Cell Oncol. (2014) 37:363–375 DOI 10.1007/s13402-014-0197-1
ORIGINAL PAPER
BMP9 regulates cross-talk between breast cancer cells and bone marrow-derived mesenchymal stem cells Shaoheng Wan & Yuehong Liu & Yaguang Weng & Wei Wang & Wei Ren & Chang Fei & Yingying Chen & Zhihui Zhang & Ting Wang & Jinshu Wang & Yayun Jiang & Lan Zhou & Tongchuan He & Yan Zhang
Accepted: 1 September 2014 / Published online: 11 September 2014 # International Society for Cellular Oncology 2014
Abstract Purpose Breast cancer cells frequently metastasize to distant organs, including bone. Interactions between breast cancer cells and the bone microenvironment are known to enhance tumor growth and osteolytic damage. Here we investigated whether BMP9 (a secretary protein) may change the bone microenvironment and, by doing so, regulate the cross-talk between breast cancer cells and bone marrow-derived mesenchymal stem cells. Methods After establishing a co-culture system composed of MDA-MB-231breast cancer cells and HS-5 bone marrowderived mesenchymal stem cells, and exposure of this system to BMP9 conditioned media, we assessed putative changes in migration and invasion capacities of MDA-MB-231 cells and concomitant changes in osteogenic marker expressionin HS-5 cells and metastases-related genes in MDA-MB-231 cells. Results We found that BMP9 can inhibit the migration and invasion of MDA-MB-231 cells, and promote osteogenesis Shaoheng Wan and Yuehong Liu these authors contributed equally to this work. S. Wan : Y. Liu : Y. Weng : W. Wang : C. Fei : Y. Chen : Z. Zhang : T. Wang : J. Wang : Y. Jiang : L. Zhou : Y. Zhang (*) Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, China e-mail: [email protected] Y. Zhang e-mail: [email protected] W. Ren Department of General Surgery, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing 400042, China T. He Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
and proliferation of HS-5 cells, in the co-culture system. We also found that the BMP9-induced inhibition of migration and invasion of MDA-MB-231 cells may be caused by a decreased RANK ligand (RANKL) secretion by HS-5 cells, leading to a block in the AKT signaling pathway. Conclusions From our data we conclude that BMP9 inhibits the migration and invasion of breast cancer cells, and promotes the osteoblastic differentiation and proliferation of bone marrow-derived mesenchymal stem cells by regulating crosstalk between these two types of cells through the RANK/ RANKL signaling axis. Keywords BMP9 . Breast cancer . Bone marrow-derived mesenchymal stem cells . Cross-talk . RANKL/RANK axis Abbreviations BMP9 Bone morphogenetic protein 9 BCC Breast cancer cells BMSC Bone marrow –derived mesenchymal stem cells MSCs Mesenchymal stem cells OPG Osteoprotegerin RANK Receptor activator of nuclear factor-kappa B RANKL The RANK ligand Ad-BMP9 Adenovirus expressing BMP9 protein Ad-GFP Adenovirus expressing green fluorescent protein ERK1/2 Extracellular signal regulated kinases p-ERK1/2 Phospho-ERK1/2 OPN Osteopontin OCN Osteocalcin IL-6 Interleukin-6 IL11 Interleukin-11 PTH-rp Parathyroid hormone-related protein MMP9 Matrix metallopeptidase 9 MMP2 Matrix metallopeptidase 2
364
DKK1 ALP MAPK GSK-3β pGSK-3β CTGF LRP-6 BMP9-CM GFR-CM
S. Wan et al.
Dickkopf WNT signaling pathway inhibitor 1 Alkaline phosphatase Mitogen-activated protein kinase Glycogen synthase kinase-3β Phospho-GSK-3β Connective Tissue Growth Factor Low density lipoprotein receptor-related protein 6 BMP9 condition medium GFP Vector control condition medium
what role BMP9 (as a secretary protein) plays in the bone microenvironment and whether it does affect the cross-talk between BCCs and BMSCs. In the past, it has been reported that BMP9 can increase the osteogenic differentiation of BMSCs [7, 12]. Here, we focused on the effect of BMP9 on the interaction between BCCs and BMSCs, aiming to unravel the role of BMP9 in decreasing the occurrence of osteolytic lesions in the BCC microenvironment. To this end, a coculture system composed of MDA-MB-231 BCCs and HS-5 BMSCs was employed to investigate the effects of BMP9 on the interaction between these two cell types, and to clarify the action of BMP9 in inhibiting BCC osteolytic metastases in the bone microenvironment.
