Role Of Volume-regulated Chloride Channel In Neuroendocrine Prostate Cancer Differentiation And Its Mechanismic Study

BackgroundThe 2021 American Cancer Report and 2018 European Cancer Statistics show that prostate cancer（PCa）is the most common malignancies and the second most lethal tumor among men.The diagnosis rate of PCa in China is continuously increased.Although Androgen deprivation therapy（ADT）and high-affinity AR antagonists such as abiraterone and enzalutamide（ENZ）are indeed effective in the short-term,their applications have been concerned as the main cause of another variant with higher levels of death rate and malignancy,neuroendocrine prostate cancer（NEPC）.NEPC patients are resistant to ENZ and sensitive to cisplatin in a period of time,lack of effective therapeutic drugs is the main puzzle.However,challenges for precision oncology still remain due to high heterogeneity inter and intra tumors as well as diverse mechanisms of resistance to treatment.New biomarkers will help to better classify disease molecular subtypes,so that clinical decisions can be made faster and more accurately.Ion channels,the hot spot in global drug development,are progressively emerging as novel potential biomarkers and therapeutic targets for a variety of human cancers such as PCa.Accumulating evidence demonstrates that the development of PCa involves aberrant ion channels expression,activity as well as functional change,including potassium channel,sodium channel,calcium channel and calcium-activated chloride channels.Compared with adenocarcinoma,NEPC contains various phenotypic features such as higher expression of neuroendocrine marker protein SYP,sensitivity to platinum drugs.Additionally,NEPC cells induced in vitro also showed dendritic synapse-like phenotypic characteristics,and the cell volume changed significantly.Moreover,studies have confirmed that the volume-regulated chloride channel（VRCC）is the main ion channel that regulates cell volume and transports cisplatin across the cell membrane.It was also mentioned that the activity of VRCC changes during the NEPC process.More importantly,recent studies have confirmed the VRCC as a multimer composed of five structurally similar proteins of LRRC8A-8E,among which leucine-rich repeat-containing protein 8A（LRRC8A）and leucine-rich repeat-containing protein 8D（LRRC8D）are the main molecules that affect the sensitivity to cisplatin.Multiple prostate cancer gene expression databases（Gene Expression Omnibus,GEO）and chip database（Oncomine）showed that expression of LRRC8D decreased in NEPC compared with adeno-PCa.In addition,it was reported that one of the targets of Repressor element-1-silencing transcription factor（REST）,a key transcription factor regulating NEPC differentiation,is ion channels.Though the regulatory relationship between REST and VRCC has not been reported,the transcription factor target prediction software suggests that there are potential binding sequences of REST upstream of LRRC8D promoter.Furthermore,regulating the expression of the channel protein can improve the sensitivity of temozolomide in the treatment of glioma,indicating that the VRCC channel component protein can be an important intervention target for tumor treatment.Therefore,in this study we will mainly explore the role of LRRC8D in the differentiation of prostate adenocarcinoma to NEPC and their molecular mechanisms,in order to provide a new target for prostate cancer research.The aimThe aim is to observe the changes of VRCC and channel component molecules LRRC8D in the neuroendocrine differentiation of prostate cancer and to explore the effect of LRRC8D on the differentiation of NEPC and the molecular mechanisms.The methods1.cAMP and SRRM4 lentivirus infection were used to induce the differentiation of NEPC model,respectively.NE markers SYP,CHGA,NSE in constructed NEPC cell models were detected by real-time quantitative PCR and immunohistochemistry stain respectively.Transmission electron microscope was used to observe and count the neurosecretory granules in a single cell section.The drug sensitivity of each cell model to enzalutamide and cisplatin was examed by CCK-8 experiment.2.The VRCC chloride current of NEPC cells was detected and recorded by whole-cell patch clamp to reflect the activity of VRCC.Bioinformatics analysis of prostate cancer databases（GEO,Oncomine,and public data）was applied to investigate the expression relationship between LRRC8A,LRRC8B,LRRC8C,LRRC8D,LRRC8E and NE marker protein SYP.Gene chip comparison analysis was conducted to screen differentially expressed genes between SRRM4-induced NEPC cells（22RV1-SRRM4）and the corresponding adenocarcinoma（22RV1-NC）cells.GO analysis and Pathway analysis of obtained differentially expressed genes were also applied to verify the phenotype of NEPC.3.Immunohistochemistry and Western Blot experiment were conducted to detect the protein expression of LRRC8A,LRRC8D,SYP in prostate adenocarcinoma and NEPC,the test samples included slices derived from 16 PCa patients,NCI-H660 cell line（NEPC cells）,LNCa P-cAMP cells（NEPC cells）,SRRM4-induced NEPC cells and NEPC mouse CDX tissues of different passages.Knockdown and over-expression of LRRC8D were achieved through Si RNA interference experiment and LRRC8D lentiviral packaging vector infection,and were applied to analyze the effects of LRRC8A or LRRC8D on expression of SYP.Meanwhile,the effect of LRRC8D on drug sensitivity was checked by CCK-8 and drug-induced apoptosis was further analysed by flow cytometry.Effect of LRRC8D over-expression on NEPC tumor growth in vivo was reflected by tumor