inhibition of the stromal p38mapk/mk2 pathway limits breast … · inhibition of the stromal...
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Inhibition of the stromal p38MAPK/MK2 pathway limits breast cancer metastases and
chemotherapy-induced bone loss
Bhavna Murali1, Qihao Ren1, Xianmin Luo1, Douglas V. Faget1, Chun Wang2, Radia Marie
Johnson3, Tina Gruosso3, Kevin C. Flanagan1, Yujie Fu1, Kathleen Leahy1, Elise Alspach1,
Xinming Su4, Michael H. Ross4, Barry Burnette5, Katherine N. Weilbaecher4, Morag Park3,
Gabriel Mbalaviele2 and Joseph B. Monahan5 and Sheila A. Stewart1,2, 6,7
Author and affiliations: 1Department of Cell Biology and Physiology, 2Division of Bone and
Mineral Diseases, 3Goodman Cancer Center, Department of Oncology, Department of
Biochemistry, McGill University, 4Department of Medicine, 5Aclaris Therapeutics, Inc., Saint
Louis, MO, USA, 6Siteman Cancer Center, 7ICCE Institute, Washington University School of
Medicine, St. Louis, MO 63110 USA.
*Corresponding Author: Sheila A. Stewart, Department of Cell Biology and Physiology,
Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8228, St.
Louis, MO 63110. Phone: 314-362-7437; Fax: 314-362-7463; E-mail:
RUNNING TITLE: Targeting stromal p38MAPK/MK2 limits metastasis
Key Words: stromal, metastasis, p38MAPK, MK2, breast cancer
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ABSTRACT
The role of the stromal compartment in tumor progression is best illustrated in breast cancer
bone metastases, where the stromal compartment supports tumor growth, albeit through
poorly defined mechanisms. p38MAPKα is frequently expressed in tumor cells and
surrounding stromal cells, and its expression levels correlate with poor prognosis. This
observation led us to investigate whether inhibition of p38MAPKα could reduce breast cancer
metastases in a clinically relevant model. Orally administered, small-molecule inhibitors of
p38MAPKα or its downstream kinase MK2, each limited outgrowth of metastatic breast cancer
cells in the bone and visceral organs. This effect was primarily mediated by inhibition of the
p38MAPKα pathway within the stromal compartment. Beyond effectively limiting metastatic
tumor growth, these inhibitors reduced tumor-associated and chemotherapy-induced bone
loss, which is a devastating comorbidity that drastically impacts quality of life for cancer
patients. These data underscore the vital role played by stromal-derived factors in tumor
progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for
metastatic disease and prevention of tumor-induced bone loss.
STATEMENT OF SIGNIFICANCE
Pharmacologically targeting the stromal p38MAPK-MK2 pathway limits metastatic breast
cancer growth, preserves bone quality, and extends survival.
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INTRODUCTION
Breast cancer is one of the leading causes of cancer-related deaths in women in the United
States (1). The mortality is largely attributed to metastasis of the disease from the primary site
to other organs. Currently, there are limited therapeutic options for breast cancer metastases
and it remains a clinical challenge. For this reason, there is a continued and unmet need to
identify novel therapeutic targets that increase disease-free survival while at the same time
limit the morbidities associated with disease progression and therapy.
Tumor progression is a complex process that is governed by both cell autonomous alterations
within tumor cells and ongoing changes in the tumor microenvironment (TME). Importantly,
work over the last decade has revealed that the TME plays a complex active and insidious role
in tumor progression (2,3). Further, it is now clear that tumor cells and stromal cells collaborate
to facilitate proliferation, migration, invasion, immune evasion, and resistance to therapy (4,5).
Tumor-associated stromal cells promote progression by expressing a plethora of tumor-
promoting factors, which display a high degree of overlap with the senescence-associated
secretory phenotype (SASP) (6). Interestingly many of these factors are regulated by the
stress-kinase p38MAPK that we previously revealed support primary tumor growth in a
stromal-dependent manner (7). Additionally, in earlier work using a novel genetic model, we
demonstrated that induction of senescence in osteoblasts induced localized
osteoclastogenesis through secretion of IL-6, a SASP factor known to be responsive to
p38MAPK signaling (8), leading to conditioning of the premetastatic niche and subsequent
increase in bone metastatic outgrowth (9).
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p38MAPK is a central regulator of the inflammatory response and its activation is vital for
expression of an array of inflammatory cytokines and chemokines (10). Orally administered,
small-molecule inhibitors of p38MAPK have been evaluated as potential therapeutic targets in
several chronic inflammatory diseases including Rheumatoid Arthritis (RA) (11), Chronic
Obstructive Pulmonary Disease (COPD) (12), Crohn’s disease (13) and cancer (14). In RA,
several p38MAPK inhibitors were discontinued from clinical trials as a result of side effects,
such as elevated liver enzymes and skin rash. Further, some inhibitors that advanced in trials
displayed only weak clinical efficacy and transient suppression of inflammatory cytokines and
systemic inflammation markers (11). Similar to their effect in RA, p38MAPK inhibitors have not
shown promising results in Crohn’s disease clinical trials (13). However, the p38 inhibitor that
showed transient efficacy in RA, when implemented in a clinical trial for COPD patients
displayed remarkable improvements in symptoms and advanced to Phase III trials (12). These
studies demonstrate that efficacy of p38MAPK inhibitors is disease-specific.
Mouse model studies also revealed that the role of p38MAPK signaling in tumor progression is
complex and variable depending on cell-type and tumor-type (15). Further, p38MAPK inhibitors
have also been investigated for oncological indications such as, multiple myeloma (14) and
advanced metastatic disease (NIH Clinical Trial # NCT01463631). While the outcomes of
these clinical trials are currently unknown, these inhibitors have proven effective in animal
models of human cancer. One study revealed a cell autonomous role for p38MAPK in p53-
deficient tumor cells (16), while others demonstrate that p38MAPK signaling within stromal
cells leads to paracrine support of tumor growth (7,17). A recent study demonstrated that
conditional global deletion of p38MAPK reduced tumorigenesis in the PyMT breast cancer
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model. The degree to which deletion of p38MAPK in tumor cells versus the stromal
compartment contributed to the reduced tumorigenesis was not investigated but this study
underscores the complexity of the p38MAPK pathway (18). In contrast, a study using the 4T1
triple negative model demonstrated that knockdown of p38MAPK within tumor cells had no
impact on orthotopic tumor growth but increased metastatic growth in the lung (19). Taken
together, these findings underscore the complexity of the p38MAPK pathway in cancer and
highlight the need to investigate the therapeutic potential of the pathway in metastatic settings.
