Molecular network, pathway and functional analysis of time-dependent gene changes associated with pancreatic cancer susceptibility to oncolytic vaccinia virotherapy

Abstract

BACKGROUNDOncolytic viral therapies have shown such success in preclinical trials as a novel cancer treatment modality that several phase 1 and 2 trials are already underway. 1 We have previously reported on the construction and generation of a novel attenuated replication-competent vaccinia virus (VACV), GLV-1h153, a derivative of parental virus GLV-1h68 engineered to carry the human sodium iodide symporter (hNIS) for the imaging of viral replication within tumors via enhanced uptake of several radionuclide probes. 2The noninvasive tracking of virus delivery may enable clinicians to correlate efficacy and therapy, monitor potential viral toxicity, and possibly provide a more sensitive and specific diagnostic technique to detect tumor origin and, more importantly, presence of metastases.3,4 GLV-1h153 facilitated enhanced dose-dependent radiouptake in cell culture and effective replication and killing of pancreatic cancer cells both in cell culture and in animal models. Furthermore, Received 27 December 2015; accepted 2 February 2016 Background: Pancreatic cancer is a fatal disease associated with resistance to conventional therapies. This study aimed to determine changes in gene expression patterns associated with infection and susceptibility of pancreatic cancer cells to an oncolyticvaccinia virus, GLV-1h153, carrying the human sodium iodide symporter for deep tissue imaging of virotherapy.Methods: Replication and susceptibility of pancreatic adenocarcinoma PANC-1 cells to GLV-1h153 was confirmed with replication and cytotoxicity assays. PANC-1 cells were then infected with GLV-1h153 and near-synchronous infection confirmed via flow cytometry of viral-induced green fluorescent protein (GFP) expression. Six and 24 hours after infection, three samples of each time point were harvested, and gene expression patterns assessed using HG-U133A cDNA microarray chips as compared to uninfected control. Differentially expressed genes were identified using Bioconductor LIMMA statistical analysis package. A fold change of 2.0 or above was used as a cutoff, with a P value of 0.01. The gene list was then analyzed using Ingenuity Pathways Analysis software.Results: Differential gene analysis revealed a total of 12,412 up-and 11,065 downregulated genes at 6 and 24 hours postinfection with GLV-1h153 as compared to control. At 6 hours postinfection. A total of 139 genes were either up or downregulated >twofold (false discovery rate < 0.05), of which 124 were mapped by Ingenuity Pathway Analysis (IPA). By 24 hours postinfection, a total of 5,698 genes were identified and 5,563 mapped by IPA. Microarray revealed gene expression changes, with gene networks demonstrating downregulation of processes such as cell death, cell cycle, and DNA repair, and upregulation of infection mechanisms (P < 0.01). Six hours after infection, gene changes involved pathways such as HMGB-1, interleukin (IL)-2, IL-6, IL-8, janus kinase/signal tranducer and activator of transcription (JAK/STAT), interferon, and ERK 5 signaling (P < 0.01). By 24 hours, pr...