Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B

Abstract

Acquired resistance to anticancer treatments is a substantial barrier to reducing the morbidity and mortality that is attributable to malignant tumors. Components of tissue microenvironments are recognized to profoundly influence cellular phenotypes, including susceptibilities to toxic insults. Using a genome-wide analysis of transcriptional responses to genotoxic stress induced by cancer therapeutics, we identified a spectrum of secreted proteins derived from the tumor microenvironment that includes the Wnt family member wingless-type MMTV integration site family member 16B (WNT16B). We determined that WNT16B expression is regulated by nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB) after DNA damage and subsequently signals in a paracrine manner to activate the canonical Wnt program in tumor cells. The expression of WNT16B in the prostate tumor microenvironment attenuated the effects of cytotoxic chemotherapy in vivo, promoting tumor cell survival and disease progression. These results delineate a mechanism by which genotoxic therapies given in a cyclical manner can enhance subsequent treatment resistance through cell nonautonomous effects that are contributed by the tumor microenvironment.

Full text

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nature medicine VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 1359
A major impediment to more effective cancer treatment is the ability
of tumors to acquire resistance to cytotoxic and cytostatic therapeu-
tics, a development that contributes to treatment failures exceeding
90% in patients with metastatic carcinomas1. Efforts focused on cir-
cumventing cellular survival mechanisms after chemotherapy have
defined systems that modulate the import, export or metabolism of
drugs by tumor cells2–6. Enhanced damage repair and modifications
to apoptotic and senescence programs also contribute to de novo or
acquired tolerance to anti-neoplastic treatments3,7,8. In addition,
the finding that ex vivo assays of sensitivity to chemotherapy do not
accurately predict responses in vivo indicate that tumor microenvi-
ronments also contribute substantially to cellular viability after toxic
insults9–11. For example, cell adhesion to matrix molecules can affect
life and death decisions in tumor cells responding to damage12–14.
Further, the spatial organization of tumors relative to the vasculature
establishes gradients of drug concentration, oxygenation, acidity and
states of cell proliferation, each of which may substantially influence
cell survival and the subsequent tumor repopulation kinetics15,16.
Most cytotoxic agents selectively target cancers by exploiting
differential tumor cell characteristics, such as high proliferation rates,
hypoxia and genome instability, resulting in a favorable therapeu-
tic index. However, cancer therapies also affect benign cells and can
disrupt the normal function and physiology of tissues and organs.
To avoid host lethality, most anticancer regimens do not rely on single
overwhelming treatment doses: both radiation and chemotherapy
are administered at intervals to allow the recover y of vital normal
cell types. However, gaps between treatment cycles also allow tumor
cells to recover, activate and exploit survival mechanisms and resist
subsequent therapeutic insults.
Here we tested the hypothesis that treatment-associated DNA
damage responses in benign cells comprising the tumor microenvi-
ronment promote therapy resistance and subsequent tumor progres-
sion. We provide in vivo evidence of treatment-induced alterations
in tumor stroma that include the expression of a diverse spectrum of
secreted cytokines and growth factors. Among these, we show that
WNT16B is activated in fibroblasts through NF-κB and promotes an
epithelial to mesenchymal transition (EMT) in neoplastic prostate
epithelium through paracrine signaling. Further, WNT16B, acting
in a cell nonautonomous manner, promotes the survival of cancer
cells after cytotoxic therapy. We conclude that approaches targeting
constituents of the tumor microenvironment in conjunction with con-
ventional cancer therapeutics may enhance treatment responses.
RESULTS
Therapy induces damage responses in tumor microenvironments
To assess for treatment-induced damage responses in benign cells
comprising the tumor microenvironment, we examined tissues col-
lected before and after chemotherapy exposure in men with prostate
1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. 2Buck Institute for Research on Aging, Novato, California, USA.
3Lawrence Berkeley National Laboratory, Berkeley, California, USA. 4Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington,
USA. 5Department of Medicine, University of Washington, Seattle, Washington, USA. 6Division of Hematology and Medical Oncology, Oregon Health and Science
University, Portland, Oregon, USA. 7Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon, USA. 8Department of Pathology, University of
Washington, Seattle, Washington, USA. Correspondence should be addressed to P.S.N. (pnelson@fhcrc.org).
Received 25 April 2011; accepted 8 June 2012; published online 5 August 2012; doi:10.1038/nm.2890
Treatment-induced damage to the tumor micro-
environment promotes prostate cancer therapy resistance
through WNT16B
Yu Sun1, Judith Campisi2,3, Celestia Higano4,5, Tomasz M Beer6,7, Peggy Porter1, Ilsa Coleman1, Lawrence True8 &
Peter S Nelson1,4,5,8
Acquired resistance to anticancer treatments is a substantial barrier to reducing the morbidity and mortality that is attributable
to malignant tumors. Components of tissue microenvironments are recognized to profoundly influence cellular phenotypes,
including susceptibilities to toxic insults. Using a genome-wide analysis of transcriptional responses to genotoxic stress induced
by cancer therapeutics, we identified a spectrum of secreted proteins derived from the tumor microenvironment that includes
the Wnt family member wingless-type MMTV integration site family member 16B (WNT16B). We determined that WNT16B
expression is regulated by nuclear factor of k light polypeptide gene enhancer in B cells 1 (NF-kB) after DNA damage and
subsequently signals in a paracrine manner to activate the canonical Wnt program in tumor cells. The expression of WNT16B in
the prostate tumor microenvironment attenuated the effects of cytotoxic chemotherapy in vivo, promoting tumor cell survival and
disease progression. These results delineate a mechanism by which genotoxic therapies given in a cyclical manner can enhance
subsequent treatment resistance through cell nonautonomous effects that are contributed by the tumor microenvironment.
npg © 2012 Nature America, Inc. All rights reserved.

articles
1360 VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 nature medicine
cancer enrolled in a neoadjuvant clinical trial combining the geno-
toxic drug mitoxantrone (MIT) and the microtubule poison docetaxel
(DOC) (Fig. 1a)17,18. After chemotherapy, we found evidence of DNA
damage in fibroblasts and smooth muscle cells comprising the pros-
tate stroma, as determined by the phosphorylation of histone H2AX
on Ser139 (γ-H2AX) (Fig. 1b). To ascertain the molecular conse-
quences of DNA damage in benign cells, we treated primary prostate
fibroblasts (PSC27 cells) with MIT, bleomycin (BLEO), hydrogen per-
oxide (H2O2) or gamma radiation (RAD), each of which substantially
increased the number of γ-H2AX foci (Supplementary Fig. 1a,b). We
used whole-genome microarrays to quantify transcripts in PSC27 cells
and determined that the levels of 727 and 329 mRNAs were commonly
increased and decreased, respectively (false discovery rate of 0.1%),
as a result of these genotoxic exposures (Supplementary Fig. 1c). To
focus our studies on those factors with the clear potential for para-
crine effects on tumor cells, we evaluated genes with at least 3.5-fold
elevated expression after genotoxic treatments that encode extracellu-
lar proteins, here collectively termed the DNA damage secretory pro-
gram (DDSP) (Fig. 1c). Consistent with previous studies, transcripts
encoding matrix metalloproteinases such as MMP1, chemokines such
as CXCL3 and peptide growth factors such as amphiregulin were sub-
stantially elevated in PSC27 fibroblasts after genotoxic damage19,20.
Notably, the expression of WNT16B increased between eightfold and
64-fold as a result of these treatments (P < 0.005) (Fig. 1c,d).
Wnt family members participate in well-described mesenchymal
and epithelial signaling events that span developmental biology,
stem cell functions and neoplasia21. Though little information links
Wnt signaling to DNA damage responses, a previous study reported
WNT16B overexpression in the context of stress- and oncogene-
induced senescence22. We confirmed that DNA damage increased
WNT16B protein expression and found elevated amounts of extra-
cellular WNT16B in conditioned medium from prostate fibroblasts
after chemotherapy or radiation (Fig. 1e,f). Transcripts encoding
other Wnt family members were not substantially altered in the pros-
tate fibroblasts we studied here (Fig. 1g). In contrast to the WNT16B
responses in fibroblasts, we observed little induction of WNT16B
expression in epithelial cells (Fig. 1h).
