ER stress negatively regulates AKT/TSC/mTOR pathway to enhance ...

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May 7, 2009 - that inhibition of mTOR activity could trigger autophagy in the. MEFs in our experiments (Fig. 2F). To detect whether mTOR is involved in ER ...
Basic Research Paper

Basic Research Paper

Autophagy 6:2, 239-247; February 16, 2010; © 2010 Landes Bioscience

ER stress negatively regulates AKT/TSC/mTOR pathway to enhance autophagy Liang Qin,1,* Zheng Wang,2 Lianyuan Tao3 and Yun Wang2 Departments of Physiology & Pathology; and 2Department of Medical Genetics; Institute of Basic Medical Sciences & School of Basic Medicine; Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing, China; 3Department of General Surgery; Peking Union Medical College Hospital; Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing, China

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Key words: ER stress, autophagy, mTOR, AKT, PDGFR, IRS1 Abbreviations: AMPK, AMP-activated protein kinase; eIF2α, eukaryotic translation initiation factor-2α; ER, endoplasmic reticulum; IRS, insulin receptor substrate; LC3, microtubule-associated protein-1 light chain-3; MEF, mouse embryonic fibroblast; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; PERK, PRK (RNA-dependent protein kinase)-like ER kinase; PI3K, phosphatidylinositol-3 kinase; PTEN, phosphatase and tensin homolog; TSC1/TSC2, tuberous sclerosis complex 1/2 heterodimer; UPR, the unfolded protein response; 4-PBA, 4-phenylbutyric acid

Disturbance to endoplasmic reticulum (ER) homeostasis that cannot be rescued by the unfolded protein response (UPR) results in autophagy and cell death, but the precise mechanism was largely unknown. Here we demonstrated that ER stress-induced cell death was mediated by autophagy which was partly attributed to the inactivation of the mammalian target of rapamycin (mTOR). Three widely used ER stress inducers including tunicamycin, DTT and MG132 led to the conversion of LC3-I to LC3-II, a commonly used marker of autophagy, as well as the downregulation of mTOR concurrently. TSC-deficient cells with constitutive activation of mTOR exhibited more resistance to ER stress-induced autophagy, compared with their wild-type counterparts. Furthermore, our studies showed that ER stress-induced deactivation of mTOR was attributed to the downregulation of AKT/TSC/mTOR pathway. Phosphatase and tensin homolog (PTEN) and AMP-activated protein kinase (AMPK) as two regulators in this pathway seemed to be absent in this regulation. As a chemical chaperone helping the correct folding of proteins, 4-phenylbutyric acid (4-PBA) partly rescued the AKT/TSC/ mTOR pathway in drug-induced acute ER stress. Moreover, constitutively-activated mTOR-induced long-term ER stress attenuated the RTK/PI3K/AKT signaling pathway in response to the stimulation by various growth factors, which could also be partly restored by 4-PBA.

Introduction The endoplasmic reticulum (ER) is an essential intracellular organelle providing apparatus for synthesizing nascent proteins as well as their further modification and correct folding, such as the formation of N-linked glycans and disulphide bonds.1 Such a process is crucial for cell survival thus requiring stringent quality control that only correctly modified and folded proteins can be transported out of ER. However, this can be disturbed by a variety of means under either physiological conditions, such as increased secretory load, or pathological conditions, such as accumulation of mutated proteins that cannot be properly folded in ER.2 To sense and respond to this imbalance, named ER stress, specific signaling pathways have evolved in eukaryotic cells that are collectively termed the unfolded protein response (UPR). Three sensors located on the membrane of ER are responsible for monitoring ER stress and initiating UPR: inositol-requiring ER-tonucleus signal kinase-1 (IRE1), PRK (RNA-dependent protein kinase)-like ER kinase (PERK) and activating transcription

factor-6 (ATF6). Once sensing ER stress at the early stage, these monitors are activated immediately from their dormant forms and perform as the brake to cause the global slowdown of protein translation. For example, activated PERK phosphorylates eukaryotic translation initiation factor-2α (eIF2α) thus inhibiting protein synthesis. Meanwhile, misfolded proteins are degraded in a proteasome-dependent manner.1-3 However, if ER stress is too severe to be relieved, several signaling pathways leading to apoptosis and autophagy would be initiated. Autophagy, named as “self-eating,” acts as a double-edged sword in mediating cell fate. Compared with the proteasome pathway, autophagy is capable of degrading folded proteins, protein complexes and even entire organelles.4 Interestingly, as a process which could result in both cell survival and cell death under nutrient starvation, autophagy, breakthroughs about which have taken place during only the past decade, has raised great interest in a variety of fields.5,6 Although a group of genes involved in this process have been identified (Atg), much remains unknown about this event that frequently occurs during development,

