Sclerostin induced tumor growth, bone metastasis and osteolysis in breast cancer

Breast cancer is the second leading cause of cancer-related deaths among women worldwide. Many patients suffer from bone metastasis. Sclerostin, a key regulator of normal bone remodeling, is critically involved in osteolytic bone diseases. However, its role in breast cancer bone metastasis remains unknown. Here, we found that sclerostin was overexpressed in breast cancer tumor tissues and cell lines. Inhibition of sclerostin by antibody (Scl-Ab) significantly reduced migration and invasion of MDA-MB-231 and MCF-7 cells in a time- and dose-dependent manner. In xenograft model, sclerostin inhibition improved survival of nude mice and prevented osteolytic lesions resulting from tumor metastasis. Taken together, sclerostin promotes breast cancer cell migration, invasion and bone osteolysis. Inhibition of sclerostin may serve as an efficient strategy for interventions against breast cancer bone metastasis or osteolytic bone diseases.


Expression of sclerostin in breast cancer cell lines.
To determine the expression profile of sclerostin in different breast cancer cell lines (MDA-MB-231, BT-549, MCF-7, MDA-MB-453 and SK-BR3), qRT-PCR assay and Western blot analysis were adopted. Sclerostin protein extracted from supernatant culture medium was detectable in all cell lines ( Fig. 2A). A higher expression level was observed in MDA-MB-231 and MCF-7 cells, whereas a lower level in SK-BR3, BT-549 and MDA-MB-453 cells. However, concerning expression of sclerostin mRNA, SK-BR3 was higher than MCF-7 (Fig. 2B).

Effect of sclerostin on migration and invasion of MDA-MB-231 and MCF-7 cells.
To explore the impact of reduced sclerostin expression on migration and invasion, breast cancer cell lines with high endogenous sclerostin were treated with blocking antibody against sclerostin. A subsequent MTT assay revealed that reduced sclerostin did not affect cell proliferation (Fig. 3A,B). Meanwhile, the migratory ability of cells was inhibited (Fig. 3C,D). Invasiveness of breast cancer cell lines treated with Sci-Ab was significantly undermined compared with control as demonstrated by Transwell chamber assay (Fig. 3E,F). These observations indicated that decreased sclerostin may suppress migration and invasion of breast cancer cells.

Effect of sclerostin inhibition on tumor growth in vivo.
To investigate whether suppression of sclerostin may lose growth advantage in vivo, MDA-MB-231 cells (5 × 10 6 in 30 μl PBS) were injected into the bone marrow space of BALB/c-nu/nu mice and tumor volume was measured starting at 1 week later. Tumors from control and PBS-treated groups were comparable in volume and weight to those from Scl-Ab treated group (Fig. 4A,C). However, a survival curve indicated Scl-Ab-treated animals lived longer (Fig. 4B). Then, Scl-Ab treated mice exhibited reduced expression of sclerostin protein in tumor tissue as shown by western blot analyses (Fig. 4D,E). Tumors were confirmed by H&E staining and tumor sections were stained immunohistochemically to determine the expression of sclerostin. Notably, Scl-Ab treated group demonstrated a lower expression of sclerostin (Fig. 5A). Compared with controls, a higher level of osteocalcin (Fig. 5B) and a lower level of OPG (Fig. 5C) were identified in Scl-Ab treated group. Finally, a lower level of sclerostin in serum was detected in Scl-Ab treated mice (Fig. 5D), suggesting that reduced sclerostin expression correlates with a better survival.

