JNK and PTEN cooperatively control the development of invasive adenocarcinoma of the prostate.
ABSTRACT The c-Jun NH(2)-terminal kinase (JNK) signal transduction pathway is implicated in cancer, but the role of JNK in tumorigenesis is poorly understood. Here, we demonstrate that the JNK signaling pathway reduces the development of invasive adenocarcinoma in the phosphatase and tensin homolog (Pten) conditional deletion model of prostate cancer. Mice with JNK deficiency in the prostate epithelium (ΔJnk ΔPten mice) develop androgen-independent metastatic prostate cancer more rapidly than control (ΔPten) mice. Similarly, prevention of JNK activation in the prostate epithelium (ΔMkk4 ΔMkk7 ΔPten mice) causes rapid development of invasive adenocarcinoma. We found that JNK signaling defects cause an androgen-independent expansion of the immature progenitor cell population in the primary tumor. The JNK-deficient progenitor cells display increased proliferation and tumorigenic potential compared with progenitor cells from control prostate tumors. These data demonstrate that the JNK and PTEN signaling pathways can cooperate to regulate the progression of prostate neoplasia to invasive adenocarcinoma.
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ABSTRACT: JNK and p38 phosphorylate a diverse set of substrates and, consequently, can act in a context-dependent manner to either promote or inhibit tumor growth. Elucidating the functions of specific substrates of JNK and p38 is therefore critical for our understanding of these kinases in cancer. ATF2 is a phosphorylation-dependent transcription factor and substrate of both JNK and p38. Here, we show ATF2 suppresses tumor formation in an orthotopic model of liver cancer and cellular transformation in vitro. Furthermore, we find that suppression of tumorigenesis by JNK requires ATF2. We identify a transcriptional program activated by JNK via ATF2 and provide examples of JNK- and ATF2-dependent genes that block cellular transformation. Significantly, we also show that ATF2-dependent gene expression is frequently downregulated in human cancers, indicating that amelioration of JNK-ATF2-mediated suppression may be a common event during tumor development. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.Cell Reports 11/2014; 9(4):1361-74. · 7.21 Impact Factor
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ABSTRACT: The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary-specific deletion of Pten via WAP-Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV-Cre, which targets basal/luminal progenitors. Combined Pten-p53 mutations accelerated formation of claudin-low, triple-negative-like breast cancer (TNBC) that exhibited hyper-activated AKT signaling and more mesenchymal features relative to Pten or p53 single-mutant tumors. Twenty-four genes that were significantly and differentially expressed between WAP-Cre:Pten/p53 and MMTV-Cre:Pten/p53 tumors predicted poor survival for claudin-low patients. Kinome screens identified eukaryotic elongation factor-2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53-deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53-deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro. Our results identify a prognostic signature for claudin-low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling.EMBO Molecular Medicine 10/2014; 6(12):1542-1560. · 7.80 Impact Factor
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ABSTRACT: The c-Jun N-terminal kinase (JNK) family, with its three members JNK1, JNK2, and JNK3, is a subfamily of mitogen-activated protein kinases. Involved in many aspects of cellular processes, JNK has been also associated with pathological states such as neurodegenerative diseases, inflammation, and cancers. In oncology, each isoform plays a distinct role depending on the context of the targeted tissue/organ, the tumor stage, and, most likely, the signaling pathway activated upstream. Consequently, the current challenge in finding new successful anti-JNK therapies is to design isoform-selective inhibitors of the JNKs. In this review, a particular focus is given to the JNK inhibitors that have been developed thus far when examining 3D structures of various JNK-inhibitor complexes. Using current data regarding structure-activity relationships and medicinal chemistry approaches, our objective is to provide a better understanding of the design and development of selective JNK inhibitors in the present and future. Copyright © 2014 Elsevier Ltd. All rights reserved.Chemistry & biology. 11/2014; 21(11):1433-1443.
