The Mechanism Of ATF4/CEMIP/PKCα Promotes Anoikis-resistance By Enhancing Protective Autophagy In Prostate Cancer Cells

Objective:The effects of nest loss on the apoptosis,invasion,migration and autophagy were investigated by constructing a cell model of anoikis-resistant in prostate cancer.Methods:Human prostate cancer PC-3,DU145 cells were cultured in suspension for one week and then re-adherent for two days to construct an anoikis-resistant cell model.The survival and proliferation ability of parental and anoikis-resistant cells in Ultra-low adhesion culture plate(ULAP)were determined by CCK-8 assay.Flow cytometry was used to determine the level of apoptosis in the parental and anoikis-resistant cells.PCa cells invasion level was assessed by transwell assay.Furthermore,mCherry-GFP-LC3B double plasmid was transfected into parent and anoikis-resistant PC-3、DU145 cells,respectively.Then,the level of autophagy flow in parental and anoikis-resistant PCa cells was detected by immunofluorescence method.The autophagosomes in PCa cells were photographed by transmission electron microscopy(TEM).Western blotting was used to detect the protein expressions of Beclinl、p-Beclinl and LC3BⅡ/Ⅰ in the parental and anoikis-resistant PCa cells,and PCa cells at the different suspension culture time points.In addition,after exogenously added autophagy activator-rapamycin and autophagy inhibitor-3-MA to parental and anoikis-resistant PCa cells for 24 hours,apoptosis level and autophagy related protein(Beclinl、p-Beclinl and LC3BⅡ/Ⅰ)levels were detected by flow cytometry and Western blotting,respectively.Results:CCK-8 assay proved that the cell survival and proliferation rate of anoikisresistant PCa cells in suspension culture were significantly higher than that of the corresponding parental cells.The results of flow cytometry showed that the apoptosis rate of anoikis-resistant PCa cells was significantly lower than that of the corresponding parental cells.Transwell assay showed that the invasion and migration ability of anoikis-resistant PC-3 and DU145 cells were significantly improved compared with parental cells.Immunofluorescence detection of autophagy flow showed that the level of autophagy flow was significantly enhanced in anoikis-resistant PC-3 and DU145 cells.Transmission electron microscopy showed that the number of autophagosomes in anoikis-resistant PCa cells was significantly increased.Western blotting showed that the expression levels of Beclin1、p-Beclinl and LC3BⅡ/Ⅰ were significantly higher in the anoikis-resistant PCa cells than in the corresponding parental cells.The results of flow cytometry showed that the exogenous addition of the autophagy activator,rapamycin,could further reduce the apoptosis rate of anoikis-resistant PCa cells,while the addition of the autophagy inhibitor,3-MA,could significantly increase the apoptosis rate.Western blotting results showed that the addition of autophagy modulator(rapamycin and 3-MA)could significantly regulate the expression levels of autophagy related proteins(p-Beclinl and LC3BⅡ/Ⅰ).Conclusions:The cell model of anoikis-resistant can be constructed in prostate cancer PC-3 and DU145 cells.The expression level of CEMIP,the ability of anoikis-resistant,the ability of invasion and migration and the level of autophagy in anoikis-resistant PCa cells were significantly increased.Objective:To investigate the effects of endoplasmic reticulum stress(ERS)on CEMIP expression,autophagy level and tumor metastasis of prostate cancer cells in suspended state.Methods:ShRNA was used to silence CEMIP in parental and anoikis-resistant prostate cancer PC-3、DU145 cells.After the autophagy activator(rapamycin)or autophagy inhibitor(3-MA)was added exogenously for 24h,flow cytometry was used to detect the apoptotic rate of PCa cells.Transwell assay and wound healing confirmed the ability of PCa cells invasion and migration after silencing CEMIP.The number of autophagic fluid and autophagosomes was detected by immunofluorescence and transmission electron microscopy.Furthermore,the stable overexpression and silencing of CEMIP plasmid were transfected into cy3 labeled PC-3 cells,and the role of CEMIP in tumor metastasis was verified by tail vein injection in nude mice.Then,qRT-PCR was used to detect the mRNA expressions of CEMIP,ER stress-related molecules and autophagy related molecules in parental and anoikis-resistant PCa cells.Western blotting was used to detect the protein expressions of endoplasmic reticulum stress-related molecules and autophagy related molecules in prostate cancer PC-3 DU145 cells at different suspension time points,as well as in the parental and anoikis-resistant PCa cells.The levels of calcium ions in parental and anoikis-resistant PCa cells were detected by immunofluorescence assay and enzyme-label assay.Moreover,overexpression of transcriptional activator 4(ATF4)plasmid was transfected,and qRT-PCR was used to verify the efficiency and regulation of CEMIP transcription.The efficiency and the regulation of CEMIP translation can be assessed by Western blot method.After stable overexpression of ATF4 and silent CEMIP,the expression levels of ATF4,CEMIP and autophagy related molecules(p-Beclinl,LC3BⅡ/Ⅰ)were detected by Western blotting.The binding between the promoter region of CEMIP and ATF4 was detected by ChIP assay,and the specific binding sites of ATF4 and CEMIP were further confirmed by dual fluorescence reporter assay.Results:Flow cytometry results showed that silent CEMIP could significantly increase the apoptosis rate of anoikis-resistant