Hypoxia-Induced Energy Stress Inhibits the mtor Pathway by Activating an AMPK/REDD1 Signaling Axis in Head and Neck Squamous Cell Carcinoma 1

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1 Volume 10 Number 11 November 2008 pp Hypoxia-Induced Energy Stress Inhibits the mtor Pathway by Activating an AMPK/REDD1 Signaling Axis in Head and Neck Squamous Cell Carcinoma 1 Abraham Schneider*,, Rania H. Younis* and J. Silvio Gutkind *Department of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA; Greenebaum Cancer Center, Program in Oncology, University of Maryland, Baltimore, MD 21201, USA; Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA Abstract The mammalian target of rapamycin (mtor) signaling network is frequently hyperactivated in patients with head and neck squamous cell carcinoma (HNSCC). Recent studies suggest that hypoxia, a common microenvironmental stress found in tumors, blocks this mitogenic pathway. Here, we demonstrate that in HNSCC cell lines, the expression of the phosphorylated forms of the mtor downstream targets S6 kinase and S6 (ps6) decreased after hypoxia. These events were associated with a marked up-regulation of the regulated in development and DNA damage 1 (REDD1), a recently characterized hypoxia-induced protein that negatively controls mtor activity. Conversely, ps6 levels were retained under hypoxia in REDD1 knock-down cells and in HNSCC cells lacking endogenous REDD1 expression. Furthermore, we observed that prolonged hypoxia induced an energy-depleting response as evidenced by decreased cellular ATP levels and AMP-activated protein kinase (AMPK) activation. Interestingly, AMPK inhibition before prolonged hypoxia prevented REDD1 expression, thereby sustaining mtor activity. These results suggest a novel mechanism by which AMPK activation after hypoxia-induced energy stress may be crucial in regulating REDD1 expression to control the mtor pathway in HNSCC. Furthermore, we found that, in some HNSCC cells, the reduced mtor activity in response to hypoxia through AMPK/REDD1 was deregulated, which hence might contribute to the persistent activation of the mtor pathway in this cancer type. Neoplasia (2008) 10, Introduction Cancer development is frequently initiated by genetic alterations affecting cell growth promoting as well as tumor-suppressive signaling networks in conjunction with epigenetic disturbances and progresses by the constant adaptation of tumor cells to microenvironmental selective pressures [1,2]. Microenvironmental stresses such as poor oxygenation or hypoxia arising in part by the structural and functional abnormalities affecting the vasculature are commonly found within the heterogeneous regions of expanding solid tumors [3]. This is particularly evident in head and neck squamous cell carcinomas (HNSCC), including those of the oral cavity and pharynx, where the extent of intratumoral hypoxia plays an important prognostic factor as it relates to cancer aggressiveness, chemoradiotherapy resistance, and overall patient survival [4 6]. The availability of molecular oxygen also plays a critical role in controlling signaling pathways responsible for relaying nutrient- and Abbreviations: HNSCC, head and neck squamous cell carcinoma; REDD1, regulated in development and DNA damage 1; mtor, mammalian target of rapamycin; ps6, phosphorylated ribosomal protein S6; ps6k, phosphorylated ribosomal protein S6 kinase; AMPK, AMP-activated protein kinase; TSC1/TSC2, tuberous sclerosis complex 1/2; ACC, acetyl CoA carboxylase; REDD1 / MEFs, REDD1-depleted mouse embryonic fibroblasts Address all correspondence to: Abraham Schneider, DDS, PhD, Department of Oncology and Diagnostic Sciences, University of Maryland, Dental School, 650 W. Baltimore St., Room 7265, Baltimore, MD aschneider@umaryland.edu 1 This study was supported in part by the Intramural Research Program, National Institute of Dental and Craniofacial Research, National Institutes of Health (J.S.G.), by funds from the National Institutes of Health/National Institute of Dental and Craniofacial Research, Training Program in Oral and Craniofacial Biology Grant 2T32 DE A1 (to A.S.) and by start-up funds from the University of Maryland Dental School (to A.S.). Received 14 May 2008; Revised 26 August 2008; Accepted 28 August 2008 Copyright 2008 Neoplasia Press, Inc. All rights reserved /08/$25.00 DOI /neo.08586

