Our study has been the first to demonstrate RSV is an oncolytic virus, and this oncolytic activity is functional in vivo both in immune-deficient nude mice and in an immune-competent host environment, since RSV inhibited prostate tumor growth in syngeneic C57BL/6J mice. RSV infectivity and the virus-induced apoptotic index in vitro were much higher in androgen-dependent LNCaP cells compared to non-tumorigenic RWPE-1 prostate cells. Aberrant type I interferon (IFN)-dependent antiviral defense response , which culminated in impaired activation of the STAT-1 transcription factor (STAT-1 is required for expression of IFN-dependent antiviral genes), associated with the high RSV burden in infected LNCaP cells. We conclude that blockade in STAT-1 activation, leading to inhibition in the expression of critical IFN-dependent antiviral genes, accounts for excessive RSV replication leading to apoptosis of LNCaP cells. This is unlike PC-3 androgen-independent prostate cancer cells for which RSV-induced oncolysis was associated with failure in a sustained NF-κB activation, which would cause failure in the induction of NF-κB dependent antiviral genes.
Using IFN-neutralizing antibody, we also provide the first direct evidence (Figure 9) that protection of non-malignant epithelial cells against virus-induced oncolysis is due to IFN-mediated antiviral defense response. Our results further revealed that the oncolytic function of RSV may remain active even when the IFN-regulated antiviral pathway is functional, provided a second defense arm involving NF-κB signaling is deregulated.
Innate immunity is the first line of defense mounted by the host to combat virus infection before an orchestrated adaptive immune response is launched [10, 20]. IFN-mediated activation of the JAK/STAT antiviral pathway is recognized as a major antiviral innate immune defense mechanism . In this regard, we have recently demonstrated that RSV-infected lung epithelial cells and immune cells (e.g. macrophages) utilize Nod2 protein as a molecular sensor to induce production of IFN-α/β from infected cells after interacting with viral single-stranded RNA genome and subsequently triggering innate antiviral response . IFN-α/β, which are potent antiviral cytokines produced in infected cells, bind to cognate cell surface receptors on uninfected cells (via autocrine/paracrine action) to induce the JAK/STAT antiviral pathway; which helps promote nuclear translocation and activation of the transcription factors STAT-1 and STAT-2 that in turn activate antiviral genes . We also reported that an IFN-independent innate defense mechanism involving TNF-α -induced activation of NF-κB can restrict virus replication in infected cells due to induction of antiviral genes [15, 26]. These two antiviral pathways mediated by IFN (via the JAK/STAT pathway) and TNF-α (via the NF-κB pathway) are activated in infected cells either individually or together to coordinate the transcriptional induction of the antiviral gene network.
A large number of cancer cells are deficient in the IFN signaling cascade [27, 28], making many types of cancer cells susceptible to apoptosis by oncolytic viruses. In the context of prostate cancer, our results suggest that both androgen-dependent prostate cancer cells (such as LNCaP cells) and androgen-independent prostate cancer cells (such as PC-3 and RM1 cells) are susceptible to RSV-induced oncolysis. We show that, while IFN production from infected LNCaP cells was normal, IFN failed to activate STAT-1 in LNCaP cells. In fact, it was reported earlier that LNCaP cells fail to express JAK1 . On the other hand, RSV infection and IFN treatment of non-tumorogenic RWPE-1 cells and PC-3 cells was associated with robust STAT-1 activation and protection against RSV-induced apoptosis. We also show that while PC-3 cells respond to IFN and induce DNA-binding activity of STAT-1 (in agreement with previous reports that IFN-treated PC-3 cells are activated for antiviral signaling; [30, 31]), impaired NF-κB activation is associated with apoptosis in RSV-infected PC-3 cells. LNCaP cells, on the other hand, were competent to activate NF-κB in response to RSV infection. We speculate that androgen dependence and/or the androgen receptor expression status of prostate cancer cells may influence RSV-mediated modulation of the innate antiviral apparatus (NF-κB activation vs. IFN-mediated JAK/STAT activation). Our results (Figures 8, 9, 10, 11)  lead us to conclude that deregulation of the IFN pathway in androgen-sensitive LNCaP prostate cancer cells accounts for loss of STAT-1 activation (and non-expression of antiviral factors), higher RSV replication, induction of apoptosis and reduced cell viability, whereas deregulation of the NF-κB-dependent antiviral defense in androgen-independent PC-3 prostate cancer cells accounts for susceptibility of these cells to RSV-induced apoptosis.
Advanced-stage cancer cells, which continue to express the androgen receptor in a majority of tumor specimens, are resistant to apoptosis from androgen ablation or from the cytotoxicity induced by chemotherapeutic agents. Development of treatment protocols that would promote prostate cancer cell apoptosis and prevent cancer cell progression to androgen independence has remained a major challenge in prostate cancer therapy. To this end, it is tempting to speculate that complete ablation of prostate cancer cells at an early stage, when the cells are still androgen-sensitive, is likely to prevent clonal emergence of androgen-independent prostate cancer cells. The anti-tumor, oncolytic activity of RSV against androgen receptor-negative prostate tumors has additional clinical significance, since reduced or non-detectable androgen receptor expression has been observed in a small percent of metastatic neoplastic foci at distant organ sites from castrate resistant prostate cancer patients [32, 33]. The observation that RSV-induced oncolysis of prostate tumors can occur in immune-competent C57BL/6J mice has obvious clinical significance. It is important to mention that systemic delivery of RSV represent a clinically feasible route for therapy. In that regard, we have previously demonstrated that intraperitoneal (i.p.) injections of RSV are effective in causing regression of PC-3 xenograft tumors  which are more aggressive than LNCaP xenograft tumors with regard to tumor growth. Thus, studies are underway to examine oncolytic efficacy of RSV against LNCaP tumors following systemic administration. The results from our study suggest that RSV-based therapy has the potential to be a viable strategy for prostate cancer treatment.