1 Introduction Breast cancer is a common malignancy in women world-wide, more than 1.38 million new cases are diagnosed and about 500,000 individuals die of the disease [1]. Breast cancer cells (BCCs) frequently metastasize to distant organs such as bone, lung and liver [2]. When BCCs metastasize to bone, they disturb the intricate equilibrium of the bone microenvironment, resulting in osteolytic injury [3]. The process of osteolytic metastasis is closely related to the bone microenvironment, and involves osteoblasts, osteoclasts and bone marrow-derived mesenchymal stem cells (BMSCs). In the past, it has been shown that the interaction between BCCs and bone microenvironment cells enhances tumor growth and osteolytic damage [4, 5]. BCCs, on one hand, not only secrete various cytokines to activate osteoclasts, but also activate osteoblasts to secrete osteoclast-activating factors, which results in the formation of osteolytic lesions. Osteoclasts and osteoblasts, on the other hand, secrete cytokines that promote the metastasis and proliferation of BCCs, which aggravates the osteolytic damage [4, 5]. It thus appears that their crosstalk generates a vicious cycle which is sustained by cytokines. Recently, several studies have focused on ways to interfere with the cross-talk between BCCs and bone microenvironment cells [3–5]. Mesenchymal stem cells (MSCs) are present in many tissues, including bone marrow, and they have the capacity to differentiate into three different lineages, i.e., osteogenic, chondrogenic and adipogenic lineages [6]. Luo et al. recently found that BMP9 can induce the osteogenic differentiation of MSCs [7]. In the bone microenvironment, BCCs suppress the differentiation and proliferation of bone marrow-derived stem cells (BMSCs) [8], and BMSCs promote the invasion and migration of BCCs [9]. Previously, we found that BMP9 promotes the apoptosis of MDA-MB-231 breast cancer cells, inhibits the proliferation and invasion of these BCCs, and decreases the occurrence of osteolytic lesions elicited by these BCCs, both in vitro and in vivo [10, 11]. As yet, however, the mechanisms underlying these phenomena remained unclear. We specifically asked
2 Materials and methods 2.1 Cell culture HCT116 colon cancer cells and HS-5 bone marrowderived mesenchymal stem cells were maintained in complete DMEM (Dulbeccos modified Eagle medium) supplemented with 10 % fetal bovine serum (FBS; GIBCO, USA) and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5 % CO2. MDA-MB231breast cancer cells (purchased from Shanghai Institute for Biological Sciences, Chinese Academy of Science) [13, 14] were maintained in complete L-15 medium supplemented with 10 % FBS and 100 units/ml streptomycin/ penicillin at 37 °C in a humidified atmosphere without CO2. 2.2 Preparation of BMP9 conditioned medium Sub-confluent HCT116 cells cultured in 75 cm2 flaks were infected with optimal titers of recombinant adenoviruses expressing BMP9 and GFP (control), respectively (Ad-BMP9 and Ad-GFP were kindly provided by Professor TC He, Chicago University, USA). 24 h after infection, the culture medium was changed to serum-free medium composed of a mix of L-15 and DMEM. Conditioned medium (CM) was collected at 48 h after infection and used immediately. The experimental group was treated with MDA-MB-231+HS-5+ BMP9 CM, while MDA-MB-231+HS-5+GFP CM treated cells served as a control group and MDA-MB-231+HS-5 CM treated cells as a blank group. 2.3 Western blotting For Western blotting, cells were collected and lysed in RIPA buffer. Total cell lysates were denatured by boiling and loaded onto an 8–15 % gradient SDS-PAGE gel. After electrophoretic separation, proteins were transferred to an Immobilon-P
BMP9 regulates cross-talk between breast cancer cells
365
