Download Clusterin Mediates TGF-b–Induced Epithelial

Published OnlineFirst August 15, 2012; DOI: 10.1158/0008-5472.CAN-12-0254
Cancer
Research
Molecular and Cellular Pathobiology
Clusterin Mediates TGF-b–Induced Epithelial–Mesenchymal
Transition and Metastasis via Twist1 in Prostate Cancer Cells
Masaki Shiota1, Anousheh Zardan1, Ario Takeuchi1, Masafumi Kumano1, Eliana Beraldi1, Seiji Naito2,
Amina Zoubeidi1, and Martin E. Gleave1
Abstract
TGF-b promotes epithelial–mesenchymal transition (EMT) and induces clusterin (CLU) expression, linking
these genes to cancer metastasis. CLU is a pleiotropic molecular chaperone that confers survival and proliferative
advantage to cancer cells. However, the molecular mechanisms by which TGF-b regulates CLU expression and
CLU affects metastasis remain unknown. In this study, we report that the transcription factor Twist1 mediates
TGF-b–induced CLU expression. By binding to E-boxes in the distal promoter region of CLU gene, Twist1
regulated basal and TGF-b–induced CLU transcription. In addition, CLU reduction reduced TGF-b induction of
the mesenchymal markers, N-cadherin and fibronectin, thereby inhibiting the migratory and invasive properties
induced by TGF-b. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, our
findings indicate that CLU is an important mediator of TGF-b–induced EMT, and suggest that CLU suppression
may represent a promising therapeutic option for suppressing prostate cancer metastatic progression. Cancer
Res; 72(20); 1–12. 2012 AACR.
Introduction
Prostate cancer is the most common nonskin cancer and
the second leading cause of cancer-related death among
males in developed countries. The incidence of prostate
cancer has been increasing as a result of an aging population
and prevalence of high-fat diets (1). Men with prostate
cancer die predominantly from metastatic disease predisposed to become resistant to androgen-deprivation therapy,
termed castration-resistant prostate cancer (CRPC). Acquisition of migratory properties is a prerequisite for cancer
invasion into surrounding tissue. In cancer, acquisition of
invasiveness requires a dramatic morphologic alteration,
termed epithelial–mesenchymal transition (EMT), wherein
cancer cells lose their epithelial characteristics of cell polarity and cell–cell adhesion, and switch to a mesenchymal
phenotype (2). EMT is a characteristic of cancer cell intravasation and metastasis and is closely associated with CRPC.
E-cadherin is a cell-to-cell adhesion molecule in which loss
of expression is a hallmark of EMT, leading to increased cell
Authors' Affiliations: 1The Vancouver Prostate Centre and Department of
Urologic Sciences, University of British Columbia, Vancouver, British
Columbia, Canada; and 2Department of Urology, Graduate School of
Medical Sciences, Kyushu University, Fukuoka, Japan
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
M. Shiota and A. Zardan contributed equally to this work.
Corresponding Author: Martin E. Gleave, The Vancouver Prostate Centre
and Department of Urologic Sciences, University of British Columbia, 2660
Oak Street, Vancouver, British Columbia V6H 3Z6, Canada. Phone: 1-604875-4818; Fax: 1-604-875-5654; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-12-0254
2012 American Association for Cancer Research.
motility and invasion (3). On the other hand, N-cadherin and
fibronectin are mesenchymal markers (4) in which expression is regulated by several transcription factors including a
basichelix–loop–helix (bHLH) transcription factor Twist1,
Slug, and Snail1 (5–7).
Clusterin (CLU) is a stress-activated and apoptosis-associated molecular chaperone functioning to protect cells from
various stressors and is highly expressed in many human
cancers. In prostate cancer, CLU expression levels are low in
low-grade tissues but increase with higher Gleason score (8).
Moreover, CLU expression is shown to be enhanced following
castration and in CRPC models (9, 10), wherein overexpression
of CLU in prostate cancer accelerates progression after androgen-deprivation therapy or chemotherapy, identifying CLU as
an antiapoptotic gene upregulated by treatment stress that
confers therapeutic resistance when overexpressed (9, 11, 12).
Previously, regulation of CLU expression by Egr-1 (13), androgen receptor (14), and Y-box binding protein-1 (15) has been
reported. Interestingly, CLU overexpression enhances invasive
and metastatic abilities in renal cancer (16) and hepatocellular
carcinoma (17), whereas CLU silencing suppresses EMT via
inhibition of Slug (18).
TGF-b is a multifunctional cytokine that inhibits the
proliferation of normal prostate epithelial cells and premalignant lesions. Increased levels of TGF-b in patients' serum
have significant prognostic value for highly aggressive metastatic disease and are considered a poor prognosis marker
(19–23). One mechanism by which TGF-b contributes to
cancer progression is through induction of EMT. Upon TGFb treatment, epithelial cells changed from a cuboidal to an
elongated spindle shape with decreased expression of epithelial markers and enhanced expression of mesenchymal
markers, such as E-cadherin and fibronectin. TGF-b induces
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both Twist1 (24, 25) as well as CLU expression in various
cells (26–28). In addition, both Twist1 and CLU are associated with EMT characteristics. However, the mechanisms by
which they drive progression through EMT remain obscure.
Here, we define molecular mechanism linking Twist1-regulation of CLU expression by TGF-b, which are important
regulators of EMT. Moreover, we investigate the therapeutic
effects of CLU knockdown in suppressing EMT and prostate
cancer metastasis.
