ArticlePDF Available

Development of a stress response therapy targeting aggressive prostate cancer


Abstract and Figures

Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic pathways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism by which these adaptive responses are established, their functional consequences for tumor development, and their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively activated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor 2–α (P-eIF2α) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models and an inhibitor of P-eIF2α activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.
Inhibition of P-eIF2 axis results in tumor regression and prolongs survival in a humanized model of metastatic PCa. (A) Schematic highlighting origin of PDX tumors from primary (pPCa) or lymph node metastasis (mPCa). Tumors from selected patients with high-risk features, based on Gleason score and clinical stage or with lymph node metastases determined by 68 Ga-PSMA-11 positron emission tomography (PET) scans, were used to generate PDXs. Primary and metastatic tumors were confirmed from tissue collected at the time of surgery and immediately implanted into immunodeficient NOD scid gamma (NSG) mice. (B) Representative IF images of MYC/CK8 (epithelial cell marker), P-AKT/CK8, or P-eIF2/CK8, co-staining with DAPI from benign (Ben) tissue adjacent to pPCa or mPCa tumors; scale bars, 50 m. Right: Quantification of protein expression as relative mean IF intensity normalized to adjacent stromal tissue. (C) Kaplan-Meier tumor survival curves for mice bearing pPCa or mPCa tumors treated with ISRIB (10 mg/kg) or vehicle (n = 8, per cohort; **P = 0.01, log-rank test). The survival curves represent mice euthanized when tumors reached an end point of 2 cm or when the mice showed clear signs of morbidity. (D) Representative tumor sizes after 10 days of treatment. (E) Representative TUNEL staining and quantification of PDX tumors treated with vehicle or ISRIB (10 mg/kg); scale bars, 100 m (n = 3, ***P < 0.001, t test). (F) Quantification of newly synthesized proteins in vivo, assessed by incorporation of OP-Puro within PDX treated with ISRIB (10 mg/kg) or vehicle (n = 3 to 4 per arm, mean ± SEM; *P < 0.05, t test). n.s., not significant. MFI, mean fluorescence intensity.
Content may be subject to copyright.
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
1 of 11
Development of a stress response therapy targeting
aggressive prostate cancer
Hao G. Nguyen,1* Crystal S. Conn,1* Yae Kye,1 Lingru Xue,1 Craig M. Forester,2 Janet E. Cowan,1
Andrew C. Hsieh,1‡ John T. Cunningham, Charles Truillet,3 Feven Tameire,4 Michael J. Evans,3
Christopher P. Evans,5 Joy C. Yang,5 Byron Hann,6 Constantinos Koumenis,4 Peter Walter,7
Peter R. Carroll,1 Davide Ruggero1,8†
Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive
cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic path-
ways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism
by which these adaptive responses are established, their functional consequences for tumor development, and
their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of
prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively ac-
tivated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor
2– (P-eIF2) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of
poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models
and an inhibitor of P-eIF2 activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis
selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.
Adaptation to cellular stress, driven by oncogenic lesions, is one of
the most fundamental and poorly understood features of cancer cells
(1, 2). Multiple oncogenes sustain uncontrolled cancer cell growth
and division by stimulating the production of molecular “building
blocks,” such as proteins and outputs of anabolic metabolism. How-
ever, this poses an onerous expenditure of cellular resources, and it
remains poorly understood how cancer cells adapt to this increased
metabolic load. One example is an increase in total proteins being
synthesized, because cancer cells need to sustain augmented growth
and division. For instance, more than 65% of the energy in the cell
is devoted to the bioenergetically expensive process of protein syn-
thesis that is greatly increased in most cancers (3). Left unchecked,
infinite increases in the cancer cell’s biosynthetic demand would tilt
the balance from continuous growth and division to cell death. There-
fore, increases of biosynthetic processes place a high demand on cancer
cells and are a source of constant stress that must be carefully regulated
by the activation of appropriate checkpoints, which remain poorly
understood. How then do cancer cells accommodate overwhelming
stress such as an increased protein burden? Are cytoprotective re-
sponses activated in aggressive disease, and do they represent a point
of vulnerability that can be exploited for cancer therapy?
Increased protein synthesis and the flux in the endoplasmic re-
ticulum (ER) create a state of proteotoxic stress associated with the
accumulation of misfolded proteins (46). This ER stress activates
the unfolded protein response (UPR). The UPR is composed of three
signaling arms: ATF6 (activating transcription factor 6) with transcrip-
tional activity to promote ER homeostasis, IRE1 (inositol-requiring
enzyme 1) to control splicing of the transcription factor XBP1 en-
hancing ER gene expression, and PERK [PKR (RNA-activated protein
kinase)–like ER-associated protein kinase], which promotes down-
stream phosphorylation of eIF2 (eukaryotic initiation factor 2–)
(P-eIF2) on serine 51 (4). Unlike the other arms of the UPR, PERK
P-eIF2 creates a direct “brake” for general protein synthesis because
of the conversion of eIF2 from a substrate of the ternary complex,
which is necessary to promote the initiation step of mRNA transla-
tion, to an inhibitor of this complex (7, 8). Although UPR activation
has been associated with cancer, it remains poorly understood which
oncogenes and/or combinations of oncogenes control distinct arms
of this pathway in vivo during the initiation or progression of tumor
development. It is also unclear whether and when the UPR is acti-
vated during the course of cancer evolution, its specific requirements
in distinct phases of tumorigenesis, and the potential druggability of
this stress adaptation pathway in human cancers.
Here, we set out to address these outstanding questions by inves-
tigating cancer development within a specialized secretory epitheli-
al tissue. The prostate is a walnut-sized conglomerate of tubular or
saclike glands, dedicated to the production of proteinaceous secre-
tory fluid. One of the early consequences of human primary pros-
tate cancer (PCa) is a major remodeling of the cancer cell proteome
associated with increases in protein biosynthesis (911). For ex-
ample, loss of the PTEN (phosphatase and tensin homolog) tumor
1School of Medicine and Department of Urology, Helen Diller Family Comprehen-
sive Cancer Center, University of California, San Francisco (UCSF), San Francisco, CA
94158, USA. 2Division of Pediatric Allergy, Immunology and Bone Marrow Trans-
plantation, UCSF, San Francisco, CA 94158, USA. 3Department of Radiology and
Biomedical Imaging, UCSF, San Francisco, CA 94158, USA. 4Department of Radia-
tion Oncology, The Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA 19104, USA. 5Department of Urology, University of California Davis
School of Medicine, Sacramento, CA 95817, USA. 6Helen Diller Family Compre-
hensive Cancer Center, UCSF, San Francisco, CA 94158, USA. 7Department of Bio-
chemistry and Biophysics, UCSF, Howard Hughes Medical Institute, San Francisco,
CA 94158, USA. 8Department of Cellular and Molecular Pharmacology, UCSF, San
Francisco, CA 94158, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: (C.S.C.); davide.ruggero@ (D.R.)
‡Present address: Fred Hutchinson Cancer Research Center and the Department of
Medicine, University of Washington, Seattle, WA 98195, USA.
§Present address: Department of Cancer Biology, University of Cincinnati College
of Medicine, Cincinnati, OH 45267, USA.
Copyright © 2018
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim
to original U.S.
Government Works
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
2 of 11
suppressor and hyperactivation of the
oncogene MYC, accounting for nearly
50% of the lethal metastatic form of hu-
man PCa (12, 13), have major effects on
protein synthesis (1417). Thus, we rea-
soned that the prostate would provide a
good model to understand the mecha-
nis ms by which oncogenic cells bu ffer the
burden of increased protein synthesis to
prevent proteotoxic stress during can-
cer formation.
MYC amplification with PTEN loss
diminish oncogenic increases of
global protein synthesis in lethal
murine PCa
We modeled distinct stages of human PCa
in the mouse, using a newly generated
conditional transgenic MYC mouse, where
the overexpression of C-MYC is driven
in a Cre-specific manner (MycTg), in com-
bination with the conditional loss of PTEN
in the prostate epithelium (Pb-cre4;Ptenfl/fl,
herein referred to as Ptenfl/fl) (fig. S1) (18).
The advantage of this mouse is that cells
overexpressing MycTg can be traced through
expression of green fluorescent protein
(GFP) present in the targeting locus,
allowing for visualization of the earliest
events in tumorigenesis (fig. S1, A and B).
In agreement with the notion that MYC
hyperactivation may be a secondary event
for human PCa development (19), we find
that MYC overexpression alone in pros-
tate epithelium (Pb-cre4;MycTg, herein
referred to as MycTg) increased prolifera-
tion but did not result in adenocarcinoma
by 1 year of age (fig. S1, C to E). This is con-
sistent with previous reports, which showed
MYC expression under the control of sim-
ilar promoters to those used here (20, 21).
MycTg mice with concomitant lo ss of PTEN
in prostate tissue (Ptenfl/fl;MycTg) showed
significant enlargement of prostate growth
by 6 weeks of age (P < 0.0003) and acceler-
ated development of high-grade prostatic
intraepithelial neoplasia (HgPIN) compared to mice with loss of PTEN
alone (Fig.1, A and B). PTEN loss and MYC amplification cooperated
to develop adenocarcinoma by 10 weeks (Fig.1B), resulting in marked
increases in Ptenfl/fl;MycTg tumor size visualized by ultrasound (Fig.1C).
This aggressive oncogenic progression significantly decreased overall sur-
vival (P < 0.05), with a mean life span of 75 weeks (Fig.1D). Collectively,
this genetically engineered mouse model (GEMM) recapitulates aggres-
sive human PCa and results in decreased survival.
To evaluate the effects of these key oncogenes on global protein
synthesis, we assessed newly synthesized proteins by incorporation
of 35S-labeled methionine in organoid cultures. We established pri-
mary mouse three-dimensional organoid cultures to recapitulate the
cellular environment of the murine prostate gland ex vivo (Fig.1E)
(22). Organoids were derived from dissociated mouse prostate tissue
containing a mixed population of luminal and basal cell types to mimic
the histology observed in vivo (23). Western blot analysis confirmed
that MycTg expression and PTEN loss were evident and associated
with increased GFP expression and AKT phosphorylation (Fig.1E).
Consistent with the known ability of these major oncogenic path-
ways to increase protein synthesis (24, 25), either loss of PTEN or
MYC hyperactivation significantly increased global protein synthesis
by about 20% (P < 0.0003 for both). On the contrary, we observed
Relative S
35 -labeled
proteins (%)
Cancer development (%)
Tumor size (mm2)
)gm( kw 6 ta sthgieW
Weights at 10 wk (mg)
35 newly synthesized
Ptenfl/+; MycTg (n = 8)
Survival (%)
Ptenfl/fl; MycTg (n = 20
(n = 12
WT (n = 8)
75 wk
Weeks1.5 years
(n = 10
7 months:
6 wk 10 wk 6 wk 10 wk
Fig. 1. MycTG and loss of PTEN cooperate for aggressive PCa development, resulting in decreased survival. (A)
Total dehydrated prostate weights from 6- and 10-week-old mice were averaged for each genotype (n = 3 to 6 mice
per arm, mean ± SEM). wild-type, WT. (B) Phenotypical penetrance percentages for low-grade prostatic intraepithelial
neoplasia (LgPIN), HgPIN, and cancer in anterior prostate tissues from 6- and 10-week-old mice evaluated by hematoxylin
and eosin (H&E) staining. (C) Left: Representative ultrasound images of prostate tumors at 7 months outlined in yellow
from indicated genotypes; scale bars, 9 mm. Right: Quantification of prostate tumor size in mice with an average age
of 8 months (n = 5 mice per arm, mean ± SEM). (D) Kaplan-Meier survival curves for mice with the indicated geno-
types. Dotted line highlights the median life span of 75 weeks for Ptenfl/fl;MycTg mice. (E) Top: Representative bright-
field images of three-dimensional organoid structures 6 days after seeding; scale bars, 50 m. Bottom: Western blot
analyzing the organoids, showing P-AKT, PTEN, GFP for MycTg, and -actin. (F) Newly synthesized proteins measured
by 35S methionine/cysteine incorporation in organoids (left panel), with quantification relative to WT littermates
(right panel) (n = 5, mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, t test.
