AMPK promotes tolerance to Ras pathway
inhibition by activating autophagy
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Citation Sanduja, S. et al. “AMPK Promotes Tolerance to Ras Pathway
Inhibition by Activating Autophagy.” Oncogene 35, 40 (April 2016): 5295–5303
As Published https://doi.org/10.1038/onc.2016.70
Publisher Nature Publishing Group
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Citable link http://hdl.handle.net/1721.1/117558
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AMPK promotes tolerance to Ras pathway inhibition by activating autophagy
Sandhya Sanduja1, Yuxiong Feng1, Robert A. Mathis1,2, Ethan S. Sokol1,2, Ferenc Reinhardt1, Ruth Halaban5, Piyush B. Gupta1-4,*
1
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
2
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3
Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139, USA
4
Harvard Stem Cell Institute, Cambridge, MA 02138, USA
5
Department of Dermatology, Yale University School of Medicine, New Haven, CT 06520, USA
*Correspondence: pgupta@wi.mit.edu Phone: 617-324-0086
Fax: 617-258-7778
Keywords: BRAF, RAS, AMPK, tolerance, autophagy
Abbreviations: BRAF, v-Raf murine sarcoma viral oncogene homolog B; RAS, rat sarcoma viral oncogene; AMPK, AMP-activated protein kinase; AICAR, 5-Aminoimidazole-4-carboxamide ribonucleotide; GFP, green fluorescent protein; LC3B microtubule-associated protein 1 light chain 3 beta; CQ, chloroquine; Baf, bafilomycin; ATG, autophagy-related protein; wt, wildtype; mut, mutant; shRNA, short hairpin RNA.
Abstract
Targeted inhibitors of oncogenic Ras-Raf signaling have shown great promise in
the clinic, but resistance remains a major challenge: 30% of tumors with pathway
mutations do not respond to targeted inhibitors, and of the 70% that do respond, all
eventually develop resistance. Before cancer cells acquire resistance, they respond to
initial drug treatment by either undergoing apoptosis ('addiction'), or by surviving
treatment albeit with reduced growth ('tolerance'). Since these drug-tolerant cells serve as
a reservoir from which resistant cells eventually emerge, inhibiting the pathways that
confer tolerance could potentially delay or even prevent recurrence. Here we show that
melanomas and other cancers acquire tolerance to Ras-Raf pathway inhibitors by
activating autophagy, which is mediated by the cellular energy sensor AMPK. Blocking
this AMPK-mediated autophagy sensitizes drug-tolerant melanomas to Ras-Raf pathway
inhibitors. Conversely, activating AMPK signaling and autophagy enables melanomas
that would otherwise be addicted to the Ras-Raf pathway to instead tolerate pathway
inhibition. These findings identify a key mechanism of tolerance to Ras-Raf pathway
inhibitors and suggest that blocking either AMPK or autophagy in combination with
these targeted inhibitors could increase tumor regression and decrease the likelihood of
Introduction
The Ras-Raf pathway is frequently activated in human cancers through mutations
in Ras or its downstream effector, BRAF. Given the central role that it plays in driving
tumorigenesis, the Ras-Raf pathway has become a major focus for the development of
targeted therapies. Although several strategies for inhibiting this pathway are being
explored, the most successful strategy to date has been to develop targeted inhibitors of
oncogenic forms of the BRAF protein. This has led to the development of vemurafenib
and other targeted inhibitors that specifically inhibit the oncogenic forms of BRAF (1-3).
Vemurafenib and other targeted drugs significantly prolong patient survival, but all
tumors eventually develop resistance after a median time of 5 to 8 months (3, 4). Tumors
that develop resistance are able to maintain MAPK phosphorylation -- a downstream
measure of Ras-Raf pathway signaling -- even in the presence of vemurafenib (5-11).
Although all tumors eventually develop resistance and restore MAPK
phosphorylation, they display significant differences in their initial responses to
treatment. While vemurafenib causes some tumors to completely regress, most tumors
only regress partially, if at all. Importantly, these differences in initial response cannot be
explained by differences in the extent to which vemurafenib inhibits Ras-Raf pathway
signaling as gauged by MAPK phosphorylation (5, 12-14).
These observations in patients appear to be analogous to observations that have
been made with populations of cancer cells in culture: inhibition of the Ras pathway
triggers rapid apoptosis in some cancer cell lines (addiction), whereas other lines do not
undergo apoptosis but rather survive pathway inhibition (tolerance) (15). The
latter is associated with re-activation of Ras pathway signaling in the presence of the
targeted inhibitor. Since resistant clones can only arise if some cancer cells survive the
initial drug treatment, inhibiting the pathways the confer tolerance could improve patient
outcomes by eliminating the reservoir of cells without which resistance would be unable
to develop. However, while several mechanisms of resistance have been reported (5-11),
how cells develop tolerance to RAS-RAF pathway inhibitors is not well understood.
Mutations in the Ras-Raf pathway provide a competitive advantage by enabling
cancer cells to increase their glucose uptake in low nutrient conditions (16, 17).
Consistent with this, vemurafenib and other inhibitors of Ras-Raf signaling reduce
glucose uptake by cancer cells, which can be measured through PET imaging with the
glucose analog fluorodeoxyglucose (FDG) (13, 18). It seems plausible that cancer cells
might cope with this rapid reduction in glucose by activating some of the same pathways
that normal cells use to adapt to the effects of nutrient starvation.
In normal cells, nutrient starvation triggers the activation of AMPK, which is a
sensor of the cellular AMP:ATP ratio (19). Upon its activation, AMPK stimulates an
internal scavenging program, termed autophagy, which provides essential nutrients by
breaking down cellular components (20, 21). When autophagy is inhibited, cells are
unable to survive even short bouts of nutrient starvation (22, 23). In this study, we
examined if autophagy might also promote tumor drug tolerance by enabling cancer cells
to survive the starvation provoked by inhibition of the Ras-Raf pathway.
Ras-Raf pathway inhibitors activate AMPK signaling in cancer cells with Ras
pathway mutations.
