Increasing evidence showed that multiple tissue microenvironment cues could play a pivotal role in the behaviour of untransformed as well as in preneoplastic/neoplastic cells. This is particularly relevant in the liver, where the functionality of non-tumoral hepatocytes and the aggressiveness of hepatocellular carcinoma cells appear strictly related to the tissue fibrosis, a pathological background on which invariably cancer develops. An important feature of liver fibrosis is the presence of environmental stimuli, in particular TGFß and mechanical signals, able to promote processes directly involved in liver cell differentiation and tumor progression, primarly the Epithelial to Mesenchymal Transition. A common downstream target of the pathways starting from these signals is the transcriptional factor HNF4, master regulator of hepatocyte differentiation and tumor suppressor in hepatocellular carcinoma.
Aim of this project is to identify molecular and biochemical mechanisms leading to HNF4 inactivation by TGFß and substrate stiffness in the attempt to further dissect the molecular bases of liver cell differentiation and of hepatocyte transformation and liver tumor progression. In particular, starting from literature and our preliminary observations, we plan to study:
1) the mechanisms responsible for the TGFß-induced HNF4 inactivation by analysing the TGFß-induced post-translational modifications (PTMs) on HNF4 protein and the TGFß-dependent epigenetic reprogramming of HNF4 target gene promoters;
2) the mechanisms responsible for the high stiffness-induced inactivation of HNF4 by analysing the involvement of the mechano-trasducer YAP on HNF4 expression and function.
The dissection of molecular mechanisms controlling the response of liver cells to environment stimuli, in terms of differentiation and transformation, appears mandatory to identify new targets and to set up efficient therapeutic protocols for liver fibrosis and hepatocellular carcinoma.
The project proposes the study of the influences exerted by the environmental factors of fibrotic liver tissue on differentiation /transformation of liver cells. In the first section, in particular, we propose the investigation of the role played by the pleiotropic cytokine TGFß, known to be a pivotal and instructive element of the liver stem cell niche as well as of the fibrotic tissue and of the neoplastic tissue at all stages of its progression. In the second section, we propose the study of the mechanisms underlying the dedifferentiation of both normal and transformed hepatocytes undergone to the mechanical stress derived from the high stiffness of ECM. Starting from our and literature results, that showed the convergence of the signalling generated from both elements described above on the master factor of the hepatocyte differentiation, HNF4, our aim is to acquire new knowledge on the molecular mechanisms controlling the hepatocyte biology in physiological and pathological conditions. This knowledge, importantly, could permit the design of HNF4 mutants insensitive to the TGFß inactivating action or to identify new therapeutic targets, with the final aim of obtaining a full recovery of HNF4 tumor suppressor activity in a very "hostile" environment, such as the fibrotic tissue on which the tumor occurs. These aspects are particularly important in the management of HCC, one of the most common and aggressive cancers worldwide.
All in vitro procedures described in the experimental plan are currently in use in our lab. Moreover, for the accomplishment of proteomics experiments described in the below section, we will take advantage of having a proteomic unit fully equipped for the proposed experiments (MALDI TOF/TOF, Nano High Pressure Liquid Chromatographer, Probot MALDI Spotter, 2DE First dimension apparatus, Spot-picker, Typhoon scanner 9400) and a person with expertise in this field (Dr. Claudia Montaldo).