Materials and Methods
Cell culture and transfection
PC-3 and 22Rv1 cells, which express the TGF-b receptor,
were obtained from and authenticated by American Type
Culture Collection and maintained in Dulbecco's Modified
Eagle's Medium (DMEM; Thermo Scientific) and RPMI-1640
(Thermo Scientific) supplemented with 5% FBS, respectively.
PC-3M-luc (C6) cells, which are stably expressing luciferase
protein, were obtained from Caliper Life Sciences and maintained according to the manufacturer's instruction. All cell lines
within 6 months after resuscitation were used. Cells were
transfected with the indicated siRNA or the indicated plasmid
as previously described (29, 30).
Antibodies and reagents
Antibodies against PCNA (sc-56), c-Myc (sc-815), CLU (sc6419), and Twist1 (sc-81417) were purchased from Santa Cruz
Biotechnology. Anti-Snail1 and anti-Slug antibodies were
obtained from Cell Signaling. Antibodies against E-cadherin,
N-cadherin, and fibronectin were purchased from BD Biosciences. Anti-b-actin antibody was purchased from Sigma.
Human TGF-b1 recombinant protein was obtained from R&D
Systems.
Plasmids
Twist1-Myc-Flag plasmid expressing C-terminally Myc-Flagtagged Twist1 protein and Egr-1-Myc-Flag plasmid expressing
C-terminally Myc-Flag-tagged Egr-1 protein were purchased
from OriGene. The Twist1 reporter plasmid (Twist–Luc)
cloned the Twist1 promoter region (969 bp) was kindly
provided from Dr. L.H. Wang (Mount Sinai School of Medicine,
New York, NY; ref. 31). Various lengths of CLU reporter plasmids (CLU–Luc 1,998/þ254, 1,998/702, 1,116/702,
and 707/þ254) containing the promoter and partial first
exon of the wild-type CLU gene were constructed as previously
described (15). Mutated CLU reporter plasmid (CLU–Luc
E1MT and E2MT) were constructed by introducing mutations
into the CLU–Luc 1,998/702 plasmid using a QuikChange II
XL Site-Directed Mutagenesis kit (Agilent Technologies) with
the following primer pairs: 50 -GGCAGGATGACCACCGGGTGACGATGCTGGGGATGGGGTAGG-30 and 50 -CCTACCCCATCCCCAGCATCGTCACCCGGTGGTCATCCTGCC-30 for CLU–Luc E1MT; 50 -GATCTGCTTAGAATATGAAAACTCTGGTGATGCATTTATGGCGC-30 and 50 -GCGCCATAAATGCATCACCAGAGTTTTCATATTCTAAGCAGATC-30 for CLU–Luc
E2MT. The underlined nucleotides indicate the mutated
sequences.
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siRNAs and antisense oligonucleotides
The following double-stranded 25-bp siRNA oligonucleotides were commercially generated (Invitrogen Life Technologies, Inc.): 50 -CUUCCUCGCUGUUGCUCAGGCUGUC-30 for
Twist1 #1; 50 -UUGAGGGUCUGAAUCUUGCUCAGCU-30 for
Twist1 #2; 50 -CCGUAUCUCUAUGAGAGUUACUCCA-30 for
Slug #1; 50 -GGCUCAUCUGCAGACCCAUUCUGAU-30 for Slug
#2; 50 -UCCCAGAUGAGCAUUGGCAGCGAGG-30 for Snail1 #1;
50 -UUUCGAGCCUGGAGAUCCUUGGCCU-30 for Snail1 #2. The
sequence of siRNA corresponding to the human CLU initiation
site, which was supplied by Dharmacon Research Inc., was 50 GCAGCAGAGUCUUCAUCAU-30 . The sequence of siRNA targeting the 30 -untranslated region of Twist1 gene (Twist1 siRNA
#3), which was supplied by Qiagen was 50 -CACCTCTGCATTCTGATAGAA-30 . Stealth RNAi Negative Control Medium
GC Duplex #2 (Invitrogen Life Technologies, Inc.) was used as a
control siRNA. Second-generation antisense (OGX-011) and
scrambled (ScrB) oligonucleotides with a 20 -O-(2-methoxy)
ethyl modification were supplied by OncoGenex Pharmaceuticals. OGX-011 (50 -CAGCAGCAGAGTCTTCATCAT-30 ) corresponds to the initiation site in exon II of human CLU. The ScrB
control sequence was 50 -CAGCGCTGACAACAGTTTCAT-30 .
Quantitative reverse transcription PCR
RNA extraction and reverse transcription PCR (RT-PCR)
were conducted as previously described (32, 33). Real-time
monitoring of PCR amplification of cDNA was conducted
using the following primer pairs and probes, Twist1
(Hs00361186_m1), CLU (Hs00156548_m1), and GAPDH
(Hs03929097_g1; Applied Biosystems) on ABI PRISM 7900 HT
Sequence Detection System (Applied Biosystems) with TaqMan Gene Expression Master Mix (Applied Biosystems). Target gene expression was normalized to GAPDH levels in
respective samples as an internal control. The results are
representative of at least 3 independent experiments.
Western blot analysis
Whole-cell extracts were obtained by lysis of cells in an
appropriate volume of ice-cold radioimmunoprecipitation
assay (RIPA) buffer composed of 50 mmol/L Tris–HCl (pH
7.4), 150 mmol/L NaCl, 0.5% sodium deoxycholate, 1% Nonidet
P-40, 0.1% SDS containing 1 mmol/L Na3VO4, 1 mmol/L NaF, 1
mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor
cocktail tablets (Complete, Roche Applied Science). Cellular
extracts were clarified by centrifugation at 13,000 g for 10
minutes, and protein concentrations of the extracts determined by a BCA protein assay kit (Thermo Scientific). Wholecell extracts (30 mg) were boiled for 5 minutes in SDS sample
buffer and separated by SDS–PAGE, and transferred onto a
polyvinylidene difluoride (PVDF) membrane. Membranes were
probed with dilutions of primary antibodies followed by incubation with horseradish peroxidase–conjugated secondary
antibodies. After extensive washing, proteins were visualized
by a chemiluminescent detection system (GE Healthcare).
