Control of Tumor Bioenergetics
and Survival Stress Signaling
by Mitochondrial HSP90s
Young Chan Chae,1M. Cecilia Caino,1Sofia Lisanti,1Jagadish C. Ghosh,1Takehiko Dohi,1Nika N. Danial,3
Jessie Villanueva,2Stefano Ferrero,4Valentina Vaira,1,7Luigi Santambrogio,6Silvano Bosari,5Lucia R. Languino,1,8
Meenhard Herlyn,2and Dario C. Altieri1,*
1Prostate Cancer Discovery and Development Program
2Melanoma Research Center
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
3Dana-Farber Cancer Institute, Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
4Department of Biomedical, Surgical and Dental Sciences, University of Milan Medical School and Division of Pathology
5Department of Clinical/Surgical Pathophysiology and Organ Transplant, University of Milan Medical School and Division of Pathology
6Department of Clinical/Surgical Pathophysiology and Organ Transplant, University of Milan Medical School and Division of Thoracic Surgery
and Lung Transplantation
Fondazione IRCCS Ca ` Granda, Ospedale Maggiore Policlinico, Milan 20122, Italy
7Division of Pathology, Fondazione IRCCS Ca ` Granda, Ospedale Maggiore Policlinico, Milan 20135, Italy
8Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA
Tumors successfully adapt to constantly changing intra- and extracellular environments, but the wirings of
this process are still largely elusive. Here, we show that heat-shock-protein-90-directed protein folding in
vates a signaling network that involves phosphorylation of nutrient-sensing AMP-activated kinase, inhibition
of rapamycin-sensitive mTOR complex 1, induction of autophagy, and expression of an endoplasmic retic-
ulum unfolded protein response. This signaling network confers a survival and proliferative advantage to
genetically disparate tumors, and correlates with worse outcome in lung cancer patients. Therefore, mito-
chondrial heat shock protein 90s are adaptive regulators of tumor bioenergetics and tractable targets for
Heat shock protein-90 (HSP90) chaperones oversee protein-
folding quality control in virtually every organism (Mayer, 2010).
This process is essential for cellular homeostasis, buffering pro-
teotoxic stress, and enabling cells to continuously adapt to
changes in their internal and external milieus (Taipale et al.,
2010). HSP90 plasticity has been traditionally linked to the
diversity of its ‘‘client proteins’’, molecules that are implicated
in multiple facets of cellular maintenance and require the
chaperone ATPase activity for proper folding, maturation, and
subcellular trafficking (Taipale et al., 2010). Successful cellular
adaptation must also encompass fine-tuning of bioenergetics,
nutrient sensing, and stress-response signaling, including
autophagy (Yang et al., 2011), although a role of HSP90 in these
pathways remains poorly defined. This may be important in
cancer, where HSP90 chaperoning is universally exploited
(Trepel et al., 2010), and it may help transformed cells thrive in
unfavorable environments that are chronically depleted of
oxygen and nutrients (Rodina et al., 2007).
Flexible adaptation to evolving environmental cues is a universal trait of human tumors, regardless of tissue of origin or
genetic makeup, and it plays an important role in disease outcome. Generally considered a part of the cellular stress
response, adaptive mechanisms influence cell metabolism, preserve proliferation, and promote cell survival. However,
whether these pathways operate as an integrated signaling network that contributes to tumor maintenance has not been
clearly delineated. The data presented here identify control of protein folding by mitochondrial, but not cytosolic,
HSP90s as a global integrator of tumor bioenergetics, autophagy, and interorganelle stress-response signaling. Exploited
to promote cell survival and cell proliferation in genetically disparate tumors, this adaptive network offers prime opportuni-
ties for cancer therapy.
Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc. 331
Adding complexity to chaperone-directed protein homeo-
stasis, or proteostasis, is the role of HSP90-like molecules com-
partmentalized in the endoplasmic reticulum (ER) (Richter et al.,
2007) and mitochondria (Leskovar et al., 2008). The function of
these specialized HSP90s in buffering the organelle protein-
folding environment is beginning to emerge (Haynes and Ron,
2010), and deregulation of these pathways may contribute to
human disease, including neurodegeneration (Gandhi et al.,
2009) and cancer (Rodina et al., 2007). Specifically, HSP90 and
protein-1 (TRAP-1), are abundantly present in mitochondria of
tumor (though not most normal) cells (Kang et al., 2007), where
they maintain organelle proteostasis (Siegelin et al., 2011) and
antagonize mitochondrial permeability transition (Kang et al.,
2007)mediated bythe matriximmunophilin,cyclophilinD(CypD)
(Green and Kroemer, 2004).
In this study, we asked whether HSP90-directed protein fold-
ing influences cellular energy production (Taipale et al., 2010),
especially in tumors.
Regulation of Tumor Bioenergetics by Mitochondrial
To begin exploring a role of mitochondrial chaperones in cellular
energy production, we used Gamitrinib, a small-molecule inhib-
itor of HSP90 and TRAP-1 ATPase activity engineered to selec-
tively accumulate in mitochondria (Kang et al., 2009). For these
studies, we used 5 hr incubations with Gamitrinib, which neither
reduce the viability of several independent tumor-cell types (Fig-
ure S1A available online) nor affect mitochondrial membrane
potential (Figure S1B) but do create nonlethal proteotoxic stress
in mitochondria, characterized by accumulation of misfolded
and insoluble proteins (Siegelin et al., 2011).
Conversely, 17-allylamino demethoxygeldanamycin (17-AAG),
which inhibits HSP90 ATPase activity in the cytsosol (Trepel
et al., 2010) but not in mitochondria (Kang et al., 2009), had no
effect (Figure 1B). The effect of Gamitrinib on bioenergetics was
selective for tumor cells, as FF2508 or MRC5 normal primary
human fibroblasts were not affected (Figure 1C). Instead, Gami-
trinib reduced glucose utilization (Figure 1D), extracellular lac-
tate levels (Figure 1E), and oxygen consumption (Figure 1F), the
latter a marker of impaired oxidative phosphorylation, in tumor
cells. Supplementation of exogenous sodium pyruvate failed
to rescue ATP production in Gamitrinib-treated tumor cells (Fig-
species (ROS), which were unaffected by different concentra-
one of the HSP90 targets of Gamitrinib in mitochondria (Kang
cells (Figure 1J). A nontargeting siRNA had no effect (Figure 1J).