2 1296 mtor Pathway Inhibition via AMPK/REDD1 in HNSCC Schneider et al. Neoplasia Vol. 10, No. 11, 2008 energy-sensing cues to control cellular programs commonly involved in human cancer such as cell proliferation, differentiation, and survival [7]. Among these, the Akt/mammalian target of rapamycin (mtor) pathway seems to be a major regulator of the cellular responses to hypoxia and other microenvironmental stresses [8]. Acting as a positive central integrator of mitogenic signals, the evolutionarily conserved serine/threonine protein kinase mtor transmits stimulatory cues for protein synthesis and cell growth by phosphorylating key downstream substrates involved in protein translation, including the eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and ribosomal p70 S6 kinase (S6K). This occurs after the sequential activation of phosphoinositide-3-kinase (PI3K) and the serine/threonine kinase Akt in response to a myriad of cell surface molecules including growth factors, hormones, and extracellular matrix components. However, in an attempt to preserve cellular energy consumption, mtor activity is attenuated in response to cellular stresses, such as hypoxia, through activation of the tuberous sclerosis complex 1/2 (TSC1/TSC2) tumor-suppressor complex. TSC2 functions as a GTPase-activating protein antagonizing the small GTPase Rheb acting upstream of mtor. During permissive conditions, the Rheb- GAP activity of TSC2 is kept inactive by an Akt-driven inhibitory phosphorylation, leading to the accumulation of active GTP-bound Rheb, which in turn phosphorylates and activates mtor [8 11]. These observations suggest that any aberration impinging on the TSC1/2 complex-activating network may result in an abnormally high mtor activity. Interestingly, emerging information points to the mtor signaling network as frequently altered in human HNSCC and its derived cell lines [12 16]. Thus, it is plausible that in those human HNSCC that are dependent on aberrantly high mtor activity, the hypoxia-driven inhibitory mechanisms responsible for down-regulating this pathway might be impaired. This possibility prompted us to explore whether hypoxic responses controlled by the TSC complex/mtor signaling cascade may be deregulated in HNSCC. In this regard, the identification of the regulated in development and DNA damage 1 (REDD1) gene as a novel repressor of mtor activity in both mammalian and Drosophila cells has provided a key component to the stress-response pathway negatively affecting mtor function. REDD1 encodes a 232 amino acid cytosolic protein ( 34 kda) with unknown functional domains. REDD1-mediated inhibition of mtor is stimulated in cells exposed to hypoxia as well as in cells undergoing energy stress and is dependent on the presence of a functional TSC1/TSC2 tumor-suppressor complex [17 20]. An additional pathway by which energy stress can result in mtor inhibition is mediated by a positive TSC2-phosphorylating event initiated by the cellular energy-sensing apparatus through the AMP-activated kinase (AMPK) [21]. The AMPK is the downstream target of the serine/threonine kinase LKB1, a product of the LKB1 gene whose hereditary inactivation is responsibleforthecancer-pronepeutz-jeghers syndrome [22]. Here, we show that, in HNSCC, these two mechanisms are highly interrelated. Indeed, we observed that AMPK activation controls REDD1 expression in response to prolonged hypoxia and that this biochemical route by which hypoxia-induced energy stress can reduce mtor activity is disrupted in some HNSCC cells. Our results suggest that under metabolic stress triggered by energy depletion in response to hypoxia, the AMPK energy-sensing apparatus and REDD1 might be linked in the regulation of mtor activity in human HNSCC and that the dysfunction of this mechanism by which hypoxia inhibits mtor activity may contribute to HNSCC progression. Materials and Methods Cell Culture and Reagents The HNSCC cell lines HN6, HN12, and HN13 [23] were grown and maintained in 10% FBS, 1% antibiotic/antimycotic DMEM. Unless otherwise stated, HNSCC cells were serum-starved for 24 hours before any treatment. When indicated, serum-deprived HNSCC cells were pretreated for 30 minutes with compound C (Calbiochem, Gibbstown, NJ), a potent AMPK inhibitor, or vehicle control and then cultured under normoxic or hypoxic conditions. When required, cells were also treated with 2-deoxy-D-glucose (2-DG; Calbiochem). Recombinant human epidermal growth factor (EGF) was purchased from PeproTech (Rocky Hill, NJ). Hypoxic conditions were generated by placing cells in a modular incubator chamber (Billups-Rottenberg, Inc., Del Mar, CA) containing 1% O 2,5%CO 2, and 94% nitrogen. Normoxic cells were grown at 37 C in the presence of 5% CO 2. Immunohistochemistry Human HNSCC tissue sections were obtained from the Head and Neck Cancer Tissue Array Repository [15] under a National Institutes of Health approved research activity involving human subjects. Tissue slides were dewaxed in xylene, hydrated through graded alcohols and distilled water, and washed thoroughly in PBS. Antigen retrieval was done using 10 mm citrate buffer (ph 6) in