Prostate-specific IL-6 transgene autonomously induce prostate neoplasm through amplifying inflammation in the prostate and peri-prostatic adipose tissue
Liu et al. Journal of Hematology & Oncology
Prostate-specific IL-6 transgene autonomously induce prostate neoplasm through amplifying inflammation in the prostate and peri-prostatic adipose tissue
Gang Liu 1
Jinyu Zhang 0
Lewis Frey 2
Xiao Gang 0 4
Kongming Wu 6
Qian Liu 6
Michael Lilly 5
r Wu 0 1 3
0 Department of Microbiology and Immunology, Medical University of South Carolina , Charleston, SC 29425 , USA
1 Department of Medicine, University of Washington , Seattle, WA , USA
2 Public Health Science, Medical University of South Carolina , Charleston, SC 29425 , USA
3 Hollings Cancer Center, Medical University of South Carolina , Charleston, SC 29425 , USA
4 Present address: Department of Laboratory Medicine, The Third Hospital of South Medical University , Guangzhou , China
5 Department of Hematology and Oncology, Medical University of South Carolina , Charleston, SC 29425 , USA
6 Department of Oncology, Tongji Medical College, Huazhong University of Science and Technology and Tongji Hospital , Wuhan , China
Background: The causative role of the pro-inflammatory cytokine IL-6 in prostate cancer progression has been well established at molecular level. However, whether and how IL-6 may play a role in prostate cancer risk and development is not well defined. One limitation factor to acquiring this knowledge is the lack of appropriate animal models. Methods: We generated a novel line of prostate-specific IL-6 transgenic mouse model. We compared the prostate pathology, tumorigenic signaling components, and prostate tumor microenvironment of the IL-6 transgenic mice with wild type littermates. Results: With this model, we demonstrate that IL-6 induces prostate neoplasm autonomously. We further demonstrate that transgenic expression of IL-6 in the prostate activates oncogenic pathways, induces autocrine IL-6 secretion and steadily-state of STAT3 activation in the prostate tissue, upregulates paracrine insulin-like growth factor (IGF) signaling axis, reprograms prostate oncogenic gene expression, and more intriguingly, amplifies inflammation in the prostate and peri-prostatic adipose tissue. Conclusions: The pro-inflammatory IL-6 is autonomous oncogene for the prostate. IL-6 induces prostate oncogenesis through amplifying local inflammation. We also presented a valuable animal model to study inflammation and prostate cancer development.
IL-6; Prostate neoplasm; Transgenic mouse; Inflammation
Emerging evidence indicated that the pro-inflammatory
cytokine IL-6 may play a causative role in prostate cancer
progression . For instance, IL-6 has been shown to
facilitate prostate cancer progression to androgen-independent
disease and potentially to promote bone metastasis and
neuroendocrine differentiation (NED) [2–7]. Elevation of
serum levels of IL-6 or activation of IL-6 signaling pathways
in the tumor tissue correlates with the shortened overall
survival and time to progression in prostate cancer [8–13].
In vitro and xenograft in vivo studies have demonstrated
that IL-6 plays a causative role to promote
oncogeneimmortalized non-tumorigenic prostate epithelial cells to
overt malignancy . Conclusions from recent clinical
studies propose that serum IL-6 can be a negative
prognostic biomarker for prostate cancer .
IL-6, a multi-functional cytokine that can be produced by
various cell types, including immune/inflammatory cells
(monocytes, macrophages, B cells, T cells, nature killer cells),
fibroblasts, keratinocytes, endothelial cells, and also tumor
cells, plays a pivotal role in controlling cell differentiation and
cancer cell survival [15, 16]. IL-6 signals through the adaptor
molecule gp130 via canonical membrane bound IL-6R and/or
alternatively soluble IL-6R trans-signaling in IL-6R-gp130+
cells to initiate the downstream activation cascade [17, 18]. It
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
has been well established that activation of the signal
transduction and activator of transcription 3 (STAT3), a key
effector protein of IL-6 signaling, is critical in initiating
oncogene transcription and cancer progression . IL-6 can
promote tumorigenic conversion of oncogene-immortalized
benign cells through STAT3-mediated trans-activating other
cellular signaling pathways, such as MAPK, PI3K/AKT, and
insulin-like growth factor I receptor (IGF-IR) signaling axis
[14, 20]. Persistent activation of STAT3 in prostate
carcinomas has been correlated with the shortened
survival of cancer patients [8–12]. Upregulation of IL-6
and activation of STAT3 autocrine pathway have been
shown to account for the major mechanisms of
cancer cell resistance to therapy [19, 21, 22]. Based on
these understandings, recent studies have been
focused on targeting IL-6 or IL-6 signaling pathways in
cancer patients [19, 23–25].
