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• W2963978412 abstract "KRAS mutant (KRASmut) lung adenocarcinoma is a refractory cancer without available targeted therapy. The current study explored the possibility to develop coxsackievirus type B3 (CVB3) as an oncolytic agent for the treatment of KRASmut lung adenocarcinoma. In cultured cells, we discovered that CVB3 selectively infects and lyses KRASmut lung adenocarcinoma cells (A549, H2030, and H23), while sparing normal lung epithelial cells (primary, BEAS2B, HPL1D, and 1HAEo) and EGFRmut lung adenocarcinoma cells (HCC4006, PC9, H3255, and H1975). Using stable cells expressing a single driver mutation of either KRASG12V or EGFRL858R in normal lung epithelial cells (HPL1D), we further showed that CVB3 specifically kills HPL1D-KRASG12V cells with minimal harm to HPL1D-EGFRL858R and control cells. Mechanistically, we demonstrated that aberrant activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and compromised type I interferon immune response in KRASmut lung adenocarcinoma cells serve as key factors contributing to the sensitivity to CVB3-induced cytotoxicity. Lastly, we conducted in vivo xenograft studies using two immunocompromised mouse models. Our results revealed that intratumoral injection of CVB3 results in a marked tumor regression of KRASmut lung adenocarcinoma in both non-obese diabetic (NOD) severe combined immunodeficiency (SCID) gamma (NSG) and NOD-SCID xenograft models. Together, our findings suggest that CVB3 is an excellent candidate to be further developed as a targeted therapy for KRASmut lung adenocarcinoma. KRAS mutant (KRASmut) lung adenocarcinoma is a refractory cancer without available targeted therapy. The current study explored the possibility to develop coxsackievirus type B3 (CVB3) as an oncolytic agent for the treatment of KRASmut lung adenocarcinoma. In cultured cells, we discovered that CVB3 selectively infects and lyses KRASmut lung adenocarcinoma cells (A549, H2030, and H23), while sparing normal lung epithelial cells (primary, BEAS2B, HPL1D, and 1HAEo) and EGFRmut lung adenocarcinoma cells (HCC4006, PC9, H3255, and H1975). Using stable cells expressing a single driver mutation of either KRASG12V or EGFRL858R in normal lung epithelial cells (HPL1D), we further showed that CVB3 specifically kills HPL1D-KRASG12V cells with minimal harm to HPL1D-EGFRL858R and control cells. Mechanistically, we demonstrated that aberrant activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and compromised type I interferon immune response in KRASmut lung adenocarcinoma cells serve as key factors contributing to the sensitivity to CVB3-induced cytotoxicity. Lastly, we conducted in vivo xenograft studies using two immunocompromised mouse models. Our results revealed that intratumoral injection of CVB3 results in a marked tumor regression of KRASmut lung adenocarcinoma in both non-obese diabetic (NOD) severe combined immunodeficiency (SCID) gamma (NSG) and NOD-SCID xenograft models. Together, our findings suggest that CVB3 is an excellent candidate to be further developed as a targeted therapy for KRASmut lung adenocarcinoma. Lung cancer is the leading cause of cancer-related death in both males and females in North America and worldwide.1Chen Z. Fillmore C.M. Hammerman P.S. Kim C.F. Wong K.K. Non-small-cell lung cancers: a heterogeneous set of diseases.Nat. Rev. Cancer. 2014; 14: 535-546Crossref PubMed Scopus (985) Google Scholar, 2Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2018.CA Cancer J. Clin. 2018; 68: 7-30Crossref PubMed Scopus (6017) Google Scholar Currently, most patients with lung cancer are diagnosed at an advanced stage when potentially curative treatment is no longer possible. Histologically, adenocarcinoma is the most common type of lung cancer.3Cancer Genome Atlas Research NetworkComprehensive molecular profiling of lung adenocarcinoma.Nature. 