*Correspondence to: Liang Qin; Email: [email protected] Submitted: 05/07/09; Revised: 12/25/09; Accepted: 12/29/09 Previously published online: www.landesbioscience.com/journals/autophagy/article/11062 www.landesbioscience.com

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tumorigenesis, neurodegeneration and other physiological and pathological processes. Two relatively well-established pathways regulating autophagy contain mTOR and IP3 cascades which seem to have little crosstalk. Inhibition of mTOR activity or decrease of IP3 level can both induce autophagy.4,7 Recent research provided increasing evidence about the relation between autophagy and ER stress, revealing a bidirectional regulation pattern.8,9 However, the role of mTOR in ER stress-induced autophagy is far from fully elucidated as far as we are aware. Receptor tyrosine kinase (RTK) such as PDGFR, IGF1R and EGFR is a family of cell-surface growth-factor receptors with intrinsic tyrosine-kinase activity. While they regulate diverse pathways in a ligand-controlled manner in normal cells, their aberrant activation always play the crucial role in tumorigenesis.10 Generally, three major downstream cascades activated by RTK are responsible for transmitting proliferative and antiapoptotic signals into cells: RAS/RAF/MEK1/2/ERK, JAK/ STAT and PI3K/AKT.11 AKT, also named as protein kinase B (PKB), is a major survival factor promoting cell proliferation and whose dysregulation has been frequently detected in many types of cancer. Two tumor-suppressor genes flanking AKT negatively regulate its functions: phosphatase and tensin homolog (PTEN) which suppresses AKT by catalyzing the conversion of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) into phosphatidylinositol (4,5)-bisphosphate (PIP2),12 and the tuberous sclerosis complex 1/2 (TSC1/TSC2) heterodimer that inhibits the activity of the mammalian target of rapamycin (mTOR).13 While AKT inhibits TSC1/TSC2 thus activating mTOR, hyperactivated mTOR is found to downregulate AKT as a negative feedback, which is considered to be responsible for the generally benign nature of tumors that develop in TSC patients.14 Several mechanisms have been proposed to elucidate this feedback: one theory is focused on the decrease of PDGFR in TSC-deficient cells,15 while another ascribes it to the degradation and subcellular redistribution of insulin receptor substrate (IRS) induced by mTOR.16-18 Here we reported that ER stress-induced autophagy is partly attributed to the downregulation of AKT/TSC/mTOR pathway. Considering the recent report that constitutive activation of mTOR triggers ER stress,19 our study may help propose a signaling pathway responsible for ER stress-induced autophagy and a novel mechanism accounting for the negative feedback from mTOR to AKT. Results Drug-induced ER stress triggers autophagy-mediated cell death. MEF cells were treated with three drugs inducing ER stress via distinct mechanisms: tunicamycin (inhibiting N-linked glycosylation), DTT (disturbing the formation of disulfide bonds) and MG132 (intervening in the function of proteasome). All of them could lead to the accumulation of misfolded proteins in ER thus triggering ER stress. The elevation of p-PERK and p-eIF2α indicated that all these drugs induced ER stress in MEFs (Fig. 1A–C). Remarkable cell death was observed via microscope after 12-hour treatment. Therefore, we examined the cleaved form of

