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Autophagy. May 2011; 7(5): 547–548.
Published online May 1, 2011. doi:  10.4161/auto.7.5.15078
PMCID: PMC3127215
Astrocyte elevated gene-1 activates AMPK in response to cellular metabolic stress and promotes protective autophagy
Sujit K Bhutia,1,5 Timothy P Kegelman,1 Swadesh K Das,1 Belal Azab,1 Zhao-Zhong Su,1 Seok-Geun Lee,4 Devanand Sarkar,1,2,3 and Paul B Fishercorresponding author1,2,3
1Department of Human and Molecular Genetics; Virginia Commonwealth University School of Medicine; Richmond, VA USA
2VCU Institute of Molecular Medicine; Virginia Commonwealth University School of Medicine; Richmond, VA USA
3VCU Massey Cancer Center; Virginia Commonwealth University School of Medicine; Richmond, VA USA
4Cancer Preventive Material Development Research Center; College of Oriental Medicine; Kyung Hee University; Seoul, Republic of Korea
5Department of Life Science; National Institute of Technology; Rourkela, Orissa India
corresponding authorCorresponding author.
Correspondence to: Paul B. Fisher; Email: pbfisher/at/vcu.edu
Received February 5, 2011; Accepted February 7, 2011.
Key words: AEG-1, protective autophagy, AMPK, ATG5
 
Although autophagy can function as a tumor-suppression mechanism, recent studies provide compelling evidence that during cancer progression, autophagy can serve as a protective mechanism in sustaining cell viability in apoptosis-defective and long-term metabolically stressed cancer cells. Recent work from our group defined a previously unrecognized role of Astrocyte elevated gene-1 (AEG-1) in promoting protective autophagy by activation of AMPK, and we unravel AEG-1's physiological significance in cell survival under restrictive/apoptosis-inducing conditions. These studies provide important insights and a new perspective on this multifunctional oncogene, AEG-1.
Our group and others demonstrated that AEG-1 expression increases in multiple cancers and plays a crucial role in oncogenic transformation and angiogenesis, which are two essential components in tumor development, growth and progression to metastasis. Moreover, AEG-1 directly contributes to resistance to chemotherapeutic drugs, another important hallmark of aggressive cancers. Our recent paper demonstrates that AEG-1 induces autophagy in immortalized primary human fetal astrocytes (IM-PHFA) in a dose- and time-dependent manner. Autophagy can be induced by the canonical or noncanonical pathway depending on the involvement of Beclin 1 and the class III phosphatidylinositol 3-kinase (hVps34). Knockdown of Beclin 1 and hVps34 does not decrease the percentage of GFP-LC3-positive cells or LC3-II levels in AEG-1 overexpressing cells. However, knockdown of ATG5 significantly inhibits Ad.AEG-1-induced LC3-II accumulation and punctate staining of GFP-LC3 vacuoles, indicating that Ad.AEG-1 triggers autophagy via the noncanonical pathway. Additionally, AEG-1 promotes autophagy in different normal human cell types derived from prostate, breast and brain tissue and in normal immortal cloned rat embryo fibroblast (CREF) cells indicating that this phenomenon is not restricted to a specific normal cell type or species, but rather represents a generalized property associated with elevated AEG-1 expression.
Next, we unraveled the detailed molecular mechanism of autophagy induction by AEG-1. Autophagy is activated in response to a decrease in the cellular ATP/AMP ratio. Our results show that IM-PHFA cells infected with Ad.AEG-1 display a dose- and time-dependent decrease in the levels of ATP. Additionally, pretreatment with methylpyruvate, a cell-permeable form of pyruvate, reduces LC3-II accumulation in IM-PHFA cells infected with Ad.AEG-1, indicating that AEG-1-induced disruption of cellular bioenergetics contributes to autophagy induction.
AMPK is sensitive to the cytosolic AMP-to-ATP ratio, and metabolic stress activates autophagy by suppression of TOR signaling. Our paper demonstrates that AEG-1 causes a significant increase of AMPK phosphorylation at Thr-172. Phospho-AMPKThr-172 phosphorylates and activates TSC2, further inhibiting the activation of downstream targets, such as mTOR and S6. Phosphorylation of mTOR and S6 proteins is substantially decreased in AEG-1-overexpressing cells. We confirmed that inhibition of AMPK activity either by compound C (a pharmacological inhibitor of AMPK) or by AMPK siRNA significantly reduces AEG-1-induced autophagy. Furthermore, inhibition of AMPK induces activation of mTOR and S6K, and inactivation of TSC1 followed by a decrease in ATG5 expression and finally a decrease in autophagy. Taken together, this study provides relevant insights into the complex nature of AMPK and downstream responses that may be involved in AEG-1-induced protective autophagy and cancer progression.
