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Published OnlineFirst May 9, 2012; DOI:10.1158/1078-0432.CCR-12-0782
Evidence for Efficacy of New Hsp90 Inhibitors Revealed by Ex Vivo
Culture of Human Prostate Tumors
Margaret M. Centenera, Joanna L. Gillis, Adrienne R. Hanson, et al.
Clin Cancer Res 2012;18:3562-3570. Published OnlineFirst May 9, 2012.
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Copyright © 2012 American Association for Cancer Research
Published OnlineFirst May 9, 2012; DOI:10.1158/1078-0432.CCR-12-0782
Clinical
Cancer
Research
Cancer Therapy: Preclinical
Evidence for Efficacy of New Hsp90 Inhibitors Revealed
by Ex Vivo Culture of Human Prostate Tumors
Margaret M. Centenera1, Joanna L. Gillis1, Adrienne R. Hanson1, Shalini Jindal1, Renea A. Taylor3,
Gail P. Risbridger3, Peter D. Sutherland2, Howard I. Scher4, Ganesh V. Raj5, Karen E. Knudsen6,
Trina Yeadon for the Australian Prostate Cancer BioResource1, Wayne D. Tilley1, and Lisa M. Butler1
Abstract
Purpose: Targeting Hsp90 has significant potential as a treatment for prostate cancer, but prototypical
agents such as 17-allylamino-17 demethoxygeldanamycin (17-AAG) have been ineffective in clinical trials.
Recently, a phase I study aimed at defining a biologically active dose reported the first response to an Hsp90
inhibitor in a patient with prostate cancer, which supports the development of new generation compounds
for this disease.
Experimental Design: The biological actions of two new synthetic Hsp90 inhibitors, NVP-AUY922 and
NVP-HSP990, were evaluated in the prostate cancer cell lines PC-3, LNCaP, and VCaP and in an ex vivo
culture model of human prostate cancer.
Results: In cell lines, both NVP-AUY922 and NVP-HSP990 showed greater potency than 17-AAG with
regard to modulation of Hsp90 client proteins, inhibition of proliferation, and induction of apoptotic cell
death. In prostate tumors obtained from radical prostatectomy that were cultured ex vivo, treatment with 500
nmol/L of NVP-AUY922, NVP-HSP990, or 17-AAG caused equivalent target modulation, determined by the
pharmacodynamic marker Hsp70, but only NVP-AUY922 and NVP-HSP990 showed antiproliferative and
proapoptotic activity.
Conclusions: This study provides some of the first evidence that new generation Hsp90 inhibitors are
capable of achieving biologic responses in human prostate tumors, with both NVP-AUY922 and NVPHSP990 showing potent on-target efficacy. Importantly, the ex vivo culture technique has provided
information on Hsp90 inhibitor action not previously observed in cell lines or animal models. This
approach, therefore, has the potential to enable more rational selection of therapeutic agents and
biomarkers of response for clinical trials. Clin Cancer Res; 18(13); 3562–70. 2012 AACR.
Introduction
The molecular chaperone Hsp90 is responsible for folding and stabilizing client proteins into their active conformation. Hsp90 has more than 200 clients, including many
known oncoproteins (1), thus pharmacologic inhibition of
Hsp90 in cancer cells has the potential to simultaneously
Authors' Affiliations: 1Dame Roma Mitchell Cancer Research Laboratories and Adelaide Prostate Cancer Research Centre, University of Adelaide
and Hanson Institute, Adelaide; 2Urology Unit, Surgical Specialties Service,
Royal Adelaide Hospital, Adelaide; 3Prostate and Breast Cancer Research
Group, Department of Anatomy and Developmental Biology, Monash
University, Clayton, Australia; 4Genitourinary Oncology Service, Memorial
Sloan-Kettering Cancer Center, New York; 5Department of Urology, UT
Southwestern Medical Center at Dallas, Dallas, Texas; and 6Kimmel Cancer
Center, Thomas Jefferson University, Philadelphia, Pennsylvania
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Lisa M. Butler, Dame Roma Mitchell Cancer
Research Laboratories, Discipline of Medicine, University of Adelaide and
Hanson Institute, Adelaide, SA 5000, Australia. Phone: 61-8-8222-3270;
Fax: 61-8-8222-3217; E-mail: [email protected]
doi: 10.1158/1078-0432.CCR-12-0782
2012 American Association for Cancer Research.
3562
target multiple signaling pathways implicated in cancer
progression. Targeting Hsp90 in prostate cancer is a particularly attractive therapeutic strategy as Hsp90 is commonly
overexpressed in prostate tumor cells compared with normal epithelium (2). Moreover, the androgen receptor, the
key driver of prostate cancer progression (3), is an Hsp90
client protein.
