Apoptin Studies Illuminate Intersection Between Lipidomics and Tumor Suppressors
Authors: Sunil Chada, Rajagopal Ramesh
Journal: Molecular Therapy (2006)

Improvements in current cancer treatment strategies have resulted in an increase in patient survival. Nevertheless, cancer remains a leading cause of death in the United States, and most cancer patients relapse and are resistant to subsequent treatments. Therefore, there is an urgent need for new treatment strategies and testing of novel therapeutic agents. Gene therapy is a promising strategy to treat malignancy. In the December 2006 issue of Molecular Therapy, Liu et al. evaluated the effects on tumors of delivery of the apoptin gene and uncovered an intersection between cancer gene therapy and bioactive lipid metabolism.1Liu X et al.Involvement of sphingolipids in apoptin-induced cell killing.Mol Ther. 2006; 14: 627-636Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 2Liu X et al.Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.Mol Ther. 2006; 14: 637-646Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar The sequencing of the human genome has facilitated identification of specific genetic defects in cancer cells, such as oncogene activation or loss of tumor suppressor gene function. These mutations result in malfunctioning of the signaling pathways that tightly regulate cell growth and death. Multiple gene therapy–based preclinical and clinical studies have been conducted, with early indications of translational success.3Roth JA Adenovirus p53 gene therapy.Expert Opin Bio Ther. 2006; 6: 55-61Crossref PubMed Scopus (105) Google Scholar However, limitations such as lack of tumor specificity, interdependence of the therapeutic gene product on other gene products for killing effect, and lack of efficient systemic gene delivery vehicles have precluded translational testing of many promising therapeutic genes in the clinic. In efforts to overcome some of these limitations, new targets and new gene-based therapies are being tested in the laboratory. A promising gene product that has been shown to have tumor specificity is apoptin.4Noteborn MHM et al.A single chicken anemia virus protein induces apoptosis.J Virol. 1994; 68: 346-351Crossref PubMed Google Scholar Apoptin (or VP3) is a virus protein of the chicken anemia virus. The protein is a 13.6-kDa serine- and threonine-rich protein of 121 amino acids that was initially shown to induce apoptosis in chicken thymocytes.4Noteborn MHM et al.A single chicken anemia virus protein induces apoptosis.J Virol. 1994; 68: 346-351Crossref PubMed Google Scholar On the basis of this ability, Danen-Van Oorschot et al. tested the effects in several human cancer and transformed cells.5Danen-Van Oorschot AA et al.Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells.Proc Natl Acad Sci USA. 1997; 94: 5843-5847Crossref PubMed Scopus (222) Google Scholar They showed that apoptin selectively induced apoptosis in such cells, with little or no effect on "normal" cells. Subsequent studies have confirmed the tumor killing properties of apoptin.6Tavassoli M Guelen L Luxon BA Gaken J Apoptin: specific killer of tumor cells?.Apoptosis. 2005; 10: 717-724Crossref PubMed Scopus (49) Google Scholar Molecular analyses show increased localization of apoptin in the nucleus of tumor cells; in contrast, normal cells do not show this nuclear localization, and differential subcellular compartmentalization may therefore be important in its tumor specificity.7Rohn JL Noteborn MHM The viral death effector apoptin reveals tumor-specific processes.Apoptosis. 2004; 9: 315-322Crossref PubMed Scopus (55) Google Scholar Furthermore, unlike other tumor suppressor or pro-apoptotic genes, the apoptin gene does not rely on the function of p53, Bcl-2, Bax, FADD, or caspase-8 (refs. 2,6). Thus, apoptin may be used to treat a broad spectrum of tumors that differ in the mutational status of p53 and the levels of expression of apoptotic regulators. In the new work, Liu et al. showed that adenovirus-mediated overexpression of apoptin significantly inhibited tumor growth effectively without apparent toxicity to normal cells.2Liu X et al.Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.Mol Ther. 2006; 14: 637-646Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar These studies suggest the safety of using apoptin-based therapy for cancer. Although the aforementioned studies have provided a proof of concept for using apoptin in the treatment of cancer, the molecular mechanisms responsible for tumor cell killing are not well understood. Liu et al. have revealed a novel mechanism for apoptin-mediated tumor cell killing.1Liu X et al.Involvement of sphingolipids in apoptin-induced cell killing.Mol Ther. 2006; 14: 627-636Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 2Liu X et al.Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.Mol Ther. 2006; 14: 637-646Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Studies showed that apoptin modulated the sphingolipid-ceramide pathway leading to increased concentrations of ceramide in human prostate tumor cells and resulting in apoptotic cell death (summarized in Figure 1). Ceramide is a second messenger signaling molecule that is increased by cellular stress induced by diverse therapeutic agents, such as tumor necrosis factor–α, Fas ligand, radiation, and chemotherapeutic drugs.8Ogretmen B Hannun YA Biologically active sphingolipids in cancer pathogenesis and treatment.Nat Rev Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1004) Google Scholar Ceramide is considered a tumor suppressor lipid, and the studies by Liu et al. provide the first evidence for the intersection between tumor suppressor gene therapeutics and bioactive lipid signaling. There is a burgeoning body of literature on the role of sphingolipids in cancer pathogenesis and treatment8Ogretmen B Hannun YA Biologically active sphingolipids in cancer pathogenesis and treatment.Nat Rev Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1004) Google Scholar. These membrane lipids are important in regulation of membrane fluidity and subdomain structure. Furthermore, sphingolipids have been shown to be key regulatory effectors in many biological processes, including inflammation, cell proliferation, and cell migration, in addition to apoptosis. Inhibition of ceramide synthesis is associated with drug resistance.8Ogretmen B Hannun YA Biologically active sphingolipids in cancer pathogenesis and treatment.Nat Rev Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1004) Google Scholar Ceramide metabolism is regulated by a complex biochemical pathway (Figure 2), and the molecular dissection of this pathway has identified several new targets for drug development. Interestingly, ceramide can be generated in multiple cellular compartments, including endoplasmic reticulum, mitochondria, nucleus, lysosomes, and plasma membrane, with key regulatory enzymes showing distinct compartmentalization. Thus, the ceramide pathway has many similarities to the p53 signaling pathway, and both serve as comprehensive sensors of cell stress. Apoptin increases ceramide by activation of acid sphingomyelinase, resulting in increased hydrolysis of sphingomyelin to ceramide (Figure 1). Additionally, apoptin was shown to inhibit concentrations if acid ceramidase, an enzyme that deacylates ceramide to yield sphingosine. The involvement of the sphingosine-ceramide pathway and regulation of acid sphingomyelinase and acid ceramidase was elegantly demonstrated in the new work by Liu et al. using specific enzyme inhibitors. Additionally the authors demonstrated that combining inhibitors of acid sphingomyelinase with apoptin gene transfer resulted in enhanced cell killing, and, conversely, addition of a ceramide analogue designed to inhibit acid ceramidase enhanced apoptosis. In the second study by the same investigators, elevated acid ceramidase concentrations were found in approximately 65% of human prostate tumor specimens, indicating a role for acid ceramidase in favoring the anti-apoptotic phenotype and identifying acid ceramidase as a target for therapeutic intervention.2Liu X et al.Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.Mol Ther. 2006; 14: 637-646Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Using a gene therapy approach, Liu et al. showed that treatment of human prostate cancer cells with an adenoviral vector containing green fluorescent protein–tagged apoptin (AdGFP-Apoptin) in vitro resulted in inhibition of cell proliferation and induction of apoptosis.2Liu X et al.Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.Mol Ther. 2006; 14: 637-646Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Furthermore, they demonstrated that AdGFP-Apoptin-mediated apoptosis was independent of various apoptotic regulators, including p53, caspase-3, Bcl2, Bax, survivin, FLIPs, XIAP, and CIAP. The authors extended their in vitro findings by demonstrating that intratumoral administration of AdGFP-Apoptin suppressed prostate tumor growth in vivo. Additionally, combination treatment with AdGFP-Apoptin plus the acid ceramidase inhibitor LCL204 enhanced the antitumor activity, resulting in increased animal survival. The authors indicate that the observed antitumor activity was greater than predicted and propose that a "bystander" tumor killing effect was occurring. Furthermore, a potential role for anti-angiogenic activity is indicated. Should these effects be demonstrated to occur in