| Prostate cancer is a common malignant tumor in men.The 2020 Global Cancer Statistical Report released by "CA:A Cancer Journal for Clinicians" in 2021 shows that the incidence of prostate cancer ranks second in male malignant tumors in the world,and the mortality rate ranks fifth.In our country,its morbidity and mortality also show an increasing trend year by year.Currently,androgen deprivation therapy(ADT)is the standard therapy for advanced prostate cancer.However,most patients develop hormone resistance after 12-18 months of treatment and become castration-resistant prostate cancer(CRPC).The main treatment drugs for these patients at this stage are enzalutamide,abiraterone,docetaxel,etc.Androgen receptor(AR)plays a key role in the progression of CRPC,and enzalutamide has received extensive attention from the medical community due to its high affinity with AR.Clinical studies have shown that enzalutamide can effectively improve the prognosis of patients with CRPC.The US Food and Drug Administration has approved enzalutamide for the treatment of patients with metastatic and non-metastatic CRPC.Although enzalutamide has good application prospects in the treatment of CRPC,it is regrettable that patients will inevitably develop enzalutamide resistance after a period of treatment.There is often no follow-up effective treatment in clinical practice.Therefore,it is extremely urgent and important to explore the pathogenesis of enzalutamide resistance in prostate cancer and find novel intervention strategies.More and more evidence-based evidence shows that the reactivation of AR signaling pathway is still the main molecular mechanism of enzalutamide resistance in prostate cancer cells,mainly manifested as AR amplification,mutation and the generation of splice variantsAR-V7 is the main splice variant of AR.Our research group has long been devoted to the study of the molecular mechanism of the occurrence and development of CRPC.We have identified some driver genes associated with CRPC progression,including PADI2 and TXNDC9,and revealed their pathogenic mechanism.To further identify the driving genes of enzalutamide resistance in CRPC and explore their molecular mechanism,we screened the differential genes between the resistant group and the sensitive group using the enzalutamide resistance-related database of prostate cancer,and detecting the expression of differential genes in enzalutamide resistance cell model constructed in our laboratory,we identified Kinesin family member 15(KIF15)may be a key gene that promotes enzalutamide resistance in prostate cancer.KIF15 is an N-terminally localized,positive-directed motor protein that is involved in the separation of the cell spindle and plays an important role in cell mitosis and neuron axon formation.Current research shows that KIF15 is highly expressed in a variety of tumor tissues and plays a role in promoting tumor progression,including pancreatic cancer,breast cancer,etc.In this study,we analyzed in clinical tissue samples,cell models and mouse tumor-bearing models,and used bioinformatics analysis to reveal the role of KIF15 in prostate cancer enzalutamide resistance from multiple dimensions and explore its molecular mechanism.It will provide new ideas for the treatment of clinical CRPC patients.This research is divided into two parts.Part 1 KIF15 promotes enzalutamide resistance in prostate cancer:Enzalutamide resistance in prostate cancer is one of the major clinical problems and challenges.Identifying the driver genes associated with enzalutamide resistance and revealing their biological roles can provide a theoretical basis for the clinical intervention strategies.In this study,we combined with bioinformatics analysis and enzalutamide-resistant cell model analysis,and identified KIF15 as a driver gene in promoting enzalutamide resistance in prostate cancer.The biological role of KIF15 was explored.The results obtained in this part are as follows:1.Construction of enzalutamide-resistant cell modelMorphological detection showed that:compared with control cells(C4-2B-Parental),enzalutamide-resistant prostate cancer cells(C4-2B-ENZR cells)had thicker and longer antennae;cell resistance index detection showed that:The IC50 value of C4-2B-ENZR cells was 5.75 of that of C4-2B-Parental cells;compared with C4-2B-Parental cells,the mRNA and protein levels of AR,AR-V7 and AKR1C3 were significantly increased in C4-2B-ENZR cells.And under the treatment of enzalutamide,the cell viability,proliferation and clone formation ability of C4-2B-ENZR were obviously enhanced.2.Identification of enzalutamide resistance driver gene KIF15 in prostate cancerWe analyzed the enzalutamide resistance-related database of prostate cancer,and selected genes with a fold change greater than 1.5 and a P value less than 0.05 in enzalutamide-resistant group compared with the sensitive group.And detect the mRNA expression levels of these differential genes using qRT-PCR in C4-2B-Parental and C4-2B-ENZR cell models.Among