membrane. This membrane was blocked with Super-Block Blocking Buffer, and probed with primary antibodies, followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The proteins of interest were detected using a SuperSignal West Pico Chemiluminescent Substrate kit. The following primary antibodies were obtained from Santa Cruz or Abcam: anti-BMP9(1:500), anti-phosphor-p38 (1:1500), anti-p38 (1:1000), anti-phosphor-ERK1/2 (1:1500), anti-ERK1/2 (1:1000),anti-phosphor-AKT (1:1500), antiAKT (1:1000), anti-β-actin (1:500), anti-RANKL (1:2000), anti-OPG (1:1000), anti-OPN (1:1000), anti-OCN (1:1000), anti-IL-6 (1:1000), anti-PTH-rp (1:1000) and antiMMP9 (1:1000). 2.4 RNA isolation and RT-PCR Total RNA was isolated using TRIZOL Reagents (Invitrogen) according to the manufacturer’s instructions, and employed to generate cDNA using a Prime Script Kit (TAKARA, Dalian, China). PCR Primers (β-actin, BMP-9, RANKL,OCN and OPN; listed in Table 1) were designed using the Primer 5 program. Gene expression levels were quantified by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) for which β-actin was used as an endogenous control. The cycling program used was: initial denaturation at 94 °C for 5 min, followed by 28–34 cycles of denaturation
Table 1 Primer sequences and product sizes of different genes
Gene
at 94 °C for 30 s, annealing for 30 s at 52–58 °C, and extension at 72 °C for 30 s. The PCR products (5 μl) were separated in 1.5 % agarose gels, which were subsequently stained with GoodView Nucleic Acid Stain. Images were acquired using the Quantity One Software package (BIORAD, USA). All samples were run in triplicate and gene expression analyses were performed using the Quantity One Software package (BIO-RAD, USA). 2.5 Quantitative real-time RT-PCR Total RNA was isolated using TRIZOL Reagents (Invitrogen) and used to generate cDNA with a Prime Script Kit (TAKARA, Dalian, China). PCR Primers (GAPDH, IL6, IL11, PTH-rp, RANK, RANKL, MMP2 and MMP9; listed in Table 1) were designed using the Primer 5 program. SYBR Green-based qPCR analyses were performed to amplify the cDNAs of interest using RG-6000 (Corbett Research, Australia). Triplicate reactions were carried out for each sample. The cycling program used was: 94 °C for 2 min for 1 cycle and 30 cycles at 92 °C for 20 s, 57 °C for 30 s, and 72 °C for 20 s, followed by a plate read at 78 °C for each cycle. The relative expression of target mRNAs was normalized to a reference (GAPDH) using the 2-△△CT method, and expressed as the foldchange relative to the control. All samples were run in triplicate.
Primer sequence
Product size (bp)
β-actin
Forward: 5′-CACCACACCTTCTACAATGAGC-3′
695
BMP9
Reverse: 5′-GTGATCTCCTTCTGCATCCTGT-3′ Forward: 5′-GAGCAGTCACGAGGAGGA-3′
322
OCN
Reverse: 5′-ACCCGCAGGGAGGTCTTT-3′ Forward: 5′-GGCAGCGAGGTAGTGAAGAG-3′
230
OPN
Reverse: 5′-CTGGAGAGGAGCAGAACTGG-3′ Forward: 5′- TTGCTTTTGCCTCCTAGGCA-3′
381
GAPDH
Reverse: 5′- GTGAAAACTTCGGTTGCTGG-3′ Forward: 5′- CAGCGACACCCACTCCTC-3′
120
IL-6
Reverse: 5′-TGAGGTCCACCACCCTGT-3′ Forward: 5′- TAGTGAGGAACAAGCCAGAG-3′
234
IL-11
Reverse: 5′-TACATTTGCCGAAGAGCC-3′ Forward: 5′- GCTGACGGGGACCACAAC-3′
124
PTH-rp
Reverse: 5′-GCCGCAGGTAGGACAGTAGG-3′ Forward: 5′- CAGCGACACCCACTCCTC-3′
217
RANKL
Reverse: 5′-TGAGGTCCACCACCCTGT-3′ Forward: 5′- TGCCAACATTTGCTTTCG-3′
127
RANK
Reverse: 5′-TCCTCCTTTCATCAGGGTAT-3′ Forward: 5′- GCAGATCGCTCCTCCAT-3′
132
MMP2
Reverse: 5′-CCACAGGGCAGACATACAC-3′ Forward: 5′- TGGCAAGGAGTACAACAGC-3′
174
MMP9
Reverse: 5′- TGGAAGCGGAATGGAAAC-3′ Forward: 5′- GGGACGCAGACATCGTCATC-3′
139
Reverse: 5′-TCGTCATCGTCGAAATGGGC-3′
366
S. Wan et al.
2.6 Wound healing assay
2.10 Alizarin Red S staining
A wound healing assay was performed to assess the migration of MDA-MB-231 cells. To this end MDA-MB-231 cells, seeded in 6-well plates, were co-cultured with HS-5 cells seeded in Anopore tissue culture plate inserts (0.4 μm, Millipore). When MDA-MB-231 confluent cell areas reached ~80 % of the culture plate, conditioned medium was added to the co-culture system and a wound was created at the center of the culture plate by a pipette tip. Photographs were taken under a microscope at indicated time points (0 to 24 h). The incision width was measured at different sites, and the average wound-closure rate was calculated as:(W1–W2)/W1× 100 %, where W1 is the 0 h incision width and W2 is the 24 h incision width. This assay was independently repeated three times.
