Altered immunolocalization of FGF23 in murine femora metastasized with human breast carcinoma MDA-MB-231 cells (original) (raw)
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PLOS ONE, 2017
Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been implicated in promoting breast cancer growth and progression. While the autocrine effects of FGFR activation in tumor cells have been extensively studied, little is known about the effects of tumor cell-derived FGFs on cells in the microenvironment. Because FGF signaling has been implicated in the regulation of bone formation and osteoclast differentiation, we hypothesized that tumor cell-derived FGFs are capable of modulating osteoclast function and contributing to growth of metastatic lesions in the bone. Initial studies examining FGFR expression during osteoclast differentiation revealed increased expression of FGFR1 in osteoclasts during differentiation. Therefore, studies were performed to determine whether tumor cell-derived FGFs are capable of promoting osteoclast differentiation and activity. Using both non-transformed and transformed cell lines, we demonstrate that breast cancer cells express a number of FGF ligands that are known to activate FGFR1. Furthermore our results demonstrate that inhibition of FGFR activity using the clinically relevant inhibitor BGJ398 leads to reduced osteoclast differentiation and activity in vitro. Treatment of mice injected with tumor cells into the femurs with BGJ398 leads to reduced osteoclast activity and bone destruction. Together, these studies demonstrate that tumor cell-derived FGFs enhance osteoclast function and contribute to the formation of metastatic lesions in breast cancer.
Fibroblast growth factors signaling in bone metastasis
Endocrine-Related Cancer, 2020
Many solid tumors metastasize to bone, but only prostate cancer has bone as a single, dominant metastatic site. Recently, the FGF axis has been implicated in cancer progression in some tumors and mounting evidence indicate that it mediates prostate cancer bone metastases. The FGF axis has an important role in bone biology and mediates cell-to-cell communication. Therefore, we discuss here basic concepts of bone biology, FGF signaling axis, and FGF axis function in adult bone, to integrate these concepts in our current understanding of the role of FGF axis in bone metastases.
Molecular Pathway for Cancer Metastasis to Bone
Journal of Biological Chemistry, 2003
SPARC by the integrins induces VEGF production to support bone remodeling and neovascularization to nourish the metastatic tumor. EXPERIMENTAL PROCEDURES Cell Lines and Materials-The LNCaP lineage-derived human prostate cancer cell line, LNCaP-C4-2, is androgen-independent and highly tumorigenic, with a proclivity to metastasize to bone (33). All human prostate cancer cell lines LNCaP, LNCaP-C4-2, PC3, and lacZ-transfected CWR22R (H-clones), kindly provided by Dr. Lloyd A. Culp (Case Western Reserve University, Cleveland, OH), were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Recombinant SPARC was expressed in insect cells and purified as described previously (34). Platelet-derived purified SPARC was obtained from Hematologic Technologies, Inc. (Essex Junction, VT). Fibronectin was purchased from Roche Diagnostics, and collagen type I was obtained from Calbiochem. Antibodies against ␣ v  3 (LM609), ␣ v  5 (P1F6), ␣ 5  1 (JBS5), and ␣ 2  1 (BHA2.1) integrins were obtained from Chemicon (Temecula, CA). Anti-VEGF and anti-VEGFR-2 were obtained from R&D Systems (Minneapolis, MN). Anti-platelet-derived growth factor (PDGF) was obtained from Sigma, and WOW-1 was provided by Dr. Shattil, La Jolla, CA. cRGDfV peptide was obtained from Peninsula Laboratories Inc. (San Carlos, CA). Preparation of Bone Extracts-SPARC-null and control mice of the same genetic background were generated as described previously (23). The SPARC-null mice do not contain detectable amounts of SPARC mRNA or protein. Bones excised from 6-week-old male wild-type and SPARC-null littermates (from pelvic region and limb) (1 g) were crushed and extracted for 24 h at 4°C, as described previously (4). Extracts were centrifuged at 25,000 ϫ g for 30 min, and the supernatant fractions were dialyzed against phosphate-buffered saline. Cell Migration Assays-Cell migration assays on Transwell plates (8-m pore size) were performed as described previously (27). Ligand (recombinant or purified SPARC or bone extract, fibronectin, and collagen) was diluted to a selected concentration, and 10 l of this solution was placed on the lower surface of a polycarbonate filter and air-dried. Cell migration was performed in the presence of blocking antibodies against ␣ v  3, ␣ v  5, ␣ 5  1 , and ␣ 2  1 , neutralizing antibodies to VEGF, VEGFR-2, and PDGF (20 g/ml each) or cRGDfV peptides (20 M). Flow Cytometry-WOW-1, a Fab fragment, reacts selectively with activated ␣ v  3 and ␣ v  5 (29). Its binding to cells was assessed by flow