To examine if inhibitors of the Ras pathway were provoking a starvation
response, we assessed AMPK activation after 48 hrs of inhibitor treatment, in a panel of
cancer cell lines with mutations in either Ras or Braf. Treatment with vemurafenib caused
a dose-dependent increase in the phosphorylation of AMPK in two melanoma lines
(YUKSI, YUSIK) and a colon cancer line (HT29) with BRAFV600 mutations (Figure 1A).
Treatment with vemurafenib also increased the phosphorylation of ULK1, a downstream
target of AMPK (Figure 1A) (20, 24). As a control, vemurafenib treatment did not
increase AMPK or ULK1 phosphorylation in cancer cells that lacked BRAF mutations
(Supplementary Figure 1A). Consistent with the established role of the Ras pathway in
promoting glucose uptake, we found that vemurafenib treatment also significantly
reduced uptake of the fluorescent glucose analog 2-NBDG (Supplementary Figure 1B).
To determine if these effects were unique to vemurafenib, we examined if other
inhibitors of the Ras-Raf pathway also induced AMPK signaling in cancer lines with
BRAF or RAS mutations. Treatment with a MEK inhibitor (AS703026) induced the
phosphorylation of both AMPK and ULK1 in YUKSI and YUSIK melanoma cells
(Figure 1B). In addition, AS703026 treatment induced AMPK and ULK1
phosphorylation in the KRAS-mutant breast cancer line, MDA-MB-231 (Figure 1B).
These findings indicated that Ras-Raf pathway inhibition in cancers with pathway
mutations was sufficient to stimulate AMPK signaling.
AMPK promotes energy homeostasis by turning off anabolic processes that
consume ATP and activating catabolic processes that generate ATP (25). One such
catabolic process is autophagy (20, 21, 26), a ‘self-eating’ program in which cells engulf
and break down their organelles and cytoplasmic proteins to generate energy. Autophagy
is mediated by engulfment within double-membraned vesicles termed autophagosomes,
which ultimately fuse with lysosomes resulting in degradation of their cargo (27).
AMPK-mediated autophagy is an essential mechanism for restoring energy homeostasis
in cells that are deprived of nutrients.
We next determined if vemurafenib treatment induced autophagy. Using western
blotting, we found that vemurafenib caused an increase in lipidated LC3B protein
(LC3B-II), a marker of autophagy, in the BRAF-mutant YUKSI and YUSIK melanoma
lines (Figure 2A). Consistent with this, direct visualization of autophagosomes through
expression of an GFP-LC3B fusion protein also showed that vemurafenib treatment led to
an increase in both autophagosome number and size; as a negative control, these changes
were not observed when cells were infected with a mutant LC3BG120A-GFP that is unable
to participate in autophagosome formation (Figure 2B and C; see methods for details).
Electron microscopy further confirmed this finding: vemurafenib treatment led to an
increase in double-membraned autophagic vesicles per cell with the average number of
6.42 (vemurafenib) vs 1.9 (DMSO) (Figure 2D).
Induction of autophagy was not a unique effect of vemurafenib, but was also
observed upon treatment with another BRAFV600 inhibitor (AZ628), as well as in
response to the MEK inhibitor AS703026. All of these inhibitors stimulated autophagy,
2A). As a negative control, vemurafenib did not inhibit MAPK phosphorylation or
stimulate LC3B-II accumulation in cancer lines that did not harbor mutations in BRAF
(Supplementary Figure 2B).
While the upregulation of autophagy can lead to an increase in autophagosome
numbers, autophagosome numbers can also increase when their clearance is blocked (28).
To distinguish between these possibilities, we used western blotting to assess the effects
of vemurafenib treatment on p62 protein, an autophagosome marker that is often (though
not always) decreased when autophagy is induced (28). Vemurafenib treatment reduced
p62 levels (Supplementary Figure 2C); immunostaining for p62 in YUKSI cells showed
co-localization of p62 with GFP-LC3B labeled autophagosomes upon vemurafenib
treatment (Supplementary Figure 2D).
To confirm that autophagosomes were being cleared, we determined if the
GFP-LC3B autophagosomes stimulated by vemurafenib co-localized with the lysosome
markers, LAMP1/LAMP2. We found that vemurafenib treatment led to a marked
increase in the co-localization of GFP-LC3B and LAMP1/LAMP2, indicating that
autophagosome clearance was not reduced by inhibitor treatment (Supplementary Figure
2D). Taken together, these observations indicate that the increase in autophagosome
numbers observed upon treatment with vemurafenib is caused by an induction of
autophagy, rather than by a block in autophagosome clearance.
To test whether induction of autophagy was specific to mutant BRAF inhibition or
was a general consequence of Ras-Raf-MAPK inhibition, we inhibited MAPK signaling
in other cancer lines with activating BRAF or RAS mutations. Autophagosomes
(Figure 2E, top panel). Autophagosomes also accumulated in the KRAS-mutant
MDA-MB-231 breast cancer cell line in response to a MEK inhibitor (Figure 2E, bottom panel).
However, MCF7 and T47D breast cancer cell lines, which lack Ras pathway mutations,
did not activate autophagy upon pathway inhibition (Supplementary Figure 2E).
Collectively, these findings indicated that cancer cells with activating BRAF or RAS
mutations respond to MAPK pathway inhibitors by upregulating autophagy.
To further confirm that Ras pathway inhibition was activating autophagy via
AMPK, we examined the kinetics of autophagy induction and AMPK activation in
response to vemurafenib treatment. We observed LC3B-II accumulation and ULK1
phosphorylation (both of which are indicative of autophagy induction) after 24 hrs of
vemurafenib treatment, which was concurrent with AMPK activation (Supplementary
Figure 2F). Next, we treated YUKSI cells with an AMP mimetic (AICAR); AICAR
induced AMPK and ULK1 phosphorylation (data not shown), and caused GFP-LC3B
autophagosome accumulation (Figure 3A). Furthermore, we examined autophagy in
YUKSI cells expressing an shRNA targeting AMPK and found that shRNA knockdown
of AMPK abolished ULK1 activation and autophagosome formation in response to
vemurafenib (Figures 3B and C). We also examined the ability of Ras pathway inhibitors
to activate autophagy through AMPK in KRAS-mutant lung cancer. Using a cell line
(T1) derived from the KRASmut/p53null lung adenocarcinoma mouse tumor model (29),
we observed that treatment with MEK inhibitor activated AMPK and autophagy
(Supplementary Figure 3A). However, in a KRASmut human NSCLC line NCI-H460 that
inhibition did not activate AMPK or autophagy (Supplementary Figure 3A). Thus, Ras
pathway inhibitors activate autophagy through AMPK.
