EGFR nuclear import in gallbladder carcinoma: Nuclear phosphorylated EGFR upregulates iNOS expression and confers independent prognostic impact

Background: The understanding of epidermal growth factor receptor (EGFR) deregulation in carcinogenesis remains incomplete. We investigated the implications of EGFR gene status and EGFR nuclear translocation in gallbladder carcinoma (GBCA). Methods: Subcellular localization of EGFR and phosphorylated EGFR (pEGFR) was analyzed by fractional immunoblotting and confocal immunofluorescence in GBCA cell lines. pEGFR binding to iNOS promoter was assessed by chromatin immunoprecipitation with iNOS promoter activity evaluated by luciferase assay. EGFR, pEGFR, and iNOS were immunohistochemically assessable for localization and level in the training set of 104 GBCAs on tissue microarrays, with 76 cases analyzed for EGFR gene by chromogenic in situ hybridization (CISH) and mutant-enriched PCR targeting exons 19 and 21. The prognostic impact of nuclear pEGFR (N-pEGFR) immunoexpression was reaffirmed on whole sections of 58 GBCAs in the test set. Results: Nuclear expression of EGFR and pEGFR was substantiated in vitro with augmented activity of iNOS promoter elicited by pEGFR binding upon EGF treatment. Despite no mutation, EGFR amplification, identified in 11 cases (15%) by CISH, strongly correlated with cytoplasmic EGFR expression (P < 0.001) but not with disease-specific survival (DSS). Immunoexpression of nuclear EGFR (N-EGFR), cytoplasmic pEGFR, and N-pEGFR was strongly related to that of iNOS (all ≤0.005). N-pEGFR independently predicted worse DSS in both training (P = 0.0468, HR = 2.024) and test sets (P = 0.0223, HR = 5.573). Conclusions: N-EGFR and N-pEGFR express in GBCA, conferring clinical aggressiveness partly through iNOS transactivation. Lacking response-predicting mutation, EGFR gene status, albeit amplified in 15% of GBCA, is neither related to nuclear EGFR translocation nor prognostically useful.