Approximately 70% of metastatic breast cancer patients develop bone metastases (20). Once
in the bone, the disease is incurable and treatment options are limited or only palliative.
Metastatic progression typically results in bone loss, leading to a variety of skeletal
complications characterized by bone pain, hypercalcemia and pathological fractures (21).
Chemotherapy is often used after surgical resection in an attempt to prevent relapse (22,23).
However, chemotherapy has many debilitating side effects including the induction of additional
bone loss that severely affects quality of life of patients (24). Because 70% of metastatic
breast cancer patients harbor bone metastases that cause severe osteolytic bone destruction,
it is imperative to explore therapies that can both reduce metastatic burden and prevent bone
loss.
Interestingly, p38MAPK signaling (particularly p38) plays a key role in regulating osteoclast
differentiation mediated by Receptor Activator of NF-κB ligand (RANKL) (25). Numerous
studies have reported that p38MAPK inhibitors are effective at preventing bone loss via
suppression of p38MAPK-induced cytokines. In addition, mitogen-activated protein kinase-
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activated protein kinase 2 (MAPKAPK2 or MK2), a kinase downstream of p38MAPK, also
plays a central role in osteoclastogenesis as evidenced by increased bone mass and
decreased osteoclast number and resorption in MK2-/- mice (26). We therefore postulated that
blocking the p38MAPK/MK2 pathway might limit the tumor-promoting activities of the bone
stromal compartment while simultaneously preserving bone quality, something the current
standard of care cannot achieve.
We sought to investigate the therapeutic benefit of stromal inhibition of the p38MAPK/MK2
pathway in limiting breast cancer metastasis and protecting against bone loss. To address this,
we utilized an aggressive murine breast cancer cell line, PyMT Bo-1 (27) in an intracardiac (IC)
model of bone metastasis. The PyMT-Bo1 cell line mimics the human Luminal B subtype of
breast cancer, for which there are few effective therapies (28). While the IC injection model
does not recapitulate every step in the metastatic cascade, it does allow us to examine tumor
growth post-seeding in the bone, which cannot be achieved in any other model. In this study,
we utilized a p38MAPK inhibitor (p38i) and a novel drug (MK2Pi) that directly disrupts the
p38MAPK-MK2 interface, and discovered that both approaches led to significant decreases in
bone and visceral metastases, similar to that observed in mice treated with the
chemotherapeutic agent, Paclitaxel. In addition, the inhibitors preserved bone density even in
the presence of chemotherapy, which is known to drive bone loss independent of tumor
growth. Our studies suggest that targeting the p38MAPK/MK2 pathway could have clinically
meaningful anti-tumor and bone preserving effects in breast cancer.
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MATERIALS AND METHODS
Mice
Wildtype, female B6(Cg)-Tyrc-2J/J (B6-albino) mice (age 6-8 weeks) were used in all
experiments involving PyMT-Bo1 cell injections and wildtype, female FVB/NJ mice (age 6-8
weeks) were used in all experiments involving Met-1 cell injections. All mice were obtained
from JAX laboratories and were housed in accordance with Washington University in St.
Louis’s Studies Committee and Institutional Animal Care and Use Committee (IACUC).
Cell lines and cell culture
MMTV PyMT-Bo1 mouse breast carcinoma cells were obtained through collaboration with Dr.
Katherine Welibaecher’s laboratory (27). Met-1 mouse breast carcinoma cells were a kind gift
from Dr. Sandra McAllister. Both PyMT-Bo1 cells and Met-1 cells were cultured in DMEM
supplemented with 10% heat-inactivated FBS (Cat#F2442, Sigma, Saint Louis, MO) and
antibiotics (100U/ml of Penicillin and 100 ug/ml of Streptomycin, Cat#P0781, Sigma, Saint
Louis, MO). Both cell lines were used at low-passage and regularly tested by PCR for
Mycoplasma.
Intracardiac injection (IC) and mammary gland injections
On the day of IC injection, six-week-old female mice were anesthetized with 100μL/ 20g body
weight of Ketamine/xylazine cocktail (17.7mg/ml of ketamine and 2.65mg/ml1 of xylazine).
When animals were completely anesthetized, cells were injected directly into the left cardiac
ventricle; either 50μL of PyMT-Bo1 (GFP/Luc) cells (5x104 cells) into B6-albino mice or 50μL of
Met-1 (GFP/Luc) cells (1x105) into FVB/NJ mice. For mammary gland injections 105 PyMT-Bo1
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cells were injected into the 4th inguinal mammary gland and tumor weights were assessed at
sacrifice after dissection.
Osteoclastogenesis and mineralization assays
Bone marrow macrophages (BMM) were obtained by culturing mouse bone marrow cells
isolated from C57BL6 mice in culture media containing a 1:10 dilution of supernatant from the
fibroblastic cell line, CMG 14-12, as a source of M-CSF (29) a mitogenic factor for BMM, for
approximately 5 days in a 10-cm dish as previously described (30). Nonadherent cells were
removed by vigorous washes with PBS, and adherent BMM were detached with trypsin-EDTA,
and cultured in culture media containing a 1:10 dilution of CMG.
To induce osteoclast formation, BMM were plated at 5x103 cells per well in a 96-well plate in
culture media containing a 1:50 dilution of CMG and 100 ng/ml receptor activator of NF-ҡB
ligand (RANKL), a required cytokine for osteoclast differentiation. CDD-450 or CDD-110
resuspended in DMSO was added to cell cultures to yield 0.5% DMSO final concentration.
Control cultures were exposed to 0.5% DMSO final concentration. Media with supplements
were changed every other day and maintained for 4 days at 37°C in a humidified atmosphere
of 5% CO2 in air.
Cytochemical staining for TRAP was used to identify osteoclasts as described previously (30).
Briefly, cells on a 96-well plate were fixed with 3.7% formaldehyde and 0.1% Triton X-100 for
10 minutes at room temperature. The cells were rinsed with water and incubated with the
TRAP staining solution (Sigma leukocyte acid phosphatase kit) at room temperature for 30
minutes. Under light microscopy, multinuclear TRAP-positive cells with ≥ 3 nuclei were scored
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as osteoclasts. MSC cells were treated with 50ug/ml ascorbic acid and 10 mM beta-
glycerophosphate to differentiate into osteoblasts and cells were stained on day 7 for alkaline
phosphatase per the manufactures protocol (Sigma).