cytometry, as described previously (16). In selected experiments, cells were preincubated for 5 min in the presence of an inhibitor (anti-VEGF or anti-VEGFR-2 neutralizing antibodies or VEGFR2/Fc chimera; 20 g/ml each). WOW-1 Fab was then added at a final concentration of 30 g/ml, followed by the addition of Alexa 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR) at 15 g/ml. After 30 min, the cells were washed and analyzed by flow cytometry. Specific binding of WOW-1 Fab was defined as that which could be inhibited by 10 mM EDTA. Flow cytometry was performed with a FACScan instrument, and the data were analyzed with the CellQuest software program (version 1.2). Proliferation Assays-These assays were performed as described (35). Briefly, cells were maintained in 1% serum for 20 h prior to experiments. Trypsinized cells were distributed into 96-well microtiter plates (2 ϫ 10 5 cells/well) in the presence or absence of inhibitors. The cells were labeled with 1 Ci of [ 3 H]thymidine per well. After 24-48 h, the cells were washed, and the radioactivity was precipitated with trichloroacetic acid and quantified by scintillation counting. Soft Agar Assays-LNCaP-C4-2 cells were trypsinized, washed, and resuspended in 0.4% Bacto-agar (Difco, Detroit, MI) prepared in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were plated on the top of 0.7% agar in the presence or absence of VEGFR-2/Fc chimera or anti-VEGFR-2 blocking antibodies (10 g/ml each). After 5 days, the colonies were photographed, and the number of colonies per power field was quantified 7 days after plating. Relative Quantitative Real Time PCR-Total RNA from two prostate cancer cell lines (LNCaP and LNCaP-C4-2) was prepared with RNeasy mini kits (Qiagen, Valencia, CA). Cells were plated in wells, which were left uncoated (control) or were coated with SPARC (200 ng/well), anti-␣ v  5 integrin antibodies (P1F6), or control nonimmune IgG (10 g/ml each). In selected experiments cRGDfV (20 M) was added to the cells; after specific time periods of incubation at 37°C, RNA was isolated. Real time PCR was performed using SYBR Green PCR core reagents (PerkinElmer Life Sciences) in an ABI Prism 7700 sequence detection system (PerkinElmer Life Sciences). The forward primer was 5Ј-GAG-GAGTCCAACATCACCATGC-3Ј, located on exon 3; the reverse primer
International Journal of Molecular Sciences, 2019
Hepatocyte growth factor (HGF) and transforming growth factor β1 (TGFβ1) are biological stimuli of the micro-environment which affect bone metastasis phenotype through transcription factors, but their influence on the growth is scarcely known. In a xenograft model prepared with 1833 bone metastatic cells, derived from breast carcinoma cells, we evaluated mice survival and Twist and Snail expression and localization after competitive inhibition of HGF with NK4, or after blockade of TGFβ1-type I receptor (RI) with SB431542: in the latter condition HGF was also measured. To explain the in vivo data, in 1833 cells treated with SB431542 plus TGFβ1 we measured HGF formation and the transduction pathway involved. Altogether, HGF seemed relevant for bone-metastatic growth, being hampered by NK4 treatment, which decreased Twist more than Snail in the metastasis bulk. TGFβ1-RI blockade enhanced HGF in metastasis and adjacent bone marrow, while reducing prevalently Snail expression at the fron...
Vascular endothelial growth factor acts as an osteolytic factor in breast cancer metastases to bone
The Women's Oncology Review, 2005
Vascular endothelial growth factor (VEGF) is a proangiogenic cytokine that is expressed highly in many solid tumours often correlating with a poor prognosis. In this study, we investigated the expression of VEGF and its receptors in bone metastases from primary human breast tumours and further characterised its effects on osteoclasts in vitro. Breast cancer metastases to bone were immunohistochemically stained for VEGF, its receptors VEGFR1 and 2 (vascular endothelial growth factor receptor 1 and 2), demonstrating that breast cancer metastases express VEGF strongly and that surrounding osteoclasts express both VEGFR1 and VEGFR2. RAW 264.7 cells (mouse monocyte cell line) and human peripheral blood mononuclear cells (PBMCs) were cultured with VEGF, RANKL and M-CSF. VEGF and RANKL together induced differentiation of multinucleated, tartrate-resistant acid phophatase (TRAP)-positive cells in similar numbers to M-CSF and RANKL. The PBMCs were also able to significantly stimulate resorption of mineralised matrix after treatment with M-CSF with RANKL and VEGF with RANKL. We have shown that VEGF in the presence of RANKL supports PBMC differentiation into osteoclast-like cells, able to resorb substrate. Vascular endothelial growth factor may therefore play a role in physiological bone resorption and in pathological situations. Consequently, VEGF signalling may be a therapeutic target for osteoclast inhibition in conditions such as tumour osteolysis.