a microwave (20 minutes). After incubation with 3% hydrogen peroxide in PBS (30 minutes), slides were washed in PBS and incubated in blocking solution (2.5% BSA in PBS) for 1 hour at room temperature. Sections were then incubated with the primary antibodies: mouse monoclonal anti Glut-1 at 1:200 (Abcam, Cambridge, MA) and rabbit monoclonal anti phospho-s6 ribosomal protein (Ser235/ 236) at 1:100 (Cell Signaling Technology, Danvers, MA) diluted in blocking solution overnight at 4 C. After washing with PBS, slides were incubated with biotinylated secondary antibodies (1 hour; 1:400), followed by the ABC complex (Vector Stain Elite, ABC kit; Vector Laboratories, Burlingame, CA) for 30 minutes. Slides were washed and developed in 3,3-diaminobenzidine under microscope control and counterstaining with Mayer s hematoxylin. Images were acquired using a microscope (Axioplan 2; Carl Zeiss, Thornwood, NY). Semiquantitative analysis was performed by two independent examiners to characterize the relative regional distribution of Glut-1 and phosphorylated ribosomal protein S6 (ps6) immunostaining in cells centrally localized in tumor islands (n = 5). ATP Assay The detection of ATP levels in whole cell extracts was assessed by using a luminescence-based ATP assay kit (Calbiochem). RNA Interference HN13 cells were plated at 15,000 cells/cm 2 overnight. The following day, transfections were performed by using the following reagents: short interfering RNA (sirna) duplexes (Qiagen, Valencia, CA) targeting REDD1 (Hs_DDIT4_1_HP sirna; SI ), hypoxia-inducible factor (HIF) 1α (Hs_HIF1A_1_1HP sirna; SI ), or HIF-2α (Hs_EPAS1_1_HP sirna; SI ) were transfected with Hiperfect transfection reagent (Qiagen) in complete medium following the manufacturer s recommendations. Likewise, a commercially available pool of three target-specific sirnas (sc-45312; Santa Cruz Biotechnology, Santa Cruz, CA) were used to knock down the α1 andα2 catalytic subunits of the human

3 Neoplasia Vol. 10, No. 11, 2008 mtor Pathway Inhibition via AMPK/REDD1 in HNSCC Schneider et al AMPK gene following the sirna transfection protocol of the manufacturer. Untransfected cells and cells transfected with a negative control sirna (Qiagen) were included in all experiments. The next day following transfections, cells were serum-starved for 24 hours and then exposed to either normoxia or hypoxia for 18 hours. Western Blot Analysis After the indicated experiments, medium was removed and HNSCC cell monolayers were rinsed with ice-cold Dulbecco s phosphatebuffered saline and rapidly lysed with protein lysis buffer [62.5 mm Tris-HCl (ph 6.8), 2% SDS, 10% glycerol, 50 mm DTT]. Lysates were sonicated for 20 seconds, and protein concentration was determined using the Bradford reagent (Sigma, St. Louis, MO). Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes (BioRad, Hercules, CA). Appropriate transfer and equivalent loading was confirmed by staining membranes with Ponceau S red solution (Sigma). Membranes were blocked in blocking buffer (5% nonfat dry milk in 0.05% Tween 20 TBS) for 1 hour and then replaced by the primary antibody diluted in blocking buffer for 2 hours at room temperature or overnight at 4 C, depending on the specific antibody. The following primary antibodies were used: rabbit polyclonal against phospho- S6 Ser240/244, phospho-akt (Ser473), phospho-p70 S6 kinase (Thr389), and phospho-acetyl CoA carboxylase (Ser79) and rabbit monoclonal against S6, phospho-akt (Thr308), and phospho-ampkα (Thr172) were purchased from Cell Signaling Technology and used at 1:1000 dilution. Rabbit polyclonal anti-redd1 (1:500) was obtained from Proteintech Group (Chicago, IL). Mouse monoclonal anti HIF- 1α (1:500) and rabbit polyclonal anti HIF-2α (1:250) were purchased from Novus Biologicals (Littleton, CO). Mouse monoclonal anti αtubulin (1:1000) was purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-gapdh (1:5000) was obtained from Sigma. After washing membranes three times in washing buffer, primary antibodies were detected using horseradish peroxidase linked sheep anti mouse or donkey anti rabbit IgG antibodies at 1:10,000 dilution (Amersham, Piscataway, NJ) and visualized by chemiluminescence. Statistics The data were analyzed using Student s t test with the Instat 3.0 biostatistics program (GraphPad Software, La Jolla, CA). Data are presented as mean ± SEM. Results Hypoxia Inhibits the mtor Signaling Pathway in HNSCC. Disruption of This Mechanism in Certain HNSCC Cells The aberrant activation of the PI3K/Akt/mTOR pathway seems to be a frequent event in human HNSCC and its derived cell lines [12,13,15,16]. Accumulating evidence indicates that mtor activity is negatively regulated by hypoxia, a common microenvironmental condition present in