Hypermutability as a mechanism of carcinogenesis

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Hypermutability as a mechanism of carcinogenesis

• Todd Druley, MD/PhD

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Definition

• Hypermutability refers to an incidence of
  mutation that occurs at a higher rate than that
  of the surrounding DNA.
• The predicted mutation rate of DNA in the human
  genome is 1 x 10-9.
• In contrast, hypermutable codons in p53 undergo
  mutation at a rate of 1 x 10-5. Strauss,
  Mutation Res (2000) 457 93-104.

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Mechanisms

• Aberrant mismatch repair
• Aberrant CpG methylation
• Often occur together
• Other mechanisms

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Mismatch Repair

• Mismatch repair mechanism corrects small, but
  relatively frequent, nucleotide transcription
  errors
• Instrumental in maintaining DNA integrity
• Failure allows rapid accumulation of mutations

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Hereditary Non-Polyposis Colorectal Cancer

• A.K.A. Lynch Syndrome named after Henry Lynch, MD
  professor of medicine at Creighton School of
  Medicine.
• First report by Albert Warthin, MD, who
  documented the colon cancer of an affected woman
  in 1895.
• Lifetime risk of colorectal cancer is 80.
  Tumors are predominantly proximal to the splenic
  flexure, poorly differentiated, occur at a young
  age (mean 45 yrs), have a high mucin content,
  signet-rings, diploid DNA and high levels of
  microsatellite instability (MSI-H).
• High prevalence of second tumors mainly
  endometrial (LR 60), ovarian, biliary, renal,
  small bowel, gastric, urothelial and brain.

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Hereditary Non-Polyposis Colorectal Cancer

• Autosomal dominant inheritance of an aberrant
  mismatch repair allele.
• gt95 of clinical presentation due to mutations in
  MLH1, MSH2 and MSH6.
• Results in microsatellite An/Tn or CAn/GTn
  instability (MSI).
• MSI can also occur from CpG hypermethylation of
  MLH1 (non-heritable).

Lynch and de la Chapelle. NEJM (2003) 34810,
919-932.
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Methylation

• Methylation of cytosine (CpG) suppresses gene
  expression
• Global DNA hypomethylation
• Chromosomal instability1
• Increased tumorigenesis1
• Oncogene activation2
• Hypermethylation of promoter sequence will
  silence gene expression (e.g. tumor suppressor
  genes)3
• Methylation patterns are heritable and subject
  to imprinting

• Eden et al. Science (2003) 300 455.
• Nishigaki et al. Cancer Res (2005) 65 2115-2124.
• Jones and Baylin. Nature Rev Genet (2002) 3
  415-428.

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Methylation reaction
DNA methyltransferase
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Mismatch repair and methylation

• Deamination of cytosine occurs via the
  apolipoprotein B mRNA-editing enzyme, catalytic
  peptide (APOBEC) gene family.
• Deamination of 5-CH3cytosine results in thymine,
  which causes a T-G mismatch.
• Aberrant mismatch repair and/or increased
  deaminase activity could result in a combination
  of point mutations and/or diffuse hypomethylation.

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Hypermutability in the p53 gene

• P53 is frequently mutated. Over 20,000 mutations
  have been reported and are catalogued in the IARC
  p53 Mutation Database.
• Variable hotspots within exons 5-8 of p53 have
  been reported from a variety of different
  cellular backgrounds.
• CpG sites are the hottest of the hotspots and
  result in transitional point mutations. Such
  sites account for 35 of all p53 mutations.
  Rodin et al. J Mol Med (1998) 1 191-199.

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Silent mutation hotspots

• Strauss identified 18 silent mutation hotspots in
  exons 5-8 of p53 through the IARC p53 Mutation
  Database.
• Choosing silent mutations eliminates the argument
  that the hotspots were the product of positive
  selection.
• In contrast to silent mutations that occur
  outside of hotspots, statistical analysis
  demonstrated that silent mutations within
  hotspots are not distributed randomly. This
  suggests that these sites are preferentially
  mutated. Strauss,
  Mutation Res (2000) 457 93-104.

