Our studies probe fundamental molecular and cellular processes that impact microbial infections and cancer. We seek a better understanding of how the mammalian cell resists viral infections and how the virus antagonizes the host response to infection. Interferons (IFNs) induce a family of 2',5'-oligoadenylate (2-5A) synthetases (OAS). In response to viral double stranded RNA, OAS produces 2-5A whose function is to activate RNase L causing degradation of viral and cellular RNA. Previously we cloned RNase L and established its antiviral activities in genetically deficient mice. We found that RNase L cleaves viral and cellular RNA to generate small RNAs that stimulate IFN synthesis through RIG-I and MDA5. We are currently studying viral and host 2',5'-phosphodiesterases that prevent activation of RNase L, thus allowing virus replication and viral pathogenesis. Our goals include elucidating fundamental events and biologic endpoints surrounding RNase L that impact on viral replication cycles and tumor biology. Another area of research concerns the roles of innate immunity and genetics in prostate cancer. The hereditary prostate cancer 1 (HPC1) gene maps to RNase L. We are investigating the potential role of RNase L in prostate cancer by focusing on basic cellular processes regulated by RNase L including autophagy and apoptosis. We are also developing an experimental protocol for treating late-stage cancer by combining sunitinib treatments with infection by an oncolytic virus.
Dr. Silverman has investigated molecular pathways of the anti-viral and anti-cancer activities of interferon for the past 34 years. His studies are mainly focused on a classical innate immune pathway known as the OAS/RNase L system. Interestingly, the RNase L gene maps to the hereditary prostate cancer 1 locus and may have a dual function in both suppressing hereditary prostate cancer and viral infections. The knowledge to be gained from these studies could eventually contribute to improved treatments for viral infections and cancer. For instance, his group has recently shown that transient suppression of the interferon-regulated proteins RNase L and PKR with the drug, sunitinib, enhances the anti-tumor efficacy of a therapeutic virus in mouse models of cancer.
Silverman, R.H., Jung, D.D., Nolan-Sorden, N.L., Dieffenbach, C.W., Kedar, V.P., and SenGupta, D. Purification and analysis of murine 2-5A-dependent RNase.J. Biol. Chem. 263, 7336-7341, 1988.
Zhou, A., Hassel, B.A., and Silverman, R.H. Expression cloning of 2-5A-dependent RNase-a uniquely regulated mediator of interferon action. Cell, 72, 753-765, 1993.
Hassel, B.A., Zhou, A., Sotomayor, C., Maran, A., and Silverman, R.H. A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon. EMBO J., 12, 3297-3304, 1993.
Zhou, A., Paranjape, J., Brown, T.L., Nie, H., Naik, S., Dong, B., Chang, A., Trapp, B. Fairchild, R., Colmenares, C., and Silverman, R.H. Interferon action and apoptosis are defective in mice devoid of 2',5'-oligoadenylate dependent RNase L. EMBO J. 16, 6355-6363,1997.
Der, S., Zhou, A., Williams, B.R.G., and Silverman, R.H.Identification of Genes Differentially Regulated by IFN-α β or γ or Using Oligonucleotide Arrays.Proc. Natl. Acad. Sci. U.S.A 95,15623-15628, 1998.
Zhou, A., Paranjape, J.M., Der, S.D., and Williams, B.R.G. and Silverman, R.H. Novel Innate Mechanisms of Interferon Action are Revealed in Triply Deficient Mice. Virology, 258, 435-440, 1999.
Carpten, J., N., et al. Germline Mutations in the Ribonuclease L (RNase L) Gene in Hereditary Prostate Cancer 1 (HPC1) -Linked Families. Nature Genetics, 30, 181-184, 2002.
Casey, G., et al.RNASEL R462Q variant is implicated in 13% of prostate cancer cases. Nature Genetics, 32, 581-583, 2002. Silverman, R.H. Implications for RNase L in Prostate Cancer Biology.Biochemistry, 42, 1805-1812, 2003.
