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, 15, 117-130
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Calcium Influx Caused by ER Stress Inducers Enhances Oncolytic Adenovirus Enadenotucirev Replication and Killing Through PKCα Activation

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Calcium Influx Caused by ER Stress Inducers Enhances Oncolytic Adenovirus Enadenotucirev Replication and Killing Through PKCα Activation

William K Taverner et al. Mol Ther Oncolytics.

Abstract

Oncolytic viruses represent an emerging approach to cancer therapy. However, better understanding of their interaction with the host cancer cell and approaches to enhance their efficacy are needed. Here, we investigate the effect of chemically induced endoplasmic reticulum (ER) stress on the activity of the chimeric group B adenovirus Enadenotucirev, its closely related parental virus Ad11p, and the archetypal group C oncolytic adenovirus Ad5. We show that treatment of colorectal and ovarian cancer cell lines with thapsigargin or ionomycin caused an influx of Ca2+, leading to an upregulation in E1A transcript and protein levels. Increased E1A protein levels, in turn, increased levels of expression of the E2B viral DNA polymerase, genome replication, late viral protein expression, infectious virus particle production, and cell killing during Enadenotucirev and Ad11p, but not Ad5, infection. This effect was not due to the induction of ER stress, but rather the influx of extracellular Ca2+ and consequent increase in protein kinase C activity. These results underscore the importance of Ca2+ homeostasis during adenoviral infection, indicate a signaling pathway between protein kinase C and E1A, and raise the possibility of using Ca2+ flux-modulating agents in the manufacture and potentiation of oncolytic virotherapies.

Keywords: ER stress; PKC; adenovirus; calcium; oncolytic.

