NOX4/NOX1 dual inhibition in cholangiocarcinoma as an effective approach to target TGF-beta protumorigenic actions in cancer associated fibroblasts

Transforming growth factor beta (TGF-ß) signalling has become an attractive therapeutic target, due to its pro-tumorigenic actions on epithelial cells and its immunosuppressive effects in the tumour microenvironment. In intrahepatic cholangiocarcinoma (iCCA), a highly aggressive malignancy of the biliary tract with poor prognosis, the latest clinical trials using TGF-ß inhibitors have failed indicating that the specific actions carried out by TGF-ß in iCCA are yet not well delineated. Here, we show that TGF-ß signalling is highly active in iCCA and exerts a prominent suppressor effect on tumour cells, that relies on a functional canonical SMAD2/3/4 signalling. Thus, TGF-ß inhibitors promote, instead of inhibit, tumour cell growth. NADPH oxidase 4 (NOX4), a downstream mediator of the TGF-ß signalling pathway, is strictly expressed in cancer-associated fibroblasts (CAF) of iCCA and acts in concert with NOX1 to regulate CAF functions. Using a dual NOX4/NOX1 inhibitor impaired CAF actions and reduced tumour growth in vitro and in vivo. Collectively, our findings reveal an actionable way to specifically target TGF-ß protumorigenic actions in CAF from iCCA without undesirable side effects on tumour cells. Overall design: To investigate the role of the TGF-ß signalling pathway in cholangiocarcinoma (CCA) tumour cells we exposed two CCA cell lines (HuCCT1 and RBE) to TGF-beta 1 (2 ng/ml) or a TGF-ß receptor I inhibitor, galunisertib (10 µM) for 16 hours and then we performed gene expression profiling analysis using RNAseq data. To investigate the effects of inhibiting the TGF-ß signalling pathway at different levels on liver myofibroblasts we exposed three cell lines of hepatic stellate cells (HSC) with different degree of activation (LX2-HSC