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A Proposal For The Study of the Role of Hbx in Hepatocellular Carcinoma

Abstract

Hepatitis B Virus (HBV) chronically infects over 350 million people worldwide, and is a major risk factor for the development of hepatocellular carcinoma, which is the fifth leading form of cancer and a major public health concern. Despite years of study, the mechanisms of HBV oncogenesis are still unclear. Recent efforts have focused on the role of HBV X protein (HBx), which has been shown to have pleiotropic effects in its host cells, many of which are suspected to be critical both to HBV replication and to hepatocellular transformation. The aim of this proposed study is to assess the importance of the HBx-induced upregulation of cellular transcription factors to the process of cellular transformation. This study will use inhibitors of specific transcription factor (TF) activators as well as HBx deletion mutants to assess the importance of these TF activities to the process of cellular transformation. The results of this experiment will derive the patterns of TF expression in the presence of various HBx deletion mutants, and will serve to test the importance of the upregulation of certain TFs to the hepatocarcinogenic process by correlating these patterns with the incidence of host cell transformation. Though these results will require further future expansion, they will serve to provide valuable insight into the roles different transcription factor pathways play in the mechanisms of HBx-induced hepatocarcinogenesis, and are thus important focus of study.

Introduction

Hepatocellular carcinoma (HCC) accounts for up to 85% of annual liver cancer cases, infecting 3.4 of every 100,000 people in the United States each year, with an estimated five year survival rate of 8% [1]. In certain areas of the world the incidence rate is significantly higher, with the disease presenting in 120 of every 100,000 people in Sub-Saharan Africa each year [2]. A 2008 study documented that in the United States the rates of HCC are rising at a time when average overall rates of cancer have stabilized, with another report indicating that HCC is the fifth most prevalent form of cancer [1,2].

The main risk factor for HCC is infection with Hepatitis B Virus (HBV), a small enveloped DNA virus which primarily infects hepatocytes. Chronic HBV infections are present in an estimated 350 million people worldwide, despite the availability of an effective HBV vaccine. Chronic infection characterized by presentation of HBV antigen (HBsAg) has been associated with a 2500-3700% increase in risk of HCC, and patients who are HBsAg- may still present low levels of HBV DNA in serum; a form of chronic infection termed occult HBV infection [3,4]. Occult HBV infections have been suggested to increase HCC risk, primarily in individuals co-infected with Hepatitis C Virus (HCV); however these results are debated [5].

The HBV genome has not been definitively found to possess viral oncogenes; however one proposed mechanism of HBV-induced HCC suggests that viral DNA insertion into the host genome results in activation of cellular oncogenes. This hypothesis is supported by the fact that HBV has been shown to contain an enhancer sequence which is active in integrated HBV DNA in vivo, and insertional activation of mevalonate kinase have been detected in HBV infected cells, demonstrating the potential for transformative effects of insertional activation via HBV [6,7]. Nonetheless, insertional activation alone does not account for the high incidence of HCC in HBV infected patients, and in recent years studies have focused on the properties of the HBV X protein (HBx). HBx was first noted during the nucleotide sequencing of HBV, at which time an open reading frame (ORF) in the viral genome with which no protein or function was formally associated was designated as the X region [8]. Since then, HBx has been identified as a 17kDa protein necessary for successful HBV replication, with its nuclear localization being essential to the viral replication process by means of uncertain mechanisms [9]. HBx has been shown to have pleiotropic effects, some of which are suspected to be directly related to hepatocarcinogenesis.

A study using transgenic mice expressing HBx found that the protein bound to p53, the ubiquitous tumor-suppressing transcription factor (TF), preventing its nuclear localization and thus its function, while another study found that the nuclear localization of p53 resulted in HBx degradation [10, 11]. Together these studies suggest that HBx alone is not responsible for the transformation of host cells, pointing toward a tenuous balance between HBx and p53 functionality, as the proteins are mutually inhibitory through different mechanisms [12]. This indicates that after mutations in p53 inhibit its nuclear localization (typically a later event in HCC progression), HBx production and stability both likely increase, further contributing to the disease process [11]. Additional studies of the interactions between HBx and p53 have suggested that HBx prevents the sequence specific DNA binding of p53, further contributing to the suppression of this tumor-suppressing protein [13].

