Androgens Induce Functional
CXCR4 through ERG Factor
Expression in TMPRSS2-ERG
Juan Cai*, Pridvi Kandagatla*,
Rajareddy Singareddy*, Anthony Kropinski*,
Shijie Sheng†,‡, Michael L. Cher*,†,‡
and Sreenivasa R. Chinni*,†,‡
*Department of Urology, Wayne State University School
of Medicine, Detroit, MI, USA;†Department of Pathology,
Wayne State University School of Medicine, Detroit,
MI, USA;‡The Barbara Ann Karmanos Cancer Institute,
Detroit, MI, USA
TMPRSS2-ERG fusion transcripts have been shown to be expressed in a majority of prostate cancer (PC) patients
because of chromosomal translocations or deletions involving the TMPRSS2 gene promoter and the ERG gene
coding sequence. These alterations cause androgen-dependent ERG transcription factor expression in PC patients.
We and others have shown that chemokine receptor CXCR4 expression is upregulated in PC tumor cells, and its
ligand, CXCL12, is expressed in bone stromal cells. The CXCL12/CXCR4 axis functions in PC progression to enhance
invasion and metastasis. To address the regulation of CXCR4 expression, we identified several putative ERG
consensus-binding sites in the promoter region of CXCR4. We hypothesized that androgen-dependent regulation
of the ERG transcription factor could induce CXCR4 expression in PC cells. Results of the current study show that
1) prostate tumor cells coexpress higher ERG and CXCR4 compared with benign tissue, 2) CXCR4 expression is in-
creased in the TMPRSS2-ERG fusion–positive cell line, 3) ERG transcription factorbinds to the CXCR4 gene promoter,
4) synthetic androgen (R1881) upregulates both ERG and CXCR4 in TMPRSS2-ERG fusion–positive VCaP cells, 5)
small interfering RNA–mediated down-regulation of ERG resulted in the loss of androgen-dependent regulation of
CXCR4 expression in VCaP cells, and 6) R1881-activated TMPRSS2-ERG expression functionally activates CXCR4 in
VCaP cells. These findings provide a link between TMPRSS2-ERG translocations and enhanced metastasis of tumor
cells through CXCR4 function in PC cells.
Translational Oncology (2010) 3, 195–203
Specific chromosomal alterations were recently discovered in prostate
cancer (PC) patients using bioinformatics analysis of microarray data
[1,2]. Prevalent chromosomal alterations due to interstitial deletion or
gene promoter with Ets transcription factor coding sequences. The Ets
family of transcription factors ERG, ETS translocation variants 1 and 4
(ETV1 and ETV4), were shown to be involved in chromosomal altera-
tions. Among these transcription factors, ERG was identified as being
commonly fused with the TMPRSS2 gene in most PC patients [1–3].
outcome [4–6] and that specific ERG isoform expression correlates with
aggressive disease characteristics . Other studies suggest that these
chromosomal alterations alone are not associated with patient outcome
. Previous reports demonstrate that prostate-specific overexpression
of prostate intraepithelial neoplasia (PIN) without progression to carci-
noma [9,10]. TMPRSS2-ERG translocations have also been identified
in the low-grade PIN lesions adjacent to cancer, suggesting that ERG
expression contributes to PIN development . Further, two recent
Address all correspondence to: Sreenivasa R. Chinni, PhD, Departments of Urology and
Pathology, Wayne State University School of Medicine, 9200 Scott Hall, 540 E Canfield
Ave, Detroit, MI 48201. E-mail: firstname.lastname@example.org
1This work was supported by Fund for Cancer Research, startup funds from Wayne
State University and Barbara Ann Karmanos Cancer Institute, and US Department of
Defense Idea Award W81XWH-09-1-0250.
2This article refers to supplementary material, which is designated by Table W1 and is
available online at www.transonc.com.
