The Effects Of GB_1e On The Malignancy Of Breast Cancer Cells

Breast cancer is the most common malignancy in women with the highest morbidity and mortality.Tumor metastasis and local recurrence are the major cause of death in breast cancer patients.The poor prognosis of breast cancer is mainly due to the lack of common diagnostic markers.Exploring the molecular mechanisms of the pathogenesis and metastasis of breast cancer can not only provide diagnostic and prognostic markers for breast cancer,but also provide new targets for the treatment of advanced breast cancer.γ-aminobutyric acid（GABA）is the main inhibitory neurotransmitter in the central nervous system（CNS）.GABA_B receptor is a heterodimer composed of two sub-units,GABA_B1（GB₁）and GABA_B2（GB₂）.GABA_BR can promote tumor cell migration by trans-activating epidermal growth factor receptor（EGFR）,but the mechanism is unclear.More than 14 alternatively spliced isoforms of GB₁ subunit have been identified,among which GB_1ee only contains the extracellular domain but lacks transmembrane and the intracellular domains.Compared with GB_1a and GB_1b,GB_1e has lower mRNA level in CNS,but it is highly expressed in peripheral tissues and various human tumor cell lines.GB_1ee may play an important role in tumor cells,but there is no report to address the biological function of GB_1e.In this study,immunoblotting analysis and qPCR revealed that GB_1e was expressed in9 human breast cancer cell lines.To investigate the function of GB_1e in breast cancer cells,MDA-MB-231 cells were infected with lentivirus containing GB_1e shRNA sequence to get silenced cells.Clonegenecity and spheriod formation assay showed that GB_1e promoted cell proliferation;Transwell experiment proved that GB_1ee promoted cell migration and invasion.In addition,GB_1e was found to protect cells from apoptosis induced by hypoxia or lapatinib（EGFR/Her2 inhibitor）in GB_1e overexpressing MCF-7 cells.Therefore,GB_1ee may correlate with the progress and metastasis of breast cancer.Based on previous studies in our lab,GB_1e can interact with various proteins including PTPN12,SEL1L and DAAM1.PTPN12 is a non-receptor tyrosine phosphatase that inhibits EGFR activity and is a tumor suppressor in breast cancer.Further studies were peformed on PTPN12 to clarify the mechanism of GB_1ee in promoting the maligant phenotype of breast cancer cells.Co-Immunoprecipitation and Western Blot results revealed that both GB_1e and EGFR can interact with PTPN12,and the binding of GB_1e to PTPN12 can competitively inhibit the binding of EGFR to PTPN12.In addition,GB_1e promoted the activation of EGFR and PI3K-Akt signaling pathway,but had no significant effect on the activity of ERK1/2,suggesting that PTPN12 may mediate the effects of GB_1e on the malignant phenotype of breast cancer cells by regulating EGFR and its downstream PI3K-Akt signaling pathways.Taken together,GB_1e promoted the proliferation,migration and invasion of breast cancer cells;meanwhile,it inhibited apoptosis induced by hypoxia or lapatinib.Moreover PTPN12 played an important role in GB_1e-induced biological effects by modulating EGFR and its downstream PI3K/Akt signaling pathways.These study provided the evidence for GB_1e as a novel target in the clinical treatment of breast cancer.