Journal of Hepatology
Volume 32, Issue 2 , Pages 344-351, February 2000

The potential of gene therapy in the treatment of hepatocellular carcinoma

  • Cheng Qian

      Affiliations

    • Division of Hepatology and Gene Therapy, Department of Medicine, School of Medicine and Clinica Universitaria, University of Navarra, Pamplona, Spain
    • ,
  • Marek Drozdzik

      Affiliations

    • Division of Hepatology and Gene Therapy, Department of Medicine, School of Medicine and Clinica Universitaria, University of Navarra, Pamplona, Spain
    • ,
  • Wolfgang H Caselmann

      Affiliations

    • Corresponding Author InformationWolfgang H. Caselmann, Dept. of Medicine I, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Tel: 49 228 287 5511 or 5507. Fax: 49 228 287 4698.
    • Department of Medicine I, University of Bonn, Bonn, Germany
    • ,
  • Jesús Prieto

      Affiliations

    • Division of Hepatology and Gene Therapy, Department of Medicine, School of Medicine and Clinica Universitaria, University of Navarra, Pamplona, Spain

Received 11 June 1999; received in revised form 25 August 1999; accepted 10 September 1999.

Article Outline

 

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide. It is the cause of death of around 1 000 000 people every year. This malignant tumor develops on a background of chronic hepatitis and/or cirrhosis in about 90% of cases (1). Surgical treatments such as resection or orthotopic liver transplantation are potentially curative, but yield best results in patients with small and localized HCCs 2., 3.. In contrast, locoregional therapies, e.g. percutaneous ethanol injection (4) or transarterial chemo-embolisation (5), are mainly performed with palliative intention. Conventional chemotherapy (6), chemohormonal treatment (7) and conformal radiotherapy have been shown to be ineffective in HCC (8). Patient survival after onset of symptoms is dismal. Therefore, new therapies are urgently needed. In this review, we will discuss novel approaches using gene delivery and gene therapy strategies to treat HCC that have been used to date mainly in pre-clinical experimental settings.

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Delivery Systems 

Efficient gene transfer is essential for successful gene therapy. A number of gene transfer vectors have been developed 9., 10., 11.. There are two major kinds of vectors, viral and non-viral vectors.

Viral vectors 

So far, viral vectors are the most efficient means for the transfer of foreign genes into target cells. Retroviral vectors transduce dividing cells with high efficiency in in vitro cell culture and stably integrate into the host genome. This property allows retroviruses to be used for in vitro transduction of tumor cells which can afterwards be injected into the host. This ex-vivo gene transfer method has been used to transduce HCC cells very efficiently with different foreign genes 12., 13., 14., 15.. One important disadvantage of retroviruses is the low titer of the vector which limits in vivo gene transfer using retroviruses.

Adenoviral vectors transduce dividing and non-dividing cells in a variety of tissues with high efficiency. Adenoviral vectors can infect normal hepatocytes in vitro and in vivo. These vectors have been used to transduce foreign genes into human HCC cells, liver tumor cells from transgenic mice, carcinogen-induced HCC cells in rats and virus-induced HCCs in woodchucks 16., 17., 18., 19., 20., 21., 22., 23.. In contrast to retroviral vectors, adenoviral vectors do not integrate their genome into host chromosomes. Therefore, the expression of the transgene is only transient. To overcome the poor vector distribution within solid tumors E1b 55 kDa attenuated, replication-competent adenoviral vectors were developed, which display additional oncolytic effects which may potentiate the cytotoxic effect (24).

Another disadvantage of adenoviruses is that they induce immune responses against adenoviral antigens expressed on transduced cells. This results in a limited duration of transgene expression, but may also exert an additive effect in genetic immunotherapy. The short-term expression of the therapeutic gene could nevertheless be sufficient for treatment of cancer, since after killing of tumor cells with suicide genes or after stimulation of an antitumor response with cytokines, expression of the transgene may no longer be needed. In addition, a recent study showed that neutralizing antibodies to adenovirus did not reduce transgene expression in the tumor cells when the adenoviral vector was repeatedly injected (tolerization) locally into the tumor nodules, while they markedly reduced the access of the virus to liver tissue (25).“Gutless” adenoviruses containing exclusively the packaging signal and ITR as well as the adenoviral E1 gene which is disrupted by transgene insertion, do not express any viral proteins and should therefore be devoid of immunogenicity (10). Finally, immunosuppressive treatment with FK506 or other agents may help to suppress host immunity sufficiently (26).