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
3 of 11
an unanticipated but significant damp-
ening in global protein synthesis in
Ptenfl/fl;MycTg mice (P = 0.01), de-
spite the fact that these mice devel-
oped more aggressive PCa (Fig.1F).
This observation revealed an inter-
esting paradox. It suggested that de-
spite the presence of two oncogenic
lesions that individually up-regulate
protein synthesis, a yet unknown adap-
tive response may take place when
protein synthesis is up-regulated be-
yond a specific threshold in aggres-
sive PCa.
Aggressive PCa activates a key
cellular stress response during
tumor development
Proteins that are synthesized in the
secretory pathway amount to about
30% of the total proteome in most
eukaryotic cells (4, 6). Although UPR
activation can be studied with phar-
macological inducers of ER stress,
under physiological processes, the
activation of the UPR may reduce
the unfolded protein load through
several prosurvival mechanisms, in-
cluding the expansion of the ER mem-
brane and the selective synthesis of
key components of the protein fold-
ing and quality control machinery (26).
To address how cancer cells respond
and adapt to a protein synthesis bur-
den in vivo and downstream of specific
oncogenic lesions, we tested wheth-
er a specific molecular signature of
the UPR may be activated in Ptenfl/fl- versus Ptenfl/fl;MycTg-derived PCa.
We performed quantitative immunofluorescence (IF) staining
for cleaved ATF6, P-IRE1, and P-PERK during tumor development
to test whether the UPR was activated during PCa progression. Vi-
sualizing UPR expression within prostatic tissue at 10 weeks of age
allowed us to directly gauge the activity of each arm during neoplasia.
Whereas the ATF6 and IRE1 branches of the UPR were relatively
equally activated in Ptenfl/fl and Ptenfl/fl;MycTg tissue (fig. S2A), PERK
phosphorylation was selectively increased by over 15-fold within
Ptenfl/fl;MycTg tissue compared to its near absence in Ptenfl/fl cells
(Fig.2A). Thus, PERK activation is a distinct response that may pro-
mote tumorigenesis in aggressive PCa driven by the cooperation of two
oncogenic lesions. To confirm the selective activation of PERK signal-
ing in Ptenfl/fl;MycTg mice, we evaluated the downstream signaling to
eIF2. P-eIF2 was also markedly increased in Ptenfl/fl;MycTg mice and
strongest within areas of PIN but remained absent within Ptenfl/fl
tissues (Fig.2, A and B, and fig. S2B). The expression of the ER-specific
molecular chaperone BiP was not changed and was also high in nor-
mal prostatic tissues in agreement with the secretory role of these
glands (fig. S2C). Collectively, this analysis reveals two independent,
yet linked mechanisms: (i) activation of each UPR pathway in PCa
in vivo and (ii) activation of a P-eIF2–dependent response selectively
in Ptenfl/fl;MycTg mice, which display more aggressive PCa progres-
sion and reduced survival.
Rebalancing protein synthesis through P-eIF2 is required
for aggressive PCa progression
A general UPR response may promote adaptation to proteotoxic and
ER stress, whereas the activation of P-eIF2 could place a direct brake
on the overwhelming burden of protein synthesis that occurs during
more aggressive tumorigenesis. To test this hypothesis, we used our
organoid cultures, which recapitulate the in vivo phenotype. The
Ptenfl/fl;MycTg cultures show increased activation of P-PERK, P-eIF2,
and expression of ATF4, which is a known target of the PERK–P-
eIF2 axis (Fig.3A). To determine whether the activation of this
adap tive response was altering global protein synthesis, we used a small-
molecule inhibitor of P-eIF2 activity, ISRIB, a compound that
selectively reverses the effects of eIF2 phosphorylation (fig. S3A)
(27, 28). Specifically, P-eIF2 binds its dedicated guanine nucleo-
tide exchanging factor (GEF), eIF2B, with enhanced affinity relative
to eIF2. Thus, P-eIF2 sequesters eIF2B from interacting with
eIF2 to exchange guanosine diphosphate with guanosine triphos-
phate, which is an essential step to form the translation preinitiation
complex. ISRIB increases eIF2B GEF activity by stabilizing it into a
Wild type MycTg
Ptenfl/fl Ptenfl/fl;MycTg
Ptenfl/fl Ptenfl/fl;MycTg Ptenfl/fl Ptenfl/fl;MycTg
Relative P-PERK
expressoin (fold)
Relative P-eIF2
expressoin (fold)
PIN - HgPIN Tumor growth
P-eIF2α expression
WT MycTg ;Myc
Fig. 2. The cooperation of MYC and loss of PTEN selectively activates the adaptive PERK–P-eIF2 arm of the UPR.
(A) Left: Representative IF images of P-PERK/cytokeratin 5 (CK5) or P-eIF2/CK5 co-staining with DAPI (4,6-diamidino-
2-phenylindole) used to visualize the nuclei within anterior prostate tissue from 10-week-old mice; scale bars, 100 m.
P-PERK or P-eIF2 expression quantified relative to DAPI (n = 3 mice per arm, with four images averaged per mouse, mean
± SEM). (B) Representative IF images of P-eIF2/CK5 co-staining with DAPI in anterior prostate tissue from 6-week-old mice
(scale bars, 100 m) (left panel) and directly within areas of PIN (right panel). Lower panel depicts a model showing the
timeline of PCa development within Ptenfl/fl;MycTg mice, highlighting when P-eIF2 is expressed. **P < 0.01, t test.
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
4 of 11
decamer holoenzyme to enhance the binding of the eIF2 factor,
thereby restoring protein synthesis regardless of eIF2 phospho-
rylation (29). In Ptenfl/fl organoid cultures, protein synthesis was not
altered by ISRIB treatment, despite the drug inhibiting P-eIF2
activity, as confirmed by a decrease in ATF4 expression (Fig.3B).
Conversely, we observed a marked increase of newly synthesized pro-
teins in Ptenfl/fl;MycTg cells, which show increased P-eIF2 signaling
(Fig.3B). Together, these experiments indicate that P-eIF2 creates
an adaptive response to relieve the burden of increased protein syn-
thesis within Ptenfl/fl;MycTg oncogenic cells.
In addition to PERK, other kinases can phosphorylate the eIF2 sub-
unit upon distinct stress signals: GCN2 (amino acid deprivation), PKR
(viral infection), and HRI (heme deprivation) (30). To assess whether
the selective adaptive response observed during aggressive PCa devel-
opment of Ptenfl/fl;MycTg mice was specific to the PERK–P-eIF2 axis,
we undertook a genetic approach, using Perkfl/fl mice to evaluate the loss
of PERK in the prostate gland (fig. S3B) (31). Ptenfl/fl;MycTg;Perkfl/fl mice
showed markedly reduced prostate growth compared to Ptenfl/fl;MycTg
mice, with weights similar to Ptenfl/fl and Ptenfl/fl; Perkfl/fl mice at 10 we eks
of age (fig. S3C). The reduction in
prostate size corresponded to a de-
crease in cancer progression and in
cell proliferation (fig. S3, D and E).
To determine the consequence of
PERK loss for P-eIF2 signaling in
PCa development, we monitored
P-eIF2 expression by IF stain ing.
The activation of P-eIF2 was reduced
by 70% in Ptenfl/fl; MycTg;Perkfl/fl tis-
sue compared to Ptenfl/fl;MycTg (fig.
S3F). These data strongly suggest
that the P-eIF2– dependent adap-
tive stress response is driven to a
large extent by PERK signaling.
Our studies demonstrated that
P-eIF2 is directly activated in the
early stage of Ptenfl/fl;MycTg tum-
origenesis, being visible in benign
tissue and increasing in HgPIN,
which may reflect a distinct point
of vulnerability for aggressive PCa
(Fig.2). To evaluate the necessity
of P-eIF2 for promoting tumor
growth or maintenance in vivo, we
conducted a preclinical trial. Mice
with developed tumors were im-
aged by magnetic resonance imag-
ing (MRI) to confirm a measurable
baseline of prostate volume per
mouse and then grouped into co-
horts for either vehicle or ISRIB
treatment daily over the course of
6 weeks (Fig.3C). Ptenfl/fl;MycTg
mice showed tumor regression
within 3 weeks of ISRIB treatment,
with no signs of toxicity, whereas all
Ptenfl/fl mice showed continued tu-
mor growth (Fig.3, D and E, fig. S4A,
and table S1). By 6 weeks, Ptenfl/fl
mice showed an approximate 40% increase in growth over individual
baseline measurements, whereas ISRIB-treated Ptenfl/fl;MycTg mice
demonstrated no progression in tumor size. In addition, we evaluated
the immune cell infiltration, marked by the pan-leukocyte antibody
CD45 after 3 weeks of ISRIB treatment and observed no significant
changes regardless of prostate tumor genotype and treatment (fig.
S4B). Further analysis of immune cell populations did not demon-
strate substantial differences in total T cell or myeloid populations, in-
cluding dendritic cells, macrophages, and neutrophils (fig. S4, C and
D). Of the intertumoral immune cells examined, less than 5% were either
CD4+ or CD8+ T cells, as expected for the Ptenfl/fl murine prostate model
(32). Although we cannot exclude the possibility that ISRIB may be re-
model ing tumor immunit y during initial treatment, this was not evident
after 3 weeks of treatment. Together, these studies reveal that P-eIF2
signaling is functionall y relevant in aggressive PCa and that this adaptive
response is therapeutically targetable in vivo using the small- molecule
inhibitor ISRIB.
To extend our observations directly to human disease, we created
human cell lines to mimic our genetic mouse models. Human RWPE-1
1 week 6 week
ISRIB treated
Imaging schedule:
Treatment 1 /day orally
2.5 mg/kg or vehicle only
Increase to 5 mg/kg
35 newly synthesized
Tumor volume relative
to baseline (fold)
Ptenfl/fl +ISRIB (n =
Ptenfl/fl;MycTg (n = 3)
Ptenfl/fl (n = 4)
+ISRIB (n = 5)
Ptenfl/fl Ptenfl/fl;MycTg
Percentage over WT (%)
3 wk 6 wk
Fig. 3. Inhibition of P-eIF2a activity rebalances protein synthesis and prevents PCa progression. (A) Representative
Western blot highlighting PERK signaling in organoid cultures. (B) Left: Total newly synthesized proteins measured by 35S
methionine/cysteine incorporation and Western blot showing P-eIF2 and ATF4 in organoids treated with dimethyl sulfox-
ide (DMSO) or ISRIB (500 nM) for 6 hours. Right: Quantification of radioactive pulse relative to loading, depicted as percent
over WT (n = 3, mean ± SEM). (C) Schematic of preclinical trial for escalating dosage and MRI schedule over 6 weeks. (D)
Representative scans of two ISRIB-treated mice after 1 and 6 weeks of treatment for comparison. Tumor is outlined in red,
and arrows highlight the seminal vesicles (SV) surrounding the tumor. (E) Quantification of tumor size as fold change relative
to baseline volume at 3- and 6-week time points (mean ± SEM). **P < 0.01, ***P < 0.001, t test.