them,the mRNA of KIF15 was most significantly elevated in C4-2B-ENZR compared with C4-2B-Parental cells.3.KIF15 is highly expressed in enzalutamide-resistant cells and enzalutamide-resistant tissues of prostate cancerThe analysis results of several public databases related to enzalutamide resistance in prostate cancer showed that the mRNA expression of KIF15 in enzalutamide-resistant prostate cancer cells,mouse xenografts,and clinical specimens of patients were increased significantly than sensitive group.At the cell line level,the mRNA and protein expressions of KIF15 in C42B-ENZR and 22Rv1 cells were significantly higher than those in C4-2B-Parental cells and LNCaP cells;LNCaP and C4-2B cells were stimulated with long-term enzalutamide(more than 1 month),the mRNA and protein expressions of KIF15 were significantly increased.4.High expression of KIF15 is associated with poor prognosis in prostate cancerThe analysis results of public databases such as GEO(Gene Expression Omnibus)database and The Cancer Genome Atlas(TCGA)showed that compared with benign prostate tissue,the mRNA expression of KIF15 in prostate cancer tissue was significantly increased;mRNA expression of KIF15 was positively correlated with biochemical recurrence,Gleason score,and clinical stage,and negatively correlated with survival of prostate cancer patients.Immunohistochemistry(IHC)detection showed that the immunohistochemical expression of KIF15 in prostate cancer tissues(n=7)was significantly stronger than that in benign tissues(n=3).The expression of KIF15 protein was higher in prostate cancer tissues with higher Gleason scores in 362 prostate cancer.5.KIF15 promotes the proliferation and migration of prostate cancer cellsThe results of MTS,EdU,colony formation assay,and Transwell assay show that compared with the control group,knockdown the expression of KIF15 in 22Rv1,VCaP and PC3 cells,cell proliferation,clone formation ability,migration and infiltration ability of the cells were all improved.Cell proliferation,clone formation ability,migration and infiltration ability of the cells were significantly up-regulated in LNCaP and C4-2B cells with KIF15 overexpressed.6.KIF15 promotes enzalutamide resistance in prostate cancer and xenograft progressionThe results of MTS and colony formation assay show that compared with the control group,the sensitivity of C4-2B-Parental cells to enzalutamide was significantly reduced when KIF15 was overexpressed,and the cell proliferation ability was significantly enhanced;the resistance to enzalutamide and cell proliferation were significantly reduced in C4-2B-ENZR and 22Rv1 cells with KIF15 knockdown.C4-2B-ENZR and 22Rv1 cells with KIF15 stably knockdown were injected to subcutaneous in nude mice.The results suggest that stable knockdown of KIF15 expression can inhibit tumor growth.In conclusion,KIF15 is highly expressed in enzalutamide-resistant prostate cancer cells and promotes the proliferation and tumor formation of enzalutamide-resistant cells.High expression of KIF15 is associated with poor prognosis of prostate cancer patients.Part II KIF15 promotes enzalutamide resistance in prostate cancer by regulating USP14mediated AR and AR-V7 protein stability:revealing the mechanism of KIF 15 promoting enzalutamide resistance in prostate cancer and finding effective intervention strategies,which will help to provide theoretical support for clinical treatment.In this part of the research,through bioinformatics analysis and a variety of molecular biology experiments,with the help of in vivo and in vitro models,the molecular mechanism was studied at multiple levels,and the following results were obtained:1.KIF15 is related to AR and AR-V7 signaling pathwaysGene Set Enrichment Analysis(GSEA)using RNAseq data of 22Rv1 and C4-2B-ENZR cells with KIF15 knockdown found that the androgen-induced gene sets,AR and AR-V7 activating gene sets were mainly enriched in the control group;while androgen inhibited gene sets,AR and AR-V7 inhibited gene sets were mainly enriched in the KIF 15 knockdown group.The results of qRT-PCR and Western blot showed that the mRNA and protein expressions of AR and AR-V7 in C4-2B-ENZR cells were significantly increased compared with C4-2BParental cells.2.KIF 15 regulates the protein expression of AR and AR-V7The results of qRT-PCR and Western blot showed that KIF 15 did not affect the mRNA levels of AR and AR-V7 in C4-2B-ENZR,22Rv 1,C4-2B-Parental and LNCaP cells,but KIF 15 could significantly promote AR and AR-V7 protein expression.Western blot and IHC experiments showed that compared with the control group,the protein expression levels of AR and AR-V7 in tumor tissues stably knocked down KIF15 were significantly decreased.With the treatment of KIF15 inhibitor(KIF15-IN-1)in C4-2B-ENZR and 22Rv1 cells,the protein expressions of AR and AR-V7 were down-regulated in a concentration-and time-dependent manner.3.KIF15 promtotes the cell proliferation of prostate cancer cells by regulating the expression of AR