Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Oncogene (2014), 1–11 © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Combined regulation of mTORC1 and lysosomal acidification by GSK-3 suppresses autophagy and contributes to cancer cell growth I Azoulay-Alfaguter1, R Elya1, L Avrahami1, A Katz and H Eldar-Finkelman There is controversy over the role of glycogen synthase kinase-3 (GSK-3) in cancer progression. Recent work has implicated GSK-3 in the regulation of mammalian target of rapamycin (mTOR), a known player in malignant transformation. Autophagy, a selfdegradation pathway, is inhibited by mTOR and is tightly associated with cell survival and tumor growth. Here we show that GSK-3 suppresses autophagy via mTOR complex-1 (mTORC1) and lysosomal regulation. We show that overexpression of GSK-3 isoforms (GSK-3α and GSK-3β) activated mTORC1 and suppressed autophagy in MCF-7 human breast cancer cells as indicated by reduced beclin-1 levels and upregulation of sequestosome 1 (p62/SQSTM1). Further, overexpression of GSK-3 increased the number of autophagosomes and inhibited autophagic flux. This activity was directly related to reduced lysosomal acidification triggered by GSK-3 (in which GSK-3β has a stronger impact). We found that lysosomal acidification is reduced in MCF-7 cells that also exhibit increased levels of autophagosomes and p62/SQSTM1 and increased activity of mTORC1. Subsequently, treating cells with GSK-3 inhibitors restored lysosomal acidification, enhanced autophagic flux and inhibited mTORC1. Furthermore, GSK-3 inhibitors inhibited cell proliferation. We provide evidence that GSK3-mediated mTORC1 activity and GSK-3-mediated lysosomal acidification occur via distinct pathways, yet both mTORC1 and lysosomes control cell growth. Finally, we show that GSK-3-reduced lysosomal acidification inhibits endocytic clearance as demonstrated by reduced endocytic degradation of the epidermal growth factor receptor. Taken together, our study places GSK-3 as a key regulator coordinating cellular homeostasis. GSK-3 inhibitors may be useful in targeting mTORC1 and lysosomal acidification for cancer therapy. Oncogene advance online publication, 15 December 2014; doi:10.1038/onc.2014.390 INTRODUCTION The serine threonine glycogen synthase kinase-3 (GSK-3) is a multi-functional enzyme.1–5 In mammals, GSK-3 is expressed as two isozymes, GSK-3α and GSK-3β.6 GSK-3 has emerged as a potential therapeutic target for treatment of various diseases including diabetes, Alzheimer’s disease and affective disorders.2,5,7,8 The role of GSK-3 in cancer and tumor progression is still under debate.9–12 The initial link of GSK-3 with cancer was based on its involvement in the canonical Wnt/β-catenin signaling pathway; GSK-3 targets β-catenin for proteosomal degradation.13–15 Inhibition of GSK-3 has been thus considered a risk for cancer. Nevertheless, growing evidence supports the idea that GSK-3 enhances cancer cell proliferation. Treatment with GSK-3 inhibitors reduces cell proliferation and inhibits tumor growth in models of prostate, colon, pancreatic and ovarian cancers and glioblastoma and leukemia.16–19 The ability of GSK-3 to modulate pro-inflammatory and anti-apoptotic processes involving nuclear factor-κB,20–22 the tumor suppressor protein p5323 and cyclin D1 levels24 also suggests that GSK-3 promotes cell growth. In previous work, we and others identified mammalian target of rapamycin (mTOR) as a GSK-3 target.25–28 mTOR is a nutrient sensor that controls protein synthesis, cell size and cell growth.29–31 mTOR is found in two complexes, mTOR complex-1 (mTORC1) and mTORC2, in which it associates with raptor and rictor, respectively.32 Signaling through mTORC1 is involved in cell proliferation and tumor progression,29–31,33 and rapamycin and its analogs, which inhibit mTORC1 function, are used in cancer therapy.34–36 mTORC1 is a major suppressor of macroautophagy (herein referred to as autophagy), a highly regulated process responsible for intracellular degradation. The autophagosome sequesters cellular materials and subsequently fuses with a lysosome, which facilitates cargo degradation.37–40 Aberrant autophagic activity has a prominent impact on cell survival and tumor growth. Autophagy may be required for tumor suppression as it preserves cellular homeostasis and limits genome damage.41–46 On the other hand, autophagy may accelerate cancer at later stages by providing nutrients under stressed or starved conditions.41–46 Hence, maintaining ‘normal’ cell proliferation requires ‘correct’ balance between biosynthesis and autophagy. Whether GSK-3 maintains this balance via regulation of mTORC1 and autophagy is unclear. In the present study, we show that GSK-3 activates mTORC1 and suppresses autophagy in breast cancer cells. We further identify a new role for GSK-3 in reducing lysosomal acidification that in turn suppresses autophagic flux and inhibits endocytic clearance. Our study places GSK-3 as a key player coordinating cellular homeostasis and identified new mechanisms linking GSK-3 with cancer cells growth. Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. Correspondence: Professor H Eldar-Finkelman, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel. E-mail: [email protected] 1 These authors contributed equally to this work. Received 31 August 2014; revised 10 October 2014; accepted 16 October 2014 GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al RESULTS GSK-3 is a positive regulator of mTORC1 We first examined whether GSK-3 regulates mTORC1 activity in cancer cells. We used human breast adenocarcinoma MCF-7 cells that show growth sensitivity to rapamycin (Figure 1a). MCF-7 cells were transiently transfected with green fluorescent protein (GFP)tagged GSK-3α or GSK-3β constructs. (GFP-tagged constructs were used because GPF tags make cell imaging experiments possible, similar results were obtained with non-tagged GSK-3.) mTOR activity was determined by evaluating the phosphorylation levels of the mTOR target p70 ribosomal S6 kinase (S6K-1, Thr389) and its target ribosomal S6 protein (S6, Ser240/244). Phosphorylation levels a of S6K-1 significantly increased and those of S6 showed a clear but modest increase in cells that overexpressed GFP-GSK-3α or GFPGSK-3β (Figure 1b). To determine which of the mTOR complexes were regulated by GSK-3, similar experiments were performed in the presence of rapamycin. Rapamycin completely abolished GSK-3-mediated phosphorylation of mTOR targets indicating that GSK-3 activates mTORC1 (Figure 1c). GSK-3 elevated mTORC1 activity even under starvation conditions such as low serum or no glucose (Figure 1d). It was difficult to assess GSK-3 activation of mTORC1 in the absence of amino acids because the signal was very weak (Figure 1d). The TSC1 and TSC2 complexes that mediate mTORC1 activity mainly respond to stimulation by growth factors b pS6K1 1.5 Fold of Ctrl Cell growth c NT Rap 2.0 S6K1 1.0 pS6 24 48 Time (Hr) S6 72 GSK3α Ctrl GSK3β pS6K1/S6K1 * * 2.0 2.0 1.5 1.5 1.0 1.0 GSK-3GFP 0.5 0.5 GSK-3α/β 0.0 0.5 Ctrl α β pS6/S6 pS6K1 * 0.0 Ctrl S6K1 * α β β-actin GFP GSK-3 Rap - + - + - + GSK-3α/β β-actin d e f pS6K1 3.0 2.0 1.0 0.0 GSK3α GSK3β 3.0 1.00 1.00 0.75 * 0.50 * 0.25 Fold of NT S6K1 -aa -Glucose Fold of Ctrl 0.1% FCS pS6K1/S6K1 pS6K1/S6K1 pS6K1 S6K1 ps6k1/s6k1 0.75 0.50 * * * * 0.25 0.0 0.00 2.0 Ctrl siα siβ β-actin 1.0 0.0 pS6/S6 h 40 pS6K1 S6K1 1.00 NT SB216783 AR-A01148 L803-mts CT99012 30 Fold of NT g Cells x 106 2 20 0.75 0.50 * * * * 0.25 0.0 10 0 β-actin TCS+/+ 24 48 Time (Hr) 72 TCS-/- Figure 1. GSK-3 regulates mTORC1 and cell growth. (a) MCF-7 cell growth is sensitive to rapamycin: MCF-7 cells were treated with 50 nM rapamycin (Rap) for 3 days and were counted every 24 h in triplicate samples as described in