title: ROR2 is epigenetically inactivated in the early stages of colorectal neoplasia and is associated with proliferation and migration.
abstract: Colorectal cancer (CRC) is closely linked to Wnt signalling, with 94 % of cases exhibiting a Wnt related mutation. ROR2 is a receptor tyrosine kinase that is thought to repress β-catenin dependent Wnt signalling. Our study aims to determine if ROR2 is epigenetically silenced in CRC and determine if in vitro silencing of ROR2 potentiates Wnt signalling, and alters the proliferative, migratory or invasive potential of cells.
  
  
  
  ROR2 expression was examined in CRC cell lines and patient adenomas using qRT-PCR, while COBRA and bisulphite sequencing was used to analyse ROR2 promoter methylation. 258 patient primary tumour samples from publicly available databases were also examined for ROR2 expression and methylation. In addition, the functional effects of ROR2 modulation were investigated in HCT116 cells following ROR2 siRNA knockdown and in RKO and SW620 cells following ectopic ROR2 expression.
  
  
  
  Reduced ROR2 expression was found to correlate with ROR2 promoter hypermethylation in colorectal cancer cell lines, carcinomas and adenomas. ROR2 expression was downregulated in 76.7 % (23/30) of CRC cell lines with increasing ROR2 promoter hypermethylation correlating with progressively lower expression. Analysis of 239 primary tumour samples from a publicly available cohort also found a significant correlation between reduced ROR2 expression and increased promoter methylation. Methylation analysis of 88 adenomas and 47 normal mucosa samples found greater percentage of adenoma samples to be methylated. Additional analysis also revealed that adenoma samples with reduced ROR2 expression also possessed ROR2 promoter hypermethylation. ROR2 knockdown in the CRC cell line HCT116 significantly decreased expression of the β-catenin independent Wnt targets genes JNK and NFATC1, increased cellular proliferation and migration but decreased invasion. When ROR2 was ectopically expressed in RKO and SW620 cells, there was no significant change to either cellular proliferation or migration.
  
  
  
  ROR2 is frequently epigenetically inactivated by promoter hypermethylation in the early stages of colorectal neoplasia and this may contribute to colorectal cancer progression by increasing cellular proliferation and migration.
authors.affiliation: Metastasis Research Group, Adult Cancer Program, School of Women's and Children's Health, Lowy Cancer Research Centre, UNSW Australia, Sydney, NSW, 2052, Australia.
authors.forename: Sean S Q
authors.initials: SS
authors.lastname: Ma
authors.affiliation: Colorectal Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, UNSW Australia, Sydney, NSW, 2052, Australia.|Department of Biotechnology, Motilal Nehru National Institute of Technology Allahabad, Uttar Pradesh, 211004, India.
authors.forename: Sameer
authors.initials: S
authors.lastname: Srivastava
authors.affiliation: Metastasis Research Group, Adult Cancer Program, School of Women's and Children's Health, Lowy Cancer Research Centre, UNSW Australia, Sydney, NSW, 2052, Australia.
authors.forename: Estelle
authors.initials: E
authors.lastname: Llamosas
authors.affiliation: Mayne Medical School, University of Queensland, 288 Herston Road, Herston, Brisbane St Lucia, Qld, 4072, Australia.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Hawkins
authors.affiliation: Colorectal Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, UNSW Australia, Sydney, NSW, 2052, Australia.
authors.forename: Luke B
authors.initials: LB
authors.lastname: Hesson
authors.affiliation: Colorectal Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, UNSW Australia, Sydney, NSW, 2052, Australia.
authors.forename: Robyn L
authors.initials: RL
authors.lastname: Ward
authors.affiliation: Metastasis Research Group, Adult Cancer Program, School of Women's and Children's Health, Lowy Cancer Research Centre, UNSW Australia, Sydney, NSW, 2052, Australia. caroline.ford@unsw.edu.au.
authors.forename: Caroline E
authors.initials: CE
authors.lastname: Ford
mesh_terms: D000236:Adenoma / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics*; D049109:Cell Proliferation / Q000235:genetics*; D015331:Cohort Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics; D050778:NFATC Transcription Factors / Q000235:genetics; D009367:Neoplasm Staging; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D057050:Receptor Tyrosine Kinase-like Orphan Receptors / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; C078717:ROR2 protein, human; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D048031:JNK Mitogen-Activated Protein Kinases
keywords: Colorectal cancer; Epigenetic silencing; Hypermethylation; ROR2; Wnt