We next sought to confirm that expression of WNT16B is induced
by genotoxic therapy in vivo. We used laser-capture microdissec-
tion to separately isolate stroma and epithelium and determined
CM WNT16B
Pre
BLEO
MIT
RAD
H2O2
IC WNT16B
β-actin
d
f g
h
c
Gene
symbol
Fold
change
IL8
MMP1
MMP10
ENPP5
EREG
BMP6
ANGPTL4
CSGALNACT
CCL26
AREG
ANGPT1
CCK
THBD
CXCL14
NOV
GAL
NPPC
FAM150B
CST1
GDNF
MUCL1
NPTX2
TMEM155
EDN1
PSG9
ADAMTS3
CD24
PPBP
CXCL3
MMP3
CST2
PSG8
PCOLCE2
PSG7
TNFSF15
C17orf67
CALCA
FGF18
BMP2
MATN3
TFPI
SERPINI1
TNFRSF25
IL23A
MMP12
SFRP2
WNT16B
SPINK1
33.7
76.1
24.7
23.9
22.8
21.6
17.3
15.5
15.0
14.0
11.7
10.6
10.1
9.3
8.7
8.5
8.0
8.0
7.7
7.3
6.7
6.6
6.6
6.4
6.4
5.8
5.6
5.6
5.4
5.2
5.0
4.6
4.5
4.5
4.5
4.4
4.4
4.3
4.1
4.1
3.8
3.8
3.8
3.8
3.8
3.6
3.5
9.8
BLEO RAD
H
2
O
2
1.5
2.0
4.0
> 16
< –16
–1.5
1.0
–4.0
–2.0
BLEO
Gene
symbol
Fold
change
RAD
WNT16B 33.7
WNT3
WNT7A
WNT3A
WNT10A
WNT4
WNT5A
WNT1
WNT10B
WNT5B
WNT6
WNT11
WNT8A
WNT9A
WNT7B
WNT8B
WNT9B
WNT2B
WNT2
H2O2
1.9
1.6
1.5
1.5
2.0
4.0
> 16
< –16
1.3
1.2
1.1
1.1
1.0
1.0
–1.0
–1.1
–1.2
–1.2
–1.5
–1.5
1.0
–4.0
–2.0
–1.5
–1.5
–2.1
–2.4
Patient 1
e
b
e
e
s
s
s
s
gl
gl
Before
Chemotherapy
After
Patient 2
Enroll
a
Prostate
biopsy
Comparative
analysis Radical
prostatectomy
Chemotherapy
4-week cycles
16 weeks
DOC
MIT
#1
DOC
MIT
#2
DOC
MIT
#3
DOC
MIT
#4
P < 0.0001
P = 0.006
P = 0.003
P =
0.0002
Pre
BLEO
MIT
RAD
H2O2
7
6
5
4
3
2
1
0
WNT16B log2 fold changeWNT16B
Pre
MIT RAD
BLEO
MIT
RAD
WNT16B
log
2
fold change
7
6
5
4
3
2
1
0
PSC27 LNCaP DU145 BPH1
EpithelialFibroblast
M12 PC3
Figure 1 Genotoxic damage to primary prostate
fibroblasts induces expression of a spectrum
of secreted proteins that includes WNT16B.
(a) Schematic of the prostate cancer treatment
regimen comprising a pretreatment prostate biopsy
and four cycles of neoadjuvant DOC and MIT
chemotherapy followed by radical prostatectomy.
(b) DNA damage foci in human prostate tissues
collected before and after chemotherapy. Tissue
sections were probed with antibodies recognizing
γ-H2AX (red and pink signals), and nuclei were
counterstained with Hoechst 33342 (blue).
Gl, gland lumen; e, epithelium; s, stroma. Scale
bars, 50 µm. (c) Analysis of gene expression
changes in prostate fibroblasts by transcript
microarray quantification. The heatmap depicts
the relative mRNA levels after exposure to
H2O2, BLEO or RAD compared to vehicle-treated
cells. Columns are replicate experiments.
WNT16B is highlighted in bold for emphasis.
(d) Measurements of WNT16B expression by
qRT-PCR in prostate fibroblasts. Shown are the
log2 transcript measurements before (Pre) or
after exposure to the indicated factors relative
to vehicle-treated control cells. Data are mean ±
s.e.m. of triplicates. The P value was calculated by
analysis of variance (ANOVA) followed by t test.
(e) WNT16B protein expression in prostate
PSC27 fibroblast extracellular conditioned
medium (CM) or in cell lysates (IC) after
genotoxic exposures. β-actin is a loading control.
(f) Immunohistochemical analysis of WNT16B
expression in prostate fibroblasts before (Pre) and
after exposure to MIT or RAD. Brown chromogen
indicates WNT16B expression. Scale bars, 50 µm.
(g) Expression of Wnt family members in prostate
fibroblasts after exposure to DNA-damaging
agents. Transcript quantification was determined
by microarray hybridization. Columns represent
independent replicate experiments. WNT16B
is listed in bold for emphasis. (h) WNT16B
expression by qRT-PCR in PSC27 fibroblasts
and prostate cancer cell lines after the indicated
genotoxic exposure relative to pretreatment
transcript amounts. Data are mean ± s.e.m.
npg © 2012 Nature America, Inc. All rights reserved.

articles
nature medicine VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 1361
by quantitative RT-PCR (qRT-PCR) that the number of WNT16B
transcripts increased by approximately sixfold in prostate stroma
after chemotherapy (P < 0.01) (Fig. 2a and Supplementary Fig. 1d).
The expression of other genes known to respond to DNA damage,
including CDKN2A (also known as p16), CDKN1A (also known as
p21) and IL8, also increased in response to chemotherapy in prostate
stroma (Fig. 2a)20,23. We next confirmed induction of WNT16B pro-
tein expression by immunohistochemistry. Compared to untreated
prostate tissue, WNT16B protein was substantially and significantly
increased after chemotherapy in the periglandular stroma, which
included fibroblasts and smooth muscle cells (P < 0.01) (Fig. 2b,c).
In contrast, we obser ved very limited WNT16B expression in
benign or neoplastic epithelium, and mRNAs encoding other Wnt
family proteins were not substantially altered in prostate stroma
(Supplementary Fig. 1d,e).
We confirmed these findings in breast and ovarian carcinomas, two
other malignancies commonly treated with cytotoxic chemotherapy.
Genotoxic treatments induced the expression of WNT16B protein in
primary human fibroblasts isolated directly from breast and ovarian
tissues and in the prostates, breasts and ovaries of mice treated with
MIT (Supplementary Fig. 2a–d). WNT16B protein expression was sig-
nificantly elevated in the stroma of human breast and ovarian cancers
treated with neoadjuvant chemotherapy compared with tumors from
patients that did not receive treatment (P < 0.001) (Fig. 2). Notably, in
each of the tumor types evaluated, a range of absent to robust WNT16B
expression was evident. Because responses to chemotherapy also var-
ied, we evaluated whether WNT16B expression was associated with
clinical outcome. In patients with prostate cancer treated with neo-
adjuvant chemotherapy, higher WNT16B immunoreactivity in pros-
tate stroma after treatment was associated with a significantly greater
likelihood of cancer recurrence (P = 0.04) (Fig. 2d). We next sought
to determine the mechanism(s) by which WNT16B could contribute
to treatment failure.