growing solid tumors. Despite the frequent hyperactivation of the mtor pathway in HNSCC, the intervening molecules affected by hypoxia in this cancer type have remained largely unexplored. Indeed, we noticed that in tumor islands from human HNSCC tissues, the pattern of regional distribution of the hypoxia-regulated glucose transporter Glut-1 stained tumor cells seems to be inversely correlated to the localization of cells positively stained with the phosphorylated active form of the ribosomal protein S6 (ps6), a downstream mtor target. Membrane-bound Glut-1 localization and staining in centrally localized tumor cells, which are most likely hypoxic, were significantly increased when compared to the periphery where more ps6-positive cells were present (Figure 1). Because the mechanisms controlling the mtor pathway in response to hypoxic stress are still not well defined, we were prompted to further explore which key intervening factors regulate the activity of the mtor pathway in HNSCC cell lines. To perform these experiments, we took advantage of the availability of HNSCC cell lines known to have increased basal mtor activity as evidenced by the high ps6 expression levels [12,13]. Unexpectedly, we observed a variable effect on the mtor pathway when these cell lines were cultured under hypoxia (1% O 2 for 18 hours) in serum-free conditions. Although hypoxia induced a marked reduction in ps6 levels in HN13 and HN12 cells, the effects on ps6 in HN6 cells were unaltered Figure 1. Intratumoral hypoxia correlates with reduced mtor activation in human HNSCC. Moderate to intense immunodetection of the hypoxia marker Glut-1 (A: subpanels a c) was frequently found in areas where the activation status of the mtor downstream target S6 (ps6) was reduced or absent (A: subpanels d f). Immunohistochemistry was performed on tissue sections from representative human HNSCC specimens using antibodies against Glut-1 and ps6 to determine the effects of regional intratumoral hypoxia on the mtor pathway in vivo. (B) In hypoxic foci, the relative distribution of Glut-1 immunostaining was significantly increased when compared to tumor cells positively stained with ps6 (*P <.05). Data represent the mean ± SEM.

4 1298 mtor Pathway Inhibition via AMPK/REDD1 in HNSCC Schneider et al. Neoplasia Vol. 10, No. 11, 2008 mtor pathway seemed to be dissimilar among HNSCC cell lines, we hypothesized that the lack of hypoxia-induced inhibition of the mtor pathway in HN6 cells might be linked to mechanisms underlying tumor cell responses to hypoxia through REDD1. As shown in Figure 3A, REDD1 expression in HN13 cells was markedly upregulated after hypoxia; moreover, the increase in hypoxia-induced Figure 2. Hypoxia inhibits the mtor pathway in HNSCC cells. (A) Representative serum-deprived HNSCC cell lines were subjected to normoxic (N) or hypoxic (H) conditions for 18 hours. Whole cell extracts were analyzed by Western blot analysis to determine the effects of hypoxia on the mtor pathway in vitro. Hypoxia-induced mtor substrate dephosphorylation in the HN13 and HN12 cell lines as judged by the reduction in ps6 expression. In HN6 cells, however, the mtor pathway remained hyperactivated under both normoxic and hypoxic conditions as demonstrated by the sustained expression of ps6 protein. Increased HIF-1α expression positively validated the response to hypoxia in all three cell lines. Total S6 and α-tubulin expression are shown as sample loading controls. (B) The HNSCC cell lines cultured in the presence or absence of 10% fetal bovine serum were exposed to normoxic or hypoxic conditions for 18 hours. Whole cell extracts were analyzed by Western blot analysis to determine the expression levels of phosphorylated Akt. Total Akt is shown as loading control. and remained similar to the levels observed under normoxic conditions, suggesting that these cells were tolerant to the inhibitory effects exerted by hypoxic stress on mtor activity (Figure 2A). Because the PI3K/Akt pathway is implicated in the control of mtor activity in response to growth factor mediated inputs, it might be possible that the effects on the mtor pathway were directly related to Akt inhibition associated with serum-deprived conditions rather than hypoxic stress per se. As shown in Figure 2B, the presence or absence of serum in the culture medium had little effect on the phosphorylated status of Akt in HNSCC cell lines. The only significant difference was observed in HN13 cells cultured under serum-repleted conditions where hypoxia induced substrate dephosphorylation of both Akt S473 and Akt T308. REDD1 Is Required to Inhibit mtor Activity in Response to Hypoxia in HNSCC Cells Recent studies have identified REDD1, a stress-induced gene acting upstream of the