Bioluminescence Imaging (BLI)
BLI was performed as previously described (9). In vivo imaging was performed on an IVIS100
or IVIS Lumina (PerkinElmer, Downers Grove, IL; Living Image 3.2, 1-60sec exposures,
binning 4, 8 or, 16, FOV 15cm, f/stop1, open filter). Mice were injected intraperitoneally with D-
luciferin (150mg/kg in PBS; Gold Biotechnology) and imaged 10 minutes later under isoflurane
anesthesia (2% vaporized in O2). Animals were sacrificed immediately following whole body
imaging and both hind limbs were isolated and imaged for 10 seconds ex vivo. For analysis,
total photon flux (photons/sec) was measured from a fixed region of interest (ROIs) over the
whole body or bones using Living Image 2.6 software.
In vitro live-cell bioluminescence imaging was performed on an IVIS 50 as previously
described (31). Briefly, in vitro live-cell bioluminescence imaging was performed on an IVIS 50
(PerkinElmer; Living Image 4.3, 5min exposure, bin8, FOV12cm, f/stop1, open filter). D-
luciferin (150mg/ml; Gold Biotechnology) was added to black-walled plates 10min prior to
imaging. Total photon flux (photons/sec) was measured from fixed regions of interest (ROIs)
over the plate or tumors using Living Image 2.6.
Bone histomorphology and IHC staining
Mouse femur bones were isolated and fixed in 10% neutral buffered formalin for 24 hours.
Bones were decalcified in 14% EDTA for 14 days at 4oC, embedded in paraffin and sectioned
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5μm thick at the histology core of the Washington University Musculoskeletal Research
Center. Standard H&E technique was used for all bone sections. Images were collected using
the Olympus NanoZoomer 2.0-HT System, Alafi Neuroimaging Laboratory.
Immunohistochemical staining was carried out on formalin-fixed, paraffin-embedded slides as
previously described (32). Slides were stained with the following antibodies: anti-IL-6 primary
antibody (ab6672, 1:100, AbCam), pMK2 primary antibody (3007, 1:50, Cell Signaling), Total
p38 primary antibody (9212, 1:100, Cell Signaling), Biotinylated Donkey anti-Rabbit IgG (H+L)
cross-adsorbed secondary antibody (Cat#:31821, 1:500, 2.2μg/ml, Thermofisher).
TMA Staining and Analysis
As previously described (32), patient-derived samples from primary breast cancer were
collected from patients without detectable bone metastases at diagnosis, and matching bone
metastases were collected at a later date (at least 6 months after initial diagnosis). Patient
samples were obtained in accordance with the guidelines established by the Washington
University Institutional Review Board (IRB #201102394; waiver of consent under this IRB#)
and WAIVER of Elements of Consent per 45 CFR 46.116 (d). All patient information was de-
identified prior to investigator use. All of the human research activities and all activities of the
IRBs designated in the Washington University (WU) Federal Wide Assurance (FWA),
regardless of sponsorship, are guided by the ethical principles in "The Belmont Report: Ethical
Principles and Guidelines for the Protection of Human Subjects Research of the National
Commission for the Protection of Human Subjects of Biomedical and Behavioral Research."
All breast cancer and matched bone metastatic samples displayed tumor cells, as determined
by analysis of serial TMA sections stained for H&E, E-cadherin and pan-cytokeratin along with
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either IL-6 or pMK2. Semi-quantitative analysis of the stained TMAs was performed using the
histoscore (H-score) system, which is a measure of extent and intensity of expression. Each
sample was assigned a staining intensity on a scale of 0 to 3 along with the percentage of cells
at that intensity level. The H-score was calculated as follows: H = [1 × (% cells 1+) + 2 × (%
cells 2+) + 3 × (% cells 3+)].
Virus production and Plasmids
Virus production was carried out as described previously (31). Briefly, HEK239T cells were
transfected with Trans-IT LT1 (Mirus) and virus was collected 48h later. Infections were
carried out in the presence of 1g/mL protamine sulfate. 48h post-infection, cells were selected
with 1μg/mL puromycin. Short hairpin RNA sequences targeting murine MK2
Mapkapk2: NM_008551, (5’-AGAAAGAGAAGCATTCCGAAAT-3’) (5’-
CCGGGCATGAAGACTCGTATT-3’ or 5’-CCAGAGAATGACCATCACAGA-3’), p38MAPKa
and control RFP were obtained from the Children’s Discovery Institute’s viral vector-based
RNAi core at Washington University in St. Louis, and were supplied in the pLKO.1-puro
backbone.
Oral Dosage of p38MAPK and MK2 Inhibitor
The p38MAPK small-molecule inhibitor CDD111 (Aclaris Therapeutics, Inc.) was compounded
as described previously (7). The p38MAPK/MK2 small-molecule pathway inhibitor ATI-450 was
compounded at 1000ppm. Female B6 (Cg)-Tyrc-2J/J (B6-albino) and FVB/NJ mice were fed ad
libitum. Mice were randomized onto inhibitor-containing or regular chow, 24 hours-post tumor
cell injection.
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Statistical analyses
All statistical analyses were carried out using Graphpad Prism. Numerical data are expressed
as mean +/- SEM. Mouse analyses were performed by Student’s t test or One-way ANOVA as
indicated in the figure legends. The Kaplan-Meier method was used to estimate empirical
survival probability by treatment and KM curves were generated for visualization. The survival
difference among/between treatment groups were compared by log rank test. Hazard ratios
(HR) between two treatments were estimated from Cox proportional hazard model with 95%
confidence interval. False discovery rate adjusted Log-rank test p values were derived to
adjust for multiple pairwise comparisons.
RESULTS
Expression of p38MAPK-dependent factors in the stromal compartments of primary
breast cancer and bone metastases
In previous work we co-injected tumor cells and activated fibroblasts expressing p38MAPK-
dependent factors and showed that p38MAPK inhibition of stromal-secreted tumorigenic
factors reduced subcutaneous tumor growth in immunocompromised mice (7). Further, we
showed that many p38MAPK-dependent factors were expressed in the stromal compartment
of primary breast cancer lesions (7). Because the stromal compartment is known to support
metastatic growth (2), these findings raised the possibility that strategies that target stromal
p38MAPK in the metastatic setting might similarly limit tumor growth. To evaluate the potential
clinical significance of targeting stromal p38MAPK in the metastatic setting, we first examined
tumor epithelial and stromal expression of IL-6, which is regulated by p38MAPK, in bones of
patients harboring metastatic lesions and compared this to expression in the primary tumors of
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the same patients. To carry out this analysis, we constructed a tissue microarray consisting of
a panel of 58 human breast cancer cases and 38 patient-matched bone metastatic biopsies
and then stained for IL-6, E-cadherin, pan-cytokeratin, and phosphorylated MK2 (pMK2) in
serial sections by immunohistochemistry (IHC). Semi-quantitative analysis of the IHC staining
revealed higher expression of IL-6 in the stromal compartment within the primary and
metastatic site relative to the tumor epithelial compartment (Fig. 1A-C), which was identified by
expression of pan-cytokeratin and E-cadherin (Fig. 1A & B). Of note, the IL-6 expression in
both the primary and metastatic stroma was coincident with the presence of activated (i.e.
phosphorylated) MK2 (Fig. 1B), a downstream target of p38MAPK that is responsible for
stabilizing the mRNAs of many proteins including IL-6 (33,34). Further, robust IL-6 expression
in the stroma was observed across all molecular subtypes of breast cancer samples including,
triple-negative, Luminal A, Luminal B and Her2+ (Fig. 1D). Together these data suggest that
therapeutically targeting the stromal compartment within metastatic lesions with p38MAPK or
MK2 inhibitors might reduce stromal-derived tumor-promoting factors including IL-6 and
metastatic tumor growth.
