The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression - PubMed (original) (raw)
The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression
Enrique Lara-Pezzi et al. J Clin Invest. 2002 Dec.
Abstract
Hepatocellular carcinoma is strongly associated with chronic infection by the hepatitis B virus (HBV) and has poor prognosis due to intrahepatic metastasis. HBx is often the only HBV protein detected in hepatic tumor cells; however, its contribution to tumor invasion and metastasis has not been established so far. In this work, we show that HBx enhances tumor cell invasion, both in vivo and in vitro. The increased invasive capacity induced by HBx is mediated by an upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, which in turn activates matrix metalloproteinase-2. Induction of both MT1-MMP expression and cell invasion by HBx is dependent on cyclooxygenase-2 (COX-2) activity. In addition, HBx upregulates the expression of COX-2, which is mediated by the transcriptional activation of the COX-2 gene promoter in a nuclear factor of activated T cell-dependent (NF-AT-dependent) manner. These results demonstrate the ability of HBx to promote tumor cell invasion by a mechanism involving the upregulation of MT1-MMP and COX-2 and provide new insights into the mechanism of action of this viral protein and its involvement in tumor metastasis and recurrence of hepatocellular carcinoma.
Figures
Figure 1
HBx induces tumor cell invasion in vivo and in vitro. (a and b) In vivo invasion. CMO and CMX (a) or HepG2 and 2.2.15 (b) were analyzed for their ability to invade the CAM of a chick embryo. The results are expressed as percentage of human DNA (left) or as number of intravasated cells (right). Experiments were carried out at least in quadruplicate. (c and d) In vitro invasion. The invasive capacity of CMO and CMX (c) or HepG2 and 2.2.15 (d) cells was tested using Matrigel-coated Transwells. Cells in the underside of the filter were stained, and eight independent fields were counted. The results are expressed as the mean value ± SE of three independent points.
Figure 2
HBx-induced tumor cell invasion is dependent on metalloproteinase activity, and HBx upregulates activated MMP-2 expression. (a) CMO and CMX cells were allowed to invade a Matrigel-coated Transwell in the presence of the MMP inhibitor BB-3103 or DMSO as a control. The invasion was quantified as in Figure 1. (b) Cells were grown for 24 hours in serum-free medium, and gelatin zymography analysis of the cell culture supernatants of PMA-stimulated human umbilical vein endothelial cells (control) and the different hepatic cell lines was performed. (c and d) CMO and CMX (c) or HepG2 and 2.2.15 (d) cell lysates and supernatants were analyzed for MMP-2 and MMP-9 expression by Western blot. Anti–α-tubulin and anti-albumin mAb’s assure equal protein load in all lanes.
Figure 3
HBx-mediated increase of activated MMP-2 and cell invasion is due to the induction of MT1-MMP expression. (a) MT1-MMP expression was analyzed by Western blot in CMO, CMX, HepG2, and 2.2.15 cells grown for 24 hours in the absence of serum. (b) CMO, CMX, HepG2, or 2.2.15 cells were allowed to invade a Matrigel-coated Transwell in the presence of the anti–MT1-MMP mAb LEM 1/58 or a control antibody. The invasion was quantified as in Figure 1. (c) CMO and CMX cells were incubated for 24 hours in the presence of the anti–MT1-MMP blocking mAb LEM 1/58 or a control mAb, and the presence of MMP-2 in the cell lysates was analyzed by Western blot. (d) The presence of MT1-MMP mRNA was studied by quantitative RT-PCR in CMO and CMX cells and in Chang liver cells (CHL) transiently transfected with an HBx-expression vector or a control plasmid.
Figure 4
Induction of MMP-2 activation, MT1-MMP expression, and cell invasion by HBx is sensitive to the COX-2 inhibitor NS398. (a) For 24 hours, CMX cells were grown in the presence of increasing amounts, and HepG2 and 2.2.15 cells were grown in the presence of 100 μM, of the COX-2–specific inhibitor NS398, along with 10 μM PGE2 where indicated. The presence of activated MMP-2 in the cell lysates was analyzed by Western blot. (b) CMO, CMX, HepG2, and 2.2.15 cells, as well as 4pX cells in which HBx expression was induced by removing tetracycline (Tet) for 24 hours, were treated or not treated with 100 μM NS398 for 24 hours and lysed, and the amount of MT1-MMP protein was studied by Western blot. (c) CMX and CMO cells were treated or not treated with 100 μM NS398 for 24 hours, and the expression of MT1-MMP transcripts was analyzed by quantitative RT-PCR. (d) CMO, CMX, HepG2, 2.2.15, or 4pX cells (with or without tetracycline) were allowed to invade Matrigel-coated Transwells in the presence of 100 μM NS398, or DMSO as a control, and 10 μM PGE2 where indicated. Cells that migrated to the lower chamber were quantified as in Figure 1.
Figure 5
Effect of different NSAIDs on MMP-2 activation, MT1-MMP expression, and tumor cell invasion induced by HBx. (a) CMO and CMX cells were grown for 24 hours in the presence of 60 μg/ml indomethacin, 60 μg/ml meloxicam, 600 μg/ml ASA, or DMSO as a control, and the amount of activated MMP-2 and MT1-MMP was analyzed by Western blot. (b) CMX and CMO cells were allowed to invade a Matrigel-coated Transwell in the presence of 60 μg/ml indomethacin, 60 μg/ml meloxicam, 600 μg/ml ASA, or DMSO. Cells that migrated to the lower chamber were quantified as in Figure 1.
Figure 6
HBx induces COX-2 expression. (a) The expression of COX-2 and COX-1 was evaluated by Western blot in CMO, CMX, HepG2, 2.2.15, and 4pX cells (treated or not treated with tetracycline). (b) The presence of COX-2 mRNA was analyzed by quantitative RT-PCR in CMO and CMX cells, and in Chang liver cells transiently transfected with pSV-X or the control vector pSV-hygro.
Figure 7
HBx induces the COX-2 promoter in an NF-AT–dependent manner. (a) Chang liver and HepG2 cells were transfected with 0.1 μg of the luciferase-based COX-2 reporter plasmid P2-1900 (–1796 to +104) along with increasing amounts (in Chang liver) or 5 μg (in HepG2) of pSV-X. The amount of luciferase was analyzed 18 hours later, and the results were expressed as fold induction over the value without pSV-X. (b) Chang liver cells were transfected as in a with reporter plasmids containing different deletions and point mutations of the COX-2 promoter, along with 5 μg of pSV-X or pSV-hygro. The results are expressed as fold induction over the value without pSV-X for each promoter construct and are representative of four independent experiments. (c) Chang liver cells were transfected as in a with 5 μg of pSV-X or pSV-hygro and 1 μg of the dominant negative NF-AT expression vector pSH102CΔ418, an NF-AT2 wild-type (NF-ATwt) expression vector, or the empty vector pBJ5 as a negative control. The values are expressed as fold induction over the value with pBJ5 and without pSV-X. Results are representative of at least three independent experiments.
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