RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis
Zhiying Yue, … , Mingyao Liu, Jian Luo
Zhiying Yue, … , Mingyao Liu, Jian Luo
Published November 30, 2021
Citation Information: J Clin Invest. 2022;132(2):e144579. https://doi.org/10.1172/JCI144579.
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Research Article Bone biology Cell biology

RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis

Abstract

Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate premetastatic niche and bone tropism are largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a premetastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interaction with their receptor LGR4, promoted osteoclastic premetastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating the WNT inhibitor DKK1 through Gαq and β-catenin signaling. DKK1 directly facilitated OP recruitment through suppression of its receptor LDL receptor–related protein 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibition of canonical WNT signaling. In clinical samples, RSPO2, LGR4, and DKK1 expression showed a positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in a mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of the premetastatic niche for BCa bone metastasis and identify RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.

Authors

Zhiying Yue, Xin Niu, Zengjin Yuan, Qin Qin, Wenhao Jiang, Liang He, Jingduo Gao, Yi Ding, Yanxi Liu, Ziwei Xu, Zhenxi Li, Zhengfeng Yang, Rong Li, Xiwen Xue, Yankun Gao, Fei Yue, Xiang H.-F. Zhang, Guohong Hu, Yi Wang, Yi Li, Geng Chen, Stefan Siwko, Alison Gartland, Ning Wang, Jianru Xiao, Mingyao Liu, Jian Luo

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Figure 3

LGR4 contributed to OP recruitment and regulated osteoclastic premetastatic niche formation and bone metastasis.

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LGR4 contributed to OP recruitment and regulated osteoclastic premetasta...
(A) Kaplan-Meier analysis of bone metastasis–free survival according to LGR4 mRNA expression in BCa patients (GEO GSE2603; LGR4 low, n = 38; LGR4 high, n = 39). Log-rank test. (B) LGR4 IHC staining in BCa patient samples and quantitative analysis. Data indicate the mean ± SD. **P < 0.01, unpaired 2-tailed Student’s t test. Primary, n = 73; bone met., n = 27. Scale bars: 50 μm/20 μm (top/bottom). (C) LGR4 expression was correlated with bone metastatic capability in MDA231 sublines with distinct bone-metastasis abilities (GEO GSE14244 and GSE16554). (D) Experimental design of the in vivo premetastatic niche mouse model. Conditioned medium from LGR4-knockdown SCP46 cells was collected and injected i.p. into nude mice every day for 21 days of pretreatment (tumor-free mice); SCP46-LUC cells were injected i.c. into mice (day 0). Forty-seven days after i.c. injection, bones of mice (tumor-bearing mice) were analyzed. (E and F) Representative images of IF double staining for RANK (green) and CD115 (red) and TRAP staining in the L3 spines of tumor-free mice (E), and quantitative analysis (F). White arrows, double-positive for RANK and CD115. Data indicate the mean ± SD. **P < 0.01, ***P < 0.001, unpaired 2-tailed Student’s t test, n = 5. Scale bars: 10 μm/5 μm (left/right, IF), 50 μm (TRAP). (G–J) Representative images of bioluminescent, radiographic, H&E, and TRAP staining in the L3 spines of tumor-bearing mice (G), and quantitative analysis (H–J). Yellow arrows, osteolytic lesions; red dotted lines, tumor zone. Data indicate the mean ± SD. **P < 0.01, (H) 2-way ANOVA, (I) log-rank test, n = 5 (H and I); (J) unpaired 2-tailed Student’s t test, n = 3. Scale bars: 1 mm (micro-CT), 200 μm (H&E), 30 μm (TRAP).