Conditionally replicating adenoviruses carrying mda-7/IL-24 for cancer therapy

Abstract

Background. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) suppresses growth and induces apoptosis in a broad range of human cancers without significant cytotoxicity to normal cells. Conditionally replicating adenoviruses (CRAds) not only have the ability to destroy cancer cells but may also be potential vectors for the expression of therapeutic genes. Methods. This review provides an overview of specifications for a novel anti-tumor approach CRAds carrying IL-24, and discusses recent progress in this field. Results. Studies in multiple laboratories report that CRAds carrying IL-24 selectively induced apoptosis in some cancer cells, and enhanced selective toxicity to cancer cells when combined with chemotherapeutic agents. Conclusion. CRAds carrying IL-24 may prove a novel and effective approach for the treatment of cancers.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a member of the IL-10 cytokine family. Previous evidence suggests IL-24 is a promising candidate for cancer gene therapy [1]. A unique property of IL-24, when delivered by a replication-defective adenoviral vector expressing IL-24 (Ad-IL-24), is selective induction of growth suppression and apoptosis in a broad spectrum of human cancers, without significant cytotoxicity to normal cells [2]. IL-24 also has antiangiogenic, immunostimulatory, and potent “bystander” anti-tumor activity, and could sensitize tumor cells to radiotherapy, chemotherapy and monoclonal antibody therapy [3]. Past studies have shown that IL-24 may act in a variety of ways through anti-tumor effects, such as angiogenesis, changing the ratio of apoptosis protein and anti-apoptotic protein, increasing in RNA-activated protein kinase (PKR)-induced apoptosis and increasing in TNF-related apoptosis induced ligand (TRAIL) [4,5].

Clinical safety and tolerance of a replication-defective adenoviral vector expressing IL-24 (Ad-IL-24) were confirmed in a phase I clinical trial in patients with advanced carcinomas and melanomas [6,7]. However, because replication-defective adenoviral vectors are used for IL-24 delivery, relatively small amounts of IL-24 can be transduced into cancer cells. Consistently, such vector systems have not yet demonstrated an advantage over standard therapy, especially for the treatment of large solid cancers [8,9].

Conditionally replicating adenoviruses (CRAds) are a novel class of therapeutic agents in cancer treatment [10]. As novel antitumor therapeutic agents, CRAds not only selectively replicate in and lyse tumor cells, but can also amplify the expression and efficacy of therapeutic genes in the tumor microenvironment without affecting adjacent normal cells. These features represent major advantages over replication-defective adenoviral vectors [9,11]. Currently, there are three major approaches to targeting CRAds specifically to cancer cells. The first strategy involves the deletion of adenoviral genes that are necessary for viral replication in normal cells but not in tumor cells [12,13]; these include CRAds ONYX-015, which were formed by deletion of the E1B-55KD gene and lack of viral late mRNA transport. Now, studies have shown that the observed oncolysis by ONYX-015 was a p53-independent event [14,15]. The other strategy is to use tumor- or tissue-selective promoters to control the expression of early viral genes essential for replication, such as E1A gene (the first gene expressed after viral infection, the most important transcriptional activator for subsequent adenoviral gene expression) [16–18]. Another strategy is by modification of viral coat proteins function in cancer cell infection, such as the Ad5/3 capsid modification, to improve viral infection [19,20].

Previous effort (e.g., modification of the E1A and E1B regions of adenovirus) has reduced the toxicity of virotherapy. However, efficacy of the treatment is still limited [21,22]. To achieve the goal of complete elimination of tumor xenograft in animal models, a new strategy called targeting gene-virotherapy of cancer has been developed, which aims to combine the advantages of both gene therapy and virotherapy [11]. This new strategy that has been explored for bolstering the anti-tumor activity of CRAds is to “arm” them with therapeutic genes. The inserted gene can target tumor cells and multiply by several 100- to several 1000-fold in parallel with the viral replication. Thus, the expression level of the therapeutic transgene in tumors could be increased significantly to trigger tumor regression [9].

Studies in multiple laboratories report that CRAds carrying IL-24 selectively induced apoptosis in some cancer cells, and enhanced selective toxicity to cancer cells when combined with chemotherapeutic agents. In this review, we summarize the recent progresses in using CRAds carrying IL-24 as a treatment for cancers.

The anti-tumor effects of IL-24

IL-24 was first identified as a gene associated with terminal differentiation of metastatic human melanoma cells [23]. Based on its biochemical properties, IL-24 has been classified as a member of the IL-10 family of cytokines that also includes IL-10, IL-19, IL-20, IL-22, and IL-26 [24]. IL-24 expression is detected in human tissues and cells associated with the immune system, such as spleen, thymus, peripheral blood leukocytes, and normal melanocytes [25]. Secreted IL-24 stimulates monocytes and specific populations of T lymphocytes and promotes proinflammatory cytokine production. When expressed at low, presumably physiological levels, IL-24 binds to currently recognized IL-24 receptor complexes consisting of two sets of heterodimeric chains, IL-20R1/IL-20R2 or IL-22R1/IL-20R2 [26–28]. Upon binding to its receptors, IL-24 rapidly triggers phosphorylation and consequently activation of STAT3 and/or STAT1 transcription factors in target tissues, such as lung, testis, ovary, keratinocytes and skin [29]. Furthermore, although it has been noted that IL-24, IL-20, and IL-19 all activated STAT transcription factors in IL-20 receptor expressing cancer cells, only IL-24 has the ability to cause cell death [30]. A phase I trial evaluating Ad-IL-24 efficacy and toxicity by intratumoral injection in patients with advanced solid tumors indicated that IL-24 could induce as much as 70% apoptosis in tumors after a single injection of recombinant viral vectors [7,31]. More impressively, Ad-IL-24 was well tolerated in these patients. The most intriguing property of IL-24 is preferential induction of growth suppression and apoptosis in a variety of cancer cells without harming normal cells [2,3]. Additional attributes of IL-24 that make it an ideal tool for cancer gene therapy include potent “anti-tumor bystander” activity, an ability to inhibit tumor angiogenesis, synergy with radiation, chemotherapy and monoclonal antibody therapies, and immune modulatory activity [2,3].

Current status of CRAds-IL-24 therapy in cancer

Evidence suggests IL-24 is a promising candidate for cancer gene therapy. Over-expressing IL-24 with CRAds causes selective growth suppression and apoptosis of a variety of tumor cells without overt toxicity against normal cells (Figure 1). In the following paragraphs, we summarize the recent work using CRAds expressing IL-24 to treat human cancer (Table 1).

Figure 1. A schematic diagram of the anti-tumor effects of CRAds-IL-24 therapy. (A) When normal cells are infected with CRAds-IL-24, neither transgene expression nor apoptosis will occur due to the inability of CRAds to replicate in the normal cells.(B) However, when tumor cells are infected with CRAds-IL-24, conditional replication of CRAds within infected tumor cells produces thousands of viral copies. In addition, the IL-24 gene can frequently be expressed at high levels and transported to the cytoplasm. The proapoptotic effects of IL-24 and viral replications lead to tumor cell lysis. Released viral particles spread and infect adjacent tumor cells, eventually leading to tumor regression or even eradication.

Table I. Targeting gene-virotherapy using CRAds armed with the therapeutic gene IL-24.

CRAds-IL-24	Modification	Tumor targeted	Studies	Reference
Ad.PEG-E1A-mda-7	the progression elevated gene-3 (PEG-3) promoter	Prostate cancer, breast cancer, melanoma	in vitro/in vivo	[32,34,35]
ZD55-IL-24	55kDa deletion in the E1B gene	*Pan	in vitro/in vivo	[37,38]
MUD55-IL-24	55kDa deletion in the E1B gene, the MUC1 promoter	Gastric cancer	in vitro/in vivo	[43]
Ad.DD3-E1A-IL-24	the minimal DD3 promoter	Prostate cancer	in vitro/in vivo	[47]
Ad.sp-E1A(Δ24)-IL-24	24-bp deletion in the E1A gene, the survivin promoter	*Pan	in vitro/in vivo	[50]
Ad.sp-E1A(Δ24)-E1B(Δ55)-IL-24	24-bp deletion in the E1A gene,	*Pan	in vitro/in vivo	[51]
	55kDa deletion in the E1B gene, the survivin promoter
AdCN205-IL-24	Deleted 6.7K/gp19Kof E3 genes	Hepatocellular carcinoma, colorectal carcinoma	in vitro/in vivo	[56]

*“Pan” stand for a panel of human cancer cells.

Sarkar et al. [32,33] constructed a CRAd, Ad.PEG-E1A-mda-7, in which expression of the adenoviral E1A gene, necessary for replication, is driven by a tumor-selective progression-elevated gene-3 promoter (PEG-Prom). This CRAd expresses high level of mda-7/IL-24 in the E3 region. Infection of breast cancer cells and melanoma cells with this construct inhibited tumor cell growth and induced apoptosis [32,34]. Injection into tumor xenograft (prostate cancer and melanoma) in nude mice completely eradicated primary tumors as well metastatic tumors at distant sites [34,35]. A replication-defective adenoviral vector carrying the mda-7/IL-24 gene or blank CRAd was much less efficacious. These findings highlight the potential of using this novel approach for the treatment of metastatic tumors.