PCa cells,and the apoptosis rate was further increased with the addition of autophagy inhibitor 3-MA,while the apoptosis rate was significantly decreased with the addition of autophagy activator rapamycin.Transwell assay and wound healing confirmed that silenced CEMIP inhibited the invasion and migration of anoikis-resistant PCa cells.Immunofluorescence and transmission electron microscopy tests showed that silenced CEMIP significantly inhibited the level of autophagic fluid and the number of autophagosomes in PCa cells.Animal experiments showed that silenced CEMIP significantly inhibited the number and volume of lung metastases in prostate cancer,while overexpression of CEMIP could highly reverse this result.Then qRT-PCR confirmed that the mRNA levels of ATF4 and CEMIP were significantly up-regulated in the anoikis-resistant PCa cells,while there was no significant difference in autophagy related ATGs.Western blotting results showed that ATF4、CEMIP、p-Beclinland LC3BⅡ/Ⅰdynamic changes at different suspension time points,and ATF4 reached its peak at 16 hours earlier than CEMIP.Rescue experiments showed that the activation of autophagy by overexpression of ATF4 was significantly inhibited by silencing CEMIP.Further,ChIP assay initially confirmed that ATF4 could bind to the promoter of CEMIP and promote the transcription of CEMIP.double fluorescent reporter gene assay confirmed that ATF4 could bind to the site 3 of CEMIP promoter to regulate the transcriptional activity of CEMIP.Conclusion:CEMIP promotes the survival and metastasis of anoikis-resistant PCa cells by activating protective autophagy.Endoplasmic reticulum stress promotes CEMIP transcriptional activity by up regulating ATF4 expression.Objective:To study the molecular biological mechanism of CEMIP activating protective autophagy promoting anoikis tolerance of prostate cancer cells.Methods: PC-3 and DU145 cells were selected as the research objects and the overexpression and silencing of CEMIP plasmids were constructed.CEMIP was silenced in anoikis-resistant PCa cells(PC-3-AR and DU145-AR)and overexpressed in parental PCa cells(PC-3-P and DU145-P).The expressions of Bcl-2 、 p-Bcl-2-ser70 、 Beclin1 and p-Beclin1 were detected by Western blotting in anoikis-resistant PCa cells overexpressing CEMIP.Bcl-2-GFP and Beclin1-GFP fluorescence plasmids were constructed,and the binding of Bcl-2 and Beclin1 was detected by double fluorescence complementation assay according to the principle that fluorescence could be presented after co-transfection and binding.In addition,the effect of overexpression of CEMIP on the binding of Bcl-2 and Beclin1 was also verified by the protein immunoprecipitation(Co IP)and immunofluorescence experiments.Sliencing endogenous Bcl-2 by si RNA and construction of Bcl-2-ser70 mutant plasmid.The protein levels of PKCα、Bcl-2 and p-Bcl-2-ser70 were detected by Western blotting in PCa cells overexpressing CEMIP.Under the premise of overexpression of CEMIP and silencing of endogenous Bcl-2 in PCa cells,Bcl-2-ser70 mutant plasmid was transfected.The expression levels of Bcl-2、p-Bcl-2-ser70、Beclin1 and p-Beclin1 were detected by Western blotting.In addition,membrane protein and cytoplasmic protein were obtained by protein isolation kit,and the intracellular distribution of PKCα protein was detected by Western blot.Furthermore,the localization of PKCα was detected by immunofluorescence after overexpression of CEMIP.Silent PKCα plasmid was constructed and transfected,and its transfection efficiency was verified by q RT-PCR.PKCα was silenced on the basis of overexpression of CEMIP,then,the expression levels of Bcl-2 and p-Bcl-2-ser70 were detected by Western blot.Furthermore,the role of PKCα in the binding of Bcl-2 and Beclin1 was detected by Co IP assay.Results: Western blotting in the parental PCa cells with overexpression of CEMIP showed that over-expression of CEMIP promoted the up-regulation of p-Bcl-2-Ser70 and p-Beclin1 expression,while the expression of Bcl-2 was not significantly changed.The double fluorescence complementation experiment showed that Bcl-2 could bind to Beclin1,and overexpression of CEMIP could significantly inhibit the binding of Bcl-2 to Beclin1.Furthermore,protein immunoprecipitation assay showed that overexpression of CEMIP could promote the dissociation of Bcl-2 / Beclin1 complex.Corresponding immunofluorescence assay also proved that Bcl-2 and Beclin1 were co-localized in the cells.Overexpression of CEMIP significantly inhibited the co-localization of Bcl-2 and Beclin1.Rescue experiments confirmed that overexpression of CEMIP promoted the dissociation of Bcl-2 / Beclin1 complex through up regulating the expression of p-Bcl-2-ser70.Western blotting showed that PKCα translocation occurred after overexpression of CEMIP.Furthermore,immunofluorescence assay confirmed that overexpression of CEMIP promoted PKCα protein membrane translocation.The q RT-PCR assay proved that the m RNA level of PKCα was significantly reduced in silent group.Rescue experiment proved that over-expression of CEMIP promoted the up-regulation of p-Bcl-2-Ser70 expression,which was significantly inhibited by silencing PKCα.Furthermore,Co IP experiments confirmed that overexpression of CEMIP promoted the dissociation of Bcl-2/Beclin1 complex,while silencing PKCα highly reversed the experimental results.Conclusion:Over-expression of CEMIP promotes the phosphorylation of Bcl-2-Ser70 by increasing PKCα membrane translocation to promote the disintegration of the Bcl-2/Beclin1 complex and activate autophagy.