Given the promising preclinical studies of Hsp90 inhibition in prostate cancer cells (4–6), the lack of efficacy
observed for these agents in prostate cancer clinical trials
has been disappointing (7–9). The ansamycin derivatives
17-allylamino-17 demethoxygeldanamycin (17-AAG) and
17-(dimethylaminotheyl-amino)-17-demethoxygeldanamycin (17-DMAG) are the most extensively characterized
Hsp90 inhibitors. Their clinical failure has been attributed
to poor solubility and pharmacokinetics, hepatotoxicity
and susceptibility to MDR (10). However, the first response
to an Hsp90 inhibitor in a patient with castration-resistant
prostate cancer (CRPC) was recently reported in a phase I
trial of 17-DMAG that aimed to define a biologically effective dose rather than maximum tolerated dose (MTD;
ref. 11). This encouraging result supports the development
Clin Cancer Res; 18(13) July 1, 2012
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Copyright © 2012 American Association for Cancer Research
Published OnlineFirst May 9, 2012; DOI:10.1158/1078-0432.CCR-12-0782
Efficacy of New Hsp90 Inhibitors in Prostate Tumors
Translational Relevance
The study uses a unique application of ex vivo human
prostate cancer tissue culture to provide the first evidence
that the new generation Hsp90 inhibitors NVP-AUY922
and NVP-HSP990 have significant potential as anticancer agents for men with prostate cancer. We also show
that induction of Hsp70, the widely used pharmacodynamic marker for this class of drugs, is not associated
with biologic response to Hsp90 inhibition. Clinically,
the ex vivo culture model used in this study has the
potential to improve preclinical development of novel
agents by assessing drug actions in the context of the
normal tumor microenvironment. This approach will
facilitate more rational selection of bioactive agents and
the identification of more robust markers of clinical
responses.
of new generation inhibitors with improved pharmacokinetics but also suggests that evaluating these agents at the
level of the tumor, rather than by MTD, may be essential for
this class of agent (12).
The chaperone activity of Hsp90 is ATP dependent (13).
Most Hsp90 inhibitors bind to the conserved ATP pocket of
Hsp90, preventing maturation of client proteins and causing their proteasomal degradation. NVP-AUY922 (hereafter
called AUY922) and NVP-HSP990 (HSP990) likewise bind
to the ATP-binding pocket of Hsp90 and represent 2 of the
most potent synthetic small molecule inhibitors reported to
date (14, 15). AUY922 is a resorcinylic isoxazole amide with
antitumor activity against a range of cancer cell lines and
animal models, including PC-3 prostate cancer cells and
xenografts (14, 16–18), and in a phase I study in patients
with advanced solid malignancies, disease stabilization
(16%) was observed (19). HSP990 is an orally available
aminopyrimidine with antiproliferative activity in a variety
of cancer cell lines and xenografts (15, 20) and is currently
in phase I clinical trials (www.clinicaltrials.gov). Unlike 17AAG, AUY922 and HSP990 are not susceptible to MDR
mechanisms such as P-glycoprotein–mediated efflux or
polymorphic metabolism by the enzyme NQO1/DT-diaphorase (14, 15).
The objective of this study was to compare the potential of
AUY922 and HSP990 with 17-AAG to modulate Hsp90
client proteins and influence proliferation and death of
prostate cancer cells, in vitro, and in ex vivo cultures of
human primary tumors.