Clinical trials with an E1B 55-kD-deleted CRAd, ONYX-015 revealed some encouraging anti-tumor activity, when used in combination with chemotherapy [21]. However, only a 15% remission rate could be obtained when using ONYX-015 alone in clinical trials [21]. Moreover, because of using replication-defective adenoviral vectors for IL-24 delivery (Ad-IL-24), relatively low amount of IL-24 can be transduced into tumor cells, the efficacy of this approach has proved to be limited [8,9].

To augment the anti-tumor efficacy, ZD55, a new E1B 55 kD-deficient CRAd that is similar to ONYX-015 but with a Bgl II cloning site to insert foreign therapeutic genes, was constructed by our collaborator Liu's laboratory [36]. As novel anti-tumor therapeutic agents, ZD55 not only selectively replicate in and lyse tumor cells but can also amplify the expression and efficacy of therapeutic genes such as IL-24. A study indicated that ZD55 carrying IL-24 (ZD55-IL-24) could induce apoptosis via Bax activation in a human colorectal cancer cell line [37]. ZD55-IL-24 had much stronger anti-tumor activity than either Ad-IL-24 or ONYX-015 in mice with SW620 colorectal cancer xenograft. ZD55-IL-24 also has significant anti-tumor activity in other tumors. For example, ZD55-IL-24 is more potent than ZD55 in a leukemic cell line [38]. Potential mechanisms in this cell line include activation of PKR and p38 MAPK, as well as ER stress.

To strengthen the anti-tumor effect, the concept of targeting dual gene-virotherapy strategy by Liu et al. was devised [11]. This strategy uses a combination of two CRAds carrying different therapeutic genes. A study indicated that simultaneous infection with ZD55-IL-24 and ZD55-TRAIL could result in significantly greater cytotoxicity in colorectal cancer than infection with only ZD55-IL-24 or ZD55-TRAIL [39].

To optimize the efficacy of CRAds, identification of tumor-selective promoter elements that can differentiate between normal and cancer cells is of immense import [40,41]. Previously, a double regulated CRAd named MUD55 was constructed, in which the native promoter of E1A was replaced by MUC1 promoter and the E1B-55KD gene was deleted [42]. The resulting virus achieved higher in vivo cancer selectivity than ZD55 or Ad-MUC1 (adding MUC1 alone). This construct was subsequently used to deliver IL-24, with promising results: much stronger anti-tumor activity in comparison with Ad-IL-24 or MUD55 alone both in vitro and in vivo [43]. The safety profile was comparable to that of Ad-IL-24. ROS (Reactive Oxygen Species) are known to regulate various cellular responses including apoptosis [44]. Their preliminary data also indicated that intracellular ROS generation is responsible for the induction of apoptosis, mitochondrial dysfunction, and caspase activation in cancer cells infected with this CRAd [43].

Prostate cancer gene 3 (PCA3), also known as DD3, is a new prostate-specific gene that is highly over-expressed in prostate cancer tissue [45]. Significantly higher promoter activity was discovered in the minimal region of DD3 promoter (214-bp) [46]. Fan et al. [47] constructed a CRAd Ad.DD3-E1A by utilizing the minimal DD3 promoter to replace the native viral promoter of E1A gene (the DD3 promoter-controlled E1A gene). In addition, Ad.DD3-E1A was armed with therapeutic gene IL-24 to enhance its anti-tumor activity. The resulting CRAds, Ad.DD3-E1A-IL-24, demonstrated prostate cancer specificity and excellent anti-tumor effect. Further analyses on its anti-tumor mechanism revealed that it could induce apoptosis in prostate cancer cells and inhibit angiogenesis.

