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CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY Targeting the KIF4A/AR Axis to Reverse Endocrine Therapy Resistance in Castration-resistant Prostate Cancer A C Qi Cao 1,2 , Zhengshuai Song 1,2 , Hailong Ruan 1,2 , Cheng Wang 1,2 , Xiong Yang 1,2 , Lin Bao 1,2 , Keshan Wang 1,2 , Gong Cheng 1,2 , TianBo Xu 1,2 , Wen Xiao 1,2 , Zhiyong Xiong 1,2 , Di Liu 1,2 , Ming Yang 3 , Diwei Zhou 3 , Hongmei Yang 4 , Ke Chen 1,2 , and Xiaoping Zhang 1,2 ABSTRACT Purpose: Emerging evidence indicates that castration-resistant prostate cancer (CRPC) is often driven by constitutively active androgen receptor (AR) or its V7 splice variant (AR-V7) and commonly becomes resistant to endocrine therapy. The aim of this work is to evaluate the function of a kinesin protein, KIF4A, in regulating AR/AR-V7 in prostate cancer endocrine therapy resistance. Experimental Design: We examined KIF4A expression in clin- ical prostate cancer specimens by IHC. Regulated pathways were investigated by qRT-PCR, immunoblot analysis, immunoprecipi- tation, and luciferase reporter and chromatin immunoprecipitation (ChIP) assays. A series of functional analyses were conducted in cell lines and xenograft models. Results: Examination of the KIF4A protein and mRNA levels in patients with prostate cancer showed that increased expression of KIF4A was positively correlated with androgen receptor (AR) levels. Patients with lower tumor KIF4A expression had improved overall survival and disease-free survival. Mechanistically, KIF4A and AR form an auto-regulatory positive feedback loop in prostate cancer: KIF4A binds AR and AR-V7 and prevents CHIP-mediated AR and AR-V7 degradation; AR binds the promoter region of KIF4A and activates its transcription. KIF4A promotes castration-sensitive and castration-resistant prostate cancer cell growth through AR- and AR-V7-dependent signaling. Furthermore, KIF4A expression is upregulated in enzalutamide-resistant prostate cancer cells, and KIF4A knockdown effectively reverses enzalutamide resistance and enhances the sensitivity of CRPC cells to endocrine therapy. Conclusions: These ndings indicate that KIF4A plays an important role in the progression of CRPC and serves as a crucial determinant of the resistance of CRPC to endocrine therapy. Introduction Prostate cancer is one of the most common malignancies and ranks second among causes of male cancer-related death worldwide (1). Androgens play an essential role in the growth of prostate cancer cells. Androgen deprivation therapy (ADT) is the main method for the treatment of advanced prostate cancer. However, after an average of 2 years of ADT treatment, the tumor often progresses from castration- sensitive prostate cancer (CSPC) to castration-resistant prostate can- cer (CRPC; ref. 2). As the androgen receptor (AR) signaling pathway is an important pathway for prostate cancer survival and progression, targeting AR expression and inhibiting its activity has become an effective way to treat CRPC. Enzalutamide and abiraterone are novel androgen receptor (AR) inhibitors that are used to treat patients with metastatic CRPC after chemotherapy (3). However, prostate cancer can become therapy resistant through upregulation of androgen synthesis, mutation, and abnormal expression of AR, AR splice variants (AR-Vs), and neuroendocrine transitions. Therefore, reveal- ing the complicated molecular mechanism of endocrine therapy resistance of prostate cancer is an urgent global concern. AR-V7 is the most common AR splice variant. Levels of AR-V7 are low before treatment with enzalutamide or abiraterone but increase signicantly after progression on either agent (4). A large-scale clinical trial found that AR-V7 expression in circulating tumor cells of patients with prostate cancer was associated with shorter survival and was also closely related to resistance to endocrine therapy drugs (5). Increased AR-V7 expression may represent one of the mechanisms of resistance to these agents. However, the precise role of AR-V7 in the progression of CRPC is still unclear. KIF4A, a member of the kinesin superfamily (KIFs), is located on chromosome Xq13.1 and encodes a protein consisting of 1232 amino acids. KIF4A is involved in multiple cellular activities, particularly spindle formation and centrosome assembly in mitosis (6), chromo- some concentration and separation (7), and DNA damage repair (8). KIF4A plays a critical role in a variety of tumors, such as lung cancer (9), oral cancer (10), liver cancer (11), and colorectal carcino- ma (12). Estrogen regulates the expression of KIF4A through the estrogen receptor (ERa) in breast cancer, whereas ANCCA, a cor- egulator of ERa, is involved in the regulation of KIF4A (13). KIF4A is abundantly expressed in prostate cancer, and this expression is associated with poor prognosis in patients with prostate cancer (14). These ndings suggest that KIF4A may have functions that contribute 1 Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 2 Insititute of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 3 Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 4 Department of Pathogenic Biology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, China. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Q. Cao, Z. Song, and H. Ruan contributed equally to this article. Corresponding Authors: Ke Chen, Huazhong University of Science and Tech- nology, Jiefang Avenue No. 1277, Wuhan 430022, China. Phone: 027-85351625; E-mail: [email protected]; Xiaoping Zhang, [email protected]; and Hongmei Yang, [email protected] Clin Cancer Res 2020;26:151628 doi: 10.1158/1078-0432.CCR-19-0396 Ó2019 American Association for Cancer Research. AACRJournals.org | 1516 on March 21, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 3, 2019; DOI: 10.1158/1078-0432.CCR-19-0396

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CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

Targeting the KIF4A/AR Axis to Reverse EndocrineTherapy Resistance in Castration-resistant ProstateCancer A C

Qi Cao1,2, Zhengshuai Song1,2, Hailong Ruan1,2, Cheng Wang1,2, Xiong Yang1,2, Lin Bao1,2, Keshan Wang1,2,Gong Cheng1,2, TianBo Xu1,2, Wen Xiao1,2, Zhiyong Xiong1,2, Di Liu1,2, Ming Yang3, Diwei Zhou3,Hongmei Yang4, Ke Chen1,2, and Xiaoping Zhang1,2

ABSTRACT◥

Purpose: Emerging evidence indicates that castration-resistantprostate cancer (CRPC) is often driven by constitutively activeandrogen receptor (AR) or its V7 splice variant (AR-V7) andcommonly becomes resistant to endocrine therapy. The aim ofthis work is to evaluate the function of a kinesin protein, KIF4A,in regulating AR/AR-V7 in prostate cancer endocrine therapyresistance.