vivo, then a combination therapy should be a basis for the treatment of prostate cancer and possibly other cancer types. Given that intravenous administration of Ad-Apoptin in mice suppressed growth of hepatocellular carcinoma, there is support for its potential as a systemic therapeutic agent9van der Eb MM et al.Gene therapy with apoptin induces regression of xenografted human hepatomas.Cancer Gene Ther. 2002; 9: 53-61Crossref PubMed Scopus (67) Google Scholar. Although these two studies provide strong evidence for involvement of the ceramide pathway in apoptin-mediated tumor cell killing, several questions remain. What is the effect of AdGFP-Apoptin on normal cells? The authors indicate that treatment of normal human prostate epithelial cells with AdGFP-Apoptin results in increased ceramide concentrations and induction of apoptosis. However, this occurs only when cells are treated at high multiplicity of infection (that is, doses that are higher than that required for treating tumor cells), because the normal cells are not easily transfected. This observation suggests that AdGFP-Apoptin should be tested in a panel of normal cells of various origins to preclude the possibility of treatment-related toxicity. The mechanism by which apoptin induces apoptosis was shown to involve caspase-3 and not caspase-8, indicating the involvement of the intrinsic apoptotic pathway. However, because caspase-3 is an effector caspase and is downstream of the mitochondrial-apoptosis pathway, it will be important to identify the upstream activator caspase. It is of significant interest to investigate whether apoptin functions as a transcription factor, given that it demonstrates nuclear localization in tumor cells as a result of its phosphorylation at threonine 108 (ref. 10Rohn JL et al.A tumor specific kinase activity regulates the viral death protein Apoptin.J Biol Chem. 2002; 277: 50820-50827Crossref PubMed Scopus (101) Google Scholar). Furthermore, multimers of apoptin bind to DNA and activate phosphorylation of p53 at serine 15. A better understanding of the molecular events that regulate the phosphorylation and binding of Apoptin to DNA may lead to insight into the means by which apoptin localized to the nucleus of tumor cells but was able to activate sphingomyelinase in the cytoplasm. It is currently unknown whether the combination of AdGFP-Apoptin with other stress-inducing agents such as radiation, chemotherapy, or biological agents will provide additive or synergistic antitumor activity, similarly to that observed when treatment combined AdGFP-Apoptin plus LCL204. Development of the acid ceramidase inhibitor LCL204 will be followed with interest, because this molecule and its analogues will probably also sensitize tumors to other gene therapies and cytotoxic drugs. Finally, the inability to treat metastatic tumors by current adenovirus-mediated gene therapy strategies warrants testing of alternate delivery vehicles that are nontoxic, nonimmunogenic, and capable of being administered systemically. Although the additional studies suggested here are warranted, it is evident that the findings on apoptin reported by Liu et al. provide an opportunity to treat a broad spectrum of cancer cells that have multiple gene or functional alterations and are not easily treatable by conventional modalities1Liu X et al.Involvement of sphingolipids in apoptin-induced cell killing.Mol Ther. 2006; 14: 627-636Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 2Liu X et al.Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.Mol Ther. 2006; 14: 637-646Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar. These studies also identify the ceramide pathway as a critical mediator of apoptin activity. Ceramide regulation has been linked to alterations in classical tumor suppressors, such as p53, Rb, and PTEN8Ogretmen B Hannun YA Biologically active sphingolipids in cancer pathogenesis and treatment.Nat Rev Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1004) Google Scholar, 11Taha TA et al.Down-regulation of sphingosine kinase-1 by DNA damage: dependence on proteases and p53.J Biol Chem. 2004; 279: 20546-20554Crossref PubMed Scopus (114) Google Scholar, 12Goswami R Singh D Phillips G Kilkus J Dawson G Ceramide regulation of the tumor suppressor phosphatase PTEN in rafts isolated from neurotumor cell lines.J Neurosci Res. 2005; 81: 541-550Crossref PubMed Scopus (35) Google Scholar, and it is probable that, given its pleiotropic functions, ceramide regulation will also be observed for other tumor suppressor genes. In fact, ceramide metabolism may provide clues to understand some of the supra-additive "bystander" effects observed after gene transfer of p53, apoptin, and other tumor suppressor genes.