The included patients did not receive neoadjuvant therapies, which can change the genetic and molecular composition of the tumor. Using next-generation sequencing technologies (NGS) at high coverage, we examined a custom panel of 26 cancer-related genes. Over the entire cohort, 337 variants were found, with the TP53 gene showing the most frequent alteration (67.27%). Poorer cancer-specific survival was associated with missense mutations in the TP53 gene (Log Rank P=0.0197). We discovered HNF1alpha gene disruptive mutations in 7 cases that were also affected by other gene changes. We started to investigate its role in EAC cell lines by silencing HNF1alpha to mimic our EAC cohort and we use Seahorse technique to analyze its role in the metabolism in esophageal cell. No significant changes were found in transfected cell lines. We conclude by finding that a particular class of TP53 mutations (missense changes) adversely impacted cancer-specific survival in EAC. HNF1alpha, a new EAC-mutated gene, was found, but more research is required to fully understand its function as a tumor suppressor gene.

Structural assessment of single amino acid mutations: application to TP53 function.

Single amino acid substitution is the type of protein alteration most related to human diseases. Current studies seek primarily to distinguish neutral mutations from harmful ones. Very few methods offer an explanation of the final prediction result in terms of the probable structural or functional effect on the protein. In this study, we describe the use of three novel parameters to identify experimentally-verified critical residues of the TP53 protein (p53). The first two parameters make use of a surface clustering method to calculate the protein surface area of highly conserved regions or regions with high nonlocal atomic interaction energy (ANOLEA) score. These parameters help identify important functional regions on the surface of a protein. The last parameter involves the use of a new method for pseudobinding free-energy estimation to specifically probe the importance of residue side-chains to the stability of protein fold. A decision tree was designed to optimally combine these three parameters. The result was compared to the functional data stored in the International Agency for Research on Cancer (IARC) TP53 mutation database. The final prediction achieved a prediction accuracy of 70% and a Matthews correlation coefficient of 0.45. It also showed a high specificity of 91.8%. Mutations in the 85 correctly identified important residues represented 81.7% of the total mutations recorded in the database. In addition, the method was able to correctly assign a probable functional or structural role to the residues. Such information could be critical for the interpretation and prediction of the effect of missense mutations, as it not only provided the fundamental explanation of the observed effect, but also helped design the most appropriate laboratory experiment to verify the prediction results.

DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene Mutations and Polymorphisms of TP53 and FHIT in Chronic Esophagitis and Esophageal Carcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genomic profile of a Li-Fraumeni-like syndrome patient with a 45,X/46,XX karyotype, presenting neither mutations in TP53 nor clinical stigmata of Turner syndrome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies

Background: Mutations in TP53 are common events during carcinogenesis. In addition to gene mutations, several reports have focused on TP53 polymorphisms as risk factors for malignant disease. Many studies have highlighted that the status of the TP53 codon 72 polymorphism could influence cancer susceptibility. However, the results have been inconsistent and various methodological features can contribute to departures from Hardy-Weinberg equilibrium, a condition that may influence the disease risk estimates. The most widely accepted method of detecting genotyping error is to confirm genotypes by sequencing and/or via a separate method. Results: We developed two new genotyping methods for TP53 codon 72 polymorphism detection: Denaturing High Performance Liquid Chromatography (DHPLC) and Dot Blot hybridization. These methods were compared with Restriction Fragment Length Polymorphism (RFLP) using two different restriction enzymes. We observed high agreement among all methodologies assayed. Dot-blot hybridization and DHPLC results were more highly concordant with each other than when either of these methods was compared with RFLP. Conclusions: Although variations may occur, our results indicate that DHPLC and Dot Blot hybridization can be used as reliable screening methods for TP53 codon 72 polymorphism detection, especially in molecular epidemiologic studies, where high throughput methodologies are required.

Arg72Pro TP53 polymorphism and cancer susceptibility: a comprehensive meta-analysis of 302 case-control studies

Arg72Pro is a common polymorphism in TP53, showing differences in its biological functions. Case-control studies have been performed to elucidate the role of Arg72Pro in cancer, although the results are conflicting and heterogeneous. Here, we analyzed pooled data from case-control studies to determine the role of Arg72Pro in different cancer sites. We performed a systematic review and meta-analysis of 302 case-control studies that analyzed Arg72Pro in cancer susceptibility. Odds ratios were estimated for different tumor sites using distinct genetic models, and the heterogeneity between studies was explored using I(2) values and meta-regression. We adopted quality criteria to classify the studies. Subgroup analyses were done for tumor sites according to ethnicity, histological, and anatomical sites. Results indicated that Arg72Pro is associated with higher susceptibility to cancer in some tumor sites, mainly hepatocarcinoma. For some tumor sites, quality of studies was associated with the size of genetic association, mainly in cervical, head and neck, gastric, and lung cancer. However, study quality did not explain the observed heterogeneity substantially. Meta-regression showed that ethnicity, allelic frequency and genotyping method were responsible for a substantial part of the heterogeneity observed. Our results suggest ethnicity and histological and anatomical sites may modulate the penetrance of Arg72Pro in cancer susceptibility. This meta-analysis denotes the importance for more studies with good quality and that the covariates responsible for heterogeneity should be controlled to obtain a more conclusive response about the function of Arg72Pro in cancer.

