Mechanism Study Of The Functional Role Of Beclin-1 On Prostate Cancer Cell Enzalutamide Sensitivity

Research Background and Objective: Prostate cancer(PCa)is a kind of malignant tumor belong to the urogenital system with high incidence,whose incidence is closely related to the factors like race and region.Most of the patients will inevitably develop into Castration Resistant Prostate Cancer(CRPC)even after several years of Androgen Deprivation Therapy(ADT),and their average life cycles are not more than two years.Enzalutamide is a new generation of selective androgen receptor(AR)antagonist,which owns greater accessibility than Bicalutamide.However,the patients who are initially sensitive to Enzalutamide will also be resistant to the drug if they use it for some time.Till now,there is no definite research to investigate whether the Enzalutamide resistance is associated with the imbalance of growth factor signal.In our work,we will determine the effects of Enzalutamide on growth factor signal,and to figure out their specific internal interaction mechanisms.Material and Method: C4-2 cells were cultivated by gradually improving the Enzalutamide treatment concentration that was medicated,and then the C4-2R,which was resistant to Enzalutamide was obtained after three months culturing.Meanwhile,a paired of C4-2B/C4-2BR cell lines was obtained from Professor Allen Gao as a gift.To investigate whether Enz-resistance was partly due to dysregulation of growth factor signals,we focused on the insulin-like growth factor-1 receptor(IGF-1R)and the epidermal growth factor receptor(EGFR),as well as their downstream substrates AKT,ERK1/2 and etc.C4-2 and C4-2B cells were pretreated with Enzalutamide for three days,and the translational phosphorylation levels of AKT and ERK1/2 were tested by western blot after EGF stimulation.Lenti-virus was used to stably establish Beclin-1 expressed PCa cell lines.After EGF stimulation,we detected the expressions of AKT and ERK1/2(basal and phosphorylation levels)in each group.MTT,Brd U staining and Trypan Blue counting experiments were conducted to detect the cell viability after interfering Beclin-1 expression or applying ERK inhibitor.The experiments like Co-Immunoprecipitation(CO-IP)and ELISA were used to reveal the specific mechanisms of Enzalutamide on growth factor signal by influencing the activity of AR-Beclin-1 complex.Results: After EGF stimulation,the phosphorylation levels of AKT(S473 and T308)could still be increased in Enz-treated and Enz-resistant groups compared to control,but could not sustain in the extended time,while the phosphorylation level of ERK was significantly increased and also sustained in response to EGF stimulation.Mechanism study found that after silencing Beclin-1 expression in C4-2 cells,the phosphorylation levels of AKT(both T308 and S473)and ERK1/2 were significantly increased and sustained longer in the sh BECN1-C4-2 cells compared with vector control in response to EGF stimulation.However,ectopic Beclin-1 expression could only reverse the Enz-enhanced ERK growth factor signal rather than the AKT signal in response to EGF stimulation after pretreatment with Enz for three days.In the following study,we found that the Beclin-1 expression was significantly lower in C4-2R and C4-2BR cells compared with C4-2 and C4-2,respectively,and Enz-sensitivity was significantly decreased after silencing Beclin-1 expression in C4-2 and C4-2B cells.In addition,interruption approaches using ERK inhibitor(ERKI)treatment also demonstrated that inhibition of ERK activation could partially rescue the Enz-sensitivity in the sh BECN1-C4-2 parental cells,as well as C4-2R/C4-2BR cells.Furthermore,we ectopically expressed Beclin-1 in C4-2/C4-2R and C4-2BR cells,and found Enz-sensitivity was significantly increased.In addition to MTT assay,we also conducted the Trypan blue and Brd U staining assays to determine the effects of Enz on cell fate,and the results were consistent with the findings above.We found the Beclin-1 could directly interact with AR via Co-Immunoprecipitation(Co-IP)assay,and the Enz treatment increased the interaction between AR and Beclin-1,as well as the formation of Beclin-1-Vps15-Vps34 complex.Consistent with the role of AR,there is no increased Beclin-1 complex formation in AR-negative cell line Du145 in response to Enz treatment.We further applied the ELISA assay to measure the PI3 P generation,as the total cellular PI3 P lipid level may represent the degree of growth factor activation.We found that Enz significantly decreased PI3 P generation,which was consistent with the ELISA data showing a prolonged suppression of PI3 P in cells with Beclin-1 knock down,supporting the conclusion that AR may interact with Beclin-1 complex to negatively impact the latter’s activity.Last,to distinguish this AR-Beclin-1 interaction from the regulation of AR/Enz on cellular macroautophagy where Beclin-1 plays a significant role,we knocked down ATG5 expression in C4-2 cells.The results suggested that the early endosome formation involved Beclin-1’s function that was independent of its role on autophagy.Conclusion: 1.Enzalutamide enhances and sustains EGF stimulated ERK growth factor signaling pathway;2.Beclin-1 regulates Enzalutamide enhanced and sustained ERK growth factor signaling pathway,which is activated by EGF stimulation;3.Regulating Beclin-1 expression influences the PCa cell adopted enzalutamide treatment sensitivity;4.Enzalutamide promotes the formation of AR-Beclin-1 complex,and inhibits their function in transferring ERK growth factor signaling activity;5.Applying ERK pathway inhibitor significantly suppresses the CRPC cell growth,providing potential target for CRPC patients.