Hepatitis B virus X (HBX) protein is a multifunctional viral protein that regulates different cellular processes such as the modulation of protein degradation pathways, transcription, apoptosis, signal transduction, cell cycle, and genetic stability, by interacting with the host factors either directly, or indirectly.
HBx is known to play a major role in the pathogenesis of viral induced hepatocellular carcinoma (HCC). The carboxy terminal of HBx has been shown to likely play a role in protein-protein interactions, transcriptional transactivation, DNA repair, cell, signaling and pathogenesis of HCC.
HBx protein structural and functional relationship
HBx is a 154-amino acid regulatory protein with a molecular mass of approximately 17 kDa. The X-protein consists of two functional domains. While the amino-terminal domain is mapped to the first 50 amino acids including the dimerization region which is required for dimerization activity, the C-terminal transactivation domain is located between amino acid 53 and 142 which interacts with Xenopus anti-photoreceptor-1 (XAP-1)/UV-damaged DNA binding protein (UVDDB) and p53.
The amino acid sequence of this viral gene is not homologous to any known protein, therefore it was assigned the name “HBx”. It is one of the most conserved proteins among different HBV sub-types and is found in almost all viruses of Hepadnaviridae. It is mostly localized within the cytoplasm and upto some extent in the nucleus of hepatic cells.
Being a multi-functional protein HBx can transactivate viral and cellular promoters and enhancers through protein-protein interactions with several well defined targets. HBX does not bind directly with DNA, instead, it activates transcription by getting involved with several nuclear transcription factors like RNA polymerase binding protein (RBP5), transcriptional factor IIB (TFIIB), transcriptional factor IIH (TFIIH), a subunit of RNA polymerase, cAMP response element-binding protein (CREB), CREB1-binding protein (CBP)/p300, activating transcription factor 2 (ATF-2), activating protein (AP)-2, AP-1, and nuclear factor kappa B (NF-κB).
HBx can also modulate cytoplasmic signal transduction pathways including Ras-Raf-mitogen activated protein kinase (Ras-Raf-MAPK), Janus kinase/STAT (JNK/STAT), focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), protein kinase C (PKC) and Src-dependent phosphatiylinositol-3 kinase (PI3K/Akt)
HBx can cause hepatic cellular proliferation when its activation of cellular processes is through transactivation of cellular signaling molecules. This also has tendency to directly inactivate or indirectly down-regulate various tumor suppressors, such as p53, or senescence-related factors.
Role of HBx Protein in hepatocellular carcinogenesis
On its own, HBx does not directly affect cancer progression. However, when coupled with some carcinogens such as diethynitrosamine or UV radiation, it plays a major role as a promoting factor in HCC development. It is also known to interfere with nucleotide excision repair (NER) pathway through both p53-dependent and independent mechanisms. In other to promote tumor progression, HBx protein inhibits cell cycle control heckpoints and facilitate accumulation of host mutations by interfering with NER. These effects are exerted by binding to several proteins involved in DNA repair pathways and inhibiting their repair capacity. HBx also de-regulate factors include human homologue of UV-DDB, XPB and XPD components of TFIIH, which are essential components for NER in both p53-proficient andp53-deficient hepatocytes.
As part of its role in HCC, HBx also directly represses XPB and XPD, and inhibit the DNA binding properties of the transcription factor Sp1. The COOH-terminus of p53 serves as a scaffold for HBx binding which ultimately leads to the localization of p53 from nucleolus to cytoplasm, which then leads to uncontrolled cell cycle progression and DNA repair. HBx also represses transcriptional activity of p53 by disrupting cross talk among several cellular factors. It also disrupts interaction of p53 with TFIIH resulting in compromised TFIIH induced helicase activity during assembly of the DNA repair complex. Thus, by binding to the carboxy (COOH) terminal domain of p53 HBx blocks its association with XPB and XPD.
The dual function of HBx in apoptosis
HBx can either inhibit or have no effect on apoptosis due to the differential pattern of HBx expression, and depends upon the cell types involved. When there is an increased quantity of HBx, it promotes apoptosis, but a low level of same inhibits this phenomenon.