Calcium deposition was detected using Alizarin Red S staining as described by Luu et al. [15]. Briefly, HS-5 cells were cultured in medium containing 50 μg/ml α-ascorbic acid, 100 mM β-glycerophosphate and 100 nM dexamethasone. On the 19th day after treatment with conditioned medium, the cells were fixed with 0.05 % (v/v) glutaraldehyde at room temperature for 10 min. After being washed with distilled water, the fixed cells were incubated in 0.4 % Alizarin Red S for 5 min, followed by extensive washing with distilled water. Finally, stained calcium mineral deposits were recorded under bright field microscopy.
2.7 Transwell invasion assay
A MTT assay was performed in quintuplicate to assess the viability of HS-5 cells in the co-culture system. To this end, HS-5 cells were seeded at a concentration of 5×104 cells per well into a 6-well culture plate and MDA-MB-231 cells were seeded at a concentration of 1×104 per well into a 25-mm Anopore tissue culture plate (0.04 μm pore size; Millipore). The cells were co-cultured in conditioned medium from 1 to 5 days. The absorbance of the HS-5 cells was measured on 5 consecutive days at 492 nm using a microplate reader, after which a growth curve was made. This assay was independently repeated in triplicate.
A transwell invasion assay was performed to assess the invasion of MDA-MB-231 cells. To this end, MDA-MB-231 cells were seeded at a density of 4×105/well into the upper chamber of type I-collagen-coated 6-well culture inserts and HS-5 cells were seeded at a density of 2×106/well in to 6-well plates. Duplicate reactions were carried out for each group. The cells were co-cultured in conditioned medium. After 24 h, MDAMB-231 cells were dried for 5 min, fixed with dehydrated alcohol, and stained with hematoxylin-eosin. Next, the cells that invaded the collagen-coated-inserts were counted. Mean values from five randomly selected fields were obtained for each well. This assay was independently repeated three times. 2.8 Quantitative ELISA Three days after BMP9 CM treatment the cytokines RANKL, IL-6, DKK1, PTH-rp and IL-11 in the culture supernatants were measured by quantitative ELISA using a RD Systems kit (R&D) following the manufacturer’s protocol. 2.9 ALP activity measurement On the indicated day after exposure to the conditioned medium, the alkaline phosphatase (ALP) activity of HS-5 cells was measured using a modified Great Escape SEAP Chemiluminescence Assay (BD Clontech, Mountain View, CA, USA). Additionally, ALP expression by HS-5 cells was assessed by a histochemical staining assay (using a mixture of 0.1 mg/ml naphthol AS-MX phosphate and 0.6 mg/ml Fast Blue BB salt), as described by Luu et al. [15]. Each assay was performed in triplicate, and the ALP activity was normalized against the total cellular protein concentrations of the respective samples.
2.11 Cell viability assay
2.12 Animal models Human femur fragments obtained from freshly discarded tissue at the time of surgery from female patients undergoing total hip replacement were washed with PBS and dissected into 1 cm×1 cm pieces. Then the femur pieces were subcutaneously implanted in female nude mice. After 1 week the embedded bone was successfully implanted, as evidenced lack of inflammation and/or necrosis. Female nude mice were randomly divided into three groups comprising three mice each. Simultaneously, MDA-MB-231 breast cancer cells were infected with Ad-BMP9/Ad-GFP virus and a mixed cell suspension of infected MDA-MB-231 cells (5×106 ~5×108) and HS-5 cells (5 × 106 ~ 5 × 108, cell ratio ~1) was injected directly onto the implanted femur fragments in the nude mice. MDA-MB-231+HS-5 and MDA-MB-231/AdGFP+HS-5 served as two control groups, whereas MDA-MB-231/ AdBMP9+HS-5 served as the treatment group. After 2 weeks, when tumors were palpable, tumor diameters were recorded every 3 days. Tumor volumes (V in cm3) were then calculated as: (4π/3)×[(a+b)/4]3, whereπ=3.14. After 5 weeks, tumors and implanted femur fragments were harvested to assess the morphological and immunohistochemical changes. All experiments were approved by the Institutional Animal Care and
BMP9 regulates cross-talk between breast cancer cells
Use Committee of the Chongqing Medical University, as well as by regional authorities. 2.13 Immunohistochemistry Paraffin sections of the subcutaneous tumors were prepared following standard methods. The expression of osteopontin (OPN), a late osteogenic marker, and p-AKT in the tissues was examined by immunohistochemistry (IHC). To this end, the sections were rehydrated and heat-treated for antigen retrieval with citric acid buffer as usual, and then incubated with the respective primary antibodies at 4 °C overnight. On the following day the sections were incubated with a secondary antibody and visualized using 3, 3-diaminobenzidine tetrachloride (DAB) until the desired brown reaction product was obtained. All slides were observed under a Nikon E400 Light Microscope and representative photographs were taken. 2.14 Statistical analyses The results are expressed as means±standard deviations (SD). All statistical analyses were performed by SPSS 17.0 using the independent sample t-test for comparing the two sample groups, where P
