Targeting syndecan-1 in breast cancer inhibits osteoclast functions through up-regulation of osteoprotegerin

AbstractBackgroundBreast cancer often metastasizes into bone and leads to osteolytic lesions. The underlying mechanisms, however, are complex and not fully understood. Syndecan-1 is a proteoglycan that has various functions relevant for tumor progression including cell–cell communication and cell–matrix interactions. Moreover, its two glycosaminoglycan-binding sites suggest that it may interfere with glycoproteins such as osteoprotegerin, a potent inhibitor of osteoclastogenesis. Thus, we hypothesize that tumor-derived syndecan-1 alters osteoclast biology by modulating osteoprotegerin.MethodsSyndecan-1 expression was down-regulated via siRNA and the cell fate of the breast cancer cell lines MCF-7, T-47D, and MDA-MB-231 was investigated. Furthermore, we determined the regulation of syndecan-1 by dexamethasone, a commonly used antiemetic in breast cancer therapy. Additionally, we analyzed the genesis and activity of osteoclasts in indirect co-culture experiments using supernatants from MCF-7 cells with deficient and sufficient levels of syndecan-1.ResultsDexamethasone time- and dose-dependently increased syndecan-1 expression up to 4-fold but did not alter cell behavior. Syndecan-1 up-regulation did not affect the survival or migration of breast cancer cells. Depletion of syndecan-1 using siRNA led to decreased vitality of progesterone receptor-positive cell lines. In MCF-7 cells osteoprotegerin production was up-regulated 2.5-fold after syndecan-1 knock-down. The culture of osteoclast precursors with the supernatant of MCF-7 cells with reduced syndecan-1 levels suppressed osteoclast formation and activity by 21% and 23%, respectively. Adding neutralizing antibodies to osteoprotegerin to the breast cancer supernatants reversed osteoclastogenesis.ConclusionThus, we identified tumor-derived syndecan-1 as a novel positive regulator of osteoclastogenesis and new player in the tumor-bone dialog

Zoledronic acid and atorvastatin inhibit αvβ3-mediated adhesion of breast cancer cells

Bone metastases represent common long term complications of patients with breast cancer. Zoledronic acid, an amino-bisphosphonate and mevalonate pathway inhibitor, is an established agent for the treatment of bone metastases. Direct antitumor effects of zoledronic acid have been proposed in breast cancer. Statins are another group of mevalonate pathway inhibitors that have been repeatedly discussed for potential anti-tumor activity. In this study, we tested the hypothesis, whether these agents regulate adhesion of breast cancer cells to extracellular matrix components. Treatment of breast cancer cells with zoledronic acid and atorvastatin, significantly impaired MDA-MB-231 breast cancer cell adhesion on the αvβ3 ligands gelatin and vitronectin, but had no effect on collagen type 1 (α2β1-ligand) and fibronectin (α5β1-ligand). Anti-adhesive effects of zoledronic acid were fully reversed by geranylgeranyl pyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Furthermore, effects of zoledronic acid and atorvastatin were mimicked by a specific inhibitor of geranylgeranylation GGTI-298. Functional (using integrin array) and quantitative (using FACS) integrin analyses on MDA-231 cells following zoledronic acid exposure revealed decreased levels of αv and αvβ3 expression. In addition to its effects on integrin mediated adhesion of breast cancer cells, the presence of zoledronic acid caused pronounced morphological changes in MDA-231 cells as seen by F-actin and vinculin rearrangement. Furthermore, phosphorylation of the focal adhesion kinase was inhibited by zoledronic acid. In both cases, changes were fully reversed by GGPP. These results emphasize the role of mevalonate pathway mediated impairment of geranylgeranylation in the anti-adhesive effects of zoledronic acid in breast cancer cells

Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer

Abstract Background The mammalian target of rapamycin inhibitor everolimus is approved as an antitumor agent in advanced estrogen receptor-positive breast cancer. Surrogate bone marker data from clinical trials suggest effects on bone metabolism, but the mode of action of everolimus in bone biology remains unclear. In this study, we assessed potential bone-protective effects of everolimus in the context of osteotropic tumors. Methods The effects of everolimus on cancer cell viability in vitro and on tumor growth in vivo were assessed. Everolimus-regulated osteoclastogenesis and osteoblastogenesis were also assessed in vitro before we assessed the bone-protective effect of everolimus in a model where bone loss was induced in ovariectomized (OVX) mice. Finally, the role of everolimus in the progression of osteolytic bone disease was assessed in an intracardiac model of breast cancer bone metastases. Results At low concentrations (1 nM) in vitro, everolimus reduced the viability of human and murine cancer cell lines and impaired the osteoclastogenesis of osteoclast progenitors as assessed by quantitative real-time polymerase chain reaction and counting tartrate-resistant acid phosphatase-positive, multinucleated osteoclasts (p < 0.001). Everolimus had little or no deleterious effect on osteoblastogenesis in vitro, with concentrations of 1 and 10 nM increasing the messenger RNA expression of osteoblast marker genes (p ≤ 0.05) and leaving mineralization in differentiated human mesenchymal stem cells unchanged. Everolimus treatment (1 mg/kg body weight/day) prevented the bone loss observed in OVX mice and concurrently inhibited the metastatic growth of MDA-MB-231 cells by 70% (p < 0.002) while preserving bone mass in an intracardiac model of bone metastasis. Conclusions These results underline the antitumor effects of everolimus and highlight its bone-protective efficacy, warranting further research on the potential implications on bone health in populations prone to osteoporosis and bone metastases, such as postmenopausal women with breast cancer

Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity

Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes

Additional file 4: Figure S4. of Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer

Standard histological sections of TRAP staining in the femur. Representative images of an OVX control-treated femur and an everolimus-treated femur stained for TRAP (a, ×2.5 magnification, scale bar 200 μm; b, ×20 original magnification, scale bar 50 μm). (PDF 3572 kb

Additional file 1: Figure S1. of Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer

Everolimus inhibits mTOR signaling in cancer cell lines. Quantification of Western blots shown in Fig. 1. Indicated cell lines were treated with everolimus for 24 h, and total and phosphorylated proteins were detected by Western blot analysis. The signals of phosphorylated mTOR (p-mTOR) and phosphorylated p70 S6 kinase (p-p70) were quantified and normalized to corresponding signals of GAPDH for a total of three experiments. Data were analyzed using one-way ANOVA and the Bonferroni posttest and are shown as mean ± SD (* p < 0.05; ** p < 0.01, *** p < 0.001). Equal volumes of DMSO used to prepare and administer everolimus treatments were used in the control conditions. (PDF 14 kb

Additional file 3: Figure S3. of Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer

Everolimus inhibits the bone-resorbing activity of osteoclasts. Murine bone marrow-derived mononuclear cells were differentiated to osteoclasts on bone slices in vitro before being treated with everolimus at concentrations of 1, 10, and 100 nM for 5 days in total. On day 5, supernatants were collected and analyzed for the levels of the bone resorption marker collagen type I cross-linked C-telopeptide (CTx). Data were analyzed using one-way ANOVA and the Bonferroni posttest, and significance between the control and everolimus concentrations is denoted (** p < 0.01). (PDF 9 kb

Dickkopf1 fuels inflammatory cytokine responses

Many human diseases, including cancer, share an inflammatory component but the molecular underpinnings remain incompletely understood. We report that physiological and pathological Dickkopf1 (DKK1) activity fuels inflammatory cytokine responses in cell models, mice and humans. DKK1 maintains the elevated inflammatory tone of cancer cells and is required for mounting cytokine responses following ligation of toll-like and cytokine receptors. DKK1- controlled inflammation derives from cell-autonomous mechanisms, which involve SOCS3- restricted, nuclear RelA (p65) activity. We translate these findings to humans by showing that genetic DKK1 variants are linked to elevated cytokine production across healthy populations. Finally, we find that genetic deletion of DKK1 but not pharmacological neutralization of soluble DKK1 ameliorates inflammation and disease trajectories in a mouse model of endotoxemia. Collectively, our study identifies a cell-autonomous function of DKK1 in the control of the inflammatory response, which is conserved between malignant and nonmalignant cells. Additional studies are required to mechanistically dissect cellular DKK1 trafficking and signaling pathways