The PI3K/AKT pathway is considered to play a major role in bladder carcinogenesis, but its relationships with other molecular alterations observed in bladder cancer remain unknown. We investigated PI3K/AKT pathway activation in a series of human bladder urothelial carcinomas (UC) according to PTEN expression, PTEN deletions and FGFR3, PIK3CA, KRAS, HRAS, NRAS and TP53 gene mutations. The series included 6 normal bladder urothelial samples and 129 UC (Ta n 5 25, T1 n 5 34, T2-T3-T4 n 5 70). Expression of phospho-AKT (pAKT), phospho-S6-Ribosomal Protein (pS6) (one downstream effector of PI3K/AKT pathway) and PTEN was evaluated by reverse phase protein Array. Expression of miR-21, miR-19a and miR-222, known to regulate PTEN expression, was also evaluated. pAKT expression levels were higher in tumors than in normal urothelium (p < 0.01), regardless of stage and showed a weak and positive correlation with pS6 (Spearman coefficient R S 5 0.26; p 5 0.002). No association was observed between pAKT or pS6 expression and the gene mutations studied. PTEN expression was decreased in PTEN-deleted tumors, and in T1 (p 5 0.0089) and T2-T3-T4 (p < 0.001) tumors compared to Ta tumors; it was also negatively correlated with miR-19a (R S 5 20.50; p 5 0.0088) and miR-222 (R S 5 20.48; p 5 0.0132), but not miR-21 (R S 5 20.27; p 5 0.18) expression. pAKT and PTEN expressions were not negatively correlated, and, on the opposite, a positive and moderate correlation was observed in Ta (R S 5 0.54; p 5 0.0056) and T1 (R S 5 0.56; p 5 0.0006) tumors. Our study suggests that PI3K/AKT pathway activation occurs in the entire spectrum of bladder UC regardless of stage or known most frequent molecular alterations, and independently of low PTEN expression.Urothelial carcinoma of the bladder (UC) is the sixth most common cancer among both sexes in Western countries, with about 200,000 new cases diagnosed each year. 1 It most often affects men (sex ratio: 3/1) and its incidence increases with age, peaking between the sixth and seventh decades. 2 Tobacco smoking is the most important risk factor. 2 The two major groups of UC are nonmuscle-and muscle-invasive UCs. 3 Nonmuscle-invasive UCs comprise carcinoma in situ (CIS), and Ta and T1 tumors. Tumors classified as Ta are papillary, noninvasive, mostly low grade, frequently recur (50-70%) and may rarely and unpredictably progress to stage T1 (lamina propria invaded) and then to muscle-invasive (stages T2, T3 or T4) tumors (5-10%). 3,4 CIS (stage Tis) is a flat lesion, always of high grade, that evolves to invasive carcinoma in about 50% of cases, and is considered to be the main precursor of muscle-invasive UC. 2,3 Locally advanced and/or metastatic UC is associated with a poor prognosis and no truly effective systemic therapies are available. 2,3 These two carcinogenesis pathways, 5 the low-grade Ta and CIS pathways, are supported by molecular studies. 5,6 Fibroblast growth factor receptor 3 (FGFR3), which encodes a tyrosine kinase-linked cell surface receptor, is mutated in up to 80% of low-grade ...

Epigenetic modification is one of the mechanisms leading to gene silencing in neoplastic cells. By methylation-specific PCR, we analyzed the promoter methylation of three cancer-related genes: Ras Association domain Family 1A (RASSF1A), Death Associated Protein kinase (DAP-kinase) and Retinoic Acid Receptor β2 (RARβ2) in two NPC xenografts (C15 and C17), 68 primary NPC tumors, and 9 normal nasopharyngeal epithelia. We showed that C15 and C17 displayed a complete promoter methylation of RASSF1A, RARβ2 and DAP-kinase genes. In primary NPC tumors, the incidence of promoter methylation was very high for all three tested genes: 91% for RASSF1A, 88% for both RARβ2 and DAP-kinase whereas all normal nasopharyngeal epithelia were unmethylated. Interestingly, our study revealed that aberrant promoter methylation of the three genes were statistically associated with the lymph node involvement (p < 0.0001). In addition, hypermethylation of RASSF1A was correlated with age at diagnosis (p = 0.047) and T stage (p = 0.037) while the RARβ2 hypermethylation was associated with histological type (p = 0.011). Taken together, our results demonstrate that silencing of RASSF1A and RARβ2 expression by promoter hypermethylation is associated with highly differentiated tumors, advanced tumor stage and the presence of lymph node metastasis.To assess the functional significance of the epigenetic silencing of RARβ2 and DAP-kinase in NPC, we analysed the expression of two downstream target genes COX-2 and p53 by reverse PCR (RT-PCR) and immunohistochemistry (IHC). We revealed a significant association between expression of COX-2 and loss of RARβ2 through aberrant methylation (p = 0.003) in NPC biopsies.We concluded that the inactivation of RASSF1A, RARβ2 and DAP-Kinase by hypermethylation is a key step in NPC tumorigenesis and progression.