Adeno-associated virus (AAV) is a safe vector, because the virus is naturally non-pathogenic and replication deficient. Previous work has shown that rAAV vector can transduce a variety of cells, including liver cells in animals. However, rAAV transduces some kinds of cells with low efficiency in the absence of helper virus (27). It has been reported that treatment of cells with DNA-damaging agents such as etoposide and γ-irradiation, caused a marked increase in vitro cell transduction by rAAV (28). This enhancement was related to conversion of single-stranded AAV DNA into transcriptionally active double-stranded forms of the AAV vector (27). Our studies have shown that rAAV can infect human, rat and mouse HCC cells and that adenovirus or DNA damaging agents (γ-irradiation and etoposide) are very efficient in enhancing transgene expression in vitro and in vivo (29). Su et al. also demonstrated that AAV can transduce human HCC cells in vitro (30).

Lentiviral vectors have emerged as a promising new tool in gene therapy. They are able to infect both dividing and non-dividing cells. A sustained expression of transgene could be obtained in the liver after injection of this vector into rodents (31). Hepatitis B virus (HBV) has been investigated as a carrier for transfer of therapeutic genes and transgene could be expressed in HCC cells based on this vector (32). Baculovirus has been used as a vector for efficient delivery of genes into cultured cells with a strong preference for hepatocytes of different origin with high level of expression (33). The potential of reconstituted Sendai viral envelopes containing only the fusion glycoprotein (F-virosomes) in targeted delivery of reporter genes to liver cells has recently been demonstrated in mice (34).

Non-viral vectors 

Apart from vectors based on recombinant viruses transgenes can be introduced into plasmids and these can be administered as naked DNA or complexed to different compounds such as cationic liposomes or polylysine. Naked DNA plasmids can be taken up efficiently by muscle cells. For the transduction of other cells plasmid DNA should be complexed to facilitate incorporation and expression into the cells. One strategy to provide non-viral vectors with affinity for certain tissues or organs is the construction of complexes of DNA with ligands for specific receptors such as the asialoglycoprotein receptor, whose expression is limited to hepatocytes and HCC cells. Other ligands that have been used in similar conjugates include insulin, lectins and transferrin. Similarly, antibodies could be incorporated to liposomes to endow these vectors with the tropism conferred by the antibody 35., 36.. The recently developed “gene gun” device uses DNA-coated gold particles that are accelerated by pressurized helium gas to supersonic velocity for DNA transfer into living cells (37).

Non-viral vectors offer some advantages as compared to viral vectors. They can transfer larger pieces of DNA of interest, they are weakly immunogenic and easier to use and to produce than viral vectors. However, a disadvantage is that gene transfer efficiency is low and the expression of the transgene is of short duration (35).

The choice of the appropriate vector depends on characteristics of target cells or tissues and specific gene therapy strategies that will be adopted.

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Selectivity of Transgene Expression 

A crucial issue of gene therapy is to deliver (tissue targeting) and activate (transcriptional targeting) a therapeutic gene to neoplastic tissue, without affecting healthy cells. Tissue targeting can be accomplished by direct intratumoral injection of the vector. Our data and other reports suggest that after intralesional adenoviral transfer, expression of the transgene is confined to an area adjacent to the tract of the injecting needle (21). Other options comprise the use of retroviral vectors, which preferentially target cancer cells by their specific integration into proliferating cells (38).

A practical system to ensure tumor-restricted expression of the transgenes is the use of tumor-specific promoters. Alpha-fetoprotein (AFP) is overexpressed in about 60 to 70% of HCCs. Several groups have used an AFP promoter or/and enhancer to control foreign gene expression in different vectors. Arbuthnot et al. 12., 17. demonstrated that retroviral and adenoviral vectors containing the lacZ reporter gene under the control of AFP regulatory sequences resulted in the specific expression of the lacZ gene only in HCC cells with AFP expression. Similar results were reported by Kanai et al. (39) who demonstrated that expression of the herpes simplex virus thymidine kinase (HSV-tk) gene by adenovirus from AFP promoter/enhancer induce the cells to be sensitive to ganciclovir (GCV) in the AFP-producing cells. This is also the case in adenoassociated virus suicide gene transfer under the control of AFP enhancer (30). Moreover, the dose required to kill cancer cells was inversely proportional to the level of AFP expression in the cells (30). The expression of genes driven by AFP promoter can be increased by glucocorticoids through a glucocorticoid-responsive element in the AFP promoter. Ido et al. (40) observed that addition of dexamethasone to HCC cells expressing HSV-tk driven by human AFP promoter increased the cytotoxicity of acyclovir.