ORIGINAL PAPER
BMP9 regulates cross-talk between breast cancer cells and bone marrow-derived mesenchymal stem cells Shaoheng Wan & Yuehong Liu & Yaguang Weng & Wei Wang & Wei Ren & Chang Fei & Yingying Chen & Zhihui Zhang & Ting Wang & Jinshu Wang & Yayun Jiang & Lan Zhou & Tongchuan He & Yan Zhang
Accepted: 1 September 2014 / Published online: 11 September 2014 # International Society for Cellular Oncology 2014
Abstract Purpose Breast cancer cells frequently metastasize to distant organs, including bone. Interactions between breast cancer cells and the bone microenvironment are known to enhance tumor growth and osteolytic damage. Here we investigated whether BMP9 (a secretary protein) may change the bone microenvironment and, by doing so, regulate the cross-talk between breast cancer cells and bone marrow-derived mesenchymal stem cells. Methods After establishing a co-culture system composed of MDA-MB-231breast cancer cells and HS-5 bone marrowderived mesenchymal stem cells, and exposure of this system to BMP9 conditioned media, we assessed putative changes in migration and invasion capacities of MDA-MB-231 cells and concomitant changes in osteogenic marker expressionin HS-5 cells and metastases-related genes in MDA-MB-231 cells. Results We found that BMP9 can inhibit the migration and invasion of MDA-MB-231 cells, and promote osteogenesis Shaoheng Wan and Yuehong Liu these authors contributed equally to this work. S. Wan : Y. Liu : Y. Weng : W. Wang : C. Fei : Y. Chen : Z. Zhang : T. Wang : J. Wang : Y. Jiang : L. Zhou : Y. Zhang (*) Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, China e-mail: [email protected] Y. Zhang e-mail: [email protected] W. Ren Department of General Surgery, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing 400042, China T. He Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
and proliferation of HS-5 cells, in the co-culture system. We also found that the BMP9-induced inhibition of migration and invasion of MDA-MB-231 cells may be caused by a decreased RANK ligand (RANKL) secretion by HS-5 cells, leading to a block in the AKT signaling pathway. Conclusions From our data we conclude that BMP9 inhibits the migration and invasion of breast cancer cells, and promotes the osteoblastic differentiation and proliferation of bone marrow-derived mesenchymal stem cells by regulating crosstalk between these two types of cells through the RANK/ RANKL signaling axis. Keywords BMP9 . Breast cancer . Bone marrow-derived mesenchymal stem cells . Cross-talk . RANKL/RANK axis Abbreviations BMP9 Bone morphogenetic protein 9 BCC Breast cancer cells BMSC Bone marrow –derived mesenchymal stem cells MSCs Mesenchymal stem cells OPG Osteoprotegerin RANK Receptor activator of nuclear factor-kappa B RANKL The RANK ligand Ad-BMP9 Adenovirus expressing BMP9 protein Ad-GFP Adenovirus expressing green fluorescent protein ERK1/2 Extracellular signal regulated kinases p-ERK1/2 Phospho-ERK1/2 OPN Osteopontin OCN Osteocalcin IL-6 Interleukin-6 IL11 Interleukin-11 PTH-rp Parathyroid hormone-related protein MMP9 Matrix metallopeptidase 9 MMP2 Matrix metallopeptidase 2
364
DKK1 ALP MAPK GSK-3β pGSK-3β CTGF LRP-6 BMP9-CM GFR-CM
S. Wan et al.
Dickkopf WNT signaling pathway inhibitor 1 Alkaline phosphatase Mitogen-activated protein kinase Glycogen synthase kinase-3β Phospho-GSK-3β Connective Tissue Growth Factor Low density lipoprotein receptor-related protein 6 BMP9 condition medium GFP Vector control condition medium
what role BMP9 (as a secretary protein) plays in the bone microenvironment and whether it does affect the cross-talk between BCCs and BMSCs. In the past, it has been reported that BMP9 can increase the osteogenic differentiation of BMSCs [7, 12]. Here, we focused on the effect of BMP9 on the interaction between BCCs and BMSCs, aiming to unravel the role of BMP9 in decreasing the occurrence of osteolytic lesions in the BCC microenvironment. To this end, a coculture system composed of MDA-MB-231 BCCs and HS-5 BMSCs was employed to investigate the effects of BMP9 on the interaction between these two cell types, and to clarify the action of BMP9 in inhibiting BCC osteolytic metastases in the bone microenvironment.