Although the significance of IL-6 in prostate cancer
progression is well established, whether IL-6 plays a causative
role in prostate cancer risk and early development is not
clearly well defined. Polymorphisms of IL-6 gene have
been associated with prostate cancer risk [26–28];
however, bona fida evidence of IL-6 as a sole factor in prostate
cancer risk is lacking. Most of pro-tumorigenic evidence
of IL-6 in prostate cancer was achieved from experiments
of oncogene-immortalized cell lines or clinical correlation
studies. To address the fundamental biological question
whether IL-6, as the major pro-inflammatory cytokine,
can initiate prostate tumorigenesis in an autologous state,
here, we developed a prostate-specific IL-6 transgenic
mouse line in which IL-6 was directed to express
specifically in the prostate by the rat probasin promoter. With
this model, we demonstrated that overexpression of IL-6
alone was sufficient to induce prostate epithelium
malignant neoplasm. We demonstrated that enforced
expression of IL-6 in the prostate activated STAT3 pathway in
the epithelium and stroma, induced an IL-6 autocrine and
insulin-like growth factor (IGF) paracrine loop,
reprogrammed prostate oncogenic gene expression, and
amplified pro-tumorigenic inflammation in the prostate
tissue microenvironment and peri-prostatic adipose
tissue. Our study suggests that IL-6 is an
unconventional “oncogene” for the prostate. Moreover, our
prostate-specific IL-6 transgenic mouse can serve as a
valuable model to study inflammation-associated
prostate cancer prevention.
IL-6 transgene induces early prostate neoplastic
Previously, we described that IL-6 facilitated prostate
tumorigenesis and progression through autocrine IL-6 loop
and re-programming oncogenic transcriptional profiles
using oncogene-immortalized benign prostate epithelial cell
lines . Inspired by these findings, in this study, we
aimed to address whether IL-6 has a causative role in de
novo spontaneous prostate tumor initiation and thus
generated prostate-specific IL-6 transgenic mouse lines. The
encoding transgene of human IL-6 (hIL-6), which has been
shown to activate downstream signaling cascade similarly
in both human and mouse cells [29, 30], was expressed in
the prostate of the C57BL/6 (B6) mice directed by the rat
probasin (rPb) promoter  (Fig. 1a). We chose to express
human IL-6 so that we could differentiate the expression of
hIL-6 transgene from the autocrine expression of mouse
IL-6 (mIL-6). The founder line that expressed the transgene
IL-6 in the prostate was designated as pbIL-6. Integration
of a single copy of an intact transgene was confirmed by
genomic PCR against a limited template dilution standard
(data not shown). Prostate-specific expression of the
transgene hIL-6 was detected by RT-PCR (Fig. 1b).
To demonstrate the consequences of prostate-specific
expression of hIL-6 in the adult prostate, we compared
the histological characteristics of pbIL-6 transgenic mice
with the wild type B57CL/6 (B6) littermates. The
prostate glands from pbIL-6 transgenic mice uniformly
exhibited varying degrees of prostate intraepithelial
neoplasia (PIN)-like lesions at the age of 16-week old as
we have examined, which were not observed in the B6
WT littermates (data not shown). By 24 weeks of age,
neoplasia was evident in the pbIL-6 prostates.
Specifically, the prostate exhibited increased epithelial tufting,
overlapping of cells, enlarged nuclei of variable sizes and
shapes with prominent nucleoli, and thickening of the
stroma (Fig. 1c). As animals age, prostate glands in the
pbIL-6 mice more predominantly exhibited
carcinomalike feature, such as increased proliferation, enlarged
nucleoli, and disappearance of basal cells (Table 1 and
Fig. 2). In two out of ten examined aged mouse
(>100week old), prostate carcinoma progressed to poorly
differentiated (Additional file 1: Figure S1). Consistent with
the progressive neoplasia development in pbIL-6 mice as
animal ages, the rate of weight increase of prostate and
genitourinary (GU) by age was significantly greater in
pbIL-6 mice than in B6 WT littermates (Fig. 1d, e).
Notably, unlike human, wild type mice only develop prostate
intraepithelial neoplasia (PIN) but not spontaneous
prostate carcinoma as they age . Overall, the pbIL-6
animals presented higher mortality due to progressive
tumor development in the prostate, with rare incidence
in the liver (Fig. 1f ).
In our previous report, we described multiple effects of
IL-6 on immortalized benign human prostate epithelial
cells . Consistent with these findings, the prostate
epithelium of the transgenic pbIL-6 mice demonstrated a
focally dramatic decrease of membranous expression of
Ecadherin (Fig. 2a, b). The loss of E-cadherin signifies a
reduction in cell-cell adhesion and a shift of the prostate
muscle spleen thymus
Fig. 1 Prostate histology and weight comparison between pbIL-6 mice and B6 WT mice. a Diagram of rPB-hIL-6 (human IL-6) expression cassette
construction which was microinjected into fertilized C57BL/6 embryos to generate B6.PbIL-6 transgenic mice. b RT-PCR showed the hIL-6 was
expressed specially in the prostate tissue in the transgenic mice. c Representative histology comparison of prostate from pbIL-6 mice and B6 WT
littermates at 24 weeks of age. d, e Genitourinary (GU) and prostate weight comparison of pbIL-6 mice and B6 WT. f Keplan-Meier curve of overall
survival by 104-week old
luminal cells from a clearly differentiated epithelial to a
more mesenchymal phenotype and also an early stage of
transformation [33, 34]. The disrupted expression of basal
cell marker p63 in the prostate gland from the pbIL-6
mice also signifies the early transformation events of the
prostate [35, 36] (Fig. 2a). Consistent with our previous
report of the tumorigenic potential of IL-6 in epithelial cell
lines , the prostate epithelium from pbIL-6 mice had a
significant higher index of Ki-67 positivity (Fig. 2a),
suggesting an active proliferation state. Moreover, in
comparison to WT littermate, not only a markedly overall
elevation of the oncogene β-catenin but also more
significantly elevated accumulation of β-catenin in the nucleus
was presented in the prostate epithelium of the pbIL-6
mice (Fig. 2a, e). Furthermore, in comparison to the B6
WT littermates, androgen receptor (AR) was not only
significantly increased in the levels of expression in the
prostate epithelium of the pbIL-6 mice, but also
predominantly translocated to the nucleus (Fig. 2a, f ), an
indication of increased AR activity. Given that IL-6 has been
known to increase AR activity in castration-resistant
prostate cancer , together, these molecule features of the
Table 1 Summary of the prostate pathology of pbIl-6 mice and
wild type littermate surveyed at various ages
signaling axis may also contribute to IL-6-induced
PIN prostate intraepithelial neoplasia, WD well-differentiated tumor, PD poorly
prostate epithelium of the pbIL-6 mice endowed its
neoplasia features and tumorigenic properties, which were
further substantiated by elevated expression of oncogenes,
such as c-Fos and k-Ras as measured by quantitative
RTPCR (Fig. 2c, d).
