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 9

Targeting LGR4-DKK1 signaling for the treatment of BCa bone metastasis.

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Targeting LGR4-DKK1 signaling for the treatment of BCa bone metastasis. ...
(A and B) Representative images of mouse primary OPs (top chamber) subjected to the Transwell migration assay with conditioned medium from MDA231 (top) or 4T1 (bottom) cells treated with RSPO2 (200 ng/mL), LGR4-ECD (5.4 μg/mL), and anti-DKK1 (1 μg/mL) (A) with quantification of the number of migrated cells (B). Data indicate the mean ± SD. ***P < 0.001, 1-way ANOVA with Tukey’s post hoc test. n = 4 biological replicates. Scale bars: 20 μm. (C and D) Representative images of mouse primary OPs (top chamber) subjected to the Transwell migration assay with conditioned medium from MDA231 (top) and 4T1 (bottom) cells treated with RANKL (200 ng/mL), LGR4-ECD (5.4 μg/mL), and anti-DKK1 (1 μg/mL) (C) with quantification of the number of migrated cells (D). Data indicate the mean ± SD. *P < 0.05, ***P < 0.001, 1-way ANOVA with Tukey’s post hoc test. n = 4 biological replicates. Scale bars: 20 μm. (E) Experimental design of the in vivo bone metastasis mouse model. SCP46-LUC cells were injected i.c. into nude mice. After 1 day, the mice were injected i.p. daily with LGR4-ECD (1 mg/kg/d) for 21 days. (F–J) Representative images of bioluminescent, radiographic, H&E, TRAP, and IF double staining for RANK (green) and CD115 (red) and IHC staining for DKK1 in the tibiae of mice (F), and quantitative analysis (G–J). Yellow arrows indicate osteolytic lesions; red dotted lines indicate tumor zones; white arrows indicate cells double-positive for RANK and CD115. Data indicate the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, (H) 2-way ANOVA, (G) log-rank test, and (I and J) unpaired 2-tailed Student’s t test. n = 7 per group. Scale bars: 1 mm (micro-CT), 25 μm (H&E), 20 μm (TRAP), 10 μm/5 μm (left/right, IF), 10 μm (IHC).