Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo

Despite the many advances achieved in cancer prevention, much improvement is desired for present treatment protocols, including surgery, chemotherapy, and radiotherapy. These procedures may be effective in the early stages of disease, but are always palliative and fall short of eradicating the various malignant subpopulations in neoplastic conditions [1]. The current realities have highlighted the need for more novel cancer therapies to induce effective responses in clinical trials. Gene therapy is a promising strategy for patients resistant to traditional therapies because they target defects in malignant cells, selectively correcting or eradicating defective tissues [2]. However, efficacy and specificity remain major challenges for existing cancer gene therapy [3]. Oncolytic virotherapy is an attractive drug platform of cancer gene therapy consistent with the both goals [4, 5]. The oncolytic viruses exhibit an ability to replicate selectively in tumor tissues to the exclusion of normal cells [5]. Furthermore, they can be genetically manipulated to express multiple cancer cell-specific therapeutic elements [6]. These characteristics demonstrate the utility of oncolytic virotherapy in the clinics and provide the basis for novel approaches to cancer gene therapy.

Adenovirus-based vectors are the most widely used platforms in gene delivery [5]. However, non-replicating adenoviruses are seldom effective in eradicating tumor cells [7]. Therefore, very high concentrations and multiple administrations are generally needed to produce significant anti-tumor responses; such regimens, however, often induce anti-viral immune responses that result in the neutralization of the viral vectors in subsequent immunizations and toxicity to the tissues [8, 9]. To circumvent these limitations, conditional replication-competent adenoviruses (CRCA) have been developed and are being extensively evaluated; these viruses replicate specifically in tumor cells with subsequent oncolysis and release of virus progeny to further infect and destroy neighboring cancer cells [7, 10]. Furthermore, it has been recently suggested that antibodies which neutralize replication-incompetent adenoviruses have limited effects on the replication-competent adenoviruses [7, 11]. It is therefore reasonable to anticipate that replication-selective tumor-specific adenoviruses would have potent effects in cancer gene therapy.

In this study, we constructed an oncolytic adenovirus using a cancer-specific promoter (human telomerase reverse transcriptase promoter) and a cancer cell selective apoptosis-inducing gene (Apoptin) that demonstrated significant anti-tumor activity toward solid tumors and metastatic nodules. Telomerase activity is strikingly higher in about 90% of cancers than in normal cells, and it has been widely used as a tumor marker. One of the three subunits of telomerase, human telomerase reverse transcriptase (hTERT), is the determinant of the telomerase activity and is highly active in immortalized cell lines and over 85% of human cancers. Therefore, its promoter has been used for tumor specific expression of transgenes. Apoptin, the product of the chicken anemia virus VP3 gene, shows specificity and efficiency toward a wide range of transformed and malignant cells of human origin, including hepatomas, lymphomas, cholangiocarcinomas, melanomas, and breast, lung, and colon carcinomas, while sparing non-transformed primary cells such as fibroblasts, keratinocytes, or smooth muscle cells [12‐14]. Preliminary studies have demonstrated the effect of Apoptin inserted in various vectors on restricting manifold tumors, which make it attractive for cancer gene therapy [15, 16].

In this study, we focused on melanoma, which has doubled over the past decades and the incidence is increasing more rapidly than any other cancer [17]. Although melanoma skin cancer can be cured by surgical excision and efforts have been carried out to develop new therapies, the prognosis of patients are poor and the 5-year survival rates range from merely 10% to 50% [17, 18]. Moreover, the patients with regional lymphatic or metastatic disease respond poorly to conventional radiation and chemotherapy [18]. Here, we describe the construction of a CRCA, hereafter referred to as Ad-hTERT-E1a-Apoptin, that can target tumors systemically and selectively induce apoptosis both in vitro and in vivo. We showed that infection of melanoma cells with Ad-hTERT-E1a-Apoptin resulted in a significant induction of apoptosis and induced growth suppression of melanoma cells. To determine whether Ad-hTERT-E1a-Apoptin can cause tumor regression, we also performed multiple intratumoral injections or systemic administrations of Ad-hTERT-E1a-Apoptin in subcutaneous homoplastic graft or lung metastases models, respectively. Our findings indicate that Ad-hTERT-E1a-Apoptin represents a potentially applicable anti-cancer agent for the treatment of primary and metastatic melanoma and may be of clinical value toward other neoplastic diseases.

