Molecular Mechanism Of Osteoclast Precursors Forming The Tumor Pre-metastasis Microenvironment

Osteoclasts have the function of maintaining bone homeostasis,and osteoclast therapy is currently a common method for the treatment of tumor bone metastasis.However,whether osteoclasts can serve as a key component of the microenvironment before tumor bone metastasis and regulate bone metastasis is still unknown.Therefore,we hypothesize that osteoclast precusors(OPs)at bone sites can be regulated by primary tumor cancer cells and formed the tumor pre-metastasis microenvironment followed with promoted bone metastasis.In order to examine whether OPs have function in the formation of tumor premetastasis microenvironment,firstly,we constructed the breast cancer(BCa)premetastasis microenvironment mouse model to observe osteoclastic microenvironment。The conditioned medium of BCa cells can promote the formation of pre-metastasis osteoclastic microenvironment and enhance tumor bone metastasis.This process mainly occurs in the spine and leg bones but not in skulls or pelvic bones.We used an in vitro OPs recruitment model for GPCR ligands/agonists screening at molecular level,and found that R-spondin 2(RSPO2)and RANKL promote the formation of premetastasis osteocalstic microenvironment and ultimately enhanced bone metastasis.This process is regulated by LGR4 and its downstream Wnt signaling pathway inhibitor DKK1.Recruitment of OPs and formation of pre-metastasis osteoclastic microenvironment could be on the one hand inhibited by LGR4 knockdown in MDAMB-231(MDA231)bone-tropic subline SCP46,but restored followed by DKK1overexpression;and on the other hand enhanced by RSPO2/RANKL treatment,but restored followed by DKK1 knockdown in SCP46.Overexpression of DKK1 in MDA231 poorly bone-tropic subline SCP6 significantly increased the burden of BCa bone metastasis in the pre-metastasis microenvironment mouse model.We further demonstrated that the expressions of DKK1 and RSPO2 in serum samples from BCa bone metastasis patients were significantly increased compared with primary breast cancer patients;the expression of DKK1 was positively correlated with RSPO2/RANKL in BCa bone metastasis patients.RSPO2/RANKL stimulates different subtypes of breast cancer cells to promote DKK1 transcription and protein expression,but restored followed by LGR4 knockdown.DKK1 recruit OPs through LRP5-Wnt/β-catenin-Rnasek signaling pathway.In addition,soluble LGR4 extracellular domain(LGR4-ECD)protein which is the decoy receptor for RSPO2 and RANKL significantly reduced the burden of BCa bone metastasis and improved osteolytic bone lesions in mice.In summary,these results provide unique insights into the functional role of OPs,which play a key role in the formation of BCa pre-metastasis osteoclastic microenvironment.Our results indicate that the RSPO2/RANKL-LGR4 signaling pathway may be a potential target for inhibiting BCa bone metastasis.