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
5 of 11
epithelial cells were created to stably knock down PTEN (shPTEN)
with or without MYC overexpression (MYCOE, Fig.4A). The combi-
nation of PTEN loss with increased MYC expression activated PERK
signaling and P-eIF2, showing that the adaptive response that we
had observed in mice is also triggered in human prostate cells.
To understand the requirement for this stress response checkpoint
in human cells, we treated each cell line with ISRIB and observed a
marked increase in apoptosis, independent of alterations in prolif-
eration, specifically in shPTEN;MYCOE
cells relative to control samples (Fig.4B
and fig. S5A).
High P-eIF2 expression with
loss of PTEN is associated with
an increased risk of metastasis
after surgery
To further examine the clinical relevance
of high P-eIF2 downstream of PTEN
loss, we built a human tissue microarray
(TMA) consisting of 424 tumors and an-
alyzed the expression of PTEN, c-MYC,
and P-eIF2. On the basis of our GEMMs,
we predicted that the combination of
PTEN loss and P-eIF2 would associate
with advanced PCa. We selected an array
of patients with PCa ranging from low to
high risk, who received surgery as a cura-
tive treatment with a median of 10 years
of follow-up to accurately evaluate the
incidence of clinical progression, a com-
posite outcome representing visceral or
bone metastasis or PCa-specific mortality
(MET/PCSM) (Table1). We used quan-
titative IF of P-eIF2, c-MYC, and PTEN
normalized to adjacent benign tissue
(fig. S6, A and B) and then evaluated as-
sociated risk for MET/PCSM. After con-
trolling for age, prostate-specific antigen
(PSA), Gleason score, and pathological
staging, the analysis showed that patients
with PTEN loss/high MYC expression
were more likely to experience metastatic
progression than patients with PTEN loss
or high MYC alone (Fig.4C).
Our data from the GEMMs and hu-
man prostatic cell lines suggested that
P-eIF2 is a targetable adaptive response
downstream of PTEN loss and MYC hy-
peractivation. Hence, we next examined
the associated risk of progression in pa-
tients with PTEN loss and high P-eIF2
at the time of surgery. The rate of MET/
PCSM-free survival was significantly lower
in patients with high P-eIF2 and PTEN
loss compared to PTEN loss alone (P < 0.01)
(Fig.4D). Only 4% of patients with PTEN
loss and low P-eIF2 succumbed to me-
tastasis or death, whereas 19% of patients
with PTEN loss and high P-eIF2 showed
MET/PCSM by 10 years after surgical in-
tervention with the intention to cure the
disease. Furthermore, patients with high
P-eIF2 and PTEN loss had a higher risk
Relative Annexin V+
apoptotic cells (xfold)
Myc OE
Myc OE
P value
Worse outcomeBetter outcome
–2.5 02.5 57.5 10 12.5 15
PTEN loss, high P-eIF2α
PTEN loss, low P-eIF2α
Gleason score 7–10
Pathologic stage 3–4
Hazard ratio (95% CI)
Metastasis/PCSM-free survival (%)
PTEN loss, high MYC
PTEN loss, low MYC
Log-rank P value < 0.01
PTEN normal, high MYC
0 1 2 3 4 5 6 7 8 9 10
PTEN normal, low MYC
Metastasis/PCSM-free survival (%)
0 1 2 3 4 5 6 7 8 9 10
Log-rank P value < 0.01
PTEN loss, high P-eIF2α
PTEN loss, low P-eIF2α
PTEN normal, high P-eIF2α
PTEN normal, low P-eIF2α
Fig. 4. High P-eIF2 expression in human prostate tumors with loss of PTEN function is associated with in-
creased risk of metastasis or death after surgery. (A) Representative Western blot showing PTEN, MYC, P-PERK,
P-eIF2, and total eIF2 expression with -actin loading control (Ctrl) in human prostatic RWPE-1 cell lines. (B) Quan-
tification of annexin V–positive cells analyzed by flow cytometry relative to control cells after treatment with DMSO
or 500 nM ISRIB for 9 hours (n = 3, mean ± SEM) *P < 0.05, t test. (C) Kaplan-Meier analysis of clinical progression–free
survival [progression defined as visceral or bone metastasis or PCSM] for patients with normal PTEN expression ver-
sus PTEN loss and relative MYC expression identified by IF from the TMA. (D) Kaplan-Meier analysis of MET/PCSM for
patients with normal PTEN expression versus PTEN loss grouped by eIF2 phosphorylation. (E) Cox proportional
hazards regression results are shown in a Forest plot of hazard ratios and 95% CI for factors associated with risk of
clinical progression after surgery. Independent factors are tumor with PTEN loss/low P-eIF2 or PTEN loss/high
P-eIF2 versus a reference group with normal PTEN expression; age in years; PSA in nanograms per milliliter; Gleason
score > 7 versus 6; and pathological stage T3-T4 versus T2 at the time of prostatectomy.
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
6 of 11
of MET/PCSM compared to patients with no PTEN loss, with a haz-
ard ratio of 5.40 [95% confidence interval (CI), 2.46 to 11.86; P < 0.01],
whereas other variables that may affect the risk were not significant-
ly different (Fig.4E). MYC overexpression with either low or high
P-eIF2 did not associate with increased risk of MET/PCSM (fig. S6C),
supporting our findings that MYC alone does not drive PCa. Notably,
high P-eIF2 expression played a role equivalent to the MYC oncogene
in combination with loss of PTEN at predicting metastatic progression
(Fig.4, C and D), yet unlike MYC, P-eIF2 may be a druggable target.
Together, the combination of P-eIF2 and PTEN loss may serve as
a predictor for cancer progression after curative treatment, which is
independent of the traditional risk assessment system using PSA, cancer
grade, and cancer stage.
We next evaluated the discriminatory properties of high P-eIF2
and PTEN loss as a prognostic marker independent from the most
commonly used risk assessment score in the clinic, CAPRA-S (Cancer
of the Prostate Risk Assessment after Surgery) (33). We used the
c-index (concordance index) to evaluate the ability of the protein
signature of high P-eIF2 with loss of PTEN to discriminate be-
tween individual patients who did or did not succumb to metastasis
or death after surgery. Currently, clinicians depend on genomic risk
to individualize treatment decisions using three available gene ex-
pression tests: Prolaris, Decipher, and OncotypeDx (34). The Prolaris
test relies on the average expression of 31 cell cycle progression (CCP)
genes and was validated using the same cohort of patients used in the
TMA (35). Within the same patients, the Prolaris-CCP panel has a com-
bined c-index of 0.77 (CAPRA-S + CCP) (35), whereas high P-eIF2
and PTEN loss has a c-index of 0.80 (fig. S6D). These findings show
that concurrent high P-eIF2 and PTEN loss serves as an indepen-
dent predictor with improved prognostic accuracy over standard
clinicopathologic testing for discriminating which individuals may
experience metastatic progression.
P-eIF2 is a targetable adaptive response in aggressive
human PCa
We next sought to functionally evaluate whether we could target the
UPR pathway, specifically through P-eIF2, in advanced human PCa.
Although it is historically difficult to generate human prostate patient-
derived xenograft (PDX) models (36), we were successful in gen-
erating models with similar characteristics to the Ptenfl/fl;MycTg mice
to assess the effects of ISRIB on cancer growth and mortality. In par-
ticular, we generated two PDX models: one derived from a primary
tumor, herein referred to as pPCa, and one derived from a lymph node
metastasis in the left internal iliac chain from the same patient, herein
referred to as mPCa (Fig.5A). The pPCa-PDX tumor had significantly
lower MYC expression than the mPCa-PDX tumor (P < 0.01), but both
showed loss of PTEN with increased P- AKT expression (Fig.5B and
fig. S7A). We also observed a significant increase in P- eIF2 only in
the mPCa (P < 0.01; Fig.5B).
To test the therapeutic efficacy of ISRIB in human PCa, we per-
formed a preclinical trial on the stably passaged PDX model. Targeting
P-eIF2 pharmacologically significantly prolonged survival in mice
bearing the metastatic tumor with high P-eIF2 (P < 0.01; Fig.5C),
whereas the effectiveness of ISRIB treatment was short-lived in pPCa
tumor. Consistent with our GEM model, the mPCa- PDX model, with
high expression of P-eIF2, displayed significant tumor regression
and cell death (P < 0.01), as demonstrated by increased terminal de-
oxynucleotidyl transferase–mediated deoxyuridine triphosphate nick
end labeling (TUNEL) staining and cleaved caspase 3 expression
after only 9 days of ISRIB treatment (Fig.5, D and E, and fig. S7B).
Conversely, the pPCa-PDX model, with low P-eIF2, did not show
regression but stabilized with eventual tumor regrowth and no sig-
nificant cell death (Fig.5, D and E, and fig. S7B). These findings demon-
strate that attenuating P-eIF2 activity with ISRIB elicits a potent
antitumor effect in a humanized model of advanced PCa.
We next determined whether a metastatic PCa tumor, harboring
high MYC and loss of PTEN activity in a complex genetic background
of human PCa, relies on eIF2 phosphorylation as an adaptive re-
sponse to restrain global protein synthesis. Therefore, we assessed
Table 1. Characteristics of patients included in the TMA. Baseline
characteristics of the TMA cohort consisting of 424 tumor samples, where
58 years is the average age at diagnosis. More than 50% of the cohort had
pathological Gleason grade 7 or higher, and 75% had organ-confined
disease (pathological stage T2). Median follow-up was 10 years.
