GastrointestinalMolecular determinants of susceptibility to oncolytic vesicular stomatitis virus in pancreatic adenocarcinoma
Introduction
Vesicular stomatitis virus (VSV) is among several oncolytic viruses currently being developed as anticancer therapies. VSV, the prototypical member of the family Rhabdoviridae, is a negative-stranded RNA virus whose genome encodes for five proteins: nucleocapsid (N), polymerase proteins (L and P), surface glycoprotein (G), and peripheral matrix protein (M). VSV is a potently cytolytic virus that selectively replicates in cancer cells that have downregulated their antiviral responses [1]. The selectivity of VSV for cancer cells can be enhanced by introducing mutations in the M protein, such as in the M51R variant of VSV (M protein mutant vesicular stomatitis virus [M51R-VSV]), which contains a single arginine for methionine amino acid substitution at position 51 in the M protein [2], [3]. The mutant M protein has a decreased ability to inhibit host cell antiviral mechanisms. As a result, normal cells are able to resist M51R-VSV infection [4], [5] by mounting antiviral defenses, such as interferon (IFN)-mediated antiviral signaling. In contrast, many cancer cells remain susceptible to M51R-VSV infection because they possess defects in antiviral pathways [2], [6]. Several reports have shown that M51R-VSV is more selective for tumor cells and causes oncolysis in a variety of cancer types, including prostate cancer [7], breast cancer [8], glioblastoma [9], colorectal cancer [5], malignant melanoma [10], and neuroendocrine tumors [11]. Several VSV variants have been developed that are similarly selective for cancers with defective antiviral responses [1], and one of these is currently in a phase 1 clinical trial for treatment of hepatocellular carcinoma (http://clinicaltrials.gov/show/NCT01628640).
Although these preliminary reports are encouraging, VSV is not universally oncolytic in all tumor subtypes, and significant variation in VSV sensitivity exists, even among cancers from the same anatomical site [6]. For example, both VSV-resistant and VSV-sensitive cell lines have been described in colorectal cancer [5], prostate cancer [7], breast cancer [8], malignant melanoma [10], malignant mesothelioma [12], and bladder cancer [13]. Based on the available data, VSV resistance may be as high as 36% in pancreatic adenocarcinoma and has been observed in cells from both primary and metastatic sites [14], [15]. Preserved intact antiviral mechanisms are thought to confer resistance in VSV-resistant cell lines, but additional mechanisms may also contribute to resistance [5], [7], [14], [15], [16].
Oncolytic virus therapy is a particularly attractive strategy for treatment of cancers for which current therapies are ineffective. As such, despite advances in many other malignancies, pancreatic adenocarcinoma remains a significant therapeutic challenge. The 5-y relative survival rate for patients with pancreatic cancer is 6%—the lowest among all cancers [17]. Clinical outcomes are so poor because most pancreatic cancer patients present with locally advanced or metastatic disease, and pancreatic adenocarcinoma is largely resistant to traditional systemic treatments. Clearly, innovative and effective therapies are critical to improving clinical outcomes in patients with pancreatic cancer.
In the work presented herein, we confirm the results of Murphy et al. [14] and Moerdyk-Schauwecker et al. [15] and extend their finding by analyzing the oncolytic effects of VSV in a panel of additional pancreatic adenocarcinoma cells with significant variability in their VSV susceptibility. Using microarray gene analysis, we explored the genetic differences between VSV-sensitive and VSV-resistant cell lines and thereby hypothesized that differences in IFN signaling and viral endocytosis are key molecular determinants of VSV susceptibility. In support of these hypotheses, we showed that resistant cells are capable of blocking VSV infection during the early stages of viral replication and possess intact IFN responses. We also found that the integrity IFN signaling can explain the VSV susceptibility seen in sensitive cell lines. In a murine xenograft model, we found that tumors from both sensitive and resistant cells responded to intratumoral M51R-VSV treatment. Histologic examination of treated tumor suggests that adaptive cellular immunity contributes to the oncolysis of the in vitro resistant xenografts. Collectively, these data establish that oncolytic VSV is a viable therapeutic option for pancreatic adenocarcinoma. More specifically, it identifies two significant molecular mechanisms of VSV resistance and provides a framework for further research into the immunologic responses to VSV.
