Targeting the KIF4A/AR Axis to Reverse Endocrine Therapy

Published OnlineFirst December 3, 2019; DOI: 10.1158/1078-0432.CCR-19-0396

CLINICAL CANCER RESEARCH | RESEARCH ARTICLE

Targeting the KIF4A/AR Axis to Reverse Endocrine Therapy Resistance in Castration-resistant Prostate Cancer 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-resistant KIF4A was positively correlated with androgen receptor (AR) levels. prostate cancer (CRPC) is often driven by constitutively active Patients with lower tumor KIF4A expression had improved overall androgen receptor (AR) or its V7 splice variant (AR-V7) and survival and disease-free survival. Mechanistically, KIF4A and AR commonly becomes resistant to endocrine therapy. The aim of form an auto-regulatory positive feedback loop in prostate cancer: this work is to evaluate the function of a kinesin protein, KIF4A, KIF4A binds AR and AR-V7 and prevents CHIP-mediated AR and in regulating AR/AR-V7 in prostate cancer endocrine therapy AR-V7 degradation; AR binds the promoter region of KIF4A and resistance. activates its transcription. KIF4A promotes castration-sensitive Experimental Design: We examined KIF4A expression in clin- and castration-resistant prostate cancer cell growth through AR- ical prostate cancer specimens by IHC. Regulated pathways were and AR-V7-dependent signaling. Furthermore, KIF4A expression investigated by qRT-PCR, immunoblot analysis, immunoprecipi- is upregulated in enzalutamide-resistant prostate cancer cells, and tation, and luciferase reporter and chromatin immunoprecipitation KIF4A knockdown effectively reverses enzalutamide resistance (ChIP) assays. A series of functional analyses were conducted in cell and enhances the sensitivity of CRPC cells to endocrine therapy. lines and xenograft models. Conclusions: These ﬁndings indicate that KIF4A plays an Results: Examination of the KIF4A protein and mRNA levels in important role in the progression of CRPC and serves as a crucial patients with prostate cancer showed that increased expression of determinant of the resistance of CRPC to endocrine therapy.

Introduction effective way to treat CRPC. Enzalutamide and abiraterone are novel androgen receptor (AR) inhibitors that are used to treat patients with Prostate cancer is one of the most common malignancies and ranks metastatic CRPC after chemotherapy (3). However, prostate cancer second among causes of male cancer-related death worldwide (1). can become therapy resistant through upregulation of androgen Androgens play an essential role in the growth of prostate cancer cells. synthesis, mutation, and abnormal expression of AR, AR splice Androgen deprivation therapy (ADT) is the main method for the variants (AR-Vs), and neuroendocrine transitions. Therefore, reveal- treatment of advanced prostate cancer. However, after an average of ing the complicated molecular mechanism of endocrine therapy 2 years of ADT treatment, the tumor often progresses from castration- resistance of prostate cancer is an urgent global concern. sensitive prostate cancer (CSPC) to castration-resistant prostate can- AR-V7 is the most common AR splice variant. Levels of AR-V7 are cer (CRPC; ref. 2). As the androgen receptor (AR) signaling pathway is low before treatment with enzalutamide or abiraterone but increase an important pathway for prostate cancer survival and progression, signiﬁcantly after progression on either agent (4). A large-scale clinical targeting AR expression and inhibiting its activity has become an trial found that AR-V7 expression in circulating tumor cells of patients with prostate cancer was associated with shorter survival and was also 1Department of Urology, Union Hospital, Tongji Medical College, Huazhong closely related to resistance to endocrine therapy drugs (5). Increased University of Science and Technology, Wuhan, China. 2Insititute of Urology, AR-V7 expression may represent one of the mechanisms of resistance Union Hospital, Tongji Medical College, Huazhong University of Science and to these agents. However, the precise role of AR-V7 in the progression Technology, Wuhan, China. 3Department of Pathology, Union Hospital, Tongji of CRPC is still unclear. Medical College, Huazhong University of Science and Technology, Wuhan, KIF4A, a member of the kinesin superfamily (KIFs), is located on China. 4Department of Pathogenic Biology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, China. chromosome Xq13.1 and encodes a protein consisting of 1232 amino acids. KIF4A is involved in multiple cellular activities, particularly Note: Supplementary data for this article are available at Clinical Cancer spindle formation and centrosome assembly in mitosis (6), chromo- Research Online (http://clincancerres.aacrjournals.org/). some concentration and separation (7), and DNA damage repair (8). Q. Cao, Z. Song, and H. Ruan contributed equally to this article. KIF4A plays a critical role in a variety of tumors, such as lung Corresponding Authors: Ke Chen, Huazhong University of Science and Tech- cancer (9), oral cancer (10), liver cancer (11), and colorectal carcino- nology, Jiefang Avenue No. 1277, Wuhan 430022, China. Phone: 027-85351625; ma (12). Estrogen regulates the expression of KIF4A through the E-mail: [email protected]; Xiaoping Zhang, [email protected]; and estrogen receptor (ERa) in breast cancer, whereas ANCCA, a cor- Hongmei Yang, [email protected] egulator of ERa, is involved in the regulation of KIF4A (13). KIF4A is Clin Cancer Res 2020;XX:XX–XX abundantly expressed in prostate cancer, and this expression is doi: 10.1158/1078-0432.CCR-19-0396 associated with poor prognosis in patients with prostate cancer (14). ﬁ 2019 American Association for Cancer Research. These ndings suggest that KIF4A may have functions that contribute