Received 29 December 2009; Revised 29 December 2009; Accepted 11 January 2010
Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1944-7124/10/$25.00
Volume 3 Number 3June 2010pp. 195–203
reports demonstrate that ERG overexpression alone is not sufficient for
PC progression; additional loss of PTEN co-operates in the develop-
ment of highly invasive prostate adenocarcinoma [12,13]. Studies with
patient tumor tissues confirmed in vivo findings that alterations in ERG
and PTEN genes in PC patients result in the development of aggressive
disease . The molecular targets related to androgen-mediated acti-
vation of TMPRSS2-ERG are currently unknown; herein, we provide
evidence that the chemokine receptor CXCR4 is one such target of an-
drogens in PC cells.
CXCR4 is a chemokine receptor that has been shown to function as a
key receptor for homing of circulating tumor cells to secondary sites; its
ligand CXCL12 is highly expressed at these metastatic sites [15–17].
CXCL12/CXCR4 signaling has been shown to be involved in the adhe-
sion, migration, invasion, and metastasis of PC cells in laboratory model
systems [16,17]. We have recently shown that CXCL12/CXCR4 sig-
naling transactivates members of the epidermal growth factor receptor
family in membrane microdomains of PC cells, and this transactivation
contributes to the expansion of intraosseous metastatic deposits .
CXCR4 has been shown to be deregulated in tumor cells through tran-
scriptional mechanisms. Prostate tumors and metastases express higher
levels of CXCR4 compared with nontumor tissue [19–21], and this
overexpression is associated with aggressive disease in patients [20,22].
A recent study suggests that CXCR4 is one of the functional target
genes for ERG transcription factor in PC cells . To determine the
link between TMPRSS2-ERG translocations and CXCR4 expression
in PC cells, we investigated the role of androgens in the activation of
TMPRSS2-ERG andthesubsequent expression ofCXCR4 inPC cells.
Herein, we show that PC cells that exhibit TMPRSS2-ERG fusions
have androgen-regulated CXCR4 expression and that knock down of
ERG abrogates androgen-induced CXCR4 expression. Furthermore,
the CXCR4 promoter contains several putative ERG binding sites,
and the ERG factor binds to the CXCR4 promoter in TMPRSS2-
ERG–positive VCaP cells. Androgens and CXCL12 independently
induced chemoinvasion of VCaP cells, and in combination, they in-
suggest that androgen-induced CXCR4 expression is functional in
TMPRSS2-ERG–positive PC cells. These studies provide an impor-
tant link between TMPRSS2-ERG chromosomal translocations and
androgen-induced CXCR4-mediated metastasis formation.
Materials and Methods
VCaP, PC-3, and LNCaP cells were purchased from American Type
Culture Collection (Manassas, VA). PC-3 andLNCaP cells were cultured
in RPMI 1640 medium, and VCaP cells were cultured in Dulbecco’s
modified Eagle medium. All cell lines were tested for Mycoplasma con-
tamination beforeuse intheexperimentswith VenorGeM Mycoplasma
detection kit from Sigma Biochemicals (St Louis, MO). The culture
medium was supplementedwith 10% fetalbovine serum and 1%peni-
cillin andstreptomycin.R1881 waspurchasedfromNEN Life Sciences
(Waltham, MA), flutamide and cycloheximide were purchased from
Sigma, and CXCL12 was purchased from Peprotech (Rocky Hill, NJ).
Quantitative Polymerase Chain Reaction
A total of 4 × 105cells were seeded in six-well plates. Each plate was
treated with a single agent of an androgen agonist R1881, antagonist
flutamide, or cycloheximide or with a combination of R1881 with
flutamide or cycloheximide as shown in figure legends. Total RNA was
isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA). For reverse
transcription–polymerase chain reaction (PCR) studies, first-strand
complementary DNA was synthesized from 2 μg of total RNA with an
oligo(dT) primer and SuperScript II Reverse Transcriptase (Invitrogen).