Materials and methods. (b) GSK-3 activates mTOR targets: MCF-7 cells were transiently transfected with GFP-GSK-3α or GFP-GSK-3β constructs. Control cells (Ctrl) were transfected with a construct encoding only GFP. Phosphorylation and total expression levels of S6K-1 (Thr389) and S6 (Ser240/244) were determined by immunoblot analyses. Levels of endogenous total GSK-3 or GFP-GSK-3 proteins were determined by staining with an anti-GSK-3 antibody. β-Actin was analyzed to ensure equal loading. (c) GSK-3 activates mTORC1: cells were treated and analyzed as describe in b, except that cells were treated with rapamycin (50 nM, 4 h). (d) GSK-3 rescues mTORC1 activity under starvation conditions: cells were treated and analyzed as describe in b, except that cells expressing GFP-GSK-3 were incubated with low serum (0.1% fetal calf serum), medium lacking glucose or medium lacking amino acids (aa) for 4 h. (e) Inhibition of expression of GSK-3 isozymes inhibits mTORC1: MCF-7 cells were transfected with siRNA targeting GSK-3α or GSK-3β. Control cells (Ctrl) were transfected with appropriate control siRNA. Levels of endogenous GSK-3 and β-actin are shown. (f) GSK-3 inhibitors inhibit mTORC1 activity: MCF-7 cells were treated with SB-216763 (SB, 10 μM), AR-A014418 (AR, 20 μM), CT99012 (CT, 10 μM) and L803-mts (L803, 40 μM) for 4 h. (g) GSK-3 activates mTORC1 via the TSC1/2 complex: ‘wild-type’ MEF or MEF-TSC− / − cells were treated with GSK-3 inhibitors as described in f. (h) GSK-3 inhibitors inhibit cell growth: MCF-7 cells were treated with SB-216763, ARA014418, CT99012 and L803-mts (as described in f for 3 days). Cells were counted every 24 h in triplicate samples. For all panels, representative gels are shown. Calculated ratios of indicated phosphorylated to total protein were evaluated by densitometry analysis and are shown in bar graphs that present means of three to five independent experiments ± s.e.m., *P o0.05. NT, non-treated. Oncogene (2014), 1 – 11 © 2014 Macmillan Publishers Limited GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al or glucose, but not to amino acids;47–50 therefore, these data suggest that GSK-3 mediates mTORC1 activity (at least in part) via the TSC1/2 complex. We next determined whether inhibition of GSK-3 produces an effect opposite to that of overexpression. Inhibition of either GSK-3α or GSK-3β expression with small interfering RNA (siRNA) reduced S6K-1 phosphorylation (Figure 1e). There was a small reduction in the phosphorylation of S6 that was not statistically significant (data not shown). It is possible that ablation of the expression of one isozyme alone was not sufficient to inhibit the phosphorylation of S6. Activity of both isozymes was then reduced by treating cells with selective GSK-3 inhibitors: the ATPcompetitive small-molecule inhibitors SB-216763, ARA0114418 and CT99012 and a cell-permeable peptide substrate competitive inhibitor L803-mts.26 The three GSK-3 inhibitors reduced the phosphorylation levels of S6K-1 and S6 (Figure 1f). To further examine whether GSK-3 mediates this effect via TSC1/2, we used MEF cells lacking TSC1/2 that express constitutively active mTORC1 (MEF-TSC− / −).51 mTORC1 activity was inhibited by GSK-3 inhibitors in ‘wild-type’ MEF-TSC+/+ cells, whereas no inhibition was observed in MEF-TSC− / − cells (Figure 1g). These results supported our notion that GSK-3 mediates mTORC1 activity via the TSC1/2 complex. Finally, MCF-7 cells were treated with SB-216763, ARA0114418, CT99012 or L803-mts and cell proliferation was determined. Treatment of cells with each of these GSK-3 inhibitors inhibited cell proliferation by about 50% as compared with non-treated cells (Figure 1h). Together, our data show that GSK-3 is a positive regulator of mTORC1, and this ability correlates with regulation of cell growth. GSK-3 inhibits autophagy and triggers accumulation of autophagosomes As mTORC1 suppresses autophagy, we asked whether GSK-3 regulates autophagy. Autophagy is initiated by de novo formation of double-membrane vesicles called autophagosomes that sequester intracellular waste.37–39 Fusion of autophagosomes with lysosomes facilitates cargo degradation.37–39 We examined levels of several markers known to correlate with the number of autophagosomes and with autophagic activity. Microtubule-associated protein 1 light chain 3-II (LC3-II) is a lipidated form of LC3-I that is primarily associated with phagophore (that is, autophagosome precursor) and autophagosome membranes.52,53 LC3-II levels thus serve as an index for the number of autophagosomes. Levels of LC3-II significantly increased in cells that overexpressed GFPGSK-3α or GFP-GSK-3β (Figure 2a, upper panel). To further confirm the formation of autophagic vesicles, cells were co-transfected with GFP-GSK-3 (green) and RFP-LC3 (red) plasmids. Consistently, GSK-3α or GSK-3β increased the number of LC3-II puncta per cell (Figure 2b, lower panel). Thus, unexpectedly, we found that GSK-3 increases the amount of autophagosomes. To further analyze autophagy, we determined levels of p62/SQSTM1, an autophagy receptor protein that interacts with ubiquitinated proteins and LC3-II.54 p62/SQSTM1 (sequestosome 1) levels thus increase on autophagy inhibition, whereas reduced p62/SQSTM1 levels indicate increased autophagic response and flux.54 Levels of p62/SQSTM11 were significantly increased in cells overexpressing GFP-GSK-3α or GFP-GSK-3β (Figure 2c, upper panel). Consistent with this, cells that were cotransfected with Cherry-GSK-3α or β (red) and GFP-p62/SQSTM1 (green) showed an increase in p62/SQSTM1 puncta that were brighter and larger than those in control cells (Figure 2d, lower panel). GSK-3β consistently gave a more robust effect on accumulation of p62/SQSTM1 puncta than did GSK-3α. To further examine alterations in autophagy, levels of beclin-1, a key factor that initiates formation of autophagosomes after suppression of mTORC1,55 was determined. Overexpression of GFP-GSK-3α or GFP-GSK-3β reduced beclin-1 levels further © 2014 Macmillan Publishers Limited 3 indicating suppression autophagy, likely via activation of mTORC1 by GSK-3 (Figure 2e). The accumulation in LC3-II/autophagosomes (Figures 2a and b) could not result from activation of mTORC1 (as mTORC1 inhibits the formation of autophagosomes). Hence, elevation in LC3-II likely reflected inhibition in autophagosomes’ turnover due to defects in lysosomes. To further verify this possibility, cells overexpressing GSK-3 were treated with chloroquine (CQ), a reagent that neutralize lysosomal pH. Although CQ increased levels of LC3-II (as expected), overexpression of either GSK-3 isozyme did not further elevate levels of LC3-II in the presence of CQ, indicating that GSK-3/ lysosome axis was responsible for the reduced flux (Figure 2f; detailed analysis of GSK-3-lysosomes is described in Figure 4). We then examined the role of mTORC1 in inhibiting autophagic flux by manipulating p62/SQSTM1 with rapamycin. Treatment with rapamycin largely blocked GSK-3 elevation of p62/SQSTM1 (Figure 2g). Taken together, GSK-3 suppresses autophagy and flux via mTORC1 and lysosomes. In addition, it is noteworthy that suppression of autophagy and upregulation of p62/SQSTM1 are important players in cancer progression,56,57 further supporting the link between GSK-3 and cancer. Autophagy and lysosomal acidification are suppressedH in MCF-7 cells The role of GSK-3 in inhibiting autophagic flux was an intriguing finding and hinted at a mechanism controlling lysosomal activity. Lysosomes are acidic organelles and their ‘correct’ acidification (pHo 4.5) is critical for proteolytic activity.58–60 Lysosome acidification can be visualized in live cells by staining of acidic organelles with LysoTracker Red dye. The LysoTracker Red signal in MCF-7 cells was