IL-6 is a pleiotropic cytokine with a predominantly pro-tumorigenic role in the context of breast
cancer and associated bone metastases (35,36). However, there is some evidence, albeit
incompletely understood, that IL-6 trans-signaling may mobilize T cell responses and therefore
display anti-tumorigenic properties (37,38). Given the potential dual faces of IL-6 in the tumor
microenvironment, we wanted to identify other p38MAPK-dependent factors in the stroma of
patients and investigate the putative role that these factors play in breast cancer metastasis.
We used gene set variation analysis (GSVA) to examine gene signatures associated with
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p38MAPK-dependent factors on 53 human breast cancer samples spanning all molecular
subtypes (39) and observed that not only IL-6 (Fig. 1D), but also other p38-dependent tumor-
promoting factors were more highly expressed in the stroma relative to the tumor epithelial
compartment. Furthermore, when stromal-specific gene signatures identified in the Finak (40),
Ma (41) and Karnoub (5) studies were overlaid with our p38MAPK-dependent gene signature,
we found a significant number of p38MAPK-dependent factors were enriched. We identified
these as the Finak/Ma/Karnoub overlap (Fig. 1E & F). These datasets were generated by
microarray comparison of normal and cancer-associated stroma from human breast tissue
following laser capture microdissection (LCM) (5,40,41). As predicted, the three overlap gene
signatures were highly expressed in the stroma relative to epithelium. Together with the IHC
data, these results demonstrate that numerous p38MAPK-dependent factors are expressed in
the stromal compartment of primary and bone metastatic lesions. This preferential expression
suggests that p38MAPK plays an important tumor-promoting role in the stroma not only in the
primary setting but also the metastatic setting.
p38MAPKα Inhibition Limits Bone and Visceral Metastases
Expression of p38MAPK-dependent factors in human primary breast lesions and
corresponding metastatic lesions, coupled with our previous findings that p38MAPK inhibition
can reduce the growth-promoting activities of stromal cells in a primary site (7), led us to
investigate whether inhibiting the p38MAPK pathway in the metastatic setting would also limit
tumor growth. Further, because the bone is the predominant site of metastasis in breast
cancer, we delivered a bone-tropic, murine breast cancer cell line, PyMT-Bo1 (27), into
immunocompetent C57BL/6 mice by intracardiac (IC) injection (Fig. 2A). The IC injection
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model synchronously delivers tumor cells to the bones and visceral organs of animals. One
day after tumor inoculation mice were randomized into a control or treatment group. A highly
selective p38MAPK inhibitor (p38i; also known as CDD111) (7,42), compounded into mouse
chow, was administered ad libitum to mice in the treatment group. To evaluate the efficacy of
single agent p38i as compared to standard chemotherapeutic approaches, we administered
paclitaxel (PTX; 10mg/kg) at 3 day-intervals via retro-orbital injections, either alone or in
combination with p38i. As expected, PTX treatment reduced tumor burden in the bone by 4-
fold as measured by bioluminescence imaging (BLI) on Day 13 post-IC. Strikingly, p38i as a
single agent reduced bone metastases (Fig. 2B) to the same extent as PTX alone. Histological
evaluation of bone metastases within femurs supported the BLI results (Fig. 2C). Furthermore,
p38i’s anti-metastatic effect was not confined to the bone and resulted in systemic reduction of
visceral metastases. Indeed, p38i reduced visceral metastases (non-bone; including lung, liver
and spleen) by 3-fold (Fig. 2D), similar to that obtained with PTX alone (4-fold). We failed to
observe any synergistic effect of p38i and PTX presumably because each as a single agent
dramatically diminished tumor cell growth in vivo.
To ensure that the effect of p38i on metastases was not limited to one cancer cell line, we
carried out the above experiment using the cell line Met-1, originally isolated from an MMTV-
PyMT primary mammary tumor in FVB/NJ mice (43). Following IC injection of Met-1 cells, mice
were randomized onto p38i or control groups and metastatic burdens were measured on Day
12. Similar to PyMT-Bo1 cells, p38i reduced bone metastasis and visceral metastasis by 9-
fold and 13-fold, respectively (Fig. 2E & F). Together, these findings indicate that p38i
significantly reduces metastatic growth in the bone and visceral organs.
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p38MAPK Inhibition Targets the Microenvironment/Stromal cells
Above we demonstrated that p38i drastically reduces the metastatic growth of luminal B breast
cancer cells. Previously in a subcutaneous xenograft setting we demonstrated that p38i
reduced tumor growth in a cell non-autonomous fashion by inhibiting secretion of
protumorigenic factors from stromal cells (7). To determine whether our treatment was
attenuating tumor growth by directly targeting the proliferative ability of tumor cells, we treated
luciferase-expressing PyMT-Bo1 tumor cells for 3 days with p38i (1μM) or PTX (25nM) in vitro
and assessed tumor cell growth by BLI. Our tumor cells express luciferase and we have
previously demonstrated that relative luciferase expression is a reliable surrogate for cell
number (31). As expected, PTX treatment significantly reduced the growth of PyMT-Bo1 cells
by 2.5-fold. In contrast, p38i as a single agent had no impact on the growth of PyMT-Bo1
tumor cells (Fig. 2G). Similarly, the growth of the Met-1 tumor cells was also unaffected by
treatment with p38i (Supplementary Fig. S1). These data demonstrated that the growth of our
luminal B breast cancer cell lines is not directly sensitive to p38i.
