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Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

ABSTRACT: The remodeling process in bone yields numerous cytokines and chemokines that mediate crosstalk between osteoblasts and osteoclasts and also serve to attract and support metastatic tumor cells. The metastatic tumor cells disturb the equilibrium in bone that manifests as skeletal complications. The Hedgehog (Hh) pathway plays an important role in skeletogenesis. We hypothesized that the Hh pathway mediates an interaction between tumor cells and osteoblasts and influences osteoblast differentiation in response to tumor cells. We have determined that breast tumor cells have an activated Hh pathway characterized by upregulation of the ligand, IHH and transcription factor GLI1. Breast cancer cells interact with osteoblasts and cause an enhanced differentiation of pre-osteoblasts to osteoblasts that express increased levels of the osteoclastogenesis factors, RANKL and PTHrP. There is sustained expression of osteoclast-promoting factors, RANKL and PTHrP, even after the osteoblast differentiation ceases and apoptosis sets in. Moreover, tumor cells that are deficient in Hh signaling are compromised in their ability to induce osteoblast differentiation and consequently are inefficient in causing osteolysis. The stimulation of osteoblast differentiation sets the stage for osteoclast differentiation and overall promotes osteolysis. Thus, in the process of developing newer therapeutic strategies against breast cancer metastasis to bone it would worthwhile to keep in mind the role of the Hh pathway in osteoblast differentiation in an otherwise predominant osteolytic phenomenon.

SUBMITTER: Das S

PROVIDER: S-EPMC3315536 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

Das Shamik S Tucker J Allan JA Khullar Shikha S Samant Rajeev S RS Shevde Lalita A LA

PloS one 20120329 3

The remodeling process in bone yields numerous cytokines and chemokines that mediate crosstalk between osteoblasts and osteoclasts and also serve to attract and support metastatic tumor cells. The metastatic tumor cells disturb the equilibrium in bone that manifests as skeletal complications. The Hedgehog (Hh) pathway plays an important role in skeletogenesis. We hypothesized that the Hh pathway mediates an interaction between tumor cells and osteoblasts and influences osteoblast differentiation ...[more]

PMID: 22479615

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Project description:MicroRNAs are emerging as critical post-transcriptional modulators in bone remodeling, regulating the functions of osteoblasts and osteoclasts. Intercellular crosstalk between osteoblasts and osteoclasts is mediated by miR-21 that controls the bone homeostasis response, providing potential targets for the maintenance of osteoblast function. The aim of this study was to investigate the effects of miR-21 on osteoblast function, and to explore the underlying mechanism. Increased alkaline phosphatase (ALP) activity and accelerated matrix mineralization was observed in mouse pre-osteoblast MC3T3-E1 cells compared with the non-induction (control) group. MiR-21 positively regulates osteogenic differentiation and mineralization by facilitating the expression of key osteogenic factors (ALP, Runx2, Osteopontin (OPN), Osterix (OSX) and Mef2c) in MC3T3-E1 cells. Furthermore, a deficiency of miR-21 suppresses the expression of those factors at both the mRNA and protein levels, indicating that miR-21 is a positive regulator of osteoblastic differentiation. H-E staining, Azan staining, Masson's Trichrome staining and Toluidine blue staining were performed in jaw and femur tissues of miR-21 knockout (miR-21KO) and wild-type (WT) mice. Immunohistochemical staining revealed substantially lower levels of ALP, Runx2 and OSX expression in jaw and femur tissues of miR-21KO mice. A similar trend was observed in femur tissues using quantitative real-time (RT) PCR. A total of 17 osteogenesis-related mRNAs were found to be differentially expressed in miR-21KO femur tissues using Mouse Gene Expression Microarray analysis. GeneSpring and Ingenuity Pathway Analysis revealed several potential target genes that are involved in bone remodeling, such as IL-1? and HIF-1?. Several important pathways were determined to be facilitators of miR-21, which provides a reliable reference for future studies to elucidate the biological mechanisms of osteoblast function. Taken together, these results lead us to hypothesize a potential role for miR-21 in regulating osteoblast function, thus representing a potential biomarker of osteogenesis. Highlights • miR-21 facilitates osteoblast differentiation.• miR-21 deficiency impairs the osteogenic activity of alveolar bone and femur.• DNA microarrays identify differentially expressed genes with miR-21 deficiency.

Project description:BackgroundHedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated.MethodsA Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology.ResultsBMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NF?B and STAT signaling.ConclusionsWe identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

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Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

Publications

Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

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