Though the fact that HBx plays an indirect role in the transformative process has been demonstrated through experiments involving p53, uncertainty remains as to whether HBx possesses any direct oncogenic properties or simply works in concert with other oncogenic factors to promote tumor formation. A recent article published in Cancer Science by Tang et al. reviews this controversy, noting that HBx seemed to inconsistently induce tumor formation, most often requiring the presence of other carcinogens. Tang et al. go on to suggest that HBx may in some cases be critical to anchorage-independent growth, but that further studies using immortalized human hepatocytes may be necessary, as the process of tumorigenesis varies between tissues [14]. At present, the general consensus is that HBx alone cannot transform cells, but that it is integral to the transformation process.

A recent study using primary rat hepatocytes found that HBx induced changes in these cells which resulted in them passing into the G1 phase of the cell cycle but which prevented them from progressing to the S phase [15]. A separate study found that HBx deregulated the G1/S phase checkpoints by interacting with cyclin-cdk complexes, stabilizing cyclin E to promote faster progression through the stages of the cell cycle [16]. Yet another study suggests that HBx production results in an extended S phase, inducing chromosomal instability which may further lead to HCC tumorigenesis [17]. Though these proposed mechanisms of cell cycle regulation by HBx are diverse, and though some are suggested to be the result of the specific experimental conditions, it is nonetheless clear that HBx likely alters the regulation of cell cycle processes in a manner which may destabilize the genome or deregulate important checkpoints, and thus may be strongly related to cellular transformation [15].

HBx has also been shown to transactivate multiple gene products by means of multiple mechanisms, leading to suggestions that these additional changes in cellular activities may be responsible in part for the incidence of hepatocarcinogenesis. One proposed mechanism behind the plethora of processes induced by HBx is that this protein leads to an upregulation in cytosolic calcium levels in host cells, a mechanism supported by recent studies [18,19]. Calcium, serving as a common second messenger, likely activates numerous signaling pathways resulting in a number of observed upregulations in transcription factor (TF) activity in HBV-infected cells. HBx has been shown to directly or indirectly activate a number of pleiotropic TFs including AP-1, AP-2, NF-kB, and NF-AT [19,20,22]. These TFs have many intracellular roles, and as such their upregulation is likely important for enhancing viral replication and host cell survival, which is necessary for the survival of transformed cells. As such, it is likely (although uncertain) that the alteration of regulation of these and other cellular transcription factors plays a role in the transformation of human liver cells, promoting progression toward HCC. It is the role of these TFs in hepatocarcinogenesis which is the primary focus of these experiments.

Statement of Problem

Despite being the subject of intensive studies for over 20 years, the role that HBx plays in the transformation of human hepatocytes remains unclear, resulting in controversy over the protein’s importance with regard to the onset of HCC. While HBx alone is not generally reported to be a significant transformer of primary hepatocytes, it has been linked to tumorigenesis when acting in concert with other carcinogens or cellular oncogenes. Furthermore, HBx is known to modulate cell cycle regulation and to transactivate many genes, altering the expression levels of the transcription factors AP-1, AP-2, NF-kB, NF-AT, and many other gene products.

In light of the myriad roles HBx is purported to play in HBV replication and HCC tumorigenesis, this proposed study endeavors to better understand which modifications to the cell cycle have an effect on the transformative process induced by the HBx protein. Specifically, this study seeks to address the question of whether modulation of TF activity by HBx is critical to the transformation of host cells. Though the likely existence of multiple HBx-associated oncogenic mechanisms may make these studies more difficult to perform, they nonetheless show promise for shedding light on HBV-associated oncogenic mechanisms.

General Methods

Cells and HBx plasmids

The cells used in these experiments will be from the L-O2 line described by Zhang et al [21]. These cells are human liver tissue samples which where immortalized with the hTERT gene, and are suggested by Zhang to be an ideal model for studying the mechanism of hepatocarcinogenesis. To this effect, these cells will be transfected with a stable HBx plasmid inserted into restriction endonuclease sites as described by Tanaka et al [20]. Additional cells will be transfected with the HBx deletion mutant plasmids also described by Tanaka et al. A control strain of cells will be transfected with an empty vector. RT-PCR will be used to ascertain the successful transfection of these cells.

HBV Expression Constructs

L-O2 cells will be transfected with either a replication-competent wild-type HBV construct or an HBx-deficient HBV construct, as previously described [23].

Calcium Chelation

Both control and HBx transfected L-O2 strains will be induced to produce HBx or its respective deletion mutants. BAPTA-AM, a calcium chelating agent, will then be co-incubated with the cells in order to arrest the calcium signaling within the cells. BAPTA-AM was selected as it has previously been used as a chelating agent for HBx studies of cell cycle proteins [15,19]. The cells will then be lysed and the levels of transcription factors and related regulatory proteins will be assessed by western immunoblotting.