Prior studies have shown that survivin promoter-driven CRAds exhibited tumor-selective cytotoxicity, suggesting the survivin promoter on transcriptional targeting is a good candidate for targeting cancer therapy [48,49]. Zhang et al. [50] constructed a CRAd carrying IL-24, Ad.sp-E1A(Δ24)-IL-24 (the survivin promoter-controlled E1A gene, a 24-bp deletion in the E1A gene targeting the Rb pathway to improve safety). Compared with E1B 55 KD gene-deficient CRAd ZD55, the E1A was double regulated by the survivin promoter and by the deletion of 24 bp in 923–946 region. Their data showed that Ad.sp-E1A(Δ24)-IL-24 efficiently expresses the IL-24 gene and induces cytotoxicity in various tumor cells but rarely in normal cells. Ad.sp-E1A(Δ24)-IL-24 achieves nearly complete inhibition (although not elimination) of NCI-H460 lung carcinoma growth in nude mice. Similarly, Xiao et al. [51] constructed a CRAd carrying IL-24, Ad.sp -E1A(D24)-E1B(D55)-IL-24 (the deletion of E1A-24 bp and E1B-55 KD gene, the survivin promoter-controlled E1A gene). They showed that Ad.sp-E1A(Δ24)-E1B(Δ55)-IL-24 exhibited much better anti-tumor effect than E1 single regulated CRAd ONYX-015 in a variety of tumor cells in vitro. Furthermore, Ad.sp-E1A(D24)-E1B(D55)-IL-24 could effectively inhibit the progression of the NCI-H460 xenograft lung carcinoma in nude mice. Therefore, the E1A and E1B triple regulated CRAds armed with IL-24 as therapeutic gene represents a novel treatment paradigm for human cancers.

Some studies have explored whether adenoviral endogenous promoters could be used for controlling therapeutic gene expression [52,53]. The endogenous E3 genes are expressed only during replication. As a result, transgenes under the control of the adenoviral endogenous E3 promoter could only be expressed in rapidly dividing cells, such as tumor cells [54,55]. Luo et al. [56] constructed a CRAd by replacement of 6.7K/gp19K of E3 genes with the IL-24 gene (AdCN205-IL-24), so that expression of IL-24 was driven by the adenoviral endogenous E3 promoter. The substitution of 6.7K/gp19Kof E3 genes with transgenes did not affect viral replication in tumor cells. Treatment of the tumors induced high level of local expression of IL-24 in tumor cells. AdCN205-IL-24 exerted much stronger cytotoxicity to tumor cells compared with that induced by control adenovirus AdCN205-EGFP or Ad-Wt. Neither AdCN205-EGFP nor AdCN205-IL-24 induced cytopathic effect to normal cells.

Preclinical trials of CRAds-IL-24 therapy in combination with chemotherapy

Preclinical studies have shown that enhanced and even synergistic anti-tumor activity can be achieved when CRAds-IL-24 therapy is used in combination with chemotherapy (Table II). These studies suggest that the administration of CRAds-IL-24 in combination with chemotherapeutic agents could maximize the benefits of combined treatment.

Table II. CRAds armed with IL-24 in combination with chemotherapeutics.

CRAds-IL-24	Chemotherapeutics	Tumor targeted	Effect	Studies	Reference
ZD55-IL-24	Dacarbazine	Melanoma	Additive or synergistic	in vitro	[57]
ZD55-IL-24	Cisplatin	*Pan	Enhanced	in vitro	[59]
ZD55-IL-24	Adriamycin or Cisplatin	Lung	Enhanced	in vitro/in vivo	[58]
ZD55-IL-24	Dichloroacetate	*Pan	Enhanced	in vitro	[61]
ZD55-IL-24	Wortmannin	Leukemia	Enhanced	in vitro/in vivo	[62]
CRAdRGDflt-IL24	Temozolomide	Glioma	Enhanced	in vitro/in vivo	[69]

*“Pan” stand for a panel of human cancer cells.

A study from our laboratory demonstrated that ZD55-IL-24 could enhance the cytotoxic and apoptosis-inducing effect of dacarbazine in melanoma cells and reduce the toxicity to normal cells [57]. Some studies indicated that ZD55-IL-24 could improve anti-tumor effects, and thereby minimize the toxic side effects of cisplatin and adriamycin by reducing the concentrations of chemotherapeutic agents [58,59]. The presented data showed that dichloroacetate treatment could induce apoptosis in several tumor cells via mitochondrial apoptotic pathway while sparing normal cells [60]. Dichloroacetate in combination with ZD55-IL-24 exhibit a remarkably increased apoptosis in cancer cells and significantly reduce the toxicity in normal cells [61]. The combination of ZD55-IL-24 with a phosphatidylinositol 3-kinase inhibitor, wortmannin, result in a significant suppression of leukemia cells growth [62].