One of the mechanisms by which HBx exhibits its anti-apoptotic feature involves a complex formation and sequestration of p53 in the cytoplasm, and blocking of its entrance into the nucleus. As a result, p53 wouls fail to up-regulate its various downstream effector molecules like Bax, p21, or Fas, which are required in the apoptotic pathway, thus leading to increased cell survival. HBx also prevents p53-induced apoptosis through HBx-P3K-Akt-Bad pathway, by inhibiting caspase 3 function associated with H-ras oncogene by induction of phosphatidyl inositol-3 kinase and AKt pathway.
The role of Cyclooxygenase-2 (COX-2) in anti-apoptotic effects of HBx protein
COX-2 plays an important function in HBx protein anti apoptic role. When activated, COX-2/PGE2 pathway leads to the elimination of p53-induced apoptosis imposed by HBX expression. However, this cannot be a therapeutic target. The reason is that beside P53 induced-apoptosis through HBx expression, HBx can also directly induce apoptosis by activating cytoplasmic cytochrome c, caspase-3-like activity and nuclear fragmentation.
Role of HBx protein in hcc cell proliferation
HBx plays major roles in promoting cell proliferation through the regulation of miRNA. By downregulating miR-132, miR-429, miR-205, miR-15b, and miR-145; and by upregulating miR-221, Hbx promotes aberrant HCC cell proliferation.
MicroRNAs (miRNAs) are noncoding RNAs which are involved in the regulation of gene expression. They also play crucial roles in numerous pathological processes, including tumor formation.
HBx also promotes hepatoma cell proliferation by downregulating the expression p16 gene. p16 is a tumor-suppressive gene, which regulates cell cycle and cell growth. To do this, p16 encodes a cyclin kinase inhibitor, which decreases the phosphorylation level of retinoblastoma (Rb) protein by binding to and inhibiting the cell cycle-dependent protein kinases CDK4 and CDK6. The silencing of p16 gene expression can lead to the unlimited proliferation of cells. When transfected into HepG2 cells, HBx induces methylation of some CpG sites in the promoter region of the tumor-suppressive gene p16, then downregulates the expression of p16 gene, and would ultimately and significantly enhanced the proliferation ability of hepatoma cells. This is carried out through the amino terminal of HBX.
HBx also promotes cell proliferation by upregulating mRNA and protein levels of b-catenin in hepatoma cells, thereby activating the Wnt/β-catenin pathway. The abnormal activation leads to initiation and progression of HCC by promoting cell proliferation and increasing the levels of angiogenesis factors, such as matrix metalloproteinase 2 (MMP2) and vascular endothelial growth factor (VEGF)-A.
HBx promotes the migration and metastasis of hepatoma cells
HBx protein promotes hepatocellular cancinoma cell invasion and metastasis through multiple mechanisms. One of such mechanism involves increasing the expression of the vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) by activating the nuclear factor κB (NF-κB) pathway. This upregulation of VEGF and MMPs promotes HCC cell invasion and metastasis.
HBx protein is also localized to peroxisomes where it increases the level of cellular reactive oxygen specie (ROS). When localized in the peroxisome, HBx increases the expressions of MMPs and decreases the expression of E-cadherin, which facilitate HCC cell invasion.
HBx also promotes HCC cell invasion through the upregulation of calpain small subunit 1 (Capn4), regulation of miRNAs and lncRNAs, and by suppressing p53-mediated activation of miR-148a, which results in the upregulation of hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP). HPIP increased the expression of mTOR through the AKT/ERK/FOXO4/ATF5 pathway, which resulted in HCC cell invasion and metastasis. HBx also downdegulate E-cadherin expression by suppressing its actviator, miR-373. This downregulation of E-Cadherin is associated with the enhancement of the cell invasion ability of HCC. HBx also promotes HCC cell through upregulation of miR-29a by targeting PTEN. It also elevates oncoprotein AEG-1 expression to promote HCC cell migration by downregulating miR-375 and miR-136.
HBx stabilizes HBV DNA in hepatoma cells
HBx protein can promote the progression of HCC by enhancing the stability of HBV cccDNA in HCC cells through activation of the positive feedback loop of HBx/MSL2/HBV cccDNA/HBV. MSL2 can regulate the level of APOBEC3B ubiquitination by acting as an E3 ubiquitination ligase, which promotes its degradation in hepatoma cells and thus enhances the stability of HBV covalently closed circular DNA (cccDNA). Interfering with MSL2 expression effectively inhibited the proliferation of hepatoma cells in vivo and in vitro; and this makes MSL2 a therapeutic target for HCC proliferation.
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