Mechanism Of Neurokinin-1 Receptor Involving The Process Of Neuroendocrine Trans-Differentiation In Prostate Cancer

Prostate cancer is the most common tumor in the male genitourinary system,and abnormal activation of androgen receptor(AR)is a key mechanism for the occurrence and development of prostate cancer.Castration-resistant prostate cancer(CRPC)is the standard treatment for advanced prostate cancer.However,most patients resist the treatment and develop to CRPC.Since most CRPC patients still maintain androgen dependence,more powerful androgen receptor pathway inhibitors(ARPIs)are used to treat these patients in clinical practice.However,with the inevitable drug resistance,the CRPC with resistance of ARPIs continue to grow by producing survival mechanisms independent of androgen signaling.A number of ARPIs-resistant tumors have turned from adenocarcinoma tissue phenotype to neuroendocrine phenotype,developing into neuroendocrine prostate cancer(NEPC).NEPC with rapid development and short survival time is a malignant lethal form of prostate cancer and lack of effective treatment drugs.Therefore,it is necessary to explore the pathogenesis of NEPC related to anti-androgen therapy and find new biomarkers and targets for the diagnosis and treatment of NEPC for improving the therapeutic level of NEPC.Neurokinin-1 Receptor(NK1R),a member of the G-protein-coupled receptor family,is expressed abnormally in a variety of tumors.It plays an important role in the growth and metastasis of tumors by mediating ERK1/2,Akt and other signal pathways involving cell proliferation and migration.In our previous studies,we found that NK1 R expression was up-regulated in prostate cancer.Here,we focused on the mechanism of neuroendocrine trans-differentiation in prostate adenocarcinoma,further investigated the role of NK1 R in treatment-related NEPC development and the potential of NK1 R as a therapeutic target for NEPC at molecular,cellular,animal levels.We detected NK1 R expression in the tissue chip of prostate cancer patients and found that NK1 R expression in tumor tissues was increased compared with adjacent tissues,positively correlating with the malignancy of prostate cancer.Survival analysis showed that NK1 R expression was associated with poor prognosis in prostate cancer patients.Compared with adenocarcinoma,NK1 R expression in NEPC samples was further increased,accompanied by low AR activity and significant neuroendocrine differentiation.In the treatment of anti-androgen therapy,AR signal loss and neuroendocrine markers in prostate cancer cells were found to be significantly increased.Meanwhile,the expression and distribution on cell membrane surface of NK1 R in t NEPC cells was significantly up-regulated,which was consistent with the analysis results in clinical samples.These data suggested that NK1 R expression was significantly increased with anti-androgen therapy,and associated with neuroendocrine trans-differentiation of prostate cancer.AR,which belongs to the nuclear receptor family,acts as a transcription factor to regulate the expression of downstream target genes after AR activation.We further studied the relationship between AR and NK1 R expression.We analyzed the CHIPSEQ data and found that there was a significant binding peak of AR protein at 9 Kb downstream of NK1 R gene.CHIP-QPCR showed that AR activation effectively induced its enrichment on Androgen Response Element1(ARE1)downstream of NK1 R gene.CRISPR-CAS9-mediated deletion of ARE1 can significantly reduce the enrichment of AR protein on NK1 R gene.Meanwhile,the transcriptional inhibition effect of AR on NK1 R is weakened,the expression of NK1 R is increased,and the m RNA expression levels of NE markers and related genes are up-regulated.The upregulation of the expression of Glucocorticoid Receptor(GR)is one of the important mechanisms of castration resistance in prostate cancer.Both GR and AR belong to the nuclear receptor family,and the target genes involved in the regulation of expression of GR and AR are highly overlapping.We found that GR was positively correlated with NK1 R expression in NEPC associated with antiandrogen therapy,and promoted NK1 R protein expression by binding ARE1.We speculated that antiandrogen therapy resulted in the inhibition of AR activity in prostate cancer cells,the increase of GR and NK1 R expression,and GR could further bind to ARE1 site to promote the expression of NK1 R.Therefore,the expression of NK1 R is regulated by AR/GR dual transcription.We continue to study the function of NK1 R in NEPC progression.In adenocarcinoma prostate cancer cells,overexpression of NK1 R significantly promoted NE differentiation,enhanced cell proliferation and invasion,significantly reduced sensitivity to enzalutamide,and significantly enhanced tumorigenic ability in nude mouse models.On the contrary,NE marker expression was decreased,cell proliferation was inhibited,and the tumorigenic ability was also decreased in NK1 R knockdown NEPC cells.Mechanism studies have shown over-expression of NK1 R can promote the expression of N-Myc and Aurora kinase A(AURKA)in prostate cancer cells,and the protein-protein interaction between N-Myc and AURKA is significantly increased.Activation of NK1 R leads to upregulation of PKCα and AURKA phosphorylation,while inhibition of NK1R-PKCα-AURKA signaling pathway leads to significant cell cycle arrest.We further explored the feasibility of targeting NK1 R or its mediated downstream signaling pathways in the treatment of NEPC.In vitro and in vivo experiments demonstrated that inhibition of the expression or activity of NK1 R could effectively suppress the growth,invasion and tumorigenic ability of NEPC cells,and reduce the expression of NE markers.Prostate cancer cells with high expression of NK1 R showed obvious sensitivity to AURKA inhibitor showing the synergistic antitumor effect in the presence of NK1 R inhibitor.Reducing NK1 R expression can enhance the sensitivity of NEPC cells to paclitaxel chemotherapy drugs.These data indicate that targeting NK1 R has potential application value in the treatment of NEPC.In summary,this study found that NK1 R is an important factor driving antiandrogen therapy-related NEPC.Upregulation of NK1 R induced by antiandrogen therapy is associated with the transition from adenocarcinoma to NEPC in prostate cancer.NK1 R mediates PKCα-AURKA/N-Myc pathway in prostate cancer cells,promoting NE transdifferentiation and tumor growth of prostate cancer cells.And targeting NK1 R inhibits NEPC progression.These studies indicate that NK1 R plays an important role in the progress of t NEPC,providing an experimental basis for NK1 R to be used as a therapeutic target and clinical biomarker of NEPC.