title: Activin signaling in microsatellite stable colon cancers is disrupted by a combination of genetic and epigenetic mechanisms.
abstract: BACKGROUND
  Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers.
  
  
  METHODS
  Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2'-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria.
  
  
  RESULTS
  Of 51 MSS colon tumors, 7 (14%) lost ACVR2, 2 (4%) ACVR1, and 5 (10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05).
  
  
  CONCLUSIONS
  Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, California, United States of America. bjung@ucsd.edu
authors.forename: Barbara
authors.initials: B
authors.lastname: Jung
authors.forename: Jessica
authors.initials: J
authors.lastname: Gomez
authors.forename: Eddy
authors.initials: E
authors.lastname: Chau
authors.forename: Jennifer
authors.initials: J
authors.lastname: Cabral
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Lee
authors.forename: Aimee
authors.initials: A
authors.lastname: Anselm
authors.forename: Przemyslaw
authors.initials: P
authors.lastname: Slowik
authors.forename: Deena
authors.initials: D
authors.lastname: Ream-Robinson
authors.forename: Karen
authors.initials: K
authors.lastname: Messer
authors.forename: Judith
authors.initials: J
authors.lastname: Sporn
authors.forename: Sung K
authors.initials: SK
authors.lastname: Shin
authors.forename: C Richard
authors.initials: CR
authors.lastname: Boland
authors.forename: Ajay
authors.initials: A
authors.lastname: Goel
authors.forename: John M
authors.initials: JM
authors.lastname: Carethers
mesh_terms: D030201:Activin Receptors, Type I / Q000235:genetics*; D030301:Activin Receptors, Type II / Q000235:genetics*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000145:classification / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019656:Loss of Heterozygosity / Q000235:genetics; D053842:Microsatellite Instability; D018895:Microsatellite Repeats / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D051899:Smad2 Protein / Q000235:genetics*; D014158:Transcription, Genetic; D047368:Tumor Burden / Q000235:genetics
chemical_list: C497782:SMAD2 protein, human; D051899:Smad2 Protein; C425423:ACVR1 protein, human; D030201:Activin Receptors, Type I; D030301:Activin Receptors, Type II; C425585:activin receptor type II-A