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Cancer Upregulated Gene 2, a Novel Oncogene, Confers Resistance to Oncolytic Vesicular Stomatitis Virus Through STAT1-OASL2 Signaling

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Cancer Upregulated Gene 2, a Novel Oncogene, Confers Resistance to Oncolytic Vesicular Stomatitis Virus Through STAT1-OASL2 Signaling Cancer Gene Therapy (2013) 20, 125–132 & 2013 Nature America, Inc. All rights reserved 0929-1903/13 www.nature.com/cgt ORIGINAL ARTICLE Cancer upregulated gene 2, a novel oncogene, confers resistance to oncolytic vesicular stomatitis virus through STAT1-OASL2 signaling W Malilas1, SS Koh2, R Srisuttee1, W Boonying1, I-R Cho1, C-S Jeong3, RN Johnston4 and Y-H Chung1 We have recently found a novel oncogene, named cancer upregulated gene 2 (CUG2), which activates Ras and mitogen-activated protein kinases (MAPKs), including ERK, JNK and p38 MAPK. Because activation of these signaling pathways has previously been shown to enhance cancer cell susceptibility to oncolysis by certain viruses, we examined whether vesicular stomatitis virus (VSV) could function as a potential therapeutic agent by efficiently inducing cytolysis in cells transformed by CUG2. Unexpectedly, NIH3T3 cells stably expressing CUG2 (NIH-CUG2) were resistant to VSV because of the activation of signal transducers and activators of transcription 1 (STAT1). The result was supported by evidence showing that suppression of STAT1 with short interference RNA (siRNA) renders cells susceptible to VSV. Furthermore, 20–50 oligoadenylate synthetase-like (OASL) 2 was the most affected by STAT1 expression level among anti-viral proteins and furthermore suppression of OASL2 mRNA level caused NIH-CUG2 cells to succumb to VSV as seen in NIH-CUG2 cells treated with STAT1 siRNA. In addition, Colon26L5 carcinoma cells stably expressing CUG2 (Colon26L5-CUG2) exhibited resistance to VSV, whereas Colon26L5 stably expressing a control vector yielded to VSV infection. Moreover, Colon26L5-CUG2 cells stably suppressing STAT1 succumbed to VSV infection, resulting in apoptosis. Taken together, we propose that VSV treatment combined with the selective regulation of genes such as STAT1 and OASL2 will improve therapeutic outcomes for CUG2-overexpressing tumors. Cancer Gene Therapy (2013) 20, 125–132; doi:10.1038/cgt.2012.96; published online 11 January 2013 Keywords: VSV; STAT1; OASL2; CUG2; virotherapy INTRODUCTION peripheral matrix protein.4 VSV causes vesicular lesions in the To discover new genes that have a crucial role in common mucous membranes of mouse and in the nasal tissues of horses, tumorigenesis regardless of tissue origin, we analyzed commonly cattle and pigs. However, VSV does not induce significant upregulated unknown genes using 242 normal and 300 tumor symptoms in humans. Moreover, evidence of human exposure to 5 samples originating from 11 different tissues with an Affymetrix VSV is generally uncommon in seroprevalence studies, which is gene chip system. An uncharacterized gene, later named cancer beneficial for the potential use of VSV as an oncolytic agent in upregulated gene 2 (CUG2), was identified as a potential human trials. Furthermore, VSV can replicate in malignant cells 6–9 candidate that was commonly upregulated in various tumor more efficiently than in normal cells as do other oncolytic viruses. tissues, including ovary, liver, lung, pancreas and colon. CUG2 was Successful treatments with VSV have been reported in laboratory 10–13 mapped to chromosome 6q22.32, extends about 8.5 kb in length models of breast, lung, brain and colon cancers. However, VSV with a 3-exon structure, encoding a 88-amino-acid polypeptide.1 is very sensitive to the anti-viral activity of interferon (IFN) induction, Further study has revealed that CUG2 protein is a novel and therefore improved strategies for the application of VSV have component of centromeres required for proper kinetochore been sought for preferential replication in tumor cells and 10,14 function during cell division.2 Of interest, CUG2 has been shown resistance to the anti-viral action of host cells. to possess an oncogenic effect in a transplanted model using IFN-induced anti-viral proteins have been extensively studied. NIH3T3 cells expressing CUG2, behaving similarly to Ras.1 Our 20–50 Oligoadenylate synthetase (OAS) stimulated by double- previous study has shown that CUG2 activates Ras and mitogen- stranded RNA polymerizes ATP to 20- and 50-linked oligomers of activated protein kinases (MAPKs) including p38 MAPK, which adenosine and activates RNaseL by inhibiting initiation of protein eventually facilitate reoviral replication and the death of virus- synthesis or degrading RNA.15 The OAS gene family contains four infected cancer cells.3 OAS members: OAS1, OAS2, OAS3 and OASL.16 IFN-stimulated gene Vesicular stomatitis virus (VSV), also known as a potential 15 (ISG15), a 