Regulation Of Pre-metastatic Niche By RSPO2/RANKL-LGR4 Signaling In Breast Cancer Bone Metastasis

The incidence and mortality of breast cancer are among the first in women cancer and metastasis is the leading cause of death in patients.The bone metastases incidence is as high as 65-80%.Researchers have shown that breast cancer bone metastasis can modulate by metastatic bone niche,witch containing variety of cells and cytokines’regulation and creating a metastatic "vicious cycle".But the upstream regulation mechanism of metastatic bone niche remains unclear.The leucine-rich repeat-containing G protein-coupled receptors 4(LGR4)and its family members have reported on tumor growth and metastases,but little is known about the regulation of LGR4 in bone metastases,and especially in pre-metastatic bone niche.Our previous study found that LGR4 is closely related to bone development,bone metabolism,mammary development and breast cancer.So our interests focus on the function of LGR4 in bone metastasis and pre-metastatic niche.Here we report RSPO2/RANKL-LGR4 regulate DKK1 secretion from breast cancer cells,thus facilitating pre-metastatic bone niche and bone metastasis.Firstly,bioinformatics analysis in breast cancer patients showed that the higher LGR4 expression was correlated with the lower bone metastases-free survival,and a higher LGR4 expression in bone metastatic tumors compared with primary tumors.Furthermore,we demonstrated the role of LGR4 in promoting bone metastasis and osteolytic by two animal models.Considering the significance of pre-metastatic bone niche in tumor bone metastasis,we studied whether LGR4 could affect breast cancer pre-metastatic bone niche.We found that breast cancer cells with LGR4 deficiency reduced the osteoclasts activity in vivo pre-metastatic niche animal model,as a result,promoted breast cancer bone metastasis and osteolytic.In vitro LGR4 in breast cancer cell regulated osteoclastogenesis and pre-osteoclast recruitment.Further screening of LGR4-regulated Wnt signaling pathway downstream genes in different breast cancer cells revealed that LGR4 mainly regulated DKK1 secretion.We also found that LGR4 and DKK1 have corelation in MDA-MB-231 derivatives with different bone metastatic ability,as well as in clinical bone metastatic and primary tumors.DKK1 promoted the activity of osteoclasts in pre-metastatic bone niche in vivo,and the osteoclastogenesis,pre-osteoclast recruitment,inhibited osteoblast differentiation in vitro.LGR4-DKK1 regulated osteoblasts secreting RANKL/OPG.Therefore,rescue experiments in vivo and in vitro showed that LGR4-DKK1 promoted breast cancer bone metastasis by pre-metastatic bone niche.In the mechanism,we screened the LGR4 ligands and found that RSPO2/RANKL-LGR4 mainly regulated DKK1 expression,and RANKL-LGR4-DKK1 independent of RANKL-RANK pathway.RSPO2/RANKL-LGR4 coupled with Gαq and further regulated the Wnt signaling pathway,promoted DKK1 secretion from breast cancer cells into pre-metastatic bone niche..In conclusion,RSPO2/RANKL-LGR4 induced DKK1 is a critical mediator of breast cancer pre-metastatic bone niche,which illuminates an upstream regulation mechanism of metastatic bone niche.