JNK and PTEN cooperatively control the development
of invasive adenocarcinoma of the prostate
Anette Hübnera,1, David J. Mulhollandb, Claire L. Standena, Maria Karasaridesa,3, Julie Cavanagh-Kyrosc,
Tamera Barrettc, Hongbo Chid, Dale L. Greinera, Cathy Tourniere, Charles L. Sawyersf, Richard A. Flavellg,2,
Hong Wub, and Roger J. Davisa,c,2
aProgram in Molecular Medicine, University of Massachusetts Medical School, andcHoward Hughes Medical Institute, Worcester, MA 01605;bDepartment
of Molecular and Medical Pharmacology and Institute for Molecular Medicine, University of California Los Angeles School of Medicine, Los Angeles, CA 90095;
dDepartment of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105;eFaculty of Life Sciences, University of Manchester, Manchester
M13 9PT, United Kingdom;fHoward Hughes Medical Institute and Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center,
New York, NY 10065; andgHoward Hughes Medical Institute and Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520
Contributed by Richard A. Flavell, June 7, 2012 (sent for review April 11, 2012)
The c-Jun NH2-terminal kinase (JNK) signal transduction pathway is
implicated in cancer, but the role of JNK in tumorigenesis is poorly
understood. Here, we demonstrate that the JNK signaling path-
way reduces the development of invasive adenocarcinoma in the
phosphatase and tensin homolog (Pten) conditional deletion
model of prostate cancer. Mice with JNK deficiency in the prostate
epithelium (ΔJnk ΔPten mice) develop androgen-independent met-
astatic prostate cancer more rapidly than control (ΔPten) mice.
Similarly, prevention of JNK activation in the prostate epithelium
(ΔMkk4 ΔMkk7 ΔPten mice) causes rapid development of invasive
adenocarcinoma. We found that JNK signaling defects cause an
androgen-independent expansion of the immature progenitor cell
population in the primary tumor. The JNK-deficient progenitor
cells display increased proliferation and tumorigenic potential
compared with progenitor cells from control prostate tumors.
These data demonstrate that the JNK and PTEN signaling path-
ways can cooperate to regulate the progression of prostate neo-
plasia to invasive adenocarcinoma.
transcription factors, including ATF2, c-Jun, JunB, and JunD
(1). These transcription factors represent an important com-
ponent of the immediate-early gene response to mitogens and
inflammatory stimuli (2). AP1 transcription factors are also
implicated in dysregulated growth and tumor development (2).
Significantly, JNK deficiency suppresses AP1-dependent gene ex-
pression and causes defects in cell proliferation, senescence, and
apoptosis (3–5). JNK may, therefore, play a role in carcinogenesis.
Studies using mouse models of cancer have confirmed that JNK
can play a key role in cancer. Thus, JNK deficiency reduces the
development of Bcr/Abl-induced lymphoma (6) and KRas-induced
lung tumors (7). Moreover, carcinogen-induced hepatocellular
carcinoma (8–10) and skin cancer (11) can be reduced by JNK
deficiency. These observations demonstrate that JNK can pro-
mote cancer. However, loss of JNK signaling can also promote
development of other tumors (2, 9, 12–15). These opposing roles
of JNK in tumor development (promotion or repression) may
represent differences in JNK function between tumor types (1).
Alternatively, these differences may reflect separate functions of
JNK in tumor cells and the tumor microenvironment (9).
Mutational inactivation of the tumor suppressor phosphatase
and tensin homolog (PTEN) frequently occurs in human prostate
cancer (16–18). Mouse models of Pten deficiency in the prostate
epithelium demonstrate that loss of PTEN expression is suffi-
cient to cause activation of the AKT signaling pathway, prostatic
intraepithelial neoplasia (PIN) lesions, and subsequent devel-
opment of castration-resistant prostate cancer (19). Loss of PTEN
function is, therefore, established to be a key step in the de-
velopment of prostate cancer. Importantly, PTEN inactivation is
associated with increased activity of the JNK signaling pathway
in human prostate cancer (20). Indeed, it has been proposed that
he c-Jun NH2-terminal kinase (JNK) signaling pathway can
target members of the activating protein 1 (AP1) group of
JNK may be an effector of the PI3K/AKT pathway in prostate
cancer with PTEN inactivation (20).