Western Blot Analysis

Cells of interest will be and the protein amounts will be equalized by means of a BCA assay. The protein will then be run through an SDS gel and transferred to a nitrocellulose membrane. The membrane will next be probed with a primary antibody, followed by a secondary antibody linked to an Alexa Fluor molecule, allowing for the quantification of the resultant fluorescence. Primary antibodies will be specific to subunits of NF-kB, AP-1, AP-2, NF-AT, and p53 transcription factors, as well as to FAK and the Ras and Jab family proteins which have been associated with HBx-related transcription factor activities [20,22].

Inhibitors of Jab/Ras Family Proteins

Cells transfected with HBV expression constructs will be treated either macrophage migration inhibitory factor (MIF) or S-trans, trans-farnesylthiosalicylic acid (FTS), which have been shown to inhibit the Jab and Ras family proteins, respectively [24,25].

Cyclosporin A Inhibition of NF-AT Activity

Cells transfected with HBV expression constructs will be treated with Cyclosporin A, which has been shown to disrupt the nuclear translocation of NF-AT as induced by HBx [26].

Assessment of Transformation

Cells will be grown in soft agar to assess whether or not they exhibit anchorage-independent growth. Transfected L-O2 cells may also be injected into nude mice, which will be killed after the tumors have been allowed to grow for at least 3 weeks [21]. Any tumors can then be removed and visualized in histological assays to confirm tumorigenesis.

Experimental Approach

The first experiments in this study will focus on the role of HBx-induced calcium signaling in the activation of the transcription factors in question. Cells will be induced to produce either HBx or their respective HBx deletion mutants, and a calcium chelating agent (BAPTA-AM) will be added to prevent the upregulation of cytosolic calcium. These cells will then be assayed by means of a Western blot to establish the levels of AP-1, AP-2, NF-AT, NF-kB, p53, and TF regulatory proteins present.

Next, the patterns of TF activity for HBx transfected cells and deletion mutants will be assessed by means of Western immunoblotting without any addition of calcium chelating agents. Cells transfected with replication-competent HBV expression vectors will also be assayed by the same means. These results can then be compared to BAPTA-AM+ TF expression patterns to understand the role of calcium signaling in HBx-induced TF regulation.

After initial TF expression patterns have been established, experiments involving the transformation of cells will be conducted, so as to determine the relationship between TF patterns and oncogenesis. L-O2 cells will be transfected with either the WT or HBx-deficient HBV expression constructs, and will be cultured for an appropriate time period. These cells will then be suspended in soft agar or injected into nude mice, so that after a period of several weeks these two sources may be examined for the presence of transformed tumor cells.

These same two strains of transfected cells will also be treated with MIF, FTS, or both of these compounds so as to inhibit the function of the Jab/Ras family proteins respectively. Since HBx has been found to regulate certain TFs through Jab/Ras-dependent pathways, inhibiting these proteins will inhibit that specific TF activity. These cells will first be analyzed by Western immunoblots to confirm the downregulation of AP-1and NF-kB (mediated by Jab and Ras proteins, respectively) as compared to cells not treated with these compounds. The cells will then be assayed for transformation in soft agar or nude mice, as previously described.

An experiment similar to that described above will also be performed using Cyclosporin A, an immunosuppressive drug which is known to inhibit NF-AT activity as induced by HBx. Assessment of the tumor formation of Cyclosporin A-pretreated cells will be conducted as with the MIF/FTS treated cells. All three compounds will be used simultaneously on cells as well so as to observe the transformative capacity of HBx in the absence of its upregulation of these three major cellular TFs.

The final experiments will consist of transfecting cells with both the HBx-deficient HBV expression construct and one of the HBx deletion mutant plasmids simultaneously. After selecting for the successfully transfected cells and establishing their functionality through PCR, these viral constructs will be tested for successful viral replication based on a plaque assay. Western blots of all the aforementioned proteins will be performed for each strain of deletion mutant transfected HBV-expressing cell to confirm TF expression patterns in the context of deletion mutant whole virus replication. These cells will then be assessed for their transformative capacity by suspension in soft agar and by injection into nude mice. They will also be exposed to MIF/FTS/Cyclosporin A and assessed for cellular transformation. This will end the experimental procedures of this study.

