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Oncogene (2014), 1–11
© 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14
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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
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2.0
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1.0
pS6
24
48
Time (Hr)
S6
72
GSK3α
Ctrl
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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
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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
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0
β-actin
TCS+/+
24
48
Time (Hr)
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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
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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
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a
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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,
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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
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Ctrl
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72
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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
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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
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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
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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
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