The purpose of this study was to examine the role of the JNK
signaling pathway in prostate cancer using a mouse model with
selective gene disruption in the prostate epithelium. Previous
studies indicate that JNK may be a positive (20) or a negative
(21) regulator of prostate cancer development. Here, we report
that JNK signaling plays a key role in the development of invasive
adenocarcinoma caused by Pten inactivation.
JNK Deficiency in the Prostate Epithelium. To test the functional
role of JNK, we used a model of prostate cancer using mice with
conditional (floxed) Pten and selective expression of Cre recom-
binase in the prostate epithelium (19). We crossed Pten condi-
tional deletion mice to Jnk1−/−and Jnk2−/−mice on the BALB/cJ
strain background. The resulting compound mutant mice de-
veloped prostatic neoplastic lesions similar to that of Pten single
deletion, indicating that JNK1 and JNK2 may be functionally
redundant in PTEN-controlled prostate cancer formation. To
test this hypothesis, we examined the effect of concomitant de-
letion of JNK1 plus JNK2 on tumor development. Because
Jnk1−/−Jnk2−/−compound mutant mice die during midembryo-
genesis (1), we used a conditional (floxed) deletion approach.
Mice with dual deficiency of Jnk1 plus Jnk2 in the prostate epi-
thelium (ΔJnk mice) were viable and fertile. Histopathological
analysis of sections prepared from the prostate gland of wild-type
(WT) mice and ΔJnk mice indicated that JNK was not required
for prostate gland development (Fig. S1).
Mice with triple deficiency of Jnk1, Jnk2, plus Pten (ΔJnk ΔPten
mice) in the prostate epithelium were also found to be viable and
fertile (Fig. 1A). However, the majority of the ΔJnk ΔPten male
mice died by age 20 wk with large prostate tumors and urethral
obstruction (Fig. 1B). In contrast, the mean lifespan of mice with
Pten deficiency alone (ΔPten mice) was greater than 80 wk.
Kaplan–Meier analysis demonstrated that the lifespan of ΔJnk
Author contributions: A.H., D.J.M., M.K., C. L. Sawyers, H.W., and R.J.D. designed research;
A.H., D.J.M., C. L. Standen, M.K., J.C.-K., and T.B. performed research; H.C., D.L.G., C.T.,
and R.A.F. contributed new reagents/analytic tools; A.H., D.J.M., C. L. Standen, M.K.,
J.C.-K., T.B., C. L. Sawyers, R.A.F., H.W., and R.J.D. analyzed data; and A.H. and R.J.D.
wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1Present address: Developmental and Molecular Pathways, Novartis Pharma AG, CH-4002
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org, or roger.
3Present address: Department of Medical Affairs, Infinity Pharmaceuticals, Boston,
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 24, 2012
| vol. 109
| no. 30 www.pnas.org/cgi/doi/10.1073/pnas.1209660109
ΔPten mice was significantly shorter than WT mice, ΔJnk mice,
or ΔPten mice (Fig. 1C). Moreover, the primary tumors present
in ΔJnk ΔPten mice were significantly larger than ΔPten mice at
age 20 wk (Fig. 1B). Microscopic analysis of sections prepared from
these primary tumors demonstrated that the ΔJnk ΔPten mice dis-
played significant disruption of prostate glandular structure com-
pared with ΔPten tumors (Fig. 1D), consistent with a more
advanced tumor phenotype. Indeed, we detected PIN lesions in the
primary tumors of ΔPten mice and invasive adenocarcinoma in the
primary tumors of ΔJnk ΔPten mice at age 20 wk (Fig. 1D). Im-
munohistochemical analysis demonstrated the presence of activated
AKT in both ΔPten and ΔJnk ΔPten primary tumors, but JNK was
detected only in ΔPten tumors (Fig. 1E). Nuclear androgen recep-
tors were detected in both ΔPten and ΔJnk ΔPten tumors (Fig. S2).
It is established that JNK can influence proliferation, senes-
cence, and apoptosis (1). The larger prostate tumors in ΔJnk
ΔPten mice compared with ΔPten mice (Fig. 1) could, therefore,
reflect increased growth and/or decreased apoptosis. The majority
of cells detected in ΔPten PIN lesions expressed senescence-
associated β-galactosidase and did not stain for the proliferation
marker Ki67 (Fig. S3). It is established that cellular senescence
in ΔPten PIN lesions limits prostate cancer progression (22, 23).