Autophagy is essential for cancer cells to tolerate Ras-Raf pathway inhibition
Although treatment with vemurafenib suppressed proliferation of the YUKSI and
YUSIK melanoma lines, it did not appear to induce death in either of these lines,
suggesting that they tolerated pathway inhibition. For instance, treatment with
vemurafenib did not lead to an increase in either the sub-G0/G1 population or cleavage of
caspase-3 (Supplementary Figures 3B and C). Since the findings above showed that
vemurafenib treatment caused these drug-tolerant lines to induce AMPK signaling and
autophagy, we next examined if this induction was contributing to their ability to survive
and tolerate BRAF inhibition.
To address this, we treated YUKSI cells with chloroquine (28) or bafilomycin
(Baf), which block autophagy by inhibiting autophagosome clearance (Figure 4A). Both
CQ and Baf strongly sensitized YUKSI cells to vemurafenib (Figure 4B and
Supplementary Figure 4A). We next used shRNA-mediated knockdown of a gene
required for autophagy as an alternate strategy for autophagy inhibition. Two different
shRNAs that targeted ATG5 blocked autophagic flux, and significantly abrogated
vemurafenib-induced LC3B lipidation (Figure 4C; Supplementary Figure 4B). Compared
to shCntrl cells, ATG5 knockdown cells were significantly more sensitive to vemurafenib
(Figure 4D; Supplementary Figure 4C).
To rule out the possibility that this effect was specific to ATG5, we performed
gene, ATG7. Two different shRNAs that targeted ATG7 blocked clearance of autophagic
flux, and also strongly sensitized YUKSI cells to vemurafenib treatment (Supplementary
Figures 4D-F).
Blocking autophagy similarly sensitized the YUSIK BRAFV600 mutant melanoma
line to vemurafenib, causing significantly reduced cell survival (Supplementary Figure
4G). Therefore, chemical or genetic ablation of autophagy sensitizes cells to
vemurafenib, indicating that autophagy promotes tolerance to BRAF inhibition.
We next examined if experimentally stimulating AMPK and autophagy would
allow cells that are addicted to Ras-MAPK signaling to tolerate pathway inhibition. The
YUSIV melanoma line is addicted to Ras pathway signaling and undergoes massive cell
death at very low doses of pathway inhibitors (Supplementary Figure 5A). To induce
AMPK signaling, we treated the YUSIV cells with the AMP mimetic AICAR, which, as
expected, activated both AMPK and autophagy (Supplementary Figure 5B). While
AICAR treatment alone did not affect cell growth or viability, AICAR significantly
increased the fraction of cells that survived upon treatment with the MEK inhibitor
AS703026 (24% vs. 7%; Figure 5A). Blocking autophagy with shRNAs against ATG5
prevented AICAR-induced tolerance to Ras pathway inhibition (Figures 5B and C);
blocking autophagy with CQ had similar effects (Supplementary Figure 5C). AICAR also
promoted tolerance to vemurafenib in a BRAFV600 mutant melanoma line (YUGEN8)
previously reported to be addicted to the MAPK pathway (Supplementary Figure 5D)
(31). We observed that while YUGEN8 cells modestly activated AMPK upon
vemurafenib treatment, co-treatment with vemurafenib and AICAR led to a much
(Supplementary Figure 5E). These observations indicated that activation of AMPK
promotes tolerance to Ras-Raf pathway inhibitors by inducing autophagy.
Blocking autophagy synergizes with vemurafenib to inhibit melanoma tumor
growth
We next examined if the effectiveness of vemurafenib could be improved by
blocking autophagy in vivo. YUKSI melanoma cells were injected into mice and allowed
to form palpable tumors (2-3 mm). Mice were then treated daily for 3 weeks with
vemurafenib, CQ, a combination of both, or with a vehicle control (Figure 6A
schematic).
Consistent with the ability of YUKSI cells to tolerate MAPK pathway inhibition
in vitro, vemurafenib reduced tumor growth but did not cause tumor regression, although
ERK signaling was abrogated (Figures 6B and C). Also in agreement with the in vitro
findings, vemurafenib activated AMPK and induced autophagy within tumors as
evaluated by immunohistochemical staining for p-AMPK and increased LC3B puncta
(Figure 6C).
While vemurafenib or CQ alone very modestly attenuated YUKSI tumor growth,
combination treatment with chloroquine almost completely blocked tumor growth
(Figure 6B). We stained tumor tissues with cleaved caspase-3 antibody and did not see an
increase in apoptosis (data not shown). However, we noticed a significant increase in
necrosis when autophagy was inhibited; vemurafenib-treated tumors in which autophagy
had been inhibited had larger regions of necrosis, relative to vemurafenib-treated tumors
Blocking autophagy in YUKSI tumors by shRNA-knockdown of ATG5 also
significantly reduced tumor growth in response to vemurafenib, relative to tumors
expressing a control shRNA (Supplementary Figure 6B). Treatment of heterogeneous
tumors (created by co-injecting GFP-positive control cells with GFP-negative ATG5
knockdown cells) with vemurafenib caused selective depletion of shATG5 cells
(Supplementary Figure 6C). Blocking autophagy also reduced tumor growth of another
BRAF-mutant melanoma line (YUSIK) (Supplementary Figure 6D). Taken together,
these observations indicate that autophagy increases the ability of BRAF-mutant
melanomas to tolerate Ras pathway inhibition in vivo.