IL-6 transgene activates autocrine IL-6 loop and
upregulates the IGF-I signaling axis in the prostate
In the previous report, we demonstrated that IL-6
stimulates the autocrine IL-6 loop in the immortalized benign
prostate epithelial cells . To investigate whether this
is a bona fida effect of IL-6 signaling event during de
novo tumorigenesis, we examined the expression of
mouse IL-6 (mIL-6) in the prostate. Quantitative
RTPCR revealed a significant induction of mouse IL-6
expression in the prostate of pbIL-6 mice in comparison
to the WT B6 littermate as we examined at 16 weeks of
age (Figs. 3a, P < 0.01). By 24 weeks of age, significantly
elevated serum levels of mIL-6 were detected in pbIL-6
mice (Figs. 3b, P < 0.01), presumably due to the more
severe destruction of the prostate architect and release
of secretory mediators. These results suggested that IL-6
in prostate tissue environment can induce bona fida
IL6 autocrine secretion. We also observed autocrine IL-6
induction in mouse prostate tumor cell lines exposed to
exogenous IL-6 (Additional file 1: Figure S2).
We and the others have previously described that IL-6
signaling trans-activates the endocrine IGF signaling axis
in cancer cells . Activation of IGF signaling axis has
been shown to play an essential role in tumor cell
survival and proliferation . In pbIL-6 mice, a
significantly increased expression IGF-I and IGF-II was
demonstrated by quantitative RT-PCR (Fig. 3c, d).
Collectively, these data suggest that trans-activating IGF
IL-6 transgene universally activates STAT3 pathway and
reprograms gene expression in the prostate tissue
Engagement of IL-6/IL-6Rα or IL-6/sIL-6Rα complex to
the signaling adaptor molecule gp130 triggers the
activation of downstream signaling cascade, most prominently
the JAK2/STAT3 pathway . Phosphorylation of
STAT3 and translocation to the nucleus are the critical
events for initiating the oncogenic transcriptional
activity . As shown in Fig. 4a, the prostate of pbIL-6 mice
exhibited high levels of phosphorylated STAT3
(pSTAT3), whereas the prostate from WT littermates
rarely presented positivity for pSTAT3. Intriguingly,
pSTAT3 was present not only in the prostate epithelium
cells but also in the stromal components of pbIL-6 mice.
These data suggest that IL-6 signaling may pose effects
on the prostate epithelium and stromal environment to
induce neoplastic transformation.
We further addressed how expression of IL-6 in the
prostate may impact prostate gene expression profiles
using Affymetrix whole-transcript RNA array analyses.
One hundred thirty genes were identified at least 2-fold
upregulated in the prostate of pbIL-6 mice in comparison
to the WT littermates at the significant level of P < 0.05.
The most significantly regulated genes are indicated in the
volcano plot of Fig. 4b and Additional file 1: Tables S1–S4.
Notably, a large portion of these upregulated genes are
associated with inflammation, oncogenesis, or metabolism.
The most relevant upregulated genes, such as cytokine
IL6 and oncogenes c-JUN, β-catenin, and Ras have been
validated by quantitative RT-PCR (Figs. 2c–e and 3a). IGF-I
and the androgen signaling downstream gene TMPRSS2
were also shown in the gene expression array to be
upregulated (Additional file 1: Table S3). Many of these
transcriptional changes are consistent with our previous
findings with in vitro IL-6 overexpression studies .
Together, these data indicate that expression IL-6 not only
intrinsically reprograms the prostate to express
protumorigenic genes but also primes a pro-inflammatory
tissue microenvironment whereby the combinatory effect
may contribute to prostate neoplasm.