Bui et al. (19) demonstrated that direct delivery of interleukin-2-recombinant adenoviruses in which transgene expression was controlled by the AFP or cytomegalovirus promoter induced HCC growth retardation in all treated animals; some of them had even complete tumor regression. These data demonstrate that selective expression of transgenes in tumor cells not only improves the outcome of the treatment, but also reduces systemic toxicity; this results in a much higher therapeutic index.

Tumor targeting can also be achieved by use of tumor-reactive, high-affinity monoclonal antibodies, such as AF-20 which recognizes a rapidly internalized 180 kDa cell surface glycoprotein that is abundantly expressed on HCC (41). Coupling to cholesteryl-spermidine allowed highly specific and efficient nonviral target gene delivery to AF-20-positive cells.

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Strategies of Gene Therapy of Hepatocellular Carcinoma 

There are numerous gene therapy strategies that have been applied for the treatment of cancer (42). They include gene replacement (e.g. tumor suppressor genes), and antisense strategies (to inhibit oncogene expression and to reestablish apoptosis), drug sensitization (suicide genes), genetic immunotherapy (cytokines, costimulatory molecules, polynucleotide vaccination), and interventions to interfere with the biology of the tumor growth (antiangiogenesis).

Gene replacement 

Replacing nonfunctional tumor suppressor genes would be an obvious strategy, particularly in tumors in which specific defects have been determined. Loss of wild-type p53 function either by its mutation or inactivation, results in the formation of a malignant tumor (43) and enhances tumor cell resistance to a variety of chemotherapeutic agents (44). Our studies have shown that liposome-mediated gene transfer of wild-type p53 to human HCC cells inhibited growth rates in HCC cells with p53 mutation or deletion (45). Similar findings were reported by Xu et al. using retrovirus carrying wild-type p53 (15). In addition, these authors found that transduced cells were sensitive to the chemotherapeutic drug cisplatin. Anderson et al. showed that daily hepatic artery dosing of adenovirus expressing p53 gene suppressed tumor growth in a rat model of HCC (46). These data suggest that gene replacement therapy with p53 is a promising modality to treat advanced stages of HCC.

To inhibit tumor growth completely using gene transfer of p53, a transduction efficiency of nearly 100% for tumor cells would be required. However, available gene transfer vectors do not allow this efficiency to be reached. Nevertheless, introduction of wild-type p53 to neoplastic cells suppressed transcription of vascular endothelial growth factor (VEGF) (47), which reduces neoangiogenesis within a tumor. Wild-type p53 also induced production of thrombospondin-1, a potent inhibitor of angiogenesis (48). These effects may account for antitumoral effects observed in non-transduced neoplastic cells. However, this approach may still not be sufficient to cure tumors, since it was recently shown in MDA-MB468 breast cancer and KM12SM colorectal carcinoma cells that mutations in the exogenously delivered wild-type p53 gene and its excision by cellular enzymes abrogated efficiently wild-type p53 function (49). Finally, p53 mutations or inactivations may only be one defect the cell encounters on its development towards the malignant state.

Antisense strategies 

Direct targeting of causative oncogenes can be another option for treatment of HCC. Transduction of HCC cells with retroviruses carrying antisense RNA directed against the N-ras oncogene resulted in an inhibition of tumor cell growth in vitro and a decrease of tumorigenicity in nude mice (14). Stable transfection of HCC cells with plasmids containing antisense RNAs targeted against fibroblast growth factor 2 (FGF-2) mRNA led to the inhibition of FGF-2 synthesis and was associated with a loss of tumorigenicity in nude mice (50). Also, antisense inhibition of insulin-like growth factor I expression, which is involved in the maintenance of the transformed phenotype of HCC, was shown to restore the significantly decreased apoptotic capability of transformed hepatocytes (51). Therefore, antisense targeting of oncogenes or growth factors may be considered useful for gene therapy of liver cancer. This approach is restricted by the difficulties of exact targeted delivery to the tumor-bearing organ and the half-lives of antisense therapeutics which usually allow only transient inhibition of gene expression. This technique may therefore be developed to represent an additional therapeutic tool in combination with standard surgical or chemotherapeutic measures.

Drug sensitization 

The principle of this procedure is to deliver a suicide gene encoding a foreign enzyme that transforms a nontoxic prodrug into a toxic compound in transfected cells (52). One key feature of this system is the “bystander effect”, by which complete tumor regression can occur after GCV administration even when only 10% of tumor cells had been transduced with herpes simplex virus-thymidine kinase (HSV-tk) (52). Both the transfer of the toxic compound to adjacent untransduced tumor cells via phagocytosis of apoptotic particles or gap junctions and the involvement of immune system may contribute to the “bystander effect” (52).