HSP90 Controls CypD Protein Folding and Hexokinase-II
Recruitment to Tumor Mitochondria
We next asked how mitochondrial HSP90s controlled tumor
bioenergetics. Mitochondrial proteotoxic stress induced by Ga-
itant accumulation in the cytosol (Figure 2A) HK-I expression and
subcellular localization were not affected (Figure 2A). HK-II teth-
ering to mitochondria is required for glycolysis (Vander Heiden
et al., 2009) and for coupling glucose metabolism to oxidative
phosphorylation. Consistent with this model, Gamitrinib-treated
tumor cells exhibited decreased hexokinase activity, whereas
17-AAG had no effect (Figure 2B). siRNA knockdown of TRAP-1
gave similar results, with detachment of HK-II from tumor mito-
chondria (Figure 2C) and loss of hexokinase activity (Figure 2D).
In mitochondria, HSP90 binds the matrix peptidyl prolyl isom-
erase (PPIase), CypD (Kang et al., 2007), a component of the
permeability transition pore (Green and Kroemer, 2004), which
has been implicated in HK-II recruitment to the organelle outer
membrane (Machida et al., 2006). Accordingly, siRNA knock-
down of CypD released HK-II from mitochondria (Figure 2C)
Mitochondria isolated from CypD?/?mouse embryonic fibro-
blasts (MEFs) showed reduced content of HK-II compared to
WT (CypD+/+) MEFs (Figure 2E). However, transfection of these
cells with a WT CypD cDNA restored binding of HK-II to mito-
chondria, whereas an H168Q CypD mutant defective in PPIase
activity, or empty vector, had no effect (Figure 2F). As a control,
the expression of voltage-dependent anion channel (VDAC) was
not affected (Figure 2F). We next asked whether CypD retention
of HK-II involved chaperone-directed protein folding. Inhibition
of mitochondrial HSP90s by Gamitrinib rendered CypD insoluble
at increasing detergent concentrations, which suggests protein
misfolding and aggregation, compared to untreated cultures
(Figure 2G). In contrast, the folding of another mitochondrial
protein, COX-IV was indistinguishable in control or Gamitrinib-
treated cells, and VDAC remained insoluble at all detergent con-
centrations used (Figure 2G).
Regulation of Energy-Sensing Pathways by
The downstream implications of defective HK-II-dependent bio-
energetics were next investigated. First, siRNA silencing of the
energy-sensing AMP-activated kinase (AMPK) (Mihaylova and
Shaw, 2011) did not affect HK-II association with tumor mito-
chondria (Figure 3A) positioning its function downstream of
HK-II-directed bioenergetics (Mihaylova and Shaw, 2011). Con-
versely, tumor cells treated with Gamitrinib, but not 17-AAG, ex-
ure 3B; Figure S2A). This response occurred within 30 min of
Gamitrinib treatment, remained sustained for 9 hr (Figure 3C),
and was quantitatively more robust than that induced by metfor-
min, a known AMPK inducer (Figure 3D). Total AMPK levels were
unaffected (Figures 3B–3D), and the combination of metformin
plus Gamitrinib did not further stimulate AMPK phosphorylation
Silencing ofAMPK (Figure S2B)or its mainupstreamactivator,
the serine/threonine kinase LKB1 (Mihaylova and Shaw, 2011)
(Figure S2C), using multiple independent siRNA sequences sup-
pressedAMPKphosphorylation mediatedbyGamitrinib (Figures
S2B and S2C). This response was selective for tumor cells, as
FF2508 or MRC5 primary human fibroblasts did not activate
AMPK in response to Gamitrinib, consistent with the absence
Mitochondrial HSP90s in Tumor Bioenergetics
332 Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc.
of TRAP-1 in normal mitochondria (Figure S2D) (Kang et al.,
2007). In complementary studies, TRAP-1 knockdown in tumor
cells using various siRNA sequences (Figure 3E) stimulated
AMPK phosphorylation and induced detachment of HK-II, but
not VDAC or COX-IV, from mitochondria (Figure 3E).
Downstream of AMPK activation, Gamitrinib treatment in-
hibited the rapamycin-sensitive mammalian target of rapamycin
complex-1 (mTORC1) in tumor cells (Wullschleger et al., 2006),
with loss of phosphorylation of mTOR and its downstream
targets, p70S6 and 4EBP1 (Figures 3F and 3G). In these exper-
iments, 17-AAG had no effect (Figure 3G), and total mTORC1
protein content was unchanged (Figures 3F and 3G). Consistent
with a selectivity of this pathway for tumor cells, Gamitrinib treat-
ment of nontransformed NIH 3T3 fibroblasts did not affect ATP
production (Figure S2E), or AMPK or mTORC1 phosphorylation
(Figure 3G). siRNA silencing of LKB1 (Figure S2F) or AMPK (Fig-
ure S2G) partially restored phosphorylation of mTOR, p70S6,
and 4EBP1 in Gamitrinib-treated tumor cells (Figures S2F and
Figure 1. Mitochondrial HSP90 Regulation of Tumor Bioenergetics
(A) Breast (MCF-7),prostate(PC3,LNCaP), lung (A549,H1473), and brain (glioblastoma,LN229) tumorcell lineswere treated with theindicated concentrationsof
Gamitrinib (Gam) for 5 hr and analyzed for ATP production. Mean ± SEM (n = 3).
(B) The indicated tumor cell lines were treated with 17-AAG (20 mM) for 5 hr and analyzed for ATP production. Mean ± SEM (n = 3).
(C)Theindicated normal(FF2508,MRC5) ortumor(LN229,PC3,BPH-1) celllineswereincubated with17-AAGorGamitrinib(10mM)for5hrandanalyzed forATP
production. Mean ± SEM (n = 3).
(D and E) LN229 cells were treated with the indicated concentrations of Gamitrinib for 5 hr and analyzed for glucose consumption (D) or extracellular lactate
content (E). Mean ± SEM (n = 3); *p = 0.015-0.022.
(F) LN229 cells were plated at the indicated number, treated with vehicle, Gamitrinib, or 17-AAG (5 mM), and analyzed for O2consumption by a fluorimetric assay.
Mean ± SEM (n = 3), *p = 0.019; **p = 0.001.
(G) PC3 or LN229 cells were incubated with sodium pyruvate (Pyr, 1 mM) in the presence (5 or 10 mM, respectively) or absence (None) of Gamitrinib for 7 hr and
analyzed for ATP production. Mean ± SD of replicates (n = 2).
(H) LN229 cells were labeled with the fluorescent dye H2-DCFA (6 mM), treated with Gamitrinib (5–10 mM), and analyzed for changes in fluorescence expression in
a luminometer, with or without the antioxidant N-acetyl-L-cysteine (10 mM, NAC). H2O2(5 mM) was used as control. Mean ± SEM (n = 4).
(I) H2-DCFA-labeled LN229 cells were treated with 10 mM Gamitrinib for the indicated time intervals and analyzed for changes in ROS production at the indicated
time intervals with or without NAC. H2O2was a control. Mean ± SEM (n = 3).