very weak compared with the signal in the ‘non-cancerous’ mammary epithelial MCF-10A cells (Figure 3a). (Treatment with CQ reduced the fluorescence of the red vesicles and confirmed dye specificity26 (data not shown).) In addition, LC3-II and p62/SQSTM1 expression levels were significantly higher in MCF-7 cells than in MCF-10A cells (Figure 3b). The lysosomal hydrolase cathepsin D (CatD) serves as a marker for lysosomal activity. CatD is proteolyzed in acidified lysosomes to produce an active mature fragment (mCatD). Consistently, mCatD levels were significantly lower in MCF-7 cells than in MCF-10A cells, verifying reduced lysosomal acidification in MCF-7 cells (Figure 3b). Lamp2, a marker for lysosomes, was abundant in both cell lines (Figure 3b). Finally, MCF-7 cells showed significantly higher levels of mTORC1 activity than did MCF-10A cells (Figure 3c). Interestingly, overexpression of GSK-3 had a similar impact on mTORC1 and autophagy in the non-cancer-derived MCF-10A cells as in the MCF-7 cells. In MCF-10A cells, overexpression of GSK-3α or GSK-3β increased mTORC1 activity and reduced beclin-1 levels (Figure 3d). Overexpression of GSK-3 also increased levels of LC3-II and p62/SQSTM1 and reduced levels of mCatD (Figure 3e). Taken together, these data show that MCF-7 cells are deficient of lysosomal and autophagic activity. In addition, we conclude that GSK-3 impacts on mTORC1 and autophagy is a likely a broader phenomenon that is not restricted to MCF-7 cells. GSK-3 regulates lysosomal acidification and reduces autophagic flux The results presented in Figure 2 suggested that GSK-3 regulates lysosomal acidification. To further explore this possibility, MCF-7 cells were transfected with GFP-GSK-3α or- GSK-3β and then stained with LysoTracker Red to evaluate the acidic pool of lysosomes. The experiments were performed in starved cells (0.1% serum), as these conditions enabled a better detection of acidified lysosomes (which are present at low levels in MCF-7 cells as indicated). The numbers of lysosomal puncta and intensities were markedly reduced in cells overexpressing GSK-3β compared with control cells (Figure 4a). The effect of GSK-3α was less pronounced than was the effect of GSK-3β: only in part of the cell population, Oncogene (2014), 1 – 11 GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al 4 GFP GSK-3 GSK-3α/β LC3- II/LC3 * p62/SQTM1 * p62/ SQTM1 1.0 GFP 0.5 GSK-3 GSK-3α/β 0.0 * * 2.0 1.5 1.0 0.5 0.0 Ctrl α β β-actin β-actin GFP Fold of Ctrl LC3-I LC3-II Fold of Ctrl 1.5 LC3 Merge Zoom Cherry p62/SQSTM1 Merge Ctrl Ctrl GSK3α GSK3α GSK3 10µm GSK3 5µm α Ctrl GFP GSK-3 GSK-3α/β * 0.75 LC3-I LC3-II * GFP 0.5 GSK-3 GSK-3α/β GFP GSK-3 GSK-3α/β 0.25 0.0 p62/ SQSTM1 Ctrl α β Rap + + 1.5 1.0 0.5 β-actin β-actin CQ - β 2.0 Fold of Ctrl Beclin1 Fold of Ctrl Beclin1 1.0 + - + - + 0.0 Rap - + - + - + + Figure 2. GSK-3 suppresses autophagy and triggers accumulation of autophagosomes. (a) GSK-3 overexpression increases levels of LC3-II: MCF-7 cells were transiently transfected with GFP-GSK-3α or GFP-GSK-3β constructs. Control cells (Ctrl) were transfected with a construct encoding only GFP. LC3-I and LC3-II levels were determined by immunoblot analysis. Calculated ratios of LC3-II/total LC3 (total LC3 = LC3-I +LC3-II) are shown in the right panel. Levels of endogenous and GFP-GSK-3 proteins are also shown. β-Actin was analyzed as a control for equal loading. (b) GSK-3 upregulates numbers of autophagosomes per cell. MCF-7 cells were co-transfected with RFP-LC3 (red) and GFPGSK-3α or GFP-GSK-3β (green) plasmids. Control cells (GFP) were transfected with a construct encoding GFP only. Live cells were imaged by confocal microscopy. Arrows indicate representative cells expressing GSK-3 (green) and RFP-LC3 (red). Zoom of the LC3-II signal in autophagosomes is indicated by rectangle and yellow arrows (c). GSK-3 elevates p62/SQSTM1 levels: cells were treated as in a, except that p62/SQSTM1 levels were determined by immunoblot analysis. (d) GSK-3 expression increases numbers of p62/SQSTM1 aggregates: MCF-7 cells were co-transfected with GFP-p62/SQSTM1 and Cherry-GSK-3α or Cherry-GSK-3β expression plasmids. Control cells (Cherry) were transfected with a construct encoding only Cherry. Live cells were imaged by confocal microscopy. Arrows indicate representative cells expressing GSK-3 (red) and GFP-p62/SQSTM1 (green). (e) GSK-3 expression reduces beclin-1 levels: cells were treated as in a, except that cell lysates were blotted for beclin-1. (f) GSK-3 upregulates LC3-II via lysosomes: MCF-7 cells were transiently transfected with GSK-3 plasmids as described in a. Cells were treated with CQ and levels of LC3-II were determined. No statistical differences were obtained in LC3-II levels in cells expressing GSK-3 that were treated or non-treated with CQ. (g) GSK-3 inhibits autophagy via mTORC1: MCF-7 cells were transiently transfected with GSK-3 plasmids as described in a. Cells were treated or not treated with rapamycin (50 nM, 4 h) and levels of p62/SQSTM1 were determined. For all panels, representative gels are shown, and bar graphs present means of three to five independent experiments ± s.e.m., *Po0.05. we observed a reduction in numbers of puncta relative to control cells (Figure 4a). It is possible that the effect of GSK-3α on lysosomes is dependent on additional cellular factors that are not known at this point. As MCF-7 cells showed deficits in acidic lysosomes, we examined whether inhibition of GSK-3 activity would restore this deficiency. The cells were treated with SB-216763, ARA0114418 or L803-mts and then stained with LysoTracker Red. Treatment with GSK-3 inhibitors increased the pool of acidified lysosomes (Figure 4b). Immunofluorescence analysis in fixed cells stained with anti-Lamp2 antibody indicated that GSK-3 inhibitors did not alter levels of Lamp2 (Figure 4c). Consistent results were obtained Oncogene (2014), 1 – 11 in western blot (Figure 4d). Thus, GSK-3 inhibitors did not increase the number of lysosomes, but rather altered acidification. We expected that improved lysosomal acidification achieved with GSK-3 inhibitors would enhance autophagic flux. It was difficult to assess autophagic flux by measuring LC3-II because inhibition in mTORC1 increases LC3-II levels, although activation of lysosomes enhances LC3-II degradation resulting in a ‘zero’ net effect. We thus used p62/SQSTM1 levels as a marker for flux: inhibition in mTORC1 activates autophagy and enhances p62/ SQSTM1 degradation and activated lysosomes accelerates autophagosmses’ turnover resulting in p62/SQSTM1 degradation as well. Changes in p62/SQSTM1 were determined by immuno© 2014 Macmillan Publishers Limited GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al 5 a MCF-7 b MCF-10A p62/SQSTM1 LC3-II Fold of MCF-7 LC3-I Lys LC3-II P62/ SQSTM1 Lamp2 1.0 1.0 0.75 0.50 0.75 * mCat D * 1.5 * 1.0 0.50 0.25 0.25 0.5 0.0 0.0 0.0 BF Cat D mCat D β-actin MCF-7 MCF-10A e pS6K1 S6K1 S6K1 Beclin1 pS6 4.0 LC3-I LC3-II 3.0 p62/ SQTM 2.0 1.0 S6 GSK-3 GSK-3α/β 0.0 β-actin β-actin 1.0 GFP pS6K1/S6K1 * * Cat D Ctrl α β * * Fold of Ctrl MCF-10A 0.75 GSK-3GFP GSK-3α/β 0.25 β-actin 0.0 Ctrl α β * 2.0 1.0 0.5 0.0 0.5 α p62/SQSTM1 * * 1.5 1.0 Ctrl β 0.0 Ctrl α β mCatD Lamp2 0.50 * 1.5 mCat D Beclin1 MCF-7 MCF-10A LC3-II/LC3 2.0 1.0 Fold of Ctrl pS6K1 Fold of Ctrl d Fold of Ctrl c * 0.75 * 0.50 0.25 0.0 Ctrl α β MCF-10A Figure 3. Autophagy and lysosomal acidification are suppressed in MCF-7 cells. (a) Lysosomal acidification is reduced in MCF-7 cells: MCF-7 cells and MCF-10A cells were stained with LysoTracker Red (Lys) and live cells were imaged by confocal microscopy. White arrows indicate acidified lysosomal puncta (upper panel). Lower panel shows merged image of bright field with LysoTracker Red (BF). (b) Comparison