EPCR promotes breast cancer progression by altering SPOCK1/testican 1-mediated 3D growth
Perurena et al. Journal of Hematology & Oncology
EPCR promotes breast cancer progression by altering SPOCK1/testican 1-mediated 3D growth
Naiara Perurena 0 4
Carolina Zandueta 0 4
Susana Martínez-Canarias 0 4
Haritz Moreno 0 4
Silvestre Vicent 0 1 4 7
Ana S. Almeida 8
Elisabet Guruceaga 3
Roger R. Gomis 5
Marta Santisteban 6
Mikala Egeblad 8
José Hermida 2
Fernando Lecanda 0 1 4 7
0 Adhesion and Metastasis Laboratory, Program Solid Tumors and Biomarkers, Center for Applied Medical Research (CIMA), University of Navarra , 31008 Pamplona , Spain
1 IdiSNA, Navarra Institute for Health Research , Pamplona , Spain
2 Cardiovascular Sciences Program, Center for Applied Medical Research, University of Navarra , Pamplona , Spain
3 Proteomics , Genomics and Bioinformatics Core Facility, Pamplona , Spain
4 Adhesion and Metastasis Laboratory, Program Solid Tumors and Biomarkers, Center for Applied Medical Research (CIMA), University of Navarra , 31008
5 Oncology Program, Institute for Research in Biomedicine , Barcelona , Spain
6 Department of Oncology, Clínica Universidad de Navarra , Pamplona , Spain
7 Department of Histology and Pathology, University of Navarra , Pamplona , Spain
8 Cold Spring Harbor Laboratory , Cold Spring Harbor, NY , USA
Background: Activated protein C/endothelial protein C receptor (APC/EPCR) axis is physiologically involved in anticoagulant and cytoprotective activities in endothelial cells. Emerging evidence indicates that EPCR also plays a role in breast stemness and human tumorigenesis. Yet, its contribution to breast cancer progression and metastasis has not been elucidated. Methods: Transcriptomic status of EPCR was examined in a cohort of 286 breast cancer patients. Cell growth kinetics was evaluated in control and EPCR and SPARC/osteonectin, Cwcv, and kazal-like domains proteoglycan (SPOCK1/testican 1) silenced breast cancer cells in 2D, 3D, and in co-culture conditions. Orthotopic tumor growth and lung and osseous metastases were evaluated in several human and murine xenograft breast cancer models. Tumor-stroma interactions were further studied in vivo by immunohistochemistry and flow cytometry. An EPCRinduced gene signature was identified by microarray analysis. Results: Analysis of a cohort of breast cancer patients revealed an association of high EPCR levels with adverse clinical outcome. Interestingly, EPCR knockdown did not affect cell growth kinetics in 2D but significantly reduced cell growth in 3D cultures. Using several human and murine xenograft breast cancer models, we showed that EPCR silencing reduced primary tumor growth and secondary outgrowths at metastatic sites, including the skeleton and the lungs. Interestingly, these effects were independent of APC ligand stimulation in vitro and in vivo. Transcriptomic analysis of EPCR-silenced tumors unveiled an effect mediated by matricellular secreted proteoglycan SPOCK1/testican 1. Interestingly, SPOCK1 silencing suppressed in vitro 3D growth. Moreover, SPOCK1 ablation severely decreased orthotopic tumor growth and reduced bone metastatic osteolytic tumors. High SPOCK1 levels were also associated with poor clinical outcome in a subset breast cancer patients. Our results suggest that EPCR through SPOCK1 confers a cell growth advantage in 3D promoting breast tumorigenesis and metastasis. Conclusions: EPCR represents a clinically relevant factor associated with poor outcome and a novel vulnerability to develop combination therapies for breast cancer patients.
Matricellular; Metastasis; Microenvironment; Sphere cultures; Extracellular matrix
Endothelial protein C receptor (EPCR) is an endothelial
type 1 transmembrane receptor that enhances the
activation of protein C (PC) by the thrombin
(IIa)-thrombomodulin (TM) complex . EPCR-dissociated activated
protein C (APC) negatively regulates the coagulation
process, while EPCR-bound APC induces cytoprotective
signaling through the proteolytic cleavage of
proteaseactivated receptor 1 (PAR1), leading to anti-inflammatory
and anti-apoptotic responses .
Recently, research in EPCR has gained considerable
momentum by the identification of new EPCR ligands
. An EPCR domain distinct from the APC binding site
was shown to interact with a specific T cell antigen
receptor with potential implications in
immunosurveillance of tumors . EPCR was also identified as the
endothelial receptor for some subtypes of the
erythrocyte membrane protein 1 (PfEMP1) on the surface of
the parasite Plasmodium falciparum, mediating its
sequestration in the blood vessels during severe malaria
. FVII/FVIIa has been shown to bind EPCR with a
similar affinity as PC/APC , whereas the binding of
FX/FXa to EPCR remains an open question .
Recently, EPCR has been identified as a marker of
multipotent mouse mammary stem cells (MaSCs). These
EPCR+ cells (accounting for 3–7% of basal cells) exhibited
a mesenchymal phenotype and enhanced colony-forming
abilities . EPCR was also shown to be necessary for cell
organization and growth of human mammary epithelial
cells in 3D cultures .