Modified Eagle’s Medium containing 10% FBS, 1% sodium
pyruvate, 1% MEM non–essential amino acids, and 0.1
nmol/L 5a-dihydrotestosterone. AUY922 and HSP990
were obtained from Novartis and dissolved in dimethyl
sulfoxide (DMSO). 17-AAG was obtained from the National Cancer Institute and dissolved in DMSO. Primary antibodies are detailed in Supplementary Table S1.
Cell viability
Trypan blue exclusion and 40 , 6-Diamidino-2-phenylindole (DAPI) staining were carried out as described previously (21).
Cell cycle
Flow cytometric analysis of cell-cycle distribution was
carried out on cells treated with vehicle (DMSO) or Hsp90
inhibitors, with or without the pan-caspase inhibitor zVADfmk (20 mmol/L; BD Biosciences), as described previously
(21). Analysis was done on a FACSCalibur (BD Biosciences)
running CellQuest software (BD Biosciences). DNA frequency histograms were obtained using FlowJo software,
using the Dean-Jett-Fox model.
Ex vivo culture of human prostate tumors
Prostate cancer tissue was obtained with written informed
consent through the Australian Prostate Cancer BioResource
from men undergoing robotic radical prostatectomy at the
Royal Adelaide Hospital. Histopathologic features of tumors
used in this study are detailed in Table 1. An 8-mm core of
tissue was dissected into 1-mm3 pieces and cultured in
duplicate on a presoaked gelatin sponge (Johnson and
Johnson) in 24-well plates containing 500 mL RPMI-1640
with 10% FBS, antibiotic/antimycotic solution, 0.01 mg/mL
hydrocortisone and 0.01 mg/mL insulin (Sigma). Tissues
were cultured at 37 C for 48 hours, then formalin-fixed and
paraffin embedded or preserved in RNAlater (Invitrogen).
Protein preparation
Whole-cell lysates were prepared as described previously
(21). Cultured prostate tumors stored in RNAlater were
lysed in ice-cold radioimmunoprecipitation assay buffer
(10 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA,
1% Triton X-100) containing protease inhibitor cocktail
(Sigma), using the Precellys24 tissue homogenizer (Bertin
Technologies).
Materials and Methods
Immunoblotting
Proteins (20 mg) were analyzed by immunoblotting as
described previously (21). Densitometry was carried out
using the Fluorchem SP Digital Imaging System (Alpha
Innotech Corporation).
Cells and reagents
LNCaP, VCaP, and PC-3 human prostate carcinoma cells
were obtained from the American Type Culture Collection.
All cell lines underwent verification by short-tandem repeat
profiling in 2010 by Cell Bank Australia. LNCaP and PC-3
cells were maintained in RPMI-1640 containing 10% or 5%
FBS, respectively. VCaP cells were maintained in Dulbecco’s
Immunohistochemistry
Sections (2 mm) on SuperFrost-plus slides underwent
immunohistochemical staining as detailed in Supplementary Table S2. Images were captured with a Nanozoomer scanner. The percentage of Ki67 and cleaved
caspase-3–positive nuclei were determined by blind
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manual counting of at least 200 malignant cells over 5 to
10 fields at 40 magnification. Due to variation in staining, areas of highest density staining were selected for
counting. Samples were removed from analysis only if
there were insufficient malignant cells present, or if the
Ki67 positivity in the DMSO-treated sample was 2% or
less. On the basis of these criteria, 6 of 19 patient samples
were removed due to lack of malignancy and 3 of 19 due
to Ki67 levels. All remaining tissues were included for
statistical analysis.
Statistical analysis
Data are displayed as the mean SE. Differences were
determined using one-way ANOVA with Tukey or Dunnett post hoc test as indicated, using GraphPad Prism
Software. Repeated measures ANOVA were used where
stated. A P value 0.05 or less was considered statistically
significant.
Results
Enhanced potency of AUY922 and HSP990 compared
with 17-AAG in human prostate cancer cell lines
The efficacy of the synthetic Hsp90 inhibitors, AUY922
and HSP990, was assessed in androgen receptor–negative
(PC-3) and androgen receptor–positive (LNCaP, VCaP)
prostate cancer cell lines and compared with that of 17AAG. In LNCaP cells, AUY922 caused a significant, dosedependent inhibition of cell proliferation (P < 0.001)
compared with vehicle, with maximal inhibition achieved
at approximately 20 nmol/L (Fig. 1A; Supplementary Fig.
