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. 2010 Dec 21;107(51):22243-8.
doi: 10.1073/pnas.1009479107. Epub 2010 Dec 2.

Astrocyte elevated gene-1 induces protective autophagy

Sujit K Bhutia  1 , Timothy P Kegelman, Swadesh K Das, Belal Azab, Zhao-Zhong Su, Seok-Geun Lee, Devanand Sarkar, Paul B Fisher
Affiliations

Astrocyte elevated gene-1 induces protective autophagy

Sujit K Bhutia et al. Proc Natl Acad Sci U S A. .

Abstract

Astrocyte-elevated gene-1 (AEG-1) expression increases in multiple cancers and plays a crucial role in oncogenic transformation and angiogenesis, which are essential components in tumor cell development, growth, and progression to metastasis. Moreover, AEG-1 directly contributes to resistance to chemotherapeutic drugs, another important hallmark of aggressive cancers. In the present study, we document that AEG-1 mediates protective autophagy, an important regulator of cancer survival under metabolic stress and resistance to apoptosis, which may underlie its significant cancer-promoting properties. AEG-1 induces noncanonical autophagy involving an increase in expression of ATG5. AEG-1 decreases the ATP/AMP ratio, resulting in diminished cellular metabolism and activation of AMP kinase, which induces AMPK/mammalian target of rapamycin-dependent autophagy. Inhibition of AMPK by siAMPK or compound C decreases expression of ATG5, ultimately attenuating AEG-1-induced autophagy. AEG-1 protects normal cells from serum starvation-induced death through protective autophagy, and inhibition of AEG-1-induced autophagy results in serum starvation-induced cell death. We also show that AEG-1-mediated chemoresistance is because of protective autophagy and inhibition of AEG-1 results in a decrease in protective autophagy and chemosensitization of cancer cells. In summary, the present study reveals a previously unknown aspect of AEG-1 function by identifying it as a potential regulator of protective autophagy, an important feature of AEG-1 that may contribute to its tumor-promoting properties.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AEG-1 induces autophagy in IM-PHFA cells. (A) IM-PHFA cells were transfected with GFP-LC3 and infected with different pfu/cell of Ad.AEG-1 for 48 h, localization of LC3 in transfected cells was examined by confocal microscopy (magnification 100×), and autophagosome formation was quantified and data presented as percentage of GFP-LC3–transfected cells with punctate fluorescence to autophagosome formation. A minimum of 100 GFP-LC3–transfected cells were counted. *P < 0.05, compared with control. (B) The MDC fluorescent intensity of Ad.AEG-1–treated IM-PHFA cells was analyzed by flow cytometry. This result was representative of three different experiments. (C) IM-PHFA cells were infected with Ad.AEG-1 or Ad.vec for 48 h, fixed, and processed for electron microscopy (single arrow, autophagosome; control and others, 2 μm). (D) The numbers of autophagosomes in IM-PHFA cells 48 h after Ad.AEG-1 infection, the values are the means ± SD of three independent experiments. Asterisk indicates statistically significant change vs. corresponding control.
Fig. 2.
Fig. 2.
AEG-1 induces accumulation of LC3-II in normal cells. (A) IM-PHFA, Primary human fetal astrocytes, P69, and MCF10A cells were infected with Ad.AEG-1 or Ad.vec for 48 h and LC3 expression was analyzed by Western blotting. (B) IM-PHFA cells were infected with Ad.AEG-1 or Ad.vec for different times and LC3 expression was analyzed by Western blotting. (C) LC3 expression was examined in stable AEG-1–overexpressing CREF clones 29 and 30 (13).
Fig. 3.
Fig. 3.
AEG-1 induces noncanonical autophagy in IM-PHFA cells. (A) IM-PHFA cells were transfected with the indicated siRNAs and LC3-GFP followed by infection with 50 pfu/cell Ad.AEG-1 or Ad.vec and cytoplasmic aggregation of LC3-GFP was determined. A minimum of 100 GFP-LC3–transfected cells were counted. *P < 0.05; **P < 0.001, compared with sicontrol. (B) LC3-II expression was determined 48 h after administration of the indicated siRNAs and Ad.AEG-1 infection (50 pfu/cell) by immunoblotting. IM-PHFA Cells were infected with Ad.AEG-1 or Ad.vec for 48 h and mRNA (C) and protein (D) expression of Beclin-1 and ATG5 were analyzed using Taqman real-time PCR and Western blotting, respectively. Values are the mean ± SD of three independent experiments and *P < 0.05; **P < 0.001 vs. control cells.
Fig. 4.
Fig. 4.
AEG-1 induces cell energy deficiency and activates the AMPK/mTOR-dependent pathway by autophagy. IM-PHFA Cells were infected with Ad.AEG-1 or Ad.vec at different doses (pfu/cell) (A) and times (h) (B) followed by measurement of ATP levels using Bioluminescence Assay Kit. Asterisks indicate statistically significant change vs. corresponding control. IM-PHFA cells were infected with 50 pfu/cell of Ad.AEG-1 or Ad.vec for 48 h and then pretreated with 1 mM MP for 2 h followed by measurement of ATP levels in cells (C) and analysis of LC3 expression by immunoblotting (D). (E) IM-PHFA Cells were transfected with siAMPK and LC3-GFP followed by infection with 50 pfu/cell of Ad.AEG-1 or Ad.vec and cytoplasmic aggregation of LC3-GFP was determined. A minimum of 100 GFP-LC3–transfected cells were counted. *P < 0.05; **P < 0.001, compared with sicontrol. (F) LC3-II expression and other protein expressions were determined 48 h after administration of the siAMPK and Ad.AEG-1 infection (50 pfu/cell) by immunoblotting. (G) IM-PHFA cells were infected with 50 pfu/cell of Ad.AEG-1 or Ad.vec in the presence of compound C (10 μmol/L for 1 h) for 48 h, followed by analysis of LC3 expression and other proteins by immunoblotting.
Fig. 5.
Fig. 5.
Inhibition of autophagy resulted in serum starvation-induced cell death. IM-PHFA cells were transfected with siATG5 and MTT assay for cell viability (A), Annexin V assay for apoptosis (B), and Caspase-Glo(R) 3/7 assay for caspase-3 expression (C) was performed 48 h after infection with Ad.AEG-1 (10, 20, or 50 pfu/cell) or Ad.vec (50 pfu/cell), followed by 4 d after serum starvation (0.1%). *P < 0.05, compared with sicontrol.
Fig. 6.
Fig. 6.
Inhibition of AEG-1–induced autophagy in cancer cells enhances sensitivity to chemotherapeutic agents. (A) Stable shAEG-1-T98G cells were transfected with GFP-LC3, localization of LC3 in transfected cells was examined by confocal microscopy (magnification 100×) and autophagosome formation was quantified and data presented as percentage of GFP-LC3–transfected cells with punctate fluorescence to autophagosome formation. A minimum of 100 GFP-LC3–transfected cells were counted. *P < 0.05, compared with control. (B) LC3 expression was analyzed in shAEG-1-T98G cells by Western blotting. T98G cells were transfected with siATG5, and MTT assays for cell viability (C), Annexin V assays for apoptosis (D), and Caspase Glo(R) 3/7 assays for caspase 3 expression (E) were performed 24 h after infection with Ad.AEG-1 (50 pfu/cell) or Ad.vec (50 pfu/cell), followed by 4-d treatment with doxorubicin. *P < 0.05, compared with sicontrol.Ad.vec. (F) Model illustrating the possible molecular mechanism of AEG-1–mediated protective autophagy, which leads to escape from apoptosis and chemotherapy toxicity.
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