The HSV-tk/GCV system has been used to kill HCC cells in vitro (16). This system was found to be effective in eliminating HCC tumors implanted subcutaneously in mice and in inducing regression in a model of multifocal HCC induced in rats by administration of diethylnitrosamine 20., 21.. Although these studies demonstrated the efficacy of HSV-tk/GCV system in treating HCC, they also showed that this procedure may cause serious liver toxicity 20., 53.. This toxicity could be controlled by adjusting the dose of the vector or by injecting the vector directly into the tumor nodule rather than using the intravascular route. On the other hand, several groups have employed AFP promoter and enhancer to control HSV-tk expression by retroviral and adenoviral vectors 39., 40., 54.. These vectors can successfully transduce AFP-producing HCC cells and make these tumor cells sensitive to GCV. Using tumor-specific promoters the expression of suicide genes can be restricted to tumoral cells. This strategy allows an efficient antitumoral effect without the toxicity from transduction of normal liver cells as it occurs when employing universal promoters.

There are several other suicide genes that can be used for treatment of HCC. One is cytosine deaminase (CD) from Escherichia coli that converts 5-fluorocytosine to 5-fluorouracil and has been used for successful treatment of HCC in the murine model (55). Another suicide gene is varicella-zoster virus thymidine kinase (VZV-tk) that transforms nontoxic prodrug 6-methoxypurine arabinonucleoside into the cytotoxic adenine arabinonucleoside triphosphate (13). Drug sensitization is definitely a potent means of tumor treatment. If tumor-specific expression can be achieved and if systemic side effects can be kept low, it may become a useful clinical therapy.

Genetic immunotherapy 

Accumulating evidence suggests that failure of the immune system to eliminate a tumor is due to the inability of tumor antigens to stimulate an effective immune response (56). Possibilities for such failure include the loss of MHC class I or co-stimulator B7 expression on tumor cells, the aberrant presentation of tumor antigens, or the inhibition of the immune system by factors secreted from or expressed on tumor cells (56). Many cytokines (IL-2, IL-4, IL-6, IL-7, IL-12, γ-INF, TNF, GM-CSF) have been employed for stimulation of immune responses against tumors (57). Murine HCC cells infected in vitro with retroviral vectors expressing TNF-alpha lost their tumorigenicity and induced anti-tumor immunity. Intratumoral administration of retroviruses resulted in a significant prolongation of survival (58). We have found that mouse HCC cells engineered to express IL-12 by retroviral vectors lost their tumorigenicity. Treatment of established tumor with irradiated IL-12-expressing tumor cells or IL-12 retroviral vector significantly inhibited tumor growth (59). Huang et al. (60) demonstrated that long-term remission was achieved in 75% of the animals with primary and disseminated HCC after treatment with adenovirus expressing IL-2 and surviving animals developed antitumoral immunity. The importance of B7 in antitumoral immunity is illustrated by reports showing that immunization of mice with B7-expressing tumor led to the regression of established B7-negative tumors. Tatsumi et al. (61) showed that transfection of HCC cells with the B7-1 gene substantially augmented primary cytolytic activity against parental human HCC cells and resulted in a significant tumor rejection or at least inhibition of tumor growth in a mouse model (62). We similarly found that mouse HCC cells partially lost their tumorigenicity after transduction with a retroviral vector encoding B7-1 (59).

Antigen-presenting cells (APCs) may be of central importance for the generation and regulation of tumor immunity (63). Dendritic cells (DC) are very efficient APCs which can be manipulated artificially to present tumor antigens by either pulsing them with tumor peptides or tumor extracts, by fusing them with tumor cells or by transfer of genes encoding the relevant tumor antigen using viral or non-viral vectors (63). Administration of these manipulated DCs can promote potent antitumor immunity. In rats immunized with hybridoma cells produced from fusion of HCC cells with activated B cells not only is a protective effect on parental tumor cell rechallenge induced but the pre-established tumors are also cured (64).

