Role of HNF4a in Liver Regeneration, Liver Cancer Pathogenesis and Global Metabolism

Abstract

Liver is the central metabolic organ and performs many functions necessary for survival. Hepatocyte Nuclear Factor 4 alpha (HNF4a) is a nuclear receptor well characterized for its role in embryonic hepatocyte differentiation and maintenance of the adult hepatocyte phenotype. HNF4a regulates genes involved in many basic hepatocyte functions and deletion or decreased expression of HNF4a results in impaired liver function. More recently, HNF4a has been shown to exhibit tumor suppressor activity and deletion of HNF4a results in hepatocyte proliferation. The objectives of these studies were to characterize the role of hepatic HNF4a in hepatocyte proliferation and metabolism during liver regeneration and to harness HNF4a activity as a prognostic tool in liver cancer. The remarkable ability of liver to regenerate is possible due to the innate proliferative potential of quiescent adult hepatocytes. Knowing the role of HNF4a in maintaining hepatocyte function and suppressing hepatocyte proliferation, we hypothesized that HNF4a activity and expression may be adjusted to navigate hepatocytes between proliferative and quiescent states during liver regeneration. Using the partial hepatectomy model of liver regeneration, we identified decreased HNF4a activity in livers during the initiation of hepatocyte proliferation. Overexpression of HNF4a delayed hepatocyte proliferation after partial hepatectomy, and we identified Src-kinase as a potential regulator of HNF4a during the initiation of regeneration. Hepatocyte-specific deletion of HNF4a in mice (HNF4a-KO mice) resulted in 100% mortality after partial hepatectomy. RNA-Seq analysis of livers from these mice revealed significant dedifferentiated, and increased activation of proliferative and carcinogenic pathways. Our results show that HNF4a is required to prevent hepatic failure after liver regeneration, is a critical regulator of termination of regeneration and provide new understanding of HNF4a position in regenerative networks. Downregulation of HNF4a has been well recognized in the progression of liver disease. We hypothesized that HNF4a activity could be used in the diagnosis and prognosis of liver diseases. Under the assumption that HNF4a activity could best be quantified by its target gene transcription, we identified a set of 44 genes by selecting only those genes that were direct HNF4a targets which were differentially regulated between WT and HNF4a-KO livers in normal and in liver tumors. qPCR analysis was used to validate the signature in mouse models of HNF4a deletion and liver cancer. Using this method, we detected lower HNF4a activity in human cirrhotic samples when compared to HCC samples. While no correlation was observed between disease state and the HNF4a staining pattern in diseased human tissues, the signature was able to accurately classify the severity of samples based on HNF4a activity. Further in silico analysis using human datasets confirmed the signatures ability to diagnose the severity of disease according to HNF4a activity. Lastly, when this analysis was performed on a dataset of human cirrhotic samples, we observed increased survival in a subset of cirrhotic samples with high HNF4a activity as determined by the signature. The role of HNF4a in regulating carbohydrate and lipid metabolism has been well studied, but how it effects overall global metabolism is not known. We used indirect calorimetry to examine changes in energy expenditure and substrate utilization between WT and HNF4a-KO mice in fed, fasted, and high fat diet-fed conditions. We observed significant decreases in energy expenditure in HNF4a-KO mice during fed conditions which were exacerbated during high fat diet feeding. The changes were accompanied by significant adipose depletion and hypoglycemia. Our findings bring focus to the central role of liver in metabolism, which is largely regulated by hepatocyte HNF4a. Altogether, these studies indicate that hepatic HNF4a is a critical regulator of liver regeneration and global metabolism. Further, hepatic HNF4a activity can be used as a prognostic tool in chronic liver diseases such as cirrhosis and cancer.