Experimental Design: We examined KIF4A expression in clin-ical prostate cancer specimens by IHC. Regulated pathways wereinvestigated by qRT-PCR, immunoblot analysis, immunoprecipi-tation, and luciferase reporter and chromatin immunoprecipitation(ChIP) assays. A series of functional analyses were conducted in celllines and xenograft models.

Results: Examination of the KIF4A protein and mRNA levels inpatients with prostate cancer showed that increased expression of

KIF4Awas positively correlatedwith androgen receptor (AR) levels.Patients with lower tumor KIF4A expression had improved overallsurvival and disease-free survival. Mechanistically, KIF4A and ARform an auto-regulatory positive feedback loop in prostate cancer:KIF4A binds AR and AR-V7 and prevents CHIP-mediated AR andAR-V7 degradation; AR binds the promoter region of KIF4A andactivates its transcription. KIF4A promotes castration-sensitiveand castration-resistant prostate cancer cell growth through AR-and AR-V7-dependent signaling. Furthermore, KIF4A expressionis upregulated in enzalutamide-resistant prostate cancer cells, andKIF4A knockdown effectively reverses enzalutamide resistanceand enhances the sensitivity of CRPC cells to endocrine therapy.

Conclusions: These findings indicate that KIF4A plays animportant role in the progression of CRPC and serves as a crucialdeterminant of the resistance of CRPC to endocrine therapy.

IntroductionProstate cancer is one of the most commonmalignancies and ranks

second among causes of male cancer-related death worldwide (1).Androgens play an essential role in the growth of prostate cancer cells.Androgen deprivation therapy (ADT) is the main method for thetreatment of advanced prostate cancer. However, after an average of2 years of ADT treatment, the tumor often progresses from castration-sensitive prostate cancer (CSPC) to castration-resistant prostate can-cer (CRPC; ref. 2). As the androgen receptor (AR) signaling pathway isan important pathway for prostate cancer survival and progression,targeting AR expression and inhibiting its activity has become an

effective way to treat CRPC. Enzalutamide and abiraterone are novelandrogen receptor (AR) inhibitors that are used to treat patients withmetastatic CRPC after chemotherapy (3). However, prostate cancercan become therapy resistant through upregulation of androgensynthesis, mutation, and abnormal expression of AR, AR splicevariants (AR-Vs), and neuroendocrine transitions. Therefore, reveal-ing the complicated molecular mechanism of endocrine therapyresistance of prostate cancer is an urgent global concern.

AR-V7 is the most common AR splice variant. Levels of AR-V7 arelow before treatment with enzalutamide or abiraterone but increasesignificantly after progression on either agent (4). A large-scale clinicaltrial found that AR-V7 expression in circulating tumor cells of patientswith prostate cancer was associated with shorter survival and was alsoclosely related to resistance to endocrine therapy drugs (5). IncreasedAR-V7 expression may represent one of the mechanisms of resistanceto these agents. However, the precise role of AR-V7 in the progressionof CRPC is still unclear.

KIF4A, a member of the kinesin superfamily (KIFs), is located onchromosome Xq13.1 and encodes a protein consisting of 1232 aminoacids. KIF4A is involved in multiple cellular activities, particularlyspindle formation and centrosome assembly in mitosis (6), chromo-some concentration and separation (7), and DNA damage repair (8).KIF4A plays a critical role in a variety of tumors, such as lungcancer (9), oral cancer (10), liver cancer (11), and colorectal carcino-ma (12). Estrogen regulates the expression of KIF4A through theestrogen receptor (ERa) in breast cancer, whereas ANCCA, a cor-egulator of ERa, is involved in the regulation of KIF4A (13). KIF4A isabundantly expressed in prostate cancer, and this expression isassociated with poor prognosis in patients with prostate cancer (14).These findings suggest that KIF4Amay have functions that contribute

1Department of Urology, Union Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Wuhan, China. 2Insititute of Urology,Union Hospital, Tongji Medical College, Huazhong University of Science andTechnology, Wuhan, China. 3Department of Pathology, Union Hospital, TongjiMedical College, Huazhong University of Science and Technology, Wuhan,China. 4Department of Pathogenic Biology, School of Basic Medicine, HuazhongUniversity of Science and Technology, Wuhan, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Q. Cao, Z. Song, and H. Ruan contributed equally to this article.

Corresponding Authors: Ke Chen, Huazhong University of Science and Tech-nology, Jiefang Avenue No. 1277, Wuhan 430022, China. Phone: 027-85351625;E-mail: [email protected]; Xiaoping Zhang, [email protected]; andHongmei Yang, [email protected]

Clin Cancer Res 2020;26:1516–28

doi: 10.1158/1078-0432.CCR-19-0396

�2019 American Association for Cancer Research.

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to prostate cancer progression, but whether KIF4A can regulate the ARsignaling pathway remains unclear.

In this study, we demonstrate for the first time that KIF4A ispositively correlated with AR levels in prostate cancer. Patients withhigher KIF4A expression have worse survival. AR binds to thepromoter region of KIF4A and activates its transcription. In addition,KIF4A binds and stabilizes the AR and AR-V7 proteins to modulatethe AR pathway in prostate cancer cells. Furthermore, our resultsindicate that KIF4A promotes human CSPC and CRPC cell growththrough AR-dependent signaling. Most importantly, we show thatdepletion of KIF4A reverses enzalutamide resistance and enhances thesensitivity of CRPC cells to endocrine therapy.

Materials and MethodsTissue microarray

A human prostate cancer tissue microarray (TMA) was purchasedfrom Alenabio (PR1921b). All detailed clinical information includingpathology, diagnosis, stage, Gleason scores, and PSA level is freelyavailable on the Web (http://www.alenabio.com/public/details?productId¼59058&searchText¼). The following antibodies wereused: anti-KIF4A (1:50; Proteintech) and anti-AR (1:100; sc-816; SantaCruz Biotechnology). The TMA was evaluated using by scoring thestaining intensity in a range of 0 to 3 (0 ¼ absent, 1 ¼ weak, 2 ¼intermediate, 3¼ strong) and the percentage of positively stained cellsin a range of 0 to 4 (0� 5%, 1¼ 5%–25%, 2¼ 25%–50%, 3¼ 50%–75%,4 � 75%). The two scores were multiplied to obtain an immunore-activity score (IRS) ranging from 0 to 12. The IHC data were evaluatedby two blinded pathologists.

Cell culture and drug treatment293T, LNCaP, C4-2, and 22Rv1 cells were purchased from ATCC.