Future strategies comprise the use of polynucleotide vaccines against tumor-associated antigens, which have successfully been used for carcinoembryonic antigen in colorectal carcinoma of mice (65) and in nonhuman primate models (66). Very recently Vollmer et al. used genetic immunization strategies such as dendritic cells engineered to express AFP and plasmid-based vaccination to generate potent T-cell responses which altered tumor growth or induced HCC rejection in a mouse model. In addition, a 9-mer peptide derived from human AFP was also shown to efficiently generate a T-cell response by the same group 67., 68.. This interesting approach may mainly be useful for the treatment of small tumors or the prevention of HCC development in premalignant conditions. Intracellular antibodies that can bind and reverse oncogenic effects as recently shown for intracellular scFv anti-ras that was able to produce sustained regression of local HCT116 colon carcinoma cells (69) may have implications for both tumor therapy and targeting, but its value can at present not yet be judged.

Antiangiogenesis 

Since tumor cells are genetically diverse and unstable, the above strategies may not achieve the complete elimination of all tumor cells. In contrast to neoplastic cells, endothelial cells in tumoral vessels are genetically more stable and less likely to develop resistance to therapeutic procedures. A large number of angiogenic factors are implicated in tumor vascularization that is essential for supporting growth of tumors. Strategies aimed at blocking these factors can reduce tumor growth rate. It has been reported that antisense oligonucleotide against VEGF mRNA was able to reduce tumor growth rate of glioblastoma (70). Another promising approach is to target the two VEGF high-affinity receptors expressed on endothelial cells, flt-1 and KDR/Flk-1. Genetically modified tumor cells producing native soluble flt-1 can inhibit VEGF by sequestering this substance and also by forming inactive heterodimers with membrane-spanning VEFG receptors. It has been shown that survival is prolonged in those tumor-bearing mice which received these genetically modified tumor cells (71). Recently Lin et al. (72) used recombinant adenovirus to deliver a soluble Tie2 receptor (endothelium-specific receptor tyrosine kinase that has crucial roles during the development of the embryonic vasculature) that blocked activation of the Tie2 receptor on endothelial cells. Treatment of tumor-bearing animals with this vector not only significantly inhibited primary tumor growth, but also almost completely inhibited neovascularization and growth of metastasis (72). Another strategy explores natural antagonists to angiogenesis, such as angiostatin and endostatin 73., 74.). They act by selectively inhibiting endothelial cells to respond to angiogenic signals. When given to mice bearing transplanted murine or human tumors, they cause marked regression of the tumors to microscopic dormant foci 73., 74.. Gene transfer of angiostatin by retroviral and adenoviral vectors has been used for treatment of different tumors in animal models (75). To our knowledge, no studies based on an antiangiogenic approach have been performed in HCC. Whether isolated respective cDNAs retain their biological activity in humans remains to be discovered and will determine the use of this concept in clinical therapy in the future.

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Clinical Protocols 

Only a few of the above-mentioned experimental strategies for the treatment of HCC are currently being investigated in clinical medicine. Habib and his group performed a phase I clinical trial, in which patients with HCC were injected intratumorally with naked wild-type p53 plasmid DNA (76). Decrease of serum AFP concentration and reduction of tumor mass was documented in some patients, including one case of complete tumor regression. There was evidence for an immune-mediated bystander effect (77). Another clinical protocol was directed by Venook et al. 78., 79., in which patients with HCC were treated by injections of recombinant adenovirus carrying wild-type p53 gene through the hepatic artery. Although approved in 1996, the results of this trial are not yet available. This may reflect the difficulties encountered, when research protocols evaluated in small animals are transferred to the clinical setting.

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Prospects for the Future 

Gene therapy is a powerful tool which holds promise for the treatment of many forms of diseases including cancer. However, many problems need to be solved in practice before gene therapy becomes a useful and efficient therapy to be applied routinely in patients with HCC. We need a better understanding of the biology of tumor lesions and of the mechanisms by which the HCC evades host defenses. We need to increase the therapeutic window of different gene therapy modalities (especially of those based on suicide genes and immunostimulatory substances) by reducing toxicity and improving specific targeting to the tumor. Finally, we need to obtain a better transduction efficiency to deliver potent transgenes to hepatic tumors. Recent studies have pointed out features of the tumor vasculature which result in the formation of a blood-tumor barrier, thereby preventing diffusion of the vectors from the capillary lumen to the neoplastic cells 22., 80.. This impermeability of the malignant tumor to gene therapy vectors should be considered in the elaboration of clinical protocols for the treatment of solid neoplasms.

In the future, new vectors will be generated, new tumor-specific promoters will be used for tumor targeting and new gene therapy approaches will be developed as efficient means to manipulate the biological features of tumors and to stimulate anti-tumor responses. These achievements may enable us to offer improved clinical treatment to HCC patients in the future.

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PII: S0168-8278(00)80082-3

Journal of Hepatology
Volume 32, Issue 2 , Pages 344-351, February 2000

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