Discussion
Our findings show that cancer cells tolerate RAS pathway inhibition by activating
AMPK and autophagy. RAS pathway inhibition activates autophagy in an
AMPK-dependent manner, and blocking autophagy sensitizes cancers that would otherwise
tolerate RAS pathway inhibitors. Conversely, cancer cells that would otherwise be
sensitive to RAS pathway inhibitors acquire tolerance when AMPK activity is
experimentally induced with an AMP mimetic. In this context, AMPK activation does not
promote tolerance if autophagy is blocked, suggesting that autophagy is the key process
activated downstream of AMPK to promote tolerance. Taken together, these findings
provide a strong rationale for combining inhibitors of autophagy with inhibitors of the
Ras pathway, a strategy being explored in several ongoing clinical trials.
Studies from the laboratories of E. White and A. Kimmelman have shown that
autophagy, which enables them to preserve mitochondrial function and resist metabolic
stress in the tumor microenvironment (32-35). These investigators have shown that
inhibiting this basal autophagy results in mitochondrial defects that impair the growth of
KRAS/BRAF-driven tumors. BRAF-driven pediatric brain tumors also show high levels
of autophagy, and increased sensitivity to autophagy inhibition (36). Recent studies from
the laboratory of J. Debnath have also suggested that the invasion of RAS-driven tumors
may depend on pro-inflammatory cytokines whose secretion is dependent on a
non-canonical form of autophagy (37). Since these studies demonstrate that autophagy is
upregulated in KRAS/BRAF cancers, and is critical for cancer cell survival, one might
have expected that inhibiting the MAPK pathway would lead to a reduction in autophagy.
We find that this does not occur in the subset of cancers that are tolerant to Ras-Raf
inhibition, which instead upregulate autophagy when the pathway is inhibited.
Prior research has also established a close connection between the RAS pathway
and AMPK. Since AMPK reduces cellular proliferation upon nutrient deprivation, its
activation often reduces cancer cell growth and is therefore being actively explored as a
therapeutic strategy. Cantley and colleagues have shown previously that oncogenic
BRAF blocks AMPK activation by inhibiting LKB1, and that vemurafenib can activate
AMPK in BRAF-mutant melanomas (38). Our study extends these findings by showing
that this AMPK activation induces autophagy, which is crucial for melanomas to tolerate
BRAF inhibitors, and more generally for cancers with RAS or BRAF mutations to
tolerate pathway inhibition. Additionally, our findings also suggest that AMPK
when applied in combination with Ras pathway inhibitors, whose effects they may well
antagonize.
Our findings concerning tolerance to BRAF inhibitors complement those of
Amaravadi and colleagues, who have shown that targeting autophagy also overcomes
resistance to these inhibitors (41). While we found autophagy is activated by AMPK in
tolerant cells, these authors have reported that AMPK does not play a role in activating
autophagy in cells that are resistant to BRAF inhibitors. They have shown that, in
drug-resistant melanomas, vemurafenib induces BRAF to bind the ER chaperone BIP, and that
this results in ER stress and autophagy. We did not find any evidence that vemurafenib
induced ER stress in our drug-tolerant melanoma lines. One possible explanation for this
difference is that MAPK inhibition may be essential for vemurafenib to activate AMPK;
since vemurafenib only inhibits the pathway in tolerant cells, AMPK may play a unique
role in this context. While the mechanisms involved are very different in these two
contexts (Figure 3; Supplementary Figure 3D), blocking autophagy would appear to be a
promising therapeutic strategy for both drug tolerance and resistance.
Materials and Methods
Cell Culture and Reagents
Early passage human melanoma cell lines, YUKSI, YUSIK, YUSIT1, YUSIV,
YUGEN8 and YUVON were provided by Dr. Ruth Halaban (Yale University School of
Medicine), and were maintained in Opti-Mem with 5% FBS, GlutaMax, Penicillin and
Streptomycin. HEK293T, HT29, MCF7, T47D, NCI-H460, and MDA-MB-231 cell lines
kindly provided by Dr. Tyler Jacks (Koch Institute for Integrative Cancer Research).
RAS and BRAF mutational status of the various lines is specified in the figures.
GFP-LC3B baculovirus infection was performed following the manufacturer’s instructions
(P36235; Life Technologies). Lentivirus production, target cell infection, and selection
were performed as previously described (42). Vemurafenib (S1267; Selleck Chemicals)
and AS703026 (S1475; Selleck Chemicals) were dissolved in DMSO, and chloroquine
(C6628; Sigma) was dissolved in PBS. Cells were treated with Raf/MAPK inhibitors for
48 hrs unless otherwise specified.
Electron Microscopy
Transmission electron microscopy was performed using the standard procedure.
Briefly, cells were fixed in a fixative solution containing 2.5% glutaraldehyde, 3%
paraformaldehyde and 5% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4). Cells
were pelleted followed by treatment with 1% OsO4 –veronal acetate, and cell pellet was
dehydrated and embedded in Embed-812 resin. 50 nm sections were cut using Reichert
Ultracut E microtome, stained with uranyl acetate and lead citrate. Sections were
examined using a FEI Tecnai spirit at 80KV and photographed with an AMT CCD
camera. Autophagosomes/cell were quantified for DMSO (n=59 cells) and vemurafenib
(n=50 cells) treatments.
Western Blotting
Preparation of cells extracts and western blotting were performed using standard
Technologies). The following antibodies from Cell Signaling Technology were used for
immunoblotting: LC3B (#2775), pERK1/2 (#4377), ERK1/2 (#9102), p62 (#8025),
ATG5 (#8540), p-AMPKα (T172, #2535), AMPKα (#5832), pULK1 (S555, #5869), ULK1 (#4773), GAPDH-HRP (#3683).
Immunofluorescence
To visualize autophagosomes, GFP-LC3B labeled cells were fixed with 4%
paraformaldehyde after indicated treatments, and washed with PBS prior to mounting.