between autophagy and lysosomes in MCF-7 and MCF-10A cells: expression levels of LC3-I and LC3-II, p62/SQSTM1, mCatD and Lamp2 were determined in MCF-7 cells and in MCF-10A cells by immunoblot analysis. Bar graphs show densitometry analysis of indicated proteins expressed in both cell lines. (c) mTORC1 activity is higher in MCF-7 cells: cells were treated as described in b, except that S6K-1 and S6 were analyzed by immunoblot analysis. (d) GSK-3 activates mTORC1 and inhibits autophagy in MCF-10A cells: MCF-10A cells were transiently transfected with GFP-GSK-3α or GFP-GSK-3β constructs. Control cells (Ctrl) were transfected with a construct encoding GFP. Phosphorylation and total expression levels of S6K-1 (Thr389) and beclin-1 were determined by immunoblot analyses. Expression levels of endogenous GSK-3 and GFP-GSK-3 proteins are also shown. β-Actin was analyzed as a control for equal loading (e). GSK-3 impacts autophagy and lysosomes in MCF-10A cells: cells were treated as in d, except that levels of LC3-II, p62/SQSTM1, CatD, mCatD and Lamp2 were determined. Bar graphs show densitometry analyses of indicated proteins. For all panels, representative gels or images are shown, and densitometry analyses bar graphs of indicated proteins present means of three to five independent experiments ± s.e.m., *P o0.05. fluorescence analyses in cells treated with GSK-3 inhibitors and stained with p62/SQSTM1 antibody. These experiments showed that numbers of p62/SQSTM1 puncta were significantly reduced by the GSK-3 inhibitors (Figure 4e). Consistently, western blotting also showed reduced p62/SQSTM1 levels on treatment of cells with GSK-3 inhibitors (Figure 4f). Thus, GSK-3 inhibitors re-acidify lysosomes and enhance autophagic flux. As suppression in autophagy contributes to progression of early-stage cancers,41–45 GSK-3 inhibitors may be useful in treating these conditions. GSK-3 controls mTORC1 and lysosomes via distinct mechanisms that both act to regulate cell growth To complete this part of our studies, we examined whether mTORC1 and lysosome pathways crosstalk61,62 via GSK-3 and whether both mTORC1 and lysosomes underlie the impact of GSK-3 inhibitors on cell growth. To determine whether GSK-3 activates mTORC1 via lysosomes, we treated MCF-7 cells with CQ. This treatment inhibited the phosphorylation of mTORC1 target, S6, indicating that dysfunctional lysosomes may inhibit mTORC1 (Figure 5a). However, CQ treatment did not prevent activation of mTORC1 by GSK-3α and GSK-3β, indicating that GSK-3 activates mTORC1 independently of lysosomal function (Figure 5a). To examine whether GSK-3 regulation of lysosomes occurs through © 2014 Macmillan Publishers Limited mTORC1, cells were treated with rapamycin and tested for lysosomal acidification. Rapamycin slightly enhanced lysosomal acidification; however, the effect of rapamycin was much weaker than that observed with L803-mts (Figure 5b) or other GSK-3 inhibitors (data not shown). From these results we conclude that GSK-3/mTORC1 and GSK-3/ lysosomes axes represent independent processes. Inhibition of growth of MCF-7 cells when treated with GSK-3 inhibitors correlated well with the abilities of these compounds to inhibit mTORC1, the results with rapamycin and previous data indicating that mTORC1 promote cell growth (see Figure 1 and Laplante and Sabatini,29 Cornu et al.,30 Proud31 and Mamane et al.33). As GSK-3 inhibitors restored lysosomal acidification independently of mTORC1 as discussed above, we asked whether lysosomal acidification also manipulates cell growth. There are no known drugs that directly enhance lysosomal acidification. We thus screened for conditions that resulted in reacidification of lysosomes in MCF-7 cells. We found that starvation (either low serum or lack of amino acids) restored lysosomal acidification (Figure 5c). Starvation increased autophagic flux as judged by reduced levels of LC3-II and p62/SQSTM1 (Figure 5d). As expected, starvation inhibited mTORC1 (Figure 5d). The fact that both rapamycin and starvation inhibited mTORC1, although only starvation showed a strong impact on lysosomal acidification, Oncogene (2014), 1 – 11 GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al 6 GSK3α Lys GSK3 Lyso GFP Ctrl Lamp2 BF NT NT SB 216763 SB 216763 10µm L803mts L803mts Lamp2 β-actin NT SB AR L803mts AR A0114418 p62/ SQTM AR A0114418 p62/SQTM1 NT SB 216763 L803mts p62/ SQTM1 AR A0114418 BF β-actin NT SB AR L803mts 1.0 0.8 0.6 0.4 0.2 0.0 NT SB AR L803mts 10µm Figure 4. GSK-3 regulates lysosomal acidification and autophagic flux. (a) GSK-3 reduces lysosomal acidification: MCF-7 cells were transiently transfected with GFP-GSK-3α or GFP-GSK-3β constructs. Control cells (Ctrl) were transfected with a construct encoding GFP only. Cells were starved with 0.1% fetal calf serum (4 h) and stained with LysoTracker Red (Lys). Live cells were imaged by confocal microscopy. Arrows indicate representative cells expressing GFP-GSK-3 (upper panel) and lysosomal puncta (lower panel). (b) GSK-3 inhibitors restore lysosomal acidification: MCF-7 cells were treated with 10 μM SB-216763, 20 μM AR-A014418 or 40 μM L803-mts for 4 h. Cells were stained with LysoTracker Red (Lys). Left panel shows live cells imaged by confocal microscopy. Right panel shows merged image of bright field with LysoTracker Red (BF). (c) GSK-3 inhibitors do not alter Lamp2 expression: cells were treated as in b, except that cells were fixed with paraformaldehyde (PFA) and subjected to immunofluorescence analysis with anti-Lamp2 antibody. Images were taken by confocal microscopy. (d) Cells were treated as in c, except that Lamp2 levels were determined by immunoblot analysis. (e) GSK-3 inhibitors decrease p62/SQSTM1 levels: MCF-7 cells were treated with GSK-3 inhibitors as described in b. Cells were fixed with PFA and subjected to immunofluorescence analysis using anti-p62/ SQSTM1 antibody. Images were taken by confocal microscopy. (f) Cells were treated as in d, except that p62/SQSTM1 levels were determined by immunoblot analysis. For all panels, representative gels or images are shown and densitometry analyses bar graphs of indicated proteins present means of three to five independent experiments ± s.e.m. NT, non-treated. promoted us to compare growth rates of MCF-7 cells in the presence of rapamycin or starvation medium. Rapamycin reduced cell growth by about 50%, however, starvation had an even stronger impact on cell proliferation (Figure 5e). Hence, enhanced lysosomal acidification could be an additional player in inhibiting cell growth. (We acknowledge the possibility that additional mechanisms activated or inhibited by starvation may be responsible for this phenomenon.) Our results suggest that by inhibiting mTORC1 and ‘correcting’ lysosomal acidification GSK-3 inhibitors inhibit cell growth. GSK-3 impairs endocytic clearance As lysosomes are located at the crossroad of autophagic and endocytic degradation, it is possible that defects in lysosomal Oncogene (2014), 1 – 11 acidification impair endocytic clearance. Epidermal growth factor receptor (EGFR) undergoes endocytosis and degradation in lysosomes.63–65 We thus used EGFR as a marker for lysosomal function. To verify that EGFR clearance is mediated by lysosomes, MCF-10A cells (that express endogenous EGFR) were treated with CQ or leupeptin (which inhibits lysosomal proteases) and EGFR levels were measured. Both CQ and leupeptin increased levels of EGFR (Figure 6a). We then examined whether GSK-3 mediates EGFR endocytic degradation. MCF-10A cells transfected with GFPGSK-3 constructs were treated with EGF. EGFR downregulation was observed after 60–240 min treatment with EGF, but overexpression of GSK-3α or GSK-3β attenuated EGFR clearance (Figure 6b). Inhibition in EGFR clearance was accompanied