WNT16B promotes cancer cell proliferation and invasion
Members of the Wnt family influence cellular phenotypes through
β-catenin–dependent and –independent pathways21. We generated
a prostate fibroblast cell strain with stable expression of WNT16B
20
a b c
fe
WNT16B
p21
Before
After
IL-8
p16
20
20
25
15
15
10
10
5
5
00
20
15
15
P = 0.01
P = 0.002 P < 0.0001
P = 0.03
P < 0.0001
P < 0.001
10
10
Patient groups
Untreated
Breast
120
0
1
2
3
WNT16B IHC
Chemotherapy
Untreated
WNT16B IHC
Chemotherapy
5
5
0
0 20 25
15
15
10
10
5
5
0
0
151050
15
10
5
0
100
80
60
Percentage of categorized
scoring levels (%)
40
20
0
Untreated
Patients = 21
Treated
Patients = 21
Patient groups
Untreated
Patients = 6
Treated
Patients = 6
120
100
80
60
Percentage of categorized
scoring levels (%)
40
20
0
P < 0.0001
Untreated
Cores = 186
Patients = 30
Patient groups
Treated
Cores = 267
Patients = 50
120
100
80
60
Percentage of categorized
scoring levels (%)
40
20
0
Ovary
0
1
2
3
0
1
2
3
Prostate
Untreated
WNT16B IHC
After chemotherapy
d0 ≤ WNT16B < 1
1 ≤ WNT16B < 2
2 ≤ WNT16B ≤ 3
100
80
60
Biochemical relapse-free
survival (%)
40
20
0
0 25 50 75 100
DFI (months)
Figure 2 Cytotoxic chemotherapy induces WNT16B expression in the tumor microenvironment. (a) Chemotherapy-induced gene expression changes in
human prostate-cancer–associated stroma measured by qRT-PCR of microdissected cells. The amounts of transcript before treatment (x axis) are
plotted against the amounts of transcript after chemotherapy (y axis) from the same individual. Each data point represents the measurements from
an individual patient. The results are shown as PCR cycle number relative to ribosomal protein L13 (RPL13), which served as the reference control.
The P values were calculated by Student’s t test. (b) IHC assessment of prostate stromal WNT16B expression in prostatectomy tissue samples from
men with prostate cancer who were either untreated (n = 30) or treated with chemotherapy (n = 50). Patients were assigned to four categories based on
their stromal WNT16B staining: 0, no expression; 1, faint or equivocal expression; 2, moderate expression; 3, intense reactivity. P < 0.0001 by ANOVA.
(c) Representative example of intense WNT16B expression in prostate stroma after in vivo exposure to MIT and DOC. The black arrows denote areas of
the stroma with fibroblasts and smooth muscle. Note the minimal WNT16B reactivity in the epithelium (gray arrows). Scale bars, 50 µm. (d) Kaplan-
Meier plot of biochemical (prostate-specific antigen) relapse-free survival based on the expression of WNT16B in prostate stroma after exposure to MIT
and DOC chemotherapy (P = 0.04 by log-rank test comparing WNT16B < 1 with WNT16B ≥ 2 survival distributions). DFI, disease-free interval from
surgery. (e,f) WNT16B staining of breast (e) and ovarian (f) carcinoma from patients receiving neoadjuvant chemotherapy or no treatment before surgical
resection. Staining is recorded on a 4-point scale: 0, no expression; 1, faint or equivocal expression; 2, moderate expression; 3, intense reactivity.
Scale bars, 50 µm. The P values were calculated by ANOVA.
npg © 2012 Nature America, Inc. All rights reserved.

articles
1362 VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 nature medicine
(PSC27WNT16B) and fibroblast strains that expressed shRNAs spe-
cific to WNT16B (shRNAWNT16B), which blocked the induction of
WNT16B expression by RAD and MIT (Supplementary Fig. 3a,b).
PSC27WNT16B-conditioned medium significantly enhanced pros-
tate cancer cell growth (P < 0.01) (Fig. 3a) and increased cellular
migration and invasion (P < 0.05) compared to conditioned medium
from PSC27 vector controls (PSC27C) (Fig. 3b and Supplementary
Fig. 3c,d), confirming that WNT16B can promote phenotypic changes
in tumor cells through paracrine mechanisms.
The DDSP comprises a diverse spectrum of secreted proteins with
the potential to alter the phenotypes of neighboring cells (Fig. 1c).
We next sought to determine to what extent WNT16B is responsible
for such effects in the context of the amalgam of factors induced by
DNA damage. Conditioned medium from irradiated PSC27 fibro-
blasts (PSC27-RAD), representing the full DDSP, increased the pro-
liferation (between 1.5-fold and twofold, P < 0.05) and invasiveness
(between threefold and fourfold, P < 0.05) of neoplastic epithelial cells
compared to conditioned medium from untreated PSC27 fibroblasts
(Fig. 3c,d). Compared to irradiated PSC27 cells expressing control
shRNAs, conditioned medium from PSC27-RAD + shRNAWNT16B
fibroblasts reduced these responses to the full DDSP by between 15%
and 35%, depending on the cell line (P < 0.05) (Fig. 3c,d).
To investigate the in vivo consequences of WNT16B expression in
the tumor microenvironment, we combined nontumorigenic BPH1 or
tumorigenic PC3 cells with PSC27WNT16B (BPH1+PSC27WNT16B and
PC3+PSC27WNT16B, respectively) or control PSC27 (BPH1+PSC27C
and PC3+PSC27C, respectively) fibroblasts and implanted the recom-
binants under the renal capsule of recipient mice (Fig. 3e). At 8 weeks
after implantation, BPH1+PSC27WNT16B grafts were larger than
BPH1+PSC27C grafts (~200 mm3 compared to ~10 mm3, respectively;
P < 0.001) (Supplementary Fig. 3e). PC3+PSC27WNT16B recombinants
generated very large poorly differentiated and invasive tumors with an
average size of 500 mm3, which was substantially larger than any of the
control tumors (P < 0.001) (Fig. 3f and Supplementary Fig. 3f).
In vivo, PC3 cells combined with PSC27-RAD cells express-
ing the full fibroblast DDSP resulted in substantially larger tumors
e
WNT16B
PFC
PC
WNT16B
WNT16B
WNT16B
WNT16B
Renal
capsule
6 weeks
PSC27C
PSC27WNT16B
PSC27WNT16B + shRNAC
PSC27WNT16B + shRNAWNT16B#1
PSC27WNT16B + shRNAWNT16B#2
Epithelial cell number × 1,000
a
BPH1 M12 PC3
80
* P < 0.05
** P < 0.01
70
*
*
****
*
60
50
40
30
20
Epithelial cell number × 1,000
cPSC27
*
*
*
*
*
**
* P < 0.05
** P < 0.01
BPH1 M12 PC3
PSC27-RAD
PSC27-RAD + shRNAC
PSC27-RAD + shRNAWNT16B#1
PSC27-RAD + shRNAWNT16B#2
80
100
60
40
20
PC3
Tumor volume (mm3)
g
P = 0.0106
P = 0.0148
500
400
300
200
100
0
PSC27 +
shRNA
C
PSC27-RAD +
shRNA
C
PSC27-RAD +
shRNA
WNT16B
#1
PSC27-RAD +
shRNA
WNT16B
#2
PSC27C
PSC27WNT16B
PC3
PC3+PSC27WNT16B
PC3+PSC27C
Tumor volume (mm3)
fP < 0.0001
650
550
450
350
250
100
50
0
+
+ +
+
+ +
+
––
–
––
–
–
–
Percentage invasion of cells
d
*
*
**
** **
***
* P < 0.05
** P < 0.01
*** P < 0.001
M12 PC3 Hela
PSC27
PSC27-RAD
PSC27-RAD + shRNAWNT16B#1
PSC27-RAD + shRNAC
PSC27-RAD + shRNAWNT16B#2
50
40
30
20
10
0
PC3
PSC27C
b
PSC27WNT16B
0 h 12 h 24 h
Figure 3 WNT16B is a major effector of the full DDSP and promotes the growth and invasion of prostate carcinoma. (a) Conditioned medium from
WNT16B-expressing prostate fibroblasts (PSC27WNT16B) promotes the proliferation of neoplastic prostate epithelial cells. shRNAC, control shRNA;
shRNAWNT16B#1 and shRNAWNT16B#2, WNT16B-specific shRNAs. (b) Scratch assay showing the enhanced motility of PC3 cells exposed to conditioned
medium from prostate fibroblasts expressing a control vector (PSC27C) or fibroblasts expressing WNT16B (PSC27WNT16B). Scale bars, 100 µm.
