The Role Of Smoothened In Neuroendocrine Transdifferentiation Of Prostate Cancer And Its Pathogenic Mechanism

BackgroundProstate cancer is the second leading cause of death from cancer in men worldwide.Prostate cancer is an androgen-dependent malignant tumor,and the androgen receptor（AR）pathway has vital effects on the occurrence and development of the tumor.Androgen deprivation therapy（ADT）is the main strategy for the treatment of advanced prostate cancer.Plenty of patients who respond to ADT for the first time will gain resistance and undergo tumor recurrence at last.These tumors are termed castrate-resistant prostate cancer（CRPC）.In recent years,with the advent of next-generation AR pathway inhibitors（APIs）,the overall survival of patients has been significantly improved.However,the remission of the disease can only last for a few years,and then the tumors gradually become resistant to APIs.A considerable number of the tumors lose the phenotype of androgen-dependence and transform into a more aggressive type of tumor with neuroendocrine characteristics.These tumors are called neuroendocrine prostate cancer（NEPC）.Researchers have found that it takes several years to develop from androgen-dependent prostate cancer（AdPC）to NEPC,and it only takes several months from NEPC to death.Therefore,it is urgent to study the process and mechanism of the conversion from AdPC to NEPC.Timely monitoring of the disease development process and finding effective therapeutic targets are extremely important.AdPC is an androgen-dependent tumor,holding features of prostate adenocarcinoma（AD）,expressing signs such as AR,KLK2,KLK3,NKX3-1,SPDEF,etc.;while NEPC is an androgen-independent tumor with NE characteristics,expressing SOX2,ENO2,CHGA,CHGB,SYP,and other markers.Lineage plasticity refers to the feature of cells that lose their original lineage characteristics and acquire the lineage characteristics of other types of cells.In recent years,it is believed that lineage switching plays an important role in the occurrence of NEPC.Primary NEPC is very rare,laboratory and clinical experiments suggest that most NEPC is transformed from AdPC.The transformation process from AdPC to NEPC is a complex and sophisticated network,in which a variety of transcription factors,cytokines,and certain tumor suppressor genes are involved.The study of molecules involved in regulating this transformation process has been a hot topic in this field in recent years.Hedgehog（Hh）signaling is a very old and conserved one,which plays an indispensable role in the embryonic development of bilaterally symmetrical animals.During the embryonic period of vertebrates,the Hh pathway plays an important regulatory role in the development of the brain,gastrointestinal tract,lungs,musculoskeletal system,and prostate.In recent years,studies have found that the Hh signaling pathway plays a vital role in maintaining the pluripotency of cancer stem cells and the development of tumors.In prostate cancer,genes related to the Hh signaling pathway are differentially expressed between cancer tissues and paracancerous tissues,between carcinoma in situ and metastatic carcinoma.Further studies have found that the Hh signaling pathway is paracrine or autocrine in prostate cancer.It enhances tumor progression by promoting tumor cell proliferation and epithelial-to-mesenchymal transition（EMT）.In addition,under the condition of androgen deprivation,the Hh pathway can be used as an alternative pathway to the AR pathway to maintain the survival of prostate cancer cells and promote the formation of CRPC.NEPC is a special variant of prostate cancer,and the relationship between Hh signaling pathway and NEPC has not been reported yet.It is known that the Hh pathway plays an important role in the occurrence of prostate cancer and the formation of CRPC,and AdPC can transform into NEPC through lineage conversion.We speculate that the Hh pathway may play a role in the progression of NEPC.PurposesBased on public data,we clarify the changes of Hh signal pathway activity in the process of NEPC pathogenesis,identify the Hh pathway-related genes that are significantly differentially expressed between AdPC and NEPC,and explore the role and mechanism of smoothened（SMO）in the process of neuroendocrine transdifferentiation in prostate cancer.We hope to provide a new theoretical basis for the diagnosis and treatment of NEPC.Methods1.Obtaining the expression profile of public datasets that containing both AdPC and NEPC cases,comparing the differences in the expression of Hh pathway-related genes between the two types of tumors.2.6 cases of AdPC,5 cases of NEPC,5 cases of AdPC with NE differentiation,2 cases of a mixture of AdPC and NEPC,and 22 cases of high-grade AdPC tumor specimens were collected,and the expression of SMO was tested by immunohistochemistry.3.Analyzing the changes in the expression of AR pathway-related genes,NE markers,NE-related transcription factors,and SMO during the transformation of AdPC to NEPC in the model LTL331/331R.4.Analyzing the genetic variations of SMO,emasculating prostate cancer cells in vitro,or treating cells with JQ1 in vitro to explore the reasons for the changes in SMO gene expression.5.Dividing the prostate cancer cases in the public database into two groups according to the expression of SMO,and speculating the signal pathways that related to SMO expression through GSEA（gene set enrichment analysis）.6.Knocking down SMO expression in prostate cancer cell lines by lentivirus.Real-time PCR and western blotting were used to detect the knockdown efficiency,and the expression of AR pathway-related genes between sh-SMO and sh-NC.Gli inhibitor（GANT61）and Hh activator（SAG）were used to inhibit or activate the Hh pathway activity,and the changes in AR and