2014; 511: 543-550Crossref PubMed Scopus (3291) Google Scholar Further subcategorization has been achieved by molecular criteria, such as specific driver mutations in genes that encode signaling proteins crucial for cellular proliferation and survival.4Ding L. Getz G. Wheeler D.A. Mardis E.R. McLellan M.D. Cibulskis K. Sougnez C. Greulich H. Muzny D.M. Morgan M.B. et al.Somatic mutations affect key pathways in lung adenocarcinoma.Nature. 2008; 455: 1069-1075Crossref PubMed Scopus (2109) Google Scholar Somatic mutations in epidermal growth factor receptor (EGFR) have been identified in ∼15% of all patients with lung adenocarcinoma, with the proportion increasing to 50% in patients who have never smoked.4Ding L. Getz G. Wheeler D.A. Mardis E.R. McLellan M.D. Cibulskis K. Sougnez C. Greulich H. Muzny D.M. Morgan M.B. et al.Somatic mutations affect key pathways in lung adenocarcinoma.Nature. 2008; 455: 1069-1075Crossref PubMed Scopus (2109) Google Scholar Although patients with EGFR mutant (EGFRmut) lung adenocarcinoma have increased sensitivity to tyrosine kinase inhibitors, primary and acquired resistance toward these agents remains a major clinical obstacle.5Lin Y. Wang X. Jin H. EGFR-TKI resistance in NSCLC patients: mechanisms and strategies.Am. J. Cancer Res. 2014; 4: 411-435PubMed Google Scholar Conversely, Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are more common in patients who had a history of cigarette smoking and account for ∼25% of lung adenocarcinomas.6Riely G.J. Kris M.G. Rosenbaum D. Marks J. Li A. Chitale D.A. Nafa K. Riedel E.R. Hsu M. Pao W. et al.Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma.Clin. Cancer Res. 2008; 14: 5731-5734Crossref PubMed Scopus (440) Google Scholar However, these patients have a poor prognosis because of the lack of survival benefit from adjuvant chemotherapy and resistance to targeted kinase inhibitors.7Kaufman J. Stinchcombe T.E. Treatment of kras-mutant non–small cell lung cancer: The end of the beginning for targeted therapies.JAMA. 2017; 317: 1835-1837Crossref PubMed Scopus (10) Google Scholar Therefore, there is an urgent need for developing new therapeutics for this subgroup of the patients. Oncolytic virus (OV) is clinically defined by its ability to induce lysis of malignant cells through a self-replication process without causing damage to normal tissues.8Kaufman H.L. Kohlhapp F.J. Zloza A. Oncolytic viruses: a new class of immunotherapy drugs.Nat. Rev. Drug Discov. 2015; 14: 642-662Crossref PubMed Scopus (686) Google Scholar, 9Lawler S.E. Speranza M.C. Cho C.F. Chiocca E.A. Oncolytic Viruses in Cancer Treatment: A Review.JAMA Oncol. 2017; 3: 841-849Crossref PubMed Scopus (289) Google Scholar Over the past decades, a better understanding of tumor biology and molecular mechanisms of viral cytotoxicity has provided a scientific rationale to develop more efficient oncolytic viruses as potent, self-amplifying cancer therapeutics.10Miest T.S. Cattaneo R. New viruses for cancer therapy: meeting clinical needs.Nat. Rev. Microbiol. 2014; 12: 23-34Crossref PubMed Scopus (190) Google Scholar As a result, several viruses including adenovirus, herpes simplex virus 1 (HSV-1), coxsackievirus A21 (CVA21), measles virus, and reovirus have demonstrated varying degrees of success in clinical trials,11Akhtar L.N. Benveniste E.N. Viral exploitation of host SOCS protein functions.J. Virol. 