15-kDa ubiquitin-like protein, has recently emerged therapeutic anticancer agent, is a non-enveloped, negative- as an important tool in the defense against viral infections. Like stranded RNA virus and belongs to the Rhabdoviridae family. ubiquitin and other ubiquitin-like proteins, ISG15 is attached to The RNA genome of VSV encodes five proteins, including target proteins through a C-terminal Gly–Gly motif, which is nucleocapsid, polymerase proteins, surface glycoprotein and referred to as ISGylation and is implicated in anti-viral activity.17 1WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, Republic of Korea; 2Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Department of Functional Genomics, University of Science and Technology, Daejeon, Republic of Korea; 3School of Biological Sciences, Ulsan University, Ulsan, Republic of Korea and 4Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada. Correspondence: Professor Y-H Chung, WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Republic of Korea. E-mail: [email protected] Received 2 October 2012; revised 13 December 2012; accepted 13 December 2012; published online 11 January 2013 CUG2 confers resistance to VSV through STAT1 W Malilas et al 126 Guanylate-binding proteins (GBPs) are a family of large IFN- Active ras capture assays 18 induced GTPases. Although it is currently unclear how GBP To capture GTP-bound Ras from cell lysates, a GST-Raf-RBD fusion protein functions in anti-viral activity, ectopic expression of GBP1 or GBP2 was used as described elsewhere.24 The cells were lysed with lysis buffer protects cells against VSV and encephalomyelitis virus infections.19 and glutathione-agarose beads (Clontech, Mountain View, CA, USA) were IFN-inducible protein-10 (IP-10), a non-ELR CXC chemokine, is a added to the supernatant, and the solutions were rocked on ice for 30 min chemoattractant for natural killer and T cells and is believed to be to exclude nonspecific binding. After centrifugation, the supernatant was an important regulator of the T-helper type 1, interleukin-12- harvested, followed by the addition of GST-Raf-RBD (Cytoskeleton, Denver, CO, USA). The solution was rocked on ice for 1 h, followed by the addition driven inflammatory responses as an inducer of cellular 20 of glutathione-agarose beads to capture active Ras, which binds to GST- infiltration. IFN-induced protein with tetratricopeptide repeat 3 Raf-RBD. After washing three times, the pellet was mixed with 40 mlof2Â (IFIT3) is significantly induced upon RNA virus infection. Recent SDS loading buffer. To detect the captured Ras, immunoblotting was study has shown that IFN-induced tetratricopeptide repeat protein performed with anti-Ras antibody (Santa Cruz Biotechnology). IFIT3 potentiates anti-viral signaling by bridging mitochondrial anti-viral signaling complex and tumor necrosis factor receptor- Qualitative reverse transcription-polymerase chain reaction associated factor family member-associated nuclear factor-kB activator-binding kinase 1.21 Total RNA was extracted from cells using the RNeasy mini kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. In Several lines of evidence have shown that Ras/Raf/MEK all, 2 mg of total RNA were converted to cDNA using Superscript II reverse signaling reduces the threshold for VSV infection by attenuation transcriptase (Invitrogen, Carlsbad, CA, USA), and polymerase chain 22,23 of IFN signaling. Our observation that CUG2 upregulates Ras reaction (PCR) was performed using the specific primers designed by the and MAPK activity3 led us to predict that NIH3T3 cells transformed Vector NTI software (Invitrogen). The cDNA products of each reaction were with CUG2 should become susceptible to VSV infection. In this diluted, and PCR was run at the optimized cycle number. Glyceraldehyde study, we tested this hypothesis by asking whether VSV efficiently 3-phosphate dehydrogenase mRNA was used as an internal standard. induces cytolysis in NIH3T3 cells transformed with CUG2. We unexpectedly found that CUG2 activates STAT1, leading to the Real-time RT-PCR upregulation of the anti-viral gene OASL2, which actually confers In addition, for a quantitative real-time reverse transcription (RT)-PCR, resistance to VSV infection. We also confirmed that activation of several sets of primers were designed by Bioneer Corporation (Daejeon, STAT1 mediated by CUG2 provides resistance to VSV in colon South Korea). Gene amplification was carried out using cDNA samples with carcinoma cells. We herein suggest that VSV treatment combined a SYBR Green-based real-time PCR. All real-time PCR amplification and with selective downregulation of genes such as STAT1 and OASL2 analysis was conducted by Accupower qPCR array using Exicycler 96 Real- will enhance therapeutic efficiency for CUG2-overexpressing Time Quantitative Thermal Block (Bioneer, Daejeon,
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