PSA expression in cells in different treatment groups were analyzed.7.LNCaP and C4-2B cells with SMO knocking down were transfected with a cDNA-Gli1 plasmid,western blotting was used to detect the expression of AR and PSA.8.Real-time PCR and western blotting were used to detect the expression of NE markers in sh-SMO and sh-NC cells;SYP and CHGA proteins were examined in sh-SMO and sh-NC with different periods of androgen deprivation treatment in vitro.9.CCK-8 test and the scratch test were used to examine the cell proliferation and migration activity in sh-SMO and sh-NC cells.Results1.SMO mRNA is significantly reduced in NEPCA total of 6 datasets both containing AdPC and NEPC specimens were obtained from the cBioportal and GEO databases.The expression of 6 key Hh pathway genes（SHH,PTHC1,SMO,Gli1,Gli2,Gli3）were compared between AdPC and NEPC.The result showed that SMO was the most significantly different expressed Hh-related gene between AdPC and NEPC,and the expression in NEPC was significantly reduced.The expression profiles of prostate cancer cell lines and PDX models were obtained from CCLE and LTL databases,whose results confirmed the observation that SMO was selectively reduced in NEPC.2.SMO protein is selectively reduced in NECP tissuesThe result of immunohistochemistry showed that SMO was positively expressed in AdPC specimens,AdPC with NE differentiation specimens,and AdPC regions of AdPC and NEPC mixed specimens;in contrast,SMO expression was absent in NEPC specimens,and NEPC regions of AdPC and NEPC mixed specimens.The AR pathway gene（AR）and NE markers（SYP and CHGA）were also immunohistochemically stained at the same time.The expression of SMO and AR overlapped obviously,but they were mutually exclusive with the expression of SYP and CHGA.3.Dynamic changes of SMO in the process of neuroendocrine transdifferentiation in the LTL331/331R modelIn the LTL331/331R model,as the tumor transforming from AdPC to NEPC,the expression of AR pathway-related genes（AR,KLK3,TMPRSS2,NKX3-1,SPDEF）decreased or even disappeared,the expression of NE markers（CHGA,ENO2,SYP,SCG3）increased to the highest peak gradually,and the level of NE-related transcription factors（ASCL1,SOX2,PEG10,BRN2,SRRM4）increased to different degrees.The expression of the SMO gene was relatively stable during the first 12 weeks and started to decline from the 12th week until the tumor relapsed and became NEPC.The dynamic changes of SMO were similar to those of REST and YAP1.4.The expression of SMO in prostate cancer may be regulated by epigenetic factorsGene mutations and copy number variations of SMO were compared between AdPC and NEPC.The result showed that gene mutations or copy number variations could not explain the difference of SMO mRNA between AdPC and NEPC.In vitro,ADT could not change the expression of SMO either.BRDs inhibitor（JQ1）down-regulated the expression of SMO in LNCaP and C4-2B cells.Therefore,changes in SMO expression in prostate cancer were not caused by gene mutations or AR pathway activity but may be regulated by epigenetic factors.5.SMO loss associated with key features of NEPCThe prostate cancer data downloaded from the above public databases were sorted in descending order of SMO expression level.Those with SMO lower than the top 1/4 were named "SMO-LO" group,and the remaining cases were named "SMO-HI" group.GSEA was used to analyze the differences in the expression profiles of the two groups of cases.The results showed that SMO expression was positively correlated with features of"ANDROGEN_RESPONSE" and "CHOLESTER_HOMEOSTASIS",and negatively correlated with features of "E2F_TARGETS","PANCREAS_BETA_CELLS",and "G2M_CHECKPOINT".6.SMO knockdown leads to reduced AR pathway activity and AR gene expressionCompared with the sh-NC group,the expression levels of Gli1,AR,and PSA in the sh-SMO group were reduced.Gli inhibitor significantly down-regulated the expression of AR and PSA in prostate cancer cells;Hh activator could restore the decrease in AR and PSA gene expression caused by SMO knockdown.This suggests that the Hh signal pathway is involved in the regulation of the AR pathway activity and AR gene expression.7.Overexpression of Gli1 up-regulates AR pathway activity and AR gene expressionThe cDNA-Gli1 plasmid was transfected into sh-SMO cells and sh-NC cells.Gli1 up-regulated the expression of PSA and AR in the sh-SMO group but did not in the sh-NC group.This result indicates that Gli1 is a downstream gene of SMO,involving the regulation of AR pathway activity and AR gene expression.8.SMO gene knockdown enhances NE-related genes expression in androgen deprived conditionCompared with the sh-NC group,the sh-SMO group showed no significant difference in the expression of NE markers.sh-SMO and sh-NC cells were cultured in androgen-deprived conditions for several days and the expression of CHGA and SYP was detected.The levels of SYP and CHGA increased with the extension of androgen deprivation in the two groups of cells,while the increased level in the sh-SMO group was higher than that of the sh-NC group.9.SMO knockdown has no significant effect on cell proliferation and migrationComparing the sh-SMO group with the sh-NC group,there was no significant difference in the results of the CCK-8 experiment and cell scratch test.In vitro experiments indicate that the SMO gene has no significant effect on the activity of LNCaP cells.ConclusionSMO loss is an important characteristic of NEPC,and immunohistochemical detection of SMO can assist clinical diagnosis of NEPC.SMO acts as an obstacle in the transformation of AdPC to NEPC.SMO regulates AR pathway activity and AR gene expression through Glil.SMO loss together with other transformation-promoting factors could synergistically promote the conversion of AdPC to NEPC.