C-Jun N-terminal kinases are required for oncolytic adenovirus-mediated autophagy - PubMed (original) (raw)
C-Jun N-terminal kinases are required for oncolytic adenovirus-mediated autophagy
S R Klein et al. Oncogene. 2015.
Abstract
Oncolytic adenoviruses, such as Delta-24-RGD (Δ24RGD), are replication-competent viruses that are genetically engineered to induce selective cancer cell lysis. In cancer cells, Δ24RGD induces massive autophagy, which is required for efficient cell lysis and adenoviral spread. Understanding the cellular mechanisms underlying the regulation of autophagy in cells treated with oncolytic adenoviruses may provide new avenues to improve the therapeutic effect. In this work, we showed that cancer cells infected with Δ24RGDundergo autophagy despite the concurrent activation of the AKT/mTOR pathway. Moreover, adenovirus replication induced sustained activation of JNK proteins in vitro. ERK1/2 phosphorylation remained unchanged during adenoviral infection, suggesting specificity of JNK activation. Using genetic ablation and pharmacological inactivation of JNK, we unequivocally demonstrated that cells infected with Δ24RGD required JNK activation. Thus, genetic co-ablation of JNK1 and JNK2 genes or inhibition of JNK kinase function rendered Δ24RGD-treated cells resistant to autophagy. Accordingly, JNK activation induced phosphorylation of Bcl-2 and prevented the formation of Bcl-2/Beclin 1 autophagy suppressor complexes. Using an orthotopic model of human glioma xenograft, we showed that treatment with Δ24RGD induced phosphorylation and nuclear translocation of JNK, as well as phosphorylation of Bcl-2. Collectively, our data identified JNK proteins as an essential mechanistic link between Δ24RGD infection and autophagy in cancer cells. Activation of JNK without inactivation of the AKT/mTOR pathway constitutes a distinct molecular signature of autophagy regulation that differentiates Δ24RGD adenovirus from the mechanism used by other oncolytic viruses to induce autophagy and provides a new rationale for the combination of oncolytic viruses and chemotherapy.
Figures
Figure 1. The canonical pathway of autophagy is not activated during adenovirus-mediated autophagy
(a) Kinetics of phosphorylated p-AMPKα1 (T174), p-AMPKα2 (T172), p-Akt (S473), and p-mTOR (S2448) protein levels in MCR5 lung fibroblasts infected with AdWT at an MOI of 50. Whole-cell lysates were analyzed by using a human phospho-kinase antibody array (Supplementary Figure 1 and Table 1) at the indicated times after infection. The graph (top panel) represents the densitometry analyses of the duplicate dots (bottom panel) normalized against the controls. (b) U87 MG malignant glioma cells (ATCC) were infected with UV-inactivated or Delta-24-RGD (Δ24RGD) adenovirus (50 MOIs) for the indicated times and then cell lysates were analyzed by Western blotting for total and phospho-protein levels of Akt (S473). Protein levels of unprocessed LC3-I and the proteolytically cleaved LC3-II are shown as markers of autophagy. Actin is shown as a loading control. Quantification of the ratio p-Akt/Akt levels, normalized with the actin levels of expression, is indicated. UVi, UV-inactivated. (c) Wild-type AMPKα1 and_AMPKα1_-null MEFs were infected with AdWT (100 MOIs) for 48 h and cell lysates were analyzed for expression of the indicated proteins. (d) U251 MG cells (ATCC) were transfected with a pool of shNC or shTSC2 for 48 h and then infected with the Delta-24-RGD adenovirus (10 MOIs) for an additional 48 h. Whole-cell lysates were analyzed with use of anti-LC3 and anti-Actin antibodies (left panel). Right panel, Western blot illustrates the effect of shTSC2 and non-coding shRNA on TSC2 protein levels in U251 MG cells. All Western blots are representative of three independent experiments.
Figure 2. Adenoviral infection induces activation of the JNK pathway
(a) Kinetics of phosphorylated p-panJNK (T183/Y185, T221/Y223) and p-ERK1/2 (T202/Y204, T185/Y187) protein levels in immortalized MCR5 lung fibroblasts infected with AdWT (50 MOIs). Whole-cell lysates at the indicated times were analyzed by using a human phospho-kinase antibody array (Supplementary Figure 1). The graph (top panel) represents the densitometry analyses of the duplicate dots (bottom panel) normalized against the controls. (band c) Whole-cell lysates from HeLa cervical cancer cells (b) (ATCC) or A549 lung cancer cells (c) infected with indicated adenoviruses at an MOI of 25 were analyzed for total and phosphorylated JNK and c-Jun protein levels. Anisomycin-treated cells (5 µM, 30 min) were used as a positive control for JNK pathway activation. E1A expression is shown as evidence of adenoviral infection. Results represent at least three independent experiments. UVi, UV-inactivated. (d) Immunofluorescence analysis of brains from intracranial-bearing glioma mice treated with UV-inactivated or Delta-24-RGD adenoviruses. Anti-phospho-panJNK (green fluorescence) and anti-hexon (red fluorescence) antibodies were used to assess the co-localization (yellow) of p-JNK with viral proteins. DAPI was used for nuclear staining (blue). All fields in brain tissue from 4 mice per treatment were scrutinized under deconvolution microscopy. The experiment was repeated twice with mounted tissue sections from each animal. Representative images for healthy brain (bottom panel) and tumor (middle panel) tissue from nude mice infected with Delta-24-RGD as well as images of tumor from UV-inactivated adenovirus-infected nude mice (top panel) are depicted. Scale bar = 25 µm (_see also_Fig. S2).
