HepaCAM Inhibits The Malignant Behavior Of Castration-resistant Prostate Cancer Cells By Down-regulating Notch Signaling

Objective:Hepatocyte cell adhesion molecule(HepaCAM),regared as tumor suppressor,is frequently and regularly down-regulated or lost in many cancer typies.The Notch signaling pathway is significantly over-activated in many tumor tissues including prostate cancer tumors.However,the expression and role of HepaCAM in castration-resistant prostate cancer(CRPC)is still unknown.In this study,we investigated the role of both HepaCAM and Notch and the relationship betweem them in the development and progression of CRPC.Methods:We collected 45 CRPC tissue samples and blocks from the First Affiliated Hospital of Chongqing Medical University from April 2008 to September 2017,and 41 blocks of matched primary prostate cancer(PPC)tissues.The expressions of HepaCAM protein and key molecules of Notch signaling pathway including Notchl and Hesl were detected by using Immunohistochemistry(IHC).The correlation between HepaCAM and Notch1,Hes1 expression in CRPC tissues was analyzed by using Pearson’s correlation.And the correlation between HepaCAM expression and clinical pathological data in CRPC and matched PPC tissues was retrospectively analyzed.The LNCaP cell were continuously cultured with 10μM bicalutamide and 10μ,M enzalutamide for 6 months to induce bicalutamide-resistant LNCaP(Bica-R)and enzalutamide-resistant LNCaP(Enza-R).Western blot was performed to detect the expression of HepaCAM in human normal prostate epithelial cell lines RWPE-1,LNCaP,Bica-R and Enza-R.Adenovirus vector containing HepaCAM gene was transfected into DU 145,LNCaP,Bica-R,Enza-R cells,respectively.CCK-8 and Clone formation experiments were performed to observe the cell proliferation ability;and invasion ability of CRPC cells was evaluated by using Wound healing assay and Transwell assay.Half maximal inhibitory concentration of enzalutamide to Enza-R cells was detected after treated the cells with HepaCAM by using CCK-8assay.LNCaP and Enza-R cells,cultured with 10 nM docetaxel for 6 months,respectively,were induced to generate docetaxel-resistant LNCaP(Doce-R)and sequential dual-resistance LNCaP to enzalutamide and docetaxel(E+DR Cells).RT-qPCR and Western blot were performed to detect mRNA and protein expression of Notch signaling pathway-associated molecules such as Jaggedl,Notch1,NICD(only protein level)and Hes1 in LNCaP,Bica-R,Enza-R,Doce-R and E+DR cells,respectively;Western blot assya was performed to detect the expression of EMT-associated proteins,such as E-cadherin,N-cadherin,and Snail proteins,after treating Enza-R cells with Ad-HepaCAM or/and PF-3084014.The expression of Notchl and Hesl were evaluated by using Immunofluorescence assay after treating the Enza-R with Ad-HepaCAM or/and PF-3084014.The mRNA levels of Jaggedl,Notchl and Hesl in Enza-R cells were detected by using RT-qPCR after trating them with Ad-HepaCAM or/and PF-3084014.The protein levels of Jaggedl,Notchl,NICD,and Hesl in both Bica-R and Enza-R cells were detected by using Western blot assay after after trating them with Ad-HepaCAM or/and PF-3084014.CCK-8 assay was performed to detect the half maximal inhibitory concentration(IC50)of docetaxel to Doce-R and E+D-R cells and their parental cells respectively after treated with Ad-HepaCAM or/and PF-3084014.The role of HepaCAM in ihibiting Doce-R and E+D-R cells was evaluated by using CCK-8 assay.The sensitivity of Enza-R cells to enzalutamide was evaluated by using CCK-8 assay after treated with Ad-HepaCAM and/or PF-3084014.The sensitivity of Doce-R cells to docetaxel was evaluated by using CCK-8 assay after treated with Ad-HepaCAM and/or PF-3084014.The sensitivity of E+D-R cells to docetaxel was evaluated using CCK-8 assay after treated with