Forward and reverse primers were designed using the Primer 3 program
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The for-
ward and reverse primers are as follows: for ERG, 5′-AAC GAG CGC
AGA GTT ATC GT-3′ and 5′-GTG AGC CTC TGG AAG TCG
TC-3′; for CXCR4, 5′-GGC CCT CAA GAC CAC AGT CA-3′ and
5′-TTA GCT GGA GTG AAA ACT TGA AG-3′; for prostate-specific
antigen (PSA), 5′-GGT GAT GAC TCC AGC CAC GA-3′ and 5′-
GCG CAC ACA CGT CAT TGG AA-3′; and for glyceraldehyde 3-
phosphate dehydrogenase (GAPDH), 5′-AAG GTC ATC CCT GAG
CTG AA-3′ and 5′-TGA CAA AGT GGT CGT TGA GG-3′. Real-
time PCR analysis was performed with SYBR Green PCR core reagents
(Stratagene, La Jolla,CA) ina Stratagene Mx4000cycler,anddata analy-
sis was performed using Mx4000 v3.01 software. All primer sets were
tested in real-time PCR and found to produce no detectable peaks in
dissociation curves due to primer-dimer amplifications. Relative message
levels were calculated with a comparative Ct(threshold cycle) method
. Briefly, message levels were normalized to endogenous GAPDH
message levels. In treated samples, relative quantitation was performed
by the comparative Ctmethod  using the formula 2−ΔΔCt, where
[Cttest gene (control sample) − CtGAPDH (control sample)]. For
resents the mean Ctvalue of each sample, and GAPDH is the endoge-
nous control used to normalize the quantification of a test gene.
Secondary Data Analysis for ERG and CXCR4 Expression
in Human Benign Prostate and Prostate Cancer Tissue
Expression profile data sets for human benign and PC tissue were
queried for ERG and CXCR4 expression using the Gene Expression
Omnibus database (http://www.ncbi.nlm.nih.gov/geo/). This record
was deposited by Yu et al. as previously described . We extracted
the gene expression values for ERG and CXCR4 for benign prostate
(n = 18) and PC tissue (n = 65) from GDS2546 record. ERG and
CXCR4 expression values were analyzed with GraphPad Prism software
version 3.0 (GraphPad, San Diego, CA).
Western Blot Analysis
Subconfluent cultures of VCaP cells were washed with phosphate-
taining 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 1 mM PMSF, and 1 ×
Protease inhibitor cocktail (Roche, Indianapolis, IN). Protein content
was quantified with a BCA protein assay (Pierce Biotechnology, Inc,
Rockford, IL), and equal amounts of protein were resolved by 10%
Cruz Biotechnology, Santa Cruz, CA), CXCR4 (Millipore, Billerica,
body incubation, chemiluminescence reaction was performed with
SuperSignal Western Femto or Pico Substrate (Pierce Biotechnology,
Inc). The band intensities were determined by quantitation of pixel
intensities using Un-Scan-It software (version 5.1; Orem, UT). Appar-
ent molecular weights of ERG forms were also determined by Un-Scan-
ERG Regulation of CXCR4 Via TMPRSS2-ERG ActivationCai et al.Translational Oncology Vol. 3, No. 3, 2010
It software using a reference position of molecular weight markers in
Fluorescence-Activated Cell Sorting Analysis
Fluorescence-Activated Cell Sorting Analysis (FACS) was performed
on VCaP cells aspreviously described . Briefly, VCaP cells grownin
culture plates were scraped and counted on hemocytometer. A total of
5 × 105cells were resuspended in phosphate-buffered saline supple-
mented with 5% fetal bovine serum and incubated with either phyco-
erythrin (PE)-conjugated anti-CXCR4 antibody (BD Pharmingen, San
Diego, CA) or isotype-matched IgG2a(BD Pharmingen) for 15 min-
utes on ice. Antibody-bound cancer cells were washed three times and
analyzed on fluorescence-activated cell sorter (Becton Dickinson, San
Diego, CA). CXCR4-positive cells were enumerated using the cell
quest software (Becton Dickinson). Data shown are percent of total
gated cells that are positive for anti–CXCR4-PE antibody binding.
VCaP cells were serum-starved for 4 hours. A total of 1.5 to 2.0 ×
105cells were seeded onto inserts in the upper chamber of transwell
culture plates (Becton Dickinson). Before seeding, the inserts were pre-
coated with Matrigel. To activate the androgen receptor (AR), 0.5 nM
of R1881 was added to the upper chamber. CXCL12 was placed in the
bottom chamber for CXCR4-mediated chemoinvasion. Cell invasion
was allowed to proceed for 24 hours. Later, the upper chambers were
cleaned with cotton swabs to remove nonmigrated/invaded cells, and
the inserts were stained with Diff-Quik stain set (Dade Behring, Inc,
Newark, DE). The total number of migrated cells in a high-power field
was counted under a microscope, and the data presented are based on
three independent experiments.