IL-6 amplifies pro-tumorigenic inflammation in prostate
Emerging evidence suggests that chronic or recurrent
inflammation may initiate and promote cancer
development, including prostate cancer [40–43]. This process
involves multiple inflammatory cells as well as a broad
array of inflammatory cytokines . IL-6, as a major
pro-inflammatory cytokine can be secreted by an array
of inflammatory cell types, are not only the growth
factor for epithelial cell, but also critical for the survival
Fig. 2 Expression of neoplasm related markers in the prostate of pbIL-6 mice and WT littermates. a Immunohistochemistry staining of panel of
neoplasm related markers. b–f Relative expression levels of neoplasm related markers assessed by quantitative RT-PCR. Data shown were representative
images and expression data of prostates from 24-week-old pbIL-6 mice and B6 WT littermates. AR androgen receptor
and proliferation of inflammatory cell types in the tissue
microenvironment. This process is frequently referred as
“smoldering” inflammation [40, 44–46].
In general, an enriched infiltration of inflammatory
cells was present in the prostate of the pbIL6 mice (data
not shown). We thus characterized these inflammatory
cell types by immunohistochemistry staining (IHC) with
specific markers. The enriched infiltration of T
lymphocytes (CD3+), macrophages (MAC-2+), and B
lymphocytes (B220+) was remarkable and significantly increased
in the prostate of pbIL6 mice in comparison to WT
littermates (Fig. 5a, b), all of which are known to be
IL6- producing cells. Intriguingly, a rich infiltration of
macrophages and T lymphocytes was also evident in the
peri-prostatic adipose tissue in the pbIL6 mice, whereas
infiltration is rare in the peri-prostatic adipose tissue of
B6 WT littermates (Fig. 6). Together, these data suggest
that enriched IL-6 expression in the prostate can amplify
the inflammatory responses in the local tissue and
periprostatic adipose tissue, which may confer as one of the
pathways to induce neoplasm in the prostate.
The link between IL-6 and prostate cancer progression
has been well established . However, whether IL-6
alone can induce de novo prostate tumor initiation in an
autologous state is unknown. Using the novel
prostatespecific IL-6 transgenic mice, we clearly demonstrate the
oncogenic property of IL-6. We show that elevated
expression of IL-6 alone in the prostate is sufficient to
induce local neoplasm. We further show that
constitutive IL-6 expression in the prostate, resembling chronic
inflammation, activates STAT3, reprograms prostate
gene transcription to pro-tumorigenic, activates the
Fig. 3 IL-6 induces autocrine IL-6 secretion and upregulates IGF
signaling axis in the prostate. a Quantitative RT-PCR demonstrating
significantly increased mouse IL-6 (mIL-6) expression in the prostate
of pbIL-6 mice in comparison to B6 WT littermates. b Significant
elevation of serum mIL-6 in pbIL-6 mice in comparison to B6 WT
littermates. c, d Significant elevation of IGF-I and IGF-II expression in
the prostate of pbIL-6 mice in comparison to B6 WT littermates. Data
shown were representatives of mice at the age of 26-week old
autocrine IL-6 and paracrine IGF signaling axis, and
amplifies inflammation in the prostate and peri-prostatic
adipose tissue. This is the first study, to our knowledge,
that established a direct link between IL-6 and de novo
tumorigenesis in the prostate. Our data conclude that IL-6
is an “unconventional” oncogene in prostate tumorigenesis.
Given that IL-6 is a common cytokine produced by
many inflammatory cell types, our study also suggests a
direct link between inflammation and carcinogenesis.
The link between inflammation and cancer has been
suggested since centuries ago by Dr. Rudolf Virchow
. However, the conventional wisdom considered
inflammatory acts as a “stimuli” to other genotoxic events
to facilitate cancer development. Our current study
clearly demonstrated that inflammation can also act as
an “ignitor” to autonomously initiate de novo tumor
development without other genomic insults, in which
context the pro-inflammatory cytokine IL-6 serves as a
critical mediator or “lynchpin” .