Studies of ΔJnk ΔPten adenocarcinoma cells demonstrated no ex-
pression of senescence-associated β-galactosidase and markedly
increased Ki67 staining (Fig. S3). No differences in apoptosis were
detected between ΔPten and ΔJnk ΔPten primary tumors (Fig. S3).
Together, these data indicate that JNK deficiency in ΔPten mice
increases prostate cancer development by increasing tumor growth.
Disruption of the JNK Pathway in the Prostate Epithelium. The in-
creased tumor burden in ΔJnk ΔPten mice compared with ΔPten
mice (Fig. 1) indicates that JNK may play an important role
mice (Cre+and Cre−) was examined by PCR using amplimers designed to detect the deleted Pten and Jnk alleles. (B) Representative prostate glands of WT,
ΔJnk, ΔPten, and ΔPten ΔJnk mice (age 20 wk) are illustrated. (C) Kaplan–Meier analysis of the survival of WT, ΔJnk, ΔPten, and ΔPten ΔJnk mice. The lifespan
of ΔPten ΔJnk mice was significantly shorter than ΔPten mice (P < 0.001). (D) Sections of the anterior prostate of ΔPten and ΔPten ΔJnk mice were stained with
H&E or with antibodies to the androgen receptor (AR) or CK5. (E) Sections were stained with antibodies to pSer473AKT or JNK1/2. (F) Sections were stained
with antibodies to p63, CD44, or Ki67.
Loss of JNK cooperates with Pten deficiency to promote prostate cancer. (A) Genomic DNA isolated from the anterior prostate gland of ΔPten ΔJnk
Hübner et al. PNAS
| July 24, 2012
| vol. 109
| no. 30
during prostate cancer development. However, it is unclear
whether these data reflect a role of JNK signaling because
functions of nonactivated JNK have been reported (24). To test
whether JNK signaling contributes to prostate cancer pro-
gression, we examined the effect of deficiency of the MAP kinase
kinases (MKK4 and MKK7) that phosphorylate and activate
JNK (1). It is established that Mkk4−/−and Mkk7−/−mice are not
viable (1). We, therefore, used conditional alleles to selectively
disrupt Mkk4 and Mkk7 in the prostate epithelium of ΔPten mice
(Fig. 2 A–D). This analysis demonstrated similar prostate cancer
in ΔMkk4 ΔPten mice and ΔMkk7 ΔPten mice. Analysis of tumor
sections indicated that the ΔMkk4 ΔPten and ΔMkk7 ΔPten
neoplastic lesions exhibited greater luminal disruption than
ΔPten tumors (Fig. 2E), but these tumors did not resemble the
invasive carcinoma detected in ΔJnk ΔPten mice (Fig. 1). MKK4
and MKK7 have partially redundant functions, and complete
ablation of the JNK pathway requires compound mutation of
both Mkk4 and Mkk7 (25). We, therefore, examined prostate
tumor development in ΔMkk4 ΔMkk7 ΔPten mice. Compound
deficiency of MKK4 plus MKK7 in the prostate epithelium of
ΔPten mice caused development of invasive adenocarcinoma that
was similar to ΔJnk ΔPten mice (Fig. 2 F–H). The similarity of
prostate tumors in ΔJnk ΔPten mice (Fig. 1) and ΔMkk4 ΔMkk7
ΔPten mice (Fig. 2) indicates that JNK signaling plays an im-
portant role in ΔPten-dependent prostate carcinogenesis.
Androgen Dependence of JNK-Deficient Prostate Tumors. A hallmark
of advanced prostate cancer is the progression to androgen in-
dependence (26). Studies of ΔPten mice demonstrate that cas-
tration (androgen withdrawal) causes dramatic tumor regression
followed by the subsequent development of castration-resistant
prostate cancer (19). In contrast, castration-induced tumor re-
gression was suppressed in ΔJnk ΔPten mice compared with
ΔPten mice (Fig. S4). The ΔJnk ΔPten tumors from castrated
mice exhibited increased glandular disruption, decreased E-
cadherin expression, and disorganized expression of α-smooth
muscle actin compared with ΔJnk ΔPten tumors (Fig. S5). The
aggressive prostate cancers detected in ΔJnk ΔPten mice there-
fore markedly differ from tumors detected in ΔPten mice in their
response to androgen withdrawal. This observation is consistent
with a more advanced tumor phenotype in ΔJnk ΔPten mice
compared with ΔPten mice.