with sustained activation of EGFR downstream targets ERK1 and ERK2 (Erks) as indicated by increased ERKs phosphorylation (Figure 6b). © 2014 Macmillan Publishers Limited GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al 7 Ctrl GSK3α b GSK3β pS6 NT Rap L803mts Rap +L803mts Lys a GSK-3GFP GSK-3α/β BF S6 β-actin - + - + - + c d Ctrl Strv e Ctrl Strv Lys Ctrl Rap Strv 2.0 LC3-I LC3-II p62/ SQSTM1 pS6K-1 Cell growth CQ 1.5 1.0 0.5 0.0 BF 24 S6K-1 48 72 Time (Hr) 96 β-actin Figure 5. GSK-3 regulates mTORC1 and lysosomes via distinct pathways and both act to regulate cell growth. (a) GSK-3 activates mTORC1 independent of lysosomes: MCF-7 cells were transiently transfected with GFP-GSK-3α or GFP-GSK-3β constructs. Cells were treated or nontreated with CQ and phosphorylation and total expression levels of S6 (Ser240/244) were determined by immunoblot analyses. Levels of endogenous total GSK-3 or GFP-GSK-3 proteins and β-actin are shown. No statistical differences were obtained in pS6/S6 in cells expressing GSK-3 that were treated or non-treated with CQ. (b) MCF-7 cells were treated with rapamycin L803-mts or combination of both reagents. Cells were stained with LysoTracker Red (Lys) and imaged in confocal microscopy (upper panel). Lower panel shows merged image of cells at bright field with LysoTracker Red (BF). (c) Starvation enhances lysosomal acidification: MCF-7 cells were incubated with starved medium (Strv, 0.1% fetal calf serum with no amino acids) for 4 h. Live cells were stained with LysoTracker Red (Lys) and imaged in confocal microscopy. (d) Cell lysates prepared from starved cells were subjected to immunoblot analysis using indicated antibodies. (e) Cell proliferation was determined in starved cells (Strv) or cells treated with rapamycin (Rap, 50 nM). Number of cells was determined by XTT cell proliferation assay. For all panels, representative gels or images are shown of three independent experiments. These results indicated that GSK-3 slows down EGFR endocytic clearance. Lysosomal acidification restored by GSK-3 inhibitors observed in MCF-7 cells could enhance endocytic degradation of EGFR. We used MCF-7 cells that were transfected with EGFR plasmid (these cells do not endogenously express EGFR). The expressed receptor was functional as indicated by EGF-induced tyrosine phosphorylation of EGFR and by activation of ERKs (Figure 6c). Treatment with the GSK-3 inhibitor L803-mts reduced ‘basal’ levels of EGFR, EGFR tyrosine phosphorylation and ERK phosphorylation (Figure 6d). To further verify that this reduction reflects EGFR endocytosis, we used MCF-7 cells that expressed GFP-EGFR, which were treated with EGF. At time 0, the EGFR signal was localized at the cell surface (Figure 6e). Over time in the presence of EGF, the receptor was internalized and gradually accumulated in endosomes as ‘green’ cytoplasmic aggregates that remained at 4 h after treatment with EGF (Figure 6e, upper panel). In the presence of the GSK-3 inhibitor L803-mts, the EGFR signal disappeared after 4 h treatment with EGF (Figure 6e, lower panel). To further verify that enhanced EGFR clearance was mediated by lysosomes, the cells were treated with CQ. Treatment with CQ elevated EGFR aggregates and prevented their degradation by L803-mts (Figure 6g). Thus, enhanced degradation of EGFR by L803-mts was mediated by lysosomes. DISCUSSION Here we show that GSK-3 networks with mTORC1, autophagy and lysosomes represent mechanisms in which GSK-3 coordinate © 2014 Macmillan Publishers Limited cellular homeostasis and regulate cell proliferation. We first determined that GSK-3 is a positive regulator of mTORC1. This pathway is known to promote cell proliferation and cancer development: elevated mTORC1 activity is detected in many types of cancers and its inhibition by the drug rapamycin provokes anticancer effects.66–68 Inhibition of cell proliferation observed with GSK-3 inhibitors is likely due to their ability to inhibit mTORC1, which mimics the anticancer activity of rapamycin;31,34,35 these data have important implications in breast cancer for which mTOR is considered a therapeutic target.69,70 Our studies are in agreement with previous work that positioned GSK-3 as an activator of the mTORC1 pathway.25,27 In contrast, results from other analyses indicate that GSK-3 inhibits mTORC1.71–73 One study revealed that a coordinated phosphorylation of TSC2 by GSK-3 and AMPK suppresses mTORC1 activity.71 A more recent study reported that GSK-3 interacts with AMPK and inhibits AMPK activity, thus preventing the suppressive effects of AMPK on mTORC1.74 Thus, the role of GSK-3 in regulation of mTORC1 may depend on cell type or cell context. This issue needs further clarification. We then broadened our focus to analyze the effect of GSK-3 on regulation of autophagy. We found that GSK-3 inhibits autophagy —as would be expected from the fact that GSK-3 activates mTORC1. However, we also observed an increase in the numbers of autophagosomes in cells that overexpressed GSK-3, suggesting inhibition in autophagic turnover due to a deficiency in lysosomal activity. Indeed, further experiments showed that GSK-3 impairs lysosomal acidification, which in turn reduces the turnover of autophagosomes. We further indicated that GSK-3 regulates Oncogene (2014), 1 – 11 GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al 8 Ctrl Leup GSK3α GSK3β EGF EGFR EGFR β-actin pERK1/2 - L803 mts + - + EGFR EGFR pEGFR MCF10A pEGFR pERK1/2 ERK MCF-7 β-actin GSK-3α/β MCF-7 β-actin 10 60 240 0 10 60 240 ** 0.5 0.0 L803 mts pERK1/2 ERK GSK-3GFP 0 1.0 + pERK2 ERK EGF (min) EGFR Fold of NT CQ Fold of NT NT 1.0 ** 0.5 0.0 L803 mts - + 0 10 60 240 MCF-10A L803mts L803mts+CQ CQ GFP NT NT BF +L803mts 10μm 10μm EGF 0 (min) 5’ 60’ 240’ MCF-7 EGF 240’ MCF-7 Figure 6. GSK-3 inhibits EGFR endocytic clearance. (a) Lysosomes regulate EGFR levels: MCF-10A cells were treated with 30 μM CQ or 40 μM leupeptin (Leup) for 4 h. EGFR levels were determined by western blot analysis. (b) GSK-3 attenuates downregulation of EGFR: MCF-10A cells were transiently transfected with GFP-GSK-3α or GFP-GSK-3β constructs. Control cells (Ctrl) were transfected with a GFP-coding construct. Cells were treated with 50 ng/ml EGF, and EGFR levels or levels of phosphorylated ERK1/2 (Thr202/Tyr204) were determined at each time point by western blot analysis. β-Actin levels were analyzed as a control equal loading. (c) Expression of EGFR in MCF-7 cells: cells were transiently transfected with EGFR and treated with EGF for 10 min. Increased tyrosine phosphorylation of EGFR and phosphorylated levels of ERK1/2 relative to control cells indicated that EGFR was functional in these cells. (d) GSK-3 inhibitor reduces EGFR levels. Cells were treated as in c, except that cells were treated with L803-mts (40 μM) for 4 h. Levels of EGFR and total and phosphorylated ERK1/2 were determined. (e) GSK-3 inhibitor accelerates EGFR endocytic clearance: MCF-7 cells were transiently transfected with of GFP-EGFR expression plasmid. Cells were treated or not with 40 μM L803-mts and then treated with 50 ng/ml EGF for indicated times. Cells were fixed with paraformaldehyde, and GFPEGFR signal was imaged by confocal microscopy. (f) As in e, except that cells were treated with L803-mts in the presence of CQ and imaged 4 h after treatment with EGF. For all panels, representative gels or images are shown, and densitometry analyses bar graphs of indicated proteins are means of three to five independent experiments ± s.e.m., **P o0.01. mTORC1 and lysosomes via independent pathways that culminate into suppressed autophagy/autophagic turnover. This paradigm is illustrated in Figure 7. Autophagy is considered a tumor-suppressive mechanism. Many human tumors are deficient in autophagy, and insufficient autophagy