In cancer, aberrant expression of EPCR is detected in
tumors of different origin including the lung , breast
, ovarian , colon , glioblastoma ,
mesothelioma , and leukemia . In lung tumorigenesis,
APC/EPCR drives an anti-apoptotic program that
endows cancer cells with increased survival ability,
enhancing their metastatic activity to the skeleton and adrenal
glands . Moreover, high expression levels of this
single gene at the primary site in early stage lung
cancer patients predict the risk of adverse clinical
progression [9, 15].
In breast cancer patients, tumor cells often
disseminate to target sites including the skeleton, lungs, brain,
and lymph nodes . This event represents a frequent
complication associated with a 5-year survival rate
~25.9%. Recent findings have unveiled novel markers in
the primary tumor that predict the development of
metastasis to target organs such as the skeleton . High
EPCR levels have been associated with poor disease
progression in the polyoma middle T (PyMT) breast cancer
model, closely similar to the luminal B type in humans
. Moreover, EPCR+ sorted MDA-MB-231 human
breast cancer cells showed stem cell-like properties and
enhanced tumor-initiating activity, an effect inhibited by
APC-EPCR blocking antibodies . In contrast,
overexpression of EPCR in MDA-MB-231 cells resulted in
reduced final tumor volumes in a xenograft model despite
favoring tumor growth at initial stages . The effect of
EPCR at different stages of tumor progression remains
In this study, we addressed the functional role of
EPCR in primary and metastatic tumor growth in breast
cancer using several human and murine xenograft
models. We found that EPCR silencing impaired
orthotopic tumor growth and metastatic activity to the
skeleton and lungs. Moreover, high EPCR expression levels
associated with a poor clinical outcome in a cohort of
breast cancer patients. Furthermore, we showed that
EPCR effects in tumor progression were APC
independent and were partially mediated by a novel mechanism
involving SPOCK1. Thus, these findings unveil a novel
mechanism mediated by EPCR in tumorigenesis and
metastasis of breast cancer with potential clinical impact on
the therapeutic management of breast cancer patients.
Cell lines and reagents
One thousand eight hundred thirty-three human breast
cancer cell line was a kind gift from Dr. Massagué
(Memorial Sloan-Kettering Cancer Center, NY, USA) .
ANV5 murine breast cancer cell line was previously
described [21, 22]. APC (Xigris®) was purchased from Eli Lilly
(Indianapolis, IN, USA). Anti-EPCR antibodies RCR252
and RCR1 were kindly provided by Dr. Fukudome (Saga
Medical School, Japan) while 1489 was kindly gifted by
Dr. Esmon (Oklahoma Medical Research Foundation,
Oklahoma City, USA). F(ab´)2 fractions of the RCR252
antibody were obtained as previously detailed .
shRNAs cloned into PLKO.1-puro vector and the
empty vector were obtained from Mission®
Cell proliferation assay
Cell proliferation was assessed using CellTiter 96®
AQueous One Solution Cell Proliferation Assay (MTS),
according to manufacturer’s recommendations
(Promega). All absorbance values were normalized with the
absorbance values from day 0 (5 h after seeding cells).
Cell cycle analysis
Cell cycle analysis was carried out with Click-iT® EdU
Flow Cytometry Assay Kit (Invitrogen). Cells were
maintained in culture for 24 or 48 h before adding 10 μM
EdU for 2 h. Next, cells were harvested, fixed in
formaldehyde (Click-iT® fixative), permeabilized in 1X Click-iT®
saponin-based permeabilization and wash reagent, and
incubated with the Click-iT® reaction cocktail for 30 min
at room temperature in the dark. After a washing step,
cells were incubated with 0.2 μg/μl RNase A
(SigmaAldrich) for 1 h at room temperature, in the dark.
7AAD was added to the tubes 10 min before the
acquisition of cells in a FACSCanto II cytometer (BD
Biosciences). Data were analyzed using FlowJo® software v9.3.
Annexin-V flow cytometry assay
Cells were seeded into 24-well plates and cultured for
24 h. Next, cells were incubated with 2 μM
staurosporine for 1 h or serum-starved overnight before the
addition of 50 nM APC for 4 h followed by 2 μM
staurosporine for 1 h next day. After staurosporine treatment,
cells were harvested, resuspended in annexin-binding
buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM
CaCl2, pH 7.4) and incubated with Alexa Fluor
647conjugated annexin-V and 7AAD (BD Biosciences) for
15 min at room temperature, in the dark. Cells were
acquired in a FACSCanto II cytometer (BD Biosciences)
and analyzed using FlowJo® software v9.3.
Cell culture in 3D
Culture media was mixed at 1:1 ratio with Growth
Factor Reduced Matrigel (BD Biosciences). One hundred
microliters of the mix were added to each well of a
96well plate and incubated at 37 °C for 30 min. Five
hundred (1833, BT-549, ANV5, MCF10A) or 1000
(MDA-MB-231) cells in medium with 10% matrigel were
added on top of the coating and maintained in culture
for 8–10 days. Medium with 10% matrigel was replaced
at day 4–5. Pictures of the spheres were taken at day 8–
10 at ×4 magnification using an inverted microscope
(Leica) and analyzed using Fiji software .