size in CDX model establishment assays.4.The knockdown of REST was achieved through the Si RNA of REST,and the Western Blot experiment was further used to analyze the effect of down-regulating REST on the expression of SYP and LRRC8D.The transcriptional regulation effect of REST on LRRC8D was verified by dual luciferase reporter experiment.Furthermore,the effect of LRRC8D on the expression of cisplatin-induced pro-apoptosis-related proteins Bax and Cleaved Caspase-3 was analyzed by Western Blot experiment.The subcutaneous tumor tissues of NEPC cells with LRRC8D overexpression（22S8DOE）and the control group were analyzed by mass spectrometry to investigate possible mechanisms of overexpression of LRRC8D in regulating the growth of NEPC tumors in vivo.The results1.We successfully constructed NEPC cell models,including LNCa P-cAMP,LNCa P-SRRM4 and 22RV1-SRRM4 cells.Compared with their NC cells（LNCa P,LNCa P-NC,22RV1-NC）,NEPC cells showed the following phenotypes:（1）SYP and CHGA are positive in NEPC cells.（2）Neuroendocrine granules increased significantly(P^**<0.01);（3）The NEPC cells were resistant to enzalutamide,which is significantly different from the corresponding adenocarcinoma cells(P^**<0.01),while the sensitivity of NEPC to cisplatin is lower than adenocarcinoma cells（P^*<0.05）.（4）The NEPC cells were transplanted subcutaneously into nude mice,and the tumor displayed more rapid growth and higher expression of neuroendocrine markers after passage.2.VRCC current decreased in NEPC cells.The bioinformatics analysis demonstrated that the expression of LRRC8D in adeno-PCa and NEPC is opposite to SYP.3.The expression of LRRC8A and LRRC8D was lower in NEPC patients,NEPC cell lines（NCI-H660 cells）,cAMP-induced NEPC model,SRRM4-induced NEPC model,and tumor tissues of NEPC CDX（P^*<0.05）,which is consistent with the decreased chloride current in SRRM4 or cAMP induced NEPC cells in hypotonic external fluid（P^*<0.05）.Knockdown of LRRC8D can increase the expression of SYP（P^*<0.05）and increase production of neurosecretory granules（P^*<0.05）,but it had no effect on sensitivity to enzalutamide and cisplatin（P>0.05）.Knockdown of LRRC8A had no effect on the expression of NE markers such as SYP,CHGA and NSE,and had no regulatory effect on the expression of LRRC8D（P>0.05）.Increasing expression of LRRC8D can significantly down-regulate SYP expression and reduce the number of neurosecretory granules（P^*<0.05）.The decrease in cell viability caused by cisplatin can be exacerbated by up-regulation of LRRC8D,while pre-treatment of DCPIB,the VRCC inhibitor,can restrain this decrease caused by cisplatin(P^**<0.01).Flow cytometry analysis demonstrated that over-expression of LRRC8D could promote the produce of apoptotic cells induced by cisplatin,especially the early apoptotic cells in 22RV1-SRRM4 cells（P^*<0.05）.The 22S8DOE cells（over-expression of LRRC8D in 22RV1-SRRM4 cells）were injected subcutaneously into nude mice,and the xenografts formed were apparently smaller compared to the 22RV1-SRRM4 group.4.The results of Western Blot confirmed that knockdown of REST can decrease the expression of SYP,CHGA,similar to the Western Blot results after LRRC8D knockdown.At the same time,the expression of LRRC8A and LRRC8D was also significantly reduced after knockdown of REST.Further application of dual-luciferase reporter gene detection experiments clarified that REST can play a transcriptional regulatory role on LRRC8D through the TCTCTGTTCATGTTCCTGT sequences of LRRC8D（P^*<0.05）.Additionally,expression of Bax and Cleaved Caspase-3 increase and was also regulated by LRRC8D.Additionally,results of mass spectrometry showed multiple differential proteins between22S8DOE and 22S8DNC were consistent with those reported in the literature,and multiple pathway molecules related to lipid metabolism were significantly down-regulated.The conclusion1.Treatment of cAMP or overexpression of SRRM4 can induce prostate adenocarcinoma cells to differentiate into neuroendocrine prostate cancer cells.After differentiation into neuroendocrine carcinoma,the expression of neuronal marker protein,the number of neurosecretory granules,and the drug sensitivity are all changed significantly.Neuroendocrine tumor displays earlier formation and more rapid growth than those of adenocarcinoma.2.The differentiation of prostate adenocarcinoma to NEPC leads to down-regulation of the expression of LRRC8A and LRRC8D,inhibition of the VRCC activity and reduced VRCC current(I_SWELL,Cl).3.LRRC8D is the molecular basis for regulating the expression of SYP,the production of neurosecretory granules and tumor drug sensitivity during NEPC differentiation.Down-regulation of LRRC8D can lead to the increase of SYP and neurosecretory granules,but has no effect on the sensitivity of enzalutamide and cisplatin.Over-expressing LRRC8D can inhibit the expression of SYP,reduce the production of neurosecretory granules and increase the sensitivity of NEPC cells to cisplatin.Additionally,LRRC8D can also inhibit the tumor growth in nude mice.4.REST can interact with a sequence upstream of the LRRC8D promoter to affect the transcription and protein expression of LRRC8D and inhibit the production of SYP.Therefore,LRRC8D regulates the expression of SYP but the specific mechanism remains to be determined by further experiments.On the other hand,the increasing sensitivity of cisplatin to NEPC cells after LRRC8D over-expression is due to elevated expression of Cleaved Caspase-3 and Bax mediated by LRRC8D.Besides,LRRC8D may inhibit tumor proliferation in vivo by regulating lipid metabolism-related molecules.