Erin J. White

Human Adenovirus Type 5 Induces Cell Lysis through Autophagy and Autophagy-Triggered Caspase Activity

ABSTRACT Oncolytic adenoviruses, such as Delta-24-RGD, are promising therapies for patients with brain tumor. Clinical trials have shown that the potency of these cancer-selective adenoviruses should be increased to optimize therapeutic efficacy. One potential strategy is to increase the efficiency of adenovirus-induced cell lysis, a mechanism that has not been clearly described. In this study, for the first time, we report that autophagy plays a role in adenovirus-induced cell lysis. At the late stage after adenovirus infection, numerous autophagic vacuoles accompany the disruption of cellular structure, leading to cell lysis. The virus induces a complete autophagic process from autophagosome initiation to its turnover through fusion with the lysosome although the formation of the autophagosome is sufficient for virally induced cell lysis. Importantly, downmodulation of autophagy genes (ATG5 or ATG10) rescues the infected cells from being lysed by the virus. Moreover, autophagy triggers caspase activity via the extrinsic FADD/caspase 8 pathway, which also contributes to adenovirus-mediated cell lysis. Therefore, our study implicates autophagy and caspase activation as part of the mechanism for cell lysis induced by adenovirus and suggests that manipulation of the process is a potential strategy to optimize clinical efficacy of oncolytic adenoviruses.

The RB-E2F1 Pathway Regulates Autophagy

Autophagy is a protective mechanism that renders cells viable in stressful conditions. Emerging evidence suggests that this cellular process is also a tumor suppressor pathway. Previous studies showed that cyclin-dependent kinase inhibitors (CDKI) induce autophagy. Whether retinoblastoma protein (RB), a key tumor suppressor and downstream target of CDKIs, induces autophagy is not clear. Here, we show that RB triggers autophagy and that the RB activators p16INK4a and p27/kip1 induce autophagy in an RB-dependent manner. RB binding to E2 transcription factor (E2F) is required for autophagy induction and E2F1 antagonizes RB-induced autophagy, leading to apoptosis. Downregulation of E2F1 in cells results in high levels of autophagy. Our findings indicate that RB induces autophagy by repressing E2F1 activity. We speculate that this newly discovered aspect of RB function is relevant to cancer development and therapy.

Chapter 13 Autophagy Pathways in Glioblastoma

Glioma cells are more likely to respond to therapy through autophagy than through apoptosis. The most efficacious cytotoxic drugs employed in glioma therapy, such as temozolomide and rapamycin, induce autophagy. Oncolytic adenoviruses, which will soon be tested in patients with gliomas at the University of Texas M. D. Anderson Cancer Center, also induce autophagy. Autophagy in gliomas thus represents a promising mechanism that may lead to new glioma therapies. In this chapter, we present the methods for studying autophagy in glioma cells, including assessment of in vitro cellular markers acidic vesicle organelles, and green fluorescent protein (GFP)-LC3 punctation; biochemical markers LC3-I/II conversion, p62 degradation, Atg12-Atg5 accumulation, and p70S6K dephosphorylation; and ultrastucture of the autophagosomes. In addition, we will address how LC3B and Atg5 up-regulation during autophagy can be examined through immunostaining in treated tumors and the potential of these proteins for use as surrogate markers to monitor therapeutic effects in clinical trials. Finally, we will discuss the challenges of studying autophagy in gliomas and the future directions of such use.

The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected cells.

The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.

Adenovirus's last trick: You say lysis, we say autophagy

The last stage of the adenovirus replication cycle, lysis, is considered not very efficient and remains poorly understood. Pathogen infection induces autophagy in eukaryotic cells. In the case of viruses, autophagy is a double-edged sword that can either facilitate or impede replication. On one hand, autophagy reduces the replication capability of the herpesviruses. On the other hand, the RNA virus poliovirus uses autophagosomes to form replication complexes. Recently we characterized the autophagy induced by the oncolytic adenovirus Delta-24-RGD in brain tumor stem cells. Late in the adenoviral infectious cycle, we observed remarkable up-regulation of the Atg12-Atg5 complex and prominent autophagy. In addition, adenovirus-induced autophagy results in disruption of the cytoplasmic structure and the continuity of the cellular membrane. We speculate that adenoviruses induce autophagy to facilitate the release of viral progeny at the end of the infectious cycle. The substitution of “autophagy” for “lysis” is not just semantic. Because autophagy is a genetically programmed process and not a passive phenomenon, it immediately suggests interactions between adenovirus proteins and autophagy regulators. Understanding the mechanism underlying adenovirus-mediated autophagy should propel the development of novel vectors with enhanced capability to release viral progeny and, as a result, more potent oncolytic effect. Addendum to: Jiang H, Gomez-Manzano C, Aoki H, Alonso MM, Kondo S, McCormick F, Xu J, Kondo Y, Bekele BN, Colman H, Lang FF, Fueyo J. Examination of the therapeutic potential of Delta-24-RGD in brain tumor stem cells: Role of autophagic cell death. J Natl Cancer Inst 2007; 99:1410-4.

C-Jun N-terminal kinases are required for oncolytic adenovirus-mediated autophagy.

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.