In vivo experiments
Athymic nude mice (Foxn1nu) were purchased from
Harlan (Barcelona, Spain) and maintained under specific
pathogen-free conditions. Five- to six-week-old mice
were used for all experiments. RAG-2−/− mice were bred
at the in-house Animal Core Facility and used for the
intratibial experiment. For the orthotopic injection, 50 μl
containing 500,000 cells resuspended in Growth Factor
Reduced Matrigel (BD Biosciences) mixed with PBS at
1:1 ratio were directly injected into the fourth mammary
fat pads of mice (2 tumors per mouse). In the second
orthotopic experiment, cells were injected resuspended
in 20 μl of PBS without matrigel. Tumor growth was
monitored regularly using a digital caliper and tumor
volume was calculated as follows: π × length × width2/6.
For intracardiac injection, 105 cells in 100 μl of PBS were
inoculated into the left cardiac ventricle, using a 29G
needle syringe . For intratibial injection, 15,000 cells in
5 μl of PBS were injected into the tibia’s bone marrow
through the femoro-tibial cartilage using a Hamilton syringe
. For intravenous injection, 100,000 cells in 100 μl of
PBS were injected through the tail vein of mice. For BLI,
animals were anesthetized and inoculated with 50 μl of
15 mg/ml D-luciferin (Promega). Images were taken during
1 min with a PhotonIMAGER™ imaging system (Biospace
Lab) and analyzed using M3Vision software (Biospace Lab).
Photon flux was calculated by using a region of interest
(ROI) or by delineating the mouse for whole-body
bioluminescence quantification. All bioluminescence signals
were normalized with values from day 0, except for the
metastasis experiment with RCR252 treatment.
Radiographic and micro-computed tomography (Micro-CT)
analyses were performed as described elsewhere .
RNA was extracted from snap-frozen mammary tumors
and hybridized to Human Gene ST 2.0 microarrays
(Affymetrix). Data were normalized with RMA (Robust
Multi-Array Average) approach. Low expression probes
were removed by filtering those that did not exceed a
level of expression of 32 in at least one of the samples
for each condition. Differentially expressed genes were
identified using LIMMA (linear models for microarray
data) method .
Statistical analysis was performed using SPSS v15.0. When
data exhibited homoscedasticity, pairwise Student’s t test
and Mann–Whitney U test were used for normally and
non-normally distributed variables, respectively. When
data exhibited heterocedasticity, Welch and Median tests
were used for normally and non-normally distributed
variables, respectively. ANOVA and posterior Bonferroni
tests were used for multiple comparisons of normally
distributed variables. Kruskal–Wallis and posterior
Bonferroni adjusted-Mann–Whitney U tests were used
for multiple comparisons of non-normally distributed
variables. Statistical significance was defined as
significant (p < 0.05, *), very significant (p <0.01, **) and highly
significant (p < 0.001, ***). Other additional methods are
included in the Additional file 1.
High EPCR expression in breast tumors correlates with
poor clinical outcome
To evaluate the association between EPCR expression
levels and risk of metastasis in breast cancer, we
performed a relapse-free survival analysis in a cohort of 286
patients, including 106 patients with distant relapses
(GSE2034) . EPCR expression levels were classified as
“high” or “low” according to the median. We found that
patients with high EPCR expression levels had
significantly shorter relapse-free survival times (Fig. 1a)
(Additional file 2: Figure S1). The clinical predictive
potential of EPCR was not related to a higher EPCR
expression in different molecular subtypes (Fig. 1b).
Overall, these results indicate that EPCR is a poor prognosis
factor in breast cancer patients.
EPCR silencing impairs breast tumorigenesis and
To study the role of EPCR, we used several
triplenegative breast cancer cell lines, including human cell
lines (MDA-MB-231 and its bone metastatic derivative
1833, and BT-549) and the ANV5 murine cell line. We
silenced EPCR expression levels by lentiviral
transduction of different shRNAs targeting human (shEPCR#1
and shEPCR#2) and murine (shEPCR#3) EPCR and a
scramble shRNA (shControl) as control (Fig. 2a and
Additional file 3: Figure S2A). EPCR knockdown did not
affect cell proliferation, cell cycle progression, or basal
and induced apoptosis of MDA-MB-231, 1833, BT-549,
and ANV5 cells in 2D cultures (Fig. 2b–d and Additional
file 3: Figure S2A–D). However, EPCR knockdown
significantly reduced the number of spheres grown in 3D
matrigel cultures in all cell lines tested (Fig. 2e and Additional
file 3: Figure S2E).
To explore the relevance of these findings in vivo, we
performed an orthotopic experiment using the highly
metastatic subpopulation 1833, as outlined in Fig. 2f.
Remarkably, EPCR silencing significantly reduced
primary tumor growth after the injection of shControl,
shEPCR#1, or shEPCR#2 1833 cells into the fourth
mammary fat pads of athymic nude mice, in two
independent experiments (Fig. 2g). Consistently, time until
resection of tumors at 300 mm3 was significantly longer
for EPCR-silenced groups, showing that control tumors
maintained higher proliferation rates over the course of
the experiment (Fig. 2h). Of note, several tumors in
EPCR-silenced groups did not reach the size established
for tumor resection by the end of the experimental
period (Fig. 2h). In addition, BLI performed after tumor
resection showed that the number of mice with
metastasis and the number of metastatic events were lower in
EPCR-silenced groups (Fig. 2i). Importantly, EPCR
inhibition was confirmed by immunohistochemistry in
resected primary tumors (Fig. 2j). Similarly, in another
xenograft model of murine ANV5 cells, EPCR silencing
reduced primary tumor growth after orthotopic injection
of shControl and shEPCR#3 cells into athymic nude mice.