S1). HSP990 and 17-AAG had lower antiproliferative activity than AUY922 at equivalent concentrations. VCaP was
the least sensitive cell line, and PC-3 cells were markedly
more sensitive than LNCaP cells with respect to inhibition
of proliferation by HSP990 and AUY922; 17-AAG displayed
the least efficacy in all cell lines (Fig. 1A). Both AUY922 and
HSP990 (10–40 nmol/L) induced a dose-dependent
increase in death of all 3 cell lines (P < 0.001; Fig. 1B).
17-AAG did not induce death in any of the cell lines with the
doses used (10–80 nmol/L; Fig. 1B).
AUY922 and HSP990 induce G2–M cell-cycle arrest and
apoptosis
Culture of PC-3 and LNCaP cells with AUY922 or
HSP990 (40 nmol/L) caused accumulation of cells in phase
G2–M of the cell cycle (Fig. 2A and B). No cell-cycle changes
were observed for cells cultured with 17-AAG (Supplementary Fig. S2). AUY922 and HSP990 induced a significant
Figure 1. AUY922 and HSP990 are more potent than 17-AAG in prostate cancer cells. PC-3 and LNCaP cells were treated for 4 days and VCaP cells for 5 days,
with the indicated doses of 17-AAG, AUY922, HSP990, or vehicle control. Cell proliferation (A) and cell death (B) were assessed by trypan blue exclusion.
Results are representative of 3 independent experiments and are presented as the mean SE of triplicate wells. ANOVA with Tukey post hoc test:
, a, b; P < 0.01 treatment versus equivalent dose of 17-AAG.
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Published OnlineFirst May 9, 2012; DOI:10.1158/1078-0432.CCR-12-0782
Efficacy of New Hsp90 Inhibitors in Prostate Tumors
Figure 2. HSP990 and AUY922
induce G2–M cell-cycle arrest and
apoptotic cell death. PC-3 (A) and
LNCaP (B) cells were cultured in
medium containing vehicle control,
40 nmol/L AUY922 or HSP990, in the
absence or presence of the pan
caspase inhibitor zVAD-fmk. Cells
were fixed following the indicated
times, stained with propidium iodide,
and analyzed by flow cytometry. Cellcycle distribution was determined
using FlowJo software. Results are
representative of 2 independent
experiments, each containing
triplicate wells. C, the percentage of
cells in the sub-G1 phase of the cell
cycle in each treatment group
presented graphically as the mean SE of triplicate wells. ANOVA with
Tukey post hoc test: , P < 0.001. D,
representative DAPI staining
showing changes in DNA
morphology (arrows) in PC-3 cells
treated with 40 nmol/L AUY922 or
HSP990 compared with vehicle
control. Graph shows percentage of
cells displaying abnormal nuclear
morphology, counted in 10 random
fields at 40 magnification.
increase in the sub-G1 peak from 3% in control cells to 27%
and 24%, respectively, in PC-3 cells treated for 72 hours (P <
0.001, Fig. 2A and C), indicative of induction of apoptosis
by these agents. In LNCaP cells, significant accumulation of
cells in sub-G1 was evident after 96 hours of treatment with
AUY922 (13%) or HSP990 (7%) compared with vehicle
(3%, P < 0.05, Fig. 2A and C). Consistent with their antiproliferative efficacy, both inhibitors induced a greater
magnitude of change in PC-3 cells compared with LNCaP
cells. Coculture of PC-3 cells with the pan-caspase inhibitor
zVAD-fmk completely prevented the observed increase in
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sub-G1 fraction in AUY922- and HSP990-treated cells and
caused a concomitant increase in cells in the G2–M phase (P
< 0.001, Fig. 2A and C). In LNCaP cells, zVAD-fmk prevented cells from entering sub-G1 in cells treated with
AUY922 (P < 0.001) but not HSP990 (Fig. 2B and C).
DAPI staining showed that PC-3 or LNCaP cells cultured for 24 hours with AUY922 or HSP990 (40 nmol/L)
exhibited chromatin condensation (Fig. 2D), which is
characteristic of apoptosis. As above, PC-3 cells showed
the greatest response to treatment with AUY922 (32.6%)
or HSP990 (23%) when compared with vehicle
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Centenera et al.