(c) The full fibroblast DDSP induced by radiation (PSC27-RAD) promotes the proliferation of tumorigenic prostate epithelial cells. The proliferative
effect is significantly attenuated by the suppression of damage-induced expression of WNT16B (PSC27-RAD+shRNAWNT16B). (d) The full paracrine-
acting fibroblast DDSP induced by radiation (PSC27-RAD) promotes the invasion of neoplastic epithelial cells. Invasion is significantly attenuated
by the suppression of damage-induced expression of WNT16B (PSC27-RAD+shRNAWNT16B). Data in a, c and d are mean ± s.e.m. of triplicates, with
P values calculated by ANOVA followed by t test. (e) Schematic of the xenograft cell recombination experiment to assess the ability of fibroblasts
expressing WNT16B to influence prostate tumorigenesis in vivo. PFC, prostate fibroblast cells; PC, prostate cancer cells. (f) Prostate fibroblasts
engineered to express WNT16B promote the growth of prostate carcinoma in vivo. Subrenal capsule grafts comprised of PC3 prostate epithelial cells
alone, PC3 cells in combination with PSC27C control fibroblasts or PC3 cells in combination with PSC27WNT16B fibroblasts are shown. The green
dashed lines denote the size of the tumor outgrowth from the kidney capsule. (g) Irradiated prostate fibroblasts (PSC27-RAD) promote the growth of
prostate carcinoma cells in vivo, and this effect is significantly attenuated by the suppression of fibroblast WNT16B using WNT16B-specific shRNAs
(PSC27-RAD+shRNAWNT16B) (P < 0.05). Shown are tumor volumes 8 weeks after renal capsule implantation of PC3 and PSC27 cell grafts. In f and g,
horizontal lines denote the mean of each group of eight tumors, and P values were determined by ANOVA followed by t test.
npg © 2012 Nature America, Inc. All rights reserved.

articles
nature medicine VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 1363
than PC3 cells combined with untreated PSC27 control fibroblasts
(P < 0.001) (Fig. 3g). Reducing the fibroblast contribution of
WNT16B attenuated the PSC27-RAD effects: grafts of PC3+PSC27-
RAD averaged 380 mm3, whereas PC3 cells combined with PSC27-
RAD+shRNAWNT16B averaged 280 mm3, a ~25% reduction in tumor
size when fibroblast WNT16B was suppressed (P < 0.02) (Fig. 3g).
Taken together, these findings show that paracrine WNT16B activity
can promote tumor growth in vivo and accounts for a substantial
component of the full DDSP effect on neoplastic epithelium.
WNT16B signals through b-catenin and induces an EMT
Having established that WNT16B can promote tumor growth through
paracrine signaling, we next sought to determine the mechanism(s)
by which it does so. PSC27WNT16B-conditioned medium activated
e
a
10 BPH1-TOPflash
BPH1-FOPflash
M12-TOPflash
M12-FOPflash
PC3-TOPflash
PC3-FOPflash
P = 0.01
P = 0.0006
P = 0.005
8
6
4
Relative luciferase activity
2
0
Epithelial
media
PSC27
C
CM
PSC27
WNT16B
CM
Constitutive
β-catenin
b
20 BPH1
M12
PC3
15
10
5
Fold change
0
Axin 2 SP5
β-catenin target proteins
c-Myc Cyclin D1
d
**
**
**
**
PSC27
WNT16B
+ XAV939
PSC27-RAD
PSC27-RAD
+ XAV939
100
90
PSC27
WNT16B
PSC27
C
* P < 0.05
** P < 0.01
M12 PC3
80
70
60
Epithelial cell number × 1,000
50
40
30
20
ih
PSC27
C
-Pre
PSC27
C
-BLEO
PSC27
IκBα
-Pre
PSC27
IκBα
-H
2
O
2
PSC27
IκBα
-BLEO
PSC27
IκBα
-RAD
PSC27
C
-RAD
PSC27
C
-H
2
O
2
P < 0.001
P = 0.003
P = 0.003
8
7
6
5
4
3
2
1
WNT16B ∆∆Ct to control
0
–1
–2
**
**
*
PSC27
C
DMEM
PSC27
WNT16B
* P < 0.05
** P < 0.01
Log
2
fold change
Vimentin
3
2
0
1
–1
BPH1
M12
PC3
****
*
PSC27
C
DMEM
PSC27
WNT16B
* P < 0.05
*** P < 0.001
Snail 2
Log
2
fold change
2.5
1.5
0.5
BPH1
M12
PC3
–0.5
**
**
*
PSC27
C
DMEM
PSC27
WNT16B
4
* P < 0.05
** P < 0.01
N-cad
Log
2
fold change
3
2
0
1
–1
BPH1
M12
PC3
**
***
PSC27
C
DMEM
PSC27
WNT16B
2* P < 0.05
** P < 0.01
E-cad
Log
2
fold change
1
0
–1
–2
BPH1
M12
PC3
NS
NS
NS
PSC27
C
DMEM
PSC27
WNT16B
NS,
P > 0.05
* P < 0.05
Twist 2
Log
2
fold change
2
1
0
BPH1
M12
PC3
*
***
PSC27
C
DMEM
PSC27
WNT16B
* P < 0.05
*** P < 0.001
Twist 1
Log
2
fold change
3
2
0
1
BPH1
M12
PC3
c
f g
WNT16B-p1
NF-κBInput No Ab
Pre
Pre
Pre
Mock
Rad
Rad
Rad
WNT16B-p2
WNT16B-p3
IL-6–p1
IL-8–p1
**
**
NS
NS
NS
NS
*
*
PSC27
IκBα
-RAD
100
75
50
25
0
BPH1 DU145 M12 PC3
PSC27
C
-RAD
PSC27
C
PSC27
IκBα
NS,
P > 0.05
* P < 0.05
** P < 0.01
Epithelial cell
number × 1,000
5
4
3
***
**
2
1
0
Low High Low High Low High Low High
Prostate carcinoma transcript
expression post/pre chemotherapy (log
2
)
Axin 2 SP5 c-Myc Cyclin D1
High
WNT16B expression in stroma
after chemotherapy: Low
PSC27
C
PSC27
C
PSC27
WNT16B
PSC27
shRNA-WNT16B
PSC27
shRNA-WNT16B
PSC27
WNT16B
M12
β-catenin
β-actin
Control
Control
E-cad
N-cad
Vimentin
PC3
Figure 4 Genotoxic stress upregulates WNT16B through NF-κB and signals through the canonical Wnt–β-catenin pathway to promote tumor cell proliferation
and the acquisition of mesenchymal characteristics. (a) Assay of canonical Wnt pathway signaling through activation of a TCF/LEF luciferase reporter construct
(TOPflash) or a control reporter (FOPflash). Epithelial cells were exposed to conditioned medium (CM) from PSC27 prostate fibroblasts expressing WNT16B
(PSC27WNT16B) or control vector (PSC27C). Data are mean ± s.e.m. of triplicates, and P values were determined by ANOVA followed by t test. (b) qRT-PCR
assessment of the expression of β-catenin target genes in prostate cancer cell lines (BPH1, M12 and PC3) before and 72 h after exposure to PSC27WNT16B-
conditioned medium. Data represent the mean ± s.e.m. fold change after as compared to before exposure for three replicates. (c) Expression of β-catenin
target genes in human prostate cancers in vivo after neoadjuvant treatment with MIT and DOC. Log2 transcript amounts in carcinoma cells after and before
chemotherapy are shown in relation to low (blue) or high (red) WNT16B expression in the prostate stroma. Each data point represents an individual patient;
n = 8 patients. Horizontal bars are group means. *P < 0.05, **P < 0.01 by ANOVA followed by t test. (d) The β-catenin pathway inhibitor XAV939 suppresses
the proliferation of prostate cancer cells in response to PSC27WNT16B CM and attenuates the response to the full DDSP in PSC27-RAD–conditioned medium.