Patient characteristics
of TMA Value n(%)
Race/ethnicity Native American 1 0
Asian/Pacific Islander 13 3
African-American 14 3
Caucasian 359 85
Mixed 25 6
Unknown 12 3
Biopsy Gleason grade 3 + 3 263 64
3 + 4 95 23
4 + 3 25 6
8 − 10 29 7
Missing 12 —
Clinical T stage T2 296 98
T3 5 2
T4 2 1
Missing 121 —
Pathologic Gleason grade 3 + 3 184 43
3 + 4 173 41
4 + 3 45 11
8 − 10 22 5
Pathologic T stage T2 313 75
T3 102 24
T4 5 1
Missing 4 —
Pathologic N stage NX 200 48
N0 208 50
N1 7 2
Missing 9 —
Surgical margins No 354 83
Yes 70 17
Adverse path (Gleason Grade
≥ 4 + 3 or pT3a/pN1)
No 291 69
Yes 133 31
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
7 of 11
newly synthesized proteins in vivo by measuring the incorporation
of O-propargyl–puromycin (OP-Puro) within the primary and meta-
static tumor–derived PDXs, which have low or high P-eIF2, re-
spectively. Upon ISRIB treatment, we observed a marked increase in
global protein synthesis specifically in the mPCa PDX, but no change
in pPCa tumors where P-eIF2 expression was not up-regulated
(Fig.5F). To further assess the functional relevance of P-eIF2 sig-
naling, we decreased ATF4 expression in vivo using intratumor knock-
down by small interfering RNA (siRNA). Within the area of intra-
tumor ATF4 loss, we observed apoptosis and decreased proliferation
Benign mPCa
Mean fluorescence intensity
lacihteot lavivruS
)%(tniopd ne
Days elapsed
0510 15 20 25
Days elapsed
0510 15 20 25
Control ISRIB
Control ISRIB
Control ISRIB
pPCa pPCa
TUNEL-positive cells (%
Vehicle ISRIB
Vehicle ISRIB
Relative OP-Puro MFI
expression (fold)
LN (mPCa)
Tumor (pPCa)
Metastasis LN (mPCa)
Primary tumor (pPCa)
Fig. 5. Inhibition of P-eIF2 axis results in tumor regression and prolongs survival in a humanized model of metastatic PCa. (A) Schematic highlighting origin of
PDX tumors from primary (pPCa) or lymph node metastasis (mPCa). Tumors from selected patients with high-risk features, based on Gleason score and clinical stage or with
lymph node metastases determined by 68Ga–PSMA-11 positron emission tomography (PET) scans, were used to generate PDXs. Primary and metastatic tumors were confirmed
from tissue collected at the time of surgery and immediately implanted into immunodeficient NOD scid gamma (NSG) mice. (B) Representative IF images of MYC/CK8
(epithelial cell marker), P-AKT/CK8, or P-eIF2/CK8, co-staining with DAPI from benign (Ben) tissue adjacent to pPCa or mPCa tumors; scale bars, 50 m. Right: Quantification of
protein expression as relative mean IF intensity normalized to adjacent stromal tissue. (C) Kaplan-Meier tumor survival curves for mice bearing pPCa or mPCa tumors treated with
ISRIB (10 mg/kg) or vehicle (n = 8, per cohort; **P = 0.01, log-rank test). The survival curves represent mice euthanized when tumors reached an end point of 2 cm or when the
mice showed clear signs of morbidity. (D) Representative tumor sizes after 10 days of treatment. (E) Representative TUNEL staining and quantification of PDX tumors treated
with vehicle or ISRIB (10 mg/kg); scale bars, 100 m (n = 3, ***P < 0.001, t test). (F) Quantification of newly synthesized proteins in vivo, assessed by incorporation of OP-Puro
within PDX treated with ISRIB (10 mg/kg) or vehicle (n = 3 to 4 per arm, mean ± SEM; *P < 0.05, t test). n.s., not significant. MFI, mean fluorescence intensity.
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
8 of 11
assessed by TUNEL and Ki67 staining of
mPCa PDX (fig. S7C). This demonstrated
that inhibition of the PERK-eIF2 axis by
a genetic or pharmacological approach
effectively results in cell death of aggres-
sive PCa in vivo.
Targeting P-eIF2 activity reduced
metastasis and prolonged survival
in a PDX model of metastatic
castration-resistant PCa
In hormone-sensitive metastatic PCa, an-
drogen deprivation therapy (ADT) re-
mains the mainstay treatment; however,
these tumors inevitably develop resistance
to ADT and progress into the lethal for m
of metastatic castration-resistant PCa
(mCRPC) (37). Characterization of the
hormone-sensitive metastatic disease
has not been predictive of outcomes
in the clinical setting of lethal mCRPC
(38, 39). To directly study the contribu-
tion of P-eIF2 to metastasis, we gener-
ated an additional PDX (herein mCRPC
PDX) derived from a patient with mCRPC
despite prolonged treatment with com-
plete androgen blockage using leupro-
lide (ADT) and antiandrogen therapy
(enzalutamide) (37). Three weeks after
implantation of the mCRPC tumor un-
der the mouse renal capsule, we observed
tumor dissemination to the liver, distant
kidney, lymph nodes, and spleen (fig. S8,
A and B). The mCRPC PDX line con-
tinued to exhibit metastatic dissemi-
nation in the mouse host after multiple
passages and retained histological and
molecular characteristics of the original
tumor. The distant metastatic lesions
exhibited loss of PTEN, high MYC, and
high P-eIF2 expression (Fig.6A and
fig. S8C).
To examine the role of P-eIF2 from
the early stages of metastatic growth to
late stages of dissemination, we used a
prostate-specific membrane antigen [68Ga–
PSMA-11 PET/computed tomography
(CT)] scan to trace the progression of very
small metastases from early to late stages
of dissemination, which were not visible
by conventional imaging modalities such
as 18F-DG PET/CT (fig. S8D) (40). Prostate-
specific membrane antigen (PSMA) is
highly expressed on the surface of PCa
cells and allows sensitive staging to evaluate
therapy response in the clinical setting
(40). We subjected mice bearing liver or distal metastasis (confirmed by
PSMA PET) to either vehicle or ISRIB treatment (Fig.6, B and C).
Inhibition of P-eIF2 with ISRIB significantly prolonged survival in
mCRPC PDX mice bearing distal metastatic lesions (P = 0.01; Fig.6C).
In contrast, mice with metastasis died within 10 days on vehicle treat-
ment. By direct imaging with PSMA PET/CT, we observed substantial
PDX tumor
Mouse kidney
PDX tumor
PDX tumor
Liver metastasis Liver metastasis
PDX tumor
Liver metastasis
0Day 7Day 14
3 weeks for growth
End study
lacihte ot lavivruS
)%( tniopdne
Days elapsed
0510 15
Control ISRIB
Visible liver metastatic
PDX tumor
Day 0 Day 7 Day 0 Day 7
Vehicle: ISRIB:
H&E liver
PDX tumor
H&E liver
Injected dose/g4.5%1.5% 1.5% Injected dose/g 4.5%
Fig. 6. ISRIB treatment decreases metastatic progression in an advanced castration-resistant PCa PDX model.
(A) Representative H&E staining and IF of P-eIF2, PTEN, and MYC expression at the primary site of implantation
(mCRPC tumor), left kidney, and distant metastatic lesions in the liver; scale bars, 200 m (top left); 100 m (bottom
left); and 50 m for IF images. (B) Schematic of preclinical trial for mCRPC tumor growth and PET/CT schedule. Repre-
sentative 68Ga–PSMA-11 PET/CT scans on day 0 (time of treatment) and on day 7 are shown for the control versus ISRIB-
treated cohorts. Uptake of the PSMA-targeted radiotracer agent is observed in the liver, lymph node, and at the site
of primary tumor implantation in the left kidney capsule. Physiologic uptake of the PSMA-targeted radiotracer is also
seen in the contralateral kidney and bladder because it is excreted in the urinary tract. Histologic confirmation of
liver metastasis is shown by H&E staining at the time of euthanasia with dashed outlines around metastatic lesions.
(C) Kaplan-Meier survival curve for mice bearing mCRPC tumors treated once per day with ISRIB (10 mg/kg) or vehicle
(n = 3, per cohort); *P = 0.02, log-rank test. The survival curves represent mice euthanized when PSMA 68Ga PET/CT
showed progression from one distant metastatic lesion to two or more sites or when the mice showed signs of be-
coming moribund. (D) Quantification of visible metastatic lesions on the left medial lobe of the liver at the time of
euthanasia in the cohorts (n = 3 per cohort); ***P = 0.001.
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
9 of 11
metastatic regression at distal sites in mice treated with ISRIB (Fig.6B).
In addition, we confirmed a difference in metastatic progression in
the liver by pathohistological analysis at time of euthanasia (Fig.6D).
Therefore, two independent PDX models of metastatic disease, one
derived from a patient with early nodal metastasis (hormone-sensitive)
and the second from a patient with castration- resistant PCa, demon-
strated that blocking the activation of the adaptive brake on global protein
synthesis via the P-eIF2 axis resulted in profound tumor regression
and inhibition of metastatic dissemination.
The biological processes that allow cancer cells to balance working
at capacity for tumor progression while dealing with stress pheno-
types induced by the overload of cellular processes underlying rapid
cell growth and division (bioenergetic processes including DNA and
protein synthesis) are still poorly understood. Our data reveal a cell-
autonomous mechanism wherein the activity of two major oncogenic
lesions, loss of PTEN and MYC overexpression, which independently
enhance protein synthesis, paradoxically, decrease global protein pro-
duction when these oncogenic events coexist. This high lights the re-
quirement for an adaptive protein homeostasis response to sustain
aggressive tumor development.
Proteostasis is essential for normal cell health and viability, and as
such is ensured by the coordinated control of protein synthesis, folding,
and degradation (41). Although the UPR enables proteostasis to be re-
stored during unfavorable conditions, we found that PCa cells have
usurped a specific branch of this pathway for tumor growth and main-
tenance. The UPR consists of three main branches, yet only the PERK–P-
eIF2 axis is selectively triggered in this pathophysiological state to
ensure continued survival of cancer cells. The mechanisms triggering the
selective activation of the PERK–P-eIF2 axis in PCa may be through
increased protein misfolding itself, as a consequence of augmented pro-
tein synthesis at the ER, or through additional cues acting indepen-
dently from the UPR (42). Nonetheless, the adaptive response involving
P-eIF2 signaling provides a barrier to uncontrolled increases in pro-
tein synthesis and creates a permissive environment for continued tumor
growth. It is also possible that P-eIF2 may affect the translation of
select transcripts that are essential for aggressive oncogenesis (4345).
It is tempting to speculate that cancer cells may have usurped mech-
anisms normally operating in certain cell types, whereby activation of
specific branches of the UPR enables cellular differentiation or main-
tenance of stem cell features (46). For example, B lymphocytes normally
induce the UPR during their differentiation into plasma cells to preemp-
tively prepare for increased antibody production and secretion (47). More-
over, skeletal muscle stem cells maintain enhanced P-eIF2 to promote
a quiescent state required for their self-renewal capacity, which requires
diminished protein synthesis (48). Such control of the UPR seen in spe-
cialized cell types may have been hijacked by specific oncogenic lesions
to promote cancer survival and metastatic behavior. Our data show the
functional relevance of targeting this adaptive brake with ISRIB treat-
ment to trigger cytotoxicity during aggressive lethal stages of advanced
and castration-resistant PCa, for which at present there is no cure.
Study design
This study was designed to evaluate how two oncogenic lesions, which
augment protein synthesis, cooperate in aggressive PCa and prevent
proteotoxic stress to support tumor growth and survival. This objective
was addressed by (i) creating mouse models, cell lines, and PDX models
that depict the loss of PTEN with or without the overexpressi on of MY C,
(ii) evaluating PCa development downstream of these oncogenes,
(iii) observing global changes in newly synthesized proteins, followed
by (iv) identifying the adaptive response responsible for our observa-
tions. Using a genetic and pharmacological approach in both GEM and
PDX models, we inhibited the identified adaptive response to observe
the effects on tumor development and growth. TMA analysis was also
conducted to investigate the clinical relevance of our findings in as-
sociation with advanced PCa.
For all experiments, our sample sizes were determined on the ba-
sis of experience and published literature, which historically show
that these in vivo models are penetrant and consistent for tumor
development. We used the maximum number of mice available for
a given experiment based on the following criteria: the number of
GEMMs available in the age range of tumor development and tumor
size availability for implantation in PDXs. All mice were randomly
assigned to each treatment group for all preclinical trials. Blinded
observers visually inspected mice for obvious signs of tumor growth
or morbidity including weight loss, hunched posture, or lethargy. MRI
tumor recognition, IF imaging, and data collection by flow cytome-
try were done by researchers blinded to the sample identification
after analysis. The number of experimental replicates is specified
within each figure legend and elaborated for specific experiments
within Supplementary Materials and Methods.
