Role of the TGFβ-NOX4 axis in the liver tumour microenvironment

Abstract

[eng] HYPOTHESIS: Considering that TGF-β is the main regulator of NOX4 in hepatocytes and that its levels are low in an important percentage of HCC patients, we hypothesized that NOX4 could be relevant for the response to TGF-β as a tumour suppressor factor. In addition, we hypothesized that the expression of NOX4, either in the tumour or in the stroma cell, could influence tumour progression, exerting actions on the tumour cell but also on the tumour microenvironment. OBJECTIVES: Objective 1. Characterization of the role of NOX4 in HCC tumour cells. 1.1 Generation of loss (CRISPR-Cas9 technology) of NOX4 function in human HCC cells. 1.2 Response to TGF-β in terms of cell proliferation and apoptosis in the new generated HCC cell lines. Molecular mechanisms involved. 1.3 Collection of the HCC samples. Analysis of the translational relevance of the findings in biopsies from HCC patients in tumoral and non-tumoral areas. Gene expression analysis. 1.4 Is the TGF-β-NOX4 axis regulating TGF-β-induced pro-tumorigenic actions? Objective 2. Role of NOX4 on liver tumorigenic progression in experimental animal models of HCC 2.1 Generation of DEN-induced hepatocarcinogenesis mice model. Design of new models of HCC induction in mice. 2.2 Macroscopic and microscopic analysis of the tumoral lesions: histologic analysis, immunohistochemistry. Detailed analysis of the effects on tumour and stromal cells. Gene and protein expression analysis. Objective 3. Impact of NOX4 in the interactome between HCC cells and immune system cells. 3.1 Set up of 3D spheroid model with HCC cells, for monoculture and multicellular culture. 3.2 NOX4 role in the interactome between tumour and immune cells. Effect of Conditioned Media from HCC cells on recruitment and phenotype of immune cells.