Xiang, Y., Wang,Z., Murakami, J., Plummer, S., Klein, E.A., Carpten, J., Trent, J., Isaacs W., Casey, G., and Silverman, R. H. Effects of RNase L mutations associated with prostate cancer on apoptosis induced by 2',5'-oligoadenylates. Cancer Res. 63: 6795-6801, 2003.
Malathi, K., Paranjape, J.M., Bulanova, E., Shim, M., Guenther-Johnson, J.M., Faber, P.W., Eling, T.E., Williams, B.R.G., and Silverman, R.H. A novel transcriptional signaling pathway in the interferon system mediated by 2'-5'-oligoadenylate activation of RNase L. Proc. Natl. Acad. Sci. U.S.A., 102, 14533-14538, 2005.
Malathi, K., Dong, B., Gale, M., and Silverman, R.H. Small Self RNA Generated by RNase L Amplifies Antiviral Innate Immunity. Nature, 448: 816-819, 2007.
Malathi, K., Saito, T., Crochet, N., Barton, D.J.,Gale, M.and Silverman, R.H. RNase L Releases a Small RNA from HCV RNA that Refolds into a Potent PAMP. RNA, 16: 2108-2119, 2010.
Jha, B.K., Polyakova, I., Kessler, P., Dong, B., Dickerman, B., Sen, G.C., and Silverman, R.H. Inhibition of RNase L and RNA-dependent protein kinase (PKR) by sunitinib impairs antiviral innate immunity. J. Biol. Chem.,286: 26319-26326. 2011.
Zhao, L., Jha, B., Wu, A., Elliott, R., Ziebuhr, J., Gorbalenya, A.E., Silverman, R.H., Weiss, S.R. 2012. Antagonism of the interferon-induced OAS-RNase L pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology. Cell Host and Microbe, 11:607-616, 2012.
Chakrabarti, A., Ghosh, P.K., Banerjee, S., Gaughan, C., and Silverman, R.H. RNase L triggers autophagy in response to viral infections. J. Virol. 86: 11311-21, 2012.
Jha, B.K., Dong, B., Nguyen, C.T., Polyakova, I. and Silverman, R.H. Suppression of antiviral innate immunity by sunitinib enhances oncolyticvirotherapy. Molecular Therapy, Epub, 2013.
Zhao, L., Birdwell, D. Wu, A., Elliott, R., Rose, K., Phillips, J., Li, Y., Grinspan, J., Silverman, R., and Weiss, S. Cell type specific activation of the OAS-RNase L pathway by a murine coronavirus. J. Virol., 87:8408-18, 2013. [Featured article in "JVI Spotlight"]
Sorgeloos, F., Jha, B.K., Silverman, R.H., and Michiels, T.Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L.PLoS Pathogens, Epub, 2013.
Zhang, R., Jha, B.K., Ogden, K., Dong, B., Zhao, L., Elliot, R., Patton, J.T., Silverman, R.H.* and Weiss, S.R.* Homologous 2',5'-phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity. Proc. Natl. Acad. Sci., Epub, 2013. [*Co-corresponding authors].
The intersection between innate immunity and tumor suppression is the focus of the Silverman Lab. Our studies probe fundamental molecular and cellular processesthat impact microbial infections and cancer. We seek a better understanding of how the mammalian cell resists viral infections and how the virus antagonizes the host response to infection. We are also investigating the role of the RNase L gene in hereditary prostate cancer. In addition, we are developing a combination therapy for late-stage metastatic cancer by combining an oncolytic virus with a drug.
The interferon (IFN) system is a frontline host defense against invading viral pathogens. The 2',5'-oligoadenylate (2-5A)synthetase (OAS)-RNase L pathway is among the most potent IFN-induced antiviral effectors, blocking viral infections by several mechanisms, including directly cleaving viral single-stranded RNA genomes, depleting viral and host mRNA available for translation, and enhancing type I IFN induction. Type I IFNs bind to the cell surface receptor IFNAR initiating JAK-STAT signaling to the OAS genes, which results in elevated levels of OAS proteins. When activated by viral double-stranded (ds)RNA, certain OAS isoforms use ATP to synthesize 5'-triphosphorylated 2-5A. Trimer and longer species of 2-5A bind with high specificity and affinity to the inactive monomeric RNase L, causing it to dimerize and become active.
