The Roles Of CIITA Gene Epigenetic Silence In The Pathogenesis Of HBV Related Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It is the fifth leading cause of cancer-related death in the world. Only in China, there are >500,000 new cases each year and >100,000 deaths annually. Chronic HBV infection is a leading risk factor of HCC. Recently, it has been estimated that about 53% of HCC cases in the world are related to HBV. HCC is generally diagnosed at an advanced stage of tumor progression, and more than 80% of the cases prove fatal, underscoring the need for better understanding of the molecular mechanisms that could be exploited for early detection and therapeutic intervention.Antitumor immunity and immune escape of tumor cells play an important role in the development and progression of HCC. Immunotherapy represents a potential therapeutic option for patients with HCC, and the development of HCC immunotherapy strategies is based on better understanding of the molecular mechanisms of HCC immune escape. T lymphocytes play a major role in the anti-tumor immune response and surveillance. Much attention has been paid to the role of CD8~+ lymphocytes in antitumor immunity, as these cells are able to lyse tumor cells directly upon recognition of peptide-MHC class I complexes expressed by the tumor. However, systemic conventional cytotoxic therapy has not demonstrated any efficacy in patients with HCC. It has been argued that this event is due, in part, to the poor tumor specific, MHC-II restricted CD4 + T cell help generated in tumor-bearing patients, as T helper (Th) cells are required for optimal induction of both humoral and cellular effector mechanisms. MHC-II molecules, are constitutively expressed only on few cell types such as B cells, thymic epithelial cells and mature dendritic cells. However, MHC-II expression can be induced in many cell types by a variety of stimuli and particularly by IFN-γ. MHC Class II-expressing tumor cells can function as antigen-presenting cells and activate specific CD4~+ T lymphocytes inducing antitumor immunity. The lack of tumor human leukocyte antigen DR (HLA-DR) expression is considered to be a key mechanism that enables some gastrointestinal cancer cells to escape immune surveillance.Class II Transactivator(CIITA) is a switch molecule of mastering HLA-DR expression, and plays a pivotal role in regulating constitutive and IFN-γinducible HLA-DR expression of cells. The epigenetic silencing of CIITA gene was closely associated with the lack of inducible HLA-DR expression in gastric and colorectal cancer cells, and was believed to be a key mechanism that enables some gastric and colorectal cancer cells to escape immune surveillance. The epigenetic silencing of CIITA gene is also a key mechanism that HepG2 cells lack constitutive and IFN-γinducible HLA-DR expression. However, little is known about the roles of CIITA gene epigenetic silence in the pathogenesis of HBV related HCC.Firstly, we examined the expression of CIITA, HLA-DR , Dnmts and ERK at both the mRNA and protein level in hepatocellular carcinomas (HCCs) and paired non-neoplastic liver tissues from 51 patients with HBV related HCC, and analyzed the association of hepatic HLA DR expression with hepatic CIITA expression. Secondly, we also detected the methylation status of CIITA promoterⅣin HCCs and paired non-neoplastic liver tissues from 51 patients with HBV related HCC, and analyzed the association of methylation status at CIITA promoterⅣwith hepatic CIITA and Dnmts expression. Thirdly, we evaluated the effects of methyltransferase inhibitor 5-Aza-CdR and IFN-γon CIITA and HLA-DR expression of primary cultured human HCC cells. Fourthly, we detected the deacetylation status of CIITA histone H3 in HCCs and paired non-neoplastic liver tissues from 4 patients with HBV related HCC. Fifthly, we evaluated the effects of histone deacetylase inhibitor TSA alone or combination with 5-Aza-CdR on CIITA and HLA-DR expression of primary cultured human HCC cells.The main results were followed as:1. Hepatic HLA-DR protein expression was detected in adjacent non-tumor liver tissues among 100%(51/51) patients, and in tumor tissues among 25.5%(13/51) patients, having a signifant difference (χ~2=60.56, P< 0.05). There was no HLA-DR protein expression in tumor cells of patients with HCC, and little HLA-DR expression