The Therapeutic Effect And Molecular Mechanism Of Targeting HDAC1/2 In The Hepatocellular Carcinoma

Objectives:Hepatocellular carcinoma（HCC）is the most prevalent form of primary liver cancer,accounting for more than 700,000 deaths annually worldwide.Hepatitis B and C,alcohol and aflatoxin have been identified as major risk factors for HCC.Despite progress in surgical techniques,chemotherapy and radiotherapy in the treatment of HCC,the 5-year relative survival rate for patients with HCC is only 7%,largely due to tumour recurrence and metastases.The paucity of effective and well-tolerated treatments for advanced HCC highlights the need for new therapeutic approaches.In the past decade,systemic administration of a multi-kinase inhibitor,sorafenib,was approved for clinical use for patients with advanced HCC.However,beneficial effects of sorafenib were observed in only approximately 30%of patients,and acquired drug resistance often develops within 6 months.Thus,there is an urgent need to develop novel and specific HCC-targeting drugs.Histone deacetylases（HDACs）are a class of enzymes that remove acetyl groups from specific lysine residues on core his-tones,thereby regulating gene transcription via histone and chromatin structure modifications.HDACs typically interact with other transcriptional co-repressors（eg mSin3,SMRT and N-CoR）to form multi-protein complexes that interact with DNA-binding factors to inhibit target gene transcription.These complexes are involved in various physiological processes,such as cell cycle progression,differentiation,apoptosis and tumorigenesis.HDAC deregulation has been detected in various cancers,and several HDAC inhibitors（HDACis）have been approved by the U.S.Food and Drug Administration（FDA）for use in treating clinical cutaneous T-cell lymphoma（CTCL）or peripheral T-cell lymphoma（PTCL）.Although HDACis have been shown to be effective in the treatment of many other types of cancer,their efficacy against HCC is still largely unknown.In mammals,a total of 18 HDAC homologues have been identified,and they are subdivided into classes I,IIa,IIb,III and IV.The functions of HDAC isoforms are not yet fully understood.Some HDAC isoforms have been found to be associated with specific diseases,such as cancer and neurodegenerative diseases.Most early HDACis,such as SAHA,TSA,VPA and butyrate,are global HDACi.Their effects in cancer therapy are unpredictable,and they have shown different side effects as well.16Therefore,selective HDACis are highly desirable for achieving a better understanding of the biological functions of different HDAC isoforms and,more importantly,for the development of agents with more precise therapeutic effects and fewer side effects.HDAC1 and HDAC2,the 2members of the class I HDAC family,are ubiquitously expressed in organs and tissues,including the liver.HDAC1 and HDAC2 typically associate with co-repressors to form transcriptional co-repressor complexes.They are also required for chromatin condensation,spindle formation and correct separation during cell mitosis and deregulation of HDAC1/2 can lead to abnormal karyokinesis.Both HDAC1 and HDAC2 play an essential role in mouse growth and development,and they play redundant roles in the regulation of cell proliferation.More than 95%of HCC patients have abnormally high levels of HDAC1/2 in the liver.Previous research by Rikimaru et al demonstrated that the survival rates after surgical resection in patients with low and high HDAC1/2 expression at 5 years were 81.8%and40.0%,respectively.Quint additionally investigated the expression of HDACs 1,2,3 and 7 in HCC and concluded that the HDAC2 expression level was associated with survival of patients with HCC.Herein,considering the similar structures of HDAC1 and 2 and their redundant functions,we hypothesized that combined inhibition of HDAC1and 2 may have better therapeutic effects on HCC.To address this hypothesis,we used 3 different selective HDACis to treat HCC cell lines and evaluated their effects on HCC cell proliferation and apoptosis.Our findings indicate that selective inhibition of HDAC1 or 2 does not affect the growth of HCC cells,but combined inhibition of HDAC1 and 2 leads to cell cycle arrest and apoptosis.Additionally,p21^Waf1/Cip1af1/Cip1 and p19^INK4d signalling was significantly activated by HDAC1/2 inhibition.Methods:HDAC1 inhibitor Tacedinaline（CI994）,HDAC2 inhibitor Santacruzamate A（CAY10683）,HDAC1/2 common inhibitor Romidepsin（FK228）and global HDAC inhibitor Vorinostat（SAHA）were used to treat HCC cells.Cell cycle,apoptosis and the protein levels of CDKs and CDKNs were performed to evaluate HCC cell growth.Inhibition of HDAC1/2 by RNAi was further investigated.Results:Combined inhibition of HDAC1/2 led to HCC cell morphology changes,growth inhibition,cell cycle blockage and apoptosis in vitro and suppressed the growth of subcutaneous HCC xenograft tumors in vivo.p19^INK4dNK4d and p21^Waf1/Cip1,which play roles in cell cycle blockage and apoptosis induction,were upregulated.Inhibition of HDAC1/2 by siRNA further demonstrated that HDAC1 and 2 cooperate in blocking the cell cycle and inducing apoptosis via p19^INK4dNK4d and p21^Waf1/Cip1 upregulation.Finally,H3K18,H3K56 and H4K12 in the p19^INK4dNK4d and p21^Waf1/Cip1 promoter regions were found to be targets of HDAC1/2.Conclusions:Pharmacological or transcriptional inhibition of HDAC1/2increases p19^INK4dNK4d and p21^Waf1/Cip1 expression,decreases CDK expression and arrests HCC growth.These results indicated a potential pharmacological mechanism of selective HDAC1/2 inhibitors in HCC therapy.