2011; 85: 1912-1921Crossref PubMed Scopus (90) Google Scholar, 12Galanis E. Atherton P.J. Maurer M.J. Knutson K.L. Dowdy S.C. Cliby W.A. Haluska Jr., P. Long H.J. Oberg A. Aderca I. et al.Oncolytic measles virus expressing the sodium iodide symporter to treat drug-resistant ovarian cancer.Cancer Res. 2015; 75: 22-30Crossref PubMed Scopus (123) Google Scholar, 13Lee C.Y. Rennie P.S. Jia W.W. MicroRNA regulation of oncolytic herpes simplex virus-1 for selective killing of prostate cancer cells.Clin. Cancer Res. 2009; 15: 5126-5135Crossref PubMed Scopus (84) Google Scholar, 14Nemunaitis J. Ganly I. Khuri F. Arseneau J. Kuhn J. McCarty T. Landers S. Maples P. Romel L. Randlev B. et al.Selective replication and oncolysis in p53 mutant tumors with ONYX-015, an E1B-55kD gene-deleted adenovirus, in patients with advanced head and neck cancer: a phase II trial.Cancer Res. 2000; 60: 6359-6366PubMed Google Scholar, 15Prestwich R.J. Ilett E.J. Errington F. Diaz R.M. Steele L.P. Kottke T. Thompson J. Galivo F. Harrington K.J. Pandha H.S. et al.Immune-mediated antitumor activity of reovirus is required for therapy and is independent of direct viral oncolysis and replication.Clin. Cancer Res. 2009; 15: 4374-4381Crossref PubMed Scopus (127) Google Scholar, 16Silk A.W. Kaufman H. Gabrail N. Mehnert J. Bryan J. Norrell J. Medina D. Bommareddy P. Shafren D. Grose M. Zloza A. Phase 1b study of intratumoral Coxsackievirus A21 (CVA21) and systemic pembrolizumab in advanced melanoma patients: Interim results of the CAPRA clinical trial.Cancer Res. 2017; 77 (CT026)Google Scholar whereas a modified HSV-1 has been approved by the US Food and Drug Administration in October 2015 for the treatment of melanoma.17Poh A. First oncolytic viral therapy for melanoma.Cancer Discov. 2016; 6: 6Crossref PubMed Scopus (29) Google Scholar On the other hand, there are still several hurdles to overcome for oncolytic viruses to become clinically effective, which includes poor tropism for targeted organs and pre-existing immunity against oncolytic virus replication in adults.10Miest T.S. Cattaneo R. New viruses for cancer therapy: meeting clinical needs.Nat. Rev. Microbiol. 2014; 12: 23-34Crossref PubMed Scopus (190) Google Scholar Coxsackievirus type B3 (CVB3), a non-enveloped, human pathogenic enterovirus of the Picornaviridae family, encompasses a 7.4-kb single-stranded, positive-sense RNA genome. Although CVB3 infection is associated with high incidence of myocarditis, pancreatitis, meningitis, and encephalitis in children and adolescents, infection in adults is generally asymptomatic or causes mild flu-like symptoms.18Fung G. Luo H. Qiu Y. Yang D. McManus B. Myocarditis.Circ. Res. 2016; 118: 496-514Crossref PubMed Scopus (239) Google Scholar, 19Huber S. Ramsingh A.I. Coxsackievirus-induced pancreatitis.Viral Immunol. 2004; 17: 358-369Crossref PubMed Scopus (97) Google Scholar, 20Pinkert S. Dieringer B. Diedrich S. Zeichhardt H. Kurreck J. Fechner H. Soluble coxsackie- and adenovirus receptor (sCAR-Fc); a highly efficient compound against laboratory and clinical strains of coxsackie-B-virus.Antiviral Res. 2016; 136: 1-8Crossref PubMed Scopus (10) Google Scholar Recently, large-scale screening of 28 enterovirus strains has identified CVB3 as one of the most potent oncolytic viruses against a panel of different human cancer cells, including non-small-cell lung cancer (NSCLC).21Miyamoto S. Inoue H. Nakamura T. Yamada M. Sakamoto C. Urata Y. Okazaki T. Marumoto T. Takahashi A. Takayama K. et al.Coxsackievirus B3 is an oncolytic virus with immunostimulatory properties that is active against lung adenocarcinoma.Cancer Res. 2012; 72: 2609-2621Crossref PubMed Scopus (129) Google Scholar In addition to its natural tropism for NSCLC cells, CVB3 also possesses two features that make it an excellent candidate for oncolytic virotherapy. First, CVB3 preferentially infects and lyses actively dividing cells over quiescent cells, thus activation of