GPR110 promotes progression and metastasis of triple-negative breast cancer

Abstract

Breast cancer is the most common type of cancer in women, and approximately 70% of all breast cancer patients use endocrine therapy, such as estrogen receptor modulators and aromatase inhibitors. In particular, triple-negative breast cancer (TNBC) remains a major threat due to the lack of targeted treatment options and poor clinical outcomes. Here, we found that GPR110 was highly expressed in TNBC and GPR110 plays a key role in TNBC progression by engaging the RAS signaling pathway (via Gαs activation). High expression of GPR110 promoted EMT and CSC phenotypes in breast cancer. Consequently, our study highlights the critical role of GPR110 as a therapeutic target and inhibition of GPR110 could provide a therapeutic strategy for the treatment of TNBC patients.

Fig. 1. GPR110 was highly expressed in triple-negative breast cancer.

A Representative IHC images of GPR110 staining in invasive ductal carcinoma (IDC) cancer tissue and the quantification graph. Image J software was used for analysis. B, C qRT-PCR analysis and Western blotting showed the expression of GRP110 in cell lines from the Luminal, Her2⁺, TNBC subtypes. D Kaplan–Meier survival analysis showed that high expression of GPR110 was associated with poor prognosis of Luminal A, Luminal B, Her2⁺, and TNBC type patients. *p < 0.05, **p < 0.001, ***p = 0.0001 and ****p < 0.0001; ns not significant; determined by two-tailed Student’s t test (95% confidence interval).

Fig. 2. GPR110 is a key regulator of the epithelial–mesenchymal transition in breast cancer.

A, B GSEA of hallmark epithelial-to-mesenchymal transition gene signature and stemness maker to GPR110 in breast cancer patients (GSE22516). C Invasion/migration assays were performed after silencing GPR110 in MDA-MB231 cells. D qRT-PCR analysis showing EMT markers and regulators after knockdown of GPR110 in MDA-MB231 cells. E Representative images of ICC staining of Vim, Fn, Slug, and Zeb1 in MDA-MB231 transfected with si-GPR110. F A positive correlation between Slug and GPR110 was observed using the public TCGA database (n = 1247). And a positive correlation between Vim and GPR110 was observed using the public TCGA database (n = 1247). G, H After injection into fat pad of female NOD/SCID mice, the image of mouse lungs and representative image of H&E staining of lung metastasis and the number of lung metastatic foci. I, J qRT-PCR analysis and Western blotting of EMT markers and regulators using mouse tissues. K IHC analysis of EMT markers and regulators in xenograft tumor of mice. Scale bar = 100 μm. β-actin was used as a control for normalization of expression. *p < 0.05, **p < 0.001, ***p = 0.0001 and ****p < 0.0001; ns not significant; determined by two-tailed Student’s t test (95% confidence interval).

Fig. 3. GPR110 promotes breast cancer stem-like cells.

A Sphere formation assay was performed using GPR110-overexpressing MCF7 and the colony size was measured and shown in a graph. Representative images of forming cells showing the growth of the sphere (left). Scale bar: 20 μm. The graph showed the size of spheres formed (right). B Flow cytometric analysis of the percentage of CD44+/CD24− cells in the GPR110-overexpressing MCF7 and GPR110-silenced MDA-MB231 cells. C in vitro Limiting dilution assay was performed using GPR110-overexpressing MCF7 cells. D, E qRT-PCR and Western blotting analysis were performed to check the CSC regulators after silencing GPR110 in MDA-MB231. F Representative ICC Image of CD44, OCT4 and NANOG in GPR110-silenced MDA-MB231 cells. G A positive correlation between CD44 and GPR110 was observed using the public GSE database (GSE11121) and a negative correlation between CD24 and GPR110 was observed using the public GSE database (GSE10780). H–J Western blotting, qRT-PCR analysis, and IHC staining of CSC regulators using mouse tissues. Scale bar = 100 μm. *p < 0.05, **p < 0.001 and ****p < 0.0001; ns not significant; determined by two-tailed Student’s t test (95% confidence interval).

Fig. 4. GPR110 induces epithelial–mesenchymal transition and cancer stem-like cells phenotype via Gαs/RAS pathway.

A GESA analysis revealed that GPR110 expression was positively correlated with the Gαs signaling (GSEA12093). B Co-immunoprecipitation with Gas antibody and western blot analysis to check the interaction between Gas and GPR110 in MDA-MB231. C Co-Ip assay to analyze GPR110 and Gαs interaction in HEK293T cells. D Representative images and quantification of in situ PLA showing the interaction between Gas and GPR110. Scale bar = 100 μm. E The invasive and migrated cell numbers were assessed in GPR110 expression alone or together with Gαs expression in MCF7 cells. F qRT-PCR analysis of EMT markers and regulators using the same rescue experiments. G Sphere forming assay were assessed in GPR110 expression alone or together with Gαs expression in MCF7 cells. H qRT-PCR analysis of CSC regulators using the same rescue experiments condition. I, J Western blotting analysis to assess active RAS signaling pathway (p-RAF, p-MEK, p-ERK) using GPR11-silencing MDA-MB231 cells or GPR110-overexpressing MCF7 cells. K GSEA of RAS protein signal transduction signature and KRAS oncogene signature to GPR110 expression in breast cancer patients (GSE54326, GSE24460). L The invasive and migrated cell numbers were assessed in GPR110 expression alone or together with K-Ras expression in MCF7 cells. M, N Western blotting analysis and qRT-PCR analysis of EMT markers and regulators using the same rescue experiments condition. O Sphere forming assay was assessed in GPR110 expression alone or together with K-Ras expression in MCF7 cells (left) and the graph showed the size of spheres formed (right). P, Q Western blotting analysis and qRT-PCR analysis of CSC regulators using the same rescue experiments condition. R IHC analysis of GTP-Gas, Active-RAS, p-RAF, and p-ERK in xenograft tumor of mice. Scale bar = 100 μm. *p < 0.05, **p < 0.001, ***p = 0.0001 and ****p < 0.0001; ns not significant; determined by two-tailed Student’s t test (95% confidence interval).

Fig. 5. GPR110 indicated a poor prognosis of breast cancer.

A Graph showing GPR110 expression in stage1 (n = 371), stage2 (n = 571), and stage3 (n = 90) of breast cancers using the database from METABRIC breast cancer databases. B Tissue microarray analysis of GPR110 expression in different stages of breast cancer (left) and the proportion is shown in the graph (right). C Graph showing GPR110 expression in grade1 (n = 170), grade2 (n = 770), and grade3 (n = 952) of breast cancers using the database from METABRIC breast cancer databases. D Tissue microarray analysis of GPR110 expression in different grades of breast cancer (left) and the proportion in shown in the graph (right). E Kaplan–Meier survival analysis showed that high expression of GPR110 was associated with a poor survival rate of breast cancer patients obtained from the GEO database (GSE25085, GSE11121). F Kapan–Meier survival analysis of metastatic breast cancer patients (GSE45255, GSE7390). G GSEA of metastasis and cancer progression signature to GPR110 expression in breast cancer patients. H Schematic of GPR110/Gαs/RAS signaling axis mechanism in breast cancer. *p < 0.05, **p < 0.001 and ****p < 0.0001; ns not significant; determined by two-tailed Student’s t test (95% confidence interval).