Luciferase reporter assay
PC-3 cells were transfected with the indicated reporter
plasmid, expression plasmid, siRNA, and pRL-TK as an internal
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CLU Suppression Inhibits Cancer Metastasis
control. In case using TGF-b, media were changed to serumfree media with or without TGF-b. The luciferase activities
were measured using a Dual-Luciferase Reporter Assay System
(Promega) and a microplate luminometer (EG&G Berthold).
The Firefly luciferase activities were corrected by the corresponding Renilla luciferase activities. The results are representative of at least 3 independent experiments.
DNA pull-down assay
The following 50 -biotinylated oligonucleotides were used; 50 ACCGGGTGCAGATGCTGGGGAT-30 and 50 -ATCCCCAGCATCTGCACCCGGT-30 for E1WT; 50 -ACCGGGTGACGATGCTGGGGAT-30 and 50 -ATCCCCAGCATCGTCACCCGGT-30 for
E1MT; 50 -ATATGAAACATCTGGTGATGCA-30 and 50 -TGCATCACCAGATGTTTCATAT-30 for E2WT; 50 -ATATGAAAACTCTGGTGATGCA-30 and 50 -TGCATCACCAGAGTTTTCATAT-30
for E2MT; 50 -CCCTAATTCAGCTGGCTAAGGC-30 and 50 GCCTTAGCCAGCTGAATTAGGG-30 for E3WT; 50 -CCCTAATTACGCTGGCTAAGGC-30 and 50 -GCCTTAGCCAGCGTAATTAGGG-30 for E3MT; 50 -TCATCGTCCAGATGGAAGAAAC-30
and 50 -GTTTCTTCCATCTGGACGATGA-30 for E4WT; and 50 TCATCGTCACGATGGAAGAAAC-30 and 50 -GTTTCTTCCATCGTGACGATGA-30 for E4MT. The underlined nucleotides
indicate the mutated sequences. Nuclear extracts were
obtained from PC-3 cells, treated with or without TGF-b, as
previously described (15). Biotinylated oligonucleotides (4 mg)
were incubated with 10 mL of streptavidin–agarose beads
(Sigma) and 400 mg of nuclear extract in immunoprecipitation
(IP) lysis/wash buffer [25 mmol/L Tris–HCl (pH 7.4), 150
mmol/L NaCl, 1 mmol/L EDTA, 0.25 mg/mL poly(dI-dC)-poly
(dI-dC), and 1% NP-40] for 2 hours at 4 C. Complexes were
washed 3 times in IP lysis/wash buffer, and associated proteins
and input (20 mg) were applied to Western blot analysis.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assay was conducted
as previously described (15). Briefly, PC-3 cells were cross-linked
with paraformaldehyde and digested with micrococcal nuclease
to achieve a DNA smear of 200 to 1,000 bp. ChIP assay was
conducted using SimpleChIP Enzymatic Chromatin IP Kit (Agarose Beads) according to the manufacturer's protocol (Cell
Signaling Technology) on the CLU and RPL30 genes. Then,
obtained samples were treated with RNase (Qiagen). The quantitative RT-PCR (qRT-PCR) assay with 2 mL of 20 mL DNA
extraction, the primer pairs (described later), and RT2 RealTime SYBR Green/Rox PCR master mix (Qiagen) was conducted
using ABI 7900HT System (Applied Biosystems). The results are
representative of at least 3 independent experiments. The primer
pairs for CLU02 (GPH025704()02A) targeting around 1,403
bp, CLU01 (GPH025704()01A) targeting around 417 bp,
and CLUþ07 (GPH025704(þ)07A) targeting around þ6,603 bp
were obtained from Qiagen. The primer pairs for RPL30 gene
(exon 3) and antihistone H3 antibody were included in SimpleChIP Enzymatic Chromatin IP Kit.
Cell migration and invasion assay
Cell migration was determined by a wound-healing assay.
After PC-3 cells transfected with the indicated siRNA reached
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confluency, a wound was made in the monolayer by pressing a
pipette tip down on the plate. The debris was removed by
washing the monolayer twice with serum-free media, and
the cells were cultured in serum-free media with or without
0.1 ng/mL of TGF-b for an additional 48 hours. Cell migration
was recorded in 6 different microscopic fields. The percentage
of wound healing was calculated by the equation; (percentage
of wound healing) ¼ average of [(gap area: 0 hours) (gap area:
48 hours)/(gap area: 0 hours)]. Cell invasion was administrated
using Matrigel invasion chambers (BD Biosciences). PC-3 cells
(5 104 cells) transfected with the indicated siRNA were
applied on the upper compartment with 500 mL of serum-free
medium, and the lower compartment was filled in 500 mL of
DMEM with or without 0.1 ng/mL of TGF-b. After 24 hours of
incubation, noninvaded cells on the upper surface of the filter
were removed carefully with a cotton swab, and cells were fixed
with 100% methanol for 2 minutes. Invading cells on the lower
side of the filter were stained with 0.5% crystal violet for 5
minutes, and eluted the dye with Sorensen's solution (9 mg
trisodium citrate, 305 mL distilled water, 195 mL 0.1 N HCl,
500 mL 90% ethanol) and optical density (OD) was measurement at 595 nm.