Statistical analyses
Statistical analyses were performed using Microsoft Excel, GraphPad
Prism, or SAS (Statistical Analysis System) 9.4 for Windows, with ad-
ditional description in Supplementary Materials and Methods. Raw
values were depicted when possible or normalized to internal con-
trols from at least three independent experiments, shown as quan-
titative values expressed as means ± SD or SEM, as indicated. Data
were analyzed applying unpaired Student’s t test to compare quan-
titative data between two independent samples, unless otherwise
specified. The Kaplan-Meier method was used for survival analy-
sis. P < 0.05 were considered significant and denoted by *P < 0.05,
**P < 0.01, or ***P < 0.001.
Materials and Methods
Fig. S1. MycTg and PTEN loss cooperate for aggressive PCa development in mice.
Fig. S2. The UPR is activated in murine PCa.
Fig. S3. PERK loss blocks PCa progression and decreases P-eIF2 expression.
Fig. S4. Loss of P-eIF2 activity by ISRIB shows no toxicity and does not substantially alter
infiltrating immune cells.
Fig. S5. Inhibition of P-eIF2 activity by ISRIB does not affect human prostatic cell lines’ growth.
Fig. S6. PCa tissue from TMA shows specificity of protein expression in benign and tumor cells.
Fig. S7. Treatment with ISRIB or ATF4 siRNA results in increased apoptosis within metastatic tumor.
Fig. S8. A PDX was generated to recapitulate mCRPC.
Table S1. MRI tumor volumes during treatment in GEMMs.
References (4954)
1. D. Hanahan, R. A. Weinberg, Hallmarks of cancer: The next generation. Cell 144, 646–674
2. J. Luo, N. L. Solimini, S. J. Elledge, Principles of cancer therapy: Oncogene and
non-oncogene addiction. Cell 136, 823–837 (2009).
3. D. Ruggero, Revisiting the nucleolus: From marker to dynamic integrator of cancer
signaling. Sci. Signal. 5, pe38 (2012).
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
10 of 11
4. D. Ron, P. Walter, Signal integration in the endoplasmic reticulum unfolded protein
response. Nat. Rev. Mol. Cell Biol. 8, 519–529 (2007).
5. M. Wang, R. J. Kaufman, The impact of the endoplasmic reticulum protein-folding
environment on cancer development. Nat. Rev. Cancer 14, 581–597 (2014).
6. C. Hetz, E. Chevet, S. A. Oakes, Proteostasis control by the unfolded protein response.
Nat. Cell Biol. 17, 829–838 (2015).
7. C. Koumenis, C. Naczki, M. Koritzinsky, S. Rastani, A. Diehl, S. Nahum, A. Koromilas,
B. G. Wouters, Regulation of protein synthesis by hypoxia via activation of the
endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation
factor eIF2. Mol. Cell. Biol. 22, 7405–7416 (2002).
8. D. R. Fels, C. Koumenis, The PERK/eIF2/ATF4 module of the UPR in hypoxia resistance
and tumor growth. Cancer Biol. Ther. 5, 723–728 (2006).
9. A. C. Hsieh, Y. Liu, M. P. Edlind, N. T. Ingolia, M. R. Janes, A. Sher, E. Y. Shi, C. R. Stumpf,
C. Christensen, M. J. Bonham, S. Wang, P. Ren, M. Martin, K. Jessen, M. E. Feldman,
J. S. Weissman, K. M. Shokat, C. Rommel, D. Ruggero, The translational landscape of
mTOR signalling steers cancer initiation and metastasis. Nature 485, 55–61 (2012).
10. A. C. Hsieh, H. G. Nguyen, L. Wen, M. P. Edlind, P. R. Carroll, W. Kim, D. Ruggero, Cell
type–specific abundance of 4EBP1 primes prostate cancer sensitivity or resistance to
PI3K pathway inhibitors. Sci. Signal. 8, ra116 (2015).
11. C. M. Koh, B Gurel, S. Sutcliffe, M. J. Aryee, D Schultz, T. Iwata, M. Uemura, K. I. Zeller,
U. Anele, Q. Zheng, J. L. Hicks, W. G. Nelson, C. V. Dang, S. Yegnasubramanian,
A. M. De Marzo, Alterations in nucleolar structure and gene expression programs in
prostatic neoplasia are driven by the MYC oncogene. Am. J. Pathol. 178, 1824–1834 (2011).
12. A. Kumar, I. Coleman, C. Morrissey, X. Zhang, L. D. True, R. Gulati, R. Etzioni, H. Bolouri,
B. Montgomery, T. White, J. M. Lucas, L. G. Brown, R. F. Dumpit, N. DeSarkar, C. Higano,
E. Y. Yu, R. Coleman, N. Schultz, M. Fang, P. H. Lange, J. Shendure, R. L. Vessella,
P. S. Nelson, Substantial interindividual and limited intraindividual genomic diversity
among tumors from men with metastatic prostate cancer. Nat. Med. 22, 369–378 (2016).
13. H. Beltran, D. Prandi, J. M. Mosquera, M. Benelli, L. Puca, J. Cyrta, C. Marotz,
E. Giannopoulou, B. V. S. K. Chakravarthi, S. Varambally, S A. Tomlins, D. M. Nanus,
S. T. Tagawa, E. M. Van Allen, O. Elemento, A Sboner, L. A. Garraway, M. A. Rubin,
F. Demichelis, Divergent clonal evolution of castration-resistant neuroendocrine prostate
cancer. Nat. Med. 22, 298–305 (2016).
14. J. T. Cunningham, M. V. Moreno, A. Lodi, S. M. Ronen, D. Ruggero, Protein and nucleotide
biosynthesis are coupled by a single rate-limiting enzyme, PRPS2, to drive cancer. Cell
157, 1088–1103 (2014).
15. M. Barna, A. Pusic, O. Zollo, M. Costa, N Kondrashov, E. Rego, P. H. Rao, D. Ruggero,
Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency. Nature
456, 971–975 (2008).
16. M. Pourdehnad, M. L. Truitt, I. N. Siddiqi, G. S. Ducker, K. M. Shokat, D. Ruggero, Myc and
mTOR converge on a common node in protein synthesis control that confers synthetic
lethality in Myc-driven cancers. Proc. Natl. Acad. Sci. U.S.A. 110, 11988–11993 (2013).
17. D. Ruggero, The role of Myc-induced protein synthesis in cancer. Cancer Res. 69,
8839–8843 (2009).
18. R. Lesche, M. Groszer, J. Gao, Y. Wang, A. Messing, H. Sun, X. Liu, H. Wu, Cre/loxP-mediated
inactivation of the murine Pten tumor suppressor gene. Genesis 32, 148–149 (2002).
19. Cancer Genome Atlas Research Network, The molecular taxonomy of primary prostate
cancer. Cell 163, 1011–1025 (2015).
20. J. Kim, I.-E. A. Eltoum, M. Roh, J. Wang, S. A. Abdulkadir, Interactions between cells with
distinct mutations in c-MYC and Pten in prostate cancer. PLOS Genet. 5, e1000542
21. K. Ellwood-Yen, T. G. Graeber, J. Wongvipat, M. L. Iruela-Arispe, J. Zhang, R. Matusik,
G. V. Thomas, C. L. Sawyers, Myc-driven murine prostate cancer shares molecular features
with human prostate tumors. Cancer Cell 4, 223–238 (2003).
22. J. Drost, W. R. Karthaus, D. Gao, E. Driehuis, C. L. Sawyers, Y. Chen, H. Clevers, Organoid
culture systems for prostate epithelial and cancer tissue. Nat. Protoc. 11, 347–358
23. W. R. Karthaus, P. J. Iaquinta, J. Drost, A. Gracanin, R. van Boxtel, J. Wongvipat,
C. M. Dowling, D. Gao, H. Begthel, N. Sachs, R. G. J. Vries, E. Cuppen, Y. Chen, C. L. Sawyers,
H. C. Clevers, Identification of multipotent luminal progenitor cells in human prostate
organoid cultures. Cell 159, 163–175 (2014).
24. J. van Riggelen, A. Yetil, D. W. Felsher, MYC as a regulator of ribosome biogenesis and
protein synthesis. Nat. Rev. Cancer 10, 301–309 (2010).
25. M. L. Truitt, D. Ruggero, New frontiers in translational control of the cancer genome.
Nat. Rev. Cancer 16, 288–304 (2016).
26. C. Hetz, The unfolded protein response: Controlling cell fate decisions under ER stress
and beyond. Nat. Rev. Mol. Cell Biol. 13, 89–102 (2012).
27. C. Sidrauski, J. C. Tsai, M. Kampmann, B. R. Hearn, P. Vedantham, P. Jaishankar, M. Sokabe,
A. S. Mendez, B. W. Newton, E. L. Tang, E. Verschueren, J. R. Johnson, N. J. Krogan,
C. S. Fraser, J. S. Weissman, A. R. Renslo, P. Walter, Pharmacological dimerization and
activation of the exchange factor eIF2B antagonizes the integrated stress response. eLife
4, e07314 (2015).
28. C. Sidrauski, A. M. McGeachy, N. T. Ingolia, P. Walter, The small molecule ISRIB reverses
the effects of eIF2 phosphorylation on translation and stress granule assembly. eLife 4,
e05033 (2015).
29. J. C. Tsai, L. E. Miller-Vedam, A. A. Anand, P. Jaishankar, H. C. Nguyen, A. R. Renslo, A. Frost,
P. Walter, Structure of the nucleotide exchange factor eIF2B reveals mechanism of
memory-enhancing molecule. Science 359, eaaq0939 (2018).
30. K. Pakos-Zebrucka, I. Koryga, K. Mnich, M. Ljujic, A. Samali, A. M. Gorman, The integrated
stress response. EMBO Rep. 17, 1374–1395 (2016).
31. P. Zhang, B. McGrath, S. Li, A. Frank, F. Zambito, J. Reinert, M. Gannon, K. Ma,
K. McNaughton, D. R. Cavener, The PERK eukaryotic initiation factor 2 kinase is required
for the development of the skeletal system, postnatal growth, and the function and
viability of the pancreas. Mol. Cell. Biol. 22, 3864–3874 (2002).
32. A. J. Garcia, M. Ruscettia, T. L. Arenzanac, L. M. Trana, D. Bianci-Friasd, E. Syberta,
S. J. Pricemana, L. Wu, P S. Nelsond, S. T. Smalec, H. Wu, Pten null prostate epithelium
promotes localized myeloid-derived suppressor cell expansion and immune suppression
during tumor initiation and progression. Mol. Cell. Biol. 34, 2017–2028 (2014).
33. M. R. Cooperberg, D. J. Pasta, E. P. Elkin, M. S. Litwin, D. M. Latini, J. DuChane,
P. R. Carroll, The University of California, San Francisco Cancer of the Prostate Risk
Assessment score: A straightforward and reliable preoperative predictor of disease
recurrence after radical prostatectomy. J. Urol. 173, 1938–1942 (2005).
34. H. G. Nguyen, C. J. Welty, M. R. Cooperberg, Diagnostic associations of gene expression
signatures in prostate cancer tissue. Curr. Opin. Urol. 25, 65–70 (2015).
35. M. R. Cooperberg,J. P. Simko, J. E. Cowan, J. E. Reid, A. Djalilvand, S. Bhatnagar, A. Gutin,
J. S. Lanchbury, G. P. Swanson, S. Stone, P. R. Carroll, Validation of a cell-cycle
progression gene panel to improve risk stratification in a contemporary prostatectomy
cohort. J. Clin. Oncol. 31, 1428–1434 (2013).