For additional staining, cells were permeabilized using 0.1% TritonX-100 and incubated
with blocking solution (PBST with 10% goat serum and 3% BSA) for 1 hr at room
temperature. β-Tubulin Alexa Fluor 594 (CST #8058) and DAPI were used to stain cytoskeleton and nuclei, respectively. SQSTM1/p62 (CST #8025), LAMP1 (CST #9091),
and LAMP2 (Abcam #25631) were detected using Alexa Fluor 555 anti-rabbit antibody
following respective primary antibody incubations. Images were captured using Zeiss
AxioPlan2 fluorescence microscope. Autophagosome quantification was performed using
CellProfiler software (43). In short, a CellProfiler pipeline was developed to identify cells
based on the GFP signal. GFP-LC3B puncta were identified, and filtered based on
intensity and size adjustments.
Flow Cytometry
To measure glucose uptake, cells were cultured with DMSO or vemurafenib in
glucose- and serum-free DMEM containing a fluorescent glucose analog, 2-NBDG
manufacturer’s protocol, and analyzed by FlowJo software. For cell cycle analysis, both
floating and adherent cells were collected, fixed, and stained with propidium iodide
(EMD Biosciences # 537059), and analyzed by ModFit software. A total of 104 events
were acquired in all cases.
Dose-Response Assays
Cells were seeded in 96-well plates, at a density of 3000 cells per well. Drugs
were added at eight different doses with three replicates per dose. The same volume of
DMSO was added in three replicates as a control. For each vemurafenib dose, CQ and
Baf were used at 10 μM and 1 nM, respectively. Cell viability was measured after 72 hrs with CellTiterGlo reagent (Promega #G7572) following the manufacturer’s protocol.
Animal Experiments and Immunohistochemistry
NOD/SCID mice were purchased from The Jackson Laboratory. All animal
studies were approved by the Animal Care and Use Committees of the Massachusetts
Institute of Technology (Cambridge, MA). One million cells (per mouse) were
resuspended in 100 μl DMEM/Matrigel (BD Biosciences #354234) mixture (1:1 volume) and subcutaneously injected into six- to eight-week-old female NOD/SCID mice (n >5
mice per group). Once palpable tumors formed, mice were administered vemurafenib
(20mg/kg), and CQ (60mg/kg), singly or in combination, and with vehicle control,
everyday for 3 weeks, by intraperitoneal injections. Tumor sizes were recorded twice a
week. Animals were randomized by weight. No blinding was applied in performing
neutral-buffered formalin, and embedded in paraffin. Serial-sections (5 μm) were used for immunohistochemical analysis of pERK1/2, ERK1/2, pAMPK, and LC3B (CST #3868).
Briefly, tissue sections were deparaffinized using xylene and series of alcohols, followed
by heating in 10 mM citrate buffer (pH 6.0) for antigen retrieval. Sections were blocked
with blocking solution (Dako North America, Inc. #K4010) for 1hr at room temperature,
incubated with the primary antibody, washed, incubated with HRP-labeled secondary
antibody, and stained using the DAB reagent. Nuclei were counterstained with
Haematoxylin (Sigma #MHS16). Hematoxylin and eosin stained sections were used to
identify necrotic regions as previously described, marked by reduced tissue staining and
patches of destroyed tumor architecture (44).
Statistical Analysis
All data unless otherwise specified represent mean + S.E.M of at least three replicates.
Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc.,
La Jolla, CA, USA). Unpaired two-tailed Student’s t-test was performed for comparison
between any two groups of data unless specified otherwise. P<0.05 were considered
significant. The assumption of normality was tested using a D'Agostino-Pearson omnibus
test when n >8. The assumption of equal variances was tested using a two-sample F Test.
Welch’s correction was applied when unequal variances were found.
For animal experiments, we used power analysis to determine the minimum number of
mice required to reach P<0.01. Assuming that difference in means/standard deviation >2,
Acknowledgements
We thank Dr. Tyler Jacks for providing us with mouse KRASmut/p53-/- NSCLC
line, the Whitehead flow cytometry facility, and Nicki Watson and Wendy Salmon at the
Whitehead Keck Imaging Facility for microscopy services. We thank Dr. David Pincus
and Dr. Luke Whitesell for critical reading of the manuscript. This research was
supported by grants from the Ellison Foundation (P.B.G) and Melanoma Research
Alliance (#311800; P.B.G), a Yale SPORE in Skin Cancer funded by the National Cancer
Institute (#1 P50 CA121974; R.H.), and funds from the NSFGRFP (#1122374; E.S.S).
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Figure Legends
Figure 1. Vemurafenib and MEK inhibitors activate AMPK signaling in
BRAF-mutant melanomas.
Western blots showing pAMPK, pULK1 and pERK1/2 levels in BRAF or KRAS mutant
cancer cells treated for 48 hrs with indicated doses of (A) vemurafenib or (B) AS703026.
GAPDH is the loading control.
Figure 2. Vemurafenib and MEK inhibitors activate autophagy in BRAF-mutant
(A) Western blot showing LC3B levels in BRAF-mutant melanoma cells treated with 1
μM vemurafenib or DMSO for 48 hrs. GAPDH is the loading control. (B) GFP-LC3B (wildtype or G120A mutant) labeled YUKSI melanoma cells were treated with
vemurafenib or DMSO for 48 hrs, and autophagosomes were visualized by fluorescence
microscopy. Alexa Fluor 594-conjugated β-Tubulin and DAPI were used to stain the cytoskeleton and the nuclei, respectively. Scale bar, 20 μm. (C) Fluorescence images showing GFP-LC3B labeled autophagosomes in YUKSI melanoma cells treated with
vemurafenib or DMSO for 48 hrs. Inset shows identification of autophagosomes (green)
inside cells (white outlines) by image segmentation analysis (see full methods). Data
represent mean + S.E.M. (DMSO n=76 cells, Vem n=54 cells; *P<0.05 log-rank test. (D)
Representative electron micrographs of DMSO- and vemurafenib-treated YUKSI cells at
specified magnifications; black arrowheads mark double-membrane autophagosomes.