To determine if p38MAPK is required within tumor cells for metastatic growth, we transduced
the PyMT-Bo1 tumor line with a p38MAPKα-specific shRNA. p38MAPKα shRNA expression
led to a significant reduction in p38MAPKα protein levels, as observed by Western blot
analysis (Supplementary Fig. S2A) and had no impact on the in vitro growth of the cells
(Supplementary Fig. S2B). In addition, there was no difference in the ability of these cells to
form tumors in the mammary gland (Supplementary Fig. S2C). We introduced these
p38MAPKα-depleted cells into mice via IC injection to examine the ability of these cells to grow
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in metastatic sites. Upon IC delivery of control (shRFP) or sh-p38MAPKα-expressing PyMT-
Bo1 cells into C57BL/6 mice, we measured tumor growth in the overall body and bones 13
days post-IC injection. We found that tumor burdens were similar in mice injected with sh-
p38MAPKα versus shRFP-expressing control cells (Fig. 2H). Histological evaluation of bone
lesions in femurs from shRFP- and sh-p38-tumor bearing mice confirmed the BLI results (Fig.
2I). To establish whether tumor cell expression of p38MAPK was requisite for metastatic
growth, we stained lesions for p38MAPK. We found that p38MAPK expression within tumor
cells was relatively heterogeneous and there were lesions that lacked p38MAPK staining (Fig.
2J). Together these data support the hypothesis that p38i primarily targets the stromal
compartment to reduce metastatic growth. This finding was not limited to the bone because
we also failed to observe a reduction in visceral metastasis in animals injected with sh-
p38MAPKα-expressing PyMT-Bo1 cells (Fig. 2K). In fact upon analysis of the visceral
metastasis, we found that tumor growth was increased in animals bearing sh-p38MAPKα cells.
While the reason for this is unclear, there are reports that p38MAPK plays an active role in
tumor cell dormancy (44,45), raising the possibility that in vivo the reduction of p38MAPK
within breast tumor cells increases tumor cell growth. Together these findings indicate that our
p38i strategy does not directly target tumor cells but rather it is the stromal compartment that is
the target of p38i.
MK2 Inhibition Reduces Bone and Visceral Metastases
p38MAPK targets a large number of downstream factors and more recent work suggests that it
plays a role in maintaining tumor cell dormancy in some models (44,45). Further, the clinical
trials using p38MAPK inhibitors in chronic inflammatory diseases have had mixed results in
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regards to the durability of the treatment, and the efficacy of p38i treatment in patients with
breast cancer remains unknown. For these reasons, we next asked if we could target the
p38MAPK-MK2 axis to limit breast cancer metastasis, since MK2 is downstream of p38MAPK
and stabilizes protumorigenic cytokine mRNAs including IL-6 (46). For this purpose, we used a
recently discovered p38MAPK-MK2 inhibitor, ATI-450 (MK2Pi) (30). To first establish whether
MK2Pi would target tumor cells we treated PyMT-Bo1 cells in vitro with MK2Pi and measured
their growth relative to vehicle control cells via BLI. PyMT-Bo1 cells were grown for 3 days in
the presence of vehicle or MK2Pi (100nM) and growth was measured by BLI. Upon treatment
with MK2Pi, tumor cell growth did not decrease compared to vehicle, indicating that MK2
inhibition alone does not directly affect tumor cell growth (Fig. 3A). To demonstrate this in vivo,
we used shRNA to deplete MK2 in PyMT-Bo1 cells. Knockdown was confirmed by western blot
revealing a 93% reduction in MK2 protein levels in PyMT-Bo1-shMK2 cells relative to control
cells (shRFP) (Supplementary Fig. S2D) In addition, there was no difference in proliferative
ability between control (shRFP) cells and shMK2-expressing PyMT-Bo1 cells in vitro
(Supplementary Fig. S2B) nor was there a difference in the ability of these cells to form
tumors in the mammary gland (Supplementary Fig. S2C). To examine the impact of MK2
depletion on tumor cell growth in vivo, control or shMK2 tumor cells were delivered into mice
by IC injection, and metastatic tumor burden was measured on Day 13 post-IC injection.
Similar to what we found with PyMT-Bo1 cells expressing sh-p38MAPKα, the shMK2 tumor
cell-bearing mice developed bone and visceral metastases comparable to those injected with
control PyMT-Bo1 cells (Fig. 3B & C) and analysis of lesions from mice injected with shMK2
tumor cells revealed lesions that lacked MK2 staining (Supplementary Fig. S2E), indicating
that metastatic lesions can grow when tumor cells have significantly reduced levels of MK2.
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These data demonstrate that inhibiting MK2 in the tumor cells has no effect on their metastatic
potential.
We next asked if MK2Pi could limit metastasis. PyMT-Bo1 tumor cells were injected IC and
twenty-four hours later mice were randomized into either an MK2Pi or control treatment group.
The drug was administered ad libitum for 12 days (Fig. 3D). We found that MK2Pi significantly
reduced metastases in the bone (5-fold) and visceral organs (2.6-fold) compared to mice
receiving control chow (Fig. 3E & F). Histology of tumor-bearing femurs confirmed reduction of
metastases seen via BLI (Fig. 3G). In addition, mice injected with an alternate tumor cell line,
Met-1, showed similar reduction in both bone and visceral metastatic outgrowth when treated
with MK2Pi (Fig. 3H & I). Taken together, these results uncover a cell-non-autonomous action
of MK2 inhibitor in limiting overall metastases.
p38MAPKα and MK2 Inhibition Extends Survival
To assess the impact of p38i versus MK2Pi on overall survival, PyMT-Bo-1 cells were
delivered to mice by IC injection and 24 hours later mice were enrolled into a single or dual
arm treatment strategy and overall survival was assessed. As shown in Figure 4, PTX, p38i
and MK2Pi significantly extended survival compared to animals receiving vehicle alone. When
p38i was combined with PTX we failed to observe a combinatorial effect. In contrast, the
combination of MK2Pi and PTX significantly extended survival compared to the single arm
treatments (Fig. 4 and Supplementary Fig. S3). The reason for this extension is not clear but
we did find that MK2Pi provided enhanced bone protection relative to p38i treatment (Fig. 5,
below), thereby preventing paralysis of hind limb and allowing the mice to remain active and
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mobile for longer. Alternatively, MK2Pi may be more effective at limiting the p38MAPK-MK2
pathway. Indeed, we found that Hsp27 phosphorylation, which plays a role in stabilizing
numerous pro-tumorigenic cytokines including IL-6 (47), was lower in the lungs of tumor
bearing mice treated with MK2Pi relative to p38i (Supplementary Fig. S4A & B).
p38MAPKα and MK2 Inhibition Maintains Bone Integrity in Tumor-Bearing Mice
Bone metastasis often leads to increased osteoclastogenesis leading to osteolytic-driven bone
destruction (21); and chemotherapy is known to exacerbate bone loss in metastatic patients.