Evaluation of spontaneous metastases in this model was
limited by the highly frequent local recurrence after tumor
resection (Additional file 3: Figure S2F, G).
Analysis of tumors in the orthotopic model of 1833
cells, either size-matched tumors resected at different
time points (Additional file 4: Figure S3) or tumors of
different size resected at the same time point (Additional
file 5: Figure S4) revealed a slight increase in apoptosis
(cleaved caspase-3) and/or necrosis and a lower
proliferation rate, assessed by Ki67 staining, in EPCR-silenced
tumors. Of note, we did not observe relevant changes in
angiogenesis and immune infiltration patterns of tumors
(Additional file 5: Figure S4 and Additional file 6: Figure
S5). Taken together, these data indicate that EPCR
contributes to primary tumor growth and the development
of spontaneous metastases in breast cancer.
EPCR silencing reduces metastasis to the bone and lungs
Next, we studied the activity of EPCR in additional
experimental models of metastasis. The effect of EPCR
silencing in bone metastasis was assessed after
intracardiac inoculation of shControl, shEPCR#1, and shEPCR#2
1833 cells into athymic nude mice (Fig. 3a). The
percentage of mice and the bones with metastases was
significantly lower in EPCR-silenced groups (Fig. 3b),
consistent with the reduced whole-body and hind limb
bioluminescence signals (Fig. 3c, d). Differences in BLI
were statistically significant from day 13 of the
experiment, suggesting that EPCR promotes tumor growth of
Perurena et al. Journal of Hematology & Oncology (2017) 10:23
Mice with mets (%)
Fig. 2 Effects of EPCR silencing in vitro and in vivo tumor growth in an orthotopic xenograft model. a Western blot analysis of EPCR protein
levels in MDA-MB-231 and 1833 cells transduced with a scramble shRNA (shControl) and two different shRNAs targeting human EPCR (shEPCR#1
and shEPCR#2). β-tubulin was used as loading control. b MTS in vitro proliferation assay of MDA-MB-231 (top) and 1833 (bottom) cells. Data were
normalized with absorbance values from day 0 and represent mean ± SD of six replicates. Experiments were repeated three times with similar
results. c Percentage of MDA-MB-231 (top) and 1833 (bottom) cells in each phase of the cell cycle, after maintaining cells in culture for 24 and
48 h. d Percentage of apoptotic MDA-MB-231 (top) and 1833 (bottom) cells in basal and staurosporine-induced conditions, measured by annexin-V
binding flow cytometry assay. Data are mean ± SD of triplicates and representative of three independent experiments. Sta staurosporine
e Quantification of spheres grown in 3D matrigel cultures. Data are mean ± SD of 8 replicates from two independent experiments. Representative
images at ×4 magnification. Scale bar 0.5 mm. f Outline of the in vivo orthotopic experiment (n = 8 per group). g Quantification of tumor volume until
day 28 post-injection. Each dot represents mean ± SEM. h Kaplan–Meier curves of resection-free survival. I Incidence of metastatic events and
representative images showing metastases (red arrows), assessed by BLI. Mets metastases. j Representative images at ×20 magnification showing
immunohistochemical staining of EPCR in formaldehyde-fixed mammary tumors. Scale bar 50 μm. **p < 0.01, ***p < 0.001
cancer cells once they have reached the target organ.
Accordingly, EPCR silencing significantly reduced bone
tumor burden and the extension of osteolytic lesions at
day 28 post-injection (Fig. 3e–g). Importantly, EPCR
inhibition by shRNAs was maintained until the end of
the experimental period (Fig. 3g, bottom panel). These
results substantiate the role of EPCR in breast cancer
and indicate that EPCR promotes metastatic activity to
shControl shEPCR#1 shEPCR#2
% mice with bone mets % bones with mets
shControl shEPCR#1 shEPCR#2
Fig. 3 Evaluation of the prometastatic activity of 1833 control and EPCR-silenced cells. a Outline of the experiment after intracardiac inoculation
(n = 8, n = 7, and n = 6 for shControl, shEPCR#1, and shEPCR#2, respectively). b Number of mice and the bones with metastasis in each group.
Whole-body (c) and hind limbs (d) photon flux quantification along the experiment. Data were normalized with BLI values from day 0. e Tumor
area quantification in H&E-stained bone sections. f Osteolytic bone area quantification in X-ray images at day 28 post-injection. g Representative
images of BLI, micro-CT scans, X-ray scans, H&E-stained bone sections, and immunohistochemical staining of EPCR in tumors, from top to bottom,
respectively. Scale bar 20 μm. h Outline of the experiment of intratibial bone colonization (n = 8 per group). i BLI quantification in the hind limbs
along the whole experimental period. Data were normalized with BLI values from day 0. j Tumor area quantification in H&E-stained bone sections.
k Osteolytic bone area quantification in X-ray images at day 25 post-injection. l Representative images of BLI, X-ray scans, and H&E-stained bone
sections, from top to bottom, respectively. All data represented are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001
the bone. Moreover, the lower incidence of metastatic
events in mice injected with EPCR-silenced cells
suggests that EPCR is required during metastatic tumor
reinitiation at the secondary site.