(J) LN229 transfected with control (Ctrl) or TRAP-1-directed siRNA were analyzed for changes in ATP production or extracellular lactate content (left) or TRAP-1
protein level (right). Mean ± SEM (n = 3); *p = 0.017; **p = 0.005.
See also Figure S1.
Mitochondrial HSP90s in Tumor Bioenergetics
Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc. 333
S2G). Conversely, knockdown of HK-II enhanced the effect of
Gamitrinib, with increased AMPK phosphorylation and mTORC1
inhibition (Figure S2H).
Further supporting a role of defective bioenergetics in this re-
sponse, exposure of tumor cells to the nonhydrolyzable glucose
(Egan et al., 2011), reproduced the effect of Gamitrinib, with
strong activation of AMPK and suppression of mTOR, p70S6,
and 4EBP1 phosphorylation (Figure 3H).
Mitochondrial Proteotoxic Stress Activates Prosurvival
The implication of mTORC1 inhibition by mitochondrial proteo-
toxic stress was next investigated. Consistent with an inhibitory
role of mTORC1 on autophagy, and in agreement with recent
observations (Siegelin et al., 2011), Gamitrinib strongly induced
autophagy in tumor cells, with conversion of microtubule-asso-
and appearance of a punctate fluorescence pattern of LC3-CFP
staining in transfected cells (Figures S3A and S3B). This induc-
tion of autophagy required AMPK, as siRNA silencing of LKB1
(Figure 4B) or AMPK (Figure 4C) suppressed LC3-II conversion
(Figures 4B and 4C) and autophagosome formation (Figures
S3Aand S3B)inducedbyGamitrinib. Ascontrol, siRNAsilencing
of the essential autophagy gene, ATG5, produced similar results
(Figure 4D; Figures S3A and S3B), consistent with recent obser-
vations (Siegelin et al., 2011).
We next asked whether autophagy activated by mitochondrial
proteotoxic stress influenced tumor cell viability (Siegelin et al.,
2011). Inhibition of phagosome formation by 3-methyadenine
(3-MA) (Figure 4E), or siRNA knockdown of ATG5 (Figure 4F)
(Siegelin et al., 2011) or LKB1 (Figure 4G), enhanced tumor cell
killing mediated by suboptimal concentrations of Gamitrinib.
Similarly, siRNA silencing of HK-II (Figure S3D) potentiated Ga-
mitrinib-induced tumor cell death (Figure 4H), as characterized
by increased Annexin V labeling, compared to control transfec-
tants (Figure 4I). In contrast, the combination of 17-AAG plus
3-MA (Figure 4E), or 17-AAG plus siRNA silencing of LKB1 (Fig-
ure 4G) or HK-II (Figure 4H), did not decrease tumor cell viability
compared to each treatment alone.
Figure 2. Mitochondrial HSP90 Control of CypD Folding and HK-II Recruitment
(A) LN229 cells were treated with Gamitrinib (Gam), and cytosolic or mitochondrial (Mito) fractions were analyzed after 5 hr by western blotting. COX-IV was
a mitochondrial marker.
(B) LN229 cells were treated with 17-AAG (10 mM) or Gamitrinib (0.2–10 mM), and mitochondrial fractions were analyzed for hexokinase activity after 5 hr. Mean ±
SD (n = 2); **p = 0.005–0.004.
(C) LN229 cells were transfected with control (Ctrl) or with CypD- or TRAP-1-directed siRNA, and isolated mitochondrial (Mito) or cytosol fractions were analyzed
by western blotting after 48 hr.
(D) Mitochondrial fractions from LN229 cells transfected as in (C) were analyzed for hexokinase activity after 48 hr. Mean ± SD (n = 2); ***p = 0.0009; **p = 0.0024.
(E) Mitochondrial (Mito) or cytosol (Cyto) fractions from WT (CypD+/+) or CypD?/?MEFs were analyzed by western blotting.
(F) CypD+/+, CypD?/?, or CypD?/?MEFs reconstituted with WT or PPIase-defective H168Q mutant CypD cDNA were fractionated in cytosol or mitochondrial
(Mito) extracts, and analyzed by western blotting.
(G) LN229 cells were left untreated (None) or incubated with Gamitrinib (5 mM) and mixed with the indicated increasing concentrations of CHAPS. Detergent-
insoluble proteins were analyzed by western blotting. The bar graph shows densitometric quantification of protein bands. AU, arbitrary units.
Mitochondrial HSP90s in Tumor Bioenergetics
334 Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc.
Metabolic Interorganelle ER Signaling by Mitochondrial
Defective mitochondrial bioenergetics impair protein posttrans-
trigger an unfolded protein response (UPR) (Hetz and Glimcher,
2009).Consistent with thismodel, tumor cells treated with Gami-
trinibexhibited increased expression of inositol-requiring-1 (IRE-
1) kinase (Figure 5A), an ER stress sensor (Hetz and Glimcher,
2009), and de novo mRNA splicing, i.e., activation, of its target,
X-box protein-1 (XBP1) (Figure 5B). This was associated with
activation of other ER UPR branches (Hetz and Glimcher,
2009), with activating transcription factor-6 (ATF-6)-mediated
and PKR-like endoplasmic reticulum kinase (PERK) induction
of transcription factors CCAAT-enhancer binding protein (C/
EBPb) and C/EBP homology protein (CHOP) (Figure 5C; Figures
sient phosphorylation of PERK-regulated eIF2a in tumor cells
(Figure 5C). Upregulation of ER stress markers by Gamitrinib
occurred within 1 hr of treatment (Figure S4A), and over a broad
range of concentrations (Figure S4B), similar to the response
induced by the ER stressor tunicamycin (Figure S4D).
Figure 3. Modulation of AMPK and mTORC1 Signaling by Mitochondrial HSP90s
(A) LN229 cells weretransfected withcontrol (Ctrl) or AMPK-directed siRNA,and totalcell extracts(top)or isolated cytosol (Cyto)or mitochondrial (Mito) fractions
(bottom) were analyzed by western blotting.
(B) The various tumor cell lines were treated with the indicated concentrations of Gamitrinib, and analyzed by western blotting after 5 hr. The bar graphs show
densitometric quantification of protein bands. RU, relative units.
(C) Gamitrinib-treated (10 mM) LN229 cells were analyzed at the indicated time intervals by western blotting.
(D) LN229 cells were treated with metformin (Met, 5 mM) in the presence or absence of Gamitrinib (Gam, 5–10 mM) and analyzed after 12 hr by western blotting.
(E) LN229 cells were transfected with control (Ctrl) or the indicated individual siRNA sequences against TRAP-1, and isolated cytosol or mitochondrial (Mito)
fractions were analyzed by western blotting.