Our prostate-specific IL-6 transgenic mice presented
several intriguingly features in the prostate: (1) activation
of STAT3 in the stroma; (2) enriched infiltration of
inflammatory cell types in the prostate; and (3) enriched
infiltration of inflammatory cells in the peri-prostatic
adipose tissue. IL-6 is the most well-known conventional
Log2 fold change
Fig. 4 IL-6 activates STAT3 pathway and reprograms prostate gene
expression to be pro-tumorigenic. a IHC demonstrating phosphorylation
of STAT3 in the prostate epithelium and stromal components in pbIL-6
mice, whereas pSTAT3 was rarely detected in prostate from the B6 WT
littermates. b A volcano plot demonstrating upregulation of genes
associated with oncogenic and inflammation in the prostate of pbIL-6
mice. The log2 fold change of genes between four samples of IL6 mice
vs. two WT littermates is plotted against the negative log10 p value in
the volcano plot. The brown and red dots are above bonferroni adjusted
p values for 22,623 tests for alpha 0.05. The yellow and green dots are
genes above an alpha of 0.1 and below 0.05. The red, green and blue
dots are genes with average fold greater than 1 for the log2 ratio. The
green and red are above the log2 ratio of 1 and are significant at the 0.1
or 0.05 alpha level, respectively. The function of the most upregulated
genes are indicated in Additional file 1: Table S1-4
activator of STAT3 [19, 47]. In normal cells, activation
of STAT3 by IL-6 is regulated by a negative feedback
loop through the activity of suppressor of the cytokine
signaling 3 (SOCS3) whose expression can be induced
by IL-6 signaling [48, 49]. SOCS3 can bind to the Y759
residue on the adaptor molecule gp130 and thus
preventing constitutive IL-6 signaling and activation of
STAT3 in a steadily state [48, 49]. When cells are
insulted by abnormal physiological conditions, STAT3
can be constitutively activated by IL-6 signaling due to
functional silencing of the SOCS3 gene through
epigenetic hypermethylation [49, 50]. Prostate cancer patients
who have methylation in the promoter region of SOCS3
T-cell B-cell Macrophage
(CD3) (B220) (Mac-2)
Fig. 5 IL-6 amplifies inflammation in the prostate tissue. a Representative
micrographs of IHC demonstrating increased infiltration of T lymphocytes
(CD3+), macrophage (MAC-2+), and B lymphocytes (B220+) in prostate
tissue of pbIL-6 mice in comparison to B6 WT littermates. b Quantitative
scoring of each inflammation cell types in the prostates of pbIL-6 mice
and B6 WT littermates. Ten random fields containing prostate glands and
stroma of each prostate section were scored. Prostates from 6 to 10
animals from each group were evaluated. *P < 0.05
presented a more aggressive phenotype . As
inflammation is known to cause epigenetic perturbation [52, 53],
cancers in this subset of patients are thus suggested to be
inflammation-originated. Although not determined in this
study, our findings warrant a future investigation on
epigenetic modifications in the prostate. Given the current
understanding that activation of STAT3 may also be
the “hub” of the interplay among adipose tissue,
inflammation, and cancer [39, 54], our observations suggest
that IL-6 may exert its “oncogenic” property through
multiple pathways (Fig. 7).
Elevation in IL-6 expression and loss of E-cadherin
have been linked with increase cancer stem cell
population in various cancer types [55–57]. In this study, we
did not observe positivity for cancer stem cells with
markers such as CD133, OCT4, or Nanog (data not
shown), suggesting that rising or re-populating of cancer
stem cells is not the major mechanism by which IL-6
induces prostate tumorigenesis in current model.
Prostate cancer is a heterogeneous neoplasm which is
regulated by factors, such as age, hormones, obesity, and dietary
habits, in addition to genomic insults common to other
cancers. Many epigenetic and clinical follow-up studies
have suggested a strong link between chronic inflammation
and prostate cancer risk [43, 58–60]. However, to date, our
understandings are limited by lacking appropriate animal
models to study the development of prostate cancer from
chronic inflammation. In the current study, we not only
demonstrated that IL-6 is an oncogene for prostate cancer,
but also present a valuable model that recapitulates the role
of inflammation in prostate cancer development.
Generation of transgenic mice
Mice were bred and housed under specific pathogen-free
conditions in the University of Washington animal facility
in accordance with the institutional guidelines. All mice
used in this study were on the C57BL/6 (B6) background.
The rPB-IL6 expression cassette was constructed by
replacing the SV40T human IL-6. The entire rPB-IL6
expression cassette was gel isolated following digestion with
Hind III and was microinjected into fertilized B6 embryos
at University of Washington Comparative Medicine
transgenic core facility. Transgenic progeny were identified by
PCR analysis of DNA extracted from tail biopsies using
the forward primer specific for rPB
(5′-acaagtgcatttagcctctccagta-3′) and the reverse primer specific for IL-6
(5′-tgtgtcttggtcttcatggc-3′). All experimental mice were
randomly assigned to cohorts and euthanized at indicated
age for evaluation of GU and the prostate.
Total RNA was extracted using TRizol (Invitrogen)
followed by treatment with DNAase I (Fermentas) to
exclude the genomic DNA before reversal transcription.
Complementary DNA (cDNA) was synthesized using the
SuperScript II kit (Invitrogen). A volume of 1 μL of cDNA
was mixed with Power SYBR Green PCR MasterMix
(Applied Biosystem, Carlsbad, CA, USA), and specific
primer sets were added to a final concentration of 400 nM in
20 μl of reaction mixture. The reaction was performed on
an ABI9700 Machine. Data were analyzed using the
Lightcycler software v3.5 (Roche Applied Science, Indianapolis,
IN, USA). Each sample was assayed in triplicates. Target
mRNA levels were normalized against mouse GAPDH.
The primers used are listed in Additional file 1: Table S5.
Fig. 6 IL-6 induces inflammation in the peri-prostatic adipose tissue. Representative micrographs of IHC demonstrating increased infiltration of
macrophage (MAC-2+) and T lymphocytes (CD3+) in the peri-prostatic adipose tissue of pbIL-6 mice
Total RNA of the prostates from four 24-week-old pbIL-6
transgenic male mice and wild type C57BL/6 littermates
was obtained as described above. After RNA quality
confirmation with a Bioanalyzer (Agilent), 300 ng of each RNA
sample was used in the Affymetrix Whole-Transcript Sense
Target Labeling Assay (Rev 3), followed by hybridization to
prostate inflammation IGF axis
Prostate tumorigenic conversion
Fig. 7 Proposed mechanism of the oncogenic property of IL-6 in the
prostate. The pro-inflammatory cytokine IL-6 in the prostate, as a result
of inflammation or peri-prostatic obesity, induce autocrine and paracrine
pathway and amplify inflammation in the prostate tissue environment
and peri-prostate adipose tissue which subsequently induces
transcriptional reprogramming in the prostate and tumorigenesis
a GeneChip Mouse Gene 1.0 ST Array. Eight GeneChips
were used to provide biological replicates of each genotype.