oncogenic signaling pathways within tumor cells creates a permissive microenvironment supporting virus replication.22Feuer R. Whitton J.L. Preferential Coxsackievirus Replication in Proliferating/Activated Cells: Implications for Virus Tropism, Persistence, and Pathogenesis.in: Tracy S. Oberste M.S. Drescher K.M. Group B Coxsackieviruses. Springer Berlin Heidelberg, 2008: 149-173Crossref Scopus (22) Google Scholar Second, CVB3 infection is profoundly inhibited by type I interferon; as a result, normal cells with intact interferon signaling are more resistant to CVB3 infection than tumor cells that often display an impaired interferon signaling.23Althof N. Harkins S. Kemball C.C. Flynn C.T. Alirezaei M. Whitton J.L. In vivo ablation of type I interferon receptor from cardiomyocytes delays coxsackieviral clearance and accelerates myocardial disease.J. Virol. 2014; 88: 5087-5099Crossref PubMed Scopus (29) Google Scholar, 24Critchley-Thorne R.J. Simons D.L. Yan N. Miyahira A.K. Dirbas F.M. Johnson D.L. Swetter S.M. Carlson R.W. Fisher G.A. Koong A. et al.Impaired interferon signaling is a common immune defect in human cancer.Proc. Natl. Acad. Sci. USA. 2009; 106: 9010-9015Crossref PubMed Scopus (176) Google Scholar, 25Feng Q. Langereis M.A. Lork M. Nguyen M. Hato S.V. Lanke K. Emdad L. Bhoopathi P. Fisher P.B. Lloyd R.E. van Kuppeveld F.J. Enterovirus 2Apro targets MDA5 and MAVS in infected cells.J. Virol. 2014; 88: 3369-3378Crossref PubMed Scopus (146) Google Scholar In this study, we showed that wild-type (WT) CVB3 specifically targets KRASmut lung adenocarcinoma cells with limited effects on normal lung epithelial cells and EGFRmut lung adenocarcinoma cells. Mechanistically, we demonstrated that enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and impaired type I interferon response contribute, at least in part, to the sensitivity of KRASmut lung adenocarcinoma cells to CVB3-induced cytotoxicity. Xenograft models of lung adenocarcinoma demonstrated that treatment with WT-CVB3 results in a significant decrease in tumor size in immunocompromised mice bearing KRASmut lung adenocarcinoma. Taken together, our findings suggest that CVB3 could be an excellent candidate for further development into a novel oncolytic virus for the treatment of KRASmut lung adenocarcinoma. The development of targeted therapies to driver oncogenes has led to a substantial benefit for NSCLC patients carrying EGFR and other specific mutations; however, KRAS mutations are currently undruggable. This evokes us to question whether CVB3-based virotherapy can be a novel approach targeting KRASmut lung adenocarcinomas. In fact, previous studies have shown that tumor selectivity of several oncolytic viruses can be enhanced by gain-of-function mutations in given oncogenes of the Ras-signaling pathways.26Noser J.A. Mael A.A. Sakuma R. Ohmine S. Marcato P. Lee P.W. Ikeda Y. The RAS/Raf1/MEK/ERK signaling pathway facilitates VSV-mediated oncolysis: implication for the defective interferon response in cancer cells.Mol. Ther. 2007; 15: 1531-1536Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar To test our hypothesis, seven patient-derived lung adenocarcinoma cell lines, including three KRASmut (H23, H2030, and A549) and four EGFRmut (H1975, H3255, PC-9, and H4006) cells were selected to examine their sensitivities to CVB3 infection. We also chose three normal lung epithelial cell lines (1HAEo, HPL1D, and BEAS2B) and primary airway epithelial cells isolated from normal donors to evaluate the specificity of CVB3 treatment in vitro. As shown in Figure 1A, CVB3 exhibited powerful cytotoxic activities against KRASmut lung adenocarcinoma cells in a dose-dependent manner. However, EGFRmut lung adenocarcinoma cells and normal lung