Intrahepatic metastasis is a main cause of HCC recurrence and poor prognosis after tumor resection (1
). Although chronic infection by HBV is responsible for the development of most HCCs, the specific role of HBV proteins in HCC progression is unknown (1
). In this work, we provide evidence, for the first time to our knowledge, that HBx, often the only viral protein expressed by transformed hepatocytes (2
), induces cell invasion, suggesting its involvement in the metastatic spreading and recurrence of hepatic tumors.
We present both in vivo and in vitro results that support the ability of HBx to induce tumor cell invasion. The chick embryo CAM invasion assay showed that HBx increases the capacity of tumor cells to degrade the ECM, cross the endothelial barrier, and reach the bloodstream. This upregulation of the metastatic abilities of tumor cells was corroborated by the Matrigel invasion assays, in which HBx-expressing cells also displayed increased invasive potential.
Cancer cell invasion requires degradation of the basement membrane, composed mainly of collagen IV, which is mediated by MMP-2 and MMP-9 (9
). Most of the MMP-2 found in the hepatic tumor is produced by the hepatic stellate cells in the surrounding stroma and is secreted as an inactive precursor (11
). Although some activated MMP-2 can be found in the fibrotic liver, the highest MMP-2 activation efficiency is found in tumor tissues (11
) and correlates with MT1-MMP expression and tumor spreading (9
). In this regard, MT1-MMP is overexpressed in highly invasive HCCs (10
), and activation of MMP-2 is associated with tumor progression and recurrence in HCC patients (11
). Moreover, MT1-MMP is also capable of activating other MMPs and directly degrading ECM components and membrane receptors (9
). This capacity has a pronounced effect on cellular invasiveness independently of MMP-2 processing (26
). Thus, the induction of MT1-MMP expression by HBx provides the cell with a powerful invasive tool that contributes to tumor spreading and may play a key role in HCC recurrence.
NSAIDs show antineoplastic activity in a number of malignancies, due to their ability to inhibit the synthesis of prostaglandins by COX-2 (13
). Expression of this enzyme is sufficient to induce tumor growth and invasion by a variety of mechanisms (13
), some of which are shared by HBx (2
). In this regard, COX-2 enhances cell migration, expression of MT1-MMP, and MMP-2 activation (12
). HBx is also capable of inducing cell motility (17
), and we show herein that it upregulates MMP-2 activation and MT1-MMP expression. Both COX-2–dependent invasion and HBx-induced migration are mediated by CD44 (17
), which is cleaved by MT1-MMP (26
). Thus, by regulating COX-2, MT1-MMP, and CD44, HBx may be activating a complex mechanism that will eventually lead to cell invasion.
COX-2–expressing cells induce neovascularization by upregulating different proangiogenic enzymes and growth factors, such as inducible nitric oxide synthase (iNOS), TGF-β, and VEGF (29
). HBx itself induces the expression of iNOS, TGF-β, and VEGF, and an angiogenic effect of this viral protein has been proposed (30
). Moreover, COX-2 expression results in increased apoptosis resistance (13
), another property shared by HBx in certain systems (8
). Thus, the induction of COX-2 expression by HBx may help to explain the variety of pro-oncogenic effects of this viral protein and may unveil a new target for the treatment of HBV-derived HCC.
Interestingly, NSAID and NS398 treatment of Chang liver and HepG2 cells results in increased response to IFN-α, suggesting that COX-2 expression may result in IFN therapy resistance (32
). In this context, the induction of COX-2 by HBx would help the virus to evade the immune system, favoring chronic HBV infection and the progression to cirrhosis and HCC.
COX-2 expression can be induced by a number of mitogenic, inflammatory, and pro-oncogenic stimuli (13
). It has been recently reported that the calcium-regulated transcription factor NF-AT is essential for the activation of COX-2 transcription by VEGF or PMA plus calcium ionophore (24
), whereas activation by LPS or TNF-α is mediated by NF-κB (13
). Our results demonstrate that NF-AT, but not NF-κB, is necessary for the induction of COX-2 by HBx. We have previously shown that HBx activates NF-AT in a calcineurin-dependent manner (25
), which is likely due to the increase in intracellular calcium and oxidative stress generated after the interaction of HBx with the mitochondrion (33
). These results suggest a role for NF-AT, oxidative stress, and calcium signaling in the induction of inflammatory mediators and tumor development.
In conclusion, our results suggest that the expression of HBx provides tumor cells with metastatic potential. Since HBx is still expressed after HCC development, we propose that this viral protein may not only have a role in carcinogenesis, as is currently believed, but may also be responsible for tumor recurrence that results from intrahepatic metastasis. The mechanisms described herein by which HBx induces cell invasion, namely induction of COX-2 and MT1-MMP expression and activity, are in agreement with our previous findings reporting that HBx induces cell migration, disruption of intercellular adhesion, and cell-matrix interactions and cytoskeleton rearrangements (16
). The involvement of HBx in tumor spreading represents a new finding in the contribution of the hepatitis B virus to HCC and provides new clues for understanding the role of HBx after tumor establishment, unveiling potential new targets in the therapy against this aggressive malignancy.