Reprogramming Of Hepatocellular Carcinoma By The Combination Of HNF1?, HNF4? And FOXA3

Hepatocellular carcinoma(HCC)is the sixth most common malignant tumor in the world and is the second only to lung cancer in mortality.Despite the enormous advances in the diagnosis and treatment of HCC during the past several decades,the prognosis of this deadly disease remains disappointing to date.Therefore,the strategy for HCC treatment needs to be adjusted effeciently.Differentiation therapy is a kind of treatment developped in recent decades,which induces the malignant cancer cell into a more benign phenotype,thus inhibiting the the proliferation and promoting the apoptosis of HCC cells so as to achieve the purpose of treating liver cancer.So far,the most effective differentiation therapy is the application of all-trans retinoic acid for the treatment of acute promyelocytic leukemia,which benefits a large number of patients.The success in leukemia differentiation treatment also promotes the research of differentiation treatment in solid tumors.Dimethyl sulfoxide and arsenic agent have been used for the treatment of breast cancer,lung cancer and colon cancer following the application of all-trans retinoic acid.But the application of differentiation therapy in liver cancer remains scarce.Hepatocyte nuclear factor(HNF)is a kind of liver-enriched transcription factors,which play an important role in the differentiation of hepatocyte and the maintenance of hepatic function.HNF family includes HNF1,HNF3,HNF4,HNF6,CCAAT/enhancer binding protein(C/EBP)and D-binding protein(DBP).It has been reported that HNF1? or HNF4? alone could induce the differentiation of liver cancer.In theory the combination of various HNFs may has a synergistic effect and posses a better differentiation effect in liver cancer treatment.In previous study,three liver-enriched transcription factors including HNF1??HNF4? and FOXA3(HNF3?)has been selected from a set of transcription factors to reprogram the human fibroblasts into functional hepatocytes successfully.These human induced hepatocytes(hi Heps)exhibit similar epithelial morphology to normal hepatocytes with hepatic markers and functions.In present study,we aim to induce the malignant HCC cells into a benign phenotype with the combination of HNF1??HNF4? and FOXA3.We hope the result of our study could faciliate the research on cancer cell reprogramming and provide a novel strategy for cancer therapy.Methods: 1.The expression of HNF1??HNF4? and FOXA3 in different liver cancer cell lines and normal hepatocytes was compared by the realtime-RCR.2.The expression of HNF1??HNF4? and FOXA3 in liver cancer tissue arrays was detected by the immunohistochemistry staining.3.The three adenoviruses expressing HNF1??HNF4? or FOXA3 were constructed respectively,and the efficiency of the infection in liver cancer cells was detected by realtime-PCR and Western blot.4.The expression of hepatic functional genes was mesured by realtime-PCR after the synchronal infection of three adenoviruses in HCC cells.5.The morphological change of HCC cells induced by the three factors was observed through microscope.6.Total RNAs of reprogramed hepatocyte-like cells and control cells were hybridized to whole-human gene expression microarray(Agilent)in accordance with the manufacturer's instruction.7.Reprogramed hepatocyte-like cells were stained by PAS,oil red,and Di I-ac-LDL.The supernatants were collected for the analysis of urea and albumin regarding the instruction of manufacturer..8.The effect of three factors on liver cancer stem cells was evaluated by flow cytometry and sphere formation assay.9.The proliferation ability was tested by CCK8 and clone formation assay.The cell migration assay was performed as the protocol of transwell experimental assay.10.The in vivo effect of the three factors on the proliferation and differetation was asseaaed using PDX HCC model.Results: 1.The expression of HNF1??HNF4? and FOXA3 was different among distinct liver cancer cell lines 2.The three factors was lowly expressed in the most of liver cancer samples.3.The combination of HNF1??HNF4? and FOXA3 was superior to one factor alone or the combination of two factors(1+4,1+F,4+F)in aspect of enhancing the expression of hepatic function-related genes.4.Reprogramed hepatocyte-like cells dispeared more broad and flat in comparison with the control cells.5.Gene expression profile analysis showed that three factors-induced liver cancer cells were clustered with primary human hepatocytes(PHH).6.The three factors-induced liver cancer cells exhibited some hepatic functions,such as glycogen synthesis,lipid accumulation,and secretion of albumin cells.7.The combination of three factors could decrease the proportion of LCSCs in liver cancer cells and suppress their self-renewal ability.8.The proliferation and migration ability of the three factors-induced liver cancer cells was significantly impaired.9.The three factors could inhibit the proliferation of PDX tumor and promte its differetation,which was reflected by the enhanced ALB expression and reduced AFP expression.Conclusion:The expression of HNF1??HNF4? and FOXA3 was different among distinct liver cancer cell lines,and the most liver cancers had low expression of theses three factors.Differention induction effct of the combination of three factors was much better than the usage of one facor alone or combination of two factors.The three factors induced-cancer cells not only have impaired malignant properties,but also showed superior hepatic function,suggesting the combination of three factor could reprogram liver cancer cells.This study might broarden our understanding on cancer reprogramming and provid a new strategy for the treatment of liver cancer.