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caspase-3 and LC3 which respectively indicate the occurrence of apoptosis and autophagy.20 As a result, the dramatic elevation of cleaved caspase-3 and the conversion of LC3-I (the upper band) to LC3-II (the lower band) reflected the concurrence of apoptosis and autophagy (Fig. 1A–C). Moreover, to exclude the possibility that the increase of LC3-II was due to the block of autophagic degradation, we co-treated the cells with bafilomycin A1, a specific inhibitor of the vacuolar type H+ -ATPase (V-ATPase), to suppress the autophagosome-lysosome fusion.21-23 As a result, ER stress inducers could still lead to the elevation of LC3-II with the presence of bafilomycin A1, which confirmed the induction of autophagy in MEFs (Fig. 1D). Interestingly, while autophagy is traditionally considered as a cytoprotective process during ER stress, the autophagy and apoptosis in our experiments seemed to progress in the same direction. To detect the role autophagy plays in regulating ER stressinduced cell death, we treated cells with two autophagy inhibitors which block autophagy via distinct mechanisms: bafilomycin A1 (inhibiting the fusion of autophagosome and lysosome) and 3-methyladenine (3-MA, suppressing the activity of class III PI3K). Both the observation under microscopy and MTT assay reflected the dramatically elevated cell viability under ER stress with the presence of the two autophagy inhibitors (Fig. 1E and F). Such data demonstrated the prominent role autophagy plays in mediating ER stress-induced cell death. However, as the ER stress extended over 24 h, the autophagy inhibitors cannot alleviate cell death apparently (data not shown). ER stress-induced autophagy is partly attributed to the downregulation of AKT/TSC/mTOR pathway. Considering the already known relation between mTOR and autophagy, we explored whether ER stress induces autophagy through the suppression of mTOR. It was found that the treatment with all three ER stress inducers resulted in the drop of mTOR activity in wildtype MEFs, as indicated by the decrease of p-S6 (a marker of mTOR activity) (Fig. 2A–C and E). Moreover, the treatment with rapamycin, an mTOR inhibitor and classical autophagy inducer, also led to the conversion of LC3 in MEFs, confirming that inhibition of mTOR activity could trigger autophagy in the MEFs in our experiments (Fig. 2F). To detect whether mTOR is involved in ER stress-induced autophagy, both TSC-deficient MEFs and their wild-type counterparts were treated with the drugs. The lesser conversion of LC3 in TSC-deficient MEFs signified markedly reduced autophagy, in comparison with their wild-type control cells (Fig. 2A–C and E). The observations above suggest that the suppression of mTOR activity contributes to ER stress-induced autophagy. Considering its pivotal role in integrating numerous upstream survival signals, we postulated AKT to be responsible for ER stress-induced inhibition of mTOR. Since AKT activates mTOR through phosphorylating thus deactivating TSC1/2 complex, both TSC1-/- and TSC2-/- MEFs were treated with ER stress inducers to detect the change of AKT/TSC/mTOR pathway. As shown in our results, while the levels of p-AKT were sharply decreased in all cell lines, the activity of mTOR in the TSC1-/- and TSC2-/cells did not drop as did that in their wild-type counterparts (Fig. 2A–C and E). As indicated by the robust p-S6, TSC-deficient

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Figure 1. ER stress inducers trigger autophagy which plays a prominent role in ER stress-induced cell death. MEF cells were treated with (A) tunicamycin (2 µg/ml), (B) DTT (200 µM) and (C) MG132 (5 µM) for the indicated time. (D–F) MEFs were treated with tunicamycin (2 µg/ml), DTT (200 µM) and MG132 (5 µM) with or without the co-incubation of bafilomycin A1 (100 nM) or 3-MA (10 mM) from the same time point for 12 h. Cell lysates were analyzed for the levels of p-PERK, p-eIF2α, eIF2α, LC3 and cleaved caspase-3 via western blot. Cell viability was determined by (E) MTT assay and (F) inverted microscope.

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Figure 2. ER stress downregulates AKT/TSC/mTOR pathway to enhance autophagy. Wild-type (wt), and TSC2 knock-out (TSC2-/-) MEFs were treated with (A) tunicamycin (2 µg/ml), (B) DTT (200 µM) and (C) MG132 (5 µM) for the indicated time. (D) Wild-type MEFs were transfected with either control siRNA (siCtrl) or siRNA against TSC2 (siTSC2) for 48 h, and then treated with tunicamycin (2 µg/ml) for the indicated time. (E) Wild-type (wt) and TSC1 knock-out (TSC1-/-) MEFs were treated with tunicamycin (2 µg/ml) for the indicated time. (F) Wild-type MEFs were treated with rapamycin (10 nM) for the indicated time. (G) Wild-type MEFs transfected with pLXIN-hyg (vector control, vec) or pLXIN-hyg-mutAKT (expressing activated AKT, mutAKT) were treated with tunicamycin (2 µg/ml) for the indicated time. Cell lysates were analyzed for the levels of TSC2, p-AKT, AKT, p-S6, S6, LC3 and β-actin via western blot.

