EPCR promotes breast cancer progression by altering SPOCK1/testican 1-mediated 3D growth

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) [20]. 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 [9]. shRNAs cloned into PLKO.1-puro vector and the empty vector were obtained from Mission® (Sigma-Aldrich).

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 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 (Sigma-Aldrich) 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.

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 647-conjugated 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.

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 96-well 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 [23].

Athymic nude mice (Foxn1 ^nu ) 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 [24]. 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 [25]. 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 [26].

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 [27].

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.

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) [28]. 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.

To study the role of EPCR, we used several triple-negative 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.

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 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 the bone. Moreover, the lower incidence of metastatic events in mice injected with EPCR-silenced cells suggests that EPCR is required during metastatic tumor re-initiation 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 osseous compartment.

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).

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: Figure S8B–F).

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 modulating SPOCK1.

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 SPOCK1-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.