(10%; Fig. 2C). In addition, phase-contrast microscopy of
cells treated with the Hsp90 inhibitors showed typical
apoptotic morphology, including membrane blebbing
and formation of apoptotic bodies (Supplementary
Fig. S3).
AUY922 and HSP990 modulate Hsp90 client proteins in
prostate cancer cell lines
Induction of Hsp70 and degradation of Cdk4 and c-Raf
are common clinical markers of Hsp90 inhibition (7, 22).
Induction of Hsp70 and a concomitant reduction in
c-Raf, Cdk4, Her2, and Akt were observed in PC-3 and
LNCaP cells treated with 40 nmol/L AUY922 and HSP990
compared with control, but AUY922 was consistently
more potent than HSP990 (Fig. 3A and B). Marked
depletion of androgen receptor and prostate-specific antigen (PSA), an androgen receptor–regulated protein, was
observed following treatment of LNCaP cells with both
inhibitors (Fig. 3B). Modulation of client proteins
was absent or modest in cells treated with 40 nmol/L
17-AAG, a concentration that was maximally effective
for AUY922 and HSP990. No change in Hsp90 expression
was observed with any treatment in either cell line
(Fig. 3A and B).
AUY922, HSP990, and 17-AAG induce Hsp70 in human
prostate tumors ex vivo
To assess the activity of the synthetic Hsp90 inhibitors
in clinical prostate cancer, we used an ex vivo methodology to culture human prostate tumors (Fig. 4A). Initially
we assessed whether the Hsp90 inhibitors could induce
Hsp70 expression in the human prostate tumors.
Matched tissues from the same patients were cultured in
medium with increasing concentrations of an Hsp90
inhibitor. A dose-dependent increase in Hsp70 levels was
observed in tumors cultured with 17-AAG, AUY922, or
HSP990 (at 100, 500, or 1,000 nmol/L) compared with
control (Fig. 4B).
On the basis of these initial studies, the ability of each
Hsp90 inhibitor to induce Hsp70 and decrease levels of 2
Hsp90 client proteins with relevance to prostate cancer
(androgen receptor and Akt) was directly compared at a
single, effective dose (500 nmol/L) in 8 independent
tumors. Compared with control, Hsp70 expression was
significantly increased in tumors treated with each inhibitor
(P < 0.01; Fig. 4C), and androgen receptor expression was
significantly decreased (P < 0.01; Fig. 4C). A similar reduction in Akt levels was also observed in tumors cultured with
the inhibitors (Fig. 4C).
As immunoblotting was carried out on whole tissue
lysates, immunohistochemistry was used to validate the
observed induction of Hsp70 in tumor cells. Significant
Hsp70 induction was seen in the tumor cells of human
prostate tissues treated with 17-AAG, AUY922, or HSP990
when compared with vehicle control (P < 0.05, Fig. 5A).
Antiproliferative and proapoptotic activity of AUY922
and HSP990, but not 17-AAG, in human prostate
tumors
We next assessed whether modulation of Hsp70 in the
human prostate tissues was associated with altered biologic
endpoints such as proliferation or apoptosis. When we
assessed Ki67 positivity in the same tumor tissues assessed
above for Hsp70, only 1 of 7 tumors cultured with 17-AAG
at a dose (500 nmol/L) that was effective in modulating
levels of Hsp70 (Fig. 5B) elicited a decrease in proliferation
compared with control tissues. Overall, there was no significant change in Ki67 positivity between 17-AAG-treated
(32.3%) and control tissues (32.5%; Fig. 5B). In contrast,
Ki67 positivity was markedly and consistently reduced to
1.8% and 0.5% in tissues cultured with 500 nmol/L
AUY922 or HSP990, respectively (P < 0.001; Fig. 5B).