293T cells were maintained in DMEM. LNCaP, C4-2, and 22Rv1 cellsweremaintained in RPMI1640medium. Allmedia were supplementedwith 10% FBS and 1% penicillin–streptomycin solution. All cells werecultured at 37�C in an incubator containing 5% CO2. Drug concen-trations (unless otherwise indicated) were enzalutamide (20 mmol/L),bicalutamide (10 mmol/L), DHT (10 nmol/L), and cycloheximide(CHX; 10 mmol/L).

Establishment of the enzalutamide-resistant cell lineC4-2-ENZ-R

The starting treatment concentration of enzalutamide in the paren-tal C4-2 cell culturemediumwas 0.2mmol/L. At this concentration, the

cells were stably passaged three times. The drug concentration wasthen increased, and the culture was continued. The concentrationgradients of enzalutamide were 0.4, 0.8, 1, 2, 4, 8, 10, 20, and 40mmol/L.After 4months of induction, the stable enzalutamide-resistant cell linewas obtained and named C4-2-ENZ-R.

TransfectionThe pLent-GFP shRNA targeting KIF4A and pENTER-Flag KIF4A

were purchased from Vigenebio. GV298-shRNA targeting CHIP,MYC-CHIP, eGFP-AR, and HA-AR-V7 were purchased from Gen-echem. pCDNA3.1-3 � Myc-Ub was purchased from miaolingbio.Briefly, 3� 105 cells were seeded in six-well plates and transfected with2 mg of plasmid using Lipofectamine 2000 (Invitrogen) according tothe manufacturer's procedure.

Lentiviral constructsLentivirus was packaged by cotransfection of the constructs with the

plasmids pMD2.G, pRRE, and pRSV/REV in 293T cells. The super-natants collected at 72 and 96 hours after transfection were filtered by a0.45-mmfilter, and thefiltrateswere then added to prostate cancer cells.After being selected with puromycin (1mg/mL), the protein expressionlevel was verified by Western blotting.

IHC and immunofluorescenceIHC was conducted as described previously (15). Tissues were

sequentially fixed in formalin, dehydrated, and embedded in paraffin.Then, IHC was conducted by incubating the tissue sections withprimary antibodies including KIF4A, PSA, or Ki67 overnight at 4�C.Subsequently, after washing three times with PBS, the sections wereincubated with the secondary antibody (1:200; GB23303; Servicebio,Inc.) at room temperature for 2 hours. Immunofluorescence (IF) wasperformed as described previously (16). Cells were fixed in 4%paraformaldehyde, permeated by 0.3% Triton X-100, and blockedwith 3% BSA for 1 hour at 37�C, followed by incubation with primaryKIF4A and AR antibodies.

ChIP assaysThe ChIP assay was performed according to the protocol of the

ChIP Assay Kit (CST). 22Rv1 and C4-2 cells were cultured in 10-cmdishes. Then, the chromatin in the cells was cross-linked by addingformaldehyde to a final concentration of 1% at room temperature for10 minutes, followed by washing twice with 2 mL of ice-cold PBScontaining protease inhibitors, lysis in ChIP lysis buffer, and sonica-tion to completely lyse nuclei. The digested, cross-linked chromatinwas diluted with ChIP buffer. A 10-mL sample of the diluted chromatinwas removed as a 2% input sample. The remaining 500 mL of dilutedchromatin was incubated with anti-AR or anti-IgG at 4�C overnightwith rotation. After elution of chromatin, reversal of cross-links andDNA purification, qRT-PCR was performed to amplify the potentialAR binding sites on the KIF4A promoter region.

Cell proliferation assayA total of 3� 103 cells were added to eachwell of a 96-well plate. The

cell proliferation rate was determined using Cell Counting Kit-8(CCK-8; Dojindo) every 24 hours. Briefly, 10 mL of CCK-8 solutionwas added to each well. After 4 hours, the absorbance was measured at450 nm.

Immunoprecipitation and Western blot analysisThe collected cells were lysed in RIPA buffer containing protease

inhibitor. The lysate was kept on ice for 30 minutes and centrifuged at

Translational Relevance

Prostate cancer often progresses to castration-resistant prostatecancer (CRPC) and becomes resistant to endocrine therapy, whichis commonly driven by the androgen receptor (AR) or its V7 splicevariant (AR-V7). Reducing the high rates of progression of thisdisease is an urgent unmet clinical need. Here, we report a protein(KIF4A) that is upregulated and transcriptionally regulated by ARin prostate cancer. Overexpression of KIF4A significantly reducesAR and AR-V7 degradation. Inhibition of KIF4A blocks cancerprogression and reverses enzalutamide resistance. We speculatethat targeting the KIF4A/AR axis could be used to increase thesensitivity of CRPC cells to endocrine therapy. KIF4A is critical forCRPC progression and endocrine therapy resistance.

KIF4A Promotes Prostate Cancer Endocrine Therapy Resistance

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12,000 rpm for 5 minutes. Then, the supernatant was collected in twoparts: a small amount of lysate was taken as input, and the remaininglysate was incubated with 2 mg of the corresponding antibody and30 mL of agarose beads at 4�C overnight. The immune complexes werecentrifuged at 3,000 rpm for 2 minutes. The supernatant was carefullydiscarded, and the agarose beads were washed three to four times with200mL of lysis buffer. Finally, 40mL of RIPA lysate and 10mLof loadingbuffer were added to the beads and boiled for 10minutes. ForWesternblotting, total proteins were separated by 10% SDS-PAGE and trans-ferred to polyvinylidene fluoride (PVDF) membranes. The PVDFmembranes were blocked and then incubated with primary antibodies.The antibodies used in Western blotting were KIF4A (ab124903;Abcam), AR (sc-7305; Santa Cruz Biotechnology), AR-V7 (ab198394;Abcam), Flag (20543-1-AP; ProteinTech), MYC (60003-2-Ig; Protein-Tech), HA (66006-2-Ig; ProteinTech), eGFP (66002-1-Ig; Protein-Tech), and b-actin (AC026; ABclonal). The proteins were visualizedusing ChemiDoc-XRsþ (Bio-Rad).

RNA isolation and RT-PCRTotal RNA was extracted from cells using TRIzol reagent (Invitro-

gen) and then reverse transcribed by the PrimeScript RT reagent Kitwith gDNA Eraser (Takara). Real-time PCR was conducted usingMaxima SYBRGreen/ROXqPCRMasterMix (Thermo Fermentas) onthe ABI-7500 qRT-PCR system. The primers used for qRT-PCR arelisted in Supplementary Table S1.

Luciferase assaysCells were seeded in a 24-well plate at 70% confluence and tran-

siently transfected with 0.8mg of expression vector plasmids and 0.4mgof promoter reporter plasmids. The fluorescence intensity was mea-sured after 48 hours. The luciferase activity of the gene promoter wasnormalized to Renilla luciferase activity as an internal standardcontrol. The plasmids of KIF4A-luc and mutated KIF4A-luc weredesigned and synthesized by Genechem.