(F) The indicated tumor cell types were treated with increasing concentrations of Gamitrinib and analyzed after 12 hr by western blotting.
(G) Tumor (LN229) or normal (NIH 3T3) cell types were treated with Gamitrinib or 17-AAG (10 mM) and analyzed after 12 hr by western blotting.
(H) LN229 cells were treated with 2-DG (25 mM) and analyzed after 12 hr by western blotting.
See also Figure S2.
Mitochondrial HSP90s in Tumor Bioenergetics
Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc. 335
Next, we asked whether this ER UPR involved de novo gene
regulation of CHOP, C/EBPb, and GRP78 mRNA levels (Fig-
ure 5D). Similarly, exposure of tumor cells to Gamitrinib, 2-DG,
or tunicamycin all resulted in transcriptional activation of IRE-1,
ATF6, and PERK response elements in luciferase promoter anal-
ysis (Figures 5E and 5F). A minimal CHOP promoter region was
also transcriptionally induced by Gamitrinib (Figure 5F).
A mechanistic link between Gamitrinib-induced ER UPR and
defective mitochondrial bioenergetics was next investigated.
First, exposure of tumor cells to the mitochondrial uncoupler
carbonyl cyanide 3-chlorophenylhydrazone (CCCP) reproduced
the effect of Gamitrinib, with time-dependent phosphorylation
of AMPK (Figure S4E), upregulation of CHOP, C/EBPb, and
GRP78, inhibition of 4EBP1 phosphorylation, and stimulation
of LC3-II conversion (Figure 5G). In contrast, incubation of tumor
cells with inhibitory concentrations of the ROS scavenger
N-acetyl-L-cysteine (NAC) did not affect Gamitrinib-induced
phosphorylation of AMPK or its target acetyl-CoA carboxylase
(ACC), or upregulation of ER stress markers (Figure 5H). Con-
versely, energy deprivation, or impaired N-linked glycosylation
(Kurtoglu et al., 2007), caused by 2-DG mimicked the effect of
Gamitrinib and resulted in concentration (Figure S4F)- and time
(Figure S4G)-dependent upregulation of ER UPR in tumor cells.
When combined with 2-DG, Gamitrinib maximally stimulated
AMPK and eIF2a phosphorylation in tumor cells (Figure S4H).
This resulted in complete translational repression, and ablation
of GRP78, CHOP, or C/EBPb levels (Figure S4H). Functionally,
this was associated with enhanced tumor cell killing by sub-
optimal concentrations of Gamitrinib, compared to each agent
alone (Figure S4I).
In parallel experiments, exposure of tumor cells to low-
ylation and increased the expression of CHOP, C/EBPb, and
GRP78 (Figure 5I). Supplementation of tumor cells with high-
glucose-containing medium partially reversed this response
and attenuated the expression of ER UPR markers and AMPK
phosphorylation in the presence of Gamitrinib (Figure 5I). Similar
to ATP production (Figure 1G), addition of exogenous sodium
pyruvate did not modulate AMPK or ER UPR signaling by Gami-
trinib (Figure 5J).
Cytoprotective Role of ER UPR Induced by Gamitrinib
We next mapped the requirements of Gamitrinib-induced ER
UPR. First, siRNA knockdown of HK-II was insufficient, alone,
to upregulate the expression of CHOP or GRP78, promote
Figure 4. Mitochondrial Proteotoxic Stress Stimulates Autophagy
(A) LN229 cells treated with Gamitrinib or 17-AAG (10 mM) for 12 hr were analyzed by western blotting.
(B–D) LN229 cells were transfected with control (Ctrl), or with LKB1-, AMPK-, or ATG5-directed (B–D, respectively) siRNA, treated with vehicle or Gamitrinib
(10 mM), and analyzed after 48 hr by western blotting.
(E) LN229 cells were treated with the inhibitor of phagosome formation, 3-MA, treated with Gamitrinib or 17-AAG (10 mM), and analyzed for cell viability by 3-(4,5-
dimethylthiazol-2-YI)-2,5-diphenyltetrazolium bromide (MTT). Mean ± SD (n = 2); **p = 0.0072.
(F and G) LN229 cells were transfected with control siRNA (Ctrl) or with ATG5- or LKB1-directed siRNA (F and G, respectively), incubated with 17-AAG or
Gamitrinib (10 mM) (G), and analyzed for cell viability by MTT. Mean ± SEM (n = 4). *p = 0.02; ***p < 0.0004.
(H) LN229 cells were transfected with control (squares) or HK-II-directed (circles) siRNA, treated with increasing concentrations of 17-AAG (black) or Gamitrinib
(purple), and analyzed after 12 hr for cell viability by MTT. Mean ± SD (n = 2); *p = 0.02.
(I) LN229 cells were transfected with control (Ctrl) or HK-II-directed siRNA, treated with vehicle (None) or Gamitrinib, and analyzed for Annexin V and propidium
iodide staining by multiparametric flow cytometry. The percentage of cells in each quadrant is indicated.
See also Figure S3.
Mitochondrial HSP90s in Tumor Bioenergetics
336 Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc.
AMPK phosphorylation, or stimulate LC3-II conversion in tumor
cells (Figure 6A). However, the combination of HK-II knockdown
plus Gamitrinib enhanced AMPK phosphorylation, ER UPR in-
duction, and autophagy in tumor cells (Figure 6A). This path-
way still depended on impaired tumor bioenergetics, as siRNA
knockdown of AMPK (Figure 6B) or LKB1 (Figure 6C) attenuated
Gamitrinib-induced phosphorylation of AMPK, ACC, and the up-
regulation of ER UPR markers (Figures 6B and 6C). Conversely,
siRNA silencing of the ER UPR effector GRP78 (Figure 6D; Fig-
ure S5) did not affect phosphorylation of AMPK or mTORC1
kinases in the presence of Gamitrinib, positioning GRP78 induc-
cally, siRNA knockdown of the ER stress sensor IRE-1 did not
significantly affect the induction of UPR markers by Gamitrinib
(Figure 6E). In contrast, knockdown of PERK, alone or in combi-
nation with IRE-1 silencing, inhibited the expression of CHOP
and C/EBPb and abolished eIF2a phosphorylation induced
by Gamitrinib, whereas GRP78 was not significantly affected,
and no changes were observed in LC3 conversion (Figure 6E).
In all silencing experiments, a nontargeting siRNA was ineffec-
tive (Figures 6A–6E).
Next, we asked whether components of this ER UPR influ-
enced tumor cell functions. Silencing of GRP78 using multiple
independent siRNA sequences inhibited tumor cell proliferation
compared to control siRNA transfectants (Figure 6F). Similar
results were obtained in different cell types (Figure 6G), indi-
cating a general requirement of GRP78 for tumor cell prolifera-
tion. In addition, GRP78 knockdown decreased the viability of
selected tumor cell types including prostate cancer PC3 or
LNCaP cells (Figure 6H). In contrast, IRE-1 or PERK knockdown,
alone or in combination, did not reduce tumor cell viability in the
presence or absence of Gamitrinib (Figure 6I).