TSC1/TSC2 tumor-suppressor complex, as a key molecule within an inhibitory cascade targeting mtor activity in response to hypoxia [18,24]. Because the effects of hypoxia on the Figure 3. Hypoxic inhibition of the mtor pathway in HNSCC cells correlates with REDD1 protein expression levels. (A) Whole cell extracts from serum-deprived HN13 and HN6 cells exposed to normoxia (N) or hypoxia (H) were subjected to Western blot analysis with an antibody against the stress-induced protein REDD1. In HN13 cells, REDD1 was markedly up-regulated by hypoxia, and this response correlated with mtor pathway inhibition as judged by ps6 dephosphorylation. In contrast, REDD1 expression in HN6 cells was not induced by hypoxia and S6 remained in its phosphorylated active form under these conditions. (B) REDD1 protein expression was knocked-down in HN13 cells through sirna to further validate the effects of REDD1 on mtor activity under hypoxia. HN13 cells were either left untransfected ( ) or transfected with a control (C) or REDD1 sirna oligonucleotide duplex. Forty-eight hours after transfection, serum-deprived cells were exposed to normoxia or hypoxia for 18 hours. Western blot analysis shows that in REDD1-depleted cells hypoxia was unable to induce ps6 dephosphorylation compared to control cells where ps6 expression levels were readily decreased. Total S6 levels are shown to assess sample loading. (C) Following a similar RNAi protocol as described in (B), Western blot analysis shows that REDD1 expression remained up-regulated in HIF-1α or HIF-2α depleted as well as untransfected ( ) and control sirna (C) HN13 cells subjected to hypoxic stress.

5 Neoplasia Vol. 10, No. 11, 2008 mtor Pathway Inhibition via AMPK/REDD1 in HNSCC Schneider et al REDD1 seemed to correlate with the down-regulation in ps6 detection levels. In contrast, however, exposure of HN6 cells to hypoxic stress did not increase REDD1, and this lack of expression was linked to a sustained mtor activity as judged by the levels of ps6, which remained unchanged when compared to cells maintained in normoxia. To further examine the role of REDD1 on the regulation of mtor activity in HNSCC, HN13 cells were transfected with sirna oligonucleotide duplexes targeting REDD1. Our findings showed that, in both untransfected and control sirna transfected HNSCC cells, REDD1 was induced after exposure to hypoxia resulting in mtor substrate dephosphorylation. However, when REDD1-depleted cells were cultured in hypoxic conditions, ps6 expression was comparable to levels detected in cells grown under normal ambient oxygen, indicating that REDD1 acted as a key negative regulator of mtor activity in HNSCC cells (Figure 3B). Previous studies have reported that in response to hypoxic stress, REDD1 is controlled at the transcriptional level by HIF-1, which acts as the master regulator of oxygen sensing in mammalian cells [25,26]. Thus, we performed experiments to challenge whether the presence of the oxygen-regulated α subunit of HIF-1 or HIF-2 was in fact required to control REDD1 expression in HNSCC cells exposed to hypoxia. Surprisingly, we found that in HN13 cells depleted of HIF-1α or HIF-2α, REDD1 protein expression remained up-regulated, suggesting that under hypoxic stress, REDD1 might also be controlled at the transcriptional level by HIF-independent mechanisms (Figure 3C). Prolonged Hypoxia Induces Energy Stress and Inhibits the mtor Pathway in HNSCC Cells by Activating the AMPK Pathway To cope with hypoxia, cells often turn on adaptive mechanisms that reversibly switch off ATP-consuming processes and switch on ATP-producing catabolic events to remain viable and survive unfavorable microenvironmental conditions [27]. These responses are exquisitely controlled by a well-orchestrated intracellular energy-sensing system governed by the activation of the evolutionarily conserved AMPK [28]. AMPK is the downstream target of a protein kinase network that is induced by cellular stresses that increase the cellular AMP/ATP ratio. To be activated, human AMPK requires to be phosphorylated at the threonine 172 residue within its catalytic α subunit [29]. As shown in Figure 4A, HN13 cells cultured under prolonged hypoxia led to the activation of AMPK and its downstream target acetyl CoA carboxylase (ACC). As expected, REDD1 expression was also increased resulting in mtor substrate dephosphorylation. Furthermore, a marked reduction in cellular ATP levels supported these results. Cellular ATP levels were significantly reduced by 74% and 57% when HN13 cells were exposed to hypoxia or to the energy-depleting AMPK activator 2-DG, respectively (Figure 4B). Overall, these findings indicated that HNSCC cells under sustained hypoxic stress triggered responses associated with cellular