Critical Review. Oncoapoptosis: A Novel Molecular Therapeutic for Cancer Treatment

Transcription

1 IUBMB Life, 62(2): 87 91, February 2010 Critical Review Oncoapoptosis: A Novel Molecular Therapeutic for Cancer Treatment John A. Blaho Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, NY Summary Many cancer cells refractory to radiation treatment and chemotherapy proliferate due to loss of intrinsic programmed cell death (apoptosis) regulation. Consequently, the resolution of these cancers are many times outside the management capabilities of conventional therapeutics. We have developed a replication defective herpes simplex virus system which triggers apoptosis specifically in transformed human cells, termed oncoapoptosis. Susceptibility to virus induced cell death is dependent on the p53 protein status in the tumor cells, indicating specific targeting of the treatment. Primary cells which produce functional p53 are resistant to oncoapoptotic killing but not to apoptosis induced by nonviral environmental factors. Thus, induction of apoptosis by nonreplicating virus is a feasible molecular therapeutic approach for killing human cancer cells. Our findings have important implications in designing novel virus-based anticancer strategies. Ó 2009 IUBMB IUBMB Life, 62(2): 87 91, 2010 Keywords programmed cell death; cancer; p53; virotherapy; herpes simplex virus; viroceuticals. Received 11 September 2009; accepted 22 September 2009 Address correspondence to: Department of Microbiology, Box 1124, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, Tel: (212) Fax: (212) john.blaho@mssm.edu INTRODUCTION Apoptosis Apoptosis, or programmed cell death, is a highly regulated cell death process in which the dying cells exhibit characteristic morphological changes including chromatin condensation, DNA fragmentation, cell shrinkage, and membrane blebbing (1 4). Apoptosis is activated during normal development and by various stimuli that disturb cell metabolism and physiology. Both cell surface receptor mediated (extrinsic) and internal, mitochondrial (intrinsic) initiated apoptotic pathways exist in cells (5). The observed characteristic apoptotic features are the consequence of regulated proteolysis processes (5). A family of cysteine proteases called caspases play a pivotal role in apoptosis induction. Active caspases cleave and activate other caspases, as well as cellular components vital for survival (reviewed in 6, 7). One effector caspase, caspase 3, chiefly cleaves cellular substrates important for maintaining the structural and biochemical integrity of the cell, such as poly(adpribose)polymerase (PARP), DNA fragmentation factor, and lamins. Caspase activation occurs by cleavage of prodomains from their precursor forms. The apoptotic potential of a cell has a profound effect on whether that cell might respond to an environmental stress. In tumor cells, modulation of apoptosis plays a significant role in the progression to malignancy (8, 9). p53 The central mediator of the cellular response to environmental stresses is the p53 protein, and it is a major determinant of whether a cell will undergo apoptosis (10, 11). Thus, it is not surprising that mutations in the p53 gene are found in greater than half of all human tumors (12, 13). Activation of p53 occurs in response to various types of stress, including DNA damage, hypoxia, and oncogenes, and results in increased levels of modified protein. Activated p53 initiates signaling pathways which lead to either cell cycle arrest or apoptosis (10, 11). The proapoptotic response of p53 to DNA damage introduced by chemotherapeutic agents or radiation is therefore the basis for much of the efficacy of these treatments (14). Conversely, cells whose p53 has been inactivated are associated with resistance to anticancer drugs (15). Herpes Simplex Virus Modulation of Apoptosis Because of the cell s innate ability to self destruct, apoptosis is also an important mechanism of host cell defense against viral infections. Accordingly, several distinct viruses have developed mechanisms to block the premature apoptosis of ISSN print/issn online DOI: /iub.274

