Wang Chaomin1, Niu Wenhao2, Hua Jialei1, Zhao Ting1, Feng Honglei1, Hao Zhuang3, Wang Yichao4, Bai Changsen5, Li Yueguo6. 1. Department of Clinical Laboratory, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Huanhuxi Road, Hexi District, Tianjin, 300060, China. 2. Department of Immunology, School of Basic Medical Sciences, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Educational Ministry of China, Tianjin Medical University, Tianjin, China. 3. Department of Hepatobiliopancreatic Surgery, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou, Henan, China. 4. Department of Clinical Laboratory Medicine, Taizhou Central Hospital, Taizhou University Hospital, No. 999 Donghai Road, Jiaojiang District, Taizhou, 318000, Zhejiang, China. wangyichaobei@126.com. 5. Department of Clinical Laboratory, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Huanhuxi Road, Hexi District, Tianjin, 300060, China. changsenbai@tmu.edu.cn. 6. Department of Clinical Laboratory, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Huanhuxi Road, Hexi District, Tianjin, 300060, China. yli08@tmu.edu.cn.
Abstract
PURPOSE: Hepatitis B virus (HBV) plays a crucial role in the progression of hepatocellular carcinoma (HCC). It is known that HBV-encoded X protein (HBx) can induce genetic alterations in some oncogenes and that SMAD4 is relevant for the development of some cancers, especially HBV-related HCC. Previously, it has been reported that HBx can promote SMAD4 protein expression in liver fibrosis and HCC but, as yet, its regulatory mechanism has not been fully elucidated. Here, we aimed to investigate the correlation between and regulatory mechanism behind HBx and SMAD4 in HCC. METHODS: mRNA and protein expression of SMAD4 in HCC tissues was detected by qRT-PCR, Western blotting and IHC. CCK-8 and colony forming assays, as well as xenograft murine models were used to evaluate the effects of HBx and SMAD4 on the proliferation and tumorigenicity of HCC cells. Luciferase reporter, immunofluorescence, Co-IP and truncation assays were performed to assess the regulatory relationship between HBx and SMAD4. RESULTS: We found that SMAD4 was highly expressed in HBV-positive HCC patient samples and correlated with a poor prognosis. The proliferation of HCC cells with a high SMAD4 expression was found to be enhanced in vitro and in vivo, and knocking down HBx while replenishing SMAD4 rescued HCC cell proliferation. Mechanically, we found that HBx regulates SMAD4 expression at the transcriptional level via TFII-I and can bind to SMAD4 to repress its ubiquitination. The binding region comprised the MH2 domain of SMAD4. Furthermore, we found that SMAD4 can promote HBx expression through a positive feedback mechanism. CONCLUSIONS: From our data we conclude that SMAD4 is modulated spatiotemporally via both transcriptional activation and protein stabilization by HBx in HCC cells. Our data shed light on the molecular mechanism underlying HBx-induced hepatocarcinogenesis.
PURPOSE: Hepatitis B virus (HBV) plays a crucial role in the progression of hepatocellular carcinoma (HCC). It is known that HBV-encoded X protein (HBx) can induce genetic alterations in some oncogenes and that SMAD4 is relevant for the development of some cancers, especially HBV-related HCC. Previously, it has been reported that HBx can promote SMAD4 protein expression in liver fibrosis and HCC but, as yet, its regulatory mechanism has not been fully elucidated. Here, we aimed to investigate the correlation between and regulatory mechanism behind HBx and SMAD4 in HCC. METHODS: mRNA and protein expression of SMAD4 in HCC tissues was detected by qRT-PCR, Western blotting and IHC. CCK-8 and colony forming assays, as well as xenograft murine models were used to evaluate the effects of HBx and SMAD4 on the proliferation and tumorigenicity of HCC cells. Luciferase reporter, immunofluorescence, Co-IP and truncation assays were performed to assess the regulatory relationship between HBx and SMAD4. RESULTS: We found that SMAD4 was highly expressed in HBV-positive HCC patient samples and correlated with a poor prognosis. The proliferation of HCC cells with a high SMAD4 expression was found to be enhanced in vitro and in vivo, and knocking down HBx while replenishing SMAD4 rescued HCC cell proliferation. Mechanically, we found that HBx regulates SMAD4 expression at the transcriptional level via TFII-I and can bind to SMAD4 to repress its ubiquitination. The binding region comprised the MH2 domain of SMAD4. Furthermore, we found that SMAD4 can promote HBx expression through a positive feedback mechanism. CONCLUSIONS: From our data we conclude that SMAD4 is modulated spatiotemporally via both transcriptional activation and protein stabilization by HBx in HCC cells. Our data shed light on the molecular mechanism underlying HBx-induced hepatocarcinogenesis.
Authors: D K Lee; S H Park; Y Yi; S G Choi; C Lee; W T Parks; H Cho; M P de Caestecker; Y Shaul; A B Roberts; S J Kim Journal: Genes Dev Date: 2001-02-15 Impact factor: 11.361
Authors: Darshan S Chandrashekar; Bhuwan Bashel; Sai Akshaya Hodigere Balasubramanya; Chad J Creighton; Israel Ponce-Rodriguez; Balabhadrapatruni V S K Chakravarthi; Sooryanarayana Varambally Journal: Neoplasia Date: 2017-07-18 Impact factor: 5.715