Pierre Hainaut

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.

Somatic TP53 Mutations in the Era of Genome Sequencing

Amid the complexity of genetic alterations in human cancer, TP53 mutation appears as an almost invariant component, representing by far the most frequent genetic alteration overall. Compared with previous targeted sequencing studies, recent integrated genomics studies offer a less biased view of TP53 mutation patterns, revealing that >20% of mutations occur outside the DNA-binding domain. Among the 12 mutations representing each at least 1% of all mutations, five occur at residues directly involved in specific DNA binding, four affect the tertiary fold of the DNA-binding domain, and three are nonsense mutations, two of them in the carboxyl terminus. Significant mutations also occur in introns, affecting alternative splicing events or generating rearrangements (e.g., in intron 1 in sporadic osteosarcoma). In aggressive cancers, mutation is so common that it may not have prognostic value (all these cancers have impaired p53 function caused by mutation or by other mechanisms). In several other cancers, however, mutation makes a clear difference for prognostication, as, for example, in HER2-enriched breast cancers and in lung adenocarcinoma with EGFR mutations. Thus, the clinical significance of TP53 mutation is dependent on tumor subtype and context. Understanding the clinical impact of mutation will require integrating mutation-specific information (type, frequency, and predicted impact) with data on haplotypes and on loss of heterozygosity.

Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal form of cancer with few therapeutic options. We found that levels of the lysine methyltransferase SMYD2 (SET and MYND domain 2) are elevated in PDAC and that genetic and pharmacological inhibition of SMYD2 restricts PDAC growth. We further identified the stress response kinase MAPKAPK3 (MK3) as a new physiologic substrate of SMYD2 in PDAC cells. Inhibition of MAPKAPK3 impedes PDAC growth, identifying a potential new kinase target in PDAC. Finally, we show that inhibition of SMYD2 cooperates with standard chemotherapy to treat PDAC cells and tumors. These findings uncover a pivotal role for SMYD2 in promoting pancreatic cancer.

Effects of insulin, insulin-like growth factor-I (IGF-I) and progesterone on glucose and amino acid uptake in Xenopus laevis oocytes.

In Xenopus oocytes, insulin or IGF-I activated glucose transport and maturation, but not amino acid transport, as measured by the uptake of alanine. Glucose transporter identical or closely related to the mammalian erythroid/brain transporter (Glut-1/EGT) were found in oocyte membranes. The EC50 for stimulation of glucose uptake and of maturation were similar (1-5 x 10(-8) M for insulin and 2-8 x 10(-10) M for IGF-I), confirming that these effects were mediated through IGF-I receptors. Other agents, such as phorbol 12-myristate 13-acetate (TPA) (0.5 microM) and vanadate (2 mM) evoked only part of the insulin effect on glucose uptake (50% and 65%, respectively), without being additive to insulin. In contrast, progesterone at 1 microM, a potent inducer of maturation, inhibited at least partially the insulin-induced glucose uptake. Uptake of alanine and glucose was decreased after prolonged incubations (3-6 h) with agents that trigger maturation, and was dramatically reduced in oocytes that have undergone maturation (unfertilized eggs). Maturation is thus accompanied by a reduction in glucose and amino acid transports. These result further document the validity of Xenopus oocytes as a model to study insulin and IGF-I signalling.

Higher-than-expected population prevalence of potentially pathogenic germline TP53 variants in individuals unselected for cancer history

Li–Fraumeni syndrome (LFS) is an autosomal‐dominant cancer predisposition disorder associated with pathogenic germline variants in TP53, with a high penetrance over an individuals lifetime. The actual population prevalence of pathogenic germline TP53 mutations is still unclear, most likely due to biased selection of cancer affected families. The aim of this study was to estimate the population prevalence of potentially pathogenic TP53 exonic variants in three sequencing databases, totaling 63,983 unrelated individuals. Potential pathogenicity was defined using an original algorithm combining bioinformatic prediction tools, suggested clinical significance, and functional data. We identified 34 different potentially pathogenic TP53 variants in 131 out of 63,983 individuals (0.2%). Twenty‐eight (82%) of these variants fell within the DNA‐binding domain of TP53, with an enrichment for specific variants that were not previously identified as LFS mutation hotspots, such as the p.R290H and p.N235S variants. Our findings reveal that the population prevalence of potentially pathogenic TP53 variants may be up to 10 times higher than previously estimated from family‐based studies. These results point to the need for further studies aimed at evaluating cancer penetrance modifiers as well as the risk associated between cancer and rare TP53 variants.