2 88 BLAHO infected cells (16). The most likely reason for this is to prevent the cellular apoptosis that occurs as a result of viral infection to prolong cell survival so that the production of the viral progeny can be maximized. Wild type (wt) herpes simplex virus (HSV) triggers apoptosis in cells and then subsequently (between 3 and 6 hpi) stimulates the synthesis of proteins, which act to prevent the process from killing the infected cells (17 19). The first report of this phenomena was in 1997, when Koyama and Adachi showed that infecting HEp-2 cells with HSV in the presence of the protein synthesis inhibitor, cycloheximide (CHX), caused apoptosis (20). This illustrated the ability of HSV to trigger apoptosis in the absence of protein synthesis. In later studies, HSV-infected cells were found to be resistant to apoptosis induced by exogenous agents (21 25), demonstrating that proteins synthesized in infected cells are capable of preventing apoptotic cell death. HSV replication is tightly regulated and occurs in an ordered cascade (26). Recombinant HSV strains which initiate but cannot complete their replication cycle because of a reduction in their expression of early and late genes have been defined as apoptotic viruses (27). Viral factors responsible for apoptotic prevention include ICP4, ICP27, ICP22, U S 3, U S 5, glycoprotein D, and the latency associated transcript (25, 28 33). In HEp-2 cells, there is a balance between the induction and prevention of apoptosis during a wt HSV infection that allows virus propagation to occur and the infected cells to die through cell lysis (17 19). When the preventers of apoptosis are not efficiently made, as is the case when the infection is carried out in the presence of CHX, the balance is upset and the infected cells die through apoptosis. Following from the balanced apoptosis model, we predict that the reason these mutant viruses cause apoptosis is that they do not efficiently produce apoptotic preventers. As we shall see, viruses which retain the ability to trigger apoptosis in human tumor cells but not prevent it are termed oncoapoptotic viruses. DISCOVERY OF ONCOAPOPTOSIS Differences in HSV Dependent Apoptotic Responses Between Immortalized and Transformed Cells In our initial report describing the phenomenon that infection with recombinant HSV strains deleted for the viral regulatory protein ICP27 results in apoptosis, we observed this cell death in three separate human tumor cell lines but not in immortalized African green monkey kidney (Vero) cells (30). Subsequent detailed experimentation indicated that Vero cells are, in fact, susceptible to HSV dependent apoptosis but this effect was much less pronounced and took much longer to manifest than that in the human tumor cells (34). Our initial attempt to determine the molecular basis of the different cellular responses focused on what was the cause of the transformation of the tumor cells. For example, we knew that HeLa cells are defective in p53 activity. Biochemical comparisons of the electrophoretic motilities of p53 in denaturing gels indicated that the abundance and migration of p53 in the tumor cells differed from that of Vero cells (35). This was the first indication that p53 might be a determinant of tumor cell susceptibility to HSV dependent apoptosis. Absence of HSV Dependent Apoptosis in Primary Cells Following up on the studies aforementioned, we determined that the p53 protein pattern of primary mouse embryonic fibroblast cells was the same as that of the immortalized Vero cells (35). Importantly, while these primary mouse cells were killed by the addition of either extrinsic or intrinsic inducers of apoptosis, they were resistant to HSV dependent apoptosis (35). We confirmed this general effect using syngeneic cells of which one was derived from breast caner tissue and the other from surrounding normal tissue from the same individual. In this instance, the tumor cells were susceptible and the normal cells resistant to HSV dependent apoptosis (36). We now refer to this process as viral oncoapoptosis, that is, the killing of cancer cells by the induction of apoptosis by virus. ONCOAPOPTOSIS IN HUMAN TUMOR CELLS Identification of Sensitive and Resistant Tumor Cells We next performed a general survey (36) of the oncoapoptotic sensitivities of various representative tumor cell lines derived from human breast, colon, prostate, cervix, and brain cancers (Table 1). All primary cells that were tested, including mammary epithelial (HMEC) and foreskin fibroblast (HFF) cells, were resistant to oncoapoptosis but sensitive to environmental apoptosis inducers through either the extrinsic or intrinsic pathways. During this analysis, it was recognized that certain tumor cells were resistant to both oncoapoptosis as well as environmental apoptosis (Table 1). Reconstitution of Oncoapoptosis Sensitivity The breast cancer MCF7 cells were of particular interest because it was known that these cells are defective for the proapoptotic