JNK and Prostate Tumor Metastasis. Pathological examination of
ΔJnk ΔPten mice indicated the presence of metastatic cells at sites
distant from the primary tumor. The lumbar and caudal lymph
nodes are established to be preferential sites for metastasis in
orthotopic mouse models of prostate cancer (27, 28). Metastasis of
ΔPten prostate tumor cells to these lymph nodes has been repor-
ted (29). We found that the lumbar lymph nodes of ΔJnk ΔPten
mice were enlarged compared with ΔPten mice at age 20 wk
(Fig. 3A). Androgen-receptor–positive metastatic cells in the lymph
ablation on ΔPten-dependent prostate
cancer. (A and B) Representative images
of prostate glands from ΔMkk4 ΔPten
and ΔMkk7 ΔPten mice (age 20 wk) are
illustrated. (C and D) Genomic DNA iso-
lated from the prostate gland and tail of
ΔMkk4 ΔPten (C) and ΔMkk7 ΔPten mice
(D) were genotyped for Mkk4, Mkk7,
and Pten alleles. (E) Representative H&E-
stained tissue sections of the anterior
prostate glands of ΔPten mice, ΔMkk4
ΔPten mice, and ΔMkk7 ΔPten mice (age
20 wk) are presented. (F–H) Representa-
tive image of a ΔMkk4 ΔMkk7 ΔPten
prostate gland (20-wk-old mouse) is il-
lustrated (F). Genomic DNA isolated
from the prostate gland and tail of
ΔMkk4 ΔMkk7 ΔPten mice were geno-
typed for Mkk4, Mkk7, and Pten alleles
(G). A representative H&E-stained tissue
prostate gland of a ΔMkk4 ΔMkk7 ΔPten
mouse (20 wk old) is presented (H).
Effect of Mkk4 and Mkk7 gene
| www.pnas.org/cgi/doi/10.1073/pnas.1209660109 Hübner et al.
nodes of ΔJnk ΔPten mice were detected (Fig. 3B). These meta-
static cells formed organized duct-like structures with basal ex-
pression of cytokeratin (CK)5 and luminal expression of CK8 that
resemble prostate epithelium (Fig. 3C). In contrast, metastasis was
not detected in ΔPten mice (Fig. 3). The failure to detect metas-
tasis in ΔPten mice is consistent with previous observations (23)
but differs from the low incidence of metastasis detected in
studies of ΔPten mice on a mixed genetic background (19).
Together, these observations are consistent with the conclusion
that ΔJnk ΔPten mice rapidly develop advanced prostate cancer.
JNK Regulates the Tumorigenic Potential of ΔPten Prostate Tumor
Cells. It is established that ablation of Pten causes an increase
in the immature cell compartment within the prostate gland (30).
However, the prostate glands of ΔJnk ΔPten mice were found to
contain a much larger population of immature prostate cells that
stained with antibodies to p63 and cluster of differentiation (CD)
44 compared with ΔPten mice at age 20 wk (Fig. 1F). This ex-
pansion of immature prostate cells was also detected in castrated
mice (Fig. S6) and is, therefore, androgen-independent.
The expansion of the immature cell population in the primary
prostate tumors of ΔJnk ΔPten mice compared with ΔPten mice is
intriguing. To test whether these cells contribute to the tumor
phenotype, we isolated lineage-negative (Lin−) Sca1+cells from
ΔJnk ΔPten and ΔPten primary tumors and cultured these cells
in vitro. Previous studies using WT mice have demonstrated that
this procedure leads to the formation of spheres (prostaspheres)
with a surface location of prostate stem cells and a luminal lo-
cation of more differentiated cells (31). Luminal prostate stem
cells may also be present in these cultures (32, 33). Comparison
of ΔJnk ΔPten and ΔPten prostaspheres demonstrated that JNK
deficiency caused a significant increase in growth during culture
in vitro (Fig. 4A). Microscopic analysis of sections prepared from
ΔJnk ΔPten and ΔPten prostaspheres indicated that JNK de-
ficiency caused altered morphology, including reduced luminal
cell–cell interactions (Fig. 4B) and increased luminal cell apo-
ptosis (Fig. S7). Immunofluorescence analysis demonstrated
strong staining of immature progenitor cell markers (p63, CK5,
nucleostemin, and integrin α6) in a distinct surface zone of ΔJnk
ΔPten prostaspheres compared with ΔPten prostaspheres (Fig. 4
C and D and Fig. S8).