The p38MAPK-MK2 pathway plays an important role in bone homeostasis, particularly
RANKL-induced osteoclast differentiation (25). Thus, it is not surprising that MK2-deficient
mice have increased trabecular and cortical bone mass and decreased osteoclast number and
function (26). Based on these reports, we tested the effects of p38i or MK2Pi on in vitro
RANKL-induced osteoclast differentiation of bone marrow derived macrophages. We found
that both p38i and MK2Pi inhibited osteoclast differentiation in a concentration dependent
manner (Fig. 5A). We also found that these inhibitors decreased osteoclast bone-resorbing
activity in vitro (Fig. 5B). Because p38MAPK has been implicated in osteoblast function (48),
we also examined the impact of our drugs on the ability of osteoblast to differentiate. In
contrast to what we observed with osteoclasts, these inhibitors had no effect on
osteoblastogenesis as measured by alkaline phosphatase activity (Supplementary Fig. S5).
The well-established role of the p38MAPK-MK2 pathway in osteoclastogenesis, coupled with
our finding that the inhibitors limited osteoclastogenesis (Fig. 5A & B) led us to ask if p38i and
MK2Pi could attenuate the devastating bone loss observed in the metastatic setting. To test
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this, we delivered PyMT-Bo1 cells by IC injection and 24 hours later, randomized the mice into
the following groups – Vehicle, PTX, p38i, MK2Pi and Zoledronic acid (ZOL). ZOL is a widely
used bisphosphonate that can effectively limit bone loss by inhibiting osteoclast activity. To
compare the activities of p38i and MK2Pi to ZOL, we administered two doses of 0.75-μg ZOL
(or vehicle) sub-cutaneously, once each week (Fig. 5C). On day 13, tumor burden in the bones
was measured by BLI. Following that, bones were processed for density analysis using micro-
computed tomography (CT). We measured trabecular bone volume of tumor-bearing mice in
all groups except vehicle because the femurs of vehicle-treated mice had large, invasive
tumors that destroyed nearly all measureable bone making them unsuitable for CT analysis.
Instead, the bone volume of the other four groups was compared to femurs from non-tumor
bearing mice. We observed that while ZOL effectively limited tumor-induced bone loss (Fig.
5D) it did not impede tumor growth in the bone (Supplementary Fig. S6). Given that
chemotherapy can induce bone loss in mice and patients, it was not surprising to find that PTX
exacerbated bone loss by 2.5-fold relative to untreated, non-tumor bearing mice. Importantly,
treatment with either p38i or MK2Pi reduced tumor burdens (Fig. 2B & 3E, respectively) and
preserved bone density to the same extent as ZOL in tumor-bearing mouse bones (Fig. 5D).
Three-dimensional reconstructions of tumor-bearing femurs from each of the groups
corroborated the CT results (Fig. 5E). Together, these findings demonstrate that p38i and
MK2Pi provide a dual benefit, in that they not only attenuate disease progression by limiting
stromal support of tumor growth but they also effectively protect against bone loss likely by
inhibiting osteoclastogenesis, even in the face of chemotherapy, making them attractive,
stromal-targeted therapies to pursue.
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DISCUSSION
Several studies have established that the stroma plays a significant role in tumor progression,
thereby, establishing a rationale for developing stroma-targeted anti-tumor therapies. In this
study, using a preclinical model of Luminal B-like breast cancer, we demonstrated that
inhibiting the p38MAPK-MK2 pathway limits visceral and bone metastases. Importantly, we
show that depleting p38MAPK or MK2 in the tumor cells had no effect on metastatic outgrowth,
providing evidence that the inhibitor’s target is indeed the stroma and not the tumor cells
directly. This is in contrast to chemotherapy, which directly targets tumor cells. Indeed,
paclitaxel, the chemotherapeutic agent used in this study, limited metastasis as effectively as
the tested p38MAPK and MK2 inhibitors. Despite paclitaxel’s ability to limit tumor growth, it
failed to provide any survival advantage to our mice, underscoring its overall toxicity and the
effectiveness of the MK2Pi, which not only reduced tumor burden but also significantly
extended survival. Given that chemotherapy directly targets tumor cells, which tend to be
genetically malleable to imposed selective pressures, leading to drug resistance (49), our
findings suggest that stromal-targeted therapies might provide a more durable response in
patients. Therefore, targeting stromal cells could help circumvent the challenge of drug
resistance. In addition, stromal status and composition of distal organs is implicated in
determining the fate of disseminated tumor cells. Stromal-targeted therapies can be used to
block the metastatic cascade at an early stage by impeding the development of fertile niches
where tumor cells tend to thrive and eventually outgrow into macrometastases. In this way,
stromal therapy has the potential to synergize with tumor-targeted therapies to ensure more
effective and widespread killing of tumor cells.
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While many studies favor the development and use of p38 inhibitors in treating cancers, some
reports provide contradictory evidence suggesting that blocking p38 may confer a growth
advantage to tumor cells (50,51). These divergent results should be evaluated in light of the
fact that p38MAPK signaling is different across cell-types and tumor-types, thereby making it
challenging to generalize findings. Our work demonstrates that limiting p38MAPK within tumor
cells without simultaneously limiting it in the stromal compartment can increase metastatic
tumor growth (Fig. 2K). Indeed, in contrast to the limited metastatic growth we observe upon
p38i, we find that visceral metastases increase in mice injected with p38MAPKα-depleted
tumor cells. This result may not be surprising given recent evidence demonstrating that
inhibition of p38MAPKα within tumor cells can increase their invasiveness (50). In addition,
work from Guiso et al., suggests that active p38MAPKα keeps tumor cells in a dormant state
by phosphorylating a number of factors including ATF2 (52) that are not substrates for MK2. In
light of this finding, inhibition of p38MAPK could be seen as deleterious as it may “awaken”
cells out of dormancy that may have otherwise continued in an indolent state. If true, the use of
our MK2Pi may be a better approach in patients with minimal residual disease rather than
those with active metastatic lesions. Another potential advantage of p38i/MK2Pi is that if they
were to drive non-dividing tumor cells – be it dormant or otherwise – into the cell cycle, they
may increase the killing potential of chemotherapies that rely on cell cycling. This is an
important area that will require further investigation.
