Pia Herbolsheimer

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Identification of New Tumor Suppressor Genes
in Breast and Prostate Cancer
Pia Herbolsheimer (formerly Huusko), MD,
PhD Georgetown University Hospital
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TSG inactivation- Two-hit hypothesis

• Biallelic inactivation is typical for tumor
  suppressor genes

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Comparative Genomic Hybridization (CGH) on cDNA
microarray
Tumor DNA
Normal DNA
Pollack et al., Nat Genet. 1999 Monni et al.,
PNAS. 2002 Hyman et al., Cancer Res. 2002
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Amplifications Activated oncogenic
genes Deletions Inactivated (tumor suppressor)
genes
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Nonsense mediated RNA decay (NMD)

• Premature termination codons (nonsense mutations)
  result in truncated transcripts
• NMD pathway monitors and destroys defective
  transcripts
• Blocking NMD results in the accumulation of
  truncated transcripts
• ?A method to screen for mutated transcripts?

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NMD microarrays
Noensie et al Nature Biotech, 2001
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Our approach
1 CGH to identify deleted regions in cancer
genome
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Our approach 2 NMD to find mutated transcripts
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Mining NMD microarray data from prostate cancer
cell lines to identify new mutated genes

• Mean NMD-ratio
• Normalized NMD-ratio (cancer cell lines vs.
  normal cell lines)
• Targeting deleted regions (microarray CGH)
• Gene ontology annotation (gene function)
• 36 genes were selected for mutational screening

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Candidate gene EphB2
43/16 000 based on mean NMD ratio
With CGH data ranked among top ten
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Sequence validation of EphB2 mutation in DU145

• Homozygous mutation Gln723Stop in DU145 cell
  line
• Mutation creates a stop codon and truncates the
  protein
• The other allele is deleted ? No functional
  protein

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Mutations in clinical tumors-approximately 10
of prostate tumors had EphB2 mutation
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Future Directions

• Identification of TSGs in breast cancer using NMD
  microarray technique
• Determine the role of EphB2 in other cancer types
• Search for other mechanisms for biallelic
  inactivation (methylation)

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Breast Cancer Candidate Genes

• Further prioritization by comparing candidate
  genes to expression data on all human genes,
  looking for genes with loss of expression
• Gene ontology
• First round of mutation analysis on cell lines,
  and if nonsense mutations found, then further
  validation with clinical tumor DNA
• Laborious, expensive, risky

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EphB2 Mutations in Other Cancer Types

• Initial screening using only few tumors/cell
  lines per each cancer type
• If hits are found, then screen a larger tumor
  material

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Other Mechanisms of Biallelic Inactivation -
Methylation

• DNA methylation is an epigenetic event that
  alters gene expression by covalent addition of a
  methyl group
• Hypermethylation occurs at CpG sites
  (cytosine-phosphate-guanine sites) often found
  near gene promoter
• Hypermethylation of CpG sites inactivates the
  remaining wild type allele
• Identification of hypermethylation done by
  pyrosequencing

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Research Collaborators

• Georgetown University Hospital
• Robert Clarke
• Leena Hilakivi-Clarke
• Nancy Dawson
• Translational Genomics Research Institute
• Spyro Mousses
• VTT Technical Research Centre and Univ. Turku,
  Finland
• Maija Wolf
• Olli Kallioniemi