MEFs exhibited constitutive mTOR activity, even after 24-hour treatment with ER stress inducers (Fig. 2A–C and E). Moreover, knockdown of TSC2 with siRNA prevented ER stress-induced decrease of mTOR activity to some extent, which validated the crucial role AKT/TSC2 plays in ER stress-induced downregulation of mTOR (Fig. 2D). To further confirm the critical role AKT plays in ER stressinduced suppression of mTOR, we introduced the vector expressing constitutively activated AKT through glutamic acid-to-lysine substitution at amino acid 17 (E17K). The transfected cells exhibited robust AKT and mTOR activity even under long-time ER stress (Fig. 2G). Furthermore, the constitutive activation of AKT also dampened autophagy induced by tunicamycin (Fig. 2G). Such observation validates AKT/TSC/mTOR as a targeted pathway in ER stress-induced autophagy. AMPK and PTEN are not involved in ER stress-induced negative regulation of AKT/TSC/mTOR pathway. In an attempt to find whether other mediators located upstream of TSC1/2 complex are also responsible for ER stress-induced mTOR inhibition, we detected AMP-activated protein kinase (AMPK) that activates TSC1/2 complex thus suppressing mTOR. As shown in our result, the level of p-AMPK did not change after the treatment with tunicamycin, which excluded its involvement in ER

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stress-induced suppression of mTOR (Fig. 3A). Considering that PTEN deactivates AKT through reversing PI3K-promoted conversion from PIP2 to PIP3, we examined whether it is also responsible for the decrease of AKT activity in ER stress. Results showed that there was no change on the level of PTEN after the treatment with tunicamycin (Fig. 3A). Furthermore, both PTEN/and the wild-type control cells were treated with tunicamycin. PTEN-deficient cells exhibited higher level of p-AKT and p-S6, yet the deletion of PTEN did not change ER stress-induced AKT downregulation (Fig. 3B). Therefore, while PTEN is involved in downregulation of PI3K/AKT pathway, it seems absent in tunicamycin-induced AKT downregulation. 4-PBA partly rescues AKT/TSC/mTOR pathway from druginduced acute ER stress. As a chemical chaperone, 4-phenylbutyric acid (4-PBA) was reported to alleviate ER stress in both cell lines and animal models.19,24 Here we introduced 4-PBA into our system to detect whether it could also restore AKT/TSC/mTOR pathway and inhibit autophagy under ER stress. Wild-type MEFs were treated with the three ER stress inducers and with or without 4-PBA from the same time point. The relatively lower level of p-eIF2α in 4-PBA-treated cells indicated the alleviation of ER stress induced by the three drugs (Fig. 4A–C). Compared with the cells treated with only ER stress inducers, the recovery

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Figure 3. AMPK and PTEN are absent in ER stress-induced downregulation of AKT/TSC/mTOR pathway. (A) Wild-type MEFs were treated with tunicamycin (2 µg/ml) for the indicated time. (B) Wild-type (wt) and PTEN-deficient (PTEN-/-) MEFs were treated with tunicamycin (2 µg/ml) for the indicated time. Cell lysates were analyzed for the levels of p-AMPKα, PTEN, p-AKT, AKT, p-S6, S6, LC3 and β-actin via western blot.

