Study Of Recombinant Human Heregulin-β1-induced GPR30Upregulation On The Biologic Behavior Of Breast Cancer

BackgroundBreast cancer is a highly heterogeneous disease, and the decision to use endocrine therapy for breast cancer remains primarily based on the presence of ERs in tumor today. However, only about60%of ERa-positive patients benefit from endocrine treatment, in which approximately40%of patients develop drug resistance during the progression. A growing number of studies have observed that the interaction between different cell signal transduction pathways plays an important role in tumor development and drug resistance. G protein-coupled receptor30(GPR30), as a newly discovered estrogen receptor, has crosstalk with members of ErbBs receptor tyrosine kinase family. ErbB3and ErbB4are the receptors for Heregulins (HRGs) and prefer to heterodimerize with ErbB2when binding to HRGs, which activates downstream signaling transduction and triggers a series of changes in biological functions. The cooperation between GPR30and HRGs signaling has not been reported, thus the subject of our research is to explore the association between GPR30and HRGs, and to investigate the effects of this interaction on the biologic behavior of breast cancer and endocrine treatment. Methods and resultsWe first examined the GPR30and HRG1expression in49cases of paraffin-embedded specimens of invasive ductal breast cancer using immunohistochemistry method. It was discovered that GPR30was expressed in69%of cases and HRG1in65%of cases respectively. A significant moderate correlation between GPR30and HRG1was disclosed (r=0.632, P=0.000). Then recombinant human Heregulin-β1(HRG-β1)-induced GPR30expression on mRNA and protein levels was detected by real-time quantitative PCR and Western blotting in breast cancer cell lines with different receptor characters. Transient transfections with an expression plasmid encoding a641bp5'-flanking sequence of the GPR30gene in SkBr3cells revealed that the luciferase expression was triggered by HRG-β1. At the same time, we also observed that HRG-β1induced an intracellular GPR30accumulation in MCF-7and SkBr3cells by immunocytochemistry, and there was a little GPR30distribution in the nuclear.We next examined the activation of ErbBl-4and the downstream signaling molecular ERK1/2and Akt after HRG-β1treatment in MCF-7and SkBr3cells through Western blotting. ErbB3, ERK1/2and Akt were activated in both of cell lines and the elevated phosphorylation of ErbB2was only detected in MCF-7cells. In ErbB4, the increased phosphorylation was only in MCF-7cells for a short time. Immunoprecipitation results showed that ErbB2/ErbB3complex was the main heterodimer after HRG-β1addition. Various chemical synthetic specific ErbBs signaling inhibitors were used to certify that HRG-β1up-regulates GPR30expression through the ErbB2/ErbB3-MAPK/ERK signaling transduction pathway.Finally, we studied the biological functions of HRG-β1-induced GPR30expression in breast cancer. In the condition that17-β-estradiol, G-1(the GPR30agonist) and4-OHT(the active metabolite of tamoxifen) existed, HRG-β1-increased GPR30inhibited the proliferation of ER-positive MCF-7cells but promoted the growth of ER-negative SkBr3cells; HRG-(31-induced migration and invasion in MCF-7and SkBr3cells were enhanced by the upregulation of GPR30expression, which could be blocked by ErbB2tyrosine inhibitor AG825and the MEK1/2inhibitor U0126.ConclusionsGPR30expression is positively associated with HRG1in breast cancer tissues. HRG-β1increases GPR30expression through activating ErbB2/ErbB3-MAPK/ERK pathway in breast cancer cells, which can improve the migratory and invasive ability of breast cancer cells. This interaction between GPR30and HRGs may be involved in endocrine therapy resistance of breast cancer. Blocking both of GPR30and HRGs signaling pathway may be a promising new strategy for breast cancer treatments.