epithelial cells displayed only minimal cytopathic effects after 48-h infection with CVB3 even at the highest MOI tested. Cell viability assays further validated that CVB3 infection resulted in a profound reduction (∼85%) of cell survival in KRASmut lung adenocarcinoma cells (Figure 1B). No significant reduction in cell viability in EGFRmut lung adenocarcinoma cells or slight decrease of cell survival in normal lung epithelial cells was observed upon CVB3 infection, especially at the lower dose of CVB3 (Figure 1B). Moreover, we examined the replication ability of CVB3 in lung adenocarcinoma and normal lung epithelial cells by plaque assay. Figure 1C showed that the virus titers in the supernatant of CVB3-infected KRASmut lung adenocarcinoma cells were significantly higher than those from EGFRmut lung adenocarcinoma and normal lung epithelial cells, suggesting that CVB3-mediated oncolytic effect is highly associated with its replicative capacity. As a positive control, we showed that WT-CVB3 infection of HeLa cells, a human cervical cancer cell line that has previously been shown to be extremely sensitive to CVB3 infection, caused substantial lysis at all concentrations tested (Figure 1D). Together, these results indicate that CVB3 specifically infects and kills KRASmut lung adenocarcinoma to exert its oncolytic effects through self-replication. It has become evident that lung adenocarcinoma is a heterogeneous disease marked with a high rate of somatic mutations.4Ding L. Getz G. Wheeler D.A. Mardis E.R. McLellan M.D. Cibulskis K. Sougnez C. Greulich H. Muzny D.M. Morgan M.B. et al.Somatic mutations affect key pathways in lung adenocarcinoma.Nature. 2008; 455: 1069-1075Crossref PubMed Scopus (2109) Google Scholar, 27Pao W. Girard N. New driver mutations in non-small-cell lung cancer.Lancet Oncol. 2011; 12: 175-180Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar In addition to the driver oncogene, each lung adenocarcinoma cell line tested in this study has multiple somatic mutations that may produce a synergistic role in supporting viral replication. Therefore, to specifically determine the effect of KRAS or EGFR mutation on CVB3 tropism, we generated isogenic cells from the normal lung cell line HPL1D expressing a single driver mutation of either KRAS (KRASG12V) or EGFR (EGFRL858R). HPL1D cells expressing GFP were used as a negative control. Western blot analysis verified overexpression of KRAS or EGFR in these cell lines (Figure 2A). We found that WT-CVB3 specifically targeted and lysed HPL1D-KRASG12V cells with very minimal harm to HPL1D-EGFRL858R and normal cells (Figures 2B–2D), and UV-inactivated CVB3 (UV-CVB3) failed to cause apparent cell death (Figure 2E). Our results indicate that KRAS mutation is a determinant of viral sensitivity. We next investigated the potential mechanism by which CVB3 preferentially replicates in KRASmut lung adenocarcinomas. Previous in vitro and in vivo evidence has demonstrated that CVB3 replication relies largely on the activation of oncogenic signaling pathways, among which the ERK1/2 signaling is the best characterized and proven to be the most important signaling pathway hijacked by CVB3 for effective replication.28Luo H. Yanagawa B. Zhang J. Luo Z. Zhang M. Esfandiarei M. Carthy C. Wilson J.E. Yang D. McManus B.M. Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.J. Virol. 2002; 76: 3365-3373Crossref PubMed Scopus (177) Google Scholar, 29Opavsky M.A. Martino T. Rabinovitch M. Penninger J. Richardson C. Petric M. Trinidad C. Butcher L. Chan J. Liu P.P. Enhanced ERK-1/2 activation in mice susceptible to coxsackievirus-induced myocarditis.J. Clin. Invest. 