Tumor xenograft studyThree- to 4-week-old castrated male nude mice were purchased

from Beijing Vital River Laboratory Animal Technology Co., Ltd.Approximately 5 � 106 C4-2-ENZ-R cells with knockdown of KIF4Aor control cells suspended in 100 mL of serum-free medium wereinjected subcutaneously into the armpit on the right side of the mice.Tumor volumes (V) were measured every 5 days based on measure-ments of length (L) and width (W) and calculated as V¼ (L�W2)/2.When the tumors reached 100 to 200mm3, the mice were randomizedequally, treated with vehicle or enzalutamide (10 mg/kg) twice perweek, and sacrificed approximately 40 days later.

Statistical analysisAll statistical analyses were performed using Prism 5.0 (GraphPad)

andSPSS22.0 (IBMCorporation).All in vitro experimentswere repeatedthree times. All data are presented as the mean � SD. Survival infor-mation was verified by Kaplan–Meier analysis and compared using thelog-rank test. A two-tailed unpaired Student t test was used to determinethe P values, which were considered significant at less than 0.05.

ResultsThe expression level of KIF4A is positively related to AR inprostate cancer

AR is a ligand-activated transcription factor that plays critical rolesin normal prostate development and prostate tumorigenesis (17). The

growth of advanced prostate cancer (both CSPC and CRPC) dependson AR signaling. In this study, by screening four independent prostatecancer gene sets (TCGA, Arredouani, Grasso, and Varambally;refs. 18–20), three genes (KIF4A, BIRC5, ATP8A2) that correlatedwith AR were selected on the basis of their significant differences inexpression in prostate cancer tissues compared with normal tissues(logFC > 1.5, P < 0.05; Fig. 1A; Supplementary Fig. 1A). Furthermore,Kaplan–Meier survival analysis and log-rank tests were conducted todetermine whether the disease-free survival (DFS) and overall survival(OS) of patients were associated with KIF4A, BIRC5, and ATP8A2expression in tumors. As shown in Fig. 1B, only patients with highlevels of KIF4AmRNA expression hadworseDFS (P¼ 0.0002) andOS(P ¼ 0.033) than those with low KIF4A expression. Higher BIRC5 orATP8A2 expression did not appear to affect OS in prostate cancer(Fig. 1B). A comprehensive consideration of these results led us tofocus on KIF4A for further study.

Previous studies have shown that increased KIF4A mRNA expres-sion is a potential prognostic factor in prostate cancer (14). KIF4Amayplay a key role in prostate cancer progression. Next, to further confirmthe previous findings and the interaction between KIF4A and AR, weexamined KIF4A and AR protein levels with IHC staining using atissue array containing 160 cases of human prostate cancer and 32adjacent normal tissue specimens. Similar to a previous result, weobserved that KIF4A was positively correlated with AR expression inhuman normal prostate tissues (r¼ 0.37,P< 0.05) and human prostatecancer tissues (r¼ 0.55,P< 0.0001;Fig. 1C). KIF4Aprotein levels weresignificantly higher in the prostate cancer tissues than in the normaltissues and were related to tumor stage and PSA levels (SupplementaryFigs. S1B–S1D). Taken together, these results suggested a functionalinteraction between KIF4A and AR during prostate cancerprogression.

AR transcriptionally activates KIF4A in prostate cancerAR acts as a transcription factor to regulate the expression of

its downstream target genes and promote prostate cancer progres-sion (21). Given that KIF4A and AR are positively correlated inprostate cancer, there is a possibility that AR may transcriptionallyregulate KIF4A expression. To explore this possibility, LNCaP cellswere treated with DHT for 12 hours. As shown in Fig. 2A, KIF4Aprotein levels were elevated in LNCaP cells upon androgen treat-ment. To confirm that this effect of androgen occurred throughAR, we directly modulated the levels of AR in PC3 and C4-2 cells.As expected, the changes in KIF4A protein levels were in accor-dance with the change in AR when we overexpressed AR inPC3 cells and C4-2 cells or downregulated AR in C4-2 cells(Fig. 2B–D). These results indicated that KIF4A is elevated byAR activation.

In the nucleus, AR binds to specific DNA sequences termedandrogen response elements (AREs) in the promoter regions of targetgenes, such as prostate-specific antigen (PSA) and transmembraneprotease serine 2 (TMPRSS2; refs. 22, 23). Next, we identified acanonical ARE �531 base pairs upstream of the KIF4A transcriptionstart site (Fig. 2E).We then cloned luciferase reporter constructs into apGL3 plasmid containing the putative ARE (ARE-luc) or its mutant(AREmut-luc; Fig. 2E). As expected, ectopic AR expression signifi-cantly activated ARE-luc activity with or without DHT treatment.However, no activation of AREmut-luc activity was observed uponoverexpression of AR (Fig. 2F). To further verify the binding sites onthe promoter region, we analyzed and designed primers coveringdifferent sequences of interest in the promoter of KIF4A, which weredenoted P1, P2, P3, P4, P5, and P6 (Fig. 2G). ChIP assay revealed a

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Figure 1.

The expression level of KIF4A is positively related to AR expression in prostate cancer. A (top), Venn diagram for shared genes in the four public prostate cancerdatasets. Bottom, In four independent prostate cancer databases, the KIF4A/AR correlation was determined by Pearson correlation. B (first column), Kaplan–MeierDFS curves of patients with prostate cancer based on BIRC5, KIF4A, and ATP8A2 expression. (second column) Kaplan–Meier OS curves of patients with prostatecancer based on BIRC5, KIF4A, and ATP8A2 expression. C, A prostate tumor microarray containing 160 prostate cancer tissues and 32 adjacent normal tissues wasstained with anti-KIF4A and AR antibodies. Spearman rank correlation analysis identified positive correlations between KIF4A and AR in the prostate cancer tumorsamples and adjacent normal samples.

KIF4A Promotes Prostate Cancer Endocrine Therapy Resistance

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significant increase in AR recruitment to the P5 region compared withthe IgG control in C4-2 and 22Rv1 cells (Fig. 2H–I). Furthermore, ARrecruitment to the P5 region was significantly increased upon DHTtreatment compared with the IgG control in castration-sensitiveLNCaP cells (Supplementary Fig. S2A). These results demonstratedthat AR directly activates KIF4A transcription in prostate cancer.

