Overexpression of p16INK4a 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 p16INK4a, 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 p16INK4a is associated with HPV infection and to identify mechanisms of p16INK4a upregulation in UCIS. Materials and Methods: In 60 tissue specimens from a total of 45 patients (UCIS and controls), we performed p16INK4a immunohistochemistry followed by detection and subclassification of HPV DNA. In a subset of samples, we tested for gene amplification of p16INK4a 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 p16INK4a as well as markers of EMT. Results: We found overexpression of p16INK4a 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 p16INK4a. 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 p16INK4a 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 p16INK4a in UCIS is neither associated with HPV infection nor p16INK4a 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. © 2013 Steinestel et al.