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Tissue specific hotspots

• Glaszko found that the p53 hotspot profile
  between tumors of different cellular origin were
  significantly different.
• Tumors from the same cellular origin at different
  stages of progression did not show a
  statistically significant profile of p53
  hotspots.
• Suggests that the cell origin of a tumor is a
  decisive factor in determining the location of
  p53 hotspots

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Tissue specific hotspots

• Glaszko et al concluded
• Solid tumors contain a higher number of hotspots
  compared to lymphomas.
• P53 hotspots were all located in highly conserved
  regions of the gene that were directly involved
  in sequence-specific DNA binding.
• A p53 hotspot profile is determined by a) a
  common and heritable set of germline hotspots and
  b) a tumor-specific set of CpG and non-CpG
  hotspots. Glaszko et al. Bioch et Bioph
  Acta (2004)1679 95-106

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Classic monoclonal model of tumorigenesis
Feinberg et al. Nat Rev Genet (2006) 7 21-33.
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Epigenetic progenitor model of tumorigenesis
1
2
3
Feinberg et al. Nat Rev Genet (2006) 7 21-33.
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Epigenetic progenitor model of tumorigenesis

• 1. Epigenetic disruption of progenitor cells
  resulting in a polyclonal population of
  neoplasia-prone precursor cells. This disruption
  is mediated by tumor-progenitor genes, which
  mediate epigenetic expansion of progenitor cells,
  increase predisposition to cancer and/or augment
  the cells capacity for pluripotency.
• Examples genes for cytosine deaminases,
  chromatin structure, transcription factors
  contributing to embryonic pluripotency, and
  others.
• Supported by five lines of evidence
• In vitro studies revealing reversibility of
  phenotype in leukemia and solid tumor
  development. Lotem and Sachs. Semin
  Cancer Biol (2002) 12 339-346.
• Global DNA methylation changes suggesting clonal
  derivation. Lorincz et al. Mol
  Cell Biol (2002) 22 7572-7580.
• Cloning an entire mouse from the nucleus of a
  murine melanoma cell. Hochedlinger et al.
  Genes Dev (2004) 18 1875-1885.
• Neoplastic clones can be maintained by a small
  population of cells with stem cell properties.
  Pardal et al. Nature Rev Cancer
  (2003) 3 895-902.
• Loss of imprinting (LOI) of IGF2 througout normal
  colonic epithelium in patients who have a
  LOI-associated colorectal cancer.
  Cui et al. Science (2003) 299
  1753-1755.

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Epigenetic progenitor model of tumorigenesis
1
2
3
Feinberg et al. Nat Rev Genet (2006) 7 21-33.
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Epigenetic progenitor model of tumorigenesis

• Initiating mutation within the subset of
  epigenetically-disrupted progenitor cells.
• Previously recognized as the first step in the
  monoclonal model.
• These mutations are specific for tumor type (e.g.
  APC for colorectal cancer BCR-ABL for CML
  etc.).
• Epigenetic alterations can substitute for
  mutation-induced oncogene activation and/or tumor
  suppressor gene silencing.

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Epigenetic progenitor model of tumorigenesis
1
2
3
Feinberg et al. Nat Rev Genet (2006) 7 21-33.
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Epigenetic progenitor model of tumorigenesis

• Genetic and epigenetic plasticity that allows for
  an evolving phenotype.
• Allows for properties such as invasion,
  metastasis and/or drug resistance to be
  expressed.
• Examples of genetic plasticity include
  chromosomal instability/rearrangement, telomere
  shortening or the accumulation of new mutations.
• Epigenetic plasticity is considered to be a
  heterogenous and transient phenomenon based on
  various factors such as the cellular milieu or
  the presence/absence/mutation of certain proteins
  involved in epigenetic processing.
• Examples of epigenetic plasticity include
  aberrant chromatin condensation, aberrant
  cell-to-cell interactions (b-catenin) or others.

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Potential impact on therapy

• Earlier detection
• Reversible phenotype
• Therapy targeted at the progenitor cell(s)
• Targeted gene activation and/or silencing
• Improved identification of at-risk persons

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Future Directions

• A detailed analysis of tumor-progenitor genes in
  human cancers.
• Determine if epigenetic alterations contribute to
  or result from hypermutable codons.
• Characterize the impact of tumor-specific
  hotspots on tumorigenesis and cellular phenotype.
• Attempt to identify in vivo epigenetically
  altered progenitor cells.