and AR-V7The results of MTS assays showed that under 20 μM enzalutamide treatment,interfering with KIF 15 in C4-2B-ENZR and 22Rv1 cells could inhibit cell proliferation,and this effect could be reversed by overexpression of AR or AR-V7;in LNCaP,Overexpression of KIF15 in C4-2B-Parental cells can promote cell proliferation,and this effect can be reversed by knockdown of AR gene.4.KIF 15 binds to the N-terminal domain of ARCo-immunoprecipitation(Co-IP)assay performed in C4-2B-ENZR and 22Rv1 cells showed that KIF15 can form complexes with AR and AR-V7;GST pull-down assay showed that KIF 15 can binds directly to AR and AR-V7.Co-IP experiments were performed in 293T cells transfected with different truncated AR plasmid,and KIF15 was found to bind to the Nterminal domain of AR.Immunofluorescence assay,nuclear cytoplasmic separation combined with Co-IP assay,showed that the AR-KIF15 complex was mainly located in the cytoplasm.5.KIF15 inhibits the ubiquitination and protein degradation of AR and AR-V7The results of Western blot assay showed that under the treatment of 10 μg/mL cycloheximide(CHX),the protein half-life of AR and AR-V7 in C4-2B-ENZR and 22Rv1 cells with KIF15 knockdown or adding KIF15-IN-1 in were shorter than the control group.And the AR protein half-life was prolonged when KIF15 was overexpressed in C4-2B-Parental cells.MG 132 reversed the protein downregulation of AR and AR-V7 caused by KIF15 reduction in C4-2B-ENZR and 22Rv1 cells.Co-IP assay showed that protein ubiquitination levels of AR and AR-V7 were increased when KIF15 was knockdown in C4-2B-ENZR and 22Rv1 cells,and AR ubiquitination was decreased in C4-2B-Parental cells with KIF15 overexpressed.6.KIF15 prevents AR and AR-V7 proteins from degradation by increasing the protein association of USP14 with AR and AR-V7Mass spectrometry analysis showed that ubiquitin specific peptidase 14(USP14)interacted with KIF15.Co-IP assay showed that in C4-2B-ENZR and 22Rv1 cells,KIF15 was endogenously bound to USP 14;USP 14 combined with AR and AR-V7 proteins and could reduce the ubiquitination of AR and AR-V7 proteins level.The results of qRT-PCR and Western blot assay showed that knockdown of KIF15 expression in C4-2B-ENZR and 22Rv1 cells or overexpression of KIF15 in C4-2B-Parental cells did not affect the mRNA and protein levels of USP 14.The results of Co-IP experiment showed that KIF15 knockdown in C4-2B-ENZR and 22Rv1 cells reduced the binding of USP 14 to AR and AR-V7,and the binding of USP 14 to AR was increased in C4-2B-Parental cells with KIF 15 overexpressed compared with the control.In addition,USP 14 could reverse the protein downregulation and the increasion of ubiquitination level of AR and AR-V7 caused by knockdown of KIF 15,while the inactive mutant USP14(C114A)did not have this effect.7.KIF15 is a target gene of ARThe GEPIA(http://gepia.cancer-pku.cn)database showed that AR was positively correlated with the mRNA expression of KIF 15 in prostate cancer.The results of qRT-PCR and Western blot showed that,the mRNA and protein levels of KIF 15 were increased in a time-and dose-dependent manner in LNCaP cells with androgen treatment.AR promoted the mRNA and protein expression of KIF 15 in C4-2B-ENZR,22Rv1 and PC3 cells.The results of chromatin immunoprecipitation assay(ChIP)showed that AR could bind to the KIF 15 promoter.The results of the dual-luciferase reporter gene assay showed that AR could activate the luciferase activity of the KIF 15 wild-type promoter but not the mutant promoter.8.KIF15 inhibition suppresses enzalutamide-resistant prostate cancer cell growth and xenograft progressionThe results of MTS assay showed that,in C4-2B-ENZR and 22Rv1 cells,cell viability was decreased with the increase of KIF 15-IN-1 concentration or prolonged treatment time,and the cell viability decreased significantly when enzalutamide was combination;KIF 15-IN-1 can increase the sensitivity of C4-2B-ENZR and 22Rv1 cells to enzalutamide,and inhibit the proliferation and colony formation of C4-2B-ENZR and 22Rv1 cells.The results of the subcutaneous tumorigenesis experiment in nude mice showed that compared with the control group,KIF15-IN-1 could significantly inhibit tumor growth in mice,and the effect of KIF 15IN-1 combined with enzalutamide in inhibiting tumor growth was more significant.These results suggest that KIF 15 inhibitors and enzalutamide may synergistically inhibit CRPC progression.Taken together,this study is the first to identify and explore the role of KIF 15 in enzalutamide resistant prostate cancer.The mechanism by which KIF 15 promotes enzalutamide resistance in prostate cancer is elaborated:KIF15 maintains the stability of AR and AR-V7 proteins in a USP14 dependent manner,and AR promotes the transcription of KIF15,they form a positive feedback loop to promote enzalutamide resistance in prostate cancer.The combination of KIF15 inhibitor and enzalutamide provides a new idea for the treatment of CRPC. |
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