Reducing metastatic tumor burden is the goal of all cancer therapies. However, the
devastating side effects of many of the therapies used negatively impact a patient’s quality of
life. In breast cancer patients the risk for skeletal-related events (SREs) – pathological
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fractures, hypercalcemia and bone pain – due to tumor-induced as well as therapy-induced
osteolysis (21) remains a significant problem. For this reason, bone-preserving therapies such
as the bisphosphonate zoledronic acid (ZOL) are now standard of care in the metastatic
setting (53). While ZOL effectively protects bone quality, there is conflicting evidence about its
efficacy at limiting tumor growth in models of bone metastases and in rare instances it can
result in significant toxicity. Studies suggest that the anti-tumor effects of ZOL depend on the
size of bone lesions and whether the treatment is preventive (more effective) versus
therapeutic (54,55). Given the severity of the skeletal complications observed in many patients,
there is a clear need for new breast cancer therapies that combat not only tumor growth but
also the associated comorbidities. Strikingly, we show that blocking the p38MAPK-MK2
pathway with either inhibitor (p38i or MK2Pi) limited osteoclastogenesis and had a significant
protective effect on the bone. The dual action of p38i and MK2Pi makes them promising
candidates to pursue for clinical trials. However, given we found that sh-p38MAPK led to
increased metastatic burden in the visceral organs and the fact that MK2Pi combined with PTX
extended survival, our data suggest that MK2 may be a more viable target. Finally, given the
potent inhibition of metastases observed with the MK2 inhibitor throughout the mouse, further
studies are warranted to investigate the specific stromal cell types targeted by the drug beyond
osteoclasts to gain a mechanistic understanding of its action that will help shed light on where
and how best to employ it in breast cancer patients.
ACKNOWLEDGEMENTS
We thank Deborah (Novack) Veis, Joshua Rubin, Daniel Link, Roberta Faccio and David
DeNardo for their valuable suggestions. We thank Lynne Marsala, Julie Prior and the ICCE
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Institute at Washington University School of Medicine for live cell and live animal imaging. We
thank Crystal Idleburg and Samantha Coleman at the Musculoskeletal Histology core for their
expert technical assistance with bone tissue sectioning and staining and Deborah Veis,
Thomas Walsh and Graham Colditz for assistance in constructing the human TMA. In addition,
we thank Daniel Leib and the Structure and Strength Musculoskeletal core for μCT imaging.
shRNA constructs were obtained from the Children’s Discovery Institute’s viral vector-based
RNAi core at Washington University in St. Louis. We thank the Genome Technology Access
Center in the Department of Genetics at Washington University School of Medicine for help
with genomic analysis. Finally, we thank Lorry Blath and Judy Johnson for their constant
support, enthusiasm and critical assessment of our work and its impact on breast cancer
patients. The Center is partially supported by NCI Cancer Center Support Grant #P30
CA91842 to the Siteman Cancer Center and by ICTS/CTSA Grant# UL1TR000448 from the
National Center for Research Resources (NCRR), a component of the National Institutes of
Health (NIH), and NIH Roadmap for Medical Research. This publication is solely the
responsibility of the authors and does not necessarily represent the official view of NCRR or
NIH.
Financial support: This work was supported by the Cancer Biology Pathway Molecular
Oncology Training Grant NIH T32CA113275 (B. Murali), NIH grants NIH 5 R01 CA130919
(S.A. Stewart), NIH Cellular Biochemical and Molecular Sciences Pre-doctoral Training Grant
T32 GM007067 (K.C. Flanagan and E. Alspach), NIH F31 CA189669 (K.C. Flanagan),
American Cancer Society Research Scholar Award (S.A. Stewart), CA100730 (K.N.
Weilbaecher), CA097250 (K.N. Weilbaecher) and training grants 5T32GM007067-39 (M.H.
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Ross), T32AR060719 (M.H. Ross). The work was supported in part by the Siteman Investment
Program (supported by The Foundation for Barnes-Jewish Hospital Cancer Frontier Fund
(FBJH CFF 3773); Barnard Trust; Washington University Musculoskeletal Research Center
(NIH P30 AR057235); Fashion Footwear Charitable Foundation of New York, Inc.; and, the
National Cancer Institute Cancer Center Support Grant P30CA091842, Eberlein, PI) (S.A.
Stewart) and the St. Louis Breast Tissue Registry (funded by The Department of Surgery at
Washington University School of Medicine). T.Gruosso has been supported by the Charlotte
and Leo Karassik Foundation oncology postdoctoral fellowship. The study involving laser
capture microdissection followed by gene expression was supported by grants to M.Park from
the Québec Breast Cancer Foundation, Genome Canada–Génome Québec, NIH (National
Institutes of Health), SU2C (Stand Up 2 Cancer) and CIHR (Canadian Institutes of Health
Research). The breast tissue and data bank at McGill University is supported by funding from
the Database and Tissue Bank Axis of the Réseau de Recherche en Cancer of the Fonds de
Recherche du Québec-Santé and the Quebec Breast Cancer Foundation (to M.Park). GM is
supported by NIH/NIAMS AR064755 and AR068972 grants. Luminescent imaging was
supported by NIH P50 CA094056. Imaging and analysis of human breast cancer and bone
biopsy slides were performed using Zeiss Axio ScanZ.1 through the use of Washington
University Center for Cellular Imaging (WUCCI) supported by Washington University School of
Medicine, The Children’s Discovery Institute of Washington University and St. Louis Children’s
Hospital (CDI-CORE-2015-505) and the National Institute for Neurological Disorders and
Stroke (NS086741). Finally, the U.S. Army Medical Research Acquisition Activity, 820
Chandler Street, fort Detrick MD 21702-5014 is the awarding and administrating acquisition
office and this was supported in part by the Office of the Assistant Secretory of Defense for
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Heath Affairs, through the Breast Cancer Research Program, under award No. W81XWH-16-
1-0728. Opinion, interpretations, conclusions, and recommendations are those of the author
and are not necessarily endorsed by the Department of Defense.
Conflicts of interest: Dr. Joseph Monahan is the Executive Vice President of R&D of Aclaris
Therapeutics, Inc., Radia Johnson is an employee of Genetech, Gabriel Mbalaviele is a
consultant for Aclaris Therapeutics Inc., and Barry Burnette is an employee of Aclaris
Therapeutics Inc.
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FIGURE LEGENDS
Figure 1: p38MAPK-dependent protumorigenic factors are more highly expressed in the
stroma relative to epithelium. (A,B) Representative images of primary breast lesions, n=53,
and bone metastatic biopsies, n=33, Scale= 15 µm (Inset scale = 100µm). Serial sections were
stained with antibodies against IL-6 (A) or phosphorylated MK2 (pMK2) (B), along with
hematoxylin and eosin (H&E), E-cadherin, or pan-cytokeratin (C) IHC for IL-6 in tumor versus
stroma on primary breast and patient-matched bone metastatic lesions. Semi-quantitative
analysis using histoscore (H-score) system. Two-tailed Wilcoxon signed-rank test, ****
p<0.0001. D) IL-6 expression across molecular subtypes of breast cancer. Two-way ANOVA,
***p≤ 0.001. (E) IL-6 expression in tumor stroma versus tumor epithelium (epi) in the pan-
Breast cancer dataset. Boxplot, t-test, **** p< 2.2e-16. Below the boxplot is a list of p38MAPK-
dependent stromal factors expressed in the three datasets analyzed. (F) GSVA analysis for
enrichment of p38MAPK-dependent protumorigenic factors in stroma and epi from breast
tumor samples (right). Gene list with overlapping genes (bottom left). Significance was
determined by comparing the GSVA enrichment scores of stroma versus epi within each
signature, one-way ANOVA with Tukey post-hoc test, ***p≤ 0.0001. All data are displayed as
mean±SEM.