Depletion of KIF4A decreases AR protein levels and inhibits theAR signaling pathway

To explore the role of KIF4A in prostate cancer, we knocked downKIF4A in C4-2 cells using KIF4A-specific shRNA and observed thatthe shRNA decreased the expression levels of AR (Fig. 3A). Further-more, the transcript levels of PSA and TMPRSS2, AR canonical target

Figure 2.

ARdirectly activates KIF4A transcription.A, Immunoblot detection of KIF4A levels after 48 hours ofDHT treatment in LNCaP cells.B, Immunoblot detection of KIF4Alevels after AR overexpression in PC3 cells, with actin as a loading control. C, Immunoblot detection of KIF4A levels after AR overexpression in C4–2 cells. D,Immunoblot detection of KIF4A levels after 48 hours of AR shRNA knockdown in C4-2 cells. E, Schematic representation of the AR response element (ARE) in theKIF4A promoter and its mutant (AREmut). Theþ1 denotes the transcription initiation site. F, Luciferase assays performed using the control pGL3, ARE, and AREmutconstructs in the presence or absence of exogenous AR or DHT treatment. The data shown represent the means� SD of triplicates. G, Full sequence of the humanKIF4A promoter. P1-6 show the regions of the KIF4A promoter detected by the paired primers. ChIP analysis of ARbinding at the P1, P2, P3, P4, P5, and P6 loci in C4-2cells (H) and 22RV1 cells (I) (� , P < 0.05; �� , P < 0.01, ��� , P < 0.001).

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Figure 3.

Depletion of KIF4A decreases AR protein levels, and the KIF4A protein interacts with the AR protein.A, Immunoblot detection of AR protein levels in C4-2 cells afterKIF4A knockdown. B, Real-time qPCR analysis of AR and representative AR target genes in C4-2 cells upon KIF4A knockdown. C,Whole-cell lysates of C4-2 cellswere immunoprecipitated with anti-AR antibodies and blotted with the indicated antibodies.D, Immunoblot detection of AR and AR-V7 protein levels in 22RV1 cellsafter KIF4A knockdown. E, Real-time qPCR analysis of AR-V7 in 22Rv1 cells upon KIF4A knockdown. F,Whole-cell lysates of 22Rv1 cells were immunoprecipitatedwith anti-KIF4A antibodies and blottedwith the indicated antibodies. 293 cells were transiently transfected with Flag-KIF4A and eGFP-AR for 2 days, andwhole-celllysates were immunoprecipitated with anti-Flag (G) or eGFP (H) antibodies and blotted with the indicated antibodies. I, 293 cells were transiently transfected withFlag-KIF4A and HA-AR-V7 for 2 days, and whole-cell lysates were immunoprecipitated with anti-HA antibodies and blotted with the indicated antibodies.Representative IF images of KIF4A and AR or AR-V7 protein localization in C4-2 (J) or 22Rv1 (K) cells. Data are presented as the mean� SD (� , P < 0.05; �� , P < 0.01,��� , P < 0.001).

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genes, were also decreased (Fig. 3B). However, the transcript level ofAR was not downregulated by KIF4A knockdown (Fig. 3B). AR-Vslacking the ligand-binding domain, such as AR-V7, have been impli-cated in the pathogenesis of CRPC and in mediating resistance to newendocrine therapies that target the androgen axis (5). Next, weknocked down KIFA in 22Rv1 cells, another CRPC cell line expressinghigh levels of AR-V7. As shown in Fig. 3D, the expression levels of ARand AR-V7 were also decreased by KIF4A knockdown in 22Rv1 cells.However, the mRNA levels of AR and AR-V7 were not decreased(Fig. 3E). Because the loss of KIF4A decreased AR and AR-V7 proteinlevels and inhibited AR signaling without downregulation of AR andAR-V7 transcript levels, we hypothesized that KIF4A regulates the ARand AR-V7 proteins and their functions through a posttranslationalmodification. To test this possibility, we first performed immunopre-cipitation experiments. AR was immunoprecipitated from C4-2 celllysates with an anti-AR antibody and analyzed for KIF4A binding byWestern blot analysis. The results showed that endogenous KIF4Acoimmunoprecipitated with AR (Fig. 3C). KIF4A interacted withAR-V7 proteins in 22Rv1 cells (Fig. 3F; Supplementary Fig. S2B). Toprove their direct interaction, Flag-KIF4A and eGFP-AR or HA-AR-V7 were coexpressed in 293T cells. Flag-KIF4A was immunoprecipi-tated from cell lysates with an anti-Flag antibody, and eGFP-AR waspulled down anddetected byWestern blot analysis (Fig. 3G).Whenweused an anti-eGFP antibody to precipitate eGFP-AR, Flag-KIF4A wasalso observed in the immunoprecipitate by using an anti-Flag antibody(Fig. 3H). Furthermore, HA-AR-V7 was pulled down by exogenousKIF4A (Fig. 3I). Moreover, IF assays confirmed that KIF4A and ARhad obvious colocalization (Fig. 3J–K).

KIF4A stabilizes the AR and AR-V7 proteins via competitiveinhibition of CHIP-mediated ubiquitination

On the basis of the above results, we expected that KIF4A controlsthe function of AR and AR-V7 by an additional mechanism that mayinvolve regulation of AR and AR-V7 expression at the posttransla-tional level. To test this hypothesis, we constructed the enzalutamide-resistant cell line C4-2-ENZ-R (Supplementary Fig. S3). We knockeddown KIF4A expression by using shRNA in C4-2-ENZ-R cells andtreated cells with CHX to block de novo protein synthesis. We foundthat downregulation of KIF4A significantly accelerated the degrada-tion ofAR (Fig. 4A). In addition, the half-life ofAR andAR-V7proteinwas shortened upon KIF4A knockdown (Fig. 4C; SupplementaryFig. S4A). Similarly, we overexpressed KIF4A in 22Rv1 cells andtreated cells with CHX for different durations. The results showedthat overexpression of KIF4A reduced the rate of degradation ofAR-V7 and AR and prolonged the half-life of the AR-V7 and ARproteins (Fig. 4B andD; Supplementary Fig. S4B). In addition, KIF4Aoverexpression decreased the ubiquitination levels of endogenouslyexpressed AR and AR-V7 in C4-2-ENZ-R and 22Rv1 cells (Fig. 4Eand F), suggesting that KIF4A contributes to AR and AR-V7 stabilityby blocking its ubiquitination.