1 Introduction Breast cancer is a common malignancy in women world-wide, more than 1.38 million new cases are diagnosed and about 500,000 individuals die of the disease [1]. Breast cancer cells (BCCs) frequently metastasize to distant organs such as bone, lung and liver [2]. When BCCs metastasize to bone, they disturb the intricate equilibrium of the bone microenvironment, resulting in osteolytic injury [3]. The process of osteolytic metastasis is closely related to the bone microenvironment, and involves osteoblasts, osteoclasts and bone marrow-derived mesenchymal stem cells (BMSCs). In the past, it has been shown that the interaction between BCCs and bone microenvironment cells enhances tumor growth and osteolytic damage [4, 5]. BCCs, on one hand, not only secrete various cytokines to activate osteoclasts, but also activate osteoblasts to secrete osteoclast-activating factors, which results in the formation of osteolytic lesions. Osteoclasts and osteoblasts, on the other hand, secrete cytokines that promote the metastasis and proliferation of BCCs, which aggravates the osteolytic damage [4, 5]. It thus appears that their crosstalk generates a vicious cycle which is sustained by cytokines. Recently, several studies have focused on ways to interfere with the cross-talk between BCCs and bone microenvironment cells [3–5]. Mesenchymal stem cells (MSCs) are present in many tissues, including bone marrow, and they have the capacity to differentiate into three different lineages, i.e., osteogenic, chondrogenic and adipogenic lineages [6]. Luo et al. recently found that BMP9 can induce the osteogenic differentiation of MSCs [7]. In the bone microenvironment, BCCs suppress the differentiation and proliferation of bone marrow-derived stem cells (BMSCs) [8], and BMSCs promote the invasion and migration of BCCs [9]. Previously, we found that BMP9 promotes the apoptosis of MDA-MB-231 breast cancer cells, inhibits the proliferation and invasion of these BCCs, and decreases the occurrence of osteolytic lesions elicited by these BCCs, both in vitro and in vivo [10, 11]. As yet, however, the mechanisms underlying these phenomena remained unclear. We specifically asked
2 Materials and methods 2.1 Cell culture HCT116 colon cancer cells and HS-5 bone marrowderived mesenchymal stem cells were maintained in complete DMEM (Dulbeccos modified Eagle medium) supplemented with 10 % fetal bovine serum (FBS; GIBCO, USA) and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5 % CO2. MDA-MB231breast cancer cells (purchased from Shanghai Institute for Biological Sciences, Chinese Academy of Science) [13, 14] were maintained in complete L-15 medium supplemented with 10 % FBS and 100 units/ml streptomycin/ penicillin at 37 °C in a humidified atmosphere without CO2. 2.2 Preparation of BMP9 conditioned medium Sub-confluent HCT116 cells cultured in 75 cm2 flaks were infected with optimal titers of recombinant adenoviruses expressing BMP9 and GFP (control), respectively (Ad-BMP9 and Ad-GFP were kindly provided by Professor TC He, Chicago University, USA). 24 h after infection, the culture medium was changed to serum-free medium composed of a mix of L-15 and DMEM. Conditioned medium (CM) was collected at 48 h after infection and used immediately. The experimental group was treated with MDA-MB-231+HS-5+ BMP9 CM, while MDA-MB-231+HS-5+GFP CM treated cells served as a control group and MDA-MB-231+HS-5 CM treated cells as a blank group. 2.3 Western blotting For Western blotting, cells were collected and lysed in RIPA buffer. Total cell lysates were denatured by boiling and loaded onto an 8–15 % gradient SDS-PAGE gel. After electrophoretic separation, proteins were transferred to an Immobilon-P
BMP9 regulates cross-talk between breast cancer cells
365
membrane. This membrane was blocked with Super-Block Blocking Buffer, and probed with primary antibodies, followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The proteins of interest were detected using a SuperSignal West Pico Chemiluminescent Substrate kit. The following primary antibodies were obtained from Santa Cruz or Abcam: anti-BMP9(1:500), anti-phosphor-p38 (1:1500), anti-p38 (1:1000), anti-phosphor-ERK1/2 (1:1500), anti-ERK1/2 (1:1000),anti-phosphor-AKT (1:1500), antiAKT (1:1000), anti-β-actin (1:500), anti-RANKL (1:2000), anti-OPG (1:1000), anti-OPN (1:1000), anti-OCN (1:1000), anti-IL-6 (1:1000), anti-PTH-rp (1:1000) and antiMMP9 (1:1000). 2.4 RNA isolation and RT-PCR Total RNA was isolated using TRIZOL Reagents (Invitrogen) according to the manufacturer’s instructions, and employed to generate cDNA using a Prime Script Kit (TAKARA, Dalian, China). PCR Primers (β-actin, BMP-9, RANKL,OCN and OPN; listed in Table 1) were designed using the Primer 5 program. Gene expression levels were quantified by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) for which β-actin was used as an endogenous control. The cycling program used was: initial denaturation at 94 °C for 5 min, followed by 28–34 cycles of denaturation
