LGR4 attenuates MGP expression and suppresses EGFR activation-induced triple-negative breast cancer metastasis

Abstract

Breast cancer has emerged as the most common cancer globally, with a significant reduction in overall survival rate after metastasis. Compared with other types of breast cancer, triple-negative breast cancer (TNBC) is more prone to metastasize, presenting substantial treatment challenges due to the lack of effective therapies. LGR4, which is highly expressed in breast cancer, has been shown to promote the proliferation and invasion of breast cancer cells. However, its specific role in TNBC remains unclear. In this study, we applied a multi-omics approach to explore the regulatory mechanism of LGR4 in TNBC metastasis. Our findings showed that LGR4 could regulate actin cytoskeletal through EGFR and curtail EGFR activation-induced TNBC metastasis by inhibiting MGP expression. These insights provide new perspectives on the role of LGR4 in breast cancer metastasis.

Keywords: Breast cancer; EGFR; LGR4; MGP.

Figure 1

LGR4 knockout reduces breast cancer cell migration. (A) QPCR analysis in MDA-MB-468 LGR4 KO cells. (B) WB analysis in MDA-MB-468 LGR4 KO cells. (C, D) Representative images (C) and quantification of transwell assay (D) showing that LGR4 knockout (KO) reduces MDA-MB-468 migration. Scale bar = 50 µm.

Figure 2

Schematics representation of the workflow for MS analysis of LGR4-dependent dynamic EGFR complexes and secretomes.

Figure 3

Proteomic landscape of dynamic EGFR-complexes in MDA-MB-468 cell lines. A. Dynamic ranges of proteomes measured at 3 time points. B. Abundance of LGR4 antigens in the MDA-MB-468 cell line. C. Multiple datasets using various filtering criteria. D. Heatmap of temporal proteomic data analyzed by unsupervised clustering and hierarchical clustering based on correlation. E. Venn diagram illustrating the overlap of proteins across 3 time points in both WT and KO groups.

Figure 4

EGFR-association dynamics after EGF treatment in MDA-MB-468 WT and KO cells. (A) Protein trajectories for the 4 clusters in LGR4 WT cells. (B) Metascape term enrichments for the proteins in each cluster from (A). (C) Protein trajectories for the 4 clusters in LGR4 KO cells. (D) The Metascape term enrichments for the proteins in each cluster from (C).

Figure 5

EGF-stimulated WT/KO cell lines at various time points. A. Protein pathway enrichment analysis. B. Line plots of protein abundance indicating representative core components over time. C. Abundance line plots of representative protein.

Figure 6

Secretory proteomic landscape of MDA-MB-468 cell lines. A. Heatmap of differential protein expression. B. Volcano plot of differential proteins. C. Enrichment analysis of down-regulated proteins. D. Histogram illustrating the fold down-regulation of MGP across three groups.

Figure 7

LGR4 knockdown reduces MGP protein expression. (A) qPCR and WB analysis in MDA-MB-468 MGP knockdown cells. (B, C) Representative images (B) and quantification (C) of transwell assay showing that MGP knockdown inhibits MDA-MB-468 migration. Scale bar = 100 µm. (D) qPCR analysis of MGP expression following LGR4 knockout. (E) WB analysis of MGP expression after LGR4 overexpression. (F) WB analysis of MGP expression levels post LGR4 knockout. (G) Changes in MGP protein following LGR4 knockout and subsequent LGR4 supplementation.