Several studies have reported that AR levels are regulated by theubiquitin-proteasome degradation pathway (24–27). Several ubiquitinE3 ligases have also been implicated in AR-V7 degradation (28, 29). Itappears that only CHIP E3 ligase interacts with both the AR and AR-V7 proteins. This finding led us to expect that CHIP is involved inKIF4A depletion-induced AR and AR-V7 degradation. First, theeffects of KIF4A on CHIP-mediated degradation were examined. In293T cells, the coexpression of KIF4A andCHIP resulted in significantincreases in the protein levels of AR and AR-V7 compared withoverexpression of CHIP alone (Fig. 4G and H). This result suggestedthat KIF4A might affect CHIP-mediated ubiquitination of AR and

AR-V7. Next, the potential role of KIF4A in AR and AR-V7 ubiqui-tination was examined. In 293T cells, CHIP-dependent ubiquitinationof AR was enhanced by the overexpression of CHIP protein (Fig. 4I),but KIF4A expression rescued CHIP-mediated AR ubiquitination(Fig. 4I). Indeed, the amount of pulled down CHIP protein wassignificantly decreased by KIF4A expression (Fig. 4I). Similarly,AR-V7 ubiquitination was inhibited by KIF4A expression, and thebinding of CHIP protein to AR-V7 was also reduced (Fig. 4J).Meanwhile, CHIP-dependent ubiquitination of AR and AR-V7 wasdecreased by the deletion of endogenous CHIP protein in 293T cells.And additional KIF4A expression further reduced the ubiquitinationlevel of AR and AR-V7 (Supplementary Figs. S5A and S5B). Takentogether, these results suggest that KIF4A inhibits CHIP-mediatedubiquitination of AR and AR-V7 by blocking the interaction betweenAR or AR-V7 and CHIP.

KIF4A knockdown reverts endocrine therapy–resistant CRPCprogression in vitro

Given that KIF4A interacts with AR and inhibits its degradation, weassumed that the inhibition of cell growth by silencing KIF4A isdependent onAR. To further investigate this possibility, we transfectedAR plasmids in LNCaP and C4-2 cells in which KIF4A was silenced.The results showed that the inhibition of cell proliferation induced byKIF4A knockdown was reversed by transient overexpression of AR inLNCaP-stable cell lines (Fig. 5A). Similarly, overexpression of ARabolished the inhibition of cell proliferation induced by downregula-tion of KIF4A in C4-2 cells (Fig. 5B). Also, there was no growthinhibitory effect upon KIF4A knockdown in AR-negative cells, PC3and DU145 (Supplementary Figs. S6A and S6B). These results dem-onstrated that AR plays a critical role in KIF4A-regulated prostatecancer cell growth.

Bicalutamide, a pharmaceutical drug commonly used as an anti-androgen therapy to treat recurrent prostate cancer, is a competitiveinhibitor of AR. Enzalutamide, a novel AR signaling inhibitor, blocksthe growth of CRPC in cellular model systems and was shown in aclinical study to increase survival in patients with metastatic CRPC.The rapid development of therapy resistance in patients with prostatecancer receiving bicalutamide and enzalutamide treatment is becom-ing a major clinical challenge (30, 31). The sustained expression ofAR and AR-V7 is a hallmark of endocrine therapy resistance inprostate cancer. To further investigate whether KIF4A contributesto bicalutamide and enzalutamide resistance in prostate cancer cells,we examined the sensitivity of prostate cancer cells to these drugsunder different conditions through CCK8 assays. As shown in Fig. 5C,we observed that knockdown of KIF4A enhanced inhibition of growthby bicalutamide in castration-sensitive LNCaP cells. Similarly, down-regulation of KIF4A increased inhibition of growth by enzalutamidein castration-resistant C4-2 cells and enzalutamide-resistant C4-2-ENZ-R cells (Fig. 5D–E). Overexpression of KIF4A alleviated theinhibition of growth by enzalutamide in C4-2 cells (Fig. 5F). Fur-thermore, we have established the stably expressed additional AR cellsnamed PC3-AR. As shown in Supplementary Fig. S6E, we observedthat knockdown of KIF4A enhanced inhibition of growth by Enza-lutamide in AR-positive PC3-AR cells. But knockdown of KIF4A didnot enhance inhibition of growth byEnzalutamide inAR-negative cells(Supplementary Figs. S6C and S6D). Finally, Enzalutamide did notinhibit the growth of C4-2-ENZ-R cells, and overexpression of KIF4Ahad same effect (Supplementary Fig. S6F). These results indicated thatinhibition of KIF4A potentiates the effects of enzalutamide andbicalutamide in prostate cancer cells. KIF4A knockdown revertsendocrine therapy-resistant CRPC progression.

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Figure 4.

KIF4Astabilizes theARandAR-V7proteins via competitive inhibitionofCHIP-mediated ubiquitination.A,C4-2-ENZ-R cellswere transiently transfectedwith shNCorsh-KIF4A-1 or sh-KIF4A-2 and then treated with 10 mmol/L CHX, and total cell lysateswere collected at 0, 4, 8, and 12 hours after treatment and subjected toWesternblot analysis.B, 22Rv1 cellswere transiently transfectedwith vector or Flag-KIF4A and then treatedwith 10mmol/LCHX, and total cell lysateswere collected at 0, 4, 8,and 12 hours after treatment and subjected toWestern blot analysis.C,Thehalf-life ofARwas calculated for C4-2-ENZ-R cells.D,Thehalf-life ofAR-V7was calculatedfor 22Rv1 cells. E, C4-2-ENZ-R cells were transiently transfected with Flag-KIF4A for 2 days. Total cell lysates were collected, immunoprecipitated with AR antibody,andblottedwith the indicated antibodies.F, 22Rv1 cellswere transiently transfectedwith Flag-KIF4A for 2days. Total cell lysateswere collected, immunoprecipitatedwith AR-V7 antibody, and blotted with the indicated antibodies. G, 293T cells were cotransfected with HA-AR-V7 with or without Flag-KIF4A and MYC-CHIP for2 days, andwhole-cell lysateswere collected and subjected toWestern blot analysis.H, 293T cells were cotransfectedwith eGFP-ARwith or without Flag-KIF4A andMYC-CHIP for 2 days, and whole-cell lysates were collected and subjected toWestern blot analysis. I, 293T cells were cotransfected with HA-AR-V7 with or withoutFlag-KIF4A, MYC-CHIP, and MYC-Ub for 2 days, and total cell lysates were immunoprecipitated with HA antibody and blotted with the indicated antibodies. J, 293Tcells were cotransfected with eGFP-AR with or without Flag-KIF4A, MYC-CHIP, and MYC-Ub for 2 days, and total cell lysates were immunoprecipitated with eGFPantibody and blotted with the indicated antibodies (�, P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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Figure 5.