Next, we investigated the physiological role of protective autophagy induced by AEG-1 in mediating cell survival. Our previous studies demonstrated that AEG-1 induces serum-independent cell growth, another property of oncogenic transformation, by inhibiting apoptosis. To investigate the role of AEG-1-induced autophagy in regulating survival of normal cells under serum starvation conditions, we analyzed cell survival by standard MTT assays of Ad.AEG-1-infected ATG5-deficient IM-PHFA cells grown in 0.1% FBS. Infection of ATG5-deficient (ATG5 siRNA-treated) IM-PHFA cells with Ad.AEG-1 prevents increased cell survival under low serum conditions as compared to Ad.AEG-1-infected parental control siRNA-treated IM-PHFA cells. Moreover, the apoptosis study shows that ATG5-deficient IM-PHFA cells infected with Ad.AEG-1 are sensitive to serum starvation-induced apoptosis, whereas AEG-1 overexpression in control siRNA-transfected IM-PHFA cells protects these cells from apoptosis. These results indicate that AEG-1 promotes cell survival by altering both autophagy and apoptotic mechanisms in normal cells grown under serum-starvation conditions. This study helps elucidate the multiple oncogenic roles of AEG-1 in protecting cancer cells in unfavorable conditions, such as nutrition deprivation.
Since AEG-1 induces ATG5-mediated autophagy in normal cells, the higher expression of AEG-1 might be associated with elevated basal levels of autophagy in many cancer cells. To investigate this hypothesis, we developed shAEG-1-T98G cells, a stable AEG-1 knockdown clone in T98G, a human malignant glioma cell line, and observed that shAEG-1-T98G cells have decreased accumulation of LC3-II as compared to control T98G cells, confirmed by confocal microscopy and western blotting. Autophagy provides resistance to therapy-mediated tumor cell death. When tumor cells induce protective autophagy, inhibition of autophagy could provide a way of sensitizing tumor cells to therapy by activating apoptosis. In this recent paper, we demonstrate that chemoresistance promoted by AEG-1 may be mediated through the ability of this gene to induce protective autophagy. Our study indicated that inhibition of AEG-1 results in a decrease in protective autophagy and causes chemosensitization of cancer cells. To determine whether AEG-1-induced chemoresistance is mediated through autophagy, we evaluated growth inhibition potential of chemotherapeutic reagents including doxorubicin in ATG5-deficient T98G cells in the presence of AEG-1. The results demonstrated that Ad.AEG-1 infection increases the resistance of T98G cells towards doxorubicin as measured by standard MTT assays, whereas ATG5-deficient cells exhibit chemosensitivity towards this drug. This finding was confirmed by measuring apoptotic cells using annexin V binding and caspase 3/7 assays. These results support previous observations that inhibiting AEG-1 expression suppresses both in vitro and in vivo tumor phenotypes and indicate that the ability of AEG-1 to confer chemoresistance is mediated by protective autophagy induced by this cancer-promoting gene.
In summary, our recent study reveals distinctive aspects of AEG-1 function and identifies the AEG-1 gene as a new regulator of protective autophagy, which may directly contribute to the tumor promoting potential of AEG-1 under metabolic stress and apoptosis-deficient conditions.
Figure 1
Figure 1
Model illustrating the possible molecular mechanism of AEG-1-mediated protective autophagy, which leads to escape from apoptosis and chemotherapy toxicity.
Acknowledgements
The present study was supported in part by National Institutes of Health grant R01 CA134721, the Samuel Waxman Cancer Research Foundation (SWCRF) and the National Foundation for Cancer Research (NFCR) (P.B.F.); National Cancer Institute Grant R01 CA138540, the Dana Foundation, and the Goldhirsh Foundation for Brain Cancer Research (D.S.); and Korea Ministry of Education, Science and Technology through NRF (2009-0063466) (S.G.L.). D.S. is the Harrison Endowed Scholar in Cancer Research in the VCU Massey Cancer Center. P.B.F. holds the Thelma Newmeyer Corman Chair in Cancer Research in the VCU Massey Cancer Center and is a SWCRF and NFCR Investigator.
Notes
Punctum to: Bhutia SK, Kegelman TP, Das SK, Azab B, Su ZZ, Lee SG, et al. Astrocyte elevated gene-1 induces protective autophagyProc Natl Acad Sci USA2010212224322248 doi: 10.1073/pnas.1009479107.
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