Genetic alteration of Keap1 confers constitutive Nrf2 activation and resistance to chemotherapy in gallbladder cancer

Background & Aims: Biliary tract cancer (BTC) is a highly malignant tumor, and identification of effective therapeutic targets to improve prognosis is urgently required. Oncogenic activation of survival genes is important for cancer cells to overcome oxidative stresses induced by their microenvironments that include chronic inflammation or exposure to anticancer drugs. We attempted to examine whether deregulation of Nrf2, a master transcriptional factor of various cytoprotective genes against oxidative stress, plays a role in the carcinogenesis of BTC. Methods: We screened genetic alteration of Keap1, a negative regulator of Nrf2, in BTC including tumors originated from gallbladder and extra- and intrahepatic bile ducts. Functional analysis of cancer-related mutant Keap1 in Nrf2 repression and the association between Nrf2 activation and resistance to 5-fluorouracil (5-FU) were investigated. Results: Recurrent (in 1/11 cell lines and 6/53 primary tumors) Keap1 gene alterations were observed in BTC and were especially frequent (4/13, 30.7%) in gallbladder cancer (GBC). These alterations led to a considerable loss of Nrf2 repression activity, caused constitutive activation of Nrf2, and promoted cell proliferation. Down-regulation of Nrf2 activity by either Keap1 complementation or Nrf2 short interference RNA increased sensitivity to 5-FU in Keap1-altered BTC cells. Conclusions: Keap1 mutation occurs frequently in GBC. Aberrant Nrf2 activation provoked by Keap1 alteration is one of the molecular mechanisms for chemotherapeutic resistance in GBC and will be a novel therapeutic target as an enhancer of sensitivity to 5-FU-based regimens.