The tumorigenic potential of the Lin−Sca1+cells isolated
from ΔJnk ΔPten and ΔPten prostate tumors was examined us-
ing renal capsule transplantation assays with immunodeficient
host mice (Fig. S9). No growth was detected in studies using
Lin−Sca1+ΔPten cells, a population of cells that includes the
tumor-initiating cells of the ΔPten prostate cancer model (34). In
nodes isolated from ΔJnk, ΔPten, and ΔPten ΔJnk mice (age, 20 wk) are
illustrated. (B) Representative sections of ΔPten ΔJnk lumbar lymph nodes
were stained with H&E or with antibodies to the androgen receptor (AR) or
E-cadherin (ECad). (C) Representative sections of ΔPten ΔJnk lumbar lymph
nodes were stained with antibodies to CK5 and CK8. The merged image
includes the DNA stain DAPI.
Loss of JNK promotes prostate tumor metastasis. (A) Lumbar lymph
(A) The cloning efficiency of Sca1+cells isolated from ΔPten and ΔPten ΔJnk
prostate tumors was measured at passage 2 in vitro. Equal numbers of Sca1+
cells were plated and the number of prostaspheres obtained after 14 d in
culture was examined (mean ± SD; n = 3). Significant differences are in-
dicated (*P < 0.05). (B) Sections of prostaspheres were stained for DNA
(DAPI) and the stem cell markers p63 and nucleostemin (NS). (C and D)
Sections of prostaspheres were stained with H&E or with DNA (DAPI) plus
basal (CK5) and luminal (CK8) differentiation markers.
Loss of JNK promotes proliferation of immature ΔPten prostate cells.
Hübner et al. PNAS
| July 24, 2012
| vol. 109
| no. 30
contrast, Lin−Sca1+ΔJnk ΔPten cells efficiently formed kidney
capsule tumors (Fig. S9).
JNK Is Not Required for the Development of Prostate Neoplasia. Two
protein kinases (MKK4 and MKK7) that phosphorylate and
activate JNK (1) are detected in benign human prostate epi-
thelial cells (35). Studies of human prostate cancer demonstrate
that the expression of MKK4 and MKK7 (but not JNK) is in-
creased in PIN lesions (35). Signaling by the JNK pathway may,
therefore, be increased during the formation of PIN lesions.
Indeed, PTEN inactivation in human prostate tumors is associ-
ated with increased JNK activity (20). Together, these data in-
dicate that JNK may promote the formation of PIN lesions and
may function as an effector of increased PI3K/AKT signaling
caused by PTEN inactivation (20). To test this prediction, we
used a mouse model with conditional deletion of Pten in the
prostate epithelium. This analysis demonstrated that JNK in
prostate epithelial cells is not essential for the formation of
neoplastic lesions. However, it remains possible that JNK in
stromal cells that support epithelial cell morphogenesis and
differentiation (36) may play a role in the development of PIN
lesions. Further studies will be required to test this hypothesis in
the context of prostate cancer, but recent studies have estab-
lished that JNK in stromal cells can promote carcinogenesis (9).
Nevertheless, JNK in prostate epithelial cells is not required for
prostate cancer development caused by Pten inactivation in mice.