To further explore the function of EPCR in bone
colonization, shControl, shEPCR#1, and shEPCR#2 1833
cells were injected into the tibiae of immunocompromised
mice. Bone colonization was analyzed by BLI, X-rays, and
histological analysis (Fig. 3h). Tumors developed in all
tibiae in shControl and shEPCR#2 mice, while two tibiae
remained tumor-free in shEPCR#1 group. Differences in
BLI became very relevant at advanced experimental time
points (Fig. 3i, l). In addition, histological and X-ray
analyses revealed reduced tumor burden and osteolytic bone
areas in EPCR-knockdown groups at the end of the
experiment (Fig. 3j–l). Of note, the magnitude of the effect
of EPCR silencing revealed by different techniques (BLI
vs. histology and X-rays) differs, an event probably related
to the fact that X-ray analysis does not detect extraosseous
tumor grown through the cortical bone on the periosteal
surface (Fig. 3l, bottom panel). Similarly, extracortical
tumor cells that contribute to tumor burden were lost
during histological processing, whereas these cells
contribute to BLI. Thus, these results indicate that
EPCR promotes metastatic tumor growth in the
Next, we evaluated the prometastatic activity of EPCR
in an intra-tail injection model. For this purpose, we
injected shControl and shEPCR#3 ANV5 cells
intravenously into athymic nude mice and analyzed lung
metastases at the end of the experiment (Additional file 7:
Figure S6A). EPCR knockdown was able to block the
development of lung metastases, assessed by BLI
(Additional file 7: Fig. S6B, D) and tumor area
quantification in H&E-stained lung sections (Additional file 7:
Figure S6C, D).
APC does not mediate effects of EPCR silencing in vitro
and in vivo
Next, we explored the mechanistic insights of EPCR
function in tumor growth and metastasis. First, we
analyzed whether the main known ligand of EPCR, APC,
could signal and mediate cellular functions to favor
tumor progression in MDA-MB-231, 1833, BT-549, and
ANV5 cells. Stimulation of cells with APC did not affect
their proliferation, cell cycle progression, and resistance
to basal and induced apoptosis (Additional file 8: Figure
S7). Accordingly, treatment with the F(ab)2´fraction of
RCR252 antibody, which blocks APC binding to human
EPCR (Additional file 9: Figure S8A), did not reduce
bone metastasis of 1833 cells inoculated into the left
cardiac ventricle of athymic nude mice (Additional file 9:
Identification of SPOCK1 as a mediator of EPCR effects
In order to explore other mechanisms mediating EPCR
effects, we interrogated Human Gene 2.0 ST microarrays
(Affymetrix) to discriminate genes associated with EPCR
silencing in size-matched mammary tumors grown in
athymic nude mice after orthotopic implantation of
shControl, shEPCR#1, and shEPCR#2 1833 cells. An
unsupervised clustering analysis revealed several genes
related to tumor progression to be downregulated in
both EPCR-silenced tumor groups (Fig. 4a). Among these
genes, SPOCK1/testican 1, a member of the SPARC family
of matricellular proteins, was also downregulated in
subcutaneous tumors derived from shEPCR#1 and shEPCR#2
1833 cells, compared to control tumors (Fig. 4b). Moreover,
breast cancer patients (GSE2034 cohort) with high EPCR
expression also had significantly higher SPOCK1 expression
levels (Fig. 4c). Importantly, high SPOCK1 expression
levels associated with a significantly shorter relapse-free
survival time in patients with luminal B, basal and
HER2+ tumors (Fig. 4d) but not luminal A (Additional
file 10: Figure S9). Interestingly, these data are
consistent with the predictive potential of EPCR levels in these
three subsets, but not in luminal A. This finding
suggests that EPCR could mediate tumor progression in
part by upregulating SPOCK1.
Next, we tested the effects of SPOCK1 in vitro, by
silencing SPOCK1 expression levels with shRNAs in
MDA-MB-231, 1833, and BT549 human cell lines
(Additional file 11: Figure S10A). Interestingly, SPOCK1
silencing did not affect cell growth kinetics in 2D
cultures (Additional file 11: Figure S10B, C), but
significantly reduced the number of spheres in 3D matrigel
cultures in all cell lines (Fig. 4e). Conversely, ectopic
expression of EPCR and SPOCK1 in non-tumorigenic
MCF10A mammary cells significantly increased the
number of spheres in 3D cultures (Fig. 4f ). These data
indicate that EPCR or SPOCK1 overexpression confers a
growth advantage in 3D cultures in a non-tumorigenic
mammary cell line, but per se EPCR or SPOCK1 are not
sufficient to confer a tumorigenic phenotype requiring
an oncogenic background. Taken together, these findings
support the role of SPOCK1 mediating EPCR effects and
suggest that EPCR could promote 3D growth of breast
cancer cells by altering tumor-matrix interactions by
SPOCK1 silencing impairs breast tumorigenesis and
Next, we explored the role of SPOCK1 in breast
tumorigenesis using the previously described orthotopic model.
ShControl, shSPOCK#1, or shSPOCK#2 1833 cells were
injected into the fourth mammary fat pads of ahtymic
nude mice, and tumor growth was evaluated (Fig. 5a).
SPOCK1 silencing resulted in a significant reduction in
tumor growth (Fig. 5b, c). Importantly, SPOCK1
inhibition by shRNAs was maintained along the whole
experimental period (Fig. 5d). Taken together, these results
indicate that SPOCK1 is a relevant factor for primary
tumor growth in breast cancer.