Chaperone-Regulated Bioenergetics Controls Tumor
Next, we asked whether the signaling pathway controlled by
mitochondrial HSP90s was important for tumor maintenance.
In a first model, we looked at melanoma cells, where a V600E
mutation of the BRAF oncogene results in ERK-mediated inhib-
itory phosphorylation of LKB1 and suppression of AMPK activa-
tion (Zheng et al., 2009). Accordingly, two BRAF mutant mela-
noma cell lines, which exhibited hyperphosphorylated ERK,
ure S6A). Conversely, Gamitrinib induced AMPK phosphoryla-
tion in WT BRAF melanoma cells with low levels of phosphory-
lated ERK (Figure 7A; Figure S6A). 17-AAG had no effect on
AMPK activation in WT or mutant BRAF melanoma cells (Fig-
ure 7A; Figure S6A). Similar to the data above, AMPK activation
by mitochondrial stress activated autophagy in WT BRAF cells,
whereas BRAF mutant cells did not increase autophagy in
response to Gamitrinib (Figure 7B). Functionally, mutant BRAF
melanoma cells exhibited increased sensitivity to Gamitrinib-
induced cell death compared to WT BRAF melanoma cells
(IC50BRAF V600E, 1.95 ± 0.21; IC50BRAF WT, 6 ± 1.4) (Fig-
ure 7C). This response was due to differential activation of
compensatory autophagy, as siRNA knockdown of AMPK sup-
pressed autophagy in WT BRAF cells (Figure 7D; Figure S6B)
and enhanced their sensitivity to Gamitrinib-mediated killing
(Figure 7E; Figure S6C). As control, an inhibitor of MEK, U0126,
partially restored AMPK phosphorylation in mutant BRAF mela-
noma cells after Gamitrinib treatment (Figure S6D). To examine
tuted melanoma cell growth in 3-D spheroids embedded in a
collagen matrix (Villanueva et al., 2010). In this system, low con-
centrations of Gamitrinib (1–3 mM) efficiently killed mutant BRAF
melanoma cells, whereas WT BRAF spheroids were resistant to
cell death (Figure 7F).
Mitochondrial HSP90-Directed Bioenergetics
Influences Tumor Outcome
To determine whether mitochondrial HSP90-directed signaling
occurred in vivo, we next examined a genetic model of prostate
cancer in immunocompetent TRAMP (transgenic adenocarci-
noma of the mouse prostate) mice treated systemically with Ga-
mitrinib (Kang et al., 2011). In this model, Gamitrinib inhibited
prostatic intraepithelial neoplasia (PIN) (Kang et al., 2011). Here,
Gamitrinib treatment was associated with increased expression
of phosphorylated AMPK, induction of autophagy, i.e., LC3 con-
version, and upregulation of GRP78 in PIN lesions, but not in
normal prostate (Figure 8A). Prostate tissues from TRAMP mice
treated with vehicle did not express these markers (Figure 8A).
We next looked at primary human tumor specimens, and we
focused on a potential role of the ER stress chaperone GRP78
in disease progression in vivo. Except for lymphoma, GRP78
was strongly and uniformly upregulated in the tumor cell popula-
tion of a large panel of genetically heterogeneous cancers, as
shown by tissue microarray (TMA; Figure 8B; Figure S7A).
Accordingly, GRP78 was abundantly expressed in non-small-
cell lung cancer (NSCLC) patients (Table S1) with adenocarci-
noma (AdCa) or squamous cell carcinoma (SCC) (Figures 8C
and 8D), regardless of tumor stage (Figure S7B), or lymph
node metastasis (Figure S7C). Conversely, GRP78 was unde-
tectable in the normal epithelium of the lung (Figures 8C and
8D). When stratified for disease outcome, patients with lung
AdCa expressing GRP78 had considerably shorter overall sur-
vival compared to those with low to undetectable GRP78
Based on these results, we asked whether deregulated ex-
pression of GRP78 influenced cell proliferation and/or survival
of lung cancer cells. siRNA silencing of GRP78 (Figure 8F)
induced loss of viability of H1299 and A549 lung cancer cells,
ure 8G). Similar to other tumor types (Figures 6F and 6G),
silencing of GRP78 suppressed proliferation of all lung cancer
cells tested (Figure 8H), whereas a nontargeting siRNA had no
effect (Figures 8G and 8H).
In this study, we have shown that HSP90s compartmentalized
in mitochondria (Kang et al., 2007) are essential regulators of
bioenergetics in tumor cells but not normal cells. This pathway
controls both glycolysis and oxidative phosphorylation and
involves chaperone-dependent retention of HK-II (Vander Hei-
den et al., 2009) to the organelle outer membrane (Majewski
et al., 2004). Interference with chaperone control of mitochon-
drial protein folding causes acute decrease in ATP production
Mitochondrial HSP90s in Tumor Bioenergetics
Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc. 337
Figure 5. Regulation of ER UPR by Mitochondrial HSP90s
(A) PC3 cells were incubated with Gamitrinib (5 mM) and analyzed at the indicated time intervals by western blotting.
(B) Gamitrinib-treated tumor cells were harvested at the indicated time intervals, and total RNA was amplified with primers to detect spliced (s) or unspliced (u)
XBP1 mRNA transcripts. GAPDH was used as a control.
(C) LNCaP cells were treated with Gamitrinib and analyzed at the indicated time intervals by western blotting.
(D) Gamitrinib-treated LNCaP cells were harvested at the indicated time intervals and analyzed for changes in CHOP, C/EBPb, or GRP78 mRNA expression by
quantitative PCR. Mean ± SEM of replicates of a representative experiment (n = 3).
(E) Schematic diagram of ER stress luciferase-promoter reporter constructs used in this study.
(F) PC3 cells were transfected with the indicated luciferase-promoter reporter constructs, or with a CHOP minimal promoter upstream of a luciferase gene,
incubated with Gamitrinib (5 mM), tunicamycin (Tun, 2.5 mg/ml), or 2-DG (25 mM), and analyzed for changes in luciferase expression in a luminometer after 20 hr.
Mean ± SEM (n = 4). None, untreated.
(G) PC3 cells were treated in the presence (+) or absence (?) of the mitochondrial uncoupler CCCP and analyzed after 6 or 16 hr by western blotting.
(H)Theindicated tumorcelltypeswereincubated without(None) orwith5mMGamitrinibinthepresenceorabsenceoftheindicated concentrationsofNAC(20or
50 mM) and analyzed after 6 hr by western blotting.