of AKT and mTOR activity in cells treated with both 4-PBA and ER stress inducers represented the restoration of AKT/TSC/ mTOR signaling pathway. Moreover, autophagy was also alleviated as indicated by the reduced conversion of LC3 (Fig. 4A–C). The cleaved caspase-3 decreased after the treatment with 4-PBA indicating the reduced apoptosis (Fig. 4A–C). Interestingly, observation via microscopy reflected that 4-PBA dramatically reduced cell death induced by tunicamycin (Fig. 4D), while the effects on DTT and MG132-induced cell death are not so significant (data not shown). 4-PBA partly rescues RTK/PI3K/AKT signaling pathway from hyperactive mTOR-induced ER stress via the increase of PDGFR and IRS1. Hyperactive mTOR was reported to downregulate PDGFR, which is critical for PI3K/AKT activation.13,15 However, the precise mechanism is far from fully known. Moreover, a large number of studies implicated the negative feedback from mTOR to AKT through IGF1R/IRS/PI3K/AKT pathway.25,26 As shown in our results, the levels of both PDGFR and IRS1 were lower in TSC2-/- cells than those in their wild-type counterparts (Fig. 5A). Recent studies reported that loss of TSC1 or TSC2 could induce ER stress and activate UPR,19 which promoted us to postulate it is ER stress induced by constitutive activation of mTOR that results in the downregulation of PDGFR in TSC-deficient MEFs. After 24-hour treatment with 4-PBA, the levels of both PDGFR and IRS1 in TSC2-/- MEFs were elevated up, although still less than those in the wild-type control cells (Fig. 5A). In order to detect the effect of 4-PBA on RTK/PI3K/ AKT signaling pathway in response to the stimulation by growth factors, we depleted serum during the cell culture with or without the pretreatment with 4-PBA, and then stimulated the cells with PDGF, EGF and IGF1. TSC2-/- MEFs exhibited remarkably

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reduced sensitivity to the stimulation of AKT by PDGF, EGF and IGF1 in comparison with the control cell lines. Again this could be partly restored by the treatment with 4-PBA (Fig. 5B). Interestingly, the level of EGFR is higher in TSC-deficient MEFs than in the wild-type control cells, even through TSC2-/- cells exhibited dramatically less sensitivity to the stimulation of AKT by EGF (Fig. 5B). Our observation suggests that the impairment of RTK/PI3K/AKT pathway in TSC2-/- MEFs is due to the decreased level of PDGFR and IRS1, and 4-PBA could partly restore them through the alleviation of ER stress (Fig. 5B). Discussion The results above suggest that acute ER stress induced by the three drugs results in the suppression of AKT/TSC/mTOR signaling pathway which contributes to ER stress-induced autophagy (Fig. 6A). In TSC-deficient cells where constitutive activation of mTOR induces long-term ER stress, the downregulation of PDGFR and IRS1 induced by ER stress attenuates the signals from growth factors to AKT as a negative feedback (Fig. 6B). Besides nutrient depletion, hypoxia and other stress condition, ER stress has emerged as a novel autophagy inducer that raises increasing attention recently. As a highly conserved process from yeast to mammals, autophagy plays a controversial role in regulating cell death and survival. In ER stress, autophagy is generally considered as a cytoprotective response to the overload of unfolded or misfolded proteins that exceed the capacity of proteasome.27 Supporting evidence comes from the report that the activation of PI3K/AKT/mTOR pathway promotes necrotic cell death via suppression of autophagy.28 Combined with our data and the previous report that TSC-deficient cells are vulnerable to

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Figure 4. 4-PBA rescues AKT/TSC/mTOR pathway in drug-induced acute ER stress. Wild-type MEFs were treated with (A and D) tunicamycin (2 µg/ ml), (B) DTT (200 µM) and (C) MG132 (5 µM) and with (+) or without (-) 4-PBA (5 mM) from the same time point for the indicated time. (D) Cell viability was assessed by inverted microscope. Cell lysates were analyzed for the levels of p-eIF2α, eIF2α, p-AKT, AKT, p-S6, S6, LC3 and cleaved caspase-3 via western blot.

ER stress-induced apoptosis, it may indicate that the loss of TSC results in the resistance to ER stress-induced mTOR downregulation, which promotes cell death via the suppression of autophagy. However, it was also reported that excessive autophagy could result in the programmed cell death.29 ∆9-tetrahydrocannabinol (THC) was reported to induce human glioma cell death through stimulation of autophagy.30 Here our data also reflect its predominant role in promoting cell death. Moreover, beyond degrading proteins as one downstream of UPR, recently reported data also revealed that autophagy could function in regulating UPR pathway. 3-MA was found to suppress UPR activation.31 An even more complicated issue is the identification of the crucial mediators and downstream pathways of UPR responsible for autophagy induction. PERK, IRE1 and increased [Ca 2+] C have been implicated as distinct initiators of ER stress-induced autophagy in mammalian cells, while the precise regulatory pathway is still enigmatic.32-34