Expression of genes related to apoptosis, cell cycle and signaling pathways are independent of TP53 status in urinary bladder cancer cells

Urinary bladder cancer is the fourth most common malignancy in the Western world. Transitional cell carcinoma (TCC) is the most common subtype, accounting for about 90% of all bladder cancers. The TP53 gene plays an essential role in the regulation of the cell cycle and apoptosis and therefore contributes to cellular transformation and malignancy; however, little is known about the differential gene expression patterns in human tumors that present with the wild-type or mutated TP53 gene. Therefore, because gene profiling can provide new insights into the molecular biology of bladder cancer, the present study aimed to compare the molecular profiles of bladder cancer cell lines with different TP53 alleles, including the wild type (RT4) and two mutants (5637, with mutations in codons 280 and 72; and T24, a TP53 allele encoding an in-frame deletion of tyrosine 126). Unsupervised hierarchical clustering and gene networks were constructed based on data generated by cDNA microarrays using mRNA from the three cell lines. Differentially expressed genes related to the cell cycle, cell division, cell death, and cell proliferation were observed in the three cell lines. However, the cDNA microarray data did not cluster cell lines based on their TP53 allele. The gene profiles of the RT4 cells were more similar to those of T24 than to those of the 5637 cells. While the deregulation of both the cell cycle and the apoptotic pathways was particularly related to TCC, these alterations were not associated with the TP53 status.

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Currently, the combination of cisplatin and gemcitabine is considered a standard chemotherapeutic protocol for bladder cancer. However, the mechanism by which these drugs act on tumor cells is not completely understood. The aim of the present study was to investigate the effects of these two antineoplastic drugs on the apoptotic index and cell cycle kinetics of urinary bladder transitional carcinoma cell lines with wild-type or mutant TP53 (RT4: wild type for TP53; 5637 and T24: mutated TP53). Cytotoxicity, cell survival assays, clonogenic survival assays and flow cytometric analyses for cell cycle kinetics and apoptosis detection were performed with three cell lines treated with different concentrations of cisplatin and gemcitabine. G(1) cell cycle arrest was observed in the three cell lines after treatment with gemcitabine and gemcitabine plus cisplatin. A significant increase in cell death was also detected in all cell lines treated with cisplatin or gemcitabine. Lower survival rates occurred with the combined drug protocol independent of TP53 status. TP53-wild type cells (RT4) were more sensitive to apoptosis than were mutated TP53 cells when treated with cisplatin or gemcitabine. Concurrent treatment with cisplatin and gemcitabine was more effective on transitional carcinoma cell lines than either drug alone; the drug combination led to a decreased cell survival that was independent of TP53 status. Therefore, the synergy between low concentrations of cisplatin and gemcitabine may have clinical relevance, as high concentrations of each individual drug are toxic to whole organisms.

Ancestry of the Brazilian TP53 c.1010G>A (p.Arg337His, R337H) founder mutation : clues from haplotyping of short tandem repeats on Chromosome 17p

Rare germline mutations in TP53 (17p13.1) cause a highly penetrant predisposition to a specific spectrum of early cancers, defining the Li-Fraumeni Syndrome (LFS). A germline mutation at codon 337 (p.Arg337His, c1010G>A) is found in about 0.3% of the population of Southern Brazil. This mutation is associated with partially penetrant LFS traits and is found in the germline of patients with early cancers of the LFS spectrum unselected for familial his- tory. To characterize the extended haplotypes carrying the mutation, we have genotyped 9 short tandem repeats on chromosome 17p in 12 trios of Brazilian p.Arg337His carriers. Results confirm that all share a common ancestor haplotype of Caucasian/Portuguese-Ibe- ric origin, distant in about 72–84 generations (2000 years assuming a 25 years intergenera- tional distance) and thus pre-dating European migration to Brazil. So far, the founder p. Arg337His haplotype has not been detected outside Brazil, with the exception of two resi- dents of Portugal, one of them of Brazilian origin. On the other hand, increased meiotic recombination in p.Arg337His carriers may account for higher than expected haplotype diversity. Further studies comparing haplotypes in populations of Brazil and of other areas of Portuguese migration are needed to understand the historical context of this mutation in Brazil.

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B.

BACKGROUND The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.