KIF4A knockdown reverts endocrine therapy-resistant CRPC progression in vitro. LNCaP (A) and C4-2 (B) cells with stable knockdown of KIF4A and ARoverexpression. Cells were analyzed for cell proliferation by the CCK8 assay.C, LNCaP cells with stable KIF4A knockdownwere treatedwith 10 mmol/L bicalutamide.Cell proliferation was determined by the CCK8 assay. C4-2 (D) and C4-2-ENZ-R (E) cells with stable KIF4A knockdown were treated with 5 and 20 mmol/Lenzalutamide, respectively. Cell proliferation was determined by the CCK8 assay. F, C4-2 cells were transiently transfected with Flag-KIF4A and then treated with20 mmol/L enzalutamide. Cell proliferation was determined by the CCK8 assay (� , P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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KIF4A knockdown reverts endocrine therapy-resistant CRPCprogression in vivo

Our previous studies showed that KIF4A knockdown effectivelyreverts endocrine therapy resistance in CRPC cells. To further confirmthis function of KIF4A, we transplanted C4-2-ENZ-R cells with stableknockdown of KIF4A into castrated male SCID mice. The mice werethen treated with enzalutamide (10 mg/kg) twice a week. As shownin Fig. 6A–C, there was no effect of enzalutamide treatment alone onthe tumor growth and tumor weight of the mice bearing tumorswithout KIF4A knockdown. By contrast, KIF4A knockdown inducedinhibition of tumor growth and tumor weight by enzalutamidetreatment in C4-2-ENZ-R tumors, indicating that KIF4A knockdowncan restore enzalutamide treatment sensitivity. The knockdown effi-ciency of KIF4Awas detected by qRT-PCR. ThemRNA level of KIF4Awas significantly downregulated upon stable KIF4A knockdown(Fig. 6D). Similarly, the protein level of KIF4A was verified in tumorxenografts by IHC assays (Fig. 6F). Next, we determined the expres-sion of the AR target genes PSA and TMPRSS2 in tumor tissues.Similar results were observed. As shown in Fig. 6E, there was no effectof enzalutamide treatment alone on PSA and TMPRSS2 expression inmice bearing tumors without KIF4A knockdown. By contrast, KIF4Aknockdown induced inhibition of PSA and TMPRSS2 expression byenzalutamide treatment in C4-2-ENZ-R tumors (Fig. 6E). Finally,IHC staining of tissue sections of tumors from the four groups wasperformed to evaluate the protein expression of KIF4A, PSA, andKi67.The representative images show that the combination of KIF4Aknockdown and enzalutamide usage resulted in weaker stainingintensity of PSA and Ki67 compared with the KIF4A knockdown-only group, indicating that deletion of KIF4A enhanced the inhibitionof AR activity and proliferation ability by enzalutamide in C4-2-ENZ-R tumors (Fig. 6G; Supplementary Fig. S7A and S7B). Taken together,these results suggested that KIF4A knockdown effectively reversedendocrine therapy resistance in CRPC and that KIF4A is a noveltherapeutic target for CRPC (Fig. 6H).

DiscussionThe KIF proteins are involved in many essential cellular biological

functions, including mitosis and transport of intracellular vesicles andorganelles (32). Increasing evidence indicates that KIF membersparticipate in the genesis and development of human cancers (33–36).KIF4A, a member of the KIF family, has been reported to be abnor-mally expressed and to play a critical role in the progression of varioussolid cancers (9–13). However, the expression and function of KIF4Ain prostate cancer have not been fully researched.

In this study, we demonstrated that KIF4A was upregulated inprostate cancer tissues compared with paired normal tissues. More-over, elevated KIF4A expression was significantly correlated withseveral clinicopathologic parameters, such as tumor stage and PSAlevels. In addition, high KIF4A expression was associated with poorerOS and DFS of patients with prostate cancer, which was partiallyconsistent with a previous study in prostate cancer (14). These findingsrevealed that KIF4A plays an essential role in the progression ofprostate cancer and could act as a potential clinical prognosticindicator for patients with prostate cancer.

The AR-mediated androgen signaling pathway plays an importantrole in the development of prostate cancer (22). Upon androgenbinding, AR dissociates from heat shock proteins and translocates tothe nucleus. TheAR dimer then binds to AREs in the promoter regionsof androgen-dependent genes, thereby activating/inhibiting theirtranscription (37). PSA and TMPRSS2 are two canonical AR target

genes. A binding site at 13 kb upstream of the TMPRSS2 transcriptionstart site is necessary for AR regulation of the TMPRSS2 gene (38). Agroup confirmed that the upstream sequence of the PSA promoter(539-320 bp) is necessary for androgen regulation. AR regulates theexpression of PSA by interacting with the 50-AGAACAgcaAGTGCT-30 sequence (39). Here, we identified a half ARE site in the promoterregion of theKIF4A gene. ARChIP analysis revealed thatKIF4Amightbe upregulated directly via AR binding to the KIF4A promoter. KIF4Aexpression is enhanced by AR upon androgen treatment. These factsstrongly indicate that KIF4A is a direct AR downstream target that isupregulated by androgens.

Infinite proliferation of cancer cells is a hallmark of progression ofprostate cancer (40). Previous studies have showed that KIF4A isinvolved in regulating the proliferation of cancer cells (11). Our datarevealed that KIF4A depletion inhibits prostate cancer cell prolifer-ation. More importantly, the regulation of prostate cancer cell prolif-eration by KIF4A is dependent on AR. These results reveal a novelmode of AR-dependent signaling that is involved in regulating cellbiological behavior. The importance of the involvement of KIF4A inoncogenic regulation was reinforced by our finding that KIF4A boundwith AR/AR-V7 and inhibited their ubiquitination and degradation.The E3 ligase CHIP forms a complex with AR/AR-V7 and participatesin protein ubiquitination. KIF4A blocked CHIP and AR/AR-V7complex formation, leading to AR/AR-V7 protein stabilization. Intro-ducing KIF4A protein restored CHIP-mediated AR/AR-V7 degrada-tion. Our findings on complex formation by KIF4A and CHIP/AR orCHIP/AR-V7 are critical as this mechanism may represent a generalubiquitin–proteasome mechanism for the regulation of AR/AR-V7protein stability that may be involved in endocrine therapy resistancein prostate cancer progression.

