The Mechanism Of GABA_BR Positive Allosteric Modulator Rac-BHFF Induced EGFR Transactivation In Prostate Cancer Cells

According to the cancer statistics published by National Cancer Center,China,prostate cancer has become the most common tumor in urinary system with the highest incidence since 2008,indicating that prevention and treatment of prostate cancer has become a big challenge for human health.Current treatments of prostate cancer include surgical removal,radiotherapy,endocrine therapy,and immunotherapy.To develop new therapeutic strategies for better treatment,it is necessary to identify the mechnism underlying the onset and development of prostate cancer.G protein coupled receptors(GPCRs)are the largest and most commonly studied family of cell surface receptors,which are divided into six families based on sequence homology and functional similarity.In the classical GPCR signaling pathway,ligands induced GPCR signalings are mediated by intracellular G proteins.In addition,GPCRs can also regulate cellular responses by transactivation of tyrosine kinase receptors(RTK),such as epidermal growth factor receptor(EGFR)in ligand-dependent or-independent pathways.β-arrestin is involved in the desensitization of GPCR signal and also functions as scaffold proteins to mediate signal transduction.γ-aminobutyric acid receptor(GABA_BR)is the metabotropic receptor for inhibitory neurotransmitterγ-aminobutyric acid(GABA),and it belongs to GPCR C family.GABA_BR is not only involved in the function of nervous system,but also expressed in tumors.It has been reported that GABA_BR can promote or inhibit tumor proliferation,migration,invasion,etc.,but the specific mechanism is unknown.My laboratory has reported that the GABA_BR agonist baclofen could transactivate EGFR and its downstream ERK1/2 signaling pathway through a ligand-dependent pathway,resulting in enhanced migration of human prostate cancer PC-3 cells.This thesis is aimed to investigate whether GABA_BR positive allosteric modulator(PAM)rac-BHFF could transactivate EGFR and its downstream PI3K/Akt signaling pathway in PC-3 cells,and identify the underlying mechanism.The data showed that rac-BHFF could transiently induce phosphorylation of Akt in a dose-dependent manner.Immunoprecipitation(IP)experiment indicated that rac-BHFF could induce EGFR transactivation.Similar to EGF,confocal microscopy experiment revealed that rac-BHFF could induce EGFR internalization.Tarceva and Iressa,the small molecule inhibitors of EGFR,decreased Akt activation induced by rac-BHFF,suggesting that rac-BHFF induced Akt activation was dependent on EGFR transactivation.To investigate its molecular mechanism,PC-3 cells were pretreated with Gαi/o inhibitor pertussis toxin(PTX)and MMPs inhibitor GM6001.The results showed that neither of the inhibitors inhibited rac-BHFF induced Akt activation,indicating that rac-BHFF induced Akt activation is indenpent of Gαi/o and MMPs mediated ligand release.It has been shown thatβ-arrestin1 played an important role in GPCR induced EGFR transactivation.To confirm whetherβ-arrestin1 mediatied rac-BHFF induced EGFR transactivation in PC-3 cells,a stable cell line withβ-arrestin1 knockdown was established.It was found that knockdown ofβ-arrestin1 markedly reduced the phosphorylation of EGFR and Akt induced by rac-BHFF,indicating that GABA_BR induced activation of EGFR and its downstream Akt was depended onβ-arrestin1.When PC-3 cells were treated with rac-BHFF for a long time,rac-BHFF induced colony formation was suppressed byβ-arrestin1silence.Moreover,rac-BHFF induced autophagy under hypoxic condition was enhanced byβ-arrestin1 silence.These observations suggested thatβ-arrestin1 has an important role in the cellular response induced by rac-BHFF.In summary,this article demonstrated for the first time that GABA_BR positive allosteric modulator rac-BHFF could transactivate EGFR and its downstream Akt signaling pathway throughβ-arrestin1.Moreover,β-arrestin1 mediated rac-BHFF induced cell proliferation and inhibited autophagy induced by rac-BHFF under hypoxic condition.These results indicated thatβ-arrestin1 played a vital role in GABA_BR induced EGFR transactivation,cell proliferation,and autophagy.This work is not only helpful for elucidating the mechanism of the development of prostate cancer,but also provides a new strategy for targeted therapy of prostate cancer.