title: The pseudokinase SgK223 promotes invasion of pancreatic ductal epithelial cells through JAK1/Stat3 signaling.
abstract: BACKGROUND
  Characterization of molecular mechanisms underpinning development of pancreatic ductal adenocarcinoma (PDAC) may lead to the identification of novel therapeutic targets and biomarkers. SgK223, also known as Pragmin, is a pseudokinase and scaffolding protein closely related to SgK269/PEAK1. Both proteins are implicated in oncogenic tyrosine kinase signaling, but their mechanisms and function remain poorly characterized.
  
  
  METHODS
  Expression of SgK223 in PDAC and PDAC cell lines was characterized using gene expression microarrays, mass spectrometry (MS)-based phosphoproteomics and Western blotting. SgK223 was overexpressed in human pancreatic ductal epithelial (HPDE) cells via retroviral transduction, and knocked down in PDAC cells using siRNA. Cell proliferation was determined using a colorimetric cell viability assay, and cell migration and invasion using transwells. Expression of markers of epithelial-mesenchyme transition (EMT) was assayed by quantitative PCR. SgK223 and Stat3 signaling was interrogated by immunoprecipitation, Western blot and gene reporter assays. The functional role of specific kinases and Stat3 was determined using selective small molecule inhibitors.
  
  
  RESULTS
  Elevated site-selective tyrosine phosphorylation of SgK223 was identified in subsets of PDAC cell lines, and increased expression of SgK223 detected in several PDAC cell lines compared to human pancreatic ductal epithelial (HPDE) cells and in PDACs compared to normal pancreas. Expression of SgK223 in HPDE cells at levels comparable to those in PDAC did not alter cell proliferation but led to a more elongated morphology, enhanced migration and invasion and induced gene expression changes characteristic of a partial EMT. While SgK223 overexpression did not affect activation of Erk or Akt, it led to increased Stat3 Tyr705 phosphorylation and Stat3 transcriptional activity, and SgK223 and Stat3 associated in vivo. SgK223-overexpressing cells exhibited increased JAK1 activation, and use of selective inhibitors determined that the increased Stat3 signaling driven by SgK223 was JAK-dependent. Pharmacological inhibition of Stat3 revealed that Stat3 activation was required for the enhanced motility and invasion of SgK223-overexpressing cells.
  
  
  CONCLUSIONS
  Increased expression of SgK223 occurs in PDAC, and overexpression of SgK223 in pancreatic ductal epithelial cells promotes acquisition of a migratory and invasive phenotype through enhanced JAK1/Stat3 signaling. This represents the first association of SgK223 with a particular human cancer, and links SgK223 with a major signaling pathway strongly implicated in PDAC progression.
authors.affiliation: Cancer Research Division, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia. carole.tactacan@gmail.com.|Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia. carole.tactacan@gmail.com.
authors.forename: Carole M
authors.initials: CM
authors.lastname: Tactacan
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia. yuwei.phua@monash.edu.
authors.forename: Yu Wei
authors.initials: YW
authors.lastname: Phua
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia. ling.liu@monash.edu.
authors.forename: Ling
authors.initials: L
authors.lastname: Liu
authors.affiliation: Cancer Research Division, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia. luxi.zhang@monash.edu.|Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia. luxi.zhang@monash.edu.
authors.forename: Luxi
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Cancer Research Division, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia. ehumphrey@biochem.mpg.de.
authors.forename: Emily S
authors.initials: ES
authors.lastname: Humphrey
authors.affiliation: Cancer Research Division, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia. m.cowley@garvan.org.au.
authors.forename: Mark
authors.initials: M
authors.lastname: Cowley
authors.affiliation: Cancer Research Division, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia. m.pinese@garvan.org.au.
authors.forename: Mark
authors.initials: M
authors.lastname: Pinese
authors.affiliation: Cancer Research Division, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia. andrew.biankin@glasgow.ac.uk.|Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Scotland, G61 1BD, UK. andrew.biankin@glasgow.ac.uk.
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Biankin
authors.affiliation: Cancer Research Division, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia. roger.daly@monash.edu.|Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia. roger.daly@monash.edu.|Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Level 1, Building 77, 23 Innovation Walk, Monash, VIC, 3800, Australia. roger.daly@monash.edu.
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Daly
mesh_terms: D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics* / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053613:Janus Kinase 1 / Q000096:biosynthesis / Q000235:genetics*; D009361:Neoplasm Invasiveness / Q000235:genetics; D010183:Pancreatic Ducts / Q000473:pathology; D050796:STAT3 Transcription Factor / Q000096:biosynthesis / Q000235:genetics*; D015398:Signal Transduction
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000607534:PRAG1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C507904:JAK1 protein, human; D053613:Janus Kinase 1