Mitochondrial HSP90s in Tumor Bioenergetics
338 Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc.
and activation of an integrated signaling network, with phos-
phorylation of AMPK (Mihaylova and Shaw, 2011), inhibition
of mTORC1 (Wullschleger et al., 2006), induction of autophagy
(Yang et al., 2011), and stimulation of ER UPR (Hetz and
Glimcher, 2009). Functionally, this pathway provides prolifera-
tive and cytoprotective compensatory signals for tumor cells,
has been recapitulated in a genetic mouse model of prostate
cancer in immunocompetent animals (Kang et al., 2011), and
correlates with shortened overall survival in patients with lung
CypD is the only known component of a mitochondrial perme-
ability transition pore (Green and Kroemer, 2004) that is required
for cell death triggered by certain stimuli, for instance, oxidative
stress (Baines et al., 2005; Nakagawa et al., 2005). How this
process is regulated is still a matter of debate, but recent evi-
dence has pointed to chaperone-directed (re)folding of CypD
as a potential mechanism to preserve mitochondrial integrity,
and to antagonize apoptosis, selectively in tumor cells (Kang
et al., 2007). The structural requirements of potential HSP90-
CypD protein complexes in mitochondria (Kang et al., 2007)
remain to be fully elucidated. However, complete suppression
of chaperone ATPase activity with Gamitrinib (Kang et al., 2009)
results in misfolding and aggregation of CypD (this study), culmi-
nating in acute permeability transition and CypD-dependent cell
erone inhibition using suboptimal concentrations of Gamitrinib
and shorter incubation times (Siegelin et al., 2011) uncovered
additional functional roles of this pathway, and in particular a
mechanism of CypD conformation-dependent retention of HK-
II to the outer mitochondrial membrane. In this context, detach-
ment of HK-II after nonlethal mitochondrial proteotoxic stress
(Siegelin et al., 2011) is expected to lower an antiapoptotic
threshold maintained by growth factor-Akt signaling (Robey
and Hay, 2006), but, even more importantly, to impair aerobic
et al., 2009). Whether mitochondrial proteotoxic stress affects
other pathways of ATP or biomass production in tumors remains
observed here after Gamitrinib treatment, combined with the
inability of exogenous pyruvate to restore ATP production under
these conditions, suggest that organelle HSP90s may also con-
tribute to oxidative phosphorylation. The details of this potential
response are presently unknown, but it is intriguing that loss of
HK-II (Vander Heiden et al., 2009) has been shown to shift tumor
bioenergetics from aerobic glycolysis toward oxidative phos-
phorylation (Wolf et al., 2011), potentially rendering tumor cells
especially sensitive to the pathway of mitochondrial proteotoxic-
ity described here.
Consistent with current models of bioenergetics, loss of ATP
production after mitochondrial proteotoxic stress resulted in
downstreamactivation of an LKB1-AMPK signaling axis in tumor
cells. These molecules participate in tumor suppression, and a
growing number of cancers harbor LKB1-inactivating mutations
(Hezel and Bardeesy, 2008). Here, however, activation of the
LKB1-AMPK pathway was exploited for tumor cell survival via
stimulation of autophagy (Mihaylova and Shaw, 2011). This
may reflect release of mTORC1 inhibition on autophagy initiation
(Gwinn et al., 2008; Inoki et al., 2003) and/or direct phosphoryla-
tion of the ULK-1-containing autophagy complex by AMPK
(Egan et al., 2011). How common is the exploitation of LKB1-
mined. However, AMPK phosphorylation has been observed in
hypoxic tumors deprived of nutrients (Laderoute et al., 2006).
with therapeutic concentrations of Gamitrinib (Kang et al., 2011;
this study). In addition, downstream activation of autophagy is
being increasingly recognized as a major driver of tumor mainte-
nance, potentially at later stages of disease progression (Yang
et al., 2011). In melanoma, where AMPK can be differentially
activated depending on the mutational status of the BRAF onco-
gene (Zheng et al., 2009), autophagy was a critical determinant
of cell survival, making BRAF mutant cells especially sensitive
to mitochondrial cell death initiated by Gamitrinib (Kang et al.,
2007). This observation may have clinical relevance, as mela-
noma patients carrying a V600E BRAF mutation become invari-
ably resistant to small-molecule BRAF inhibitors, pressing the
need for alternative therapeutic targets to restore treatment re-
sponses in these settings.
ATP depletion results in insufficient energy available for pro-
tein posttranslational modifications (Kaufman et al., 2002), and
in the case of mitochondrial proteotoxic stress, this triggered
a canonical ER UPR (Hetz and Glimcher, 2009). This pathway
was reproduced by pharmacologic uncoupling of mitochondrial
membrane potential, in keeping with directional mitochondria-
to-ER signaling, was partially reversed by high glucose, con-
sistent with a causal role of defective ATP production in this
process (Kaufman et al., 2002), and required LKB1-AMPK acti-
vation as part of bioenergetics signaling (Mihaylova and Shaw,
2011). The role of ER stress in cancer is complex, and prolonged
activation of this pathway culminates with apoptosis contrib-
uted, at least in part, by transcriptional modulation of Bcl-2
proteins (Tabas and Ron, 2011). However, low-level, chronic
ER stress may be beneficial for tumor growth (Ma and Hender-
shot, 2004), and the inducible ER chaperone GRP78 was identi-
fied here as an effector of tumor cell survival and proliferation
during bioenergetics ER stress (Pfaffenbach and Lee, 2011).
Cytoprotection by GRP78 may involve modulation of multiple
antiapoptotic thresholds, including differential assembly of Bcl-
2 homodimers (Zhou et al., 2011), and, as shown here, this path-
way may become broadly exploited in genetically disparate
cancers, correlating with shortened overall survival in patients
with lung adenocarcinoma (this study), or prostate cancer (Tan
et al., 2011). Regarding a potential role of other ER markers in
disease outcome, high levels of eIF2a have been associated
with improved survival in stage I, but not stages II–IV, NSCLC
patients (He et al., 2011).
(I) LN229 cells were cultivated in the presence of the indicated increasing concentrations of glucose-containing medium without (None) or with Gamitrinib (5 mM)
and analyzed by western blotting.
(J) LN229 cells were treated with the indicated concentrations of Gamitrinib in the presence (+) or absence (?) of sodium pyruvate (Pyr, 1 mM), and analyzed after
7 hr by western blotting.
See also Figure S4.