Figure 3. JNK1 and JNK2 mediate Bcl-2 phosphorylation and dissociation of the Bcl-2/Beclin 1 complex upon adenoviral infection
(a) Whole-cell lysates from U87 MG cells infected with UV-inactivated or Delta-24-RGD adenoviruses (50 MOIs) for 24 h or 48 h were analyzed for expression of the indicated proteins. Quantification of the ratio p-Bcl-2/Bcl-2 levels, normalized with the actin levels of expression, is indicated. (b) Immunofluorescence analysis of brains from intracranial-bearing glioma mice treated with UV-inactivated or Delta-24-RGD adenoviruses. Anti-phospho-Bcl-2 (red fluorescence) and anti-hexon (green fluorescence) antibodies were used to assess the co-localization of these two proteins (yellow). DAPI was used for nuclear staining (blue). All fields in brain tissue from 4 mice per treatment were examined under deconvolution microscopy. The experiment was repeated twice with mounted tissue sections from each animal. Representative images for healthy brain (bottom panel) and central tumor (middle panel) tissue from nude mice infected with Delta-24-RGD as well as images of central tumor tissue from UV-inactivated infected nude mice (top panel) are depicted. (c) Whole-cell lysates from A549 cells pretreated with DMSO or SP600125 (25 µM) 30 min before infection with AdWT (25 MOIs) for 48 h were analyzed for the expression levels of total and phospho-Bcl-2, and total and phospho-JNK. Actin is shown as a loading control. (d) Cell lysates from wild-type JNK(JNK wt) and _JNK1/2_−/− MEFs mock-infected, or infected with UV-inactivated or AdWT adenoviruses (100 MOIs) were immunoprecipitated with anti-Bcl-2 antibody and analyzed for total and phospho-Bcl-2 protein levels. Input samples (5%) for total and phospho-JNK, and E1A are shown. Actin was used as a loading control. (e) Cell lysates from _JNK wt, JNK1_−/−, _JNK2_−/−, or_JNK1/2_−/− MEFs infected with AdWT (100 MOIs) for 48 h were immunoprecipitated with anti-Beclin1 antibody and analyzed for Bcl-2 with Beclin1 protein levels. Input sample (5%) was analyzed for p-JNK, JNK1, and JNK2 expression. Actin is shown as a loading control. All Western blots are representative of three independent experiments. UVi, UV-inactivated.
Figure 4. JNK1 and JNK2 may have redundant roles in the regulation of autophagy
(a) Whole-cell lysates from A549 cells pretreated with DMSO or SP600125 (25 µM) 30 min before infection with AdWT (25 MOIs) for 48 h were analyzed for expression levels of autophagy markers p62 and LC3-I to LC3-II conversion. Actin is shown as a loading control. (b) U87 MG cells transfected with an EGFP-LC3, as reported previously and treated with SP600125 or DMSO for 30 min before infection with Delta-24-RGD adenovirus (50 MOIs) for 48 h. Left panel, representative images of the GFP-LC3 puncta distribution visualized with use of deconvolution microscopy. Right panel, quantification of the percentage of cells presenting >10 GFP-LC3 puncta in 15 fields. * P< 0.05 (unpaired, two-tailed Student’s t-test). (c) Whole-cell lysates from _JNK wt, JNK1_−/−, _JNK2_−/−, and_JNK1/2_−/− MEFs infected with UV-inactivated or AdWT adenoviruses (100 MOIs) for 24 h or 48 h were analyzed for p62, p-JNK, JNK1, and JNK2 levels of expression. Actin is shown as loading control. (d) Whole-cell lysates from_c-Jun_−/− MEFs that were mock-infected, or infected with UV-inactivated or AdWT adenoviruses for 24 or 48 h (100 MOIs) were analyzed for expression of p62 and c-Jun. Actin is shown as loading control. UVi, UV-inactivated.
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