2002; 109: 1561-1569Crossref PubMed Scopus (96) Google Scholar To determine the potential contribution of ERK1/2 activation in permissiveness to CVB3-mediated cell death, we examined ERK1/2 activation or phosphorylation status in different lung adenocarcinoma cells and isogenic HPL1D cells expressing different mutant oncogenes. We found that the ERK1/2 was activated or phosphorylated in KRASmut cells to a greater degree as compared with EGFRmut and normal lung epithelial cells (Figures 3A and 3B ). We further showed that inhibition of ERK1/2 using a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor (U0126) decreased viral protein levels and virus titers in a dose-dependent manner in both patient-derived KRASmut H2030 (Figures 3C and 3D) and HPL1D-KRASG12V cells (Figures 3E and 3F). Together, our data suggest that enhanced ERK1/2 activation contributes, at least in part, to the susceptibility of KRASmut lung adenocarcinoma cells to CVB3-induced cytotoxicity. Because type I interferon plays a key role in the innate immune response against CVB3 infection, we also questioned whether increased CVB3 susceptibility of KRASmut cells could be a result of compromised type-I interferon response. HPL1D cell lines stably expressing GFP (control), KRASG12V, or EGFRL858R were used to determine Ifnb1 (IFN-β gene) expression upon sham or CVB3 infection at an MOI of 1.0 for different time points (Figure 4A) or at various MOIs for 7 h (Figure 4B). As expected, infection with CVB3 resulted in an upregulation of Ifnb1 (IFN-β) gene in HPL1D-GFP control cells in a time- and dose-dependent manner (Figure 4). This induction of Ifnb1 gene was significantly suppressed in HPL1D-KRASG12V cells, but further enhanced in HPL1D-EGFRL858R cells (Figure 4). Our results suggest that impaired type I interferon production in KRASG12V cells may serve as an additional factor contributing to selective CVB3 replication and consequent oncolysis in these cells. To further understand the mechanism by which KRASG12V inhibits CVB3-induced Ifnb1 gene production, we examined the phosphorylation status of eukaryotic initiation factor 2α (eIF2α) as a marker of the activation of the double-stranded RNA (dsRNA)-activated protein kinase R (PKR). As a pattern recognition receptor for viral dsRNA, PKR has been shown to be significant for type I interferon production during viral infection.30Diebold S.S. Montoya M. Unger H. Alexopoulou L. Roy P. Haswell L.E. Al-Shamkhani A. Flavell R. Borrow P. Reis e Sousa C. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers.Nature. 2003; 424: 324-328Crossref PubMed Scopus (500) Google Scholar As shown in Figure 4C, we found that the levels of phosphorylated eIF2α were markedly increased at 7 h after CVB3 infection in all three cell types, consisting with our early findings.31Fung G. Ng C.S. Zhang J. Shi J. Wong J. Piesik P. Han L. Chu F. Jagdeo J. Jan E. et al.Production of a dominant-negative fragment due to G3BP1 cleavage contributes to the disruption of mitochondria-associated protective stress granules during CVB3 infection.PLoS ONE. 2013; 8: e79546Crossref PubMed Scopus (66) Google Scholar However, neither KRASG12V nor EGFRL858R appeared to affect the phosphorylation status of eIF2α, indicating that suppression of Ifnb1 production in cells expressing KRASG12V is not through inactivation of the PKR-eIF2α pathway. The coxsackievirus-adenovirus receptor (CAR) is the primary receptor responsible for CVB3 internalization.32Bergelson J.M. Cunningham J.A. Droguett G. Kurt-Jones E.A. Krithivas A. Hong J.S. Horwitz M.S. Crowell R.L. Finberg R.W. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2584) Google Scholar We next sought to determine whether CAR expression is a determining factor for the sensitivities of lung adenocarcinoma to CVB3-induced cell death. Western blot results showed that protein levels of CAR were noticeably higher in H23 and H2030 (KRASmut) cells as compared with H1975, HCC4006, and H3255 (EGFRmut) and normal lung epithelial cells, indicating a potential relationship between KRAS status and CAR expression (Figure 5A). To assess the effects of KRAS activation on CAR, we examined protein levels of CAR in tetracycline-inducible HPLID-KRASmut stable cells. Of interest, we found that addition of doxycycline resulted in decreased CAR levels (Figure 5B). This finding was further confirmed with the experiment of KRAS inhibition, which showed that treatment with ARS853, a KRASG12C inhibitor, increased protein levels of CAR in both H23 and H2030 KRASmut lung adenocarcinomas cells (Figure 5C). CAR expression has been previously reported to be associated with ERK1/2 signaling.33Anders M. Christian C. McMahon M. McCormick F. Korn W.M. Inhibition of the Raf/MEK/ERK pathway up-regulates expression of the coxsackievirus and adenovirus receptor in cancer cells.Cancer Res. 2003; 63: 2088-2095PubMed Google Scholar We further questioned whether ERK activation plays a role in KRAS-induced downregulation of CAR. Consistent with the early report,33Anders M. Christian C. McMahon M. McCormick F. Korn W.M. Inhibition of the Raf/MEK/ERK pathway up-regulates expression of the coxsackievirus and adenovirus receptor in cancer cells.Cancer Res. 2003; 63: 2088-2095PubMed Google Scholar we found that inhibition of ERK1/2 with U0126 caused an upregulation of CAR in both H23 and H2030 KRASmut cells (Figure 5D). Moreover, through the assessment of expression levels for the gene (CXADR) encoding CAR across a panel of 230 lung adenocarcinoma tumors profiled by The Cancer Genome Atlas (TCGA), we found that the transcriptional levels of CXADR between KRASmut and EGFRmut (and KRASmut and KRAS/EGFRWT) tumors were statistically insignificant (Figure 5E). Finally, we performed virus uptake assay using HPL1D stable cell lines expressing GFP, KRASG12V, or EGFRL858R to determine whether increased viral particle uptake is a factor contributing to viral sensitivity of KRASmut cells. As shown in Figure 5F, we observed no significant differences in virus uptake between control and KRASmut or EGFRmut cells. Taken together, our results suggest that KRAS negatively regulates CAR expression via ERK1/2 activation, and that enhanced protein levels of CAR detected in H23 and H2030 cells are independent of KRAS activation. Thus, CAR expression and/or altered virus entry are unlikely major determinants for the hypersensitivity of KRASmut lung adenocarcinomas to CVB3-induced cell death. We also examined the protein levels of decay-accelerating factor, the co-receptor for CVB3,34Bergelson J.M. Mohanty J.G. Crowell R.L. St John N.F. Lublin D.M. Finberg R.W. Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55).J. Virol. 1995; 69: 1903-1906Crossref PubMed Google Scholar in various lung adenocarcinomas cells. However, no apparent changes were observed (data not shown). We next conducted xenograft animal experiments using KRASmut H2030 cells to determine the anti-tumor effects of CVB3 in vivo. We first used non-obese diabetic (NOD) severe combined immunodeficiency (SCID) gamma (NSG)-immunodeficient mice, where immunity is completely abolished because of the lack of mature T cells, B cells, and functional natural killer (NK) cells.35Zhang B. Duan Z. Zhao Y. Mouse models with human immunity and their application in biomedical research.J. Cell. Mol. Med. 2009; 13: 1043-1058Crossref PubMed Scopus (34) Google Scholar A pilot study was performed with four different dosages of CVB3 (i.e., 5 × 104, 5 × 105, 5 × 106, and 5 × 107 plaque-forming units [PFUs]), demonstrating similar results in terms of tumor regression and mortality