caspase 3 gene (37). We reasoned that perhaps reconstituting caspase 3 into these cells might cause them to gain sensitivity to oncoapoptosis. The experimental results indicated that MCF7 cells which constitutively express caspase 3 following stable transduction with a retroviral vector were susceptible to both environmental apoptosis and oncoapoptosis (37). These results provide a very important proof of principle that oncoapoptotic resistant tumor cells may be made into susceptible ones simply by adding back in trans the necessary proapoptotic machinery component. Tumor Cell Requirement for Susceptibility to Oncoapoptosis The oncoapoptosis reconstitution study aforementioned indicated the need for us to fully understand the molecular mechanism

3 ONCOAPOPTOSIS THERAPY FOR HUMAN TUMOR CELLS 89 Table 1 Cancer cell susceptibilities to oncoapoptosis Cell name Type Oncoapoptosis Environmental apoptosis RESISTANT CELLS HMEC Primary epithelial 2 1 Hs578Bst Primary epithelial 2 1 HFF Primary fibroblast 2 1 MEF Primary fibroblast 2 1 U373 Brain cancer 2 2 PC3 Prostate cancer 2 2 HEK293 Kidney tumor 2 2 HEK293T Kidney tumor 2 2 MCF-7 Breast cancer 2 2 SENSITIVE CELLS HeLa/HEp-2 Cervix cancer 1 1 HeLa/S3 Cervix cancer 1 1 HT-29 Colon cancer 1 1 RKO Colon cancer 1 1 RKO-E6 Colon cancer 1 1 Hs578T Breast cancer 1 1 MCF-7/C3 Breast cancer 1 1 SK-N-SH Brain cancer 1 1 Vero Immortalized fibroblast 1 1 through which HSV induces apoptosis in tumor cells. The goal was to determine what process preceded caspase 3 activation. Through a combination of biochemical protein studies and specific caspase inhibitor assays, we discovered that oncoapoptosis results in caspase 9 but not caspase 8 activation (38). We confirmed that oncoapoptosis occurs through the intrinsic pathway by measuring mitochondrial cytochrome c release. Importantly, we discovered that cytochrome c is released during oncoapoptosis in the absence of prior caspase activation, thus precluding the possibility of cross-talk from the extrinsic pathway via caspase 8 activation to the mitochondria (38). Taken together, all of these findings imply that in order for a tumor cell to be susceptible to killing by oncoapoptosis treatment, it must possess intact features of the intrinsic apoptotic machinery. ONCOAPOPTOSIS IS p53 DEPENDENT Exploiting the Cervical Tumor HeLa Cell Model System Inspection of Table 1 indicates that most of the tumor cells that were tested were sensitive to oncoapoptotic killing. The question remained as to what was the determinant that made tumor cells sensitive, whereas primary cells were not. To address this, we turned again to the HeLa system. Our model system exploits the fact that continuous oncogene expression is essential for HeLa cells to maintain their tumorigenic properties (39 41). HeLa cells harbor integrated human papillomavirus type 18 (HPV 18) genomes (42) and express two viral oncogenes, E6 and E7. E6 and E7 of high risk papillomaviruses like HPV 18 and 16 are known to inactivate the p53 and p105 Rb tumor suppressor proteins, respectively (43 46). These activities are essential for HPV to cause the postmitotic keratinocytes, which it infects, to enter the S-phase of the cell cycle and replicate viral genomes. In a typical nontumor HPV infection, E6 and E7 expression is limited by the HPV E2 transcriptional repressor. However, in HeLa cells, the viral genome is integrated into the host genome such that the E2 open reading frame is disrupted (42). The consequence of this is unrestrained oncogene expression and rampant proliferation of these tumor cells. It has been possible to reconstitute HeLa cells with E2 using transduction with a SV40-based recombinant virus. This treatment inhibits E6 and E7 expression, reactivates p53 and p105 Rb, and represses cell growth (41, 47, 48). In effect, the expression of E2 in trans untransforms the HeLa cells. Utilizing this system in a complex, but detailed, series of experiments, we demonstrated the essential role that p53 plays in determining the susceptibility of cancer cells to oncoapoptosis (49). Implication of our Results It is generally accepted that mutations in p53 are, perhaps, the single most negative indicator of recurrence and death in many cancer cases. It is known that selecting therapies based

