title: A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.
abstract: Dysregulated WNT signaling dominates adrenocortical malignancies. This study investigates whether silencing of the WNT negative regulator DKK3 (Dickkopf-related protein 3), an implicated adrenocortical differentiation marker and an established tumor suppressor in multiple cancers, allows dedifferentiation of the adrenal cortex.
  
  
  
  We analyzed the expression and regulation of DKK3 in human adrenocortical carcinoma (ACC) by qRT-PCR, immunofluorescence, promoter methylation assay, and copy number analysis. We also conducted functional studies on ACC cell lines, NCI-H295R and SW-13, using siRNAs and enforced DKK3 expression to test DKK3's role in blocking dedifferentiation of adrenal cortex.
  
  
  
  While robust expression was observed in normal adrenal cortex, DKK3 was down-regulated in the majority (>75%) of adrenocortical carcinomas (ACC) tested. Both genetic (gene copy loss) and epigenetic (promoter methylation) events were found to play significant roles in DKK3 down-regulation in ACCs. While NCI-H295R cells harboring β-catenin activating mutations failed to respond to DKK3 silencing, SW-13 cells showed increased motility and reduced clonal growth. Conversely, exogenously added DKK3 also increased motility of SW-13 cells without influencing their growth. Enforced over-expression of DKK3 in SW-13 cells resulted in slower cell growth by an extension of G1 phase, promoted survival of microcolonies, and resulted in significant impairment of migratory and invasive behaviors, largely attributable to modified cell adhesions and adhesion kinetics. DKK3-over-expressing cells also showed increased expression of Forkhead Box Protein O1 (FOXO1) transcription factor, RNAi silencing of which partially restored the migratory proficiency of cells without interfering with their viability.
  
  
  
  DKK3 suppression observed in ACCs and the effects of manipulation of DKK3 expression in ACC cell lines suggest a FOXO1-mediated differentiation-promoting role for DKK3 in the adrenal cortex, silencing of which may allow adrenocortical dedifferentiation and malignancy.
authors.affiliation: Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Joyce Y
authors.initials: JY
authors.lastname: Cheng
authors.affiliation: Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Taylor C
authors.initials: TC
authors.lastname: Brown
authors.affiliation: Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Murtha
authors.affiliation: Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, CCK, Stockholm, Sweden.
authors.forename: Adam
authors.initials: A
authors.lastname: Stenman
authors.affiliation: Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, CCK, Stockholm, Sweden.
authors.forename: C Christofer
authors.initials: CC
authors.lastname: Juhlin
authors.affiliation: Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, CCK, Stockholm, Sweden.
authors.forename: Catharina
authors.initials: C
authors.lastname: Larsson
authors.affiliation: Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Healy
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Manju L
authors.initials: ML
authors.lastname: Prasad
authors.affiliation: Department of Surgery, Medical School, Heinrich Heine University, University Hospital Düsseldorf, Düsseldorf, Germany.
authors.forename: Wolfram T
authors.initials: WT
authors.lastname: Knoefel
authors.affiliation: Department of Surgery, Medical School, Heinrich Heine University, University Hospital Düsseldorf, Düsseldorf, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Krieg
authors.affiliation: Department of Nephrology, Medical School, Heinrich Heine University, University Hospital Düsseldorf, Düsseldorf, Germany.
authors.forename: Ute I
authors.initials: UI
authors.lastname: Scholl
authors.affiliation: Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Reju
authors.initials: R
authors.lastname: Korah
authors.affiliation: Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT, USA. tobias.carling@yale.edu.|Department of Surgery, Yale University School of Medicine, 333 Cedar Street, FMB130A, New Haven, CT, 06520, USA. tobias.carling@yale.edu.
authors.forename: Tobias
authors.identifier: 0000-0002-1308-187X
authors.initials: T
authors.lastname: Carling
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000306:Adrenal Cortex Neoplasms / Q000235:genetics / Q000378:metabolism*; D000368:Aged; D002448:Cell Adhesion; D054337:Cell Dedifferentiation; D045744:Cell Line, Tumor; D002465:Cell Movement; D018925:Chemokines; D019175:DNA Methylation; D015536:Down-Regulation; D005260:Female; D000071161:Forkhead Box Protein O1 / Q000235:genetics* / Q000378:metabolism*; D018628:Gene Dosage; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D011401:Promoter Regions, Genetic
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018925:Chemokines; C498624:DKK3 protein, human; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D036341:Intercellular Signaling Peptides and Proteins
keywords: Adrenocortical carcinogenesis; DKK3; FOXO1