36. D. Lin, A W. Wyatt, H. Xue, Y. Wang, X. Dong, A. Haegert, R. Wu, S. Brahmbhatt, F. Mo,
L. Jong, R. H. Bell, S. Anderson, A. Hurtado-Coll, L. Fazli, M Sharma, H. Beltran, M. Rubin,
M. Cox, P. W. Gout, J. Morris, L. Goldenberg, S. V. Volik, M. E. Gleave, C. C. Collins, Y. Wang,
High fidelity patient-derived xenografts for accelerating prostate cancer discovery and
drug development. Cancer Res. 74, 1272–1283 (2014).
37. M. S. Cookson, W. T. Lowrance, M. H. Murad, A. S. Kibel; American Urological Association,
Castration- resistant prostate cancer: AUA guideline amendment. J. Urol. 193, 491–499 (2015).
38. G. Gundem, P. Van Loo, B. Kremeyer, L. B. Alexandrov, J. M. C. Tubio, E. Papaemmanuil,
D. S. Brewer, H. M. L. Kallio, G. Högnäs, M. Annala, K Kivinummi, V. Goody, C Latimer,
S. O’Meara, K. J. Dawson, W Isaacs, M R. Emmert-Buck, M. Nykter, C. Foster, Z. Kote-Jarai,
D. Easton, H. C. Whitaker; ICGC Prostate UK Group, D. E. Neal, C. S. Cooper, R A. Eeles,
T Visakorpi, P. J. Campbell, U. McDermott, D. C. Wedge, G. S. Bova, The evolutionary
history of lethal metastatic prostate cancer. Nature 520, 353–357 (2015).
39. D. Robinson, E. M. Van Allen, Y.-M. Wu, N. Schultz, R. J. Lonigro, J.-M. Mosquera,
B. Montgomery, M.-E. Taplin, C. C Pritchard, G. Attard, H. Beltran, W. M. Abida,
R. K. Bradley, J. Vinson, X. Cao, P. Vats, L. P. Kunju, M. Hussain, F. Y. Feng, S. A. Tomlins,
K. A. Cooney, D. C. Smith, C. Brennan, J. Siddiqui, R. Mehra, Y. Chen, D. E. Rathkopf,
M. J. Morris, S. B. Solomon, J. C. Durack, V. E. Reuter, A. Gopalan, J. Gao, M. Loda, R. T. Lis,
M. Bowden, S. P. Balk, G. Gaviola, C. Sougnez, M. Gupta, E. Y. Yu, E. A. Mostaghel,
H. H. Cheng, H. Mulcahy, L. D. True, S. R. Plymate, H. Dvinge, R. Ferraldeschi, P. Flohr,
S Miranda, Z. Zafeiriou, N. Tunariu, J. Mateo, R. Perez-Lopez, F. Demichelis, B. D. Robinson,
M. A. Schiffman, D. M. Nanus, S. T. Tagawa, A. Sigaras, K. W. Eng, O. Elemento, A. Sboner,
E. I. Heath, H. I. Scher, K. J. Pienta, P. Kantoff, J. S. de Bono, M. A. Rubin, P. S. Nelson,
L. A. Garraway, C. L. Sawyers, A. M. Chinnaiyan, Integrative clinical genomics of advanced
prostate cancer. Cell 161, 1215–1228 (2015).
40. K. Bouchelouche, P. L. Choyke, Advances in prostate-specific membrane antigen PET of
prostate cancer. Curr. Opin. Oncol. 30, 189–196 (2018).
41. W. E. Balch, R. I. Morimoto, A. Dillin, J. W. Kelly, Adapting proteostasis for disease
intervention. Science 319, 916–919 (2008).
42. E. Karali, S. Bellou, D. Stellas, A. Klinakis, C. Murphy, T. Fotsis, VEGF Signals through ATF6
and PERK to promote endothelial cell survival and angiogenesis in the absence of ER
stress. Mol. Cell 54, 559–572 (2014).
43. M. Holcik, N. Sonenberg, Translational control in stress and apoptosis. Nat. Rev. Mol. Cell Biol.
6, 318–327 (2005).
44. N. Robichaud, N. Sonenberg, Translational control and the cancer cell response to stress.
Curr. Opin. Cell Biol. 45, 102–109 (2017).
45. M. Bi, C. Naczki, M. Koritzinsky, D. Fels, J. Blais, N. Hu, H. Harding, I. Novoa, M. Varia,
J. Raleigh, D. Scheuner, R. J. Kaufman, J. Bell, D. Ron, B. G. Wouters, C. Koumenis, ER
stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor
growth. EMBO J. 24, 3470–3481 (2005).
46. P. Walter, D. Ron, The unfolded protein response: From stress pathway to homeostatic
regulation. Science 334, 1081–1086 (2011).
47. D. J. Todd, L. J. McHeyzer-Williams, C. Kowal, A.-H. Lee, B. T. Volpe, B. Diamond,
M. G. McHeyzer-Williams, L. H. Glimcher, XBP1 governs late events in plasma cell
differentiation and is not required for antigen-specific memory B cell development.
J. Exp. Med. 206, 2151–2159 (2009).
by guest on May 3, 2018 from
Nguyen et al., Sci. Transl. Med. 10, eaar2036 (2018) 2 May 2018
11 of 11
48 . A. Sendoel, J. G. Dunn, E. H. Rodriguez, S. Naik, N. C. Gomez, B. Hurwitz, J. Levorse,
B. D. Dill, D. Schramek, H. Molina, J. S. Weissman, E. Fuchs, Translation from
unconventional 5 start sites drives tumour initiation. Nature 541, 494–499 (2017).
49. T. Matsuda, C. L. Cepko, Electroporation and RNA interference in the rodent retina in vivo
and in vitro. Proc. Natl. Acad. Sci. U.S.A. 101, 16–22 (2004).
50. J.-i Miyazaki, S. Takaki, K. Araki, F. Tashiro, A. Tominaga, K. Takatsu, K.-i. Yamamura,
Expression vector system based on the chicken -actin promoter directs efficient
production of interleukin-5. Gene 79, 269–277 (1989).
51. E. L. Jackson, N. Willis, K. Mercer, R. T. Bronson, D Crowley, R. Montoya, T. Jacks,
D. A. Tuveson, Analysis of lung tumor initiation and progression using conditional
expression of oncogenic K-ras. Genes Dev. 15, 3243–3248 (2001).
52. S. Rees, J. Coote, J. Stables, S. Goodson, S. Harris, M. G. Lee, Bicistronic vector for the
creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to
express recombinant protein. Biotechniques 20, 102–110 (1996).
53. A. Okada, R. Lansford, J. M. Weimann, S. E. Fraser, S. K. McConnell, Imaging cells in the
developing nervous system with retrovirus expressing modified green fluorescent
protein. Exp. Neurol. 156, 394–406 (1999).
54. D. G. Altman, L. M. McShane, W. Sauerbrei, S. E. Taube, Reporting recommendations for
tumor marker prognostic studies (REMARK): Explanation and elaboration. PLOS Med. 9,
e1001216 (2012).
Acknowledgments: We thank the Mouse Pathology, Preclinical Therapeutics, and
Biomedical Imaging Core facilities at University of California, San Francisco (UCSF) for their
assistance in our study. We are grateful to J. Simko at UCSF for building the TMA, for
providing them for our use, and for assistance in selecting human tumor tissue used in
construction of the pPCa and mPCa PDX models. In addition, we thank M. Barna at Stanford
University for the helpful comments provided. Funding: H.G.N. is supported by the U.S.
Department of Defense (W81XWH-15-1-0460), AUA-SUO-Prostate Cancer Foundation
Young Investigator Award (16YOUN14), and AUA Urology Care Foundation Rising Star in
Research Award (A130596). C.S.C. is funded by the American Cancer Society (PF-14-212-01-
RMC) and the AACR-Bayer Prostate Cancer Research Fellowship (17-40-44-CONN). C.M.F. is
funded by the Campini Foundation, The Leukemia and Lymphoma Foundation Career
Development Grant, and UCSF Department of Pediatrics K12 (5K12HDO72222-05). A.C.H.
was supported by NIH (1K08CA175154-01) and the Burroughs Wellcome Fund Career Award
for Medical Scientists. M.J.E. and D.R. are supported by the American Cancer Society
(130635-RSG-17-005-01-CCE). C.K. and D.R. are supported by NIH (P01-CA1659970). P.W. is
supported by Calico Life Sciences LLC, the Weill Foundation, and the Howard Hughes
Medical Institute. D.R. is a Leukemia and Lymphoma Society Scholar, and this research was
funded by NIH grants (R01-CA140456 and R01-CA154916). C.K. and D.R. are supported by
NIH (P01-CA165997), and C.K. by NIH (2R01-CA094214). F.T. was supported by NIH
(F31CA183569). Author contributions: H.G.N., C.S.C., and D.R. provided oversight of the project.
H.G.N., P.R.C., and L.X. primarily collected and analyzed data from PDX models and TMA.
C.S.C. and Y.K. primarily collected and analyzed data from GEMMs and cell lines used. C.M.F.
collected flow data for GEMMs and PDX models. J.E.C. discussed and performed statistical analysis
for the TMA supplied under the supervision of P.R.C. The MycTg mouse model was created
by J.T.C., and initial GEMM colonies were started by A.C.H. C.T. and M.J.E. assisted for in vivo
imaging experiments. C.P.E. and J.C.Y. created and initially characterized the mCRPC PDX
model. B.H. aided in preclinical studies for mouse models. F.T. and C.K. provided materials
and discussion for studying PERK signaling. P.W. provided materials and discussion for
studying P-eIF2 with ISRIB. C.S.C. and D.R. prepared the manuscript with edits from H.G.N.
and Y.K. Additional authors provided edits for their specific contributions. Competing
interests: P.W. is an inventor on patent 9708247 held by the Regents of the University of
California that describes ISRIB and its analogs. Rights to the invention have been licensed
by UCSF to Calico. D.R., H.G.N., P.R.C., C.S.C., and L.X. are inventors on patent application
pending: Case no, SF2018-128 held the Regents of the University of California that covers
development of a novel two protein biomarker analysis for PCa and a clinical version of
ISRIB that can be used to target tumor and metastatic progression in patients. All other
authors declare that they have no competing interests. Data and materials availability:
Requests for materials should be addressed to C.S.C. or D.R. and will be provided with
the potential of a material transfer agreement. The mCRPC PDXs are available from C.P.E.
under a material agreement with UC Davis.
Submitted 12 October 2017
Resubmitted 24 January 2018
Accepted 6 April 2018
Published 2 May 2018
Citation: H. G. Nguyen, C. S. Conn, Y. Kye, L. Xue, C. M. Forester, J. E. Cowan, A. C. Hsieh,
J. T. Cunningham, C. Truillet, F. Tameire, M. J. Evans, C. P. Evans, J. C. Yang, B. Hann, C. K oumenis,
P. Walter, P. R. Carroll, D. Ruggero, Development of a stress response therapy targeting aggressive
prostate cancer. Sci. Transl. Med. 10, eaar2036 (2018).
by guest on May 3, 2018 from
Development of a stress response therapy targeting aggressive prostate cancer
Constantinos Koumenis, Peter Walter, Peter R. Carroll and Davide Ruggero
Cunningham, Charles Truillet, Feven Tameire, Michael J. Evans, Christopher P. Evans, Joy C. Yang, Byron Hann,
Hao G. Nguyen, Crystal S. Conn, Yae Kye, Lingru Xue, Craig M. Forester, Janet E. Cowan, Andrew C. Hsieh, John T.