energy depletion orchestrated through the activation of the AMPK pathway. mtor Substrate Dephosphorylation by REDD1 in Response to Prolonged Hypoxia Requires AMPK Activation in HNSCC Cells Recent studies have found that REDD1 is also up-regulated in fibroblastic cells undergoing energy stress after either glucose starvation or pharmacologically induced ATP depletion, leading to atsc1/tsc2 dependent reduction in mtor activity [19]. It Figure 4. Hypoxia induces an energy-depleting response in HNSCC cells by activating the AMPK pathway. (A) Western blot analysis of whole cell lysates harvested from serum-deprived HN13 cells grown under normoxic (N) versus hypoxic (H) conditions for 18 hours shows a marked increase in the phosphorylated status of AMPK and its downstream target ACC. In addition, hypoxia-induced energy stress up-regulates REDD1 and affects mtor activity by preventing S6 phosphorylation. (B) Cellular ATP levels were determined by luminescence in total whole cell lysates derived from HN13 cells cultured under similar conditions as described in (A). The energydepleting reagent 2-DG (25 mm for 18 hours) was used as a positive control for AMPK activation. Representative data from two independent experiments performed in triplicates and expressed as the mean ± SEM; *P <.01 and **P <.02 versus normoxic conditions, respectively. seems, however, that in these and other cell types, REDD1-mediated effects on mtor in response to hypoxia or energy stress do not depend on AMPK activation [18,20]. For example, when AMPK is directly activated, REDD1 / mouse embryonic fibroblasts (MEFs) failed to down-regulate mtor activity compared with control wild type cells, but blocking AMPK activation seems to have no effect on mtor substrate dephosphorylation promoted by REDD1 [18 20]. Moreover, REDD1 activation and the subsequent mtor substrate dephosphorylation in MEFs exposed to hypoxia is rapid (within 15 minutes) and occur before any effects on the cellular energy status [24]. Because the regulatory mechanisms by which cellular stresses such as hypoxia and energy depletion control mtor activity seem to be mediated by two parallel, TSC-dependent pathways, we decided to explore whether similar mechanisms were also operative in HNSCC cells. As shown in Figure 5A, hypoxia led to a marked inhibition of the direct mtor substrate S6 kinase and its downstream target S6 within 2 hours of hypoxic exposure. These negative effects on the mtor pathway correlated with REDD1 up-regulation. Activation of AMPK, however, occurred solely after prolonged hypoxia as demonstrated by increased phosphorylated forms of AMPK and ACC. These results suggested that the inhibitory mechanisms affecting mtor in HNSCC cells exposed to hypoxia are operating relatively early and that these processes might be regulated through REDD1 in an AMPK-independent manner. However, prolonged exposure to low oxygen levels, a situation likely to occur in the tumor microenvironment, results in an energy-depleting response leading to AMPK activation, thus raising the possibility that hypoxia-induced energy stress may use AMPK to enhance REDD1 expression, thereby inhibiting mtor function. Indeed, we found that when HN13 cells were pretreated with compound C, a potent ATP-competitive inhibitor of AMPK activation [20], this caused a dramatic inhibition

6 1300 mtor Pathway Inhibition via AMPK/REDD1 in HNSCC Schneider et al. Neoplasia Vol. 10, No. 11, 2008 Figure 5. Inhibition of AMPK activation in HNSCC cells blocks hypoxia-induced REDD1 expression and retains mtor activity. (A) Serumdepleted HN13 cells were exposed to hypoxia for the indicated times. Western blot analysis showed that AMPK activation and increased pacc levels occurred only after prolonged hypoxia (18 hours), whereas REDD1 expression and mtor substrate dephosphorylation, as judged by ps6k and ps6 levels, were relatively early events. Total S6 served as loading control. (B) HN13 serum-depleted cells pretreated with compound C, a potent AMPK inhibitor (10 μm; 30 minutes), or vehicle control and then cultured under normoxic or hypoxic conditions in the presence or absence of EGF (100 ng/ml) for 18 hours. Note that REDD1 expression under hypoxia was completely blunted in the absence of AMPK activation, thus sustaining mtor-activating status. α-tubulin was used as loading control. (C, D) HN13 cells were either left untransfected ( ) or transfected with a control (C), AMPKα1/α2 or REDD1 sirna. Forty-eight hours after transfections, serum-deprived cells were exposed to normoxia or hypoxia for 18 hours. Western blot analyses show that REDD1 expression is dependent on the activation status of AMPK (C) but that the activation of the AMPK pathway occurred in a REDD1-independent fashion (D). GAPDH