Figure 3. New Hsp90 inhibitors are
more potent modulators of Hsp90
client proteins than 17-AAG. PC-3
(A) and LNCaP (B) cells treated for
24 hours with the indicated doses
of 17-AAG, AUY922, or HSP990
were analyzed by Western blot for
modulation of Hsp90, Hsp70, PSA,
or Hsp90 client proteins HER2,
androgen receptor, c-RAF, AKT,
and CDK4. Glyceraldehyde-3phosphate dehydrogenase
(GAPDH) was used as a loading
control.
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Clinical Cancer Research
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Efficacy of New Hsp90 Inhibitors in Prostate Tumors
Figure 4. Hsp90 inhibitors induce
Hsp70 expression in ex vivo cultured
human prostate tumors. A, ex vivo
tissue culture protocol. Within 60
minutes of being removed by robotic
radical prostatectomy, human
prostate tumor samples were
3
obtained and dissected into 1-mm
pieces that were cultured on gelatin
sponges inside wells containing
medium. B, tumors cultured with
medium containing increasing doses
of 17-AAG, AUY922, or HSP990 for
48 hours were analyzed by
immunoblotting for levels of Hsp70.
GAPDH was used as a loading
control. Graph represents
densitometry analysis of
immunoblots in which levels of
Hsp70 are normalized to GAPDH. C,
densitometry analysis of
immunoblots from tissues cultured
with vehicle control (C), 500 nmol/L
17-AAG (17), AUY922 (A) or HSP990
(H) for 48 hours in which levels of
Hsp70, androgen receptor, or Akt
have been normalized to GAPDH.
Representative immunoblots are
shown below each graph. Repeated
measures ANOVA with Dunnett post
hoc test: , P < 0.05 treatment versus
control. GAPDH, glyceraldehyde-3phosphate dehydrogenase.
Tissues were then assessed for induction of cell death by
immunohistochemical detection of cleaved caspase-3.
Consistent with the proliferative data, we observed low
levels of cleaved caspase-3 positivity in control (8.7%)
and 17-AAG–cultured tissues (7.5%), whereas both
AUY922 (22%) and HSP990 (23%) significantly
increased the number of positive cells (P < 0.01; Fig.
5C), indicating that these agents induce apoptotic death
of human prostate cancer cells.
Extended dose-ranging studies (100–1,000 nmol/L), carried out for each agent on a subset of tumors in which there
was sufficient tissue available, further showed the enhanced
potency of AUY922 and HSP990 compared with 17-AAG
(Supplementary Fig. S4).
Discussion
In this study, we report that the new synthetic Hsp90
inhibitors AUY922 and HSP990 are potent anticancer
agents using human prostate cancer cell lines and ex vivo
cultured human prostate tumors. Our findings are con-
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sistent with recent preclinical studies showing superior
antitumor activity of other novel Hsp90 inhibitors, such
as the nonquinone PF-04928473 and the mitochondriatargeted gamitrinibs, compared with 17-AAG in cell lines
and animal models of prostate cancer (23–25), and
highlight the potential of new generation inhibitors.
Importantly, our findings using the human ex vivo
approach provides the first evidence that these new agents
are capable of achieving significant biologic responses in
human prostate tumors and indicate that this class of
agent may well achieve their full potential as a new
therapeutic for prostate cancer.
Although both AUY922 and HSP990 displayed greater
potency than 17-AAG in vitro, it is clear from earlier
studies of 17-AAG that preclinical efficacy does not necessarily equate to clinical response. This may reflect the
limitations of the current prostate cancer models, which
do not take into account the tumor microenvironment or
heterogeneous nature of the disease. In addition, most
cell lines and xenograft models typically represent only
late-stage disease. Consistent with clinical trial results
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Table 1. Pathologic characteristics of tumors used in this study
Patient
ID
Patient
age (y)
PSA
(ng/mL)
Gleason
grade
Pathologic
stage
AR status
(%)
28
31
32
34
35
36
39
40
41
45
63
51
70
48
71
49
50
70
65
68
14
3
6
10
8.3
6
6
15
10
3.8
3þ4
3þ4
3þ4
3þ4
4þ3
3þ4
3þ4
3þ4
3þ4
3þ4
pT3a
pT3a
pT3c
pT3a
pT3a
pT2c
pT3a
pT2c
pT2c
pT3a
95%
95%
90%
90%
95%
95%
95%
90%
90%
95%
NOTE: All tumors are adenocarcinoma of acinar type and pathologic stages are as per AJCC TNM 7th Edition.