Scale bar, 400 nm. Data below the images represent average autophagosomes per cell for
respective treatments. (E) Fluorescence images showing GFP-LC3B labeled
autophagosomes in HT29 colon cancer cells and MDA-MB-231 breast cancer cells
treated with vemurafenib and AS703026, respectively. Scale bar, 20 μm.
Figure 3. AMPK signaling is required for the activation vemurafenib-induced
autophagy.
(A) Fluorescence images and quantification of GFP-LC3B labeled autophagosomes in
YUKSI cells treated with DMSO, 1 μM vemurafenib, 1 mM AICAR, or both vemurafenib and AICAR. Data represent mean + S.E.M. across three fields of view (>75
cultured with or without vemurafenib for 24 and 48 hrs. Levels of pAMPK, pULK1, and
LC3B (examined on a 12% Bis-Tris gel) are shown. (C) Fluorescence images and
quantification showing GFP-LC3B labeled autophagosomes in shCntrl or shAMPK
expressing YUKSI cells treated with DMSO or vemurafenib. Data represent mean +
S.E.M. (shCntrl/DMSO n=55 cells, shCntrl/Vem n=41 cells, shAMPK/DMSO n=36
cells, shAMPK/Vem n=61 cells; *****P<0.00001 log-rank test).
Figure 4. Autophagy inhibition sensitizes BRAF-mutant melanoma cells to
vemurafenib in vitro
(A) Western blots showing LC3B-II and p62 accumulation in YUKSI melanoma cells
treated with autophagy blockers (CQ or Baf) for 72 hrs. (B) YUKSI melanoma cells were
treated with DMSO or 1 μM vemurafenib, alone or in combination with autophagy blockers (CQ or Baf) for 72 hrs; Left- Cells remaining attached to the plate were fixed
and DAPI stained to visualize the surviving population. Right- Data represent mean +
S.E.M. across 4 different fields of view. (C) Western blots showing LC3B and p62 levels
in YUKSI melanoma cells expressing a control (shCntrl) or 2 different shATG5 hairpins
(shATG5-1 and shATG5-2). (D) shCntrl or shATG5 YUKSI melanoma cells were treated
with DMSO or 1 μM vemurafenib, alone or in combination for 72 hrs; Left- Cells remaining attached to the plate were fixed and DAPI stained to visualize the surviving
population. Right- Data represent mean + S.E.M. across 8 different fields of view.
***P<0.001; ****P<0.0001).
YUSIV BRAFwt melanoma cells treated with 10 nM AS703026 with or without 1 mM
AICAR for 72 hrs. Left- Cells remaining attached to the plate were fixed and DAPI
stained to visualize the surviving population for (A) shCntrl, (B) shATG5-1, and (C)
shATG5-2. Right- Data represent mean + S.E.M. across at least 4 different fields of view.
***P<0.001; ns - not significant.
Figure 6. Autophagy inhibition synergizes with vemurafenib to prevent melanoma
growth in vivo.
(A) YUKSI BRAFmut melanoma cells were injected into mice and allowed to form
palpable tumors (4-5 mm) followed by indicated drug treatments (see full methods). (B)
Top- Images of representative tumors from mice treated with control, vem, CQ, or
vem+CQ; scale bar 5 mm. Bottom- Data represent mean tumor volume + S.E.M. (n=5
mice per treatment group; *P<0.05). (C) Immunohistochemical staining showing levels
of pAMPK, pERK1/2, ERK1/2, and LC3B puncta in tumors from vemurafenib- vs
control-treated mice; scale bar 100 μm.
Supplementary Figure 1.
(A) Western blots showing pAMPK, AMPK, and pULK1 levels in
vemurafenib-treated BRAFV600wt cancer cells. (B) Uptake of fluorescent glucose analog (2-NBDG) in
YUKSI cells treated with DMSO (blue) or 1 μM vemurafenib (red). Data represent mean fluorescence intensity + S.E.M. for 104 events; ****P< 0.0001 Mann-Whitney test.
(A) Fluorescence images showing GFP-LC3B labeled autophagosomes in YUKSI
melanoma cells treated with DMSO, vemurafenib, AZD628 or AS703026 for 48 hrs.
Inset shows identification of autophagosomes in green by image segmentation analysis.
(B) Western blots showing LC3B and pERK1/2 levels in vemurafenib-treated
BRAFV600wt cancer cells. (C) p62 and LC3B levels in BRAFV600mut melanoma cells
treated with DMSO or vemurafenib for 48 hrs. (D) Fluorescence images showing
co-localization of GFP-LC3B labeled autophagosomes with LAMP-1 (top), LAMP-2
(middle), or p62 (bottom) in YUKSI melanoma cells; scale bar 20 μm. (E) Fluorescence images showing GFP-LC3B labeled breast cancer cells, MCF7 and T47D, treated with
DMSO or MEKi for 48 hr; scale bar 20 μm. (F) Western blots showing the kinetics of AMPK activation (as gauged by p-AMPK levels), and LC3B-II induction (examined on a
12% Bis-Tris gel) in YUKSI melanoma cells treated with 1 μM vemurafenib for indicated times.
Supplementary Figure 3.
(A) Western blots showing pAMPK and LC3B levels in AS703026 treated KRAS-mut
NSCLC cells. (B) YUKSI and YUSIK BRAFV600mut melanoma cells treated with DMSO
or 1 μM vemurafenib for 48 hrs were stained with propidium iodide, and analyzed by flow cytometry for cell cycle profiles. (C) Western blot showing levels of pro- and
cleaved caspase-3 in YUKSI cells treated with DMSO, vemurafenib or staurosporine
(+Cntrl). (D) Western blots showing levels of different ER stress markers (BIP, ATF4
Thapsigargin activates ER stress as seen by increased expression of BIP and ATF4, and
phosphorylation of PERK as seen by mobility shift on SDS-PAGE.
Supplementary Figure 4.
