Metastatic breast cancer cells inhibit osteoblast differentiation through the Runx2/CBFβ-dependent expression of the Wnt antagonist, sclerostin.
BACKGROUND
Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction by stimulating osteoclasts to resorb bone and by preventing osteoblasts from producing new bone. The Runt-related transcription factor 2, Runx2, is an important determinant of bone metastasis in breast cancer. Runx2 is known to mediate activation of osteoclast activity and inhibition of osteoblast differentiation by metastatic breast cancer cells. However, while Runx2-regulated genes that mediate osteoclast activation have been identified, how Runx2 determines inhibition of osteoblasts is unknown.


METHODS
The aim of this study was to determine how Runx2 mediates the ability of metastatic breast cancer cells to modulate the activity of bone cells. We have previously demonstrated that Runx2 requires the co-activator core binding factor beta (CBFβ) to regulate gene expression in breast cancer cells. We, therefore, performed independent microarray analyses to identify target genes whose expression is dependent upon both Runx2 and CBFβ. Common target genes, with a role in modulating bone-cell function, were confirmed using a combination of siRNA, quantitative reverse transcriptase PCR (qRT-PCR), ELISA, promoter reporter analysis, Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The function of Runx2/CBFβ-regulated genes in mediating the ability of MDA-MB-231 to inhibit osteoblast differentiation was subsequently established in primary bone marrow stromal cell cultures and MC-3T3 osteoblast cells.


RESULTS
We show that Runx2/CBFβ mediates inhibition of osteoblast differentiation by MDA-MB-231 cells through induction of the Wnt signaling antagonist, sclerostin. We demonstrate that MDA-MB-231 cells secrete sclerostin and that sclerostin-expression is critically dependent on both Runx2 and CBFβ. We also identified the osteoclast activators IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as new target genes of Runx2/CBFβ in metastatic breast cancer cells.


CONCLUSIONS
This study demonstrates that Runx2 and CBFβ are required for the expression of genes that mediate the ability of metastatic breast cancer cells to directly modulate both osteoclast and osteoblast function. We also show that Runx2-dependent inhibition of osteoblast differentiation by breast cancer cells is mediated through the Wnt antagonist, sclerostin.
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK.
Daniel
D
Mendoza-Villanueva
Leo
L
Zeef
Paul
P
Shore
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D050658:Core Binding Factor beta Subunit / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005819:Genetic Markers / Q000235:genetics*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017370:Interleukin-11 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000378:metabolism* / Q000473:pathology*; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D051153:Wnt Proteins / Q000037:antagonists & inhibitors*
D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; C493692:CBFB protein, human; D050659:Core Binding Factor Alpha 1 Subunit; D050658:Core Binding Factor beta Subunit; D005819:Genetic Markers; C508611:IL11 protein, human; D017370:Interleukin-11; C493709:RUNX2 protein, human; C422903:SOST protein, human; D051153:Wnt Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor