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.

Materials and 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.