Zymography
PC-3 cells transfected with the indicated siRNA were incubated in serum-free media with or without 0.1 ng/mL of TGF-b
for 24 hours, and the proteins in the conditioned medium were
concentrated with Amicon Ultra-4 (Millipore) at 13,000 g for
30 minutes at 4 C. Proteins (20 mg) were loaded in nonreducing
conditions on a 10% polyacrylamide gel containing 0.1% gelatin
(Sigma). After electrophoresis, gels were incubated in Triton X100 exchange buffer composed of 20 mmol/L Tris–HCl (pH
8.0), 150 mmol/L NaCl, 5 mmol/L CaCl2, and 2.5% Triton X-100,
for 30 minutes followed by a 10 minutes wash 3 times with the
incubation buffer (same buffer without Triton X-100). Gels
were incubated in buffer overnight at 37 C, stained with 0.5%
Coomassie blue R250 (Sigma) for 2 hours, and destained
overnight with 30% methanol and 10% glacial acetic acid.
Gelatinolytic activity was shown as clear areas in the gel.
In vivo tumor metastasis model
PC-3M-luc cells (2 106 cells) were injected into tail vein of
6- to 8-week-old male athymic nude mice (Harlan SpragueDawley, Inc.) via a 30-gauge needle. The following day, mice
were then randomly divided into 2 groups for treatment with
Scr ASO or OGX-011. Each experimental group consisted of 10
mice. After randomization, 12.5 mg/kg of Scr ASO or OGX-011
was injected intraperitoneally (i.p.) once daily for 7 days
followed by 3 weekly treatments thereafter. To quantify in vivo
tumor burden, mice were then imaged on weeks 2, 4, 6, 8, and 10
in an IVIS200 Imaging System (Caliper Life Sciences; see later).
Animal procedures were conducted according to the guidelines of the Canadian Council on Animal Care.
Bioluminescence imaging
Tumors in mice were imaged using an IVIS200 camera
(Caliper Life Sciences) as previously described (34). Briefly,
mice were injected i.p. with 150 mg/kg D-luciferin (Caliper
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Figure 1. TGF-b mediated induction of Twist1 regulates CLU expression. A, PC-3 cells were treated with 0.1 ng/mL of TGF-b for the indicated duration and qRTPCR was conducted using the primer pairs and probes for Twist1, CLU, and GAPDH. Each transcript level from nontreated cells was set as 1. Boxes, mean;
bars, SD; , P < 0.05 (compared with no treatment). Whole-cell extracts were analyzed by SDS–PAGE and analyzed by Western blot analysis with specified
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CLU Suppression Inhibits Cancer Metastasis
Life Sciences), anesthetized with isoflurane 5 minutes later,
and imaged in the supine position at 11 and 15 minutes after
injection. Data were acquired and analyzed using Living
Image software version 3.0 (Caliper Life Sciences). The
results are the average of bioluminescence between 11 and
15 minutes.
Statistical analysis
All data were assessed using the Student t test. Levels of
statistical significance were set at P < 0.05.
Results
TGF-b induces Twist1 and CLU in prostate cancer cells
We first confirmed Twist1 and CLU are upregulated upon
TGF-b treatment in prostate cancer PC-3 cells. As shown
in Fig. 1A, Twist1 mRNA was induced and peaked at 24 hours
by TGF-b, which was followed by CLU mRNA induction.
Similarly, TGF-b increased Twist1 and CLU protein expression levels, suggesting an induction of CLU by Twist1
(Fig. 1A). Moreover, mesenchymal markers N-cadherin and
fibronectin also increased after TGF-b treatment, although
E-cadherin expression was undetectable in PC-3 cells. Similarly, Twist1 as well as CLU mRNA and protein were
induced by TGF-b in another prostate cancer cell line 22Rv1
with concurrent induction of N-cadherin and fibronectin, as
well as reduction of E-cadherin (Fig. 1B).
Twist1 regulates CLU expression in PC-3 cells
To define functional links between Twist1 and CLU, we
examined the effects of Twist1 knockdown on CLU expression.
As shown in Fig. 1C, Twist1 knockdown suppressed CLU
expression at both transcript and protein levels in PC-3 cells.
Inversely, Twist1 overexpression induced CLU upregulation at
both transcript and protein levels (Fig. 1D). To further confirm
the regulation of CLU expression by Twist1, we used rescue
method using Twist1-specific siRNA (Twist1 siRNA #3) targeting 30 -untranslated region of Twist1 gene, which does not
inhibit exogenous Twist1 expression by Twist1 expression
plasmid. The result showed that Twist1 overexpression recovered CLU downregulation by Twist1 knockdown both at
transcript and protein levels (Fig. 1E). In contrast, knockdown
of other EMT-related transcription factors, Snail1 and Slug, did
not decrease CLU expression at transcript or protein levels
(Supplementary Fig. S1A and S1B), directing further investigation on Twist1 as the key transcription factor mediating
TGF-b–induced CLU and EMT.
Twist1 regulates CLU transcription by binding to distal
promoter region of CLU
Because Twist1 knockdown reduces CLU expression at
both mRNA and protein levels, we explored whether Twist1 is
a transcription factor for CLU gene. To investigate mechanisms
by which Twist1 regulates CLU transcription, the CLU promoter
was analyzed, and 8 E-boxes were found that are known to be
Twist1 binding sites (6, 35). We then used various lengths of CLU
reporter plasmids (CLU–Luc 1,998/þ254, 1,998/702,
1,116/702, and 707/þ254) to identify regions associated
with transcriptional activity, as shown in Fig. 2A. CLU–Luc
1,998/702 exhibited most luciferase activity in PC-3 cells
compared with other CLU–Luc plasmids. Because CLU–Luc
1,116/702 plasmid did not exhibit much activity, the CLU
promoter region between 1,998 bp and 1,116 bp seems to be
most responsible for Twist1-activated CLU transcription (Fig.
