title: Overexpression of p16(INK4a) in urothelial carcinoma in situ is a marker for MAPK-mediated epithelial-mesenchymal transition but is not related to human papillomavirus infection.
abstract: BACKGROUND
  The role of human papillomavirus (HPV) in bladder carcinogenesis remains controversial. Overexpression of p16(INK4a), a surrogate marker for infection with oncogenic HPV in other tumours, has been described for urothelial carcinoma in situ (UCIS). Our goal was therefore to evaluate whether overexpression of p16(INK4a) is associated with HPV infection and to identify mechanisms of p16(INK4a) upregulation in UCIS.
  
  
  METHODS
  In 60 tissue specimens from a total of 45 patients (UCIS and controls), we performed p16(INK4a) immunohistochemistry followed by detection and subclassification of HPV DNA. In a subset of samples, we tested for gene amplification of p16(INK4a) applying fluorescence in situ hybridization (FISH). RAS/MAPK signalling and epithelial-mesenchymal transition (EMT) was assessed using immunohistochemistry. Finally, we transfected urothelial carcinoma cells with KRAS and examined the expression of p16(INK4a) as well as markers of EMT.
  
  
  RESULTS
  We found overexpression of p16(INK4a) in 92.6% of UCIS and in all cervical intraepithelial neoplasia (CIN) controls. In contrast, we detected high-risk HPV DNA in 80% of CIN, but none in UCIS. There was no gene amplification of p16(INK4a). High levels of phosphorylated kinases and urokinase plasminogen activator (uPA) and loss of membraneous E-cadherin were detected in UCIS. KRAS transfection of urothelial carcinoma cells led to upregulation of p16(INK4a) and uPA accompanied by loss of E-cadherin that could be inhibited by application of the kinase-inhibitor Sorafenib.
  
  
  CONCLUSIONS
  Our results show that overexpression of p16(INK4a) in UCIS is neither associated with HPV infection nor p16(INK4a) gene amplification but is a consequence of enhanced RAS/MAPK signalling that promotes EMT, possibly due to Sorafenib-sensitive paracrine secretion of the EMT activator uPA. These findings might open a novel therapeutic option for localized but aggressive urothelial cancer.
authors.affiliation: Department of Urology, University of Ulm, Ulm, Germany.
authors.forename: Julie
authors.initials: J
authors.lastname: Steinestel
authors.forename: Marcus V
authors.initials: MV
authors.lastname: Cronauer
authors.forename: Johannes
authors.initials: J
authors.lastname: Müller
authors.forename: Andreas
authors.initials: A
authors.lastname: Al Ghazal
authors.forename: Peter
authors.initials: P
authors.lastname: Skowronek
authors.forename: Annette
authors.initials: A
authors.lastname: Arndt
authors.forename: Klaus
authors.initials: K
authors.lastname: Kraft
authors.forename: Mark
authors.initials: M
authors.lastname: Schrader
authors.forename: Andres J
authors.initials: AJ
authors.lastname: Schrader
authors.forename: Konrad
authors.initials: K
authors.lastname: Steinestel
mesh_terms: D000368:Aged; D002278:Carcinoma in Situ / Q000201:enzymology / Q000473:pathology* / Q000821:virology; D045744:Cell Line, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D027383:Papillomaviridae / Q000187:drug effects / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000201:enzymology / Q000473:pathology* / Q000821:virology; D010671:Phenylurea Compounds / Q000494:pharmacology; D000077157:Sorafenib; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D019459:Urothelium / Q000473:pathology* / Q000821:virology; D002583:Uterine Cervical Neoplasms / Q000201:enzymology / Q000473:pathology / Q000821:virology; D002578:Uterine Cervical Dysplasia / Q000201:enzymology / Q000473:pathology / Q000821:virology
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D010671:Phenylurea Compounds; D009536:Niacinamide; D000077157:Sorafenib; D020928:Mitogen-Activated Protein Kinases; D014568:Urokinase-Type Plasminogen Activator