Table 1 Primer sequences and product sizes of different genes
Gene
at 94 °C for 30 s, annealing for 30 s at 52–58 °C, and extension at 72 °C for 30 s. The PCR products (5 μl) were separated in 1.5 % agarose gels, which were subsequently stained with GoodView Nucleic Acid Stain. Images were acquired using the Quantity One Software package (BIORAD, USA). All samples were run in triplicate and gene expression analyses were performed using the Quantity One Software package (BIO-RAD, USA). 2.5 Quantitative real-time RT-PCR Total RNA was isolated using TRIZOL Reagents (Invitrogen) and used to generate cDNA with a Prime Script Kit (TAKARA, Dalian, China). PCR Primers (GAPDH, IL6, IL11, PTH-rp, RANK, RANKL, MMP2 and MMP9; listed in Table 1) were designed using the Primer 5 program. SYBR Green-based qPCR analyses were performed to amplify the cDNAs of interest using RG-6000 (Corbett Research, Australia). Triplicate reactions were carried out for each sample. The cycling program used was: 94 °C for 2 min for 1 cycle and 30 cycles at 92 °C for 20 s, 57 °C for 30 s, and 72 °C for 20 s, followed by a plate read at 78 °C for each cycle. The relative expression of target mRNAs was normalized to a reference (GAPDH) using the 2-△△CT method, and expressed as the foldchange relative to the control. All samples were run in triplicate.
Primer sequence
Product size (bp)
β-actin
Forward: 5′-CACCACACCTTCTACAATGAGC-3′
695
BMP9
Reverse: 5′-GTGATCTCCTTCTGCATCCTGT-3′ Forward: 5′-GAGCAGTCACGAGGAGGA-3′
322
OCN
Reverse: 5′-ACCCGCAGGGAGGTCTTT-3′ Forward: 5′-GGCAGCGAGGTAGTGAAGAG-3′
230
OPN
Reverse: 5′-CTGGAGAGGAGCAGAACTGG-3′ Forward: 5′- TTGCTTTTGCCTCCTAGGCA-3′
381
GAPDH
Reverse: 5′- GTGAAAACTTCGGTTGCTGG-3′ Forward: 5′- CAGCGACACCCACTCCTC-3′
120
IL-6
Reverse: 5′-TGAGGTCCACCACCCTGT-3′ Forward: 5′- TAGTGAGGAACAAGCCAGAG-3′
234
IL-11
Reverse: 5′-TACATTTGCCGAAGAGCC-3′ Forward: 5′- GCTGACGGGGACCACAAC-3′
124
PTH-rp
Reverse: 5′-GCCGCAGGTAGGACAGTAGG-3′ Forward: 5′- CAGCGACACCCACTCCTC-3′
217
RANKL
Reverse: 5′-TGAGGTCCACCACCCTGT-3′ Forward: 5′- TGCCAACATTTGCTTTCG-3′
127
RANK
Reverse: 5′-TCCTCCTTTCATCAGGGTAT-3′ Forward: 5′- GCAGATCGCTCCTCCAT-3′
132
MMP2
Reverse: 5′-CCACAGGGCAGACATACAC-3′ Forward: 5′- TGGCAAGGAGTACAACAGC-3′
174
MMP9
Reverse: 5′- TGGAAGCGGAATGGAAAC-3′ Forward: 5′- GGGACGCAGACATCGTCATC-3′
139
Reverse: 5′-TCGTCATCGTCGAAATGGGC-3′
366
S. Wan et al.
2.6 Wound healing assay
2.10 Alizarin Red S staining
A wound healing assay was performed to assess the migration of MDA-MB-231 cells. To this end MDA-MB-231 cells, seeded in 6-well plates, were co-cultured with HS-5 cells seeded in Anopore tissue culture plate inserts (0.4 μm, Millipore). When MDA-MB-231 confluent cell areas reached ~80 % of the culture plate, conditioned medium was added to the co-culture system and a wound was created at the center of the culture plate by a pipette tip. Photographs were taken under a microscope at indicated time points (0 to 24 h). The incision width was measured at different sites, and the average wound-closure rate was calculated as:(W1–W2)/W1× 100 %, where W1 is the 0 h incision width and W2 is the 24 h incision width. This assay was independently repeated three times.
Calcium deposition was detected using Alizarin Red S staining as described by Luu et al. [15]. Briefly, HS-5 cells were cultured in medium containing 50 μg/ml α-ascorbic acid, 100 mM β-glycerophosphate and 100 nM dexamethasone. On the 19th day after treatment with conditioned medium, the cells were fixed with 0.05 % (v/v) glutaraldehyde at room temperature for 10 min. After being washed with distilled water, the fixed cells were incubated in 0.4 % Alizarin Red S for 5 min, followed by extensive washing with distilled water. Finally, stained calcium mineral deposits were recorded under bright field microscopy.