Antiviral Mechanisms of 2-5A Dependent RNase L:
RNase L is a principal mediator of the innate antiviral response and is thus critically important for human health. Previously, we purified RNaseL(JBC, 1988, PMID: 3366783), cloned RNase L (Cell, 1993, PMID: 7680958), and knocked out the RNase L gene in mice (EMBO J, 1997, PMID: 9351818). We showed that RNA cleavage products of self-RNA produced by RNase L function as PAMPs in amplifying innate immune signaling (Nature, 2007, PMID: 17653195). In a continuation of these studies, we are investigating how RNase L amplifies production of antiviral cytokines and how it eliminates virus particles through a process known as autophagy (Journal of Virology, 2012, PMID: 22875977). Our long-term objectives are to probe fundamental events and biologic endpoints surrounding RNase L that impact on viral lifecycles, spread and pathogenesis. The knowledge to be gained from the proposed studies could contribute to improved treatments for viral infections and cancer.
Control of Viral Pathogenesis by Regulation of 2-5A Levels: (a collaborative project with Susan Weiss, University of Pennsylvania)
The ability of viruses to evade the IFN antiviral response plays an important role in viral tropism and disease pathogenesis. Relevant to this project, the murine coronavirus ns2 protein has 2’,5’-phosphodiesterase (PDE) activity that antagonizes the IFN regulated OAS-RNase L antiviral pathway by eliminating 2-5A thereby promoting hepatitis; several other viruses and host cells have proteins with similar activities (Cell Host & Microbe, 2012, PMID: 22704621). Recently, with the teams of Susan Weiss and John Patton (NIAID, NIH), we reported group A rotavirus, an important cause of acute gastroenteritis in children worldwide, encodes a similar 2’,5’-PDE (PNAS, 2013, PMID: 23878220). The discovery and use of inhibitors of viral and cellular PDEs to enhance RNase L activity selectively in virus-infected cells potentially provides a novel mode of regulation of the antiviral response, with broad implication for the control of viral infections.
Role of RNase L in Prostate Cancer:
Prostate cancer is the leading cause of non-cutaneous malignancies and the second leading cause of cancer-related deaths among American men. Hereditary prostate cancer (HPC), which accounts for 43% of early onset cases and about 9% of all cases, is due to germline mutations in HPC genes. In 2002, we co-authored with the group of Jeffrey Trent and others a study a report that the HPC1 locus maps to the RNase L gene (Nature Genetics, 2002, PMID: 11799394). We are currently investigating the potential role of RNase L as a suppressor of prostate cancer by focusing on basic cellular processes regulated by RNase L including autophagy and apoptosis.
Combination OncolyticVirotherapy for Late-Stage Cancer:
The use of lytic viruses to preferentially infect and eliminate cancer cells while sparing normal cells is a promising experimental therapeutic approach for treating cancer. However, the efficacy of oncolyticvirotherapy is often limited by two innate immunity pathways, the protein kinase PKR and the 2’,5’-OAS/RNase L systems, which are widely present in many but not all tumor cell types. We reported that the anti-cancer drug, sunitinib, an inhibitor of VEGF-R and PDGF-R, has off-target effects against both PKR and RNase L(JBC, 2012, PMID: 21636578). Recently, we showed that combining sunitinib treatments with infection by an oncolytic virus, vesicular stomatitis virus, led to the elimination of prostate, breast and kidney malignant tumors in mice (Molecular Therapy, 2013, PMID: 23732991). In contrast, either virus or sunitinib alone slowed tumor progression but did not eliminate tumors. Results indicate that transient inhibition of innate immunity with sunitinib enhances oncolyticvirotherapy allowing the recovery of tumor-bearing animals. We are currently extending these findings to other oncolytic viruses.
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