promotes tumorigenesis in mouse models.75–77 Autophagy constrains cancer development by inhibiting prooncogenic pathways such as oxidative stress, genome instability and improper protein clearance.37,38,44,45,78 Our results are thus in agreement with the paradigm that GSK-3 promotes cell growth by suppressing autophagy. Furthermore, GSK-3-mediated elevation of p62/SQSTM11 supports this view. p62/SQSTM11 acts as an oncoprotein by activating oxidative stress, preventing repair of genome damage and by enhancing Ras-induced cell transformation.56,57 It should be noted that the role of autophagy is complex and may have opposing effects: autophagy is protective in early stages of pathogenesis but appears to support malignancies with increased metabolic demand in advanced stages of cancer progression.41–45 It is noteworthy that MCF-7 cells represent an early stage of cancer (as compared with other breast cancer cell lines), a feature that is well correlated with the suppressed autophagy observed in these cells (Figure 3). Hence, Oncogene (2014), 1 – 11 GSK-3 inhibitors may be particularly useful in treating breast cancers at early stage. However, the ‘pro-cancer’ or ‘anticancer’ activity of GSK-3 inhibitors as a function of stage and tumor aggressiveness should be taken into consideration. Previous studies have linked GSK-3 with autophagy. Impaired autophagy was found in hearts of GSK-3α knockout mice,79 and inhibition of GSK-3 blocked serum deprivation-induced autophagy via activation of the acetyltransferase TIP60.80 It is possible that under serum starvation, conditions in which mTORC1 activity is suppressed, GSK-3 inhibition has no further effect. Alternatively, under certain conditions, TIP60 may not be required for activated autophagy as argued previously.79 An additional study showed that GSK-3 target, β-catenin, is a negative regulator of autophagy by regulating TCF4.81 Accordingly, GSK-3 may enhance autophagy by its ability to reduce β-catenin levels. However, in our cell system, GSK-3 suppressed autophagy suggesting that combined activity of GSK-3 toward its multiple targets likely dictates the ‘net’ effect on autophagy. The role of GSK-3 in regulation of lysosome function is an important finding of this work. Of particular significance is the ability of GSK-3 inhibitors to restore lysosomal acidification in MCF-7 cells that show low levels of lysosomal acidification. © 2014 Macmillan Publishers Limited GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al 9 Plasmids GSK-3 mTORC1 LC3II Beclin1 lysosomes p62/SQSTM Autophagy Autophagic flux Endocytic degradation Figure 7. GSK-3 suppresses autophagy via mTORC1 and lysosomes. GSK-3 activation of mTORC1 results in suppressed autophagy as indicated by reduced beclin-1 and elevated levels of p62/SQSTM1 (marked by dashed lines). Note that LC3-II levels are decreased by mTORC1. GSK-3 reduces lysosomal acidification that in turn inhibits autophagic flux (lysosomes enhance autophagic activity and flux). This is pronounced by elevation in LC3-II and p62/SQSTM1 (marked by dashed lines). Reduced lysosomal acidification may inhibit endocytic clearance as was demonstrated by reduced levels of EGFR. Arrows indicate activation, blocked lines indicate inhibition. Deficiency in lysosomal acidification has been described in various pathological disorders,59,60 although its role in cancer has not been clear.82 A previous study indicated reduced lysosomal acidification in cancer cells,83 and a recent study using proteomic tools correlated reduced lysosomal acidification in breast cancer cells with the degree of cell transformation and aggressiveness.84 These studies thus support our notions that, by ‘correcting’ lysosomal acidification, GSK-3 inhibitors restore cellular homeostasis and reverse, at least in part, uncontrolled cell proliferation. This was further indicated in our experiments with starved cells that showed enhanced lysosomal acidification and cells’ growth inhibition that exceeded the growth inhibition achieved with rapamycin (Figure 5). GSK-3 inhibitors improved lysosomal-mediated endocytic clearance (Figure 6). Disruption of the endocytic machinery is observed in many types of cancers.63,85 Impaired deactivation of receptor tyrosine kinases, such as EGFR, is a well-recognized cause of neoplastic growth.85–88 Receptor trafficking from early endosomes to lysosomes is critical for downregulation of the EGFR mitogenic signal. Hence, poor lysosomal activity resulting from defects in lysosomal acidification or maturation had an inhibitory impact on endocytic clearance. Here we showed that inhibition of GSK-3 facilitated lysosome-mediated EGFR endocytic clearance. It is noteworthy that increased expression of EGFR is detected in 16% of breast cancer tumors,89 and enhancement of EGFR clearance is considered a promising therapeutic approach in treating breast cancer as well as other cancer types.88 Furthermore, a combination of EGFR and mTORC1 inhibitors has been successfully used as an anticancer treatment.90,91 Hence, GSK-3 inhibition may be a useful approach in cancers associated with elevated mTORC1 activity and limited lysosomal acidification. MATERIALS AND METHODS Materials AR-A014418, SB-216763 and CT99012 were purchased from Sigma (Rehovot, Israel). L803-mts peptide was synthesized by Genemed Synthesis, Inc. (San Antonio, TX, USA). Antibodies against phopsho-S6K-1 (Thr389), S6K-1, phospho-S6 (Ser240/244), S6 protein, phospho-ERK1/2 (Thr202, Tyr204) and LC3-I/II were from Cell Signaling Technologies (Beverly, MA, USA). Antibodies against GSK-3α/β, β-actin, cathepsin D, Lamp2 and EGFR were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-p62/ SQSTM1 was from Medical and Biological Laboratories (Nagoya, Japan). All other reagents were from Sigma. © 2014 Macmillan Publishers Limited GFP- or Cherry-tagged GSK-3α and GSK-3β in which GFP or Cherry were fused to the C-terminus of the enzymes were generated as described previously.92 RFP-LC3-II was a kind gift from Dr Ehud Cohen (Hebrew University of Jerusalem). GFP-p62SQSTP was obtained from Dr Zvulun Elazar from the Weizmann institute of Science, Rehovot. Human EGFR plasmids were a kind gift from Dr Ronit Pinkas-Kamaraski at Tel Aviv University and Dr Yosef Yarden at the Weizmann institute of Science, Rehovot. Cell culture and transfections MCF-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 5 mg/ml glutamine and 1% penicillin and streptomycin. MCF-10A cells were grown in DMEM-F12 medium (1:1) supplemented with 5% fetal calf serum, 5 mg/ml L-glutamine, 0.02 μg/ml EGF, 10 μg/ml insulin, 0.5 μg/ml hydrocortisol, 0.1 g/ml choleratoxin and 1% penicillin and streptomycin. Mouse embryonic fibroblast cells deficient in TSC1/2 were generously provided by Dr Kwiatkowski (Harvard Medical School) and were maintained in DMEM supplemented with 10% fetal calf serum as described for MCF-7 cells. MCF-7 or MCF-10A cells were transiently transfected with the indicated constructs (3–7 μg) using Lipofectamine 2000 (Invitrogen, Camarillo, CA, USA) for MCF-7 cells and using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) for MCF-10A cells. For experiments in which GSK-3 expression was silenced, cells were transfected with 50 nM GSK-3α or GSK-3β siRNA or with a negative control siRNA (Thermo Scientific/Dharmacon, Waltham, MA, USA) using the transfection reagent Dharmafect (Thermo Scientific/Dharmacon) according to the manufacturer's instructions. In some experiments, cells were treated with GSK-3 inhibitors 40 μM L803-mts, 20 μM AR-A014418, 10 μM SB-216763, 30 μM chloroquine or 50 nM rapamycin for 4 h or as indicated. To monitor cell growth, exponential growth phase was harvested and seeded at 20 000 cells per six-well plates. Cells were incubated with GSK-3 inhibitors as indicated. Fresh