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Growing evidence implicates the regulation of mTOR in response to UPR and its role in ER stress-induced autophagy. THC could activate ER stress response that promotes autophagy via tribbles homolog 3 (TRB3)-dependent inhibition of the AKT/mTOR pathway.30 Another well-established mechanism is CaMKKβ/AMPK/TSC1/2/mTOR cascade.32 It was also reported that ER stress induced by DL-homocysteine, DTT or tunicamycin inhibits mTOR activity via activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein-β (C/ EBP-β) in HeLa cells.35 Research on both cell lines and mouse models showed that ER stress results in the serine phosphorylation and subsequent degradation of IRS1 through the hyperactivation of c-Jun N-terminal kinase (JNK), thus diminishing the PI3K/AKT/mTOR pathway.36,37 Data from rat hippocampal neurons showed that ER stress induced by tunicamycin and thapsigargin downregulates the activity of AKT and mTOR,

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Figure 5. 4-PBA partly rescues RTK/PI3K/AKT pathway from hyperactive mTOR-induced long-term ER stress. (A) TSC2-deficient (TSC2-/-) MEFs were treated with (+) or without (-) 4-PBA (5 mM) for 24 h. (B) Wild-type (wt) and TSC2-deficient (TSC2-/-) MEFs pretreated with (+) or without (-) 4-PBA (5 mM) for 24 h were starved for 24 h and then stimulated with IGF1 (20 nM), EGF (100 µg/ml) or PDGFbb (50 µg/ml) for 10 min. Cell lysates were analyzed for the levels of p-PERK, p-eIF2α, eIF2α, PDGFRβ, IRS1, p-AKT, AKT, p-S6, S6, IGF1Rα and EGFR via western blot.

and triggers apparent apoptosis.38 More evidence of the crosstalk between UPR and mTOR comes from hypoxia signaling pathways. As two major cascades frequently activated in this stress condition, they collaborate to regulate various downstream events.39 Our study here revealed that the deactivation of AKT, as a downstream event of ER stress, led to the suppression of mTOR thus inducing autophagy. Interestingly, while large number of evidence support that ER stress downregulates AKT activity,38,40-43 it was also reported that AKT and ERK are rapidly activated and act as downstream effectors of PI3K in acute ER stress.44 Considering that AKT and MAPK pathways are two major survival signaling cascades, cells may activate them through UPR to transmit survival signals and overcome the adverse condition, when ER stress is moderate and recoverable. In comparison, when ER stress is prolonged, thus too severe to be relieved such as experiencing long-time treatment with drugs, the deactivation of AKT/TSC/mTOR pathway leads to the autophagy and apoptosis. Another question to be answered is how mTOR negatively regulates AKT. While many observations show that hyperactive mTOR inhibits AKT,14 the precise mechanism is still unclear. One model attributes this negative feedback to the degradation of IRS1 induced by mTOR. This is supported by the observation that ER stress triggered by hyperactivated mTOR degrades IRS1 by activating JNK which increases the

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serine phosphorylation of IRS1.19,36,37 Therefore it is reasonable to postulate that mTOR downregulates AKT through mTOR/ ER stress/IRS1/AKT pathway. However, another evidence that should not be neglected is that PDGFR is essential for AKT activation by not only PDGF but also EGF and IGF.15 Since IRS1 is only responsible for transmitting stimulating signals of insulin and IGF, the mTOR/ER stress/IRS1/AKT pathway is hard to utilize to explain the impaired sensitivity of AKT to the stimulation by PDGF and EGF in TSC-deficient cells (Fig. 5B). Based on our data that 4-PBA could restore PDGFR as well as the activation of AKT by the stimulation of EGF, PDGF and IGF, we tend to believe that there is another pathway responsible for the negative feedback: mTOR/ER stress/PDGFR/PI3K/ AKT which may play a more important role in downregulating AKT activity. However, what should be taken with caution is that the occurrence of ER stress in our research was induced by several drugs that result in acute and severe stress which may be different from the real condition in vivo. A major discrepancy may be the extent of stress that may play the crucial role in deciding the direction of UPR and thus the cell fate. It is a little surprising that the level of EGFR is higher in TSCdeficient MEFs than in the wild-type controls (Fig. 5B). It signifies the different regulatory pattern of these RTKs. Another interesting observation is the insensitivity of TSC-deficient cells

