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

BackgroundThe 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 MethodsIn 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.ResultsWe 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.ConclusionsOur 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.

Risk Stratification of Early Stage Oral Tongue Cancers Based on HPV Status and p16 Immunoexpression

“…Previous studies have shown p16 expression independent of HPV infection to be associated with epithelial to mesenchymal transitions mediated by MAPK pathway (Steinestel et al, 2013). Our report is also similar to recent studies showing the possibility of p16 overexpression without HPV presence in tumours concluding that it need not always be interpreted as a defacto HPV marker (Alexander et al, 2014;Kim et al, 2014).…”

Rho GTPases RhoA and Rac1 Mediate Effects of Dietary Folate on Metastatic Potential of A549 Cancer Cells through the Control of Cofilin Phosphorylation

“…Numerous cellular pathways involved in migration depend on the nucleotide supply and methylation processes, which require folate. For example, in rodents, the depletion of folate leads to the hypermethylation and inactivation of the p16 gene (46), a tumor suppressor implicated in EMT and metastasis (47,48). In line with the above assumption, a positive correlation between the migratory ability of cancer cells and folate supplementation as well as the inhibitory effect on such ability of antifolates has been demonstrated (49,50).…”

Low prevalence of HPV detection and genotyping in non-muscle invasive bladder cancer using single-step PCR followed by reverse line blot

“…Interestingly, p16INK4a overexpression is common in different histological subtypes of bladder cancer including adenocarcinoma (67 %), urothelial carcinoma with squamous differentiation (45.7 %), high-grade urothelial cancers (85.9 %) and urothelial CIS (92.6 %). However, contrary to gynecological tumors, p16INK4a is not a surrogate marker for evidence of HPV infection in bladder cancer [6,[16][17][18].…”