2B). When Twist1 was overexpressed, the reporter plasmids
containing the region between 1,998 bp and 1,116 bp were
again most active (Fig. 2C). Also, Egr-1 overexpression increased
luciferase activity by CLU reporter plasmid as previously
reported (Supplementary Fig. S2; ref. 13). DNA pull-down assays
were conducted to investigate which E-box is functional in CLU
promoter region between 1,998 bp and 1,116 bp; Fig. 2D
indicates that Twist1 preferentially bound to E1 (E1WT) and E2
(E2WT) compared with E3 (E3WT) and E4 (E4WT), and not to
mutated E-box (E1MT, E2MT, E3MT, and E4MT). To further
confirm the regulation of CLU transcription via Twist1-binding
to E-box, we introduced mutations in 2 E-boxes (E1 and E2) into
CLU–Luc 1,998/702 plasmid, and produced CLU–Luc E1MT
and E2MT reporter plasmids as shown in Fig. 2A. We then
examined Twist1-activated CLU transcription by using these Ebox–mutated reporter plasmids. As shown in Fig. 2E, mutations
in E1 and E2 reduced CLU transcription and blunted activation
of CLU transcription by Twist1 overexpression. ChIP assays were
then used to further confirm Twist1 binding to CLU promoter
region around 1,400 bp from transcription start site represented by CLU 02. No binding was apparent in the CLU gene
around þ6,600 bp from transcription start site and PRL30 gene
(Fig. 2F).
TGF-b induces CLU expression by enhancing Twist1
binding to CLU promoter region
To further define mechanisms by which TGF-b regulates
Twist1 and CLU, we examined the Twist1 and CLU transcription activity using luciferase reporter plasmids. As shown
in Fig. 3A and B, both Twist1 and CLU transcription were
antibodies. B, 22Rv1 cells were treated with 5 ng/mL TGF-b for the indicated duration and qRT-PCR was conducted using the primer pairs and probes for
Twist1, CLU, and GAPDH. Each transcript level from nontreated cells was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with no treatment). Wholecell extracts were analyzed by SDS–PAGE and analyzed by Western blot analysis with specified antibodies. C, PC-3 cells were transfected with 40 nmol/L of
the indicated siRNA. Seventy-two hours after transfection, qRT-PCR was conducted using the primers and probes for Twist1, CLU, and GAPDH. Each
transcript level from cells transfected with control siRNA was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with control siRNA). Whole-cell extracts
were analyzed by SDS–PAGE and Western blot analysis with specified antibodies. D, PC-3 cells were transfected with 1.0 mg/mL of the indicated expression
plasmid. At 72 hours after transfection, qRT-PCR was conducted using the primers and probes for CLU and GAPDH. Each transcript level from cells
transfected with mock plasmid (Myc-Flag) was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with mock). Whole-cell extracts were analyzed by
SDS–PAGE and Western blot analysis with specified antibodies. E, PC-3 cells were transfected with 20 nmol/L of the indicated siRNA and 0.5 mg/mL of the
indicated expression plasmid. At 72 hours after transfection, qRT-PCR was conducted using the primers and probes for CLU and GAPDH. Each transcript
level from cells transfected with control siRNA and mock plasmid (Myc-Flag) was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with mock). Wholecell extracts were analyzed by SDS–PAGE and Western blot analysis with specified antibodies.
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Figure 2 Twist1 regulates CLU transcription by binding to distal promoter region of CLU. A, schematic representation of the promoter region and 50 end of the
CLU gene. Black boxes, E-boxes (50 -CANNTG-30 ); gray box, HSF1-binding site; white box, AP-1-binding site; rail, primer pairs used in ChIP assays. CLU–Luc
plasmids (1,998/þ254, 1,998/702, 1,116/702, 707/þ254, E1MT and E2MT) used in B, C, and E are also shown. B, PC-3 cells were transfected with
0.5 mg/mL of the various CLU–Luc plasmids shown in A and 0.05 mg/mL of pRL-TK. The luciferase activity of CLU–Luc 1,998/þ254 was set as 1.
Boxes, mean; bars, SD; , P < 0.05 (compared with CLU–Luc 1,998/þ254). C, PC-3 cells were cotransfected with 0.5 mg/mL of the various CLU–Luc
plasmids shown in A, 0.5 mg/mL of the indicated expression plasmid, and 0.05 mg/mL of pRL-TK. The luciferase activity of each CLU–Luc with mock plasmid
(Myc-Flag) was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with mock). D, nuclear extracts from PC-3 cells were incubated with specified 50 biotinylated oligonucleotides and input and pulled-down samples analyzed by SDS–PAGE and Western blot analysis with specified antibodies. E, PC-3 cells
were cotransfected with 0.5 mg/mL of the various CLU–Luc plasmids shown in A, 0.5 mg/mL of the indicated expression plasmid, and 0.05 mg/mL of pRL-TK.
