Oncogenic roles of GPR176 in breast cancer: a potential marker of aggressiveness and a potential target of gene therapy

Abstract

Background: Belonging to the G-protein coupled receptor 1 family, G protein-coupled receptor 176 (GPR176) is associated with the Gz/Gx G-protein subclass and is capable of decreasing cAMP production.

Methods: GPR176 expression was detected by qRT-PCR, bioinformatics analysis, Western blot and immunohistochemistry, and compared with clinicopathological characteristics of breast cancer. GPR176-related genes and pathways were subjected to bioinformatic analysis. We also explored the effects of GPR176 on the phenotypes of breast cancer cells.

Results: Lower expression of GPR176 mRNA was seen in breast cancer than in normal tissues, but the opposite pattern was found for its protein (p < 0.05). GPR176 mRNA was associated with female sex, low T staging, non-Her-2⁺ subtypes, non-mutant p53 status in breast cancer (p < 0.05). GPR176 methylation was negatively correlated with its mRNA level and T staging in breast cancer, and was higher in breast cancer than normal tissues (p < 0.05). GPR176 protein expression was positively correlated with older age, small tumor size, and non-luminal-B subtype of breast cancers (p < 0.05). The differential genes of GPR176 were involved in receptor-ligand interaction, RNA maturation, and so forth (p < 0.05). GPR176-related genes were categorized into cell mobility, membrane structure, and so on (p < 0.05). GPR176 knockdown weakened the proliferation, glucose catabolism, anti-apoptosis, anti-pyroptosis, migration, invasion, and epithelial-mesenchymal transition of breast cancer cells.

Conclusion: These results indicate that GPR176 might be involved in the tumorigenesis and subsequent progression of breast cancer by deteriorating aggressive phenotypes. It might be utilized as a potential biomarker to indicate the aggressive behaviors and poor prognosis of breast cancer and a potential target of genetic therapy.

Keywords: Aggressiveness; Breast cancer; GPR176; Prognosis; Target therapy.

Fig. 1

The clinicopathological significances of GPR176 mRNA expression in breast cancer. We evaluated the GPR176 mRNA expression in pan-cancer using Timer (A). GPR176 mRNA was analyzed in breast cancer using xiantao platform (B) and real-time PCR (C). It was also compared with clinicopathological features of breast cancer using UALCAN database (D). N normal tissue, T tumor, M metastasis, BRCA breast invasive carcinoma, CHOL cholangiocarcinoma, COAD colon adenocarcinoma, ESCA esophageal adenocarcinoma, GBM glioblastoma multiform, HNSC head and neck squamous cell carcinoma, HPV human papillomavirus, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LIHC liver hepatocellular carcinoma, PAAD pancreatic adenocarcinoma, PCPG pheochromocytoma and paraganglioma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, UCEC uterine corpus endometrial carcinoma, TNBC triple-negative breast cancer. ***p < 0.01; **p < 0.01; *p < 0.05

Fig. 2

The prognostic significance of GPR176 mRNA expression in breast cancer by Kaplan–Meier plotter. ER estrogen receptor, PR progesterone receptor, HR hazard ratio

Fig. 3

The relationship between GPR176 mRNA expression and immune infiltration in breast cancer. The correlation was studied between the infiltration of immune cells and GPR176 mRNA expression in different subtypes of breast cancer using Timer (A). The enrichment of immune cells was explored between low and high expression of GPR176 in breast cancer using xiantao (B). BRCA breast cancer

Fig. 4

The clinicopathological significance of GPR176 methylation in breast cancer. The negative relationship between GPR176 mRNA expression and methylation was analyzed in breast cancer using cBioPortal database (A). Its methylation was higher in breast cancer than normal tissues (B). We also compared GPR176 methylation with clinicopathological characteristics of breast cancer (C)

Fig. 5

The differential genes and their signal pathways between low and high GPR176 expression in breast cancer. The hotmap of the differential genes was shown between low and high GPR176 expression in breast cancer (A). These genes were subjected to the signal pathway analysis using KEGG (B) and GSEA (C)

Fig. 6

The hub genes of GPR176 in breast cancer. Both string and cytoscape were employed to screen the hub genes of GPR176 in breast cancer (A). The hotspot hub genes were selected (B) and compared between breast cancer and normal tissues (C)

Fig. 7

The GPR176-related genes and signal pathways in breast cancer. The positively-related genes of GPR176 were screened (A), and were classified into the signal pathway using xiantao database (B). The negatively-related genes of GPR176 were screened (C), and were classified into the signal pathway using xiantao database (D). The genes were compared between breast cancer and normal tissues using xiantao platform (E)

Fig. 8

The clinicopathological significance of GPR176 protein expression in breast cancer. Western blot was used to detect GPR176 protein level in breast cancer (A). Densitometric analysis showed its higher expression in breast cancer than normal tissues (B, p < 0.05). Immunohistochemically, GPR176 protein was positively expressed in smooth muscle cell around breast ducts and lobules, and ductal and lobular adenocarcinoma, but weakly in ductal and lobular epithelium (C). Statistically, GPR176 protein expression was higher in breast cancer than normal tissues (D, p < 0.05). N normal, T tumor

Fig. 9

The effects of GPR176 expression on the phenotypes of breast cancer cells. GPR176 expression was screened in breast cancer cells by western blot (A). After transfection of sh-GPR176, GPR176 expression became weak in SK-BR-3 cells by Western blot (B). The transfectants (sh-GPR176#2) showed a slow proliferation, glycolysis and mitochondrial oxidation, a high apoptosis, lower migration and invasion in comparison with the control, evidenced by CCK-8 (C), seahorse energy examination (D), Annexin-V-APC/7-AAD staining (E), wound healing (F), and transwell assays (G). The phenotype-related proteins were screened by Western blot (H). Note: *, compared with the control, p < 0.05