Ad-HepaCAM and/or PF-3084014.The proliferation abilities of Enza-R,Doce-R,E+D-R cells were investigated by using CCK-8 assay after treatment with different concentrations of PF-3084014(5,10,20,30,40,60,80,and 100μM)respectively for 48 hoursResults:Compared with the matched PPC tissues,HepaCAM expression in CRPC tissues was significantly down-regulated or lost(P=0.0336);Hesl expression was significantly up-regulated in CRPC tissues(P=0.0237);there was no difference in Notchl expression between matched tissues(P=0.063).In CRPC tissues,the expression of HepaCAM was negatively correlated with Notchl(r=-0.652,P<0.01)and Hesl(r=-0.442,P=0.02).Western blot results showed that the expression of HepaCAM was negatively correlated with Notchl(r=-0.572,P=0.033)and Hesl(r=-0.728,P=0.003)in 14 CRPC specimens.Kaplan-Meier survival analysis showed that the median progression-free survival(FPS)was 27 months in CRPC patients with HepaCAM-negative,and the median FPS in CRPC patients with HepaCAM-positive was 39 months(P=0.039).HepaCAM was normally expressed in human normal prostate epithelial cells RWPE-1,HepaCAM expression is negative in Bica-R,Enza-R cells and its parental cell LNCaP.CCK-8 assay showed that HepaCAM significantly inhibited the proliferation of DU145,LNCaP and CRPC cells(Bica-R,Enza-R).Clone formation assay showed that HepaCAM inhibited the proliferation of Bica-R and Enza-R cells.Transwell and wound healing assay showed that HepaCAM inhibited the migration and invasion of CRPC cells.CCK-8 assay showed that HepaCAM did not change the sensitivity of Enza-R cells to enzalutamide.RT-qPCR and Western blot assay showed that Notch signaling pathway associated-molecules including Jaggedl,Notchl,NICD(protein level),Hes1 expression were significantly up-regulated in Bica-R,Enza-R,Doce-R cells compared with that in LNCaP cells.CCK-8 assay showed that HepaCAM and PF-3084014 have a synergistic effect on the inhibition of Enza-R cell growth.Western blot assay showed that HepaCAM significantly up-regulated the expression of E-cadherin,down-regulated the expression of N-cadherin and Snail,these effects were enhanced when combined with PF-3084014.Immunofluorescence assay showed that HepaCAM down-regulated the expression of Notchl and Hesl,and PF-3084014 showed similar results.When combined HepaCAM with PF-3084014,the effects on Notch signaling became more significant than either agent alone.Western blot assay showed that both HepaCAM and PF-3084014 could significantly decreased the protein and mRNA levels of Jaggedl,Notchl,NICD(protein levels),Hes1,and they had a synergistic effect when used in combination.CCK-8 assay showed that overexpression of HepaCAM significantly inhibited the proliferation of both Doce-R and E+D-R cells.When combined with PF-3084014,the effect became more significant.Howerver,overexpression of HepaCAM had no significant effect on the change of docetaxel sensitivity of Doce-R and E+D-R cells.PF-3084014 partially reversed the resistance of Doce-R and E+D-R cells to docetaxel.When in combined with HepaCAM,there was no synergistic effect on the resistance of these cells to docetaxel.When cells were treated with 20μM PF-3084014 alone,the anti-tumor effect of it began to appear with a dose-dependent effect.Conclusion:In most CRPC tissues,HepaCAM was negative and Notch 1 and Hes1 are overactivated.And there is a negative correlation between HepaCAM and Notch 1,Hesl.Overexpression of HepaCAM suppresses the malignant biological behavior of CRPC Cells significantly by down-regulating Notch signaling.Overexpression of HepaCAM failed to reverse the resistance of Enza-R cells to enzalutamide,as well as Doce-R and E+D-R cells to docetaxel;PF-3084014 partly reversed the resistance of Enza-R cells to enzalutamide,as well as Doce-R,E+D-R cells to docetaxel.