The Affymetrix Expression Console (v 1.1) was used to
normalize data and determine signal intensity
(RMASketch). Analysis was performed using DAVID
Bioinformatic software and R2 statistical software with bonferroni
Histological and immunohistochemical examination
The mouse prostate tissues were fixed in 10% formaldehyde
and embedded in paraffin wax. Five-micrometer sections
were cut and stained with H&E for pathological evaluation.
Sections were also stained with antibodies specific for: (1)
E-cadherin (Santa Cruz); (2) p63 (Thermo Scientific); (3)
Ki67 (Thermo Scientific); (4) β-catenin antibody (AbCAM);
(5) AR (Santa Cruz); (6) pSTAT3 (Cell signaling); (7)
macrophage (F4/80 or Mac-2; eBioscience); (8) B cells (B220,
eBioscience); and (9) anti-CD3 (Thermo Scientific). The
staining procedure has been previously described .
Briefly, sections were deparaffinized and incubated for
10 min in 10 mM citrate buffer (pH 6.0) at 95 °C for antigen
retrieval. Endogenous peroxidase activity was quenched
with 3% hydrogen peroxide in methanol. After quenching
endogenous peroxidase activity and blocking nonspecific
binding, slides were incubated with specific primary
antibody overnight at 4 °C followed by subsequent incubation
with the appropriate biotinylated secondary antibody
provided with Vectastain Elite ABC Kit. Color was developed
with DAB as the perioxidase substract. All slides were
counterstained with hematoxylin and mounted with Permount.
Ten randomly selected fields of IHC-stained sections of the
prostates from individual mice were counted for the
positively stained cells and used for statistical analysis.
All results are expressed as the mean ± SEM. Differences
between the mean of groups were analyzed using
student’s t test with one-way ANOVA analyses. In most
cases, P < 0.05 was considered as significant.
Additional file 1: Figure S1. Representative histology of a 103-week-old
mouse (#1825) that developed poorly differentiated tumor in the prostate.
CD45 stains for all leukocyte antigens in the infiltrates. Supplement Figure 2.
Exogenous human IL-6 induces paracrine mouse IL-6 expression in two
mouse prostate tumor cell lines, the TRAMP-C2 and MyC-Cap. Fifty
nanogram per milliliter of human IL-6 was supplemented in the culture
media. Cells were harvested at 24 and 48 hr of culture. Relative mouse IL-6
expression was assessed by quantitative RT-PCR. Table S1. Function of the
most significantly upregulated genes with more than log2 fold changes in
the volcano plot. Table S2. Upregulated inflammation-associated genes in
the prostate of pbIL-6 mice that are not highlighted on the volcano plot.
Table S3. Upregulated oncogenic genes in the prostate of pbIL-6 mice that
are not highlighted on the volcano plot. Table S4. Upregulated
metabolismassociated genes in the prostate of pbIL-6 mice that are not highlighted on
the volcano plot. Table S5. List of primers used for qRT-PCR. (PDF 329 kb)
We thank the University of Washington Transgenic Core Facility for generating
the founder line of the transgenic mice. We acknowledge John Jarzen for the
technical support for some part of the immunohistochemistry staining.
This work was supported by NIH-NCI grant 1R01CA149405, R01 CA204021, and
A. David Mazzone-Prostate Cancer Foundation Challenge Award (to J.Wu).
Availability of data and materials
All data generated or analyzed during this study are included in this
published article and its Additional file 1.
JW conceived the concept and prepared the manuscript. GL generated the
transgenic construct and characterized the pathology of transgenic mice. JZ
further performed the characterization of inflammatory pathways. GX
performed part of the β-catenin staining. LF performed the cDNA array data
analyses. QL, KW, and ML provided valuable discussion in the interpretation
of the experiment data. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
1. Culig Z. Proinflammatory cytokine interleukin-6 in prostate carcinogenesis . Am J Clin Exp Urol . 2014 ; 2 ( 3 ): 231 - 8 .
2. Ara T , Declerck YA . Interleukin-6 in bone metastasis and cancer progression . Eur J Cancer . 2010 ; 46 ( 7 ): 1223 - 31 .
3. Corcoran NM , Costello AJ . Interleukin-6: minor player or starring role in the development of hormone-refractory prostate cancer? BJU Int . 2003 ; 91 ( 6 ): 545 - 53 .
4. Ishiguro H , et al. aPKClambda/iota promotes growth of prostate cancer cells in an autocrine manner through transcriptional activation of interleukin-6 . Proc Natl Acad Sci U S A . 2009 ; 106 ( 38 ): 16369 - 74 .
5. Paule B , et al. The NF-kappaB/IL-6 pathway in metastatic androgenindependent prostate cancer: new therapeutic approaches ? World J Urol . 2007 ; 25 ( 5 ): 477 - 89 .