Abbreviation: AR, androgen receptor.
(26), 17-AAG was ineffective in our cultured human
prostate tumors, despite the fact that significant induction
of Hsp70 was observed. In contrast, both AUY922 and
HSP990 exhibited remarkable antiproliferative and proapoptotic activity. Notably and contrary to our observations in the cell lines, treatment with 17-AAG achieved
comparable changes in Hsp70 expression to AUY922 and
HSP990 in the ex vivo cultured tumors, but a biologic
response (i.e., reduced proliferation and induction of
apoptosis) was only consistently observed with the 2 new
Hsp90 inhibitors. This is a significant finding for this class
of agent for a number of reasons. First, we have shown
efficacy in the context of an intact tumor microenvironment in which the complex tissue architecture, cell-to-cell
signaling, and hormone responsiveness are maintained.
All of these tissue parameters may be necessary for optimal responses to certain drugs, including Hsp90 inhibitors. Second, these findings suggest that the antitumor
activity of AUY922 and HSP990 cannot simply be a
consequence of enhanced potency for inhibition of
Hsp90 but, rather, attributed to additional properties or
targets. One prospect could be the fact that both AUY922
and HSP990 are not affected by the activity of NQO1/DTdiaphorase or P-glycoprotein (14, 15) and therefore not
susceptible to metabolism or efflux in the same way as 17AAG, however further studies would be needed to investigate this on an individual patient basis. Third, as with all
new therapeutic agents entering clinical trial, Hsp90
inhibitors have previously only been tested in a setting
of metastatic CRPC in which the disease course and
behavior are influenced by prior treatments. That we
showed significant efficacy of AUY922 and HSP990 in
cultured tumors obtained from radical prostatectomy
suggests that Hsp90 inhibitors may be an effective treatment option in a neoadjuvant setting or following biochemical relapse, and that clinical trials to assess these
possibilities are warranted.
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In addition, our results not only support the view that
modulation of Hsp70 is not a clinically informative
biomarker of biologic response in tumors (9, 27) but
also indicates that despite the prosurvival actions of
Hsp70 (28), its induction does not preclude an antiproliferative response in human tumors. Although
induction of Hsp70 has been widely used to monitor
efficacy of Hsp90 inhibitors in clinical trials (7, 22),
mainly in peripheral blood mononuclear cells (PBMC),
increasing evidence suggests that Hsp70 induction is not
sufficient to predict clinical activity of these agents. For
example, in clinical trials of 17-DMAG in patients with
advanced malignancies, Hsp70 levels measured in
PBMCs showed no correlation with clinical response
(9, 27). Measuring Hsp70 in serum, rather than PBMCs
(22), detection of the Her-2 extracellular domain or
IGFBP-2 (29) or use of FDG-PET imaging (19), have
been suggested as possible alternatives, but our findings
strongly argue that evaluation of functional endpoints
such as proliferative response in human tumor tissues
may allow more accurate monitoring of drug efficacy
and be a preferable discovery platform to identify new
biomarkers of response.
The ultimate use of Hsp90 inhibitors as prostate cancer
therapy will depend on identifying agents that have
clinically acceptable pharmacologic and toxicologic profiles and are effective in human prostate tumors. This
study shows that ex vivo culture techniques can provide
important information on drug efficacy in human tissues
that is not possible with cell lines or animal models. The
incorporation of this method into future studies may
enable more robust preclinical development of novel
agents, by providing a better approach for the prediction
of drug effects and drug targeting that allows for more
rational selection of bioactive agents from a library of
compounds, and the identification of more relevant and
robust markers of drug response.
Clinical Cancer Research
Downloaded from clincancerres.aacrjournals.org on November 18, 2012
Copyright © 2012 American Association for Cancer Research
Published OnlineFirst May 9, 2012; DOI:10.1158/1078-0432.CCR-12-0782
Efficacy of New Hsp90 Inhibitors in Prostate Tumors
Figure 5. AUY922 and HSP990
exhibit potent antitumor activity in
cultured human prostate tumors.