Clinical & Experimental Metastasis, 2010
Bone likely provides a hospitable environment for cancer cells as suggested by their preferential localization to the skeleton. Previous work has shown that osteoblast-derived cytokines increased in the presence of metastatic breast cancer cells. Thus, we hypothesized that osteoblast-derived cytokines, in particular IL-6, MCP-1, and VEGF, would be localized to the bone metaphyses, an area to which breast cancer cells preferentially traffic. Human metastatic MDA-MB-231 breast cancer cells were inoculated into the left ventricle of the heart of athymic mice. Three to four weeks later, tumor localization within isolated femurs was examined using lCT and MRI. In addition, IL-6, MCP-1, and VEGF localization were assayed via immunohistochemistry. We found that MDA-MB-231 cells colonized trabecular bone, the area in which murine MCP-1 and VEGF were visualized in the bone matrix. In contrast, IL-6 was expressed by murine cells throughout the bone marrow. MDA-MB-231 cells produced VEGF, whose expression was not only associated with the breast cancer cells, but also increased with tumor growth. This is the first study to localize MCP-1, VEGF, and IL-6 in bone compartments via immunohistochemistry. These data suggest that metastatic cancer cells may co-opt bone cells into creating a niche facilitating cancer cell colonization.
Effects of Human Tumor Cell Lines on Local New Bone Formation In Vivo
Calcified Tissue International, 1997
Although some tumors cause osteolytic lesions, there are some that stimulate new bone formation. This is an important phenomenon because the responsible mechanisms probably represent an aberration of normal physiological bone formation, and identifying the factors involved in the process may lead to new therapies for various bone diseases. To clarify our understanding of the potential mechanism responsible, we compared and quantitated the extent of new bone formation stimulated by human tumors (HeLa, Hep-2, AV-3, FL, WISH and KB), some of which have osteogenic activity in vivo [2]. Tumor cells were injected over the calvaria of nude mice to examine formation of new bone. The tumor cells produced three histologically distinct patterns of new bone growth: (1) WISH and KB stimulated appositional bone growth adjacent to periosteal bone surfaces; (2) HeLa and Hep2 induced new bone growth over calvarial surface even when distant from the tumor mass; (3) FL stimulated bone formation adjacent to periosteum as well as ectopic bone formation in sites distant from bone. All tumors except AV3 induced mean new bone thickness >100 m, and Hep-2 cells produced bone 330 m thick. PCR and Northern blot analysis of mRNA isolated from cultured tumor cells revealed that all cell lines expressed mRNA for TGF, (fibroblast growth factor) FGF-1, FGF-2, and IGF-I, and most cell lines produced mRNA for PDGF. Only FL expressed large amounts of mRNA for BMP2. In serum-free conditioned media from Hep2 and HeLa cells purified by heparin affinity chromatography, we have identified FGF-1, FGF-2, and PDGF by immunodetection with specific antibodies. Our results show that new bone growth caused by these tumors is likely due to the production of bone growth factors by the tumor cells, and that the overall effects on bone may be due to several factors working in concert.
TGF-β in the Bone Microenvironment: Role in Breast Cancer Metastases
Breast cancer is the most prevalent cancer among females worldwide. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ∼70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by bone destruction, bone fractures, pain, and hypercalcemia, causing severe morbidity and hospitalization. In the bone matrix, transforming growth factor-β (TGF-β) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-β, in turn, stimulates bone metastatic cells to secrete factors that further drive osteolytic destruction of the bone adjacent to the tumor, categorizing TGF-β as a crucial factor responsible for driving the feedforward vicious cycle of cancer growth in bone. Moreover, TGF-β activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-β signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-β in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-β inhibitors in clinical practice to treat breast cancer bone metastases.
Oncogene, 1997
FGF-1 is expressed in a high proportion of breast tumors. While overexpression of FGF-4 in the MCF-7 breast carcinoma cell line confers the ability to form spontaneously metastasizing tumors in ovariectomized nude mice without estrogen supplementation and in mice that receive tamoxifen pellets, the response of a cell to individual FGFs can be controlled at multiple levels, and the signi®cance of FGF-1 expression in human breast tumors is uncertain. To study the role of FGF-1, MCF-7 human breast cancer carcinoma cells, previously transfected with bacterial b-galactosidase, were retransfected with FGF-1 expression vectors. FGF-1 transfectants formed large, vascularized tumors in ovariectomized nude mice without estrogen supplementation as well as in mice that received tamoxifen pellets. Lymphatic and pulmonary micrometastases were detected as deposits of X-galstained cells as early as 17 days after cell inoculation whereas no metastases were detected in estrogensupplemented mice bearing similar-sized control tumors. When compared with controls, both clonal and polyclonal populations of FGF-1 overexpressing cells exhibited increased anchorage-independent growth and decreased population doubling times in estrogen-depleted or 4-hydroxytamoxifen containing medium. These results suggest that FGF signaling may be important in the transition of breast cancer cells from hormone-dependent to hormone-independent and from nonmetastatic to metastatic.