title: Comparative analysis of 14-3-3 isoform expression and epigenetic alterations in colorectal cancer.
abstract: BACKGROUND
  The 14-3-3 family is a group of intracellular proteins found in all eukaryotic organisms. Humans have seven isoforms that serve as scaffolds to promote interactions of regulatory phospho-proteins involved in many vital cellular processes and previous studies have shown that disturbances in native 14-3-3 levels can contribute significantly to the development of various cancers.
  
  
  METHODS
  DNA and RNA was extracted from frozen tissue samples collected by the Human Cooperative Tissue Network. RNA samples were reverse transcribed and subjected to qRT-PCR analysis using fluorescently labelled probes. Genomic DNA was treated with bisulfite and cloned into bacterial vectors for subsequent high-resolution sequencing. Mammalian NIH3T3 cells were transformed with 14-3-3 eta and Ras expression vectors synthesized from cDNA. Colonies were counted and transforming capability assessed after 21 days of growth. Cell lysates were analyzed by western blot to verify protein expression.
  
  
  RESULTS
  Here we examined normal and cancerous 14-3-3 expression levels of all seven isoforms in a cohort of sporadic colorectal adenocarcinomas and in a group of tumors and their matched normals using qRT-PCR analysis. We found a statistically significant decrease in the levels of 14-3-3 sigma, eta, and zeta observed among adenocarcinomas compared to normal tissue. A parallel analysis of microarray data from the TCGA dataset confirmed that expression of sigma and eta were down-regulated in colon tumors. To explore the mechanisms behind 14-3-3 expression changes, we examined the methylation status of the sigma, eta, and zeta gene promoters in selected samples. Our data identified novel CpG methylation sites in the eta promoter consistent with epigenetic silencing of both 14-3-3 sigma and eta isoforms during colon tumorigenesis. Because epigenetic silencing is the hallmark of a tumor suppressor we tested eta in focus formation assays and found that it is capable of suppressing ras-induced transformation of NIH3T3 cells.
  
  
  CONCLUSIONS
  To our knowledge, this is the first study to identify the 14-3-3 eta gene as a tumor suppressor and that its expression is suppressed in colon tumors by DNA hypermethylation. These data suggest a link between 14-3-3 expression levels and the development of colon cancers.
authors.affiliation: Undergraduate Biomedical Research Program, University of Arizona Cancer Center, 1515 N. Campbell Ave, Tucson, 85724, Arizona, USA. gyoung@email.arizona.edu.
authors.forename: Gavin M
authors.initials: GM
authors.lastname: Young
authors.affiliation: Department of Pediatrics, Steele Children's Research Center, University of Arizona Cancer Center, 1515 N. Campbell Ave, Tucson, 85724, Arizona, USA. vijaymr@email.airzona.edu.
authors.forename: Vijayababu M
authors.initials: VM
authors.lastname: Radhakrishnan
authors.affiliation: Cancer Biology Graduate Interdisciplinary Program, University of Arizona Cancer Center, 1515 N. Campbell Ave, Tucson, 85724, Arizona, USA. smb4@email.arizona.edu.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Centuori
authors.affiliation: Cancer Biology Graduate Interdisciplinary Program, University of Arizona Cancer Center, 1515 N. Campbell Ave, Tucson, 85724, Arizona, USA. jrgomes07@email.arizona.edu.|University of Arizona Cancer Center, 1515 N. Campbell Ave, Tucson, 85724, Arizona, USA. jrgomes07@email.arizona.edu.
authors.forename: Cecil J
authors.initials: CJ
authors.lastname: Gomes
authors.affiliation: Department of Cell & Molecular Medicine, University of Arizona Cancer Center, 1515 N. Campbell Ave, Tucson, 85724, Arizona, USA. jmartinez@uacc.arizona.edu.|University of Arizona Cancer Center, 1515 N. Campbell Ave, Tucson, 85724, Arizona, USA. jmartinez@uacc.arizona.edu.
authors.forename: Jesse D
authors.initials: JD
authors.lastname: Martinez
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics*; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D005810:Multigene Family; D041681:NIH 3T3 Cells; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D055815:Young Adult
chemical_list: D048948:14-3-3 Proteins; D020033:Protein Isoforms