DOI: 10.1126/scitranslmed.aar2036
, eaar2036.10Sci Transl Med
prostate cancer.
that blocks this protective mechanism and has therapeutic activity against aggressive and otherwise untreatable αprotects them from excessive protein synthesis. To target this pathway, the authors identified an inhibitor of eIF2
, whichαwith a specific combination of mutations can override this stress by activating a protein called eIF2
. discovered, prostate cancer cellset alcan be toxic to the cells because it promotes cellular stress. As Nguyen
drive cancer growth. This is not a benign adaptation, however, and unchecked up-regulation of protein synthesis
synthesis is one of the cellular processes that is altered in cancer cells, because its continued activation helps
As tumors grow, they undergo a variety of metabolic changes that facilitate their proliferation. Protein
Stressing out prostate cancer
This article cites 54 articles, 18 of which you can access for free
Terms of ServiceUse of this article is subject to the
is a registered trademark of AAAS.Science Translational Medicinetitle
licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. The
Science, 1200 New York Avenue NW, Washington, DC 20005. 2017 © The Authors, some rights reserved; exclusive
(ISSN 1946-6242) is published by the American Association for the Advancement ofScience Translational Medicine
by guest on May 3, 2018 from
... The role of cellular stress in cancer is an area of increased research interest as cancer cells display increases in energy-demanding processes, such as protein synthesis, during tumorigenesis that must be delicately balanced by adaptive stress responses for sustained cell growth and survival [46,47]. In prostate cancer, therapeutically targeting this stress response via inhibition of phosphorylated eukaryotic initiation factor 2 α (P-eIF2α) promoted a cytotoxic response in metastatic prostate PDXs [48]. Based on our findings, further investigation regarding how increased cellular stress in CAFs participates in prostate tumour development is required. ...
Full-text available
Prostate cancer is the second most common cause of cancer death in males. A greater understanding of cell signalling events that occur within the prostate cancer tumour microenvironment (TME), for example, between cancer-associated fibroblasts (CAFs) and prostate epithelial or cancer cells, may identify novel biomarkers and more effective therapeutic strategies for this disease. To address this, we used cell-type-specific labelling with amino acid precursors (CTAP) to define cell-type-specific (phospho)proteomic changes that occur when prostate epithelial cells are co-cultured with normal patient-derived prostate fibroblasts (NPFs) versus matched CAFs. We report significant differences in the response of BPH-1 benign prostate epithelial cells to CAF versus NPF co-culture. Pathway analysis of proteomic changes identified significant upregulation of focal adhesion and cytoskeleton networks, and downregulation of metabolism pathways, in BPH-1 cells cultured with CAFs. In addition, co-cultured CAFs exhibited alterations in stress, DNA damage, and cytoskeletal networks. Functional validation of one of the top differentially-regulated proteins in BPH-1 cells upon CAF co-culture, transglutaminase-2 (TGM2), demonstrated that knockdown of this protein significantly reduced the proliferation and migration of prostate epithelial cells. Overall, this study provides novel insights into intercellular communication in the prostate cancer TME that may be exploited to improve patient management.
... Additionally, prostate cancer organoids are available as alternative models to PDXs 35,36,[42][43][44][45][46][47][48][49] . Some prostate cancer organoids grow for multiple passages and form tumours when grafted into immunocompromised mice. ...
Patient-derived xenografts (PDXs) are generated by engrafting human tumours into mice. Serially transplantable PDXs are used to study tumour biology and test therapeutics, linking the laboratory to the clinic. Although few prostate cancer PDXs are available in large repositories, over 330 prostate cancer PDXs have been established, spanning broad clinical stages, genotypes and phenotypes. Nevertheless, more PDXs are needed to reflect patient diversity, and to study new treatments and emerging mechanisms of resistance. We can maximize the use of PDXs by exchanging models and datasets, and by depositing PDXs into biorepositories, but we must address the impediments to accessing PDXs, such as institutional, ethical and legal agreements. Through collaboration, researchers will gain greater access to PDXs representing diverse features of prostate cancer. This Perspective covers existing patient-derived xenografts (PDXs) of prostate cancer, and their features and uses in basic and preclinical research. The authors also discuss the need for additional PDXs, and how collaboration in prostate cancer PDX research can be improved.
... Despite the marked therapeutic efficacy, the on-target toxicity PERK inhibitors still needs for further investigation (136). In addition, ISRIB, a potent eIF2a inhibitor, significantly suppresses PTEN-deficient and MYCoverexpressing prostate cancer progression, extending the survival of tumor-bearing mice (137,138). One study shows that pro-inflammatory cytokine intervention significantly upregulates the expression of IRE1-a and PERK, but does not change the expression of ATF6 (12). ...
Full-text available
Endoplasmic reticulum (ER) is an important player in various intracellular signaling pathways that regulate cellular functions in many diseases. Intervertebral disc degeneration (IDD), an age-related degenerative disease, is one of the main clinical causes of low back pain. Although the pathological development of IDD is far from being fully elucidated, many studies have been shown that ER stress (ERS) is involved in IDD development and regulates various processes, such as inflammation, cellular senescence and apoptosis, excessive mechanical loading, metabolic disturbances, oxidative stress, calcium homeostasis imbalance, and extracellular matrix (ECM) dysregulation. This review summarizes the formation of ERS and the potential link between ERS and IDD development. ERS can be a promising new therapeutic target for the clinical management of IDD.
... Although the traditional approach to cancer targeted therapy focused on inhibiting the driver oncogene, pharmacological forcing of irremediable oncogenic stress has been suggested as a viable alternative, especially in the cancers where oncogene-targeted therapy is not feasible (e.g. MYCdriven cancers) or where tumors have gained resistance to the oncogene-targeting agent [8][9][10][11]. We have previously shown that strong oncogenic signaling through Her2 amplification imposes a proteotoxic stress on the mammary epithelial cell that has to be mitigated by the activation of compensatory stress relief systems to allow for the tumor cell to survive [12]. Her2 + breast cancer cells are characterized by increased protein synthesis load due to chromosomal amplifications and hyperactive Her2/mTOR signaling, which creates dependence on the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to maintain protein homeostasis and prevent proteotoxic stress [12]. ...
Full-text available
Oncogenic kinase inhibitors show short-lived responses in the clinic due to high rate of acquired resistance. We previously showed that pharmacologically exploiting oncogene-induced proteotoxic stress can be a viable alternative to oncogene-targeted therapy. Here, we performed extensive analyses of the transcriptomic, metabolomic and proteostatic perturbations during the course of treatment of Her2+ breast cancer cells with a Her2 inhibitor covering the drug response, resistance, relapse and drug withdrawal phases. We found that acute Her2 inhibition, in addition to blocking mitogenic signaling, leads to significant decline in the glucose uptake, and shutdown of glycolysis and of global protein synthesis. During prolonged therapy, compensatory overexpression of Her3 allows for the reactivation of mitogenic signaling pathways, but fails to re-engage the glucose uptake and glycolysis, resulting in proteotoxic ER stress, which maintains the protein synthesis block and growth inhibition. Her3-mediated cell proliferation under ER stress during prolonged Her2 inhibition is enabled due to the overexpression of the eIF2 phosphatase GADD34, which uncouples protein synthesis block from the ER stress response to allow for active cell growth. We show that this imbalance in the mitogenic and proteostatic signaling created during the acquired resistance to anti-Her2 therapy imposes a specific vulnerability to the inhibition of the endoplasmic reticulum quality control machinery. The latter is more pronounced in the drug withdrawal phase, where the de-inhibition of Her2 creates an acute surge in the downstream signaling pathways and exacerbates the proteostatic imbalance. Therefore, the acquired resistance mechanisms to oncogenic kinase inhibitors may create secondary vulnerabilities that could be exploited in the clinic.
... Previously, we showed that the PERK-eIF2α pathway, which is part of the integrated stress response (ISR), is activated in CD34+ CML-BP cells and is correlated with disease progression and resistance to TKIs [14]. The ISR is a highly conserved signaling pathway responsible for cell adaptation and survival under stress conditions [15][16][17][18]. This is achieved by phosphorylation of the eukaryotic translation initiation factor eIF2α that attenuates the guanine nucleotide exchange factor eIF2B, leading therefore to remodeling of translation and transcription of stress response effector genes, including CHOP and GADD34, which are ISR markers [19]. ...
Full-text available
The integrated stress response (ISR) facilitates cellular adaptation to unfavorable conditions by reprogramming the cellular response. ISR activation was reported in neurological disorders and solid tumors; however, the function of ISR and its role as a possible therapeutic target in hematological malignancies still remain largely unexplored. Previously, we showed that the ISR is activated in chronic myeloid leukemia (CML) cells and correlates with blastic transformation and tyrosine kinase inhibitor (TKI) resistance. Moreover, the ISR was additionally activated in response to imatinib as a type of protective internal signaling. Here, we show that ISR inhibition combined with imatinib treatment sensitized and more effectively eradicated leukemic cells both in vitro and in vivo compared to treatment with single agents. The combined treatment specifically inhibited the STAT5 and RAS/RAF/MEK/ERK pathways, which are recognized as drivers of resistance. Mechanistically, this drug combination attenuated both interacting signaling networks, leading to BCR-ABL1- and ISR-dependent STAT5 activation. Consequently, leukemia engraftment in patient-derived xenograft mice bearing CD34+ TKI-resistant CML blasts carrying PTPN11 mutation responsible for hyperactivation of the RAS/RAF/MAPK and JAK/STAT5 pathways was decreased upon double treatment. This correlated with the downregulation of genes related to the RAS/RAF/MAPK, JAK/STAT5 and stress response pathways and was associated with lower expression of STAT5-target genes regulating proliferation, viability and the stress response. Collectively, these findings highlight the effect of imatinib plus ISRIB in the eradication of leukemic cells resistant to TKIs and suggest potential clinical benefits for leukemia patients with TKI resistance related to RAS/RAF/MAPK or STAT5 signaling. We propose that personalized treatment based on the genetic selection of patients carrying mutations that cause overactivation of the targeted pathways and therefore make their sensitivity to such treatment probable should be considered as a possible future direction in leukemia treatment.
... This is in line with recent evidence associating alterations in the phosphorylated eIF2␣ translational pathway with cancer, a process highly linked to the cellular stress response (7). Moreover, altered phosphorylation of eIF2␣ has been observed to occur as an adaptive stress response in both murine and humanized models of aggressive and resistant PCa (8). Perturbations in translation regulation may therefore represent key indicators of PCa severity. ...
Full-text available
Emerging evidence associates translation factors and regulators to tumorigenesis. However, our understanding of translational changes in cancer resistance is still limited. Here, we generated an enzalutamide-resistant prostate cancer (PCa) model, which recapitulated key features of clinical enzalutamide-resistant PCa. Using this model and poly(ribo)some profiling, we investigated global translation changes that occur during acquisition of PCa resistance. We found that enzalutamide-resistant cells exhibit an overall decrease in mRNA translation with a specific deregulation in the abundance of proteins involved in mitochondrial processes and in translational regulation. However, several mRNAs escape this translational downregulation and are nonetheless bound to heavy polysomes in enzalutamide-resistant cells suggesting active translation. Moreover, expressing these corresponding genes in enzalutamide-sensitive cells promotes resistance to enzalutamide treatment. We also found increased association of long non-coding RNAs (lncRNAs) with heavy polysomes in enzalutamide-resistant cells, suggesting that some lncRNAs are actively translated during enzalutamide resistance. Consistent with these findings, expressing the predicted coding sequences of known lncRNAs JPX, CRNDE and LINC00467 in enzalutamide-sensitive cells drove resistance to enzalutamide. Taken together, this suggests that aberrant translation of specific mRNAs and lncRNAs is a strong indicator of PCa enzalutamide resistance, which points towards novel therapeutic avenues that may target enzalutamide-resistant PCa.