Tumors were cultured in medium
containing vehicle control,
500 nmol/L 17-AAG, AUY922, or
HSP990. A, representative Hsp70
immunostaining. Staining in the
negative control (neg) is inset. Hsp70
levels were quantified by video image
analysis of 10 random fields at 40
magnification. Data are presented as
the mean integrated optical density SE for the indicated number of
patients in each treatment group.
B, representative Ki67
immunostaining. Quantification of
Ki67 immunostaining presented as
the percentage of positively stained
nuclei in at least 200 cells counted at
40 magnification. Data represents
the mean SE for the indicated
number of patients in each treatment
group. C, representative cleaved
caspase-3 immunostaining.
Quantification of cleaved caspase-3
presented as the percentage of
positively stained nuclei in at least
200 cells counted at 40
magnification. Bar ¼ 50 mm. ANOVA
with Dunnett post hoc test: , P < 0.05
treatment versus control.
Disclosure of Potential Conflicts of Interest
H.I. Scher is a paid consultant for Novartis. G.V. Raj is a recipient of
honoraria from Speakers Bureau of Amgen and is a consultant/advisory
board member of Johnson and Johnson. No potential conflicts of interest
were disclosed by the other authors.
Authors' Contributions
Conception and design: M.M. Centenera, J.L. Gillis, P.D. Sutherland, H.I.
Scher, W.D. Tilley, L.M. Butler
Development of methodology: M.M. Centenera, A. Hanson, R.A. Taylor,
T. Yeadon, W.D. Tilley, L.M. Butler
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): M.M. Centenera, J.L. Gillis, A. Hanson, G.
Risbridger, P.D. Sutherland, T. Yeadon
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.M. Centenera, J.L. Gillis, A. Hanson, S.
Jindal, R.A. Taylor, H.I. Scher, G.V. Raj, K.E. Knudsen, T. Yeadon, W.D. Tilley,
L.M. Butler
Writing, review, and/or revision of the manuscript: M.M. Centenera, J.L.
Gillis, A. Hanson, G. Risbridger, P.D. Sutherland, H.I. Scher, G.V. Raj, K.E.
Knudsen, T. Yeadon, W.D. Tilley, L.M. Butler
www.aacrjournals.org
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.M. Centenera, J.L. Gillis, A.
Hanson
Study supervision: L.M. Butler
Acknowledgments
The authors thank Ms. Lauren Giorgio and Ms. Shelley Hedwards for
assistance with immunohistochemistry, Ms. Raima Amin for carrying out
video image analysis, Ms. Margaret McGee for assistance with statistical
analysis, and Dr. Luke Selth for critical reading of the manuscript and also
thank the study participants, the urologists, nurses, histopathologists, and
the South Australian Coordinators of the APCC, Ms. Helen Hughes and Ms
Pamela Saunders, who kindly assisted in the recruitment and collection of
patient material and information.
Grant Support
This work was supported by grants from the Prostate Cancer Foundation of Australia/Cancer Australia (ID 627229 to L.M. Butler, W.D.
Tilley, and H.I. Scher), the Royal Adelaide Hospital Research Committee
(to L.M. Butler and M.M. Centenera), the National Health and Medical
Clin Cancer Res; 18(13) July 1, 2012
Downloaded from clincancerres.aacrjournals.org on November 18, 2012
Copyright © 2012 American Association for Cancer Research
3569
Published OnlineFirst May 9, 2012; DOI:10.1158/1078-0432.CCR-12-0782
Centenera et al.
Research Council of Australia (ID 627185 to W.D. Tilley, L.M. Butler and
H.I. Scher). The Adelaide Prostate Cancer Research Centre is supported by
an establishment grant from the Prostate Cancer Foundation of Australia
(ID 2011/0452). The Australian Prostate Cancer BioResource is supported
by the National Health and Medical Research Council of Australia
(enabling grants 290456 and 614296) and Prostate Cancer Foundation
of Australia. L.M. Butler holds a senior research fellowship from the
Cancer Council of South Australia.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received March 6, 2012; revised April 26, 2012; accepted May 1, 2012;
published OnlineFirst May 9, 2012.
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Clinical Cancer Research
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