Cell numbers were determined 72 h after treatment. (e) Quantification of transcripts in neoplastic prostate epithelial cells encoding proteins associated with
a phenotype of EMT. Measurements are from epithelial cells exposed to control media (DMEM), media conditioned by PSC27 fibroblasts (PSC27C) or PSC27
fibroblasts expressing WNT16B (PSC27WNT16B). E-cad, E-cadherin; N-cad, N-cadherin. (f) Analysis of EMT-associated protein expression by western blot. M12 or
PC3 cells were exposed to control media, media conditioned by PSC27 fibroblasts (PSC27C), PSC27 fibroblasts expressing WNT16B (PSC27WNT16B) or PSC27
fibroblasts expressing WNT16B and shRNA targeting WNT16B (PSC27shRNA-WNT16B). (g) Chromatin immunoprecipitation assays identified NF-κB binding sites
within the proximal promoter of the WNT16 gene. PCR reaction products from Mock (no DNA loading), NF-κB immunoprecipitation, input control DNA and no
antibody (Ab) control before treatment (Pre) and after irradiation (Rad). p1, p2 and p3 indicate primer pairs corresponding to putative NF-κB binding regions in
WNT16, IL-6 and IL-8, respectively (see the Supplementary Methods for the primer sequences). (h) Analysis of WNT16B transcript expression by qRT-PCR in
PSC27 prostate fibroblasts with (PSC27IκBα) or without (PSC27C) inhibition of NF-κB signaling before and after DNA-damaging exposures. (i) Inhibition of
NF-κB signaling in fibroblasts responding to DNA damage attenuates the effect of the DDSP on tumor cell proliferation. Cell numbers were determined 72 h
after RAD exposure to conditioned medium from fibroblasts with (PSC27IκBα) or without (PSC27C) inhibition of NF-κB signaling. Data in d, e, h and i are
mean ± s.e.m. of triplicates, and P values were determined by ANOVA followed by t test.
npg © 2012 Nature America, Inc. All rights reserved.

articles
1364 VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 nature medicine
canonical Wnt signaling in BPH1, PC3 and M12 prostate cancer
cells, as measured by assays of β-catenin–mediated transcription
through T cell factor/lymphoid enhancer binding factor (TCF/LEF)
binding sites (Fig. 4a). Known β-catenin target genes, including
AXIN2 and MYC, were upregulated (approximately fivefold and
over tenfold, respectively) after exposure to WNT16B-enriched
conditioned medium (Fig. 4b). In human prostate cancers treated
with chemotherapy, β-catenin localized in the nucleus of tumor
cells (Supplementary Fig. 4a). We also found that β-catenin tar-
get genes were expressed more highly in tumors with elevated
stromal WNT16B expression relative to those with low WNT16B
expression (P < 0.05) (Fig. 4c). To confirm that β-catenin signaling
contributed to the epithelial phenotypes resulting from exposure
to PSC27-RAD–conditioned medium, we treated prostate cancer
cells with the tankyrase inhibitor XAV939, which stabilizes axin
and inhibits β-catenin–mediated transcription24. XAV939 com-
pletely suppressed the proliferative and invasive responses induced
by WNT16B and markedly attenuated the effects of the PSC27-RAD
DDSP (Fig. 4d and Supplementary Fig. 4b).
Wnt signaling is known to promote the acquisition of mesenchymal
cell characteristics that can influence the migratory and invasive behav-
ior of epithelial cells through an EMT25–27. Loss of CDH1 (also known
as E-cadherin), the prototypic epithelial adhesion molecule in adher-
ens junctions, and gain of CDH2 (also known as N-cadherin) expres-
sion are among the main hallmarks of an EMT28,29. After exposure
of PC3 cells to PSC27WNT16B-conditioned medium, the number
of E-cadherin transcripts decreased 64%, whereas the number of
N-cadherin transcripts increased fourfold (P < 0.05). Similar altera-
tions occurred in M12 and BPH1 cells (Fig. 4e,f). Inhibiting β-catenin
pathway signaling with XAV939 in epithelial cells blocked the
WNT16B-induced EMT-associated gene expression (Supplementary
Fig. 4c). Exposure to PSC27WNT16B-conditioned medium also pro-
moted mesenchymal characteristics in MDA-MD-231 breast cancer
and SKOV3 ovarian cancer cells (Supplementary Fig. 4d).
Genotoxic stress induces WNT16B expression through NF-kB
A key pathway linking DNA damage with apoptosis, senescence and
DNA repair mechanisms involves activating the NF-κB complex30,31.
NF-κB is also pivotal in mediating the stress-associated induction of
inflammatory networks, including the upregulation and secretion
of interkeukin-6 (IL-6) and IL-8 (refs. 23,32). We therefore sought
to determine whether DNA-damage–induced WNT16B expres-
sion is mediated by NF-κB. We identified NF-κB binding motifs
in the WNT16B promoter region and confirmed their function
using WNT16B promoter constructs. Compared to untreated cells,
both RAD and tumor necrosis factor α (TNF-α), which are known
NF-κB activators, induced WNT16B reporter activity (P < 0.01) (Fig. 4g
and Supplementary Fig. 5a–d). We next generated PSC27 prostate
fibroblasts with stable expression of a mutant nuclear factor of κ light
polypeptide gene enhancer in B cells inhibitor, α (IκBα) (PSC27IκBα),
1.0
a db
c e
1,000
100
Average tumor volume
(mm3)
10
1
PC3 +
–––
– ––
––––
–
+
+
+
+ +
+
+
+ + +
+
+
Chemotherapy
PSC27C
PSC27WNT16B
PC3+PSC27CPC3+PSC27WNT16B
DMEM
PSC27C
PSC27WNT16B
MIT at IC50
P = 0.02
P = 0.02
P = 0.03
P = 0.04
P = 0.04 P = 0.0004
P < 0.0001
P < 0.0001 P < 0.0001
P < 0.0001
0.9
0.8
0.7
Epithelial viability
normalized to untreated
0.6
0.5
0.4
500 75
50
25
Tumor cell staining (%)
0
C MIT C MIT C MIT
PC3 PC3+
PSC27CPC3+
PSC27WNT16B
Control
MIT (IC50)
** P < 0.01
*** P < 0.001
NS, P > 0.05
* P < 0.05
** P < 0.01
NS, P > 0.05
*** *** *** ***
**
*
NS γ-H2AX
C-caspase 3
**
400
NS
300
200
100
0
PSC27C
PSC27WNT16B
PSC27WNT16B +
shRNAWNT16B#1
PSC27WNT16B +
shRNAWNT16B#2
PSC27WNT16B +
XAV939
LNCaP
Vehicle control
PSC27C CMPSC27WNT16B CM
Apoptosis (RLU, × 1,000)
MIT (IC50)
Placebo-
treated
MIT-
treated
DU145 M12 PC3 BPH1
Figure 5 Paracrine-acting WNT16B promotes the resistance of prostate carcinoma to cytotoxic chemotherapy. (a) Viability of prostate cancer cells 3 d
after treatment with a half-maximal inhibitory concentration (IC50) of MIT and medium conditioned by fibroblasts with (PSC27WNT16B) or without
(PSC27C) WNT16B. (b) Bright field microscopic view of PC3 cells cultured with control or PSC27WNT16B-conditioned medium photographed 24 h after
exposure to vehicle or the IC50 of MIT. Arrowheads denote apoptotic cell bodies. Scale bars, 50 µm. (c) Acute tumor cell responses to chemotherapy
in vitro. Quantification of apoptosis by assays reflecting combined caspase 3 and 7 activity measured 24 h after the exposure of PC3 cells to vehicle or
the IC50 of MIT. Data in a and c are mean ± s.e.m. of triplicate experiments, and P values were determined by ANOVA followed by t test. RLU, relative
luciferase unit. (d) In vivo responses of PC3 tumors to MIT chemotherapy. Grafts were comprised of PC3 cells alone or PC3 cells combined with either
PSC27 prostate fibroblasts expressing a control vector (PC3+PSC27C) or PSC27 prostate fibroblasts expressing WNT16B (PC3+PSC27WNT16B). MIT
was administered every 2 weeks for three cycles, and grafts were harvested and tumor volumes determined 1 week after the final MIT treatment. Each
data point represents an individual xenograft. Horizontal lines are group means of ten tumors, with P values determined by ANOVA followed by t test.