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cells were stably passaged three times. The drug concentration was Translational Relevance then increased, and the culture was continued. The concentration Prostate cancer often progresses to castration-resistant prostate gradients of enzalutamide were 0.4, 0.8, 1, 2, 4, 8, 10, 20, and 40 mmol/L. cancer (CRPC) and becomes resistant to endocrine therapy, which After 4 months of induction, the stable enzalutamide-resistant cell line is commonly driven by the androgen receptor (AR) or its V7 splice was obtained and named C4-2-ENZ-R. variant (AR-V7). Reducing the high rates of progression of this disease is an urgent unmet clinical need. Here, we report a protein Transfection (KIF4A) that is upregulated and transcriptionally regulated by AR The pLent-GFP shRNA targeting KIF4A and pENTER-Flag KIF4A in prostate cancer. Overexpression of KIF4A signiﬁcantly reduces were purchased from Vigenebio. GV298-shRNA targeting CHIP, AR and AR-V7 degradation. Inhibition of KIF4A blocks cancer MYC-CHIP, eGFP-AR, and HA-AR-V7 were purchased from Gen- progression and reverses enzalutamide resistance. We speculate echem. pCDNA3.1-3 Myc-Ub was purchased from miaolingbio. that targeting the KIF4A/AR axis could be used to increase the Brieﬂy, 3 105 cells were seeded in six-well plates and transfected with sensitivity of CRPC cells to endocrine therapy. KIF4A is critical for 2 mg of plasmid using Lipofectamine 2000 (Invitrogen) according to CRPC progression and endocrine therapy resistance. the manufacturer's procedure.

Lentiviral constructs Lentivirus was packaged by cotransfection of the constructs with the to prostate cancer progression, but whether KIF4A can regulate the AR plasmids pMD2.G, pRRE, and pRSV/REV in 293T cells. The super- signaling pathway remains unclear. natants collected at 72 and 96 hours after transfection were filtered by a In this study, we demonstrate for the first time that KIF4A is 0.45-mm filter, and the filtrates were then added to prostate cancer cells. positively correlated with AR levels in prostate cancer. Patients with After being selected with puromycin (1 mg/mL), the protein expression higher KIF4A expression have worse survival. AR binds to the level was verified by Western blotting. promoter region of KIF4A and activates its transcription. In addition, KIF4A binds and stabilizes the AR and AR-V7 proteins to modulate IHC and immunofluorescence the AR pathway in prostate cancer cells. Furthermore, our results IHC was conducted as described previously (15). Tissues were indicate that KIF4A promotes human CSPC and CRPC