Despite substantial progress in the development of gene therapies and traditional treatments in recent years, the prognosis for many patients with neoplastic diseases remain poor. Cancer gene therapy based on adenoviruses has been extensively studied in pre-clinical and clinical trials. In particular, CRCA has gained increased attention for a number of reasons [6, 7]. Because the promoters in these vectors are selective for cancer cells [6, 7], these oncolytic viruses have the ability to replicate and to spread to adjacent tumor cells [19]. Furthermore, it has been shown that infection with CRCA generates anti-tumoral immune responses [20] which can complement chemo- and radiotherapies [19]. Importantly, given the proper therapeutic transgenes, CRCA are capable of achieving destruction of primary and distant tumors [7].

In vitro studies showed that a core region of hTERT containing two E boxes and several Sp1 sites is sufficient for the major tumor-selective promoter activity. Many strategies including CRCA have been developed using the hTERT core promoter, containing two E boxes and several Sp1 sites [21], to selectively target tumor cells. One approach being evaluated uses CNHK300, a replicative adenovirus that targets telomerase positive cancer cells [22]. A similar replication-competent adenovirus, AdEHT2, in which hTERT promoter was used to control the expression of the adenoviral E4 gene, was capable of tumor selective replication and oncolysis [23]. Analogous results were also obtained in other replicating adenoviruses, such as Adv-TERTp-E1a [24], hTERT-Ad [25], and Ad/GT-Bax [26], which appear to be promising treatment agents for cancer.

The applicability of cancer therapies is not only determined by their efficiency in eliminating tumor cells; specificity is an equally important prerequisite [3]. Apoptin has such properties which could potentially achieve these objectives. Various research groups have reported that more than 70 analyzed tumor cell lines were proven to be susceptible to Apoptin whereas it does not affecting variety of normal, non-transformed cells such as human endothelial cells, hepatocytes, hematopoietic stem cells, keratinocytes, or smooth muscle cells [27, 28]. On the other hand, Apoptin become activated in SV40-transformed normal human fibroblasts or UV-irradiated cells with hereditary cancer-prone syndromes [28, 29]. Although, Guelen et al provided data of toxicity of Apoptin towards non-cancerous cells, this study proved cell death only in a fetal cell type and not in other non-transformed cell types [28, 30]. However, the safety of apoptin is underlined by the fact that continuous expression of Apoptin under the H2-Kb promoter in transgenic mice does not interfere with lymphocyte development and proliferation [28, 31]. While the mechanism by which Apoptin is able to distinguish between tumor and normal cells remains unclear but seems to correlate with its cellular localization. Recently, it was shown that Apoptin-induced apoptosis essentially depends on abnormal phosphatidylinositol 3-kinase (PI3-kinase)/Akt activation, resulting in the activation of the cyclin-dependent kinase CDK2 [32]. Maddika et al indicated that inhibitors of PI3-kinase or Akt not only inhibited CDK2 activation but also protected cells from Apoptin-induced cell death, and Akt-mediated activation of CDK2 was caused by direct phosphorylation as well as by the phosphorylation-induced degradation of its inhibitor p27 (Kip1) [32]. They also identified CDK2 as the principal kinase that phosphorylates apoptin and is crucially required for apoptin-induced cell death [32]. Besides the tumor-selective destruction properties, Apoptin has several important features indicating its application as a novel antitumor agent. One of these characteristics is the ability of Apoptin to induce tumor-specific apoptosis independently of p53 [28, 33]. Thus, apoptin is similarly effective in killing tumor cells that are p53-deficient or either express wildtype or mutant p53 [28, 33]. Although the role of anti-apoptotic molecules such as bcl-2 in Apoptin-induced apoptosis is still a matter of debate, another important feature of Apoptin is that in certain tumor cell lines it mediated cell death is independent of the Bcl-2 status and is even stimulated by bcl-2 or insensitive to bcr-Abl and bcl-xl [28, 29, 34]. The main controversy as to the role of bcl-2 may focus on the involvement of Nur77 that can bind to bcl-2 and change its properties from an anti-apoptotic to a proapoptotic molecule [28]. Accordingly, the different expression levels of Nur77 in various cell types might explain the opposite effects of Bcl-2 on Apoptin induced apoptosis. Based on these concepts, it therefore reasonable to anticipate that Apoptin can be used to complement radiotherapeutic and chemotherapeutic approaches.