The experiment was performed using a kit from Active Motif, Inc
(Carlsbad, CA). As per the manufacturer’s recommendations, sub-
confluent cultures of VCaP cells were fixed with 1% formaldehyde
solution, sonicated to shear the chromatin, and incubated with anti-
ERG or isotype IgG antibodies and protein G magnetic beads. The
immunoprecipitates were washed to remove nonspecific complexes.
Chromatin was then eluted by reverse cross-linking and was treated
with proteinase K. PCR was performed with primers designed with
5′ sequences of CXCR4 and GAPDH promoters. The forward primer
5′-GGA TCC CCA ACG CCT AGA AC-3′ and reverse primer 5′-
CAG CCC ATT CAG GAG GTA AA-3′ were used for CXCR4,
and the forward primer 5′-TAC TAG CGG TTT TAC GGG CG-3′
and reverse primer 5′-TCG AAC AGG AGG AGC AGA GAG CGA-
3′ were used for GAPDH in the PCR. PCRs were analyzed on 2%
For CXCR4 and ERG expression, the Mann-Whitney test was per-
formed between benignand PC tissuedata. The Pearson correlation test
was performed for CXCR4 and ERG expression in tumor samples, and
the correlation coefficient, r, was determined using GraphPad Prism
software version 3.0 (GraphPad). For in vitro chemoinvasion study,
statistical significance was determined by the nonparametric analysis of
variance test followed by the Tukey posttest to compare all pairs of a
column. P ≤ .05 was considered statistically significant.
ERG and CXCR4 Coexpressed in PC Cells
CXCR4 expression in PC cells has been shown to contribute to
secondary metastasis formation in bone tissue [18,25]. We identified
several Ets transcription factor binding sites, including several consen-
sus ERG transcription factor binding sites  in the promoter region
of CXCR4 (Table W1). Binding of Ets factors to these sites potentially
contribute to CXCR4 expression. To determine the role of TMPRSS2-
ERG translocations on CXCR4 expression, we assessed ERG and
CXCR4 expression in TMPRSS2-ERG fusion–positive VCaP cells,
TMPRSS2-ERG fusion–negative PC-3 cells, and human prostate
Secondary analysis of public domain expression array profile data of
benign prostate and prostate tumor tissue  shows that both ERG
and CXCR4 are expressed significantly higher in prostate tumor tissue
samples (Figure 1A). Correlation studies with ERG and CXCR4 in
prostate tumor samples show a moderate association between ERG
and CXCR4 gene expressions (r = 0.4238 and P < .001). Similar
ERG and CXCR4 expression patterns have been observed in the lim-
ited number of PC patient tumor tissues and the adjacent nontumor
tissue available for investigation (data not shown). Together, these data
suggest a concerted up-regulation of ERG and CXCR4 in tumor cells.
Gene expression studies with cell lines show that both ERG and
CXCR4 transcript levels are higher in VCaP cells compared with
PC-3 cells (Figure 1B). Western blot analysis showed that ERG expres-
sion is not detectable in PC-3 cells, whereas in VCaP cells, ERG is ex-
is significantly higher in VCaP cells compared with that in PC-3 cells
(Figure 1C). To address whether ERG can regulate CXCR4 gene ex-
pression, we performed chromatin immunoprecipitation experiments
with VCaP cells. These studies demonstrated that in VCaP cells anti-
plexes in VCaP cells (Figure 1D). Together, these data suggest that in
TMPRSS2-ERG–positive cells, ERG and CXCR4 are highly expressed,
and ERG binds with the CXCR4 promoter sequences in VCaP cells.