Finally, we evaluated the bone metastatic activity of
control and SPOCK1-silenced 1833 cells after intracardiac
inoculation into athymic nude mice (Fig. 5e). All mice in
the shControl and shSPOCK1#1 groups developed bone
metastases, but only 3 mice in the shSPOCK1#2 group.
Moreover, the number of bones with metastases was
significantly lower in both SPOCK1-silenced groups
compared to shControl (Fig. 5f ). Consistently, BLI and H&E
staining revealed a lower tumor burden in
silenced groups, associated with a lower osteolytic area
(Fig. 5g–j). These results indicate that SPOCK1 silencing
recapitulates the effects observed by EPCR silencing in
vivo and further support the role of SPOCK1 as an
effector of EPCR.
In this work, we unveiled a novel molecular
mechanism of EPCR contributing to breast cancer
progression favoring tumor growth and metastatic activity to
target organs. EPCR endowed cells with advantageous
growth in 3D, an effect partially mediated by the
extracellular matrix proteoglycan SPOCK1. These cell
functions were correlated with increased metastatic
risk and poor clinical outcome in breast cancer
patients. Importantly, this association was relevant in all
the molecular subtypes, except luminal A, indicating
that EPCR could be useful as a potential prognostic
marker in these patient subsets.
Previous studies identified EPCR as a marker of
human breast cancer stem cells with enhanced
tumorinitiating and growth abilities in immunodeficient mice
. In addition, EPCR deficiency attenuated
spontaneous tumor growth in the PyMT murine breast cancer
model . In agreement with these findings, we showed
that EPCR silencing impaired orthotopic tumor growth
of highly metastatic 1833 cells. In this model, differences
in tumor size between EPCR+ and EPCR− tumors
became more relevant at advanced experimental time
points. In contrast in another study, although EPCR
overexpression increased initial orthotopic growth of
MDA-MB-231 cells, it resulted in smaller final tumor
shControl shSPOCK1#1 shSPOCK1#2
Fig. 5 Effects of SPOCK1 silencing in primary tumor growth and metastasis in 1833-derived xenograft models. a Outline of the orthotopic
experiment to assess primary tumor growth. b Tumor volume measurements along the whole experimental period. c Weight of resected tumors
at day 37 post-injection. d mRNA levels of SPOCK1 in resected tumors (n = 3 per group), assessed by RT-qPCR. e Outline of the intracardiac
inoculation experiment to assess bone metastasis. f Number of mice and bones with metastases in each group. g BLI quantification in hind limbs
at day 28 post-injection. h Tumor area quantification in H&E-stained bone sections. i Osteolytic bone area quantification in micro-CT scans from
day 28 post-injection. j Representative images of BLI, micro-CT scans, and H&E-stained bone sections, from top to bottom, respectively. All data
are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. ns, non-statistical significance
volumes , a finding possibly related to EPCR loss in
evolving tumors. Thus, EPCR could display different roles
at different stages of breast cancer progression such as
initiation, maintenance, and target organ colonization.
Future experiments will help to characterize its role in
each of these stages in different histological subtypes.
Besides its role in tumorigenesis, EPCR also displayed
a marked prometastatic activity to target organs, events
that cooperatively support its contribution to prognosis.
The consistent results obtained in both metastatic
models indicate that EPCR confers a functional
advantage at late stages of the metastatic process. Moreover,
differences in metastatic tumor burden became more
relevant at advanced experimental time points,
indicating an effect more pronounced during the colonization
of target organs, as evidenced by the overt osseous
colonization observed in the intratibial model.
In contrast with previous findings in lung cancer , EPCR
did not markedly contribute to tumor cell survival in the
circulation and engraftment in secondary sites. The prominent
effect in breast cancer during colonization was associated
with its role in 3D growth and based on the low number of
tumor nodules in shEPCR mice in both models (the bone
and lung) of experimental metastasis, EPCR may also
modulate metastatic tumor re-initiation at the target organ.
Tumors are organ-like structures composed of tumor
cells and stromal cells embedded in a complex ECM within
the tumor microenvironment . Components of the
ECM such as tenascin C have been shown to promote
breast cancer progression and metastasis [30–32]. In the
same line, our study identified SPOCK1, a secreted
matricellular protein as a markedly downregulated gene in
EPCR-silenced tumors. SPOCK1 belongs to the
Ca2+-binding proteoglycan family which includes SPARC, a
wellstudied tumor-associated component involved in regulating
adhesion, matrix cellular interactions, and cell growth
[33, 34]. Recently, SPOCK1 has been shown to promote
epithelial-mesenchymal transition (EMT) and
metastasis in other tumors including lung and gallbladder
cancer and hepatocarcinoma [35–37]. Interestingly, high
SPOCK1 expression levels were associated with adverse
clinical outcome in the same subsets of breast cancer
patients predicted by EPCR levels. Therefore, EPCR
could promote tumor growth in vivo, in part, by
modulating tumor-matrix interactions through SPOCK1
favoring an advantageous 3D growth of tumorigenic cells.
Indeed, SPOCK1 silencing in breast cancer cells
impaired the number of 3D spheres and primary and
metastatic tumor growth, an effect that phenocopied
EPCR silencing. Accordingly, EPCR has been required
for cell organization and growth of mammary epithelial
cells in 3D cultures  In agreement with these
findings, EPCR/SPOCK1 axis activation in non-tumorigenic
mammalian cells increased the number of spheres
grown in 3D matrigel cultures. However, it was not
sufficient to confer a tumorigenic phenotype.