4 90 BLAHO on the molecular features of the tumor yields the most efficacious treatment. Primary resistance to therapy is presumed to arise through selection of resistant clones, which may indeed be micrometastatic. What we have identified is a situation in which we can kill tumor cells, which are defective for p53, as well as other factors. Thus, we have specifically discovered a method for killing cancer cells which does not rely on functional p53; the virus itself is capable of triggering the apoptotic cell death process. Our approach has significant added value over approaches which attempt to simply induce genotoxic and cytotoxic stress in tumor cells. PERSPECTIVE With few exceptions, fully transformed tumor cells are exquisitely sensitive to the oncoapoptotic death stimulus (35 37). In contrast, cells derived from normal tissue are resistant to oncoapoptosis. Immortalized but nontransformed cell lines display an intermediate susceptibility (34 36). Our ability to restore oncoapoptotic resistance to HeLa cells by HPV oncogene repression indicates that the tumorigenic-related changes that confer susceptibility to oncoapoptosis are reversible. Our system provides a mechanism to identify these changes. In the long term, we hope to fully exploit this novel molecular therapeutic approach to inhibit the unrestrained growth of human tumor cells. ACKNOWLEDGEMENTS The author thanks all the individuals in laboratory whose hard work has set the basis for developing this interesting new research project. Individuals who played significant roles in generating the data that served as the basis of this review include Martine Aubert, Jennifer O Toole, Lisa Pomeranz, Renee Baranin, Christine Sanfilippo, Renzo Lambardozzi, Natalie Chirimuuta, Margot Goodkin, Elise Morton, Jamie Yedowitz, Marie Nguyen, Rachel Kraft, Kristen Pena, Elisabeth Gennis, Leah Kang, Christopher Cotter, and Fatima Manzoor. J.A.B. thanks the Lucille P. Markey Charitable Trust and the National Foundation for Infectious Diseases for their support. The study was supported in part by grants from the United States Public Health Service (AI38873 and AI48582 to J.A.B.) and the American Cancer Society (JFRA 634 to J.A.B.). REFERENCES 1. Wyllie, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, Wyllie, A. H., Kerr, J. F., and Currie, A. R. (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68, Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, Kerr, J. F. R. and B. V. Harmon. (1991) Definition and incidence of apoptosis: an historical perspective. In Apoptosis: The Molecular Basis of Cell Death (Cope, L. D. T. a. F. O., ed.). Pp. 5 29, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 5. Sanfilippo, C. M. and Blaho, J. A. (2003) The facts of death. Int Rev Immunol 22, Salvesen, G. S. and Dixit, V. M. (1997) Caspases: intracellular signaling by proteolysis. Cell 91, Villa, P., Kaufmann, S. H., and Earnshaw, W. C. (1997) Caspases and caspase inhibitors. Trends Biochem Sci 22, Lowe, S. W. and Lin, A. W. (2000) Apoptosis in cancer. Carcinogenesis 21, Green, D. R. and Evan, G. I. (2002) A matter of life and death. Cancer Cell 1, Prives, C. and Hall, P. A. (1999) The p53 pathway. J Pathol 187, Levine, A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88, Hainaut, P., Hernandez, T., Robinson, A., Rodriguez-Tome, P., Flores, T., Hollstein, M., Harris, C. C., and Montesano, R. (1998) IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools. Nucleic Acids Res 26, Hainaut, P. and Hollstein, M. (2000) p53 and human cancer: the first ten thousand mutations. Adv Cancer Res 77, Lowe, S. W., Ruley, H. E., Jacks, T., and Housman, D. E. (1993) p53- dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74, Lowe, S. W., Bodis, S., McClatchey, A., Remington, L., Ruley, H. E., Fisher, D. E., Housman, D. E., and Jacks, T. (1994) p53 status and the efficacy of cancer therapy in vivo. Science 266, Koyama, A. H., Fukumori, T., Fujita, M., Irie, H., and Adachi, A. (2000) Physiological significance of apoptosis in animal virus infection. Microbes Infect 2, Nguyen, M. L. and Blaho, J. A. (2007) Apoptosis during herpes simplex virus infection. Adv Virus Res 69, Goodkin, M. L., Morton, E. R., and Blaho, J. A. (2004) Herpes simplex virus infection and apoptosis. Intl Rev Immunol 23, Aubert, M., and Blaho, J. A. (2001) Modulation of apoptosis during herpes simplex virus infection in human cells. Microbes Infect 3, Koyama, A. H. and Adachi, A. (1997) Induction of apoptosis by herpes simplex virus type 1. J Gen Virol 78, Galvan, V. and Roizman, B. (1998) Herpes simplex virus 1 induces and blocks apoptosis at multiple steps during infection and protects cells from exogenous inducers in a cell- type-dependent manner. Proc Natl Acad Sci USA 95, Koyama, A. H. and Miwa, Y. (1997) Suppression of apoptotic DNA fragmentation in herpes simplex virus type 1-infected