Therapeutics, Targets, and Chemical Biology Oncolytic Viral Therapy for Prostate Cancer: Efficacy of Reovirus as a Biological Therapeutic Chandini M. Thirukkumaran 1,5 , Michael J. Nodwell 9 , Kensuke Hirasawa 10 , Zhong-Qiao Shi 5 , Roman Diaz 5 , Joanne Luider 7 , Randal N. Johnston 1,2 , Peter A. Forsyth 1,5 , Anthony M. Magliocco 1,3,5,7 , Patrick Lee 11 , Sandra Nishikawa 2 , Bryan Donnelly 4 , Matt Coffey 8 , Kiril Trpkov 3,7 , Kevin Fonseca 6 , Jason Spurrell 5 , and Don G. Morris 1,5 Abstract Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcino- ma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infec- tion as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus–treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatec- tomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of in- creased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor. Cancer Res; 70(6); 2435–44. ©2010 AACR. Introduction Metastatic prostate carcinoma is the second leading cause of cancer-related deaths in North American males (1). In 2009, it is estimated that >185,000 new cases and 28,500 deaths will result from this disease in the United States (1). Globally, prostate cancer accounts for >220,000 deaths annu- ally (2). Current therapeutic options for advanced prostate cancer are limited to androgen deprivation and/or cytotoxic chemotherapy with the goals of treatment to extend survival while maintaining quality of life. Clearly more efficacious and/or novel treatments are required. Over the past decade both naturally occurring and genet- ically modified oncolytic viruses have been investigated as a potential treatment for a myriad of cancers. One such virus, reovirus (respiratory, enteric, orphan), is a common non- attenuated environmental double-stranded RNA virus that has been shown to have oncolytic potential against many cancers, including lymphoid, colon, ovarian, breast, pan- creatic, and high-grade glioma cancer cell lines, as well as their ex vivo correlates (3–7). Moreover, only mild symptoms are experienced by healthy adults during infection due to its tropism for transformed cells with aberrant signaling pathways (8–10). The underlying mechanism(s) behind the preferential cytotoxicity of reovirus toward transformed cells has only recently been described and seems to be at the level of intracellular signaling and not at cell surface attachment (11). When reovirus-resistant murine NIH 3T3 cells are transformed with oncogenes, such as v-erbB 2 , sos, and ras, reovirus susceptibility is conferred (9–11). Reovirus likely exploits an activated Ras/oncogenic signaling pathway, taking advantage of the inhibition of the double-stranded RNA–activated protein kinase found in these cells (11). Re- cent data has implicated the Ras/RalGEF/p38 pathway in an NIH 3T3 model system of reovirus oncolysis (12). Although the initial malignant transformation of prostate epithelial cells is not generally felt to be a ras mutation– dependent event, there are several other pathways felt to be important in both malignant transformation and involved Authors' Affiliations: Departments of 1 Oncology, 2 Medical Biochemistry, 3 Pathology, and 4 Urology, University of Calgary; 5 Tom Baker Cancer Center; 6 Provincial Laboratory for Public Health; 7 Calgary Laboratory Services; 8 Oncolytics Biotech Inc., Calgary, Alberta, Canada; 9 Bio-Rad Laboratories (Canada) Ltd., Mississauga, Ontario, Canada; 10 Memorial University of Newfoundland, St John's, Newfoundland, Canada; 11 Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada Note: Primary authorship is shared by C.M. Thirukkumaran, M.J. Nodwell, and K. Hirasawa. Corresponding Author: Don G. Morris, Department of Oncology, Tom Baker Cancer Centre, 1331, 29 Street NW, Calgary, Alberta, Canada T2N 4N2. Phone: 403-521-3437; Fax: 403-283-1651; E-mail: donmorri@cancerboard.ab.ca. doi: 10.1158/0008-5472.CAN-09-2408 ©2010 American Association for Cancer Research. Cancer Research www.aacrjournals.org 2435 Research. on April 16, 2017. © 2010 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 9, 2010; DOI: 10.1158/0008-5472.CAN-09-2408