Results

The results of the experiments involving calcium chelation are expected to confirm that HBx-induced expression of AP-1 is not dependent on calcium signaling as has been previously described [22]. Additionally, these results will ideally demonstrate that the expression of FAK is dependent on calcium signaling, another previously demonstrated characteristic of HBx transfected cells [19]. The results will also establish whether or not calcium signaling is important to the expression levels of AP-2, NF-AT, NF-kB, p53, Jab, and Ras family proteins; results which have not been previously published. These data, when compared with data of BAPTA-AM- cell lysates, will serve as a baseline in the study of patterns of TF expression and will determine whether or not the calcium signaling induced by HBx needs to be taken into account when considering the TF activation in HBV infected cells.

Additionally, these preliminary data will suggest which areas of the HBx protein are critical to the upregulation of certain TF activities, as different HBx deletion mutants will not exhibit the same TF patterns, and thus the deleted amino acid sequences can be predicted to be integral to the activation pathways of the TFs which exhibited altered expression patterns. Cells transfected with replication-competent HBV constructs will serve to confirm that these observed patterns hold true in the context of whole virus replication, confirming that they are not an artifact of HBx transfection. As HBx is important both for viral and transformative activity, it is expected that L-O2 cells transfected with an HBx-deficient HBV construct will not be transformed at a significant rate, whereas those transfected with a WT-HBV construct will have higher rates of transformation relative to controls.

With the addition of FTS/MIF, it is expected that there will be a significant decrease in HBx-associated upregulation of AP-1 and NF-kB, as the mechanisms of HBx’s interaction with these two TFs will be blocked by these inhibitors. Adding these compounds in concert with cyclosporin A will provide a broader view of the roles these three TFs (AP-1, NF-kB, NF-AT) play; the results will allow for the assessment of the transformative capacity of HBV in the absence of these TF upregulation mechanisms, and will thus provide insight into the role of these TF pathways in the production of transformed HCC cells. Adding all three compounds together may result in widespread TF downregulation, potentially resulting in deleterious effects for cells, however this will need to be experimentally assessed. Cells will be simultaneously treated with a minimum of two of these compounds to assess the roles of their respectively inhibited TFs in transformative processes.

The final experiments will serve to synthesize the results of the previous experiments in order to establish the validity of the previous findings in the context of HBV replication. By transfecting cells with both HBx deficient HBV expression vectors and HBx deletion mutant plasmids, it will be possible to induce cells to both produce normal HBV proteins and abnormal HBx proteins simultaneously (alternatively, new plasmids may be engineered which inherently contain deletions or point mutations in the HBx sequence). Western blots of the TFs and related proteins of interest in these cells will seek to confirm the previously derived TF expression patterns, much as the assessment of transformative capacity both in the presence and absence of FTS/MIF/cyclosporin A will confirm the validity of the previously determined transformative abilities of the differently pretreated HBx expressing cells.

In summary, these concluding experiments will endeavor to determine to what degree specific TF pathways play critical roles in the transformation process by selectively altering which pathways are active using inhibitory compounds and HBx deletion mutants. While it is wholly possible that these compounds/mutants will impair other aspects of viral function important to oncogenesis, these experiments will still serve as important indicators of foci for future studies of HBx induced changes in TF activity and the role those changes play in hepatocarcinogenesis.

Discussion

Though not definitive, the results of these experiments will yield important knowledge regarding the mechanisms of HBV-induced hepatocarcinogenesis. From these deletion mutant experiments, it will be possible to correlate HBx sequences to the upregulations of the TFs of interest in the context of both HBx expression and of whole HBV replication. These patterns of TF activity will be indicative of which factors may be important to the transformation process, allowing for future studies to examine more comprehensively the roles they in oncogenesis.

It may prove difficult to analyze the results of these experiments as oncogenic processes are generally multi-faceted, and thus even if one mechanism is downregulated others may remain active. Nonetheless, it is important to complete this study and to carefully analyze the data, seeking any statistically significant differences in transformative rates among the surveyed cell strains and pretreatment conditions; doing so will ultimately prove necessary to the establishment of future related areas of study.

Based on the outcomes of these experiments, the avenues for prospective future research are variable. For one, though studied by means of Western immunoblots, more investigation needs to be done regarding the role of the AP-2 TF complex in the transformative process. Additionally, luciferase reporter gene assays may be used to determine which TF-regulated genes are active in transformed as opposed to in non-transformed hepatocytes. To produce greater external validity, these experiments may also be repeated in primary rat hepatocytes; this will likely provide more applicable results regarding the role of HBx-induced TF activation in hepatocarcinogenesis.

While these experiments alone will not answer the complex question of how HBV induces HCC, they will shed light on one mechanism of HBx-induced oncogenesis, and as such these results will ideally serve as a stepping stone for more in-depth future studies, with the ultimate goal of producing drugs which can effectively combat these oncogenic mechanisms, saving countless lives.

References

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