Abstract 2871: JNK1-phosphorylation of Bcl-2 is essential for adenoviral-induced autophagic cell death in glioma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Delta-24-RGD is a highly selective and potent oncolytic adenovirus that is currently being tested in a clinical trial for patients with malignant gliomas. The mechanism of oncolysis for Delta-24-RGD is defined as autophagic cell death, yet the signal transduction and molecular interactions responsible remain uncharacterized. Methods: Utilizing JNK1- and JNK2-null mouse embryonic fibroblasts, we analyzed the involvement of JNK in Bcl-2 phosphorylation and degradation, and adenoviral-induced autophagy. Mutations at the phosphorylation sites of Bcl-2 were employed to determine which are necessary for release of Beclin 1, degradation of Bcl-2, and induction of autophagy by adenovirus. Results: In this report, we reveal that activation of c-Jun N-terminal kinase 1 (JNK1) is required to initiate and maintain autophagy induced by wild-type and Delta-24-RGD adenoviruses in the host cell. In addition, JNK1-dependent phosphorylation of Bcl-2 at serine 87 (but not serine 70) and subsequent inhibition of Bcl-2:Beclin 1 heterodimerization is necessary for autophagy to occur in infected cells. Upon phosphorylation and release from Beclin 1, Bcl-2 is degraded by the proteosome. Finally, the activation of JNK leads to its translocation to the nucleus and results in the transcription of autophagy-related (ATG) genes for the maintenance of autophagy, as yet undocumented in mammalian cells. Conclusion: Adenoviruses have thus evolved a highly complex mechanism of regulating autophagy which differs from the typical cellular stress response that may be exploited in the treatment of gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2871. doi:10.1158/1538-7445.AM2011-2871

Abstract 3340: Salinomycin displays more potent anticancer effect against brain tumor stem cells than temozolomide

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Salinomycin has recently proven to be highly effective against a model of breast cancer stem cells in vitro and in vivo. Moreover, treatment with this compound induced epithelial differentiation of tumor cells. A population of Brain Tumor Stem Cells (BTSCs) has been identified in malignant brain tumors. This population of cells has been found responsible for the initiation of these malignancies and may constitute the cause of resistance of malignant brain tumors to chemotherapy and radiotherapy and therefore responsible for the lethal recurrence after surgery. We hypothesize that salinomycin could be successfully implemented for the treatment of brain tumors and specifically for the eradication of BTSCs. Material and Methods: In vitro cytotoxic effect of salinomycin or TMZ was assessed in a panel of BTSCs lines (n=11), established glioma cell lines (n=4) and normal human astrocytes (NHA) by MTT. Self-renewal was assayed by single cell plating and limited dilution assay. Cell cycle was evaluated by flow cytometry using PI staining. In addition, we performed QT-RT-PCR and western blot analysis to assess the expression of different stem cells markers before and after the treatment. In addition, global gene expression analysis was performed using an affymetrix array. Survival studies in animal models are in progress. Results: Our results showed that salinomycin presents a potent antitumoral effect against BTSCs and glioma cell lines. Importantly, salinomycin displayed an IC50 ranging from 100 to 10000 folds lower than TMZ (currently the gold standard treatment for malignant brain tumors) in the same cell lines. Interestingly, salinomycin reduced the number of self-renewal cells in treated-BTSCs. This was confirmed with the decreased in the expression of stem cell markers such as Sox2 and Bmi1. Cell cycle analysis showed that unlike TMZ treated cells, which arrest in G2, salinomycin treated cells did not experienced growth arrest and progress through the cell cycle to end up dying. Conclusions: Altogether our data show that salinomycin is more potent than temozolomide for the treatment of brain tumor stem cells and therefore could be a promising agent against malignant brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3340.

Abstract 4836: RB/E2F1 in the crossroad of autophagy and apoptosis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Autophagy is a protective mechanism that renders cells viable in stressful conditions. Mounting evidence suggests that this cellular process is also a tumor suppressor pathway. We hypothesized that retinoblastoma protein (RB), a key tumor suppressor, induces autophagy. To test this hypothesis, we first transduced RB into RB-defective human cancer cells: sarcoma osteogenic (Saos-2) cells, hepatoma (Hep3B) cells, and brain tumor stem cells (MDNSC23). The ectopic RB induced autophagy as demonstrated by significantly increased percentage of cells with acidic vesicular organelles (from less than 10% to more than 30%, P < 0.05), and the lipidation of LC3-I leading to the formation of autophagosomes/autolysosomes that were visualized by EGFP-LC3 punctation (from less than 20% to more than 80%, P = 0.003). These double-membraned vesicles were clearly shown by ultrastructural study via transmission electron microscopy. However, when Beclin 1 was silenced with siRNA in Saos-2 cells, the RB-mediated autophagy was blocked. In addition, study of autophagy flux with Saos-2 cells expressing double-tagged mRFP-EGFP-LC3 fusion protein revealed that RB stimulated the formation and maturation of autophagosomes. Consistently, RB activators p16INK4a and p27/kip1 caused autophagy in an RB-dependent manner. Importantly, we found RB mutants Delta-22 and R661W deficient for binding E2F failed to induce autophagy. Moreover, E2F1 overexpression antagonized RB-mediated autophagy, leading to apoptosis in Saos-2 cells. In agreement with the above observations, silencing E2F1 with siRNA resulted in autophagy in U-87 MG cells; and autophagy levels increased from 4% in wild-type MEFs to 40% in E2F1 knockout MEFs (P = 0.002). Collectively, our data reveal that RB/E2F1 plays a key role in the decision of a cell to undergo autophagy or apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4836.