Figure 2: p38MAPK inhibition in the stromal compartment reduces metastatic outgrowth
as effectively as a standard chemotherapy agent. PyMT-Bo1 cells were injected into the left
cardiac ventricle. Tumor burden was analyzed by BLI on day 13-post injection and is
represented as photons per second. (A) Schematic of experimental timeline and dosing
regimen for Paclitaxel (PTX; 10mg/kg) and p38i. (B) Ex vivo bone metastatic tumor burden
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(left), representative images (right). n7 per group. (C) Hematoxylin and eosin (H&E) staining
of vehicle, PTX, p38i and PTX+p38i-treated mouse femurs. Black outline marks the tumor
area. Scale bar = 250μm. n = 4-6. (D) Tumor burden in visceral organs (top), representative
images (bottom). n7 per group (B,D) Significance was determined by one-way ANOVA with
Tukey post-hoc test, as compared to vehicle, ***p ≤ 0.001, **p≤ 0.01. (E,F) Met-1 cells were
injected into the left cardiac ventricle. Tumor burden was analyzed by BLI on day 13-post
injection. Unpaired, two-tailed t-test (compared to vehicle). **p=0.0013, ***p≤ 0.0001, n9 per
group. (E) Bone metastatic and (F) visceral organ tumor burden. (G) PyMT-Bo1 cells
expressing luciferase were cultured in vitro in the presence of PTX (25nM), p38i (1μM) or
DMSO control. Following 72 hours of treatment, luciferase expression was measured by BLI to
evaluate tumor cell proliferation. One of two biological replicates, each in technical octuplicate
is shown. One-way ANOVA with Tukey post-hoc test, ***p≤ 0.0001, ns = not significant. (H-K)
Mice were injected with shp38α-expressing PyMT-Bo1 tumor cells and metastatic burden was
analyzed on day 13 by BLI. (H) Ex vivo Bone metastatic (I) H&E staining of femurs from mice
injected with shRFP-expressing or shp38MAPKα-expressing PyMT-Bo1 cells. Scale bar =
250μm. n≥4. All data are represented as mean±SEM. (J) Representative bone sections with
shRFP and sh-p38MAPK tumors stained with anti-p38MAPK. Scale = 50 um. (K) in vivo
visceral organ tumor burden with representative images. Unpaired, two-tailed t-test (compared
to vehicle). **p=0.0074, ns = not significant, n≥5 mice per group.
Figure 3: MAPKAPK2 (MK2) inhibition reduces metastatic outgrowth in bone and
visceral organs. (A) PyMT-Bo1 tumor cells were cultured in vitro for 72 hours in the presence
of PTX (25nM), MK2Pi (100nM) or DMSO control. Growth was assessed by BLI. One of two
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biological replicates, each in technical octuplicate is shown. One-way ANOVA with Tukey post-
hoc test, ***p≤ 0.0001. (B, C) Mice were injected with shpMK2-expressing tumor cells. Day 13
metastatic burden was analyzed by BLI. (B) Ex vivo Bone metastatic, (C) in vivo visceral organ
tumor burden with representative images. Unpaired, two-tailed t-test, ns = not significant, n≥5
mice per group. All data are represented as mean±SEM. (D) Schematic representation of
experimental timeline and dosing of MK2Pi. (E-G) PyMT-Bo1 cells, 13 days post-injection, BLI
analysis of (E) ex vivo bone metastatic burden (F) and in vivo visceral organ metastatic
burden. Representative images are shown. Unpaired, two-tailed t-test, *** p=0.0005, *
p=0.0374, n≥5 mice per group. (G) H&E staining of femurs from vehicle and MK2Pi-treated
mice. Black outline indicates tumor area. Scale bar = 250μm. n≥4. (H,I) Met-1 cells, 13 days
post-injection, BLI analysis with representative images. Significance was determined by two-
tailed Mann Whitney U-test. n≥7 per group. (H) Bone metastatic burden, *** p = 0.0007. (I)
Visceral organ tumor burden, *** p = 0.0002.
Figure 4: MK2 and paclitaxel increase overall survival. Survival analysis of mice injected
IC with PyMT-Bo1 tumor cells and administered Vehicle (Veh), paclitaxel (PTX), p38i, MK2Pi,
p38i + PTX, or MK2Pi + PTX. p38i and MK2Pi were administered ad libitum. Log-rank (Mantel-
cox) test, ** p = 0.0095, n≥15 mice per group.
Figure 5: p38 and MK2 inhibitors maintain bone density. (A) Bone marrow derived
macrophages were treated with RANK ligand to induce differentiation and stained with
Tartrate-resistant acid phosphate (TRAP) in the presence of p38i or MK2Pi. Left are
representative images (magnification = 10X) of cells treated with vehicle, 0.01 uM p38i or 0.01
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uM MK2Pi and the right is quantification of TRAP positive cells treated with increasing
concentration of inhibitors (p38i or MK2Pi were used at 0.01, 0.1, 1 or 10 uM). One-way
ANOVA with Tukey post-hoc test, ***p≤ 0.0001 (B) Osteoclast bone-resorbing activity was
assessed by measuring pit area and number. Representative images are shown on the right
of osteoclasts treated with vehicle, p38i (0.01 uM) or MK2Pi (0.01 uM) and quantification of pit
area and number of pits. Significance was determined by unpaired, two-tailed t-test (compared
to vehicle). Pit area stats: *p=0.0138 (p38i), *p=0.0167 (MK2Pi); Pit number stats: **p=0.0003
(p38i), **p=0.0062 (MK2i) (C) Schematic representation of experimental set up and dosing
regimen for Zoledronic acid (Zol; 0.75μg). (D) Mouse femurs were scanned by μCT and
trabecular bone volume (BV/TV) was calculated. One-way ANOVA with Tukey post-hoc test, *
p≤ 0.05, ** p≤ 0.001. (E) Representative 3D reconstructions, generated using OsiriX, of 0.9mm
thick section of femur right below the growth plate for each of the treatment groups. n=5 per
group. All data are represented as mean±SEM.
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Published OnlineFirst August 9, 2018.Cancer Res Bhavna Murali, Qihao Ren, Xianmin Luo, et al. cancer metastases and chemotherapy-induced bone lossInhibition of the stromal p38MAPK/MK2 pathway limits breast
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