(A) Dose-response curves showing sensitivity to vemurafenib in the absence (blue) or
presence of chloroquine (red) or bafilomycin (green). (B) YUKSI melanoma cells
expressing shCntrl or shATG5 hairpins were treated with or without vemurafenib. Levels
of LC3B, p62 and ATG5 are shown. (C) Dose-response curves for vemurafenib treatment
of shCntrl and shATG5 cells. (D) p62 autophagic flux in YUKSI melanoma cells
expressing shCntrl, shATG5 or shATG7. (E) Dose-response curves for vemurafenib
treatment of shCntrl and shATG7 cells. (F) Data represent average cell viability + S.E.M.
for DMSO or vemurafenib-treated shCntrl and shATG7 cells. (G) YUSIK melanoma
cells were treated with DMSO or vemurafenib, alone or in combination with CQ for 72
hrs. Surviving cells were imaged (left) and quantified (right). Data represent mean +
S.E.M. across 4 different fields of view.
*P<0.05; ****P< 0.0001.
Supplementary Figure 5.
(A) Dose-response curves showing sensitivities of BRAFV600wt YUSIV melanoma cells to
vemurafenib (black) and other Ras pathway inhibitors (red); AZD628, AS703026 and
AZD6244. (B) YUSIV cells were treated with 1mM AICAR or DMSO for 48 hrs. Left-
Western blots showing pAMPK and pULK1 levels; Right- Fluorescence images showing
AS703026 with or without 1mM AICAR + CQ for 72 hrs. Data shows average cell
number + S.E.M across 4 different fields of view; ***P<0.001, ns is not significant. (D)
Phase-contrast images showing YUGEN8 BRAFV600mut melanoma cells treated with 1
μM vemurafenib with or without 1mM AICAR for 72 hrs. Data shows average cell count + S.E.M. for surviving cells from 4 different fields; *** p-value <0.001. (E) pAMPK
levels in YUGEN8 cells treated with 1 μM vemurafenib with or without 1mM AICAR for 24 hrs.
Supplementary Figure 6.
(A) Left- Hematoxylin and & eosin stained tumor sections showing regions of necrosis
{N}, marked by reduced tissue staining and patches of destroyed tumor architecture,
Right- Data represent percentage of necrotic tissue in the different conditions. (B) Left-
Images of representative tumors of YUKSI-shCntrl and YUKSI-shATG5 (hairpin 1)
treated with vemurafenib; scale bar 5 mm, Right- quantification of mean tumor volume
and tumor weight + S.E.M. (n=5 mice per treatment group; **P<0.01 Mann-Whitney
test. (C) Heterogeneous YUKSI tumors formed by co-injecting shCntrl (GFP+) cells with
shATG5 (GFP-) cells were treated with vehicle or vemurafenib as indicated. Data represent GFP-negative and GFP-positive tumor fractions in vehicle- vs
vemurafenib-treatment; **P<0.01. (D) YUSIK BRAFV600mut melanoma cells were injected into mice
and allowed to form palpable tumors (4-5 mm). Mice were then treated for 2.5 weeks,
daily, with vemurafenib and CQ, singly or in combination, or with a vehicle control. Data
shows tumor progression across indicated time interval (each point represents an average
Vemurafenib [μM] 48hrs pERK1/2 pULK1 pAMPKα ERK1/2 AMPKα GAPDH YUKSI A B
Figure 1
HT29 0 0.1 1 YUSIK 0 0.1 1 pULK1 pERK1/2 ERK1/2 GAPDH AMPKα pAMPKα YUKSI 0 0.1 1 MDA-MB-231 0 0.1 1 AS703206 [μM] 48 hrs YUSIK 0 0.1 1 0 0.1 1A
B
DMSO Vem DMSO Vem
pERK1/2 ERK1/2 GAPDH LC3B-II LC3B-I DMSO Vemurafenib DMSO Vemurafenib Figure 2 YUKSI YUSIK Avg 1.9 (n=59 cells) DMSO MDA-MB-231 (KRAS mut ) Vemurafenib DMSO HT29 (BRAF V600mut ) E C D Avg 6.42 (n=50 cells) Autophagosomes/ infected cell DMSO Vem GFP-LC3 (G120A) /ȕ-Tubulin/DAPI GFP-LC3/ȕ-Tubulin/DAPI GFP-LC3/ȕ-Tubulin/DAPI GFP-LC3/DAPI GFP-LC3/DAPI AS703026 DMSO GFP-LC3/DAPI GFP-LC3/DAPI GFP-LC3/DAPI GFP-LC3/DAPI Vemurafenib 0 2 4 6 *
pULK1 S$03.Į $03.Į LC3B-I LC3B-II 0 24 48 Vemurafenib [hr] YUKSI_shCntrl <8.6,BVK$03.Į A 0 24 48 YUKSI_shCntrl <8.6,BVK$03.Į DMSO V emurafenib C GAPDH DMSO Vemurafenib 0 2 4 6 A u to p h ag o so m es /infected c el l ***** shCntrl shAMPKĮ Figure 3
DMSO Vemurafenib AICAR Vemurafenib+AICAR
0 2 4 6 8
DMSO Vem AICAR Vem +AICAR
Autophagosomes/cell
B