Androgens Regulate CXCR4 Gene through ERG Transcription
Factor Expression in PC Cells
To determine whether R1881 regulation of CXCR4 gene expres-
sion was mediated through the activation of TMPRSS2-ERG fusions,
gene expression studies with VCaP, LNCaP, and PC-3 cells were per-
formed. In the absence of R1881 stimulation, CXCR4 was expressed in
all cell types, whereas ERG expression was higher in VCaP cells com-
pared with PC-3 and LNCaP cells (Figure 2A). R1881 treatment of
VCaP cells induced both ERG and CXCR4 messenger RNA expression
(Figure 2B). As expected, synthetic androgens induced PSA expression
inAR–positive VCaPand LNCaP cells but not in PC-3 cells that lack a
functional AR. Synthetic androgen treatment enhanced both ERG and
CXCR4 expression in VCaP cells but not in AR–positive LNCaP cells.
As expected, theantiandrogen flutamideabrogatedthesynthetic andro-
gen induction of PSA in VCaP and LNCaP cells. Similarly, flutamide
treatment also abrogated the androgen-induced ERG and CXCR4
expression in VCaP cells and CXCR4 expression in LNCaP cells
Translational Oncology Vol. 3, No. 3, 2010ERG Regulation of CXCR4 Via TMPRSS2-ERG Activation Cai et al.
(Figure 2C). The absence of androgen-induced ERG expression in
LNCaP cells suggests that androgen-induced ERG transcriptionally
regulates CXCR4 in VCaP cells.
To determine whether androgens regulate CXCR4 expression, we
treated VCaP cells with different concentrations of the synthetic andro-
analysis. R1881 treatment upregulated cell surface CXCR4 expression,
and a higher concentration of R881 further enhanced CXCR4 cell sur-
face expression (Figure 3).
To further determine that CXCR4 is an indirect target of androgens,
we treated VCaP cells with the translational inhibitor cycloheximide.
Vehicle- and cycloheximide-treated cells were analyzed for R1881 in-
duction of CXCR4 and PSA expression in VCaP cells. As expected,
cycloheximide did not abrogate R1881 induction of PSA but inhibited
the R1881 induction of CXCR4 expression in VCaP cells (Figure 4).
These data imply thatR1881-inducednewproteinsynthesisisrequired
for CXCR4 expression in VCaP cells. Together, these data support the
notion that R1881 activation of TMPRSS2-ERG translocations in-
duces CXCR4 expression in PC cells.
ERG Is Required for CXCR4 Gene Expression in VCaP Cells
lates CXCR4 gene expression, we tested the effect of small interfering
RNA (siRNA)–mediated down-regulation of the ERG gene. SiERG
transfection resulted in the down-regulation of both ERG and CXCR4
gene expression compared with scrambled siRNA transfection (Fig-
ure 5A). Western blot analysis show that a 60% inhibition of ERG
protein expression compared with scrambled siRNA transfection (Fig-
ure 5B). To assess the role of androgens in the regulation of CXCR4
expression, we treated the scrambled and siERG-transfected VCaP cells
with synthetic androgens and measured the CXCR4 gene expression
(Figure 5, B and C). Synthetic androgens upregulated the CXCR4
gene expression in scrambled siRNA-transfected cells but were un-
able to upregulate the CXCR4 gene in siERG-transfected cells. As ex-
pected, synthetic androgens upregulated PSA expression in both cells
(Figure 5D). These data imply that, although PSA is not a target for
R1881-induced ERG expression, CXCR4 is regulated by this mecha-
expression of ERG transcription factor regulates CXCR4 expression.
Figure 1. ERG and CXCR4 were highly expressed in TMPRSS2-ERG fusion–positive cell and prostate tumor cells, and ERG binds to
CXCR4 promoter. (A) Expression array data for ERG and CXCR4 were obtained from GDS2546 record from Gene Expression Omnibus
database. Mann-Whitney test was performed between samples to determine statistical significance. (B) Quantitative PCR analysis of
ERG and CXCR4 genes was performed with messenger RNA prepared from PC-3 and VCaP cells. The relative expressions of genes were
shown after normalization with the housekeeping gene GAPDH. (C) Total cellular proteins were isolated from PC-3 and VCaP cells and
immunoblotted with anti-ERG, anti-CXCR4, and anti-GAPDH antibodies. A representative radiograph of chemiluminescence detection is
shown with multiple independent Western blot analyses. Apparent molecular weights of ERG forms are shown for VCaP cells. (D) Chro-
matin immunoprecipitation assay was performed with VCaP cell DNA with anti-ERG and isotype antibodies. Immunoprecipitated chro-
matin was amplified with CXCR4 and GAPDH gene primers in the 5′region. Ethidium bromide–stained gel analysis of PCR-amplified DNA
fragments is also shown.
