Usefulness of bile as a biomarker via ferroptosis and cysteine prenylation in cholangiocarcinoma; role of diagnosis and differentiation from benign biliary disease

Background: Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract with a poor prognosis. Herein, we investigated possible mechanism of extrahepatic CCA (eCCA) by dysregulated iron metabolism and post-translational modifications (PTMs). Further, we evaluated potential biomarkers in the bile fluid for diagnosis of eCCA and differentiation between eCCA and benign biliary disease.

Methods: From August 2018 to April 2019, we obtained bile fluids from 46 patients; 28 patients with eCCA (eCCA group) and 18 patients with common bile duct stone (Control group) via percutaneous transhepatic biliary drainage. We examined the levels of reduced glutathione (GSH), peroxide, ferrous iron [Fe⁺²], glutathione peroxidase (GP_X) and farnesyl transferase/geranylgeranyl transferase type-1 subunit alpha (FNTA) concentration in bile fluids to clarify the mechanism of ferroptosis and prenylation.

Results: The remarkable difference of PTMs was that FNTA which means prenylated cysteine as regulator was significantly decreased in eCCA than that of control. In addition, level of GSH, peroxide, GP_X and ferrous iron [Fe⁺²] were significantly depleted in eCCA than control. These results demonstrate that PTM, dysregulated iron metabolism and GP_X-regulated ferroptosis with GSH depletion through cysteine modification in bile are possible mechanisms of eCCA. Liquid Chromatography (LC)-Mass Spectrometry (MS) analysis, several oncogenic pathways including MYC target, apoptosis, fatty acid metabolism, P53 and mTORC1 were enriched in eCCA.

Conclusions: In conclusion, redox-dependent modification of cysteine and ferroptosis in bile fluids are possible mechanisms of eCCA. Several protein and oncogenic pathways related to PTM which are seen in eCCA tissues were also enriched in bile fluids. It suggests that bile fluid represents the oncogenic characteristics of eCCA tissues. Therefore, bile fluids have a role of a biomarker for diagnosis in eCCA, especially, differentiation of eCCA from benign biliary stricture.

Keywords: Biliary system; Cholangiocarcinoma (CCA); Ferroptosis; Post-translational modification (PTM); Prenylated cysteine.