Section snippets
Cells and viruses
Panc 1, MiaPaCa2, BxPC3, Panc 03.27, and Panc 10.05 cell lines were obtained from the American Type Culture Collection and were grown in DMEM (Panc 1 and MiaPaCa2; Manassas, VA) or RPMI 1640 (BxPC3, Panc 03.27, and Panc 10.05) supplemented with various additives depending on the cell line according to American Type Culture Collection's specifications. The recombinant VSV viruses, rwt-VSV and M51R-VSV, were isolated from infectious VSV complementary DNA clones, and virus stocks were prepared
Variable susceptibility of pancreatic adenocarcinoma cells to VSV
The oncolytic activity of VSV in pancreatic adenocarcinoma was evaluated using a panel of five cell lines (Panc 1, MiaPaCa2, BxPC3, Panc 03.27, and Panc 10.05). Susceptibility to VSV was evaluated using either wild type or M protein mutant virus (rwt-VSV and M51R-VSV, respectively). Comparing the two viruses reveals the effect of host cell responses to VSV because rwt-VSV suppresses, whereas M51R-VSV induces host cell responses [2]. At low MOIs, a small percentage of cells are initially
Discussion
The results presented here are consistent with those of Murphy et al. [14], who found significant heterogeneity in VSV susceptibility among 13 pancreatic cancer cells and described similar results in terms of IFN responsiveness [14]. Given the aggressiveness of pancreatic adenocarcinoma and its insensitivity to traditional chemotherapy, it is encouraging that most cell lines tested to date are sensitive to VSV. Our results support previous findings that VSV-sensitive cancer cells possess
Conclusion
M51R-VSV is a viable option for the future treatment of pancreatic adenocarcinoma. The integrity of IFN-mediated antiviral mechanisms explains VSV sensitivity, although there is evidence that different defects exist at various stages of IFN signaling that inhibit the cells' ability to resist VSV oncolysis. Although VSV-resistant pancreatic cancer cells appear to possess intact IFN-mediated pathways, these cells also block the early stages of viral replication likely by inhibiting viral
Acknowledgment
This work was supported from the National Cancer Institute (J.S.) by grant number K08-CA131482, Robert Wood Johnson Foundation Harold Amos Faculty Development Award (J.S.) by grant number 63527, National Institute of Allergy and Infectious Diseases (D.L.) by grant number R01-AI32983, and the Bradshaw Surgical Resident Research Endowment (A.B.).
The authors thank Hermina Borgerink (Department of Comparative Medicine, Wake Forest School of Medicine) for staining of tissue sections and Lou Craddock
References (49)
- et al.
Identification of a consensus mutation in M protein of vesicular stomatitis virus from persistently infected cells that affects inhibition of host-directed gene expression
Virology
(1997) - et al.
VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents
Cancer Cell
(2003) - et al.
Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses
Virology
(2004) - et al.
Variation in susceptibility of human malignant melanomas to oncolytic vesicular stomatitis virus
Surgery
(2013) - et al.
Resistance of pancreatic cancer to oncolytic vesicular stomatitis virus: role of type I interferon signaling
Virology
(2013) - et al.
Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection
Immunity
(2000) - et al.
Inhibition of the IFN-beta response in hepatocellular carcinoma by alternative spliced isoform of IFN regulatory factor-3
Mol Ther
(2008) - et al.
The RAS/Raf1/MEK/ERK signaling pathway facilitates VSV-mediated oncolysis: implication for the defective interferon response in cancer cells
Mol Ther
(2007) - et al.
Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3 gamma
J Biol Chem
(1997) - et al.
Interferon-alpha resistance in a cutaneous T-cell lymphoma cell line is associated with lack of STAT1 expression
Blood
(1998)
Vesicular stomatitis virus as an oncolytic vector
Viral Immunol
Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis
J Virol
Oncolytic vesicular stomatitis virus for treatment of orthotopic hepatocellular carcinoma in immune-competent rats
Cancer Res
Vesicular stomatitis virus as a treatment for colorectal cancer
Cancer Gene Ther
Susceptibility of breast cancer cells to an oncolytic matrix (M) protein mutant of vesicular stomatitis virus
Cancer Gene Ther
Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo
J Virol
Oncolytic vesicular stomatitis virus as a treatment for neuroendocrine tumors
Surgery
Evaluation of an attenuated vesicular stomatitis virus vector expressing interferon-beta for use in malignant pleural mesothelioma: heterogeneity in interferon responsiveness defines potential efficacy
Hum Gene Ther
Oncolytic vesicular stomatitis viruses are potent agents for intravesical treatment of high-risk bladder cancer
Cancer Res
Vesicular stomatitis virus as an oncolytic agent against pancreatic ductal adenocarcinoma
J Virol
Early steps of the virus replication cycle are inhibited in prostate cancer cells resistant to oncolytic vesicular stomatitis virus
J Virol
Cancer statistics, 2010
CACancer J Clin
Matrix protein and another viral component contribute to induction of apoptosis in cells infected with vesicular stomatitis virus
J Virol
A comparison of normalization methods for high density oligonucleotide array data based on variance and bias
Bioinformatics
Cited by (19)
Immune Effects of M51R Vesicular Stomatitis Virus Treatment of Carcinomatosis From Colon Cancer
2020, Journal of Surgical ResearchCitation Excerpt :Such M protein mutants appear to be strong candidates for OV therapy as they are attenuated for replication in normal tissues but replicate as well as recombinant viruses with wild-type M protein in cancers that have defective antiviral responses. Previous work by our group has described the oncolytic effects of M51R VSV against a variety of malignancies including colon cancer.13-16 We therefore propose that the treatment of PSD from CRC with M51R VSV has the potential to improve patient outcomes.
Preclinical Development of Oncolytic Immunovirotherapy for Treatment of HPV<sup>POS</sup> Cancers
2018, Molecular Therapy OncolyticsCitation Excerpt :This virus was also attenuated by combining N gene translocation to position 4 in the viral genome with G gene truncation.30 The mutant M protein in VSV-M51R-E7/6 has a decreased ability to inhibit host cell antiviral mechanisms, allowing the expression of type I IFN, which is a potent VSV inhibitor, thereby helping to protect normal cells from cytolytic damage by the virus.31–33 VSV-E7/6-mouse and human (m/h)IFNβ has an intact viral M protein and incorporates the mouse or human IFNβ gene,34 which results in rapid and robust expression of this cytokine.
Phase 1 study of intratumoral Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus, in pediatric cancer patients
2015, Molecular TherapyCitation Excerpt :Pexa-Vec (pexastimogene devacirepvec, JX-594) is a replication-competent vaccinia virus derived from the commonly used Wyeth vaccine strain (Dryvax; Wyeth Laboratories). Three genetic modifications are included in Pexa-Vec: (i) a thymidine kinase gene deletion to enable more selective replication in cancer cells, (ii) GM-CSF gene insertion under the control of the synthetic early-late promoter to induce a systemic antitumoral immune response, and (iii) lac-Z gene insertion under control of the p7.5 promoter.10,11,12 Numerous studies have demonstrated the safety of Pexa-Vec in rodent models, with side effects including treatment-related inappetence and reversible changes in hematology and clinical chemistry parameters.
Oncolytic Viruses in the Therapy of Lymphoproliferative Diseases
2022, Molecular Biology