ERG Regulation of CXCR4 Via TMPRSS2-ERG Activation Cai et al. Translational Oncology Vol. 3, No. 3, 2010
Figure 3. CXCR4 is an androgen-responsive gene in VCaP cells. VCaP cells were treated with different concentrations of R1881, and cell
surface expression of CXCR4 was determined by FACS analysis.
Figure 2. Thesynthetic androgen, R1881, induces ERG and CXCR4expression in PCcells. (A) Relative geneexpressionsof ERG, CXCR4, and
PSA are shown in PC-3, LNCaP, and VCaP cells. (B) R1881- and vehicle-treated VCaP cells were analyzed for ERG, CXCR4, and GAPDH gene
expression. The PCR-amplified gene products were analyzed on ethidium bromide agarose gel. (C) PC-3, LNCaP, and VCaP cells treated with
vehicle, R1881, flutamide, and a combination of both reagents were analyzed for ERG, CXCR4, PSA, and GAPDH gene expressions.
Translational Oncology Vol. 3, No. 3, 2010ERG Regulation of CXCR4 Via TMPRSS2-ERG ActivationCai et al.
ERG-Mediated CXCR4 Expression Regulates PC
We have previously shown that bone tissue associated CXCL12 is
active in CXCR4-dependent PC cell chemoinvasion through the ex-
pression of matrix metallopeptidase-9 . To assess the functional
significance of ERG-mediated CXCR4 expression in VCaP cells, we
sure to both agents additively enhanced chemoinvasion of VCaP cells,
suggesting that androgen-induced CXCR4 is active in the chemoinva-
sion of VCaP cells toward a CXCL12 gradient. To determine whether
R1881-induced CXCR4 enhances VCaP cell chemoinvasion, we treated
VCaP cells with the CXCR4 antagonist AMD3100. AMD3100 down-
to the levels of R881 treatment (Figure 6B). Together, these data sug-
gest that the androgen activation of TMPRSS2-ERG translocations
contributes to PC cell chemoinvasion through CXCR4 expression
Herein, we demonstrate that androgens induce CXCR4 gene expres-
sion in TMPRSS2-ERG–positive VCaP cells. To our knowledge, this
isthefirstreportidentifying theCXCR4 gene asa targetfor TMPRSS2-
ERG activation in PC cells. In this study, we show that androgen-
responsive VCaP cell lines coexpress higher levels of CXCR4 and ERG
compared with androgen-unresponsive PC-3 cells. ERG protein expres-
cells (Figure 1B) as was previously shown by Tomlins et al. . The two
ERG species expressed in VCaP cells are most likely due to the alterna-
tive splicing of the fusion transcript. There is significant heterogeneity in
the expression of fusion transcripts in tumor cells with TMPRSS2-ERG
Figure 4. Cycloheximide abrogates R1881 induction of CXCR4 gene
expression. VCaP cells were treated with vehicle, R1881 (0.5 nM),
cycloheximide (5 μg/ml), and a combination of cycloheximide and
R1881. In the combination experiment, cycloheximide was initially
treated with cells, and 1 hour later, R1881 was added. CXCR4 and
PSA gene expressions were determined by quantitative PCR. Fold
differencesin the geneexpression were determined after normaliza-
tion with GAPDH expression.
Figure 5. ERG factor mediates CXCR4 expression in VCaP cells. (A) ERG, CXCR4, and GAPDH gene expressions were determined
in scrambled (Scr) and ERG siRNA-transfected VCaP cells, and ERG and CXCR4 gene expressions shown after normalization for GAPDH.