Mitochondrial HSP90s in Tumor Bioenergetics
Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc. 339
Figure 6. Functional Requirements of ER UPR Induced by Mitochondrial Proteotoxic Stress
(A–E)The indicated tumorcell linesweretransfected withcontrol siRNA(Ctrl), orwithsiRNAdirected toHK-II(A), AMPK (B),LKB1(C), GRP78 (D),or theERstress
sensors IRE-1 or PERK, alone or in combination (E), incubated in the presence or absence (None) of Gamitrinib (5 mM), and analyzed 24–48 hr after siRNA
transfection by western blotting. The bar graphs in (E) show densitometric quantification of normalized C/EBPb, CHOP, GRP78, LC3-II, or phosphorylated eIF2a
bands in the presence of Gamitrinib. Basal eIF2a levels in the absence of Gamitrinib were also calculated.
(F) A549 or PC3 cells were transfected with control siRNA (Ctrl) or the indicated individual siRNA sequences to GRP78, and analyzed after 48 hr by western
blotting. Bottom, siRNA-transfected cells as in the top images were analyzed for cell proliferation by direct cell counting. Mean ± SEM of three independent
experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
(G and H) The indicated tumor cell types were transfected with control siRNA (Ctrl) or GRP78-directed siRNA and analyzed for cell proliferation by direct cell
counting (G) or cell viability by MTT (H). Mean ± SEM (n = 8 in [G] and 3 in [H]); *p = 0.016; **p = 0.017–0.0055; ***p < 0.0001.
(I) siRNA-transfected PC3 cells as in (E) were treated in the absence (None) or presence of 5 mM Gamitrinib and analyzed for cell viability by MTT. Mean ± SEM of
replicates of a representative experiment from two independent determinations.
See also Figure S5.
Mitochondrial HSP90s in Tumor Bioenergetics
340 Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc.
Together, the results presented here add complexity to
HSP90 homeostasis (Taipale et al., 2010), uncovering a role of
the mitochondrial pool(s) of these chaperones (Kang et al.,
2007) in selective bioenergetics (Vander Heiden et al., 2009)
and stress-response signaling (Hetz and Glimcher, 2009; Yang
et al., 2011) in tumors. Although these mechanisms are critical
for tumor growth, the pathophysiological context in which mito-
getics and compensatory stress response in vivo remains to be
fully elucidated. It may be speculated that the microenvironment
of tumor growth, typically deprived of oxygen and nutrients, may
produce a chronic degree of mitochondrial proteotoxic stress
(Siegelin et al., 2011) that is further exacerbated by the higher
biosynthetic needs of transformed cells and the unique struc-
tural environment of mitochondria (Haynes and Ron, 2010). In
this context, the selective recruitment of HSP90s to tumor mito-
chondria (Kang et al., 2007) appears ideally poised to buffer
organelle proteotoxic stress in general, and specifically to
control the (re)folding of CypD (Green and Kroemer, 2004). In
turn, this prevents permeability pore opening, especially against
oxidative stimuli (Baines et al., 2005; Nakagawa et al., 2005),
maintains ATP production via HK-II tethering (Vander Heiden
et al., 2009), and connects to downstream survival mechanisms
and Glimcher, 2009; Yang et al., 2011). While this adaptive
network promotes tumor maintenance in vivo, it may also offer
tangible therapeutic prospects, as subcellular targeting of mito-
chondrial HSP90s is feasible and may selectively affect tumor
cells, but not normal tissues (Kang et al., 2009).
Mitochondrial Protein Folding
Mitochondrial protein folding assays were performed as described (Moisoi
et al., 2009). Briefly, mitochondrial fractions were isolated from vehicle or
Gamitrinib-treated LN229 cells (5 mM for 12 hr) and suspended in equal
volume of mitochondrial fractionation buffer containing increasing concen-
trations of CHAPS (0, 0.05, 0.1, 0.2, 0.5, 1, or 2%). Samples were incu-
bated for 20 min on ice, and detergent-insoluble protein aggregates were
Figure 7. Regulation of Tumor Cell Survival by Mitochondrial Proteotoxic Stress
(A)WT orV600EmutantBRAFmelanomacelllinesweretreated with17-AAG (10mM)orGamitrinib (1,2.5,5,or10mM)andanalyzedafter 5hrby western blotting.
(B) The indicated melanoma cell types were incubated with Gamitrinib (5 mM) and analyzed after 9 hr by western blotting. None, untreated.
(C) WT (WM852, WM1366) or mutant BRAF (Me1617, 451Lu) melanoma cells were treated with Gamitrinib and analyzed after 16 hr for cell viability by MTT.
Mean ± SD (n = 2).
(D) WM852 BRAF WT melanoma cells were transfected with control (Ctrl) or AMPK-directed siRNA, treated with Gamitrinib (5 mM), and analyzed by western
(E) WM852 melanoma cells transfected as in (D) were analyzed by MTT for Gamitrinib (10 mM)-mediated cell killing. Mean ± SEM (n = 3); **p = 0.002.
(F) Melanoma cells with the indicated BRAF genotype were grown as organotypic spheroids in 3D collagen-embedded matrices, incubated with the indicated
concentrations of Gamitrinib, stained after 72 hr with calcein-AM (live cells; green) and Topro-3 (dead cells; blue), and analyzed by confocal laser scanning
microscopy. Representative images were collected from one of two independent determinations.
See also Figure S6.
Mitochondrial HSP90s in Tumor Bioenergetics
Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc. 341
Figure 8. Activation of Mitochondrial Bioenergetics Signaling during Tumor Progression In Vivo
(A) Prostate samples from TRAMP mice treated systemically with vehicle or Gamitrinib were analyzed by immunohistochemistry with antibodies to phosphor-
ylated AMPK (p-AMPK), GRP78, or LC3-II. The histological diagnosis of each prostate tissue section is indicated. Right: quantification of staining intensity for
each condition. Scale bars, 50 mm.
(B) Expression of GRP78 in a universal tumor microarray was quantified by an immunohistochemistry (IHC) score. Each bar quantifies expression in the indicated
tumor sites. CNS, central nervous system. Mean ± SEM of IHC score in each individual TMA core (n = 7).
(C) Immunohistochemical reactivity of GRP78 expression in a representative NSCLC-TMA. The bottom images show areas of normal lung parenchyma negative
for GRP78 expression (arrows) adjacent to GRP78-positive lung cancer. Scale bars, 50 mm and 10 mm (bottom left).
(D) Summary of GRP78 expression in NSCLC or normal lung examined in this study. The number of cases for each histologic condition is indicated. In this series,
13 cases (6%) were not evaluable and 17 cases (8%) were negative for GRP78 expression. Bars correspond to median expression values of IHC scores with
interquartile range. AdCa, adenocarcinoma; SCC, squamous cell carcinoma; IHC, immunohistochemistry. The statistical analysis for GRP78 expression in the
various cohorts (t test) is as follows: NSCLC versus normal, p = 1.37 3 10?32; AdCa versus normal, p = 1.49 3 10?21; SCC versus normal, p = 3.9 3 10?13; AdCa
versus SCC, p = 0.051.