The upregulation of AR protein is a hallmark of CRPC and seems tobe an adaptive response to ADT (41). An increase in AR protein levelsis observed in most refractory cases (41). Several mechanisms accountfor increased AR levels because some factors confer stability of AR inCRPC or, importantly, in therapy-resistant prostate cancer. There isevidence that AR proteins are degraded by the ubiquitin–proteasomesystem (42). A number of E3 ligases have been implicated in ARregulation by protein degradation. RNF6, an E3 ligase, is involved inregulating the AR protein and induces AR ubiquitination to increaseAR transcriptional activity (43). Speckle-type POZ protein (SPOP) is aubiquitin E3 ligase that regulates AR protein stability (26). Mutationsin SPOP cause failure of SPOP to interact with AR, leading tostabilization of AR in prostate cancer cells. Other E3 ligases thatregulate ubiquitination of AR include Siah2, PIAS1, MDM2, SKP2,and CHIP (24, 44–47). Apart from E3 ligases, some proteins affect ARstability. Deleted in breast cancer 1 (DBC1), for instance, binds andstabilizes AR (48). PC-1 reduces AR stability by enhancing theinteraction between AR and CHIP, resulting in degradation of ARby proteasomes (49). BMI1, a polycomb group protein (PcG), stabi-lizes AR by competitive inhibition of MDM2 and thereby decreasesproteasome degradation (50). Our present data suggest that KIF4Astabilizes AR by physical interaction with AR and inhibiting the CHIPbinding activity required for degradation.Wehypothesize that bindingKIF4A could mask the CHIP binding site on AR. However, themechanism bywhich KIF4A stabilizes AR needs to be further clarified.In summary, our results further reveal the regulatory networks con-ferring AR protein stability.

AR-V7, a major splice variant of AR, is constitutively expressed inrefractory prostate cancer and can drive prostate cancer progressioneven under enzalutamide treatment (51). AR-V7 mRNA is generatedfrom an alternative RNA splicing process that is enhanced under

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Figure 6.

KIF4A knockdown reverts endocrine therapy–resistant CRPC progression in vivo.A andB,Nudemice bearing C4-2-ENZ-Rwith stable KIF4A knockdown xenograftswere treatedwith vector control or enzalutamide (10mg/kg p.o.) for approximately 7weeks (n¼ 4). Tumor volumesweremeasured every 5 days. Data are shown asthe means � SD. C, Tumors were weighed after resection at the end of the experiment. D and E, mRNA levels of KIF4A, PSA, and TMPRSS2 from tumorswere determinedby qRT-PCR. IHC detection of the expression of KIF4A (F) and Ki67 (G) in each groupwas performed.H, Schematic representation of the KIF4A andAR/AR-V7 positive feedback loop in prostate cancer progression and enzalutamide resistance (� , P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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castration conditions (52). The AR-V7 protein is then stabilized andperform its functions in prostate cancer cells. However, little is knownabout the regulatory mechanism of the expression and stability of AR-V7 protein. Protein phosphatase-1 (PP-1) and AKT kinase can governAR-V7 phosphorylation status. PP-1 can phosphorylate AR-V7 atserine 213, preventing MDM2-mediated AR-V7 protein degradationby the ubiquitin–proteasome pathway (28). BMI1 can also stabilizeAR-V7 by physical interaction at the N-terminus of AR and therebyinhibit MDM2-mediated degradation (50). A recent study demon-strated that DBC1 could stabilize AR-V7 by facilitating the DNA-binding activity of AR-V7 and inhibiting CHIP-mediated ubiquitina-tion and degradation of AR-V7 by competing with CHIP for AR-V7binding (53). Our functional studies show that KIF4A binding to AR-V7 acts as a positive regulator for AR-V7 stability and functions byinhibiting the E3 ligase activity of CHIP. As a consequence, down-regulation ofKIF4Adecreases AR-V7protein levels. In addition, CHIPcan block the interaction of the AR/V7 co-chaperone protein, HSP70,with AR-V7 in enzalutamide- and abiraterone-resistant prostatecancer cells, thus leading to AR-V7 protein degradation (54). Thisregulatory pathway provides an in-depth understanding of the AR-V7regulatory network.

In the past few years, much effort has been made to improve ADT.The development of novel anti-androgenic drugs such as abirateroneand enzalutamide has resulted in promising effects (55, 56). However,resistance to these drugs, especially enzalutamide, occurs, and modelshave been developed to investigate the underlying mechanisms.Herein, we investigated the potential of KIF4A inhibition to improvecurrent prostate cancer treatment strategies. Functional experimentsshowed that KIF4A knockdown increases the inhibition of the growthof prostate cancer cells by ADT drugs, including bicalutamide andenzalutamide.Most importantly, using our enzalutamide-resistant cellmodel, the in vivo study revealed that targeting KIF4A significantlyinhibits CRPC tumor growth, and the use of enzalutamide in com-bination with KIF4A knockdown further attenuated tumor growthcompared with a single treatment.

In conclusion, the present data provide a strong theoretical basis forclinical KIF4A targeting either alone or in combination with ADTdrugs, such as bicalutamide or enzalutamide, to treat CRPC over-expressing AR/AR-V7 and to improve enzalutamide treatment inprostate cancer. Targeting of the KIF4A/AR axis could be used toreverse endocrine therapy resistance in CRPC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors’ ContributionsConception and design: Q. Cao, X. ZhangDevelopment of methodology: Q. Cao, C. Wang, X. ZhangAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): Q. Cao, H. Ruan, C. Wang, X. Yang, K. Wang, G. Cheng, T. Xu,W. Xiao, Z. Xiong, D. Zhou, X. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Chen, Q. Cao, Z. Song, H. Ruan, C. Wang, M. YangWriting, review, and/or revision of the manuscript: K. Chen, Q. Cao, H. Ruan,H. Yang, X. ZhangAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Q. Cao, Z. Song, L. Bao, D. Liu, X. ZhangStudy supervision: Q. Cao, H. Yang, X. Zhang

AcknowledgmentsThis work was supported by grant from National Natural Sciences Foundation of

China (No. 81672524, No. 81672528, No. 31741032, No. 81874090), Hubei ProvincialNatural Sciences Foundation of China (2018CFA038), Foundation of Health andFamily Planning Commission of Hubei Province in China (WJ2017M124), Inde-pendent Innovation Foundation of Huazhong University of Science and Technology(2016YXZD052, No. 118530309) and Clinical Research Physician Program of TongjiMedical College, Huazhong University of Science and Technology (No. 5001530015).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 2, 2019; revised May 25, 2019; accepted November 26, 2019;published first December 3, 2019.

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2020;26:1516-1528. Published OnlineFirst December 3, 2019.Clin Cancer Res   Qi Cao, Zhengshuai Song, Hailong Ruan, et al.   Resistance in Castration-resistant Prostate CancerTargeting the KIF4A/AR Axis to Reverse Endocrine Therapy

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