cells. J Virol 71, Jerome, K. R., Fox, R., Chen, Z., Sears, A. E., Lee, H., and Corey, L. (1999) Herpes simplex virus inhibits apoptosis through the action of two genes, Us5 and Us3. J Virol 73, Goodkin, M. L., Ting, A. T., and Blaho, J. A. (2003) NF-kappaB is required for apoptosis prevention during herpes simplex virus type 1 infection. J Virol 77, Aubert, M., O Toole, J., and Blaho, J. A. (1999) Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1. J Virol 73, Roizman, B., Knipe, D. M., and Whitley, R. J. (2007) Herpes simplex viruses. In Virology, 5th edn. (Knipe, D. M. a. P. M. H., ed.). pp , Lippincott-Raven, Philadelphia, PA. 27. Aubert, M., Rice, S. A., and Blaho, J. A. (2001) Accumulation of herpes simplex virus type 1 early and leaky-late proteins correlates with apoptosis prevention in infected human HEp-2 cells. J Virol 75,

5 ONCOAPOPTOSIS THERAPY FOR HUMAN TUMOR CELLS Leopardi, R., Van Sant, C., and Roizman, B. (1997) The herpes simplex virus 1 protein kinase US3 is required for protection from apoptosis induced by the virus. Proc Natl Acad Sci USA 94, Leopardi, R. and Roizman, B. (1996) The herpes simplex virus major regulatory protein ICP4 blocks apoptosis induced by the virus or by hyperthermia. Proc Natl Acad Sci USA 93, Aubert, M. and Blaho, J. A. (1999) The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells. J Virol 73, Jerome, K. R., Chen, Z., Lang, R., Torres, M. R., Hofmeister, J., Smith, S., Fox, R., Froelich, C. J., and Corey, L. (2001) HSV and glycoprotein J inhibit caspase activation and apoptosis induced by granzyme B or Fas. J Immunol 167, Zhou, G., Galvan, V., Campadelli-Fiume, G., and Roizman, B. (2000) Glycoprotein D or J delivered in trans blocks apoptosis in SK-N-SH cells induced by a herpes simplex virus 1 mutant lacking intact genes expressing both glycoproteins. J Virol 74, Perng, G. C., Jones, C., Ciacci-Zanella, J., Stone, M., Henderson, G., Yukht, A., Slanina, S.M., Hofman, F.M., Ghiasi, H., Nesburn, A.B., and Wechsler, S. L. (2000) Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript. Science 287, Nguyen, M. L., Kraft, R. M., and Blaho, J. A. (2005) African green monkey kidney Vero cells require de novo protein synthesis form efficient herpes simplex virus 1 dependent apoptosis. Virology 336, Aubert, M. and Blaho, J. A. (2003) Viral oncoapoptosis of human tumor cells. Gene Ther 10, Nguyen, M. L., Kraft, R. M., and Blaho, J. A. (2007) Susceptibility of cancer cells to herpes simplex virus-dependent apoptosis. J Gen Virol 88, Kraft, R. M., Nguyen, M. L., Yang, X. H., Thor, A. D., and Blaho, J. A. (2006) Caspase 3 activation during herpes simplex virus 1 infection. Virus Res 120, Aubert, M., Pomeranz, L. E., and Blaho, J. A. (2007) Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells. Apoptosis 12, Laimins, L. A. (1993) The biology of human papillomaviruses: from warts to cancer. Infect Agents Dis 2, Lee, W. H., Bookstein, R., Hong, F., Young, L. J., Shew, J. Y., and Lee, E. Y. (1987) Human retinoblastoma susceptibility gene: cloning, identification, and sequence. Science 235, Goodwin, E. C. and DiMaio, D. (2000) Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. Proc Natl Acad Sci USA 97, Schwarz, E., Freese, U. K., Gissmann, L., Mayer, W., Roggenbuck, B., Stremlau, A., and zur Hausen, H. (1985) Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314, Scheffner, M., Munger, K., Byrne, J. C., and Howley, P. M. (1991) The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines. Proc Natl Acad Sci USA 88, Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J., and Howley, P. M. (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, Werness, B. A., Levine, A. J., and Howley, P. M. (1990) Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, Munger, K., Werness, B. A., Dyson, N., Phelps, W. C., Harlow, E., and Howley, P. M. (1989) Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J 8, Hwang, E. S., Naeger, L. K. and DiMaio, D. (1996) Activation of the endogenous p53 growth inhibitory pathway in HeLa cervical carcinoma cells by expression of the bovine papillomavirus E2 gene. Oncogene 12, Goodwin, E. C., Naeger, L. K., Breiding, D. E., Androphy, E. J., and DiMaio, D. (1998) Transactivation-competent bovine papillomavirus E2 protein is specifically required for efficient repression of human papillomavirus oncogene expression and for acute growth inhibition of cervical carcinoma cell lines. J Virol 72, Nguyen, M. L., Kraft, R. M., Aubert, M., Goodwin, E., DiMaio, D., and Blaho, J. A. (2007) p53 and htert determine sensitivity to viral apoptosis. J Virol 81,