6. Santer FR , et al. Interleukin-6 trans-signalling differentially regulates proliferation, migration, adhesion and maspin expression in human prostate cancer cells . Endocr Relat Cancer . 2010 ; 17 ( 1 ): 241 - 53 .
7. Zhu Y , et al. Interleukin-6 induces neuroendocrine differentiation (NED) through suppression of RE-1 silencing transcription factor (REST). Prostate . 2014 ; 74 ( 11 ): 1086 - 94 .
8. Chen MF , et al. IL-6 expression regulates tumorigenicity and correlates with prognosis in bladder cancer . PLoS One . 2013 ; 8 ( 4 ): e61901 .
9. Knupfer H , Preiss R. Serum interleukin-6 levels in colorectal cancer patients-a summary of published results . Int J Colorectal Dis . 2010 ; 25 ( 2 ): 135 - 40 .
10. Milicevic N , et al. Comparison between clinical significance of serum proinflammatory protein interleukin-6 and classic tumor markers total PSA, free PSA and free/total PSA prior to prostate biopsy . Coll Antropol . 2014 ; 38 ( 1 ): 147 - 50 .
11. Nakashima J , et al. Serum interleukin 6 as a prognostic factor in patients with prostate cancer . Clin Cancer Res . 2000 ; 6 ( 7 ): 2702 - 6 .
12. Tam L , et al. Expression levels of the JAK/STAT pathway in the transition from hormone-sensitive to hormone-refractory prostate cancer . Br J Cancer . 2007 ; 97 ( 3 ): 378 - 83 .
13. Waldner MJ , Foersch S , Neurath MF. Interleukin-6-a key regulator of colorectal cancer development . Int J Biol Sci . 2012 ; 8 ( 9 ): 1248 - 53 .
14. Rojas A , et al. IL-6 promotes prostate tumorigenesis and progression through autocrine cross-activation of IGF-IR . Oncogene. 2011 ; 30 ( 20 ): 2345 - 55 .
15. Kishimoto T. The biology of interleukin-6 . Blood . 1989 ; 74 ( 1 ): 1 - 10 .
16. Yu SH , et al. A paracrine role for IL6 in prostate cancer patients: lack of production by primary or metastatic tumor cells . Cancer Immunol Res . 2015 ; 3 ( 10 ): 1175 - 84 .
17. Jones SA , et al. IL-6 transsignaling: the in vivo consequences . J Interferon Cytokine Res . 2005 ; 25 ( 5 ): 241 - 53 .
18. McLoughlin RM , et al. IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation . Proc Natl Acad Sci U S A . 2005 ; 102 ( 27 ): 9589 - 94 .
19. Yu H , et al. Revisiting STAT3 signalling in cancer: new and unexpected biological functions . Nat Rev Cancer . 2014 ; 14 ( 11 ): 736 - 46 .
20. Heinrich PC , et al. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J . 2003 ; 374 (Pt 1): 1 - 20 .
21. Lu K , et al. The STAT3 inhibitor WP1066 reverses the resistance of chronic lymphocytic leukemia cells to histone deacetylase inhibitors induced by interleukin-6 . Cancer Lett . 2015 ; 359 ( 2 ): 250 - 8 .
22. Yang , Z. , et al., Acquisition of resistance to trastuzumab in gastric cancer cells is associated with activation of IL-6/STAT3/Jagged-1/Notch positive feedback loop . Oncotarget . 2015 ; 6 ( 7 ): 5072 - 87
23. Bournazou E , Bromberg J. Targeting the tumor microenvironment: JAKSTAT3 signaling . JAKSTAT . 2013 ; 2 ( 2 ): e23828 .
24. Guo Y , et al. Interleukin-6 signaling pathway in targeted therapy for cancer . Cancer Treat Rev . 2012 ; 38 ( 7 ): 904 - 10 .
25. Middleton K , et al. Interleukin-6: an angiogenic target in solid tumours . Crit Rev Oncol Hematol . 2014 ; 89 ( 1 ): 129 - 39 .
26. Zhang K , et al. Association between interleukin-6 polymorphisms and urinary system cancer risk: evidence from a meta-analysis . Onco Targets Ther . 2016 ; 9 : 567 - 77 .
27. Chen J , et al. Association between polymorphisms in selected inflammatory response genes and the risk of prostate cancer . Onco Targets Ther . 2016 ; 9 : 223 - 9 .
28. Chen CH , et al. Role of interleukin-6 gene polymorphisms in the development of prostate cancer . Genet Mol Res . 2015 ; 14 ( 4 ): 13370 - 4 .
29. Coulie PG , Stevens M , Van Snick J. High- and low-affinity receptors for murine interleukin 6 . Distinct distribution on B and T cells. Eur J Immunol . 1989 ; 19 ( 11 ): 2107 - 14 .
30. Hammacher A , et al. Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding . Protein Sci . 1994 ; 3 ( 12 ): 2280 - 93 .
31. Greenberg NM , et al. The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice . Mol Endocrinol . 1994 ; 8 ( 2 ): 230 - 9 .
32. Greenberg NM , et al. Prostate cancer in a transgenic mouse . Proc Natl Acad Sci U S A . 1995 ; 92 ( 8 ): 3439 - 43 .