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Figure 6. Suppression of AKT/TSC/mTOR pathway contributes to ER stress-induced autophagy. (A) Drug-induced acute ER stress leads to the suppression of AKT/TSC/mTOR pathway which enhances UPR-induced autophagy. (B) Long-term ER stress induced by the hyperactivation of mTOR reduces the levels of both PDGFR and IRS1 thus attenuating RTK/PI3K/AKT cascade. This pathway is responsible for the negative feedback from hyperactive mTOR to AKT.

to the stimulation of AKT by EGF, although they have relatively higher level of EGFR. This contradiction may indicate the involvement of other regulators in this pathway. Our observations may provide therapeutic implications for the diseases related to aberrant activation of AKT/TSC/mTOR pathway. As a central regulator integrating various survival and proliferative signals, AKT has great involvement in tumorigenesis. Aberrant activation of RTK and PI3K as well as the loss-of-function mutation of PTEN are frequently detected in human malignancies. Diverse chemotherapy strategies have been developed to inhibit this pathway. However, as drug resistance ultimately occurs, patients with poor prognosis have made up a considerable part.42,45 Our data showed a significant deactivation of this signaling pathway, which may imply a more widely downregulatory effect. Thus, chemotherapy triggering ER stress for the cancer induced by hyperactivated AKT may be helpful for overcoming drug resistance. However, since nontransformed cells are also sensitive to ER stress-induced cell death, development of more selective and less toxic drugs to induce ER stress is a more reasonable choice. Materials and Methods Reagents. Reagents were obtained from commercial sources: rapamycin (R0395), tunicamycin (T7765), 4-phenylbutyric acid (P21005), IGF1 (I8779), EGF (E4127), MTT (M2128) and PDGFbb (P4056) were from Sigma-Aldrich; DTT (#P1171) was from Promega; bafilomycin A1 (196000), MG-132 (474790) and 3-methyladenine (189490) were from Merck; Dulbecco’s modified Eagle’s medium (07-02I) was from Neuronbc; Fetal Bovine Serum (SV30087.02) was from Thermo Scientific; 4–12%

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Bis-Tris Nu-PAGE gels (NP0323BOX) and Lipofectamine 2000 (11668-019) were from Invitrogen. Antibodies. phospho-S6 (Ser235/236) and S6 have been described previously;15 TSC2 (SC-893), β-actin (SC-47778) and phospho-PERK (Thr981, SC-32577) were from Santa Cruz Biotechnology Inc.; PDGFRβ (#06-498), PTEN (#07-1372), EGFR (#06-847) and IRS1 (#06-248) were from Upstate USA Inc.; phospho-AMPKα (Thr172, #2535), AKT (#9272), phosphor-AKT (Ser473, #9271), IGF1Rα (#3022), eIF2α (#9722), phospho-eIF2α (Ser51, #9721) and cleaved caspase-3 (#9661) were from Cell Signaling Technology; LC3 (ab48394) was from Abcam. Cell culture. All MEF cells used here have been described previously.13,15,46 Cells were cultured in DMEM with 10% FBS, penicillin/streptomycin in 5% CO2 at 37°C. Small interfering RNA (siRNA) knockdown. All the siRNA were synthesized by GuangZhou RiboBio. Cells were seeded in 12-well plates and transfected with 100 nM siRNA using Lipofectamine 2000 following the manufacturer’s instructions. The cells were used for immunoblotting after 48 h. Immunoblotting. Whole cell extracts were prepared by boiling for 10 minutes after the harvest using lysis buffer (2% SDS, 10% glycerol, 10 mM Tris, pH 6.8, 100 mM DTT), and then subjected to immunoblotting as described previously.46 Cell viability assay. Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test. 2,000 cells were plated in 100 µl media per well in a 96-well plate, and incubated overnight. Cells were treated with drugs for the indicated time. 20 µl MTT (5 mg/ml in PBS) was added to each well, and the media were discarded after incubation for 3 to 4 hours. 100 µl DMSO was added into each well

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to resuspend and dissolve the MTT metabolic product. Finally, the optical density was read at 560 nm with the subtract of background at 670 nm. Acknowledgements

We thank Dr. Haiyong Peng for kindly providing the plasmid expressing constitutively activated AKT (E17K). We thank Dr. Jianhui Ma for assistance and helpful discussions. References

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