(e) Acute tumor cell responses to chemotherapy in vivo. Quantification of apoptosis by cleaved caspase 3 (C-caspase 3) IHC and of DNA damage by
γ-H2AX immunofluorescence in PC3 and fibroblast xenografts measured 24 h after in vivo treatment with vehicle (C) or MIT. Values represent a
minimum of 100 cells counted from each of 3–5 tumors per group. Data are mean ± s.e.m., and P values were determined by ANOVA followed by t test.
npg © 2012 Nature America, Inc. All rights reserved.

articles
nature medicine VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 1365
which prevents IκB kinase (IKK)-dependent degradation of IκBα
and thus attenuates NF-κB signaling. After irradiation of PSC27 cells,
NF-κB translocated to the nucleus and induced NF-κB reporter activ-
ity >100-fold (Supplementary Fig. 5d,e). In comparison, the amount
of nuclear NF-κB in PSC27IκBα-RAD cells was markedly lower.
The PSC27IκBα cells with impaired NF-κB activation had a significant
attenuation of induction of WNT16B expression after treatment with
H2O2, BLEO or RAD (P < 0.05) (Fig. 4h).
Epithelial viability
normalized to untreated
LNCaP DU145 M12 PC3 BPH1
MIT at IC50
NS, P > 0.05
* P < 0.05
** P < 0.01
1.0 NS
* * * ** *
NS NS NS NS
0.9
PSC27C
PSC27C-RAD
PSC27C-MIT
PSC27IκBα
PSC27IkBa-RAD
PSC27IkBa-MIT
0.8
0.7
0.6
0.5
DMEM
100
PSC27-RAD
PSC27-RAD + shRNAC
PSC27-RAD + shRNAWNT16B#1
PSC27-RAD + shRNAWNT16B#2
PSC27-RAD + XAV939
80
60
40
Percentage viability
20
–8 –7
Concentration of MIT (log M)
–6 –5
0
a
PSC27-RAD
PSC27-RAD + shRNAC
PSC27-RAD + shRNAWNT16B#1
PSC27-RAD + shRNAWNT16B#2
1.0
PSC27-RAD + XAV939
LNCaP
Epithelial viability
normalized to untreated
DU145 M12 PC3 BPH1
MIT at 2× IC50
* P < 0.05
** P < 0.01
*** P < 0.001
0.9
** ** **
*
***
***
0.8
0.7
0.6
0.5
0.4
b c
PSC27-RAD
PSC27
C
PSC27-RAD +
shRNA
WNT16B
#1
PSC27-RAD +
shRNA
WNT16B
#2
PSC27-RAD +
XAV939
* P < 0.05
** P < 0.01
*** P < 0.001
NS, P > 0.05
500
***
*****
** **
*
*
*
400
Control
MIT (IC50)
NS
300
200
Apoptosis (RLU, × 1,000)
100
0
d
PC3
tumor volume (mm
3
)
****
***
***
PSC27C + shRNAWNT16B#2
PSC27C + shRNAWNT16B#1
PSC27C + shRNAC
PC3
Chemotherapy
+++
+
+
+
+
+
+
+
+
40%
30
–
–
–
–
–
–
–
–
–
20
10
0
f
Genotoxic therapy
cycle 1
Genotoxic therapy
cycle 2
CEC
BEC
FC
+DDR +DDR
+DDR+DDR
+DDR
+DDR
+DDR
+DDR
-DDR
DDSP
DDSP
DDSP
DDR activated
Apoptotic cell
DDSP
DDSP
(WNT16B)
(WNT16B)
Senescence
reinforcement
-DDR
+EMT
+EMT
+Repopulation
Therapy
resistance
+DDR+DDR
-DDR
-DDR
g
PSC27-RAD + shRNAWNT16B#2
PSC27-RAD + shRNAWNT16B#1
PC3
tumor volume (mm
3
)
400
300
200
80
60
40
***
***
**
**
20
Chemotherapy
PSC27-RAD
PSC27-RAD + shRNAC
0
+ +
+
+
+
+
+ +
+
–
– –
––
–
– – –
–
–
–
–
– –
–– – –
–– – – –
–
–
e
Figure 6 Chemotherapy resistance promoted by
damaged fibroblasts is attenuated by blocking
WNT16B, β-catenin or NF-κB signaling. (a) Viability
of prostate cancer cells across a range of MIT
concentrations with (PSC27-RAD+shRNAWNT16B)
or without (PSC27-RAD+shRNAC) the suppression
of WNT16B in irradiated-fibroblast–conditioned
medium or with the addition of the β-catenin
pathway inhibitor XAV939. Data are mean ± s.e.m.
of triplicates. (b) Viability of prostate cancer
cells 3 d after treatment with two times the IC50 of
MIT in the context of conditioned medium from
irradiated prostate fibroblasts (PSC27-RAD)
expressing shRNAs targeting and suppressing
WNT16B (shRNAWNT16B), a vector control (shRNAC)
or combined with the β-catenin pathway inhibitor XAV939. (c) Viability of prostate cancer cells 3 d after treatment with the IC50 of MIT in the context of
conditioned medium from prostate fibroblasts pretreated with radiation (PSC27-RAD) or MIT (PSC27-MIT) and with (PSC27IκBα) or without (PSC27C)
the suppression of NF-κB signaling. (d) Acute tumor cell responses to chemotherapy in vitro. Quantification of apoptosis by caspase 3 and 7 activity
measured 24 h after the exposure of PC3 cells to vehicle or the IC50 of MIT. Data for b, c and d are mean ± s.e.m. of triplicates, and P values were
determined by ANOVA followed by t test. (e,f) In vivo effects of MIT chemotherapy in the context of suppressing the induction of the expression of
fibroblast WNT16B. Tumors comprised PC3 cells in combination with irradiated (PSC27-RAD) fibroblasts (e) or unirradiated (PSC27C) (f) prostate
fibroblasts expressing shRNAs targeting WNT16B (shRNAWNT16B) or a vector control (shRNAC). MIT was administered every 2 weeks for three cycles,
and grafts were harvested and tumor volumes determined 1 week after the final treatment. Each data point represents an individual xenograft. Tumor
volumes of PSC27C+shRNAC grafts in f averaged 20 mm3, and tumor volumes of PSC27C+shRNAWNT16B grafts averaged 12 mm3 (P < 0.001).
Horizontal lines are group means, with n = 10 in e and n = 8 in f. P values were determined by ANOVA followed by t test. The bracket boundaries in
f are the group means for PSC27C+shRNAC grafts compared to PSC27C+shRNAWNT16B grafts showing a 40% difference in size. Asterisks, as for the
previous panel. (g) Model for cell nonautonomous therapy-resistance effects originating in the tumor microenvironment in response to genotoxic cancer
therapeutics. The initial round of therapy engages an apoptotic or senescence response in subsets of tumor cells and activates a DNA damage response
(DDR) in DDR-competent benign cells (+DDR) comprising the tumor microenvironment. The DDR includes a spectrum of autocrine- and paracrine-
acting proteins that are capable of reinforcing a senescent phenotype in benign cells and promoting tumor repopulation through progrowth signaling
pathways in neoplastic cells. Paracrine-acting secretory components such as WNT16B also promote resistance to subsequent cycles of cytotoxic
therapy. CEC, cancer epithelial cell; BEC, benign epithelial cell; FC, fibroblast cell; –DDR, DDR-incompetent benign cells.
npg © 2012 Nature America, Inc. All rights reserved.

