title: A novel p53 mutant found in iatrogenic urothelial cancers is dysfunctional and can be rescued by a second-site global suppressor mutation.
abstract: Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A→T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.
authors.affiliation: Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, United Kingdom. Electronic address: a.f.odell@leeds.ac.uk.
authors.forename: Adam F
authors.initials: AF
authors.lastname: Odell
authors.affiliation: Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, 75123 Uppsala, Sweden.
authors.forename: Luke R
authors.initials: LR
authors.lastname: Odell
authors.affiliation: Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Jon M
authors.initials: JM
authors.lastname: Askham
authors.affiliation: Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, 75123 Uppsala, Sweden.
authors.forename: Hiba
authors.initials: H
authors.lastname: Alogheli
authors.affiliation: School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Sreenivasan
authors.initials: S
authors.lastname: Ponnambalam
authors.affiliation: Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, United Kingdom; Department C016, German Cancer Research Centre, 69120 Heidelberg, Germany. Electronic address: m.hollstein@dkfz.de.
authors.forename: Monica
authors.initials: M
authors.lastname: Hollstein
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D034341:Aristolochic Acids / Q000009:adverse effects* / Q000494:pharmacology; D014408:Biomarkers, Tumor; D002461:Cell Line, Transformed; D006801:Humans; D007049:Iatrogenic Disease; D051379:Mice; D009153:Mutagens / Q000009:adverse effects* / Q000494:pharmacology; D020125:Mutation, Missense; D028321:Plant Preparations / Q000009:adverse effects* / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism; D014523:Urethral Neoplasms / Q000139:chemically induced* / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019459:Urothelium / Q000378:metabolism / Q000473:pathology
chemical_list: D034341:Aristolochic Acids; D014408:Biomarkers, Tumor; D009153:Mutagens; D028321:Plant Preparations; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C000228:aristolochic acid I
keywords: Biomarkers; Cancer Biology; Cellular Senescence; Molecular Cell Biology; Mouse Genetics; Mutagenesis Mechanisms; Site-specific Recombination; Suppressor Mutations; Tumor Suppressor Gene