was used as loading control. of REDD1 expression in response to prolonged hypoxia resulting in hyperphosphorylated levels of S6 similar to the one observed under basal normoxic conditions (Figure 5B). Similar results were obtained when both α1 and α2 catalytic subunits of AMPK were depleted by RNA interference in cells exposed to hypoxia (Figure 5C). These findings pointed to the possibility that in our system, AMPK activation, because of an energy-depleting response triggered by prolonged hypoxia, is required to fully support REDD1 expression and function to suppress mtor activity in HNSCC cells. We also show evidence, in agreement with previous studies [19,20], that in fact REDD1 expression in response to hypoxia is not required for AMPK activation as measured by the increased phosphorylated status of AMPK and ACC in REDD1-depleted HNSCC cells (Figure 5D). Thus, in HNSCC cells, AMPK activation after hypoxia-induced energy stress seems to control an upstream signaling mechanism to promote the essential inhibitory role of REDD1 on mtor activity. These observations indicate that, in HNSCC cells under hypoxic stress, REDD1 activation is responsible for mediating the inhibitory mechanisms on the mtor pathway through AMPK-independent and -dependent pathways. In response to energy stress induced by prolonged hypoxia, AMPK activity seems to be critical in controlling REDD1 expression through a novel yet to be elucidated mechanism (Figure 6). This activation eventually triggers a response mediated by the TSC complex to inhibit mtor activation and, as a consequence, prevents the phosphorylation of its downstream targets 4EBP1 and S6K. It is then plausible that within hypoxic foci in human HNSCC, the AMPK energy-sensing apparatus and REDD1 may be linked in the control of mtor activity. Therefore, any disturbance in the proper function of these key components, as exemplified by our findings in HN6 cells, may drive the persistent hyperactivation of the mtor pathway in HNSCC and explain in part the tolerance to the inhibitory actions of hypoxic stress on this pathway. Discussion Significant progress has been achieved in recent years to identify key molecular signatures involved in the pathogenesis of human HNSCC [30]. These remarkable efforts have certainly provided a better understanding of how deregulated signaling pathways involving oncogenic and tumor-suppressive protein products contribute to

7 Neoplasia Vol. 10, No. 11, 2008 mtor Pathway Inhibition via AMPK/REDD1 in HNSCC Schneider et al Figure 6. Proposed model for the effects of hypoxia-induced energy stress on the mtor pathway in HNSCC cells. In response to ATP depletion associated with prolonged hypoxia, REDD1 plays a key role integrating yet to be elucidated signals that depend on the activation of the AMPK energy-sensing apparatus to promote mtor substrate dephosphorylation in a TSC-dependent fashion. In addition, REDD1 acts as a critical negative regulator of mtor activity in response to acute hypoxia independently on the cellular energy status. HNSCC development and progression. More importantly, these advances offer exciting opportunities to implement in the clinical setting novel molecularly targeted anticancer therapies [31]. The epidermal growth factor receptor (EGFR)/Akt/mTOR pathway has received considerable attention owing to its frequent overactivation in a large number of HNSCC patients and their derived cell lines [13,14,16,32]. For instance, the mtor downstream effector eif4e seems to be overexpressed and functionally active in the surgical margins of HNSCC patients and has been regarded as an independent risk factor for recurrent disease [16]. Tissue microarray technology has also provided compelling evidence on the widespread activation status of the mtor pathway as judged by an increase in ps6 in hundreds of human HNSCC specimens [15,33]. Of interest, in a large subset of cases, mtor activation occurs independently of the activation status of the EGFR. Aberrantly activated EGFR often transduces signals through the Akt/mTOR pathway in several cancer types. In particular, overexpressed, hyperactive, or mutated EGFRs have been frequently associated with the development and progression of HNSCC [32,34 36]. However, it seems that, in a significant fraction of patients, mtor activation takes place bypassing EGFRdependent growth-stimulatory pathways. These emerging results present a critical challenge to further explore alternative routes that may contribute to the hyperactivation of this pathway in HNSCC. Among multiple intracellular signaling cascades, the mtor pathway positions itself as an intricate network of signaling molecules that respond to a diversity of environmental and nutritional cues to coordinate appropriate