Systems medicine disease maps: community-driven comprehensive representation of disease mechanisms

The development of computational approaches in systems biology has reached a state of maturity that allows their transition to systems medicine. Despite this progress, intuitive visualisation and context-dependent knowledge representation still present a major bottleneck. In this paper, we describe the Disease Maps Project, an effort towards a community-driven computationally readable comprehensive representation of disease mechanisms. We outline the key principles and the framework required for the success of this initiative, including use of best practices, standards and protocols. We apply a modular approach to ensure efficient sharing and reuse of resources for projects dedicated to specific diseases. Community-wide use of disease maps will accelerate the conduct of biomedical research and lead to new disease ontologies defined from mechanism-based disease endotypes rather than phenotypes.

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 history. 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-Iberic origin, distant in about 72–84 generations (2000 years assuming a 25 years intergenerational 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 residents 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.

Variable population prevalence estimates of germline TP53 variants: A gnomAD-based analysis

Reports of variable cancer penetrance in Li–Fraumeni syndrome (LFS) have raised questions regarding the prevalence of pathogenic germline TP53 variants. We previously reported higher‐than‐expected population prevalence estimates in sequencing databases composed of individuals unselected for cancer history. This study aimed to expand and further evaluate the prevalence of pathogenic and likely pathogenic germline TP53 variants in the gnomAD dataset (version r2.0.2, n = 138,632). Variants were selected and classified based on our previously published algorithm and compared with alternative estimates based on three different classification databases: ClinVar, HGMD, and the UMD_TP53 database. Conservative prevalence estimates of pathogenic and likely pathogenic TP53 variants were within the range of one carrier in 3,555–5,476 individuals. Less stringent classification increased the approximate prevalence to one carrier in every 400–865 individuals, mainly due to the inclusion of the controvertible p.N235S, p.V31I, and p.R290H variants. This study shows a higher‐than‐expected population prevalence of pathogenic and likely pathogenic germline TP53 variants even with the most conservative estimates. However, these estimates may not necessarily reflect the prevalence of the classical LFS phenotype, which is based upon family history of cancer. Comprehensive approaches are needed to better understand the interplay of germline TP53 variant classification, prevalence estimates, cancer penetrance, and LFS‐associated phenotype.

P1-09-07: Contribution of TP53 p.R337H Mutation to Breast Cancer Incidence in Brazil.

Background: The exact contribution of TP53 germline mutations, associated with Li Fraumeni Syndrome — LFS, to the overall burden of cancer is still only partially known. Studies in Southern and Southeastern Brazil have shown that a particular mutant, TP53 p.R337H, has incomplete penetrance and may be present in a significant number of subjects (estimated frequency at the populational level of 1:300 individuals). In an exploratory approach, the aim of this study is to assess the prevalence of the TP53 p.R337H mutation in women with breast cancer diagnosed before 46 and after 55 years of age, unselected for family history of cancer and resident in Southern and Southeastern Brazil. Methods: Formalin-fixed paraffin-embedded (FFPE) non-tumoral tissue of 521 women diagnosed with breast cancer between 2000 and 2010 in two pathology laboratories were obtained retrospectively and consecutively, and analyzed after anonimization. Genomic DNA was isolated with the QIAamp DNA Tissue Kit and genotyping performed by allelic discrimination using a TaqMan assay. Confirmation of all mutation-positive and a sample of mutation-negative cases was done by TP53 exon 10 sequencing. Results: Analysis of the first 299 cases identified the TP53 p.R337H mutation in the germline of 15 (5,0%) cases: 13/142 (9,2%) before 46 years and 2/157 (1,3%) diagnosed after 55 years. The p53 expression pattern assessed by immunohistochemistry in the breast tumors was not different between p.R337H mutation carriers and non-carriers. Conclusion: Preliminary analysis in a sample of women with breast cancer in Southern Brazil indicates that the germline TP53 p.R337H founder mutation is present in a high proportion of cases, especially those diagnosed at a young age. Germline TP53 mutations are considered rare, occurring in about 1:5000 individuals of the general population. The occurrence of this founder mutation at such a high frequency in a particular geographic region has important implications for disease management and cancer risk counseling for these patients and families. This mutation likely contributes to a significant proportion of the health burden associated with breast cancer in Southern Brazil. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-09-07.