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. 2018;19(12):1174-1184.
doi: 10.1080/15384047.2018.1491503. Epub 2018 Aug 1.

The role of JNK phosphorylation as a molecular target to enhance adenovirus replication, oncolysis and cancer therapeutic efficacy

Stephen L Wechman  1   2 , Xiao-Mei Rao  3   4 , Jorge G Gomez-Gutierrez  3   4 , Heshan Sam Zhou  3   4   5 , Kelly M McMasters  1   3   4
Affiliations

The role of JNK phosphorylation as a molecular target to enhance adenovirus replication, oncolysis and cancer therapeutic efficacy

Stephen L Wechman et al. Cancer Biol Ther. 2018.

Abstract

Oncolytic adenoviruses (Ads) are cancer selective tumoricidal agents; however their mechanism of Ad-mediated cancer cell lysis, or oncolysis, remains undefined. This report focuses upon the autophagy mediator c-JUN n-terminal kinase (JNK) and its effects upon Ad oncolysis and replication. Previously, E1b-deleted Ads have been used to treat several hundred cancer patients with limited clinical efficacy. We hypothesize that by studying the potential interactions between E1b and JNK, mechanisms to improve oncolytic Ad design and cancer therapeutic efficacy may be elucidated. To test this hypothesis, E1b was selectively deleted from the Ad genome. These studies indicated that Ads encoding E1b induced JNK phosphorylation predominately occurred via E1b-19K. The expression of another crucial Ad gene E1a was then overexpressed by the CMV promoter via the replication competent Ad vector Adhz69; these data indicated that E1A also induced JNK phosphorylation. To assess the effects of host cell JNK expression upon Ad oncolysis and replication, siRNA targeting JNK1 and JNK2 (JNK1/2) were utilized. The oncolysis and replication of the E1b-19K wild-type Ads Ad5 and Adhz63 were significantly attenuated following JNK1/2 siRNA transfection. However the oncolytic effects and replication of the E1b-19K deleted Ad Adhz60 were not altered by JNK1/2 siRNA transfection, further implicating the crucial role of E1b-19K for Ad oncolysis and replication via JNK phosphorylation. This study has demonstrated for the first time that JNK is an intriguing molecular marker associated with enhanced Ad virotherapy efficacy, influencing future Ad vector design.

Keywords: E1a; E1b; JNK; autophagy; lung cancer; molecular targets; oncolysis.

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Figures

Figure 1.
Figure 1.
The effect of Ad5 upon JNK phosphorylation. A549 cells were mock treated or infected with Ad5 at 10 PFU/cell and then observed from 0 to 72 h post-infection. (a) A549 cell morphology following mock or Ad5 treatment was photographed at the indicated time-points. Mock treated cells were non-infected, while only Ad5 treated A549 cells displayed CPE. Pictures were taken using an inverted microscope at 200x total magnification. 600 µm scale bar is located in the top right corner. (b) A549 cell lysates were treated and harvested at the time-points indicated and observed for protein production via Western blot analysis. (c) These Western blot data were quantified via densitometric analysis using Gel-pro analyzer 4.0 software and these IOD values were normalized to actin. These values were then converted to fold changes by dividing the values by the values for mock treated cells. Cell viability was determined by crystal violet staining and plotted on the secondary y-axis. Results are expressed as the average of 3 experiments plus or minus the standard deviation. Statistical analysis was performed using one-way ANOVA by Dunnett’s test for multiple comparisons relative to the mock control (1 fold). * indicates p-value < 0.05, † indicates p-value < 0.01, ‡ indicates p-value < 0.001.
Figure 2.
Figure 2.
The effect of selective E1b-deleted Ads upon JNK phosphorylation at 36 h. A549 cells were mock-treated or infected with Ad5, Adhz63, and Adhz60 at 1, 3 and 10 PFU/cell respectively. (a) Crystal violet staining of A549 cells treated as indicated at 36 h post-infection. (b) A549 cell lysates were prepared at 36 h post-infection and observed for the production of the indicated proteins via Western blot analysis. (c) These Western blot data were quantified via densitometric analysis using Gel-pro analyzer 4.0 software and these IOD values were normalized to actin. These values were then converted to fold changes by dividing the values by the values for mock treated cells. Results were expressed as the average of 3 experiments plus or minus the standard deviation.
Figure 3.
Figure 3.
The effect of E1b-19K upon JNK phosphorylation. A549 cells were mock treated or infected with AdGFP, Ad5, Adhz63 and Adhz60 at 10 PFU/cell. (a) CPE induction in mock, AdGFP, Ad5, Adhz63 and Adhz60 treated cells. Pictures were taken using an inverted microscope at 200x total magnification at 48 and 72 h post-infection. 600 µm scale bar is located in the top right corner. (b) A549 cell lysates were observed for the production of the indicated proteins via Western blot analysis. (c) These Western blot data were quantified via densitometric analysis using Gel-pro analyzer 4.0 software and these IOD values were normalized to actin. These values were then converted to fold changes by dividing the values by the values for mock treated cells; mock = 1-fold. One of three representative experiments is shown. (d) Cell viability was determined by crystal violet staining and expressed as the percent cell viability relative to non-treated cells. One of three representative experiments is shown. (e) A549 cells were mock transfected, or were transfected with GFP or CMV-E1b19K plasmids as indicated. These cells were then lysed and observed for the production of the indicated proteins. * indicates p-value < 0.05, † indicates p-values < 0.01.
Figure 4.
Figure 4.
The effect of E1a expression upon JNK phosphorylation. A549 cells were mock-treated or infected with AdGFP, Ad5, Adhz63, and Adhz69 at a PFU of 10. (a) Cell viability was determined by crystal violet staining and expressed as the percent cell viability relative to non-treated cells at 48 h post-infection. (b) A549 cell lysates were treated as indicated at 48 h post-infection and observed for the protein production of the indicated proteins via Western blot analysis. (c) A549 cells were mock treated or infected with AdGFP, Ad5, Adhz63, and Adhz69 at a PFU of 10 as in Figure 4B; Adhz60 was included as a negative control for E1b-19K. E1b-19K protein production was determined via Western blot analysis. These Western blot data were quantified via densitometric analysis using Gel-pro analyzer 4.0 software and these IOD values were normalized to actin. These values were then converted to fold changes by dividing the values by the values for mock treated cells.
Figure 5.
Figure 5.
The effect of JNK expression upon A549 cell oncolysis and oncolytic Ad replication. (a) The concentration of JNK1/2-siRNA that efficiently repressed JNK1 and JNK2 expression was determined at 48 h by Western blot analysis using JNK1 and JNK2 selective antibodies. (b) The effect of JNK1/2-siRNA upon cell viability was assessed using crystal violet staining. Statistical analysis was performed by one-way ANOVA using Tukey’s post-test for multiple comparisons. (c) The effect of JNK1/2-siRNA upon Ad replication was assessed via the TCID50 method. Statistical analysis was performed by one-way ANOVA using Tukey’s post-test for multiple comparisons. † indicates p-value < 0.01, ‡ indicates p-value < 0.001.
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