... Phosphorylation of the eIF2 subunit regulates the heterotrimer activity [10]. Oncogenic roles of eIF2α are well-known in chronic myeloid leukemia [11], colorectal cancer [12], prostate cancer [13], and so on. eIF2β forms part of the nucleotide-binding pocket in eukaryotes and appears to bind GTP or GDP [14]. ...
Full-text available
Somatotroph adenomas are the leading cause of acromegaly, with the nearly sparsely granulated somatotroph subtype belonging to high-risk adenomas, and they are less responsive to medical treatment. The integrated stress response (ISR) is an essential stress-support pathway increasingly recognized as a determinant of tumorigenesis. In this study, we identified the characteristic profiling of the integrated stress response in translocation and translation initiation factor activity in somatotroph adenomas, normal pituitary, or other adenoma subtypes through proteomics. Immunohistochemistry exhibited the differential significance and the priority of eukaryotic translation initiation factor 2β (EIF2β) in somatotroph adenomas compared with gonadotroph and corticotroph adenomas. Differentially expressed genes based on the level of EIF2β in somatotroph adenomas were revealed. MetaSape pathways showed that EIF2β was involved in regulating growth and cell activation, immune system, and extracellular matrix organization processes. The correlation analysis showed Spearman correlation coefficients of r = 0.611 (p < 0.001) for EIF2β and eukaryotic translation initiation factor 2 alpha kinase 1 (HRI), r = 0.765 (p < 0.001) for eukaryotic translation initiation factor 2 alpha kinase 2 (PKR), r = 0.813 (p < 0.001) for eukaryotic translation initiation factor 2 alpha kinase 3 (PERK), r = 0.728 (p < 0.001) for GCN2, and r = 0.732 (p < 0.001) for signal transducer and activator of transcription 3 (STAT3). Furthermore, the invasive potential in patients with a high EIF2β was greater than that in patients with a low EIF2β (7/10 vs. 4/18, p = 0.038), with a lower immune-cell infiltration probability (p < 0.05). The ESTIMATE algorithm showed that the levels of activation of the EIF2 pathway were negatively correlated with the immune score in somatotroph adenomas (p < 0.001). In in vitro experiments, the knockdown of EIF2β changed the phenotype of somatotroph adenomas, including cell proliferation, migration, and the secretion ability of growth hormone/insulin-like growth factor-1. In this study, we demonstrate that the ISR is pivotal in somatotroph adenomas and provide a rationale for implementing ISR-based regimens in future treatment strategies.
... Moreover, other drugs that killed FBXW7-null cells, albeit with diverse primary mechanisms of action, were all shown to induce ISR signaling [305]. These observations are in apparent contrast with the known function of the ISR as one of those adaptive mechanisms induced by oncogenic stress that favor cancer cell survival and expansion, as shown in diverse tumor models [307][308][309][310], including MYC-driven lymphoma [311,312]. This apparent paradox may be readily rationalized, however, based on the well-documented dual role of the ISR in cell survival and death [284,285]. ...
Full-text available
The MYC transcription factor, encoded by the c-MYC proto-oncogene, is activated by growth-promoting signals, and is a key regulator of biosynthetic and metabolic pathways driving cell growth and proliferation. These same processes are deregulated in MYC-driven tumors, where they become critical for cancer cell proliferation and survival. As other oncogenic insults, overexpressed MYC induces a series of cellular stresses (metabolic, oxidative, replicative, etc.) collectively known as oncogenic stress, which impact not only on tumor progression, but also on the response to therapy, with profound, multifaceted consequences on clinical outcome. On one hand, recent evidence uncovered a widespread role for MYC in therapy resistance in multiple cancer types, with either standard chemotherapeutic or targeted regimens. Reciprocally, oncogenic MYC imparts a series of molecular and metabolic dependencies to cells, thus giving rise to cancer-specific vulnerabilities that may be exploited to obtain synthetic-lethal interactions with novel anticancer drugs. Here we will review the current knowledge on the links between MYC and therapeutic responses, and will discuss possible strategies to overcome resistance through new, targeted interventions.
Clinical responses to second generation androgen signaling inhibitors (e.g., enzalutamide) in metastatic castration-resistant prostate cancer (mCRPC) are variable and transient, and are associated with dose limiting toxicities, including rare but severe CNS effects. We hypothesized that changes to iron metabolism coincident with more advanced disease might be leveraged for tumor-selective delivery of antiandrogen therapy. Using the recently described chemical probes SiRhoNox and 18F-TRX in mCRPC models, we found elevated Fe2+ to be a common feature of mCRPC in vitro and in vivo. We next synthesized ferrous-iron activatable drug conjugates of second and third-generation antiandrogens and found these conjugates possessed comparable or enhanced antiproliferative activity across mCRPC cell line models. Mouse pharmacokinetic studies showed that these prototype antiandrogen conjugates are stable in vivo and limited exposure to conjugate or free antiandrogen in the brain. Our results reveal elevated Fe2+ to be a feature of mCRPC that might be leveraged to improve the tolerability and efficacy of antiandrogen therapy.
Androgen receptor, which regulates diverse biological processes for cell fate decisions, forms a homodimer in the cytoplasm and is monomerized by activation for nuclear translocation. Ser815 phosphorylated AR is expressed in mature prostates, with levels decreased by castration in mice or prostate cancer progression in humans. Here, we have examined the functional and biological roles of phosphorylation. AR phosphorylation at Ser815 stabilized homodimer formation in the cytoplasm, interrupting DHT-response nuclear translocation. cDNA microarray studies in castrated mouse prostates implied castration attenuates ER stress responses, suggesting AR phosphorylation acts on ER stress responses. In addition, AR Ser815Asp phospho-mimetic mutant expression augmented ER stress-induced death in PC-3 cells. These results suggested that phosphorylation at AR Ser815 modulates AR functions for maintaining the prostate.
Full-text available
In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four members of the eIF2α kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro-survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.
ISRIB mechanism of action In rodents, a druglike small molecule named ISRIB enhances cognition and reverses cognitive deficits after traumatic brain injury. ISRIB activates a protein complex called eIF2B that is required for the synthesis of new proteins. Tsai et al. report the visualization of eIF2B bound to ISRIB at near-atomic resolution by cryo–electron microscopy. Biochemical studies revealed that ISRIB is a “molecular staple” that promotes assembly of the fully active form of eIF2B. Zyryanova et al. report similar structures together with information on the binding of ISRIB analogs and their effects on protein translation. Science , this issue p. eaaq0939 , p. 1533
Purpose of review: In recent years, a large number of reports have been published on prostate-specific membrane antigen (PSMA)/PET in prostate cancer (PCa). This review highlights advances in PSMA PET in PCa during the past year. Recent findings: PSMA PET/computed tomography (CT) is useful in detection of biochemical recurrence, especially at low prostate-specific antigen (PSA) values. The detection rate of PSMA PET is influenced by PSA level. For primary PCa, PSMA PET/CT shows promise for tumour localization in the prostate, especially in combination with multiparametric MRI (mpMRI). For primary staging, PSMA PET/CT can be used in intermediate and high-risk PCa. Intraoperative PSMA radioligand guidance seems promising for detection of malignant lymph nodes. While the use of PSMA PET/MRI in primary localized disease is limited to high and intermediate-risk patients and localized staging, in the recurrence setting, PET/MRI can be particularly helpful when the lesions are subtle. PSMA PET/CT is superior to choline PET/CT and other conventional imaging modalities. Summary: Molecular imaging with PSMA PET continues to pave the way for personalized medicine in PCa.However, large prospective clinical studies are still needed to fully evaluate the role of PSMA PET/CT and PET/MRI in the clinical workflow of PCa.
Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.
The evidence for the importance of aberrant translation in cancer cells is overwhelming. Reflecting the wealth of data, there are excellent reviews delineating how ribosomes and initiation factors are linked to cancer [1-3], and the therapeutic strategies being devised to target them [4]. Changes in translational efficiency can engender a malignant phenotype without the need for chromatin reorganization, transcription, splicing and mRNA export [5,6]. Thus, cancer-related modulations of the translational machinery are ideally suited to allow cancer cells to respond to the various stresses encountered along the path of tumorigenesis and organism-wide dissemination [7(•),8,9,10(•)]. Emerging findings supporting this notion are the focus of this review.
We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5' untranslated regions in cancer, and expose new targets for therapeutic intervention.
The past several years have seen dramatic leaps in our understanding of how gene expression is rewired at the translation level during tumorigenesis to support the transformed phenotype. This work has been driven by an explosion in technological advances and is revealing previously unimagined regulatory mechanisms that dictate functional expression of the cancer genome. In this Review we discuss emerging trends and exciting new discoveries that reveal how this translational circuitry contributes to specific aspects of tumorigenesis and cancer cell function, with a particular focus on recent insights into the role of translational control in the adaptive response to oncogenic stress conditions.
Tumor heterogeneity may reduce the efficacy of molecularly guided systemic therapy for cancers that have metastasized. To determine whether the genomic alterations in a single metastasis provide a reasonable assessment of the major oncogenic drivers of other dispersed metastases in an individual, we analyzed multiple tumors from men with disseminated prostate cancer through whole-exome sequencing, array comparative genomic hybridization (CGH) and RNA transcript profiling, and we compared the genomic diversity within and between individuals. In contrast to the substantial heterogeneity between men, there was limited diversity among metastases within an individual. The number of somatic mutations, the burden of genomic copy number alterations and aberrations in known oncogenic drivers were all highly concordant, as were metrics of androgen receptor (AR) activity and cell cycle activity. AR activity was inversely associated with cell proliferation, whereas the expression of Fanconi anemia (FA)-complex genes was correlated with elevated cell cycle progression, expression of the E2F transcription factor 1 (E2F1) and loss of retinoblastoma 1 (RB1). Men with somatic aberrations in FA-complex genes or in ATM serine/threonine kinase (ATM) exhibited significantly longer treatment-response durations to carboplatin than did men without defects in genes encoding DNA-repair proteins. Collectively, these data indicate that although exceptions exist, evaluating a single metastasis provides a reasonable assessment of the major oncogenic driver alterations that are present in disseminated tumors within an individual, and thus may be useful for selecting treatments on the basis of predicted molecular vulnerabilities.
An increasingly recognized resistance mechanism to androgen receptor (AR)-directed therapy in prostate cancer involves epithelial plasticity, in which tumor cells demonstrate low to absent AR expression and often have neuroendocrine features. The etiology and molecular basis for this 'alternative' treatment-resistant cell state remain incompletely understood. Here, by analyzing whole-exome sequencing data of metastatic biopsies from patients, we observed substantial genomic overlap between castration-resistant tumors that were histologically characterized as prostate adenocarcinomas (CRPC-Adeno) and neuroendocrine prostate cancer (CRPC-NE); analysis of biopsy samples from the same individuals over time points to a model most consistent with divergent clonal evolution. Genome-wide DNA methylation analysis revealed marked epigenetic differences between CRPC-NE tumors and CRPC-Adeno, and also designated samples of CRPC-Adeno with clinical features of AR independence as CRPC-NE, suggesting that epigenetic modifiers may play a role in the induction and/or maintenance of this treatment-resistant state. This study supports the emergence of an alternative, 'AR-indifferent' cell state through divergent clonal evolution as a mechanism of treatment resistance in advanced prostate cancer.