Inhibition of the stromal p38MAPK/MK2 pathway limits breast … · Inhibition of the stromal p38MAPK/MK2 pathway limits breast cancer metastases and chemotherapy-induced bone loss

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:

[email protected]

RUNNING TITLE: Targeting stromal p38MAPK/MK2 limits metastasis

Key Words: stromal, metastasis, p38MAPK, MK2, breast cancer

on July 6, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0234

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http://cancerres.aacrjournals.org/

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.




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).




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




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-




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.




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




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




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




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




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.




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




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




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




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.




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




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




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.




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




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




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.




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.




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




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




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.




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




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




(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




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




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.



















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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Inhibition of the stromal p38MAPK/MK2 pathway limits breast … · Inhibition of the stromal p38MAPK/MK2 pathway limits breast cancer metastases and chemotherapy-induced bone loss