title: Nonsense mediated decay resistant mutations are a source of expressed mutant proteins in colon cancer cell lines with microsatellite instability.
abstract: BACKGROUND
  Frameshift mutations in microsatellite instability high (MSI-High) colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD), but nonsense transcripts with a cMS in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay (NMD). NMD-resistant transcripts are therefore a likely source of expressed mutant proteins in MSI-High tumours.
  
  
  METHODS
  Using antibodies to the conserved N-termini of predicted mutant proteins, we analysed MSI-High colorectal cancer cell lines for examples of naturally expressed mutant proteins arising from frameshift mutations in coding microsatellites (cMS) by immunoprecipitation and Western Blot experiments. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA) knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines.
  
  
  RESULTS
  Mutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK), but not NMD-sensitive transcripts (BAX, CASP5, MSH3). Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12) were found to be mutated in at least 5 of 11 (45%) of the MSI-High cell lines tested.
  
  
  CONCLUSIONS
  NMD-resistant transcripts can give rise to expressed mutant proteins in MSI-High colon cancer cells. If commonly expressed in primary MSI-High colon cancers, MSI-derived mutant proteins could be useful as cancer specific immunological targets in a vaccine targeting MSI-High colonic tumours.
authors.affiliation: Epithelial Biochemistry Laboratory, Ludwig Institute for Cancer Research, Melbourne Branch, Parkville, Victoria, Australia. David.Williams@austin.org.au
authors.forename: David S
authors.initials: DS
authors.lastname: Williams
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Bird
authors.forename: Robert N
authors.initials: RN
authors.lastname: Jorissen
authors.forename: Yen Lin
authors.initials: YL
authors.lastname: Yu
authors.forename: Francesca
authors.initials: F
authors.lastname: Walker
authors.forename: Franscesa
authors.initials: F
authors.lastname: Walker
authors.forename: Hui Hua
authors.initials: HH
authors.lastname: Zhang
authors.forename: Edouard C
authors.initials: EC
authors.lastname: Nice
authors.forename: Antony W
authors.initials: AW
authors.lastname: Burgess
mesh_terms: D045744:Cell Line, Tumor; D018389:Codon, Nonsense; D003110:Colonic Neoplasms / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D004260:DNA Repair; D018604:Epitope Mapping; D016368:Frameshift Mutation; D015972:Gene Expression Regulation, Neoplastic; D016678:Genome; D015234:HLA-A Antigens / Q000235:genetics*; D006801:Humans; D053842:Microsatellite Instability; D018895:Microsatellite Repeats; D009154:Mutation; D017434:Protein Structure, Tertiary
chemical_list: D018389:Codon, Nonsense; D015234:HLA-A Antigens