rate (data not shown). Figures 6A and 6B showed that intratumoral injection of WT-CVB3 at 5 × 104 PFUs resulted in a dramatic reduction in KRASmut xenograft tumor volumes, whereas tumor sizes continued to increase with the treatment of UV-CVB3. The tumor volume of mice exposed to WT-CVB3 on day 15 was ∼12-fold smaller than that of mice exposed to UV-CVB3, suggesting that CVB3 potently kills KRASmut lung adenocarcinoma in vivo, irrespective of immune response. Despite significant regression of KRASmut xenograft tumors, as shown in Figure 6C, mice obtained no survival benefit after WT-CVB3 treatment, and all mice were euthanized on days 12–15 because of sickness (according to the endpoints approved by the Animal Care Committee at the University of British Columbia). To examine possible comorbidities associated with WT-CVB3, we compared virus loads in the xenograft tumors and different organs. Figure 6D showed that, in addition to tumor, viral replication was also detected in various mouse organs, in particular the heart, suggesting an active systemic viral infection following intratumoral injection of WT-CVB3. It is well documented that the host innate immune response plays a crucial role in limiting viral spread.36McNab F. Mayer-Barber K. Sher A. Wack A. O’Garra A. Type I interferons in infectious disease.Nat. Rev. Immunol. 2015; 15: 87-103Crossref PubMed Scopus (1144) Google Scholar To determine whether partial recovery of innate immunity could attenuate the capability of CVB3 in killing tumors, we carried out the xenograft experiments using NOD-SCID mice, which have residual innate immunity including defective NK cells, macrophages, granulocytes, and complement.35Zhang B. Duan Z. Zhao Y. Mouse models with human immunity and their application in biomedical research.J. Cell. Mol. Med. 2009; 13: 1043-1058Crossref PubMed Scopus (34) Google Scholar To investigate whether CVB3 has local and/or systemic oncolytic effects on tumors, lung adenocarcinoma cells were injected subcutaneously into the bilateral flanks of the mice, and a subsequent one-dose injection of WT-CVB3 was administered only in the left flank tumor. We found that KRASmut tumor volume significantly decreased on both flanks of mice, suggesting a possible systemic effect of local intratumoral injection of CVB3 on distant tumors (Figures 7A and 7B ). However, the survival curve showed no improvement of mouse survival after WT-CVB3 treatment (Figure 7C). Viral quantitation demonstrated active viral replication in tumors at both sides and in multiple organs, particularly the heart (Figure 7D). Future research is required to further reduce the toxicity. CVB3 is known to be a common causative agent for viral myocarditis and pancreatitis, especially in children and those who have defective anti-viral immunity.18Fung G. Luo H. Qiu Y. Yang D. McManus B. Myocarditis.Circ. Res. 2016; 118: 496-514Crossref PubMed Scopus (239) Google Scholar, 19Huber S. Ramsingh A.I. Coxsackievirus-induced pancreatitis.Viral Immunol. 2004; 17: 358-369Crossref PubMed Scopus (97) Google Scholar As expected, in immunodeficient NSG mice, WT-CVB3 caused a significant cytotoxicity in heart and pancreas, as characterized by myocardial injury and inflammatory infiltration, as well as destruction of acinar cells of the pancreas, when compared with UV-CVB3 treatment in both groups (Figures 8A and 8B ). In mice infec" @default.
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• W2963978412 title "Coxsackievirus Type B3 Is a Potent Oncolytic Virus against KRAS-Mutant Lung Adenocarcinoma" @default.
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• W2963978412 doi "https://doi.org/10.1016/j.omto.2019.07.003" @default.
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