articles
1366 VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 nature medicine
We next determined whether suppressing fibroblast NF-κB signaling
in response to DNA damage would attenuate the pro-proliferative effects
of the PSC27-RAD DDSP. Whereas PSC27-RAD–conditioned medium
promoted prostate epithelial cell proliferation, conditioned medium
from PSC27IκBα-RAD cells failed to do so (Fig. 4i). These experiments
identify WNT16B as a new member of the cellular genomic program
that is regulated by NF-κB signaling in response to DNA damage.
Paracrine WNT16B attenuates the effect of cytotoxic therapy
The preceding experiments suggested that in addition to tumor-
promoting effects, paracrine-acting WNT16B may influence the
responses of tumors to genotoxic cancer therapeutics. To evaluate
this possibility, we studied MIT, a type 2 topoisomerase inhibi-
tor that produces DNA strand breaks, leading to growth arrest,
senescence or apoptosis, which is in clinical use for the treat-
ment of advanced prostate cancer. Prostate cancer cells exposed to
PSC27WNT16B-conditioned medium compared to control medium
consistently showed significant attenuation of chemotherapy-
induced cytotoxicity across a range of MIT concentrations after 3 d
(P < 0.05) (Fig. 5a and Supplementary Fig. 6a). Short-term cell
viability assays confirmed that, compared to controls, PSC27WNT16B-
conditioned medium improved cancer cell survival after acute 12-h
exposures to MIT (P < 0.01) (Supplementary Fig. 6b). Apoptotic
responses measured after 24 h of MIT exposure were substantially
attenuated by PSC27WNT16B-conditioned medium (P < 0.01), an
effect that was blocked by treatment with XAV939 (Fig. 5b,c). To
determine whether these obser vations were of relevance to tumor
therapy in vivo, we treated mice with tumor grafts comprised of PC3
cells plus PSC27WNT16B or PSC27C fibroblasts with three cycles of
MIT given every other week. MIT treatment significantly reduced
the tumor volumes (P < 0.001). However, grafts of tumor cells with
PSC27WNT16B fibroblasts attenuated the tumor inhibitory effects of
MIT compared to tumor cells grafted with control PSC27 fibroblasts:
PC3+PSC27C and PC3+PSC27WNT16B tumors averaged 13 mm3
and 78 mm3, respectively (P < 0.001) (Fig. 5d). Experiments using
MDA-MB-231 breast cancer cells plus breast fibroblasts produced
similar results (Supplementary Fig. 6c). To evaluate the influence of
WNT16B on the acute effects of chemotherapy, we examined cohorts
of PC3+PSC27C and PC3+PSC27WNT16B xenografts 24 h after MIT
treatment to quantify DNA damage using γ-H2AX immunofluores-
cence and apoptosis using cleaved caspase 3 immunohistochemistry
(IHC). Compared to PC3+PSC27C grafts, there was no difference in
the number of DNA damage foci in PC3+PSC27WNT16B tumors, but
significantly fewer apoptotic cells were present (34% compared to
14%, respectively; P < 0.05) (Fig. 5e).
The conditioned medium from PSC27-RAD cells, representing the
full fibroblast DDSP, significantly increased the viability of PC3 can-
cer cells exposed to MIT concentrations ranging between 0.1–1 µM
in vitro (P < 0.01) (Fig. 6a). In comparison to PSC27-RAD–
conditioned medium, PSC27-RAD+shRNAWNT16B or PSC27IκBαRAD
fibroblasts, engineered to suppress WNT16B expression or
NF-κB activation, respectively, substantially augmented the effects of
MIT, further increasing apoptosis and reducing tumor cell viability
by 30–40%. Blocking β-catenin signaling in carcinoma cells with
XAV939 also attenuated the effects of PSC27-RAD–conditioned
medium on promoting tumor cell survival (Fig. 6b–d and
Supplementary Fig. 7a,b). This effect of WNT16B was also evident
in vivo. PC3+PSC27-RAD tumor grafts averaged 300 mm3 in size
compared to 25 mm3 for grafts of PC3 cells alone (P < 0.001). MIT
chemotherapy suppressed the growth of the PC3+PSC27-RAD grafts,
though residual tumors were still readily detectable and averaged
55 mm3 in size (Fig. 6e). However, after MIT treatment, residual
tumors of PC3 cells with PSC27-RAD + shRNAWNT16B fibroblasts,
with attenuated WNT16B induction, were on average ~33% smaller
than PC3+PSC27-RAD tumors (P < 0.001) (Fig. 6e). Experiments
with MDA-MD-231 cells and breast fibroblasts produced similar
results (Supplementary Fig. 7c). To more accurately mimic the
clinical situation of cancer therapy, we also grafted tumor cells with
unirradiated PSC27 fibroblasts (PSC27C) and followed the same treat-
ment schema of three MIT cycles. Tumors from mice treated with
MIT were substantially smaller than tumors from untreated mice
(P < 0.001). Attenuating the induction of WNT16B further enhanced
the effects of chemotherapy: after MIT treatment, grafts of PC3 cells
and PSC27C + shRNAWNT16B were on average 40% smaller than grafts
of PC3 cells combined with PSC27C cells without shRNAWNT16B
(P < 0.001) (Fig. 6f and Supplementary Fig. 7d).
DISCUSSION
Optimizing radiotherapy and chemotherapy for the treatment of
malignant neoplasms has relied on the iterative development and
testing of models involving tumor growth dynamics, mutation rates
and cell-kill kinetics. However, the most theoretically effective tumor-
icidal strategies must usually be tempered because of detrimental
effects to the host. This reality has led to the development of regimens
in which therapies are administered at intervals or cycles to avoid
irreparable damage to vital host functions. However, the recovery and
repopulation of tumor cells between treatment cycles is a major cause
of treatment failure15,16. Interestingly, rates of tumor cell repopula-
tion have been shown to accelerate in the intervals between succes-
sive courses of treatment, and solid tumors commonly show initial
responses followed by rapid regrowth and subsequent resistance to
further chemotherapy. Our results indicate that damage responses in
benign cells comprising the tumor microenvironment may directly
contribute to enhanced tumor growth kinetics (Fig. 6g).
The autocrine- and paracrine-acting influences of genotoxic stress
responses can exert complex and potentially conflicting cell non-
autonomous effects33,34. Overall, our findings are in agreement with
studies of DNA damage in which the execution of a signaling program
culminating in a senescence phenotype is accompanied by elevated
concentrations of specific extracellular proteins termed a ‘senescence
messaging secretome’ or a ‘senescence-associated secretory pheno-
type’33,34. DNA damage responses and senescence programs can clearly
operate in a cell autonomous ‘intrinsic’ manner to arrest cell growth
and inhibit tumor progression, as has been observed in premalignant
nevi35. Secreted factors such as insulin-like growth factor binding
protein 7 (IGFBP7) and the chemokine (C-X-C motif ) receptor 2
(CXCR2) ligands IL-6 and IL-8 participate in a positive feedback loop
to fortify the senescence growth arrest induced by oncogenic stress and
also promote immune responses that clear senescent cells and enhance
tumor regression23,32,36,37. However, in addition to proinflammatory
cytokines, the damage response program comprises proteases and
mitogenic growth factors, such as MMPs, hepatocyte growth factor
(HGF), vascular endothelial growth factor (VEGF) and epidermal
growth factor receptor (EGFR) ligands that have clear roles in pro-
moting tumor growth, inhibiting cellular differentiation, enhancing
angiogenesis and influencing treatment resistance19,20,38. This con-
cept is supported by reports of tissue-specific chemoresistant survival
niches involving hematopoietic neoplasms, such as lymphomas39. The
situation also has parallels with studies of radiation and chemotherapy
paradoxically promoting tumor dissemination40.
npg © 2012 Nature America, Inc. All rights reserved.