(B) Scrambled and ERG siRNA-transfected VCaP cell lysates were immunoblotted with anti-ERG, anti-CXCR4 and anti-GAPDH antibodies.
(C) Scr and ERG siRNA-transfected VCaP cells were treated with vehicle and R1881. Quantitative PCR analysis of CXCR4 and GAPDH was
performed. (D) PSA gene expression was analyzed in scrambled and ERG siRNA-transfected VCaP cells.
ERG Regulation of CXCR4 Via TMPRSS2-ERG ActivationCai et al. Translational Oncology Vol. 3, No. 3, 2010
alterations [1,7,27,28], and the translation of these transcripts could
give rise to several ERG species. For example, these ERG forms can lack
39 amino acids at the N-terminus , fusion with the first five amino
acids of TMPRSS2 protein and lack the N-terminus of ERG , have
a deletion of the C-terminus Ets binding domain . A recent study
blet has been the translation product from the first AUG codon in the
fourth exon. This translation product lacks 39 N-terminal amino acids.
Overexpression studies in cell culture and animal models with several of
these ERG forms, with the exception of the C-terminus Ets domain de-
letion,demonstrate thatthey play a key role in PC cellproliferation ,
invasion [9,10], and progression [12,13]. Overall, the data strongly indi-
cate that activation and alterations of TMPRSS2-ERG contribute to the
lethal characteristics of PC development in patients.
ent in both primary tumor cells and disseminated metastatic cells to
several secondary sites , which suggest that downstream target genes
of TMPRSS2-ERG fusions facilitate the tumor cell invasion and dis-
semination process. Recent studies suggest that the chemokine receptor
CXCR4 in tumor cells and its ligand CXCL12 expressed in secondary
metastatic sites play a key role in the metastasis of primary tumor cells
[16–18,25]. Our data support the notion that TMPRSS2-ERG activa-
tion in PC cells regulates CXCR4 expression and subsequent metastasis
to secondary sites.
Our study shows that the synthetic androgen upregulates the ERG
expression in fusion-positive VCaP cells, which is in line with findings
previously reported by Tomlins et al. . Interestingly, we found that
CXCR4 is regulated in parallel with ERG in a panel of PC cells. In
LNCaP cells, the synthetic androgen induced a very modest degree
of CXCR4 gene expression in (Figure 2C). Because LNCaP cells have
very low levels of ERG in the absence of TMPRSS2-ERG transloca-
tions, this regulation could be mediated indirectly by additional AR-
dependent processes. Alternatively, other Ets factors could contribute
to CXCR4 expression in these cells. In support of the potential regula-
tion by other Ets factors, ETV1 has been shown to be expressed in
LNCaP cells  and could mediate CXCR4 expression through Ets
binding sites in the CXCR4 promoter. Studies are in progress to iden-
tify the specific ERG and Ets binding sites in the CXCR4 promoter.
The moderate levels of CXCR4 expression induced by R1881, coupled
with the presence of ETV1 in these cells, support the notion that ERG
and ETV1 translocationscould bemutually exclusive inprostate tumor
samples and could mediate prometastatic CXCR4 gene expression, as
well as the subsequent invasion and metastasis of tumor cells. In VCaP
cells, androgen-induced CXCR4 expression is mediated by the over-
expression of ERG rather than by the general growth effects of andro-
gens because we did not observe changes in the CXCR4 expression in
VCaP cells in the presence of serum (data not shown).
thegrowth factor throughhypoxicandnuclearfactor κBtranscrip-
tion factor activities . In contrast with those models, our data with
cycloheximide (Figure 3) suggest that androgen-mediated protein syn-
requirement is only present in PC cells exhibiting the TMPRSS2-ERG
moter does not contain consensus AR binding sites , and thus, this
regulation is most likely to be mediated indirectly. Conversely, Akashi
et al. have shown that overexpression of AR inDU145 cells down-
regulated CXCR4 expression, although it is not clear whether the over-
site in the CXCR4 promoter region suggests that such down-regulation
could be indirectly mediated by AR activation, which would be inde-
pendent of ERG function in these cells, overexpression of ERG in
fects on CXCR4 expression.