Luciferase activity of CLU–Luc 1,998/702 with mock plasmid (Myc-Flag) was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with
CLU–Luc 1,998/702). F, ChIP assays were conducted on nuclear extracts from PC-3 cells using 2.0 mg of the indicated antibodies and 20 mL of Protein G
agarose. The qRT-PCR was conducted using immunoprecipitated DNAs, soluble chromatin and specific primer pairs for the CLU and RPL30 genes. The
results of immunoprecipitated samples were corrected for the results of the corresponding soluble chromatin samples. Boxes, mean; bars, SD; , P < 0.05
(compared with IgG).
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Figure 3. TGF-b induces CLU expression by enhancing Twist1 binding to CLU promoter region. A, PC-3 cells were transfected with 0.5 mg/mL of Twist–Luc
plasmid and 0.05 mg/mL of pRL-TK. After 24 hours, cells were treated 0.1 ng/mL TGF-b for 24 hours. The luciferase activity of Twist–Luc treated without
TGF-b was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with TGF-b). B, PC-3 cells were transfected with 0.5 mg/mL of CLU–Luc 1,998/þ254
plasmid and 0.05 mg/mL of pRL-TK, and were treated 0.1 ng/mL TGF-b for 48 hours. The luciferase activity of CLU–Luc 1,998/þ254 treated
without TGF-b was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with TGF-b). C, PC-3 cells were cotransfected with 0.5 mg/mL of the various
CLU–Luc plasmids shown in Fig. 2A and 0.05 mg/mL of pRL-TK, and were treated 0.1 ng/mL TGF-b for 48 hours. The luciferase activity of CLU–Luc 1,998/
702 treated without TGF-b was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with CLU–Luc 1,998/702). D, nuclear extracts from
PC-3 cells treated 0.1 ng/mL TGF-b for 24 hours were incubated with specified 50 -biotinylated oligonucleotides and input and pulled-down samples
analyzed by SDS–PAGE and Western blot analysis with specified antibodies. E, ChIP assay was conducted on nuclear extracts from PC-3 cells 0.1 ng/mL
TGF-b for 24 hours using 2.0 mg of the indicated antibodies and 20 mL of Protein G agarose. The qRT-PCR was conducted using immunoprecipitated DNAs,
soluble chromatin, and specific primer pairs for the CLU gene. Boxes, mean; bars, SD; , P < 0.05 (compared with TGF-b).
increased by TGF-b. However, luciferase activity of CLU–Luc
E1MT and E2MT was significantly reduced and less affected by
TGF-b (Fig. 3C). These findings were supported by DNA pulldown assay, which suggests that Twist1 binding to wild-type Ebox (E1WT and E2WT) was augmented by TGF-b (Fig. 3D). To
further confirm that TGF-b induced CLU via increases in
Twist1, we conducted ChIP assays after TGF-b treatment. As
shown in Fig. 3E, Twist1 binding to CLU promoters (CLU02)
were enhanced after TGF-b treatment in PC-3 cells, indicating
increased Twist1 binding to CLU promoter after TGF-b treatment, leading to induction of CLU transcription.
www.aacrjournals.org
TGF-b induces EMT via Twist1-CLU pathway
To further evaluate the role of Twist1 in TGF-b–induced
CLU and EMT, we examined effects of Twist1 silencing
on TGF-b–induced CLU expression. As shown in Fig. 4A,
TGF-b–induced stimulation of Twist1 as well as CLU transcript expression was potently suppressed by Twist1 silencing. In addition, luciferase reporter assays using CLU–Luc
showed that Twist1 knockdown repressed TGF-b–induced
CLU transcription (Fig. 4B). Twist1 knockdown also
repressed TGF-b–induced increases in CLU protein levels
in PC-3 cells (Fig. 4C). Moreover, Twist1, as well as CLU,
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Shiota et al.
silencing suppressed the TGF-b induction of EMT markers,
such as N-cadherin and fibronectin (Fig. 4C). Similarly,
using in 22Rv1 prostate cancer cells, Twist1 or CLU knockdown reduced N-cadherin and fibronectin, whereas increasing E-cadherin expression confirms in another cell line
that CLU silencing can suppress TGF-b induction of EMT
(Fig. 4D).
Twist1 and CLU suppression inhibits TGF-b–induced
migration and invasive
The biologic consequences of the role of Twist1 and CLU in
TGF-b regulation of invasion and migration were examined
using cell biology assays. Scratch assays showed that TGF-b
increased cell migration, which was almost abolished by
Twist1 and CLU knockdown (Fig. 5A, Supplemental Fig. S3).
Figure 4 TGF-b induces EMT via a
Twist1-CLU–dependent pathway.
A, PC-3 cells were transfected with
40 nmol/L of the indicated siRNA
and incubated for 24 hours. After
cells were treated with 0. 1 ng/mL of
TGF-b for the indicated durations,
qRT-PCR was conducted using the
primer pairs and probes for Twist1,
CLU, and GAPDH. Each transcript
level from nontreated cells was set
as 1. Boxes, mean; bars, SD;
, P < 0.05 (compared with no
treatment). B, PC-3 cells were
cotransfected with 0.5 mg/mL of the
CLU–Luc 1,998/þ254 plasmid,
20 nmol/L of the indicated siRNA
and 0.05 mg/mL of pRL-TK, and
treated with or without 0.1 ng/mL of
TGF-b for 48 hours. The luciferase
activity of CLU–Luc 1,998/þ254
transfected with control siRNA and
treated without TGF-b was set as 1.
Boxes, mean; bars, SD; , P < 0.05
(compared with TGF-b). C, PC-3
cells were transfected with
40 nmol/L of the indicated siRNA
and incubated for 24 hours. After
cells were treated with 0.1 ng/mL of
TGF-b for the indicated duration,
whole-cell extracts were analyzed
by SDS–PAGE, and Western blot
analysis with specified antibodies.