JNK Regulates Cancer Progression to Invasive Adenocarcinoma. Al-
though JNK in the prostate epithelium is not essential for the
formation of neoplastic lesions in the conditional Pten deletion
mouse model, it is possible that JNK may contribute to PIN le-
sion maintenance by contributing to the cellular senescence
program that limits prostate cancer progression (22, 23). Indeed,
the senescence of primary ΔPten PIN lesions was not detected in
the prostate glands of ΔJnk ΔPten mice. These data indicate that
JNK signaling may maintain PIN lesions by reducing growth and
inducing senescence and that loss of JNK signaling promotes the
development of invasive adenocarcinoma.
The concept that JNK may restrain progression to adenocarci-
noma is consistent with the observation that MKK4 is down-
regulated in advanced stage human prostate cancer (21). The
mechanism of down-regulation is caused, in part, by translation
inhibition (37) that may be mediated by microRNA pathways, in-
cluding miR15b, miR-24, miR-25, and miR-141 (38). Intriguingly,
increased MKK4 caused by decreased microRNA expression is
associated with senescence (38). Together, these data indicate that
decreased JNK signaling in PIN lesions may contribute to cancer
progression and the formation of adenocarcinoma.
The MKK4 gene has been identified previously as a putative
human metastasis suppressor in prostate cancer (21, 39–41) and
other forms of cancer (42–44). Moreover, mutations in JNK
pathway genes (MKK4, MKK7, JNK1, and JNK2) have also been
identified in human prostate cancer (45–47). A functional role
for MKK4 deficiency is consistent with the finding that decreased
JNK signaling in the ΔPten mouse model promotes the de-
velopment of invasive adenocarcinoma. However, the effect of
MKK4 or MKK7 deficiency on murine prostate cancer was re-
duced compared with JNK deficiency (Figs. 1 and 2). This ob-
servation is consistent with previous reports that deficiency of
MKK4 or MKK7 reduces JNK activity (25). Compound de-
ficiency of MKK4 plus MKK7 is required to ablate JNK signaling
(25) and to fully promote adenocarcinoma development (Fig. 2).
Additional mechanisms may therefore contribute to the pro-
posed effects of MKK4 deficiency in humans (41). Thus, the
dominant-negative activity of catalytically inactive MKK4 pro-
teins (caused by cancer-associated MKK4 gene mutations) may
cause greater suppression of JNK activity than MKK4 gene
ablation (42, 45). Moreover, MKK4 gene mutation in human
prostate cancer may cooperate with other genetic alterations (e.g.,
increased DUSP1 expression) that suppress JNK activity (48).
JNK and PTEN Cooperate to Regulate the Development of Invasive
Adenocarcinoma. It is established that PTEN inactivation is an
important event in the development of human prostate cancer.
Loss of PTEN in the prostate epithelium causes activation of the
PI3K/AKT pathway (19) and inhibition of androgen receptor
signaling (49, 50). Reciprocal regulation of PI3K/AKT and an-
drogen receptor signaling represents a mechanism of signaling
cooperation that regulates prostate cancer development. Simi-
larly, dysregulated Smad4 (23), Erg (51, 52), cMyc (53), and
Trp53 (22) cooperate with PTEN/AKT to promote prostate
cancer. The results of this study identify JNK as another sig-
naling pathway that cooperates with PTEN deficiency to regulate
cancer progression in the prostate gland. The effects of JNK
deficiency to increase both tumor size and invasive adenocarci-
noma/metastasis more closely resembles dysregulated Smad4
than dysregulated cMyc, Erg, or Trp53. This observation indicates
that crosstalk between JNK and TGF-β signaling (54) may con-
tribute to tumor progression. PTEN deficiency therefore promotes
carcinogenesis by altering the cell signaling network. Perturbation
of this signaling network can promote or repress tumorigenesis. In
this context, JNK cooperates with PTEN to regulate progression
to invasive adenocarcinoma.
Compound JNK deficiency in epithelial cells of the prostate
(this study), breast (2), and liver (9) causes an increase in carci-
nogenesis. This observation suggests that JNK may act to reduce
tumor development in these epithelial tissues.