Micro-CT analysis in vivo.
In this study, micro-CT was performed to explore the presence of micro-structure of femoral bone in mice, and to evaluate sclerostin induced bone osteolysis in breast cancer. Furthermore, potential mechanism underlying osteolysis was investigated through micro-structure. In both

Discussion
Almost 80% of breast cancer patients have evidence of bone loss at baseline 15 and 65-75% of patients suffer from metastases to bone 16 . Breast cancer-related bone disease results from aberrant activation of osteoclasts and impaired function of osteoblasts 17,18 . A better understanding of risk factors and signaling pathways involved in the development and progression of BCBM may provide new biomarkers for early detection of metastasis and, eventually, improve patients' quality of life. Sclerostin, a regulator of normal bone remodeling, reduces bone formation by inhibiting Wnt signaling. Although several facets of function have been discovered, pathologic role of sclerostin in bone metastasis remain undefined.
In this study, we observed a significantly increased level of sclerostin in the plasma of patients with breast cancer osseous metastasis compared with that of localized breast cancer and breast benign tumor. Increased expression of sclerostin was detected in breast cancer tumor tissues and cell lines. Scl-Ab suppressed migration and invasion of MDA-MB-231 and MCF-7 cells. In a xenograft nude mouse model, inhibition of sclerostin significantly improved overall survival of mice. Importantly, suppressed sclerostin prevented osteolytic lesions resulting from tumor metastasis in vivo. Our data suggest the role of sclerostin in breast cancer cell migration, invasion and metastasis to bone.
Aberrant activation of Wnt/β-catenin signaling contributes to tumor development and progression [19][20][21][22][23][24] . Wnt/β-catenin signaling is involved in bone homeostasis and regeneration 22 . A high level of circulating sclerostin, a potent inhibitor of osteoblastogenesis, was detected in patients with BCBM. Sclerostin gene expression is activated in multiple myeloma and associated with carcinogenesis, disease progression, and prognosis of cancer patients 8,12 . As previous reported 10 , increased sclerostin secretion by myeloma cells can suppress the function of osteoblasts. In our study, high expression of sclerostin is associated with the severity of osteolytic lesions. Moreover, Scl-Ab treatment prolongs survival of nude mice.
Pathological changes caused by BCBM include growth condition of trabecular bone, bone mineralization, micro-injury, and abnormal morphology such as decreased volume of both cortical and trabecular bones 25,26 . Development of micro-CT technique has contributed to a better understanding of micro-architecture of cancer-induced bone diseases. A recent study by Florio et al. 27 used a bispecific heterodimeric antibody targeting sclerostin and DKK-1 contributing to bone formation, bone mass and bone strength in intact bones in mice, and fractured bones in rats. Our data demonstrate that Sci-Ab may prevent osteolytic lesions and prevent  25,26 , a higher number of trabeculae, thinner inter-trabecular space, and more integral connectivity of the trabecular bone structure were identified in Scl-Ab-treated mice. Increased BV/TV, Tb.N., Tb.Th. and BS/BV values may result from reduced expression of sclerostin in bone. Moreover, Tb.Sp. and BS/BV were significantly lower in controls than in the Sci-Ab treated group, suggesting a positive therapeutic effect of Scl-Ab on cancer-induced bone destruction. This indicates that increased bone mass potentially results from recovered function of Wnt/β-catenin [28][29][30][31] . Consistent with precious reports 32-34 , a lower level of OPG (p < 0.01) and a higher level of osteocalcin (p < 0.01) were detected in Scl-Ab treated group compared with controls. However, further experiments are required to determine how Scl-Ab mechanistically affects secretion of OPG and OCN involved in cancer induced bone loss.
In conclusion, we have identified increased secretion of sclerostin by breast cancer cells. Inhibition of sclerostin by Scl-Ab suppresses migration and invasion of breast cancer cells. Furthermore, Scl-Ab may prevent osteolytic lesions as demonstrated by BCBM murine model bearing tumors generated by human breast cancer cells. Our study paves a way for further investigation into potential therapeutic targets for cancer-induced bone diseases.

Methods
Patients. This study investigated surgical resection samples from 15 patients with BCBM, as well as localized breast cancer and benign breast tumor (n = 15 per group). All patients were diagnosed and treated at Beijing Shijitan Hospital (Beijing, China). This study was approved by Institutional Review Boards at Beijing Shijitan Hospital. Informed consent was obtained from all participants and all methods were performed in accordance with the relevant guidelines and regulations.