D, 22Rv1 cells were transfected
with 40 nmol/L of the indicated
siRNA, and incubated for 24 hours.
After cells were treated with
5 ng/mL of TGF-b for the indicated
duration, qRT-PCR was conducted
using the primer pairs and probes
for Twist1, CLU, and GAPDH. Each
transcript level from nontreated
cells was set as 1. Boxes, mean;
bars, SD; , P < 0.05 (compared
with no treatment). Whole-cell
extracts were analyzed by SDS–
PAGE and Western blot analysis
with specified antibodies.
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CLU Suppression Inhibits Cancer Metastasis
Moreover, we found that Twist1 or CLU silencing significantly
reduced basal and TGF-b–induced matrix metalloproteinase-9
(MMP-9) activity (Fig. 5B) as well as TGF-b induced increases
in cell invasion (Fig. 5C).
CLU knockdown using OGX-011 suppresses prostate
cancer metastasis in vivo
On the basis of the previous findings that CLU inhibition
reduced TGF-b induction of markers of EMT as well as cell
invasion and migration, we next investigated the effects of CLU
suppression on prostate cancer metastatic ability in vivo using
the CLU inhibitor, OGX-011, which has been previously shown
to suppresses CLU expression in vivo in xenograft tissue (32,
33, 36), as well as in human prostate tissue (37) and sera (38). To
establish a metastatic cancer model in vivo, highly metastatic
PC-3M-luc cells stably expressing luciferase were injected into
tail vein of male nude mice. Beginning the next day, mice were
treated i.p. with 12.5 mg/kg of OGX-011 or scramble control,
Figure 5. Suppression of Twist1 or CLU inhibits TGF-b–induced migration and invasion. A, PC-3 cells were transfected with 40 nmol/L of indicated siRNA. The
scratch widths were measured after 48 hours of additional incubation 0.1 ng/mL TGF-b. Boxes, mean; bars, SD; , P < 0.05 (compared with control siRNA).
Representative figures of each experiment are shown in the right part. B, PC-3 cells were transfected with 40 nmol/L of the indicated siRNA. For zymography,
the media was changed to serum-free media 0.1 ng/mL TGF-b 48 hours after transfection. Twenty-four hours later, conditional media was concentrated
and subjected to zymography. The MMP-9 activity when transfected with control siRNA and treated without TGF-b was set as 1. Boxes, mean; bars, SD;
, P < 0.05 (compared with control siRNA). Representative figures of each experiment are shown in the right part. C, PC-3 cells were transfected with
40 nmol/L of the indicated siRNA. Invasion assay was conducted as described in Material and Methods. The invasive cell count when transfected with control
siRNA and treated without TGF-b was set as 1. Boxes, mean; bars, SD; , P < 0.05 (compared with control siRNA). Representative figures of each experiment
are shown in the right part.
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and a bioluminescent signal was monitored every 2 weeks. A
bioluminescent signal first became detectable at 6 week after
tail vein injection. At 6 weeks, bioluminescent signals were
lower in OGX-011–treated group compared with those in
control group. Moreover, bioluminescent signals in control
group increased over time, whereas those in OGX-011–treated
group remained suppressed (Fig. 6A and B), suggesting that
OGX-011 can inhibit prostate cancer metastasis in this preclinical study.
Discussion
Promoter sequences of CLU are conserved during evolution
and include several stress-associated sites. For example, heat
shock factor-1 (HSF-1) is a well-known regulator of CLU
expression by binding CLE sites in CLU promoter regions
Figure 6 CLU knockdown using OGX-011 suppresses prostate cancer
metastasis in vivo. A, in vivo imaging of prostate cancer metastatic model
after OGX-011 treatment by IVIS imaging system. Intact male mice were
injected via their tail vein with PC-3M-luc cells and then treated with 12.5
mg/kg of OGX-011 or Scr ASO daily for first week, then 3 times weekly
thereafter. Bioluminescence indicates luciferase activity at 10 weeks after
tail vein injection. B, effect of OGX-427 treatment on metastasis of
prostate cancer. Changes in bioluminescence signals from mice treated
with OGX-011 or Scr ASO were measured using IVIS200 Imaging System.
Boxes, mean; bars, SEM; , P < 0.05 (compared with Scr ASO).
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Cancer Res; 72(20) October 15, 2012
(39). In addition, AP-1 and Egr-1 also regulate CLU expression
(13, 40). Furthermore, we recently reported that Y-box binding
protein-1 is a key transcriptional factor that induces CLU
expression during endoplasmic reticulum stress (15). In this
study, we report that, in addition to these stress-related
transcription factors, the EMT-related transcription factor
Twist1 also regulates CLU expression in response to TGF-b.