Data from experimental and clinical studies indicate that glioma is a strongly angiogenesis-dependent cancer [63]. The overexpression of the vascular endothelial growth factor (VEGF) in cancer cells promotes tumor angiogenesis by activation of the VEGF receptor expression in tumor vessels [64–66]. Recent data showed that the employment of the VEGFR-1/flt-1 promoter restricts CRAd replication to cancer cells and tumor-associated microvessels overexpressing VEGF [67,68]. Kaliberova et al. [69] developed a retargeted conditional replicating adenovirus (CRAdRGDflt-IL24) employing the VEGFR-1/flt-1 promoter to control E1A gene expression with an RGD peptide inserted into the HI-loop of the Ad fiber knob domain and encoding the IL-24 gene. They investigated its anti-tumor activity together with chemotherapy. The combination of CRAdRGDflt-IL24 and temozolomide significantly enhanced cytotoxicity in vitro, inhibited glioma cell growth and prolonged survival of mice harboring intracranial human glioma xenografts in comparison with CRAdRGDflt-IL24 or temozolomide alone.

Although the mechanism of the enhanced effects of combining conditionally replicating virotherapy and chemotherapy is unknown, several hypotheses exist. One is that the virus may increase the cell killing effects of chemotherapy through its ability to induce p53-dependent and -independent apoptotic pathways [22,70]. In addition, because most chemotherapeutic agents are immunosuppressive, this may allow a higher dose of viral particles to reach the tumor when injected systemically and maintain viral spread intratumorally due to a decrease in the production of neutralizing antibodies [71,72]. Furthermore, each CRAds mediated gene therapy or chemotherapeutic agent may be working independently on different cell populations within the tumor mass in a complementary manner, because no overlapping resistance between CRAds and chemotherapy is anticipated [22,73].

CRAds carrying IL-24 therapy in combination with chemotherapy thus holds promising potentials for further clinical developing an effective approach to treat cancers.

Current issues and future direction of CRAds-IL-24 gene therapy

Recent studies showed that administering IL-24 using CRAds represents a potentially viable strategy for increasing the therapeutic efficacy of this novel cytokine. In our opinion, further research into the following issues may provide clues for moving the field forward.

First, increased infectivity of viruses to tumors could decrease adverse reactions caused by virus administration. Attachment of viruses to target cells is primarily dependent on the binding of the fiber-knob portions to the cellular receptors expressed and secondly on the interaction between Ad penton bases and integrins (CD51) on the targets [74]. Since the major cellular receptor of the Ad5 is the coxsackie adenovirus receptor (CAR), the infectivity of Ad5 is often influenced by the expression level of the CAR on target cells [8,75]. The levels on tumors are however inconsistent irrespective of the tumor types and the expression is sometimes down regulated; consequently, these tumors are relatively resistant to Ad5-mediated gene transfer. One method is to replace the fiber-knob region with those of another type of Ad, which consequently changes the tropism of Ad infectivity [76,77].

Secondly, it has been shown that the immune response can reduce the anti-tumor efficacy of CRAds, when used alone [70,78]. A promising approach to systemic delivery, Carrier cells, can shield CRAds from neutralizing antibodies during delivery, providing a simple and effective means to enhance therapy in the face of sterilizing antiviral immunity [79,80]. In addition, the immunosuppressive effects of chemotherapeutic agents may increase CRAds efficacy by maintaining viral spread among cancer cells because of a decrease in the production of neutralizing antibodies [81,82].

Finally, as described above, most in vivo experiments have been performed with human cancer cell xenografts in immunodeficient mice. However, the full potential of these strategies could not be realized in the case of those adenoviruses that express immunostimulatory molecules [9,83]. The use of immunocompetent mice as well as other models such as the cotton rat [84], Syrian hamster [85] and pig models [86] seems reasonable for study of the interaction between CRAds and an intact immune system. A better understanding of replicating adenoviruses in immunocompetent hosts will lead to improvements in viruses for clinical use.

Conclusion

CRAds not only have the ability to destroy cancer cells but may also be potential vectors for the expression of therapeutic genes. IL-24 is a promising candidate for cancer gene therapy. Previous studies demonstrated that CRAds carrying IL-24 gene could achieve high level of IL-24 expression in tumor cells and produce significant oncolytic effects under both in vitro and in vivo conditions. Besides, recent studies have shown that the combination of CRAds carrying IL-24 and chemotherapeutic agents may have complementary or synergistic effects, leading to a greater anti-tumor effect than either treatment alone. Additionally, as CRAds and chemotherapeutic agents act by different mechanisms, cross-resistance is theoretically unlikely, thereby minimizing the development of treatment-resistant tumor cells. Based on these studies presented here, delivery of IL-24 gene by CRAds may prove a novel and effective approach for the treatment of cancers.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.