articles
nature medicine VOLUME 18 | NUMBER 9 | SEPTEMBER 2012 1367
Collectively, these studies support several conclusions: first, the out-
comes of genotoxic exposures to any specific benign or neoplastic cell
depend on the integration of innate damage response capabilities and
the context that is dictated by the composition of the tumor microenvi-
ronment; second, although intrinsic drug resistance is clearly operative
in some cancers, acquired resistance can also occur without alterations
in intrinsic cellular chemosensitivity41, and our results provide strong
support for previous studies that implicate constituents of the tumor
microenvironment as important contributors to this resistance42–44;
and third, specific microenvironment DDSP proteins that promote
therapy resistance such as WNT16B are attractive targets for augment-
ing responses to more general genotoxic therapeutics. However, the
complexity of the damage response program also supports strategies
that are focused on inhibiting upstream master regulators, such as
NF-κB45, that may be more efficient and effective adjuncts to cytotoxic
therapies, provided their side effects are tolerable.
METHODS
Methods and any associated references are available in the online
version of the paper.
Accession codes. Microarray data are deposited in the Gene
Expression Omnibus database with accession code GSE26143.
Note: Supplementary information is available in the online version of the paper.
ACKNOWLEDGMENTS
We thank J. Dean and D. Bianchi-Frias for helpful comments, A. Moreno
for administrative assistance and N. Clegg for bioinformatics support.
S. Hayward, Vanderbilt University, and J. Ware, Medical College of Virginia,
provided BPH1 and M12 cells, respectively. Primary human prostate (PSC27),
ovarian (OVF28901) and breast (HBF1203) fibroblasts were provided by
B. Knudsen, Cedars Sinai Medical Center, E. Swisher, University of Washington,
and P. Porter through the Seattle Breast SPORE (P50 CA138293), Fred
Hutchinson Cancer Research Center, respectively. B. Torok-Strorb, Fred
Hutchinson Cancer Research Center, provided HS5 and HS27A HPV E6/E7
immortalized human bone marrow stromal cells. We thank the clinicians
who participated in the trials of neoadjuvant chemotherapy : M. Garzotto,
T. Takayama, P. Lange, W. Ellis, S. Lieberman and B.A. Lowe. We are also grateful
for the participation of the patients and their families in these studies. Breast
cancer specimens were obtained from the Fred Hutchinson Cancer Research
Center/University of Washington Medical Center Breast Specimen Repository.
We thank N. Urban, Fred Hutchinson Cancer Research Center, for providing
ovarian cancer biospecimens funded through the POCRC SPORE grant
P50CA83636. This work was supported by a fellowship from the Department
of Defense (PC073217), R01CA119125, the National Cancer Institute Tumor
Microenvironment Network U54126540, the Pacific Northwest Prostate Cancer
SPORE P50CA097186 and the Prostate Cancer Foundation.
AUTHOR CONTRIBUTIONS
Y.S. designed and conducted experiments, and wrote the manuscript. J.C. provided
reagents and technical advice. C.H., T.M.B. and P.P. provided clinical materials for
the assessments of treatment responses. I.C. analyzed data. L.T. analyzed tissue
histology and immunohistochemical assays. P.S.N. designed experiments, analyzed
data and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/nm.2890.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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nature medicine
doi:10.1038/nm.2890
ONLINE METHODS
Cell cultures and treatments. We obtained epithelial cell lines from the
American Type Culture Collection and cultured them according to the recom-
mended protocols. Fibroblasts were grown until they were 80% confluent and
were then treated with 0.6 mM hydrogen peroxide (PSC27-H2O2), 10 µg ml−1
bleomycin (PSC27-BLEO), 1 µM mitoxantrone (PSC27-MIT) or ionizing radia-
tion by a 137Cesium source at 743 rad min−1 (PSC27-RAD). Additional details of
the cell culture methods are provided in the Supplementary Methods.
Gene expression analysis. We extracted total RNA from PSC27 cells using the
RNeasy kit (QIAGEN), converted mRNAs to complementary DNAs (cDNAs) and
amplified the cDNAs for one round using the MessageAmp aRNA Kit (Ambion),
followed by aminoallyl-UTP incorporation into a second-round of amplification
of the RNA. Samples were labeled with fluorescence dyes and hybridized to 44K
Whole Human Genome Expression Microarray slides in accordance with the
manufacturer’s instructions (Agilent Technologies). Additional assays of tran-
script abundance were performed by qRT-PCR (Supplementary Methods).
Immunohistochemistry. We used a mouse monoclonal antibody to WNT16B
(product number 552595, clone F4-1582, BD Pharmingen) at a dilution of
1:16,000 to immunolocalize WNT16B protein using an indirect three-step
avidin-biotin-peroxidase method according to the manufacturer’s instructions
(VECTASTAIN Elite ABC Kit, Vector Labs). The expression of WNT16B by
epithelium or fibromuscular stromal cells in each tissue section was recorded on
a 4-point scale as follows: 3 for intensely expressed, 2 for moderately expressed,
1 for faintly or equivocally expressed and 0 for no expression of WNT16B by any
stromal cells. Additional details are provided in the Supplementary Methods.
Characterization of cell phenotypes. We assessed cell proliferation using the
CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS), with signals
being captured using a 96-well plate reader. Serum-starved cells for transwell
migration and invasion assays were added to the top chambers of Cultrex 24-
well Cell Migration Assay plates (8 µm pore size) coated with or without base-
ment membrane extract prepared as 0.5× of stock solution. After 12 h or 24 h,
migrating or invading cells in the bottom chambers were stained, and the plate
absorbance was recorded. Chemoresistance assays were performed using epi-
thelial cells cultured with either DMEM and low serum (0.5% FCS) (denoted
here as ‘DMEM’) or conditioned medium generated from PSC27 cells expressing
vector controls, WNT16B or shRNAs. Cells received mitoxantrone treatment
for 12 h, 24 h or 72 h at concentrations near the IC50 of each individual cell line.
The percentage of viable cells was calculated by comparing the results of each
experiment to the results from vehicle-treated cells. Each assay was repeated a
minimum of three times, with results reported as means ± s.e.m.
In vivo studies. The Institutional Animal Care and Use Committee (IACUC)
of Fred Hutchinson Cancer Research Center reviewed and approved the animal
protocols and procedures, with surgeries carried out per the US National
Institutes of Health Guide for laboratory animals. To prepare tissue recom-
binants, 250,000 fibroblasts (PSC27 series) and epithelial cells were mixed
at a 1:1 ratio in collagen gels. ICR–severe combined immunodeficient (SCID)
male mice, obtained from Taconic, Inc, were anesthetized with isoflurane,
and an oblique incision (<1 cm) was made on the kidney capsule surface par-
allel and adjacent to the long axis of each kidney. Cells were injected under
the capsule with a blunt 25-gauge needle and a glass Hamilton syringe. The
kidney was returned to the retroperitoneal space, and the skin was closed with
surgical staples. The growth of the xenografts was assessed at weekly intervals,
and the mice were killed at 8 weeks after transplantation. Each xenograft arm
comprised 5–8 mice per xenograft type, either of individual cells or combina-
tions of fibroblasts and epithelial cells. Additional details are provided in the
Supplementary Methods.
For the chemotherapy studies, mice received cell grafts as described above and
were followed for 2 weeks to allow tumor take. Starting from the third week after
grafting, mice received mitoxantrone at a dose of 0.2 mg per kg intraperitoneally
on day 1 of week 3, week 5 and week 7 (ref. 46). In total, three 2-week cycles
were given, after which the mice were killed and their kidneys were removed
for tumor measurements and histological analysis. Each experimental arm com-
prised 5–8 mice per treatment cohort. Additional details are provided in the
Supplementary Methods.
Statistical analyses. All in vitro experiments were repeated at least three
times, and data are reported as means ± s.e.m. Differences among groups
and treatments were determined by ANOVA followed by t tests. P ≤ 0.05 was
considered significant.
Additional methods. Detailed methodology is described in the Supplementary
Methods.
46. Alderton, P.M., Gross, J. & Green, M.D. Comparative study of doxorubicin,
mitoxantrone, and epirubicin in combination with ICRF-187 (ADR-529) in a chronic
cardiotoxicity animal model. Cancer Res. 52, 194–201 (1992).
npg © 2012 Nature America, Inc. All rights reserved.