A surprising finding of our study was the lack of effects
mediated by APC, despite the fact that anti-EPCR blocking
antibodies (1535) reduced orthotopic growth of
MDA-MB231 cells in previous studies . Although, we did not
specifically address APC/EPCR effects in orthotopic tumors,
we explored its contribution in vitro and during the
development of bone metastases. EPCR-blocking antibodies in
this model could not reduce the metastatic activity of 1833
cells, suggesting that EPCR triggered APC-independent
effects. In this experiment, we used the F(ab´)2 fractions of
the anti-EPCR blocking antibody to avoid any interference
of the activated complement system, whereas Schaffner et
al.  employed whole-body antibodies. Furthermore, the
use of the same strategy of F(ab´)2 fractions showed a
significant effect on a model of lung cancer metastasis
underscoring the validity of this approach . Complementary to
this view, other ligands different than APC binding to
different regions of EPCR in each tumor type or accessible
in specific microenvironments could account for these
differences. Based on these findings, future experiments
should address other mechanisms that could be mediated
by EPCR in different tumor types and metastatic sites.
In summary, our study unveils a novel role of EPCR as a
clinically relevant factor in breast cancer, which
promotes primary tumor growth and metastatic activities in
target organs. Unexpectedly, EPCR modulates tumor
cell-ECM interactions involved in 3D growth required
for tumor progression and metastasis, in part by
upregulating SPOCK1. These findings underscore a novel role
of EPCR as a novel prognostic factor and a potential
therapeutic target in a subset of breast cancer patients.
Additional file 1: Supplementary material and methods. (DOCX 38.8 kb)
Additional file 2: Figure S1. Kaplan–Meier analysis in different molecular
subtypes of breast cancer patients based on EPCR expression levels.
Relapsefree survival curves for each molecular subtype of breast cancer. Log-rank test
was used to determine p values in all cases. (PPTX 273 kb)
Additional file 3: Figure S2. Effects of EPCR silencing in vitro and in vivo
tumor growth in an orthotopic model. A. Western blot analysis of EPCR
protein levels in human BT-549 (top) and murine ANV5 (bottom) cells
transduced with a scramble shRNA (shControl) and shRNAs targeting human
(shEPCR#1 and shEPCR#2 in BT-549) and murine (shEPCR#3 in ANV5) EPCR.
βtubulin was used as loading control. White line indicates that the membrane
was cut. B. MTS in vitro proliferation assay of BT-549 (top) and ANV5 (bottom)
cells. Data were normalized with absorbance values from day 0 and represent
mean ± SD of six replicates. C. Percentage of BT549 (top) and ANV5 (bottom)
cells in each phase of the cell cycle after maintaining cells in culture for 24
and 48 h. Sta, staurosporine. D. Percentage of apoptotic BT-549 (top) and
ANV5 (bottom) cells in basal and staurosporine-induced conditions, measured
by annexin-V binding flow cytometry assay. E. Quantification of spheres grown
in 3D matrigel cultures. Data are mean ± SD of 8 replicates. Representative
images at ×4 magnification. Scale bar 0.5 mm. F. Outline of the in vivo
orthotopic experiment (n = 8 per group). G. Quantification of tumor volume at day 15
post-injection. H. Kaplan–Meier curves of resection-free survival. (PPTX 9850 kb)
Additional file 4: Figure S3. Immunohistochemical analysis of several
markers in control and EPCR-silenced size-matched mammary tumors
resected at different time points. A. Representative images showing H&E
staining (×2.5 magnification) and the immunohistochemical staining of Ki67,
cleaved caspase-3, CD31, and F4/80 (×20 magnification) in
formaldehydefixed tumors. Scale bars 80 μm (H&E) and 10 μm (Ki67, caspase-3, CD31, and
F4/80). T. mass, tumor mass. T. border, tumor border. B. Quantification of the
percentage of immunoreactive cells. Each dot represents one tumor. Data
are mean ± SEM. ns means non-statistical significance. (PPTX 2780 kb)
Additional file 5: Figure S4. Effects of EPCR silencing in cell growth
kinetics and immune infiltration of control and EPCR-silenced mammary
tumors resected at the same time point. A. Outline of the experiment (n
= 5 per group). B. Tumor volume at the end of the experimental period
(day 32 post-injection). Each dot represents one tumor. Data are mean ±
SEM. C. Representative images showing H&E staining (×2.5 magnification)
and the immunohistochemical staining of Ki67, cleaved caspase-3, CD31,
and F4/80 (×20 magnification). T. mass, tumor mass. T. border, tumor
border. Scale bars 80 μm (H&E) and 10 μm (Ki67, caspase-3, CD31, and
F4/80). D. Quantification of the percentage of immunoreactive cells. Each
dot represents one tumor. Data are mean ± SEM. *p < 0.05. ns means
non-statistical significance. (PPTX 2260 kb)
Additional file 6: Figure S5. Analysis of immune cells infiltrating
control and EPCR-silenced mammary tumors. A. Flow cytometry gating
strategy. Arrows of the same color indicate simultaneous detection of
markers. MDSCs, myeloid derived suppressor cells. NK, natural killer cells.
DCs, dendritic cells. B. Quantification of the percentage of immune
subpopulations infiltrating the tumors. Each dot represents one tumor.