More than a quarter (27%) of human gene transfer protocols registered with the the Recombinant DNA Advisory Committee (RAC) use adenovirus vectors [35]. The most extensively used first generation human adenovirus (hAd) vectors are replication-incompetent viruses deleted in early region 1 (E1A and E1B) genes. E1 genes that are expressed rapidly upon adenovirus penetration into host cells, are responsible for inducing expression of further viral genes, orchestrating modifications of cellular gene expression and protein activity to favor viral replication [36]. Two genes of this group, E1A and E1B, act in inactivating tumor suppressor Rb and p53 genes that are frequently mutated in cancer cells [7]. Following the generation of E1 deleted hAd vectors, others hAd vectors (e.g., E1 & E3, E2, E4, E2 & E4, or E1, E2 & E4-deleted vectors) were constructed [37]. The products of the E3 gene have been described as nonessential to viral infection and play a role in modulation of the host immune response against virus-infected cells [36]. E2 and E4 genes are involved in multiple processes, such as transcriptional regulation, DNA recombination and virus assembly [36]. Various deleted strategies such as ONYX-015 (dl1520) [38] have been evaluated to be effective in in vitro and animal models and led to clinical trials [8, 19, 39]. Until a fatality case and other reports of inflammation related to adenovirus vector, the use of adenovirus-mediated gene transfer in humans was thought to be fairly benign [35, 40]. Because of the short circulatory half life of naked adenoviruses and the neutralizing antibodies existed in most adults, attempts to increase antitumor efficacy through the administration of high doses of adenovirus vectors can lead to liver toxicity and immune response [41‐43]. For these reasons, Ad has seen limited clinical use as a systemically administered gene therapy vector. To overcome the potential increased toxicity and reduced vector efficacy during the application of adenovirus vectors, it is important that identifying means to evade innate and pre-existing immunity is a major necessity. Current strategies include use of alternative adenovirus serotypes, modification or chimerism of capsid hexon proteins, generation of hybrid vectors that combine viral and non-viral elements, coating virus with PEG or similar polymers, targeting adenovirus to specific organs, tissues or cell types and so on.

We also observed anti-tumor activity in vivo in primary and metastatic tumors, which confirmed and extended the results of the in vitro studies. Although the infection of Ad-hTERT-E1a-Apoptin did not lead to complete elimination of the tumors, effective inhibition was observed in both primary and metastatic tumor models. It is plausible that the application of the hTERT promoter allows the adenovirus replication, viral dispersion and transgene expression in any tumor tissues in the animal, regardless of receiving intratumoral injections or not. Additionally, apoptotic tumor cells may trigger dendritic cells to process and present cancer-specific antigens to responding T lymphocytes, resulting in a cytotoxic response and inducing apoptosis in the unaffected tumor cells [44]. Ad-hTERT-E1a and Ad-CMV-E1a also inhibited primary transplantated tumors, but the effects on metastatic tumors were very limited. In the in vivo experiments described here, we did not observe any toxic effects after injection of Ad-hTERT-E1a-Apoptin. Thus, our data indicate that there is great potential for improving the safety and efficacy of adenovirus vectors for wide application for treatment of neoplastic diseases.

Gene therapy with Apoptin offers unique advantages over current approaches for cancer therapy. The fact that Apoptin does not need a functional p53 pathway, is not hindered by the commonly occurring blockage of apoptosis by bcl-2 or bcr-abl, apparently acts downstream of most other factors, and has unparalleled potency suggests that it will be applicable to a wide range of tumors. In addition, the CRCA we engineered induces apoptosis selectively in various cancer cells without adverse effects on normal cells. Furthermore, the in vivo and in vitro data of this study show that the CRCA expressing Apoptin is more effective but also show that the adenovector dependent on the strong hTERT promoter does not reduce the efficacy of the strategy, thus showing an improvement in the global safety of the CRCA. All these highlights warrant further evaluation for implementation of this strategy in clinical trials.