While both Twist1 and CLU are known to be induced by TGF-b
(24–28), any functional interaction between Twist1 and CLU in
regulating TGF-b–induced EMT is undefined. This study indicates that CLU is an important downstream mediator of TGFb- and Twist1-induced EMT. To date, many studies have shown
that Twist1 plays a critical role in EMT of both normal and
cancer cells, mediated by EMT regulators, such as E-cadherin,
N-cadherin, and fibronectin (41). Consistent with these prior
reports, this study confirms that Twist1 silencing reduces TGFb induction of N-cadherin and fibronectin along with biologic
actions, such as migration and invasion in prostate cancer
cells. Moreover, CLU was identified as a Twist1-induced chaperone that also regulates these EMT-related markers and
phenotype in prostate cancer cells. This finding is consistent
with, and adds mechanistic insight into, previous reports of
CLU in EMT and cancer. For example, Chou and colleagues
reported CLU involvement in EMT through extracellular signal-regulated kinase (ERK)/Slug pathway in lung cancer cells
(18). Furthermore, in monocytes and macrophages, CLU was
shown to regulate MMP-9 expression via ERK1/2 and PI3K/
Akt/NF-kB pathways, which may contribute to the tissue
reorganization by serving as a modulator for extracellular
matrix degradation (42). CLU overexpression augmented metastatic potential of renal cancer cells as indicated by increased
lung metastasis in a mice tail vein model (16). Similarly, CLU
overexpression in hepatocellular carcinoma increased migratory and metastatic abilities in vitro and in vivo (17). Recently,
orthotopic primary tumors derived from CLU-overexpressing
breast cancer cells grew more rapidly and metastasized more
often than tumors derived from parental cells (43). Collectively,
these findings link CLU overexpression to promotion of metastases in several types of cancer.
CLU has been shown to interact with the C-terminus of TGFb receptors (44), and to regulate Smad2/3 stability (45).
Because TGF-b–Smad2/3 signaling is a critical regulator of
EMT, CLU may regulate EMT and metastasis through TGFb–Smad2/3 signaling. In addition, we and others (18, 42)
showed that TGF-b and CLU regulated MMP-9 activity, which
also contributes to cancer invasiveness. Either dependent on,
or in addition to, these mechanisms described earlier, CLU
knockdown suppresses EMT-related molecules N-cadherin
and fibronectin. N-cadherin plays a critical role in EMT and
castration resistance of prostate cancer, and N-cadherin–specific monoclonal antibodies can suppress metastasis and
castration resistance in preclinical model (46). Because CLU
(9, 47), N-cadherin (46), and EMT (48) are functionally associated with progression to castration resistance, this study
identifies and defines Twist1-activated CLU as a mediator of
TGF-b–induced N-cadherin, EMT, and metastases in CRPC.
Recently, anti-CLU antibody therapy was shown to repress
metastasis in breast cancer models (28). While the
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CLU Suppression Inhibits Cancer Metastasis
antimetastatic activity using anti-CLU antibodies has not been
directly compared with CLU knockdown using OGX-011, antiCLU antibody therapy targets only secretary CLU, which is
unlikely to affect TGF-b–Smad2/3 signaling because cytoplasmic CLU interacts with TGF-b receptors (44). While
anti-N-cadherin antibody therapy is another promising
approach to target EMT, metastasis, and treatment resistance,
N-cadherin is more downstream than CLU. For example, CLU
inhibition decreases both N-cadherin as well as fibronectin
expression in addition to TGF-b signaling and MMP-9 activity.
Therefore, CLU may be more attractive "node" to target
because of its many clients and pleiotropic actions. Indeed,
many preclinical studies report that inhibition of CLU
enhances treatment-induced cancer cell death, inhibits metastases, and delays progression in prostate and other cancer
models. Clinical studies reported successful inhibition of CLU
in human prostate cancer tissues (37) and 7-month survival
benefit in randomized trials when OGX-011 was combined
with docetaxel in patients with CRPC (38, 49). OGX-011 is now
in phase III trials (50) in metastatic CRPC.
In conclusion, we found that TGF-b–regulated CLU expression via Twist1 transcription factor, which induced a mesenchymal phenotype represented by increased N-cadherin and
fibronectin expression in prostate cancer cells. Targeted inhibition of Twist1 or CLU reversed EMT markers, and reduced
invasive and metastatic abilities in prostate cancer cells. These
data support targeting CLU using OGX-011 as a rational
strategy for suppressing the role of EMT in progression of
metastatic CRPC.
Disclosure of Potential Conflicts of Interest
The University of British Columbia has submitted patent applications listing
M.E. Gleave as inventor on the antisense sequence described in this article. This
international patent has been licensed to OncoGenex Technologies, a Vancouver-based biotechnology company in which M.E. Gleave has founding shares. S.
Naito has honoraria from Speakers Bureau as moderator. No potential conflicts
of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: M. Shiota, A. Zoubeidi
Development of methodology: A. Takeuchi, A. Zoubeidi
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): M. Shiota, A. Zardan, A. Takeuchi, M.E. Gleave
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): M. Shiota, A. Zardan, A. Takeuchi, S. Naito, M.E.
Gleave
Writing, review, and/or revision of the manuscript: M. Shiota, A. Zardan, S.
Naito, A. Zoubeidi, M.E. Gleave
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Kumano, E. Beraldi
Study supervision: S. Naito
Acknowledgments
The authors thank Dr. Lu-Hai Wang (Mount Sinai School of Medicine, New
York, NY) providing the Twist–Luc reporter plasmid. The authors also thank
Mary Bowden and Virginia Yago for their excellent technical assistances.
Grant Support
This study was supported by the Terry Fox New Frontiers Program and the
Canadian Institutes of Health Research, the Pacific Northwest Prostate Cancer
SPORE, and the Japanese Postdoctoral Fellowship for Research Abroad.
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 January 26, 2012; revised August 1, 2012; accepted August 10, 2012;
published OnlineFirst August 15, 2012.
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Research.
Published OnlineFirst August 15, 2012; DOI: 10.1158/0008-5472.CAN-12-0254
Clusterin Mediates TGF-β−Induced Epithelial−Mesenchymal
Transition and Metastasis via Twist1 in Prostate Cancer
Cells
Masaki Shiota, Anousheh Zardan, Ario Takeuchi, et al.
Cancer Res Published OnlineFirst August 15, 2012.
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