medium and inhibitors were added every 24 h to the cell cultures. Cells were trypsinized and counted in Cellometer Auto T4 (Nexcelom Biosciences, Lawrence, MA, USA). Gel electrophoresis and immunoblotting Cells were collected and lysed in an ice-cold buffer G (20 mM Tris-HCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.5 mM orthovanadate, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM NaF, 1 mM benzaminidine and protease inhibitors aprotenin, leupeptin and pepstatin A). Cell extracts were centrifuged at 14 000 g for 20 min and supernatants were collected. Protein concentrations were determined by Bradford analysis, and equal amounts of protein (20 μg) were subjected to gel electrophoresis and western blot analysis using indicated antibodies. Analysis of β-actin levels demonstrated equal protein loading. Live-cell imaging To stain acidified lysosomes, cells grown on coverslips were incubated with 50 nM LysoTracker Red (Molecular Probes, Eugene, OR, USA) for 15 min at 37 °C. The cells were washed with growing medium and were immediately taken for live-cell microscopy. For other experiments, the cells were growth on coverslips and treated as indicated. Live-cell images were taken using a 63.0 × 1.40 OIL UV objective lens on a laser scanning confocal microscope (Leica, Solms, Germany, TCS-SP5 II) with spatial resolution of 50–70 nM. Images were generated using LAS-AF Lite software (Leica microsystems). Immunofluorescent analyses Cells were grown on coverslips and fixed with 4% paraformaldehyde for 15 min at room temperature. After fixation, cells were permabilized (0.2% Triton X-100), blocked with 3% bovine serum albumin and incubated with indicated antibodies (0.1% bovine serum albumin, 0.05% Triton X-100 in phosphate-buffered saline) overnight at 4 °C. Cells were then washed three times in phosphate-buffered saline, and secondary Alexa488-conjugated antibody (Invitrogen) was applied for 1 h at room temperature. Confocal images were acquired on the Leica TCS-SP5 II confocal microscope. Statistical analyses All experiments were repeated at least three times. The data are expressed as means ± s.d. (s.e.m.). Statistical analysis was performed using Student's t-test (two-tailed). The criterion for statistical significance was Po 0.05. Oncogene (2014), 1 – 11 GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al 10 CONFLICT OF INTEREST The authors declare no conflict of interest. 26 ACKNOWLEDGEMENTS This research was supported by the Israel Science Foundation grant No. 341/10 and the Finghort Fund for Cancer Research at Tel Aviv University. We thank Dr Dan Klionsky and Dr Zvulun Elazar for their constructive comments. 27 28 REFERENCES 1 Grimes CA, Jope RS. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol 2001; 65: 391–426. 2 Eldar-Finkelman H. Glycogen synthase kinase-3: an emerging therapeutic target. Trend Mol Med 2002; 8: 126–132. 3 Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 2003; 116: 1175–1186. 4 Hur EM, Zhou FQ. GSK3 signalling in neural development. Nat Rev Neurosci 2010; 11: 539–551. 5 Jope RS, Yuskaitis CJ, Beurel E. Glycogen synthase kinase-3 (GSK3): inflammation, diseases, and therapeutics. Neurochem Res 2007; 32: 577–595. 6 Woodgett JR. Molecular cloning and expression of glycogen synthase kinase-3/ factorA. EMBO J 1990; 9: 2431–2438. 7 Eldar-Finkelman H, Martinez A. GSK-3 inhibitors: preclinical and clinical focus on CNS. Front Mol Neurosci 2011; 4: 32. 8 Hernandez F, Lucas JJ, Avila J. GSK3 and tau: two convergence points in Alzheimer's disease. J Alzheimers Dis 2013; 33: S141–S144. 9 Ougolkov AV, Billadeau DD. Targeting GSK-3: a promising approach for cancer therapy? Future Oncol 2006; 2: 91–100. 10 Patel S, Woodgett J. Glycogen synthase kinase-3 and cancer: good cop, bad cop? Cancer Cell 2008; 14: 351–353. 11 Birch NW, Zeleznik-Le NJ. Glycogen synthase kinase-3 and leukemia: restoring the balance. Cancer Cell 2010; 17: 529–531. 12 McCubrey JA, Davis NM, Abrams SL, Montalto G, Cervello M, Basecke J et al. Diverse roles of GSK-3: tumor promoter-tumor suppressor, target in cancer therapy. Adv Biol Regul 2014; 54: 176–196. 13 Manoukian AS, Woodgett JR. Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways. Adv Cancer Res 2002; 84: 203–229. 14 Wu D, Pan W. GSK3: a multifaceted kinase in Wnt signaling. Trends Biochem Sci 2011; 35: 161–168. 15 Polakis P. The many ways of Wnt in cancer. Curr Opin Genet Dev 2007; 17: 45–51. 16 Kotliarova S, Pastorino S, Kovell LC, Kotliarov Y, Song H, Zhang W et al. Glycogen synthase kinase-3 inhibition induces glioma cell death through c-MYC, nuclear factor-kappaB, and glucose regulation. Cancer Res 2008; 68: 6643–6651. 17 Zhu Q, Yang J, Han S, Liu J, Holzbeierlein J, Thrasher JB et al. Suppression of glycogen synthase kinase 3 activity reduces tumor growth of prostate cancer in vivo. Prostate 2011; 71: 835–845. 18 Aguilar-Morante D, Morales-Garcia JA, Sanz-SanCristobal M, Garcia-Cabezas MA, Santos A, Perez-Castillo A. Inhibition of glioblastoma growth by the thiadiazolidinone compound TDZD-8. PLoS ONE 2010; 5: e13879. 19 Remsing Rix LL, Kuenzi BM, Luo Y, Remily-Wood E, Kinose F, Wright G et al. GSK3 alpha and beta are new functionally relevant targets of tivantinib in lung cancer cells. ACS Chem Biol 2013; 9: 353–358. 20 Ougolkov AV, Fernandez-Zapico ME, Savoy DN, Urrutia RA, Billadeau DD. Glycogen synthase kinase-3beta participates in nuclear factor kappaB-mediated gene transcription and cell survival in pancreatic cancer cells. Cancer Res 2005; 65: 2076–2081. 21 Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 2000; 406: 86–90. 22 Fishman P, Bar-Yehuda S, Ohana G, Barer F, Ochaion A, Erlanger A et al. An agonist to the A3 adenosine receptor inhibits colon carcinoma growth in mice via modulation of GSK-3 beta and NF-kappa B. Oncogene 2004; 23: 2465–2471. 23 Pluquet O, Qu LK, Baltzis D, Koromilas AE. Endoplasmic reticulum stress accelerates p53 degradation by the cooperative actions of Hdm2 and glycogen synthase kinase 3beta. Mol Cell Biol 2005; 25: 9392–9405. 24 Kim HM, Kim CS, Lee JH, Jang SJ, Hwang JJ, Ro S et al. CG0009, a novel glycogen synthase kinase 3 inhibitor, induces cell death through cyclin D1 depletion in breast cancer cells. PLoS ONE 2013; 8: e60383. 25 Karyo R, Eskira Y, Pinhasov A, Belmaker R, Agam G, Eldar-Finkelman H. Identification of eukaryotic elongation factor-2 as a novel cellular target Oncogene (2014), 1 – 11 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 of lithium and glycogen synthase kinase-3. Mol Cell Neurosci 2010; 45: 449–455. Avrahami L, Farfara D, Shaham-Kol M, Vassar R, Frenkel D, Eldar-Finkelman H. Inhibition of GSK-3 ameliorates beta-amyloid(A-beta) pathology and restores lysosomal acidification and mTOR activity in the alzheimer's disease mouse model: in vivo and in vitro studies. J Biol Chem 2013; 288: 1295–1306. Shin S, Wolgamott L, Yu Y, Blenis J, Yoon SO. Glycogen synthase kinase (GSK)-3 promotes p70 ribosomal protein S6 kinase (p70S6K) activity and cell proliferation. Proc Natl Acad Sci USA 2011; 108: E1204–E1213. Shin S, Wolgamott L, Tcherkezian J, Vallabhapurapu S, Yu Y, Roux PP et al. Glycogen synthase kinase-3beta positively regulates protein synthesis and cell proliferation through the regulation of translation initiation factor 4E-binding protein 1. Oncogene 2013; 33: 1690–1699. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012; 149: 274–293. Cornu M, Albert V, Hall MN. mTOR in aging, metabolism, and cancer. Curr Opin Genet Dev 2013; 23: 53–62. Proud CG. mTOR Signalling in Health and Disease. Biochem Soc Trans 2013; 39: 431–436. Kwiatkowski DJ, Manning BD. Tuberous sclerosis: a GAP at the crossroads of multiple signaling pathways. Hum Mol Genet 2005, 14 Spec No. 2: R251–R258. Mamane Y, Petroulakis E, LeBacquer O, Sonenberg N. mTOR translation initiation and cancer. Oncogene 2006; 25: 6416–6422. Wander SA, Hennessy BT, Slingerland JM. Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy. J Clin Invest 2011; 121: 1231–1241. Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer. Trends Mol Med 2007; 13: 433–442. Voss MH, Molina AM, Motzer RJ. mTOR inhibitors in advanced renal cell carcinoma. Hematol Oncol Clin North Am 2011; 25: 835–852. Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N. Engl J Med 2013; 368: 1845–1846. Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW et al. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 2010; 90: 1383–1435. Klionsky D. An overview of autophagy: Morphology, mechanism and regulation. Antioxid Redox Signal 2013; 20: 460–473. Schneider JL, Cuervo AM. Autophagy and human disease: emerging themes. Curr Opin Genet Dev 2014; 26C: 16–23. Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev 2011; 25: 1999–2010. Chen N, Karantza V. Autophagy as a therapeutic target in cancer. Cancer Biol Ther 2011; 11: 157–168. Mathew R, White E. Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night. Curr Opin Genet Dev 2011; 21: 113–119. Eisenberg-Lerner A, Kimchi A. The paradox of autophagy and its implication in cancer etiology and therapy. Apoptosis 2009; 14: 376–391. Wu WK, Coffelt SB, Cho CH, Wang XJ, Lee CW, Chan FK et al. The autophagic paradox in cancer therapy. Oncogene 2011; 31: 939–953. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008; 451: 1069–1075. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003; 115: 577–590. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 2010; 141: 290–303. Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, Hafen E et al. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev 2004; 18: 2893–2904. Smith EM, Finn SG, Tee AR, Browne GJ, Proud CG. The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses. J Biol Chem 2005; 280: 18717–18727. Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, Bajraszewski N et al. Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 2003; 112: 1223–1233. Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy 2007; 3: 542–545. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000; 19: 5720–5728. Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 2005; 171: 603–614. He C, Levine B. The Beclin 1 interactome. Curr Opin Cell Biol 2012; 22: 140–149. © 2014 Macmillan Publishers Limited GSK-3 promotes breast cancer cell growth I Azoulay-Alfaguter et al 11 56 Duran A, Linares JF, Galvez AS, Wikenheiser K, Flores JM, Diaz-Meco MT et al. The signaling adaptor p62 is an important NF-kappaB mediator in tumorigenesis. Cancer Cell 2008; 13: 343–354. 57 Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 2009; 137: 1062–1075. 58 Saftig P, Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol 2009; 10: 623–635. 59 Luzio JP, Pryor PR, Bright NA. Lysosomes: fusion and function. Nat Rev Mol Cell Biol 2007; 8: 622–632. 60 Boya P. Lysosomal function and dysfunction: mechanism and disease. Antioxid Redox Signal 2012; 17: 766–774. 61 Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, Imarisio S et al. Lysosomal positioning coordinates cellular nutrient responses. Nat Cell Biol 2011; 13: 453–460. 62 Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science 2011; 334: 678–683. 63 Haglund K, Dikic I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J Cell Sci 2012; 125: 265–275. 64 Sorkin A, Goh LK. Endocytosis and intracellular trafficking of ErbBs. Exp Cell Res 2009; 315: 683–696. 65 Avraham R, Yarden Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nat Rev Mol Cell Biol 2011; 12: 104–117. 66 Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 2010; 12: 21–35. 67 Harris TE, Lawrence JC Jr. TOR signaling. Sci STKE 2003; 2003: re15. 68 Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006; 124: 471–484. 69 Wazir U, Wazir A, Khanzada ZS, Jiang WG, Sharma AK, Mokbel K. Current State of mTOR Targeting in Human Breast Cancer. Cancer Genomics Proteomics 2014; 11: 167–174. 70 Lauring J, Park BH, Wolff AC. The phosphoinositide-3-kinase-Akt-mTOR pathway as a therapeutic target in breast cancer. J Natl Compr Canc Netw 2013; 11: 670–678. 71 Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X et al. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 2006; 126: 955–968. 72 Gulen MF, Bulek K, Xiao H, Yu M, Gao J, Sun L et al. Inactivation of the enzyme GSK3alpha by the kinase IKKi promotes AKT-mTOR signaling pathway that mediates interleukin-1-induced Th17 cell maintenance. Immunity 2012; 37: 800–812. 73 Huang J, Zhang Y, Bersenev A, O'Brien WT, Tong W, Emerson SG et al. Pivotal role for glycogen synthase kinase-3 in hematopoietic stem cell homeostasis in mice. J Clin Invest 2009; 119: 3519–3529. 74 Suzuki T, Bridges D, Nakada D, Skiniotis G, Morrison SJ, Lin JD et al. Inhibition of AMPK catabolic action by GSK3. Mol Cell 2013; 50: 407–419. © 2014 Macmillan Publishers Limited 75 Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K, Degenhardt K et al. Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev 2007; 21: 1367–1381. 76 Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999; 402: 672–676. 77 Takamura A, Komatsu M, Hara T, Sakamoto A, Kishi C, Waguri S et al. Autophagydeficient mice develop multiple liver tumors. Genes Dev 2011; 25: 795–800. 78 Yang Z, Klionsky DJ. Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol 2010; 22: 124–131. 79 Zhou J, Freeman TA, Ahmad F, Shang X, Mangano E, Gao E et al. GSK-3alpha is a central regulator of age-related pathologies in mice. J Clin Invest 2013; 123: 1821–1832. 80 Lin SY, Li TY, Liu Q, Zhang C, Li X, Chen Y et al. GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy. Science 2012; 336: 477–481. 81 Petherick KJ, Williams AC, Lane JD, Ordonez-Moran P, Huelsken J, Collard TJ et al. Autolysosomal beta-catenin degradation regulates Wnt-autophagy-p62 crosstalk. EMBO J 2013; 32: 1903–1916. 82 Kallunki T, Olsen OD, Jaattela M. Cancer-associated lysosomal changes: friends or foes? Oncogene 2013; 32: 1995–2004. 83 Altan N, Chen Y, Schindler M, Simon SM. Defective acidification in human breast tumor cells and implications for chemotherapy. J Exp Med 1998; 187: 1583–1598. 84 Harris RA, Yang A, Stein RC, Lucy K, Brusten L, Herath A et al. Cluster analysis of an extensive human breast cancer cell line protein expression map database. Proteomics 2002; 2: 212–223. 85 Bache KG, Slagsvold T, Stenmark H. Defective downregulation of receptor tyrosine kinases in cancer. EMBO J 2004; 23: 2707–2712. 86 Yarden Y. The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer 2001; 37: S3–S8. 87 Arteaga CL. ErbB-targeted therapeutic approaches in human cancer. Exp Cell Res 2003; 284: 122–130. 88 Davis NM, Sokolosky M, Stadelman K, Abrams SL, Libra M, Candido S et al. Deregulation of the EGFR/PI3K/PTEN/Akt/mTORC1 pathway in breast cancer: possibilities for therapeutic intervention. Oncotarget 2014; 5: 4603–4650. 89 Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JM. Expression of the HER1-4 family of receptor tyrosine kinases in breast cancer. J Pathol 2003; 200: 290–297. 90 Doherty L, Gigas DC, Kesari S, Drappatz J, Kim R, Zimmerman J et al. Pilot study of the combination of EGFR and mTOR inhibitors in recurrent malignant gliomas. Neurology 2006; 67: 156–158. 91 Fan QW, Cheng CK, Nicolaides TP, Hackett CS, Knight ZA, Shokat KM et al. A dual phosphoinositide-3-kinase alpha/mTOR inhibitor cooperates with blockade of epidermal growth factor receptor in PTEN-mutant glioma. Cancer Res 2007; 67: 7960–7965. 92 Azoulay-Alfaguter I, Yaffe Y, Licht-Murava A, Urbanska M, Jaworski J, Pietrokovski S et al. Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: functional role in calcium/calpain signaling. J Biol Chem 2011; 286: 13470–13480. Oncogene (2014), 1 – 11