cell growth and proliferative responses [37]. As a consequence, cellular anabolic metabolism is tightly regulated by the surrounding microenvironment where oxygen availability plays a profound effect in part by efficiently supporting mtor pathway dependent protein synthesis and mass accumulation [9 11]. In the case of solid tumors and HNSCC, in particular, hypoxia is a common microenvironmental stress playing a significant role in promoting treatment resistance and poor patient survival [32,38,39]. If we draw a parallel between what actually occurs in human tumors, it seems likely to hypothesize that in a fraction of tumors with aberrantly high mtor pathway dependency, the inhibitory mechanisms responsible for down-regulating mtor activity in response to hypoxia are not functioning properly. Our observations confirmed previously published genetic and biochemical studies reporting that, in response to hypoxia, REDD1 is a key component within a pathway critical for human tumor suppression [18 20,24]. However, in contrast to what it has been reported by these groups in MEFs or HEK293 cells, we observed that, in HNSCC cells, REDD1 expression is associated with the activation of AMPK after hypoxia-induced energy stress, supporting the notion that in HNSCC cells, prolonged hypoxia generates an energydepleting response orchestrated by the AMPK signaling apparatus. Hypoxia-induced energy stress has been recently linked to mrna translation and cell growth in part by impinging on the mtor pathway [11]. These effects seem to be mediated by the activation of AMPK and TSC2 in an HIF-independent fashion. For example, under serum-depleted conditions, Liu et al. observed AMPK activation in HEK293 cells within minutes and up to 20 hours. This was not the case in our system; on the contrary, we were able to detect AMPK activation only after prolonged hypoxic stress (18 hours). Although REDD1 expression and function was not assessed in the study conducted by Liu et al., our data are similar in that the effects of prolonged hypoxia on mtor inhibition can occur through AMPK activation after ATP depletion and independently of HIF-1 accumulation. Our results, however, indicate that under prolonged hypoxia, AMPK activation is necessary to inhibit mtor as long as REDD1 is intact, with the assumption that the TSC complex is functional, because AMPK activation alone did not result in reduced mtor activity. We suggest the possibility that the discrepancy observed with other studies on the dependency of active AMPK on REDD1 function may be related to the timing of hypoxic exposure as well as the origin of the cellular system being investigated [18]. The biochemical mechanism by which the TSC complex and REDD1 interact to inhibit mtor function has remained elusive. In a recent study, DeYoung et al. [24], provided compelling evidence that the mtor inhibitory mechanisms triggered by hypoxia are in part mediated by the interaction of TSC2 with proteins. By using genetic and biochemical approaches, they show that in the presence of growth factor stimulated conditions, proteins associate with TSC2 after the activationofthepi3k/aktsignaling pathway. Under hypoxic conditions, however, REDD1 specifically prevents PI3K/Akt induced TSC2/ association by binding , which leads to the release of TSC2 from this complex and promotes mtor substrate dephosphorylation. Indeed, this novel mechanism may in part explain the resistance to hypoxia-induced mtor inhibition observed in HN6 cells, a HNSCC cell line with constitutively and robust Akt activation [13]. Emerging studies also reveal REDD1-independent mechanisms for triggering mtor inhibition in response to hypoxia. It has been recently identified that by

8 1302 mtor Pathway Inhibition via AMPK/REDD1 in HNSCC Schneider et al. Neoplasia Vol. 10, No. 11, 2008 acting directly on Rheb, which is the direct downstream target of TSC2 and upstream mtor activator, the hypoxia-inducible Bnip3, a Bcl-2 homology 3 domain-containing protein, can prevent mtor activation by Rheb [40]. Further studies are definitely warranted in general but especially in the context of HNSCC cancer biology where the interplay between hypoxia and the mtor pathway is a common element affecting the progression of the disease. In this regard, the elucidation of the basic mechanisms affecting this network of signaling molecules will offer essential information to better understand and support the pharmacological mechanism-based application of mtor inhibitors, alone or in combination with other targeted therapies as well as conventional treatments to halt the development and progression of HNSCC. Acknowledgments The authors thank Xin Wang for technical assistance. 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