2.7 Transwell invasion assay
A MTT assay was performed in quintuplicate to assess the viability of HS-5 cells in the co-culture system. To this end, HS-5 cells were seeded at a concentration of 5×104 cells per well into a 6-well culture plate and MDA-MB-231 cells were seeded at a concentration of 1×104 per well into a 25-mm Anopore tissue culture plate (0.04 μm pore size; Millipore). The cells were co-cultured in conditioned medium from 1 to 5 days. The absorbance of the HS-5 cells was measured on 5 consecutive days at 492 nm using a microplate reader, after which a growth curve was made. This assay was independently repeated in triplicate.
A transwell invasion assay was performed to assess the invasion of MDA-MB-231 cells. To this end, MDA-MB-231 cells were seeded at a density of 4×105/well into the upper chamber of type I-collagen-coated 6-well culture inserts and HS-5 cells were seeded at a density of 2×106/well in to 6-well plates. Duplicate reactions were carried out for each group. The cells were co-cultured in conditioned medium. After 24 h, MDAMB-231 cells were dried for 5 min, fixed with dehydrated alcohol, and stained with hematoxylin-eosin. Next, the cells that invaded the collagen-coated-inserts were counted. Mean values from five randomly selected fields were obtained for each well. This assay was independently repeated three times. 2.8 Quantitative ELISA Three days after BMP9 CM treatment the cytokines RANKL, IL-6, DKK1, PTH-rp and IL-11 in the culture supernatants were measured by quantitative ELISA using a RD Systems kit (R&D) following the manufacturer’s protocol. 2.9 ALP activity measurement On the indicated day after exposure to the conditioned medium, the alkaline phosphatase (ALP) activity of HS-5 cells was measured using a modified Great Escape SEAP Chemiluminescence Assay (BD Clontech, Mountain View, CA, USA). Additionally, ALP expression by HS-5 cells was assessed by a histochemical staining assay (using a mixture of 0.1 mg/ml naphthol AS-MX phosphate and 0.6 mg/ml Fast Blue BB salt), as described by Luu et al. [15]. Each assay was performed in triplicate, and the ALP activity was normalized against the total cellular protein concentrations of the respective samples.
2.11 Cell viability assay
2.12 Animal models Human femur fragments obtained from freshly discarded tissue at the time of surgery from female patients undergoing total hip replacement were washed with PBS and dissected into 1 cm×1 cm pieces. Then the femur pieces were subcutaneously implanted in female nude mice. After 1 week the embedded bone was successfully implanted, as evidenced lack of inflammation and/or necrosis. Female nude mice were randomly divided into three groups comprising three mice each. Simultaneously, MDA-MB-231 breast cancer cells were infected with Ad-BMP9/Ad-GFP virus and a mixed cell suspension of infected MDA-MB-231 cells (5×106 ~5×108) and HS-5 cells (5 × 106 ~ 5 × 108, cell ratio ~1) was injected directly onto the implanted femur fragments in the nude mice. MDA-MB-231+HS-5 and MDA-MB-231/AdGFP+HS-5 served as two control groups, whereas MDA-MB-231/ AdBMP9+HS-5 served as the treatment group. After 2 weeks, when tumors were palpable, tumor diameters were recorded every 3 days. Tumor volumes (V in cm3) were then calculated as: (4π/3)×[(a+b)/4]3, whereπ=3.14. After 5 weeks, tumors and implanted femur fragments were harvested to assess the morphological and immunohistochemical changes. All experiments were approved by the Institutional Animal Care and
BMP9 regulates cross-talk between breast cancer cells
Use Committee of the Chongqing Medical University, as well as by regional authorities. 2.13 Immunohistochemistry Paraffin sections of the subcutaneous tumors were prepared following standard methods. The expression of osteopontin (OPN), a late osteogenic marker, and p-AKT in the tissues was examined by immunohistochemistry (IHC). To this end, the sections were rehydrated and heat-treated for antigen retrieval with citric acid buffer as usual, and then incubated with the respective primary antibodies at 4 °C overnight. On the following day the sections were incubated with a secondary antibody and visualized using 3, 3-diaminobenzidine tetrachloride (DAB) until the desired brown reaction product was obtained. All slides were observed under a Nikon E400 Light Microscope and representative photographs were taken. 2.14 Statistical analyses The results are expressed as means±standard deviations (SD). All statistical analyses were performed by SPSS 17.0 using the independent sample t-test for comparing the two sample groups, where P