Figures

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Figure 1
Figure 1
Effect of ER Stress-Inducing Chemicals on EnAd and Ad5 Transgene Expression SK-OV-3 ovarian cancer cells (A) or DLD-1 colorectal cancer cells (B) were plated in a 96-well plate, infected with EnAd-SA-GFP, EnAd-CMV-GFP, or Ad5-CMV-GFP and treated with Tg (0.1 μM), Im (1.25 μM), or Tm (1 μg/mL). Cells were imaged on a Celigo image cytometer and GFP expression quantified at 72 h (A) or 24 h (B) postinfection. Data represent average fold change in the percentage of GFP-positive cells from 60 replicates relative to DMSO vehicle control-treated cells; error bars represent ± SEM. Broken line represents ± SEM of the DMSO control. Significance was evaluated using one-way ANOVA. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, nonsignificant. (C) Representative images of virus-encoded GFP expression for the conditions in (A). Image fields represent 1 μm. (D) Representative images of virus-encoded GFP expression for the conditions in (B).
Figure 2
Figure 2
Thapsigargin Treatment Enhances Parameters of Infection of EnAd and Ad11p but Not of Ad5 (A and B) qPCR measurement of viral genomes in DLD-1 cells 24 h postinfection (p.i.) (A) and in SK-OV-3 cells at 72 h p.i. (B) in the presence or absence of Tg (0.1 μM) or vehicle control DMSO. (C and D) TCID50/mL of infectious virus particles in DLD-1 cells 24 h p.i. (C) and SK-OV-3 cells 72 h p.i. (D). Error bars represent ± SEM. Significance was evaluated by two-tailed t test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, nonsignificant. (E) Virus killing of DLD-1 cells was monitored by xCELLigence in the presence or absence of Tg (0.1 μM) after virus infection. Impedance was measured every 10 minutes. Data show the mean of four replicates with the weight of the line spanning ± SEM. An infectious dose of 100 VPC was used in all experiments. (F) Virus-induced cell cytotoxicity was assessed by MTS. DLD-1 cells were infected with EnAd, Ad11p, or Ad5. Cell viability was measured at 48 h p.i. Mean of six replicates is displayed per treatment group; error bars represent ± SEM. Significance was tested by two-way ANOVA. ***p ≤ 0.001; ns, non-significant.
Figure 3
Figure 3
Thapsigargin Treatment Enhances Early Viral Gene mRNA and Protein Levels (A and B) Quantification of EnAd mRNA levels of E1A (A) and E2B (B) by qRT-PCR. Data represent the mean of three biological replicates, each an average of three technical replicates. Significance was assessed by two-way ANOVA using Bonferroni post-test. ***p ≤ 0.001; ns, non-significant. Error bars represent ± SEM. (C) Western blot of FLAG-E1A protein levels in DLD-1 cells 12 h postinfection with EnAd-FLAG-E1A (MOI, 3). Numbers indicate average normalized fold upregulation relative to DMSO control of three independent replicate experiments. (D) DLD-1 cells were infected with Ad5-E1A-Luc (MOI, 3) and treated with Tg (0.1 μM) or Im (1.25 μM). Cells were lysed to measure luciferase expression 12 h post-infection. Significance was assessed by one-way ANOVA. ***p ≤ 0.001; ns, non-significant. Error bars represent ± SEM.
Figure 4
Figure 4
UPR Induction Is Not Responsible for the Increase in Early Viral Gene Expression or Virus Activity (A) XBP-1 splice status in SK-OV-3 or DLD-1 cells treated with the indicated chemical for 12 h. XBP-1(u) was observed at 283 bp; XBP-1(s), 257 bp. (B) qRT-PCR quantification of EnAd E1A mRNA in DLD-1 cells 9 h postinfection. The mean of three replicates is plotted. (C) Quantification of E1A-Luc reporter protein levels in DLD-1 cells 12 h post-infection with Ad5-E1A-Luc. (D and E) SK-OV-3 (D) and DLD-1 (E) were infected with EnAd-CMV-GFP, EnAd-SA-GFP, or Ad5-CMV-GFP and subsequently treated with SubAB. Data show the percentage of GFP-positive cells 72 h (D) and 24 h (E) postinfection, as quantified by Celigo. Significance was assessed by one-way ANOVA. ***p ≤ 0.001; **p ≤ 0.01; ns, non-significant. The following concentrations were used: Tg, 0.1 μM; Tm, 1 μg/mL; SubAB, 0.5 μg/mL; and 4μ8C, 100 μM. Error bars indicate ± SEM.
Figure 5
Figure 5
Influx of Extracellular Calcium Mediates Enhancement to Virus Activity Seen upon Treatment of DLD-1 Cells with Tg and Im (A) Measurement of cytosolic Ca2+ levels of DLD-1 cells over time. Drug treatments were injected into wells at 5 min, and Ca2+ was injected back into the media after 10 min. One representative experiment is shown. RFU, relative fluorescence units. (B) DLD-1 cells were infected with EnAd-SA-GFP in the presence of 10 μM BAPTA and subsequently exposed to Tg, Im, or vehicle control DMSO. Cells were imaged by Celigo 24 h postinfection. (C and D) DLD-1 cells were infected with EnAd-SA-GFP and subsequently treated with Tg (C), Im (D), or vehicle control DMSO. Treatment media contained variable concentrations of Ca2+ and were supplemented with 2% dialyzed (Ca2+ free) FCS. Cells were imaged by Celigo 24 h postinfection. (E and F) DLD-1 cells were infected with EnAd-SA-GFP in the presence or absence of BTP2. Cells were subsequently exposed to Tg (E) or Im (F) and imaged by Celigo 24 h postinfection. (G and H) Western blot of E1A levels in DLD-1 cells 12 h postinfection with (G) EnAd-FLAG-E1A (MOI, 3) or (H) wild-type Ad5 (100 VPC) and treated with ER stress inducers in the presence or absence of extracellular Ca2+. Numbers represent fold change relative to DMSO control within the relevant Ca2+ environment. (I) E1A-Luc levels were measured in DLD-1 cells 12 h postinfection with Ad5-E1A-Luc. Cells were treated with ER stress inducers in the presence and absence of extracellular calcium. Significance of differences in GFP expression levels relative to the DMSO control group were assessed by one-way ANOVA (B and I) or two-way ANOVA (C–F). ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, nonsignificant. Error bars represent ± SEM.
Figure 6
Figure 6
PKC Activation by Thapsigargin Mediates Enhancement of EnAd Activity (A) Western blot of phosphorylated PKC in DLD-1 cells after 30 min of stimulation with Tg, PMA (5 nM), or vehicle control DMSO. (B–F) DLD-1 cells were infected with EnAd-SA-GFP prior to exposure to Tg: (B) GF 109203X; (C) Gö 6976; (D) Enzastaurin; (E) Gö 6983 and (F) various PKC inhibitors. Treatments were performed in normal DMEM (supplemented with 2% FCS) or Ca2+-free DMEM (supplemented with 2% dialyzed Ca2+-free FCS) as indicated. Cells were imaged at 24 h postinfection using Celigo. (G) Western blot of PKCα protein levels 48 h after transfection of DLD-1 cells with either siRNAs for the targeted knockdown of PKCα or nontarget (NT) control siRNA. (H) DLD-1 cells were transfected with siRNA targeting PKCα or NT control siRNA. At 48 h post-transfection, cells were infected with EnAd-SA-GFP (100 VPC) and subsequently treated with either Tg or vehicle control DMSO. At 24 h postinfection, the proportion of GFP-positive cells was measured by Celigo. Significance was assessed by two-way ANOVA (B–F) or one-way ANOVA (H). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, nonsignificant.

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References

    1. Russell S.J., Peng K.-W., Bell J.C. Oncolytic virotherapy. Nat. Biotechnol. 2012;30:658–670. - PMC - PubMed
    1. Seymour L.W., Fisher K.D. Oncolytic viruses: finally delivering. Br. J. Cancer. 2016;114:357–361. - PMC - PubMed
    1. Workenhe S.T., Mossman K.L. Oncolytic virotherapy and immunogenic cancer cell death: sharpening the sword for improved cancer treatment strategies. Mol. Ther. 2014;22:251–256. - PMC - PubMed
    1. Keller B.A., Bell J.C. Oncolytic viruses-immunotherapeutics on the rise. J. Mol. Med. (Berl.) 2016;94:979–991. - PubMed
    1. Dyer A., Di Y., Calderon H., Illingworth S., Kueberuwa G., Tedcastle A., Jakeman P., Chia S.L., Brown A., Silva M.A. Oncolytic Group B Adenovirus Enadenotucirev Mediates Non-apoptotic Cell Death with Membrane Disruption and Release of Inflammatory Mediators. Mol. Ther. Oncolytics. 2016;4:18–30. - PMC - PubMed

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