NPRL2 Promotes Docetaxel Chemoresistance In Castration Resistant Prostate Cancer Cells By Regulating Autophagy

BackgroundProstate cancer(PCa)is a most common genitourinary malignancy among men,though the incidence in China is lower than US and Europe,but is increasing by years.Approximately 15% of newly diagnosed prostate cancers are invasive or metastatic disease and receive androgen deprivation therapy(ADT)as their basic treatment.However,most cases invariably develop to hormonal resistance and progress to castration-resistant prostate cancer(CRPC),and with poor prognosis.Docetaxel-based chemotherapy is recommended as the first line therapy for CRPC,but most patients will eventually progress because of inherent or acquired resistance to drugs after several rounds of treatment.Nitrogen permease regulator-like2(NPRL2)gene is defined as a tumor suppressor gene and our group has been working on it for a long time.Our pervious study has accidentally demonstrated that NPRL2 is overexpressed in PCa and the high level of expression is linked to clinical stage,Gleason’s score and poor prognosis,particularly in CRPC.Furthermore,NPRL2 promotes the growth ofprostate cancer cells and reduces the cytotoxicity of docetaxel to PC3 cells.Besides,our previous study found that overexpressed NPRL2 could affect the expression of autophagy related proteins LC3Ⅱ/I ratio and p62,and enhance autophagy.We also verified the relationship between NPRL2 and mTORC1 signaling pathway in prostate cancer,and found that NPRL2 can down-regulate the expression of effector proteins p-mTOR and p-s6 k in the mTORC1 signaling pathway,and inhibit the mTORC1 signaling pathway.NPRL2 is highly expressed in CRPC,NPRL2 enhances autophagy and inhibits mTOR,MTOR negatively regulates autophagy.Therefore,we could hypothesize that NRPL2 regulates autophagy through the mTOR signaling pathway to participate in the resistance of castration-resistant prostate cancer to docetaxel.Methods1.To investigate docetaxel resistance in PCa,stable docetaxel resistant CRPC cell lines were first established.Stable docetaxel resistant CRPC cell lines(LNPER-DR and PC3-DR)were established by continuous culture under different concentrations of docetaxel for up to 6 months.Cell apoptosis rates and cell proliferation rates of resistant and non-resistant groups under condition of 10 nmol/L docetaxel were detected by flow cytometry and CCK-8.2.In order to study the effect of NPRL2 on the growth of docetaxel resistant CRPC cells and its correlation with autophagy,the expression ofNPRL2,autophagy related proteins and mTOR related proteins in resistant and non-resistant groups were detected by Western blotting,and LC3 puncta was photographed by confocal laser fluorescence microscope,and autophagosomes were imaged by electron microscopy.After silencing NPRL2 and/or inhibiting autophagy,cell apoptosis rates and cell proliferation rates of individual groups under condition of 10 nmol/L docetaxel were detected by flow cytometry and CCK-8.Expression of autophagy related proteins(LC3-Ⅱ,LC3-Ⅰ and p62)of individual groups were tested by Western blotting.3.To investigate the mechanism of how NPRL2 involves in docetaxel resistance in docetaxel resistant CRPC cells,after silencing NPRL2 and/or inhibiting mTOR,cell apoptosis rates and cell proliferation rates of individual groups under condition of 10 nmol/L docetaxel were detected by flow cytometry and CCK-8,and expression of NPRL2,autophagy related proteins and mTOR related proteins was tested by Western blotting.LC3 puncta was photographed by confocal laser fluorescence microscope,and autophagosomes were imaged by electron microscopy.4.At last,the xenograft model of nude mice was conducted,and NPRL2 silenced LNPER-DR cells were used for animal experiments to observe whether silencing NPRL2 increased the sensitivity of docetaxel in drug resistant cells.Results1.Based on our previous work,we established docetaxel resistant PC3 cells by continuous culture under different concentrations of docetaxel.CCK-8 assay demonstrated that the cell proliferation rates of drug resistant cell lines were significantly higher than non-resistant groups under condition of 10 nmol/L docetaxel,and flow cytometry assay showed the cell apoptosis rates of drug resistant cell lines were significantly lower than non-resistant groups under condition of 10 nmol/L docetaxel.2.Western blot demonstrated that the expression levels of NPRL2 in LNPER-DR and PC3-DR were significantly higher than in LNPER and PC3 cells,the autophagy related protein LC3-Ⅱ/LC3-Ⅰratio was increased and P62 was decreased,while the mTOR related protein p-mTOR and p-s6 k was decreased.These data suggest that NPRL2 is overexpressed,autophagy is enhanced and mTOR signaling is inhibited in LNPER-DR and PC3-DR cells.Immunofluorescence and electron microscopy also confirmed that autophagy was significantly enhanced in LNPER-DR and PC3-DR cells.Silencing NPRL2 or inhibiting autophagy increased the sensitivity of drug-resistant cell lines to docetaxel,and autophagy flux assay showed autophagy flows freely.3.Torin1,an mTOR inhibitor,reduced the sensitivity of NPRL2 silenced drug-resistant cell lines to docetaxel,and silencing NPRL2 affected mTOR signaling pathway and inhibited autophagy of drug-resistant cell lines.MTOR inhibitor enhanced autophagy of NPRL2 silenced drug-resistant cell lines.These data suggest that NPRL2 regulates autophagy through the mTOR signaling pathway.4.After silencing NPRL2,the growth of xenograft tumors in nude mice with LNPER-DR cells was significantly inhibited by docetaxel.Western blot showed that mTOR pathway was enhanced and autophagy was inhibited after silencing NPRL2,which was consistent with cell experiments.ConclusionsNPRL2 regulates autophagy through mTOR and participates docetaxel resistance in CRPC.Targeted regulation of NPRL2 may improves the chemotherapy sensitivity of CRPC and prolongs the duration of chemotherapy.