(E) Patients with diagnosis of lung adenocarcinoma (AdCa) with no expression (negative) or high expression (positive) of GRP78 were analyzed for overall survival
by the Kaplan-Meier method.
(F) The indicated lung adenocarcinoma cell types were transfected with control (Ctrl) or GRP78-directed siRNA and analyzed by western blotting.
(G and H) The indicated lung cancer cell lines were transfected as in (F) and analyzed for cell viability by MTT (G) or cell proliferation by direct cell counting (H).
Mean ± SEM (n = 6). *p = 0.035; ***p = 0.0001–0.0002.
See also Figure S7 and Table S1.
Mitochondrial HSP90s in Tumor Bioenergetics
342 Cancer Cell 22, 331–344, September 11, 2012 ª2012 Elsevier Inc.
isolated by centrifugation (20,000 3 g) for 20 min and processed for further
Glucose and Lactate Determination
Glucose concentrations in cell culture media were determined using a glucose
kit (Sigma-Aldrich). Briefly, 2 3 106cells were seeded in 10 cm tissue-culture
(0–20 mM) for 4 hr; 200 ml aliquots of culture medium were then incubated with
1 ml assay mixture containing 1.5 mM NAD, 1 mM ATP, 1 U/ml HK, and 1 U/ml
glucose-6-phosphate dehydrogenase (G6PDH). The glucose concentration
was determined by measuring the amount of NAD reduced to NADH by
G6PDH, and quantified spectrophotometrically at 340 nm wavelength. Extra-
cellular lactate was measured by a colorimetric assay kit (Abcam). For these
experiments, culture medium was replaced with DMEM containing vehicle
(DMSO) or Gamitrinib (5 mM) for 5 hr. Changes in lactate concentrations
were measured by analysis of lactate-dependent conversion of NADP to
NADPH in the presence of excess lactate dehydrogenase (LDH) and were
quantified by absorbance at 450 nm. All assays were performed at 25?C under
conditions of linear lactate-limited NADPH formation.
Treated tumor cells were analyzed using a fluorescence oxygen-sensitive-
probe-based oxygen measuring kit (Luxcel Bioscience). For these experi-
ments, LN229 cells were plated at increasing cell density (10–60 3 104/ml)
onblack-body,clear-bottom 96-wellplates. Theculturemediumwasreplaced
with 150 ml of phenol-free DMEM containing 10% fetal bovine serum in
the presence of 17-AAG or Gamitrinib (10 mM). Cells were further incubated
with an oxygen-sensing probe (10 pmol/well), and 100 ml of heavy mineral
oil was added to each well to seal the samples from ambient oxygen. After
2 hr incubation at 37?C, oxygen consumption was determined by quantifying
the probe fluorescence signal in each well using a plate reader (Beckman
Coulter) with excitation and emission wavelengths at 370 nm and 625 nm,
Intracellular ATP concentrations were determined by a luciferin–luciferase
method (Biochain) in a microplate luminometer (Beckman Coulter) against
standard ATP solutions used as reference. In some experiments, PC3 or
LN229 cells were incubated with sodium pyruvate (1 mM) for 7 hr in the pres-
ence of vehicle or Gamitrinib before determination of ATP production.
HK activity was measured as the total glucose-phosphorylating activity using
a standard G6PDH-coupled assay kit (BioVision). Briefly, mitochondria iso-
lated from Gamitrinib- or 17-AAG-treated LN229 cells, or cultures transfected
with various siRNAs, were homogenized in cold PBS and then centrifuged at
1000 3 g for 10 min at 4?C. HK activity was determined by analysis of
G6PDH-dependent conversion of NADP to NADPH in the presence of excess
G6PDH and 2 mM glucose, followed by quantification of absorbance at
450 nm. All assays were performed at 25?C under conditions of linear HK-
limited NADPH formation.
Genetic Model of Prostate Cancer
All experiments involving vertebrate animals were approved by an Institutional
Animal Care and Use Committee at the University of Massachusetts Medical
School and The Wistar Institute. Activation of mitochondrial HSP90 signaling
was investigated in immunocompetent TRAMP mice, as described (Kang
et al., 2011).
A series of 217 consecutive patients surgically treated for non-small-cell lung
cancer (NSCLC) at Fondazione IRCCS Ca ` Granda Hospital (Milan, Italy)
between 2000 and 2004 was available for this study. Informed consent was
obtained from all patients enrolled, and the study was approved by an Institu-
tional Review Board of the Fondazione IRCCS Ca ` Granda, Milan, Italy. Repre-
sentative tissue blocks from consenting patients with various cancer diag-
noses were used under IRB approval to construct the cancer universal TMA
(CaU-TMA) and NSCLC TMAs. For the NSCLC-TMAs, four cores of each
patient were included in the blocks, along with 16 cores of nonneoplastic
Data were analyzed by means of two-sided unpaired t tests using a GraphPad
software package (Prism 4.0) for Windows. For analysis of patient samples,
groups were compared using the Wilcoxon signed-rank or Student’s t test
as univariate statistics. For overall survival analysis, the Kaplan-Meyer method
was used. Patients negative for GRP78 (immunoreactivity score <0.25) were
plotted separately from GRP78-positive cases (score R0.25), and the two-
sided log-rank test was used to compare the two curves. Data are expressed
as the mean ± SD or mean ± SEM of multiple independent experiments.
A p value of %0.05 was considered statistically significant.
Supplemental Information includes seven figures, one table, and Supple-
mental Experimental Procedures and can be found with this article online at
We thank Drs. Winklhofer and Fafournoux for providing luciferase reporter
constructs and James Hayden for assistance with confocal microscopy.
This work was supported by National Institutes of Health (NIH) Grants
CA140043, CA78810, HL54131, and CA118005 (to D.C.A.) and Fondazione
Cariplo (to S.B.). Support for Core Facilities utilized in this study was provided
by Cancer Center Support Grant (CCSG) CA010815 to The Wistar Institute.
Y.C.C., M.C.C., S.L., J.C.G., T.D., and J.V. designed and carried out exper-
iments. N.N.D. provided CypD knockout MEFs. L.S. provided clinical follow-
up data. V.V., S.F., and S.B. provided pathological evaluation of the patient
series and analysis of patient data. Y.C.C., M.C.C., S.L., N.D.D., J.V., M.H.,
L.R.L., S.B., and D.C.A. analyzed results. Y.C.C., M.C.C., S.L., and D.C.A.
wrote the manuscript.
Received: October 21, 2011
Revised: April 23, 2012
Accepted: July 24, 2012
Published: September 10, 2012
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