higher functional CXCR4 expression. Our data also demonstrate that
CXCR4 is a target gene for the ERG transcription factor, and our results
with the synthetic androgen regulation of CXCR4 (Figure 2) further
Figure 6. R1881 induced CXCR4 active in the chemoinvasion of VCaP cells: (A) VCaP cells were serum-starved, and chemoinvasion was
performed using Matrigel-coated filters. CXCL12 was included in the bottom chamber as a chemoattractant. The cells were treated
either with vehicle or with R1881 at 0.5 nM. (B) R1881-treated VCaP cells were further exposed to the CXCR4 antagonist AMD3100,
and chemoinvasion was performed in the presence of CXCL12.
Translational Oncology Vol. 3, No. 3, 2010ERG Regulation of CXCR4 Via TMPRSS2-ERG Activation Cai et al.
suggest that CXCR4 is a physiological target of androgens in prostate
tumor cells and that this process could facilitate the pathological progres-
sion of tumor cell metastasis through the CXCL12/CXCR4 axis. Carver
promoter of CXCR4, whereas the data from chromatin immunoprecip-
itation analysis in this study identified ERG binding sites in the CXCR4
promoter. We identified eight potential ERG binding sites in the 1-kb
CXCR4 promoter, and three of these putative sites were present in the
 reported that Ets1 factor binds to −397 to −412 and −478 to −481
positive cells, it is highly likely that ERG could be the relevant factor in-
teractingwiththeCXCR4promoterinPCcells.The sequences between
−513 and −996 have four potential ERG binding sites, but our attempts
to design primers to amplify this region have been unsuccessful so far
because of the high percentage of GC content at this region. Studies
are in progress to determine the relative contribution of these putative
ERG binding sites in the regulation of the CXCR4 promoter.
proliferative functions in VCaP cells. Our previous data demonstrated
that the CXCL12/CXCR4 axis promotes PC cell invasion through
activation of signaling pathways leading to protease expression .
CXCR4 expression also has been shown to contribute to the growth
of tumor cells in bone metastatic sites . Our present data demon-
strate that ERG knockdown attenuates androgen-dependent CXCR4
expression without significantly changing PSA expression. These re-
sults suggest that the TMPRSS2-ERG fusionfacilitatestumor cell inva-
sion and metastasis through the regulation of CXCR4 expression and
function in PC cells. To test this concept, our data with an in vitro in-
vasionassay (Figure6)demonstratethatR1881alone caninduceVCaP
cell invasion. This supports previously published reports that R1881-
induced ERG expression contributes to in vitro invasion of VCaP cells
and is mediated by the expression of proteases [9,10]. Interestingly,
the R1881-treated cells invaded more efficiently in the presence of
CXCL12. CXCR4 inhibition suppressed the CXCL12 effect, suggest-
ing that androgen-induced CXCR4 expression is functional in VCaP
cells and contributes to PC cell invasion. These data are in line with
previous reports, demonstrating the role of ERG in CXCR4 function
in PC cells .
In summary, we show that TMPRSS2-ERG activation in fusion-
positive cancer cells induces the expression of the prometastatic gene
CXCR4,which isfunctionally active inthechemoinvasion process.Tar-
geting CXCR4, a relevant target for androgen activation of TMPRSS2-
ERG, could be an advantageous strategy for lethal phenotypes associated
with these chromosomal translocations in PC patients.
The authors thank Stephen Ethier and Raymond Mattingly of Wayne
State University for critically reading the manuscript and Jason St. John
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Translational Oncology Vol. 3, No. 3, 2010 ERG Regulation of CXCR4 Via TMPRSS2-ERG ActivationCai et al.
Table W1. ETS/ERG Binding Sites in CXCR4 Promoter. Download full-text
Nucleotide Position Relative to Transcription
Start Site in CXCR4 Promoter
−119 to −126
−234 to −241
−420 to −417
−513 to −520
−700 to −707
−850 to −857
−889 to −896
−919 to −926
−2517 to −2525
−2664 to −2656
ETS core sequence: GGA(A/T).
ERG consensus sequence: (C/A)GGAA(G/A)T.