Materials and Methods
Mice. Mice with ablation of the Jnk1 (55) and Jnk2 (56) genes and also
mice with conditional (floxed) alleles of Jnk1 (5), Mkk4 (57), and Pten (58)
have been described. BALB/cJ and NOD.Cg-PrkdcscidIl2rgtm1Wjll/SzJ mice were
obtained from The Jackson Laboratory. Mice with floxed alleles of Mkk7 were
created using homologous recombination in embryonic stem cells, blastocyst
injection of ES cells to create chimeric mice, and breeding to obtain germ-line
transmission of the mutated Mkk7 allele with LoxP sites inserted in intron 3
and intron 7. PB-Cre4 mice were provided by Dr. P. Roy-Burman (59). These
mice were back-crossed (ten generations) to the BALB/cJ strain background.
These mice were crossed to obtain compound mutants: ΔPten (PB-Cre4+
PtenLoxP/LoxP); ΔJnk (PB-Cre4+Jnk1LoxP/LoxPJnk2−/−); ΔJnk ΔPten (PB-Cre4+
Jnk2−/−); ΔMkk4 ΔPten (PB-Cre4+
Mkk4LoxP/LoxP); ΔMkk7 ΔPten (PB-Cre4+PtenLoxP/LoxPMkk7LoxP/LoxP); and ΔMkk4
ΔMkk7 ΔPten (PB-Cre4+PtenLoxP/LoxPMkk7LoxP/LoxPMkk4LoxP/LoxP). Studies of
ΔPten mice demonstrated that tumor development in BALB/cJ mice was similar
to previous reports using mixed strain background mice (19), although pro-
gression to PIN lesions and invasive adenocarcinoma was slower in the BALB/cJ
strain background. Mice were castrated using a surgical procedure (19). The
animal studies were approved by the Institutional Animal Care and Use
Committees of the University of Massachusetts Medical School and the Uni-
versity of California, Los Angeles. The mice were housed in facilities approved
by the American Association of Laboratory Animal Care (AALAC).
Analysis of Tissue Sections. Histology was performed using tissue fixed in 10%
(vol/vol) formalin for 24 h, dehydrated, and embedded in paraffin. Sections
(7 μm) were cut and stained using hematoxylin and eosin (H&E) (American
Master Tech Scientific). Apoptotic cells were detected using the ApopTag
reagent (Millipore). Immunohistochemistry was performed by staining with
a primary antibody [androgen receptor (Santa Cruz; sc-816), CK5 (Covance;
PRB-160), CD44 (eBioscience; 14–004), E-cadherin (BD Transduction; 610181),
P-AKT-Ser473 (Cell Signaling; 3787), JNK1/2 (BD Pharmingen; 554285),
smooth muscle actin (Sigma; A5228), and Ki67 (Vector; VP-RM04)], a bio-
tinylated secondary antibody (Biogenex), streptavidin-conjugated horse-
radish peroxidase (Biogenex), and the substrate 3,3′-diaminobenzidene
(Vector Laboratories), followed by brief counter staining with Mayer’s he-
matoxylin (Sigma). Immunofluorescence analysis was performed by staining
with antibodies to CK5 (Covance; PRB-160) and CK8 (Covance; MMS-162) and
detection using fluorescence-conjugated secondary antibodies (Invitrogen).
Fluorescent images were examined using a fluorescence microscope.
| www.pnas.org/cgi/doi/10.1073/pnas.1209660109Hübner et al.
ACKNOWLEDGMENTS. We thank Dr. P. Roy-Burman for providing PB-Cre4
mice; Dr. D. Garlick for examination of mouse pathology; J. Reilly, L. Paquin,
and V. Benoit for assistance with mouse assays; and K. Gemme for adminis-
trative assistance. This work was supported by National Institutes of Health
Grants CA065861 (to R.J.D.), AI046629 (to D.L.G.), CA112988 (to D.J.M.),
CA107166 (to H.W.), and CA121110 (to H.W.), a California Institute for
Regenerative Medicine training grant (to D.J.M.), and an award from the
Prostate Cancer Foundation (to H.W.). R.J.D. is a member of the National
Institute of Diabetes and Digestive and Kidney Diseases Diabetes and
Endocrinology Research Center (Grant DK032520) at the University of
Massachusetts Medical School. R.A.F., C. L. Sawyers, and R.J.D. are Investigators
of the Howard Hughes Medical Institute.
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Hübner et al.PNAS
| July 24, 2012
| vol. 109
| no. 30