A Research Of The Mechanism Of NPRL2 Involved In The Drug Resistance Of Everolimus In Castration-resistant Prostate Cancer

Background and objectiveIn recent years,the number of patients with prostate cancer(PCa)has increased significantly in China,and a considerable number of PCa patients have been diagnosis as the advanced stages when they see the doctors at the first time.Androgen deprivation therapy is an important method for the treatment of advanced PCa.PCa patients will almost inevitably progress into a disease status named castration-resistant prostate cancer(CRPC)after a period of androgen deprivation therapy(ADT),to which prone develop into multiple drug-resistant,with a poor prognosis.Basic and clinical studies have confirmed that targeting to androgen receptor signaling pathways(ARSPs)alone is not sufficient for the treatment of CRPC,while targeting to non-androgen receptor signaling pathways(NARSPs)probably play an important role in the treatment of CRPC.The PI3K-Akt-mTOR signaling pathway is closely related to the development and drug resistance of CRPC.Although everolimus,an inhibitor of mTOR signal pathway,with less side effects,its potency were found unsatisfactory in clinical trials of CRPC patients.It is worth studying how to improve its efficacy and mitigate its resistance.Our team previous focused on some researches on nitrogen permease regulator-like 2,named NPRL2,a tumor suppressor candidate gene.NPRL2 have reported be involved in the mTOR signaling pathway.It is well known that the mTORCl negatively regulate autophagy,a conserved process to maintain homeostasis by recycling macromolecules and organelles to form double-membrane autophagosome vesicles.This“device”can recover and reuse cytoplasmic "waste" to help maintain the survival of cells in an adverse microenvironment such as hypoxia,nutrient deficiency,radiotherapy or chemotherapy,etc.Therefore,a suppositional scientific assumption that "NPRL2-everolimus resistance_autophagy" probably exist in CRPC.However,as of now,there has been no report about NPRL2 in prostate cancer(including CRPC).This study intends to preliminary understand the partial biological function of NPRL2 in prostate cancer,especially in castration-resistant prostate cancer.In additon,we explored the relationship between NPRL2 and everolimus resistance in CRPC and analyzed the potential mechanisms of role of NPRL2 in everolimus resistance.Methods1.The clinical specimens of patients with benign prostatic hyperplasia(BPH),non-CRCP prostate cancer(PCa)and CRPC were collected and immunohistochemically stained with anti-NPRL2.Then we observe the differences in expression of NPRL2 in BPH,PCa and CRPC.The correlation between NPRL2 expression and clinicopathological parameters such as patient age,prostate-specific antigen(PSA),pathological T stage(p T),Gleason classification,and be metastasis-or not were be evaluated.Kaplan-Meier analysis was use to analysis relationship between expression of NPRL2 and survival time of some PCa patients.Furthermore,Cox proportional regression analysis was performed to evaluate the prognostic role of NPRL2.2.Expression levels of NPRL2 in normal immortalized prostate epithelial RWPE-1 cells,LNCaP,LNCaP tolerance to enzalutamide cells(called LNPER cells)which simulates castration-resistant prostate cancer,and non-androgen-dependent PC3 cells were evaluated.LNCaP,LNPER,and PC3 cells were transfected by lentiviral for knocking-down/upregulating expression of NPRL2,and growth of cells were further detected by CCK-8.Flow cytometry was used to observe the apoptosis in LNPER and PC3 cells.3.Western blot was use to analysis the changements of mTOR signaling pathway in LNPER and PC3 cells which were be knocked-down/forced reexpressed NPRL2 via observeing expression of the mTOR,p-mTOR,p70S6K(S6K)and p-S6K.We also investigated the effect of NPRL2 on autophagy in LNPER and PC3 cells,and evaluated the survival rate of LNPER and PC3 cells at different concentrations of everolimus.4.On the other hand,autophagy of PC3 and LNPER were inhibited by chloroquine,an autophagy inhibitor.Then cells’ viability and expression of apoptosis-related proteins such as Bax,Bcl-2 and PARP were detected.5.Furthermore,a nude mouse tumorigenesis model was constructed to observe the effect of everolimus on tumor growth and the expression of autophagy-related and apoptotic proteins such as LC3-Ⅱ/Ⅰ,p62,Bcl-2,Bax and PARP.Results1.The identity of NPRL2 in present study is different from a tumor suppressor gene that shows a previous phenomenon that its expression level is highly in various tumors such as lung cancer,renal cancer,glioma,breast cancer and colon cancer,while lower expressed in corresponding normal tissues.This study found that NPRL2 was lowly expressed in BPH,while NPRL2 expression was abundant in PCa and CRPC tissues.The expression of NPRL2 was positively correlated with a high Gleason group,a high pathological stage(pT stage)and lymph node metastasis in patients with PCa.To a certain extent,the prognosis of PCa with high expression of NPRL2 is slightly worse,and the survival period is shorter than that of patients with low NPRL2 expression.But there is no significant difference between the expression of NPRL2 and prognosis in patients with pT3 and above.2.In vitro,it showed that NPRL2 protein levels were lower in RWPE-1 cells,while NPRL2 expression was abundant in LNCaP,LNPER,and PC3 cells.Forced reexpression of NPRL2 could significantly increase the growth of LNCaP,LNPER,and PC3 cells,and vice versa.What’s more,knock-down of NPRL2 can increase a few apoptosis rate of PC3 and LNPER cells.3.NPRL2 can regulate mTOR signaling pathway in CRPC.Knock-down of NPRL2 significantly up-regulate the expression of p-mTOR and p-S6K in LNPER and PC3 cells,and vice versa.4.Re-expression of NPRL2 can promote autophagy in PC3 and LNPER cells,while silencing of NPRL2 decreases autophagy in PC3 and LNPER cells.Knock-down of NPRL2 and(or)autophagy inhibitor can significantly increase the apoptosis rates when cells were treated with everolimus.On the contrary,re-expression of NPRL2 can significantly reduce the cytotoxicity of everolimus to PC3 and LNPER cells.5.Everolimus significantly inhibited the growth of subcutaneous tumors in nude mice with NPRL2-knocked-down LNPER cells.Western blot analysis also showed that a downregulated autophagy level and a inceased apoptosis in NPRL2-knocked-down LNPER cells subcutaneous tumors.ConclusionsTo some extent,the expression of NPRL2 was negatively associated with the poor prognosis of PCa patients.Not as a tumor suppressor gene,NPRL2,probably play a growth-promoted role in PCa and CRPC.NPRL2 enhances autophagy and the resistance to everolimus in CRPC.Targeted to NPRL2 or inhibition of autophagy may increase the efficacy of everolimus on CRPC.