Data are mean ± SEM. (PPTX 932 kb)
Additional file 7: Figure S6. Effects of EPCR silencing in the ability of
murine ANV5 cells to metastatize to the lungs. A. Outline of the intra-tail injection
tumor area (C) in the lungs at the end of the experimental period (day 28
postinjection). Each dot represents one mouse. D. Representative images of
H&Estained lung sections (top) and BLI (bottom). *p < 0.05, **p < 0.01. (PPTX 1080 kb)
Additional file 8: Figure S7. Cell growth kinetics of APC-stimulated breast
cancer cell lines. A. MTS proliferation assay of cells stimulated with increasing
doses of APC. Data were normalized with absorbance values from day 0. Each
dot represents mean ± SD of six replicates. B. Percentage of cells in each phase
of the cell cycle in control and 50 nM APC-stimulated cells for 24 and 48 h, in
serum-free and 4% serum medium. C. Percentage of apoptotic cells in basal
and staurosporine-induced conditions, measured by annexin-V binding flow
cytometry assay. Cell lines are MDA-MB-231,1833, BT-549, and ANV5, from the
left to the right, in all figure sections. (PPTX 325 kb)
Additional file 9: Figure S8. Effects of the pharmacological EPCR blockade
in the prometastatic activity of 1833 cells. A. Specificity of anti-EPCR antibodies,
RCR252, and its F(ab´)2 fraction, by surface plasmon resonance (SPR). EPCR (500
RU) was immobilized through the anti-EPCR antibody RCR2 (that does not bind
in the ligand-receptor domain) on a CM5 chip. The binding of 250 nM of
RCR252 and its F(ab´)2 fraction to the EPCR were monitored. A representative
experiment is shown. RU resonance units; s, seconds. B. Outline of the
experiment (n = 8 per group). C. Photon flux quantification in hind limbs. D. Tumor
area quantification in H&E-stained bone sections. E. Osteolytic bone area
quantification in X-ray images from day 28 post-injection. F. Representative
images of BLI (top), X-rays (middle), and H&E staining (bottom) at day 28
post-injection. All data are represented by mean ± SEM. (PPTX 1540 kb)
Additional file 10: Figure S9. Clinical relevance of SPOCK1 in different
breast cancer subtypes. A. Relapse-free survival analysis of all patients
included in the GSE2034 cohort (n = 286), classified into “high SPOCK1”
and “low SPOCK1” based on median expression value of SPOCK1. B.
SPOCK1 mRNA expression levels in the primary tumors, classified by
molecular subtypes. Whiskers represent minimum and maximum values.
AU, arbitrary units. C. Relapse-free survival curves for each molecular
subtype of breast cancer. Log-rank test was used to determine p values
in all cases. ns, non-statistical significance. (PPTX 288 kb)
Additional file 11: Figure S10. Cell growth kinetics in 2D cultures of
human breast cancer cell lines after SPOCK1 silencing. A. Analysis of
SPOCK1 expression levels by RT-qPCR in human cells transduced with a
scramble shRNA (shControl) and two different shRNAs (shSPOCK#1 and
shSPOCK#2) targeting human SPOCK1. B. MTS in vitro proliferation assay
of MDA-MB-231 (top), 1833 (middle), and BT549 (bottom) cells. Data were
normalized with absorbance values from day 0 and represent mean ± SD
of six replicates. Experiments were repeated three times with similar
results. C. Percentage of MDA-MB-231 (top), 1833 (middle), and BT549
(bottom) cells in each phase of the cell cycle, after maintaining cells in
culture for 24 and 48 h. (PPTX 223 kb)
3D: 3-dimension; APC/EPCR: Activated protein C/endothelial protein C
receptor; BLI: Bioluminescence imaging; MaSCs: Mammary stem cells;
PAR1: Protease-activated receptor 1; PfEMP1: Erythrocyte membrane protein
1; PyMT: Polyoma middle T; SPOCK1: SPARC/osteonectin, Cwcv and kazal-like
We are grateful to Carmen Berasain for helpful discussions. We thank
members of the Morphology, Genomics and Bioinformatics and Animal Core
Facilities for their helpful assistance, especially, L. Guembe, D. Corbacho, and
M. Ariz. We thank Dr. Esmon and Dr. Fukudome for anti-EPCR antibodies.
N.P. holds a FPU fellowship from the Spanish Ministry of Education. This
work was supported by “UTE project” from the Foundation for Applied
Medical Research and a grant from the Spanish Ministry of Economy and
Competitiveness to F.L. (SAF2012-40056).
Availability of data and materials
The datasets supporting the conclusions of this article are included within
the article and its additional files. Microarray datasets are included in
FL and NP designed the study, discussed the results, and wrote the manuscript.
NP, CZ, SMC, and HM performed in vitro and in vivo experiments and discussed
the results. RRG and MS provided some reagents and discussed the results. ME
and ASA directed some critical experiments and discussed the results. SV
discussed the results and provided expertise for some experiments. EG
interpreted and performed expression arrays and bioinformatics analysis. JH
performed critical experiments, provided reagents, and discussed the results. FL
coordinated all tasks and edited the manuscript. All authors participated in
drafting the article and discussing the results. All authors read and approved
the final manuscript.
Authors declare that there are no competing interests.
Consent for publication
All animal procedures (protocol 161–14) were approved by the CEEA (Ethical
Committee for Animal Experimental Research).
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