RNA isolation and qRT-PCR amplification.
Total RNA was extracted from frozen and paraffin-embedded samples using TRIzol ® reagent (Thermo) according to the manufacturer's instructions. RNA from MDA-MB-231 and MCF-7 cells was reverse-transcribed using SuperScript ® cDNA Synthesis Kit (Thermo).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assay was
conducted to evaluate inhibitory effect of sclerostin antibody (Scl-Ab, obtained from Central Laboratory at People's hospital of Peking university) on proliferation of breast cancer cells. MDA-MB-231 and MCF-7 cells were seeded into 96-well plates with the density of 6 × 10 3 cells/well and then incubated for 12-24 hours. Next, sclerostin antibody (at a concentration of 0, 1 and 4 µg/ml, respectively) was added to cells and incubated at 37 °C for 5-7 days. At the end of treatment, 20 μL of MTT solution (5 mg/ml) was added into each well and then plates were incubated for an additional 4 hours. After removing the medium, 160 μL/well of DMSO was added to each well. The optical density (OD) value of each well was measured spectrophotometrically at 490 nm.
Migration assay. Migration assays were performed in 48-well Transwell ® plates (8.0-μm pore size; Costar, Corning Incorporated, Corning, NY). After treatment with 1 or 4 µg/ml sclerostin antibody (Scl-Ab) for 48 hours, breast cancer cells from control and treatment groups were seeded into the upper chamber of Transwell system at a concentration of 5 × 10 3 cells/well in 100 μl medium containing 5% FBS. The lower chamber was filled with 500 μl medium containing 10% FBS. After 24 hours of incubation at 37 °C with 5% CO 2 , the upper surface of each filter was carefully washed with phosphate-buffered saline (PBS) and the remaining cells were removed with a cotton wool swab. Cells that had migrated to the bottom side of Transwell membrane inserts were fixed with 4% paraformaldehyde and stained with crystal violet. Migrated cells were counted (3 wells per group, 5 central fields per Transwell) at 100 × magnification using an inverted microscope (FM-600, Shanghai Puda Optical Instrument, Shanghai, China). At least five random fields of vision were counted per well to quantify migrated cells. The entire assay was repeated at least three times.
Invasion Assay. The invasive capacity of cancer cells was confirmed in vitro using a Transwell chamber coated with Matrigel ® (Becton Dickinson, Franklin Lakes, NJ), in accordance with the manufacturer's recommendation. Sci-Ab (0, 1 or 4 µg/ml) was added to MDA-MB-231 and MCF-7 cells for 24 hours. After collection and resuspension, cell suspension (5 × 10 4 cells/ml) of 50 ul was added into the upper chamber. Next, Transwell system was incubated for 1 day at 37 °C, and then cells were washed carefully with PBS to clear off those remaining on the upper side of Transwell membrane. Then, cells invaded to the bottom side of the membrane were fixed and stained with crystal violet. The entire experiment was conducted independently and repeated at least three times.
Experimental murine model of breast cancer bone metastasis. BABL/C female mice (aged at [6][7][8] weeks, approximately weighed at 30 g) were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China) and housed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. This study was reviewed and approved by IACUC at Beijing Shijitan Hospital. All experiments were conducted according to IACUC guidelines. Mice were housed for 1 week before they were anesthetized by intraperitoneal injection of chloral hydrate (10%, 0.1 ml/g). An incision was made in anesthetized mice along the right knee. The patellar tendon and muscle were split longitudinally to expose the distal femur. A surgical scalpel was used to drill a tiny hole on the cortex and MDA-MB-231 cells (5 × 10 6 cells in 30 μl PBS) were injected into the bone marrow space through the hole with a 100-μl Hamilton microsyringe (Shanghai Linbo Scientific Instruments, Shanghai, China). Breast cancer cells resuspended in 30 μl PBS were slowly introduced into the marrow space to avoid extravasation. The needle was removed, the hole sealed with bone wax, the patellar tendon reapproximated, and the wound sutured.