**
GFP-LC3/DAPI GFP-LC3/DAPI GFP-LC3/DAPI LC3-GFP/DAPI
GFP-LC3/DAPI
GFP-LC3/DAPI
GFP-LC3/DAPI
A D Figure 4 DMSO CQ (10ȝM) Baf (1nM) CQ Baf DMSO 0 50 100 150 *** *** -Vem +Vem Cell number/field DAPI ATG12-ATG5 LC3B-I GAPDH shCntrl shA TG5-1 shA TG5-2 C -Vem +Vem 0 50 100 150 Cell number/field shATG5-1 shATG5-2 shCntrl **** **** shCntrl shATG5-1 shATG5-2 p62 LC3B-II GAPDH DMSO CQ Baf (10ȝM) (1nM) p62 B LC3B-I LC3B-II DAPI -V emurafenib +V emurafenib -V emurafenib +V emurafenib
B
Figure 5
A AS703026 DMSO -AICAR +AICAR C YUSIV Control YUSIV shATG5-1 YUSIV shATG5-2 0 40 80 120 0 40 80 120 0 40 80 120 DMSO AICAR AS703026 AS703026+AICAR % C e ll v ia b ilit y DMSO AICAR AS703026 AS703026+AICAR % C e ll v ia b ilit y % C e ll v ia b ilit y ns ns *** AS703026 DMSO -AICAR +AICAR AS703026 DMSO -AICAR +AICAR DAPI DAPI DAPIB
Cntrl Vemurafenib
C
Cntrl Vem CQ Vem+CQ
Mean tumor volume (mm
3) 0 50 100 150 200 * Cntrl Vem CQ Vem+CQ
Figure 6
YUKSI (BRAFV600mut)
A Subcutaneous injection Drug treatment 3-4 weeks Assess tumor size 3 weeks Tumor growth LC3B pAMPK pERK1/2 ERK1/2
MDA-MB-231 0 0.1 1 YUSIV 0 0.1 1 Vemurafenib [μM] 48 hrs AMPKα pAMPKα pULK1
Supplementary Figure 1
B A DMSO VemurafenibGlucose analog (2-NBDG) uptake
(fluor . a.u.) 0 200 400 600 **** BRAFV600WT
Supplementary Figure 2
Vemurafenib
DMSO Vem (magnified)
A GAPDH p62 ERK1/2 LC3B-I LC3B-II pERK1/2
Vem [1μM, 48hrs] YUKSI YUSIK YUSIT- + - + - +
B MCF7 (BRAF WT/RAS WT) AS703026 DMSO T47D (BRAF WT/RAS WT) F E Vem [μM] 48 hrs pERK1/2 ERK1/2 LC3B-I LC3B-II YUSIV 0 0.1 1 YUVON 0 0.1 1 MDA-MB-231 0 0.1 1 DMSO Vemurafenib
AZD628 (BRAFi) AS703206 (MEKi)
BRAFV600WT
GFP-LC3/β-Tubulin/DAPI GFP-LC3/β-Tubulin/DAPI
GFP-LC3/β-Tubulin/DAPI GFP-LC3/β-Tubulin/DAPI
GFP-LC3/LAMP1/DAPI GFP-LC3/LAMP2/DAPI GFP-LC3/p62/DAPI GFP-LC3/LAMP1/DAPI GFP-LC3/LAMP2/DAPI GFP-LC3/p62/DAPI BRAFV600mut GFP-LC3/DAPI GFP-LC3/DAPI 1μM Vem [hr] pULK1 pAMPKα AMPKα 0 2 4 8 24 48 LC3B-I LC3B-II
YUSKI (BRAFV600mut)
C D
LAMP-1
LAMP-2
Supplementary Figure 3
AS703026 [μM] 48 hrs pAMPKα AMPKα GAPDH pERK1/2 ERK1/2 LC3B-I LC3B-II 0 0.1 1 NCI-H460 0 0.1 1 T1KRASmut NSCLC lines
G0/G1 G2/M S G0/G1 G2/M S G2/M S G0/G1 G2/M S G0/G1 Number FL3H (Propidium Iodide) A YUKSI DMSO Vem YUSIK DMSO Vem +Cntrl Ve m DMSO Pro caspase-3 Cleaved caspase-3 D BIP ATF4 PERK GAPDH +Tg DMSO Vem B C
-Vem +Vem
Relative cell survival
0.0 0.4 0.8 1.2 shATG7-1shATG7-2 shCntrl p62 ATG12-ATG5 ATG7 shCntrl shATG5 shATG7 1 2 1 2 GAPDH Fr ac tion su rv iv in g shCntrl shATG5-1 shATG5-2 Vemurafenib [M] 10-7 10-6 10-5 -0.5 0.0 0.5 1.0 1.5 Fr ac tion su rv iv in g -0.5 10-7 10-6 10-5 0.0 0.5 1.0 1.5 shCntrl shATG7-1 shATG7-2 Vemurafenib [M] p62 ATG12-ATG5 GAPDH LC3B-I LC3B-II Vem - + - + - + shCntrl shATG5-1 shATG5-2 10-5 Fr ac tion su rv iv in g 10-7 10-6 -0.5 0.0 0.5 1.0 1.5 Vemurafenib [M] DMSO CQ Baf A C E B D F Cntrl CQ
YUSIK (BRAFV600mut)
-V emuraf enib G CQ Cntrl 100 200 300
Supplementary Figure 4
YUKSI (BRAFV600mut)
*
A 10-7 10-6 10-5 10-4 -0.5 0.0 0.5 1.0 1.5 Drug [M] Fr ac tio n su rv iv ing Vem AZD628 AS703026 AZD6244 YUSIV (BRAFV600WT) B DMSO GAPDH pULK1 AMPKα ERK1/2 pERK1/2 DMSO AICAR pAMPKα AICAR -CQ +CQ 100 0 25 50 75
DMSO AS703026 AS703026 +AICAR Cell number/field C *** ns GAPDH AMPKα ERK1/2 pERK1/2 pAMPKα DMSO Vemurafenib
YUGEN8 (BRAFV600mut)
0 100 200 300 400 500 AICAR DMSO -Vem +Vem Cell number/field
***
-AICAR +AICAR Vemurafenib - + - + - - + + AICARSupplementary Figure 5
D ESupplementary Figure 6
A 0 10 20 30 Cntrl Vem CQ Vem+CQ Neccrotic tissue (%)YUKSI (BRAFV600mut)
Cntrl Vem CQ Vem+CQ
B
YUKSI_shCntrl
YUKSI_shATG5 Mean tumor volume (mm
3) * 0 25 50 75 100 125 shCntrl shATG5
Mean tumor weight (mg)
0 25 50 75 100 125 * shCntrl shATG5
YUSIK (BRAFV600mut)
m ali ze d tu m or gro w th 1 2 3 4 Cntrl Vem CQ Vem+CQ * 1.0 Vemurafenib 0.0 0.2 0.4 0.6 0.8 Tumor fraction GFP+ (shCntrl) GFP- (shATG5) ** Vehicle Assess cell fraction Tumor growth Vemurafenib
3-4 weeks 3 weeks GFP+ (shCntrl) (shATG5)GFP -C D N N N