Leucine-rich repeat-containing G-protein-coupled receptors 4/5/6 (LGR4/5/6) have crucial functions in embryonic development, adult tissue homeostasis, and diseases. LGR4/5/6 also play important roles in cancer initiation and progression. LGR4 is highly expressed in multiple types of cancer and associated with poor patient outcome. LGR4 promotes tumorigenesis and metastasis and modulates cancer stem cells, while LGR5 and LGR6 mark cancer stem cells and progenitor cells that contribute to tumor initiation. The biological functions of LGR4/5/6 are primarily attributed to their roles in potentiating Wnt signaling, which requires the binding of their four R-spondin ligands (RSPO1-RSPO4). However, it is unclear whether these proteins have Wnt signaling-independent functions. We have previously reported that LGR4 is important in mammary gland development and breast cancer progression and metastasis. Here, we demonstrate that LGR4 enhances triple-negative breast cancer (TNBC) metastasis independently of Wnt signaling. We first found that LGR4 was highly expressed in the TNBC subtype and associated with poor metastasis-free survival in TNBC patients, while a well-validated Wnt signature failed to correlate with metastasis-free survival of this same subset of TNBC patients. These bioinformatic data suggested divergent functions of LGR4 versus Wnt signaling in TNBC progression. Then we showed that depletion of LGR4 inhibited, while inducible expression of LGR4 promoted TNBC cell migration and invasion, without added exogenous ligands and any detectable impact on baseline Wnt signaling. Importantly, blockade of Wnt signaling by Wnt inhibitors or by genetic depletion of Wntless cannot abolish the effect of LGR4 on TNBC cell metastatic ability. Furthermore, we have generated non-RSPO-binding LGR4 mutants that failed to activate Wnt signaling. These LGR4 mutants that are uncoupled from Wnt signaling can still promote TNBC cell migration and invasion in vitro and TNBC lung metastasis and bone metastasis in vivo, as potently as wild-type LGR4. To identify the molecular mechanisms by which LGR4 promotes TNBC metastasis, we did computational analyses of TCGA data to search for candidate proteins associated with LGR4 and proteomics screenings to uncover protein pathways regulated by LGR4 knockdown in TNBC cells. Both in silico and proteomic analyses identified EGFR as a downstream component of LGR4 signaling in TNBC. We confirmed LGR4 enhanced EGFR signaling by immunoblot analyses and found that EGFR was the crucial mediator of LGR4’s role in TNBC metastasis. Ectopic EGFR rescued migration and invasion of LGR4 knockout cells, while EGFR knockdown and the EGFR inhibitor erlotinib attenuated LGR4-induced cell migration and invasion. In an intra-iliac injection model of bone metastasis, erlotinib also suppressed LGR4-induced TNBC bone metastasis in vivo. Mechanistically, LGR4 did not affect EGFR mRNA levels but EGFR protein stability. LGR4 interacted with EGFR and blocked EGFR ubiquitination and degradation, resulting in persistent EGFR activation. Together, these data uncover a new signaling pathway controlled by LGRs with broad implications for cancer progression and targeted therapy.

Citation Format: Fei Yue, Weiyu Jiang, Amy T Ku, Adelaide IJ Young, Weijie Zhang, Eric P Souto, Yankun Gao, Zhihan Yu, Yi Wang, Chad J Creighton, Chandandeep Nagi, Tao Wang, Xin-Hua Feng, Shixia Huang, Cristian Coarfa, Xiang H.-F. Zhang, Qingyun Liu, Xia Lin, Yi Li. LGR4 engages a wnt-independent mechanism to enhance EGFR signaling and promote metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-03.