33. Cervantes-Arias A , Pang LY , Argyle DJ . Epithelial-mesenchymal transition as a fundamental mechanism underlying the cancer phenotype . Vet Comp Oncol . 2013 ; 11 ( 3 ): 169 - 84 .
34. Fawcett J , Harris AL. Cell adhesion molecules and cancer . Curr Opin Oncol . 1992 ; 4 ( 1 ): 142 - 8 .
35. Abdulkadir SA , et al. Conditional loss of Nkx3.1 in adult mice induces prostatic intraepithelial neoplasia . Mol Cell Biol . 2002 ; 22 ( 5 ): 1495 - 503 .
36. Kim MJ , et al. Nkx3 . 1 mutant mice recapitulate early stages of prostate carcinogenesis . Cancer Res . 2002 ; 62 ( 11 ): 2999 - 3004 .
37. Schweizer MT , Yu EY . Persistent androgen receptor addiction in castrationresistant prostate cancer . J Hematol Oncol . 2015 ; 8 : 128 .
38. Garbers C , Aparicio-Siegmund S , Rose-John S. The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition . Curr Opin Immunol . 2015 ; 34 : 75 - 82 .
39. Taniguchi K , Karin M. IL-6 and related cytokines as the critical lynchpins between inflammation and cancer . Semin Immunol . 2014 ; 26 ( 1 ): 54 - 74 .
40. Candido J , Hagemann T. Cancer-related inflammation . J Clin Immunol . 2013 ; 33 Suppl 1: S79 - 84 .
41. Guven Maiorov E , et al. The structural network of inflammation and cancer: merits and challenges . Semin Cancer Biol . 2013 ; 23 ( 4 ): 243 - 51 .
42. Janakiram NB , Rao CV . The role of inflammation in colon cancer . Adv Exp Med Biol . 2014 ; 816 : 25 - 52 .
43. Taverna , G. , et al., Inflammation and prostate cancer: friends or foe? Inflamm Res , 2015
44. Aggarwal BB , et al. Inflammation and cancer: how hot is the link ? Biochem Pharmacol . 2006 ; 72 ( 11 ): 1605 - 21 .
45. Balkwill F , Charles KA , Mantovani A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease . Cancer Cell . 2005 ; 7 ( 3 ): 211 - 7 .
46. Colotta F , et al. Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability . Carcinogenesis . 2009 ; 30 ( 7 ): 1073 - 81 .
47. Zarogoulidis P , et al. Interleukin-6 cytokine: a multifunctional glycoprotein for cancer . Immunome Res . 2013 ; 9 ( 62 ): 16535 .
48. Kubo M , Hanada T , Yoshimura A. Suppressors of cytokine signaling and immunity . Nat Immunol . 2003 ; 4 ( 12 ): 1169 - 76 .
49. Murakami M , Hirano T. The pathological and physiological roles of IL-6 amplifier activation . Int J Biol Sci . 2012 ; 8 ( 9 ): 1267 - 80 .
50. Isomoto H. Epigenetic alterations in cholangiocarcinoma-sustained IL-6/ STAT3 signaling in cholangio-carcinoma due to SOCS3 epigenetic silencing . Digestion . 2009 ; 79 Suppl 1 : 2 - 8 .
51. Pierconti F , et al. Epigenetic silencing of SOCS3 identifies a subset of prostate cancer with an aggressive behavior . Prostate . 2011 ; 71 ( 3 ): 318 - 25 .
52. Cutolo M , Paolino S , Pizzorni C. Possible contribution of chronic inflammation in the induction of cancer in rheumatic diseases . Clin Exp Rheumatol . 2014 ; 32 ( 6 ): 839 - 47 .
53. Shanmugam MK , Sethi G. Role of epigenetics in inflammation-associated diseases . Subcell Biochem . 2013 ; 61 : 627 - 57 .
54. Tilg H , Moschen AR. Adipocytokines: mediators linking adipose tissue, inflammation and immunity . Nat Rev Immunol . 2006 ; 6 ( 10 ): 772 - 83 .
55. Jayachandran A , Dhungel B , Steel JC . Epithelial-to-mesenchymal plasticity of cancer stem cells: therapeutic targets in hepatocellular carcinoma . J Hematol Oncol . 2016 ; 9 ( 1 ): 74 .
56. Li X , et al. Lung tumor exosomes induce a pro-inflammatory phenotype in mesenchymal stem cells via NFkappaB-TLR signaling pathway . J Hematol Oncol . 2016 ; 9 : 42 .
57. Yin X , et al. Coexpression of gene Oct4 and Nanog initiates stem cell characteristics in hepatocellular carcinoma and promotes epithelialmesenchymal transition through activation of Stat3/Snail signaling . J Hematol Oncol . 2015 ; 8 : 23 .
58. MacLennan GT , et al. The influence of chronic inflammation in prostatic carcinogenesis: a 5-year followup study . J Urol . 2006 ; 176 ( 3 ): 1012 - 6 .
59. Nakai Y , Nonomura N. Inflammation and prostate carcinogenesis . Int J Urol . 2013 ; 20 ( 2 ): 150 - 60 .
60. Sfanos KS , Hempel HA , De Marzo AM . The role of inflammation in prostate cancer . Adv Exp Med Biol . 2014 ; 816 : 153 - 81 .