in lymphocytes of tumor tissues among 13 patients.The different levels of HLA-DR expression on hepatocytes were observed in adjacent non-tumor liver tissues from 51 patients with HCC.Hepatic CIITA protein expression was detected in adjacent non-tumor liver tissues among 100%(51/51) patients, and in tumor tissues among (27.5%, 14/51) patients, having a signifant difference (χ~2=58.06, P<0.05).The levels of hepatic HLA-DR expression were positively correlated with the levels of hepatic CIITA protein expression among patients with HCC (rs=0.886, P<0.05). HCC cells lacking CIITA protein expression also did not express HLA-DR.2. Methylation of CIITA promoterⅣwas detected in tumor tissues among 68.6% (35/51) patients, and in djacent non-tumor liver tissues among 25.5% (13/51) patients with HCC, having a signifant difference (χ~2=19.05, P<0.05). 35.30% (18/51) patients were detected to have supermethylation of CIITA promoter IV, and 33.30% (17/51) patients have semimethylation, and 31.37%(16/51) patients have unmethylation in tumor tissues.In adjacent non-tumor liver tissues, only semimethylation of CIITA promoterⅣwas detected among 25.49% (13/51) patients with HCC, and unmethylation was detected among 74.51% (38/51) patients. The level of hepatic CIITA mRNA expression was 5.90±2.50 in supermethylation tissues, and significantly lower than that in semimethylation tissues (7.09±3.16,P=0.045). The level of hepatic CIITA mRNA expression in unmethylation tissues was 13.95±4.83, and significantly higher than that in semimethylation tissues (P<0.05)Hepatic Dnmt1, Dnmt3a and Dnmt3b protein expression were detected in adjacent non-tumor liver tissues among 17.65% (9/51), 19.61% (10/51) and 21.57% (11/51) patients with HCC respectively, and in tumor tissues among 100% (51/51), 94.12% (48/51), and 94.12% (48/51) patients respectively, having a signifant difference (P < 0.05). The differences of the levels of three kinds of Dnmts protein expression in adjacent non-tumor liver tissues had no statistical significance (P>0.05), while in tumor tissues, the levels of Dnmt1 protein expression were higher than that of Dnmt3a and Dnmt3b (P<0.05).The levels of Hepatic Dnmt1, Dnmt3a and Dnmt3b protein expression were positively correlated with the status of methylation of CIITA promoterⅣ(r_s=0.512, 0.507 and 0.621,P<0.05).Human HCC cells primary cultured did not express CIITA and HLA-DR, not express after dealed with IFN-γ, however, it can express CIITA and HLA-DR through the handling of 5-Aza-CdR.3. Hepatic ERK1 protein expression was detected in adjacent non-tumor liver tissues among 31.37% (16/51) patients with HCC, and in tumor tissues among 100% (51/51)patients, having a signifant difference (χ~2=54.28, P<0.05). Hepatic ERK mRNA expression was detected in tumor tissues among 100% (51/51) patients with HCC, significantly higher than that in adjacent non-tumor liver tissues (0,χ~2=102.00, P<0.05).Deacetylation of CIITA histone H3 was detected in tumor tissues from 4 patients with HCC, and in adjacent non-tumor liver tissues from the same 4 patients acetylation of CIITA histone H3 was detected. In tumor tissues with deacetylation of CIITA histone H3, ERK expression were all detected and CIITA expression were no detected, while in adjacent non-tumor liver tissues with acetylation of CIITA histone H3, ERK expression were no detected and CIITA expression were all detected.Human HCC cells primary cultured can express CIITA and HLA-DR by the treatment of TSA singly or combination with 5-Aza-CdR.Conclusion1. Lacking HLA-DR expression in HCC cells of HBV related HCC patients is associated with the lack of CIITA expression, and may play an important role in immune escaping of HCC cells.2. CIITA promoterⅣin HCC cells of HBV related HCC patients is in the status of hypermethylation. CIITA gene epigenetic silence caused by aberrant hypermethylation of CIITA promoterⅣis one of the main mechanisms that cause HCC cells lacking CIITA expression, and may play an important role in the pathogenesis of HBV related HCC.3. Overexpression of DNA methyltransferase in HCC cells of HBV related HCC patients is the main reason that causes aberrant hypermethylation of CIITA promoterⅣ.4. CIITA histone H3 in HCC cells of HBV related HCC patients is in the status of deacetylation, and may have relation with CIITA gene epigenetic silence in HCC cells.