Tumor growth was monitored every 3 days and tumor was weighed. Tumor volume was calculated by the equation V (mm 3 ) = (a × b 2 )/2, where a is equal to the largest diameter and b is equal to the perpendicular diameter. When tumors reached a size of ~100 mm 3 (20 days), mice were randomly distributed into three groups (12 mice per group). The first group was used as a blank control (non-treated). The second group was intratumorally administered 20 μl of PBS, and the third group was intratumorally treated with sclerostin antibody (1 μg antibody in 20 μl of PBS). These treatments were repeated once after 5 days.
Micro-CT analysis. After animals were sacrificed, femoral condyles including the implants were harvested and excess tissue was removed and immediately fixed in 10% neutral-buffered formalin solution. Samples (n = 36) randomly selected from each group were used for high-resolution micro-CT (Inveon MMCT, Siemens, Munich, Germany) at 60 kV and 220 mA with a 0.5 mm aluminum filter. The scanning protocol required 440 exposures of 360° with 1500 ms per projection and an effective pixel width of 8.99 μm. Cobra software (Exxim, Pleasanton, CA) was used to reconstruct images with a down-sampling of one and a beam hardening correction. The Inveon Acquisition Workplace (Siemens) utilized a standard CT camera with a high resolution, 12-bit X-ray imaging detector with 2048 × 3072 pixels configured for mouse imaging. Three-dimensional (3-D) analysis and reconstruction of trabecular bone was performed on femurs by Inveon Research Workplace (Siemens). Consequently, constant regions of interest (ROIs) were drawn using a ROI tool at the region 0.8-2.6 mm to distal femoral metaphyses. Using ROI boxes of the same size, data analysis tools were used to calculate the trabecular bone compartment, which was manually delineated from the cortical bone. The following variables were determined: trabecular bone volume fraction (BV/TV), trabecular number (Tb.N; 1/μm), bone mineral density (BMD; gm/cm 2 ), trabecular thickness (Tb.Th; μm), cortical bone volume/tissue volume ratio (cortical BV/TV), trabecular separation (Tb.Sp; μm) and bone surface/bone volume ratio (BS/BV).
Enzyme-linked immunosorbent assay. Osteocalcin (OCN), osteoprotegerin (OPG) and sclerostin were detected in mice using enzyme-linked immunosorbent assay (ELISA) kits (Inova Diagnostics, San Diego, CA) in accordance with the manufacturer's recommendations. ELISA plates (BD Biosciences, San Jose, CA) were coated for 2 h at 37 °C with 1 g/ml GRP78 in coating buffer. Then, plates were washed with PBS four times and blocked with PBS containing 2% skim milk overnight. On the following day, QUANTA Lite ® reagents (Inova Diagnostics) were used to perform ELISA according to the supplier's protocol. Plates were read at 405 nm on an fluorometer and all samples were analyzed simultaneously. Absorption was determined with an ELISA reader at 450 nm (550 Microplate Reader; Bio-Rad) and the results were expressed as mean ± SEM.
Immunohistochemistry. Tissues were fixed in 10% neutral-buffered formalin and subsequently embedded in paraffin. Paraffin-embedded tissues were cut at 4 µm, deparaffinized with xylene, and rehydrated for further hematoxylin and eosin (H&E) or 3,3′-diaminobenzidine peroxidase (DAB) immunohistochemical staining. Briefly, following a brief proteolytic digestion and peroxidase blocking of tissue slides, slides were incubated overnight with primary antibodies against sclerostin (ab63097, Abcam) at a dilution of 1:100 at 4 °C, respectively. Visualization employed a Dako EnVision System (Dako Diagnostics, Zurich, Switzerland). Statistical analysis. Differences were compared between control and treatment groups were evaluated by one-way ANOVA followed by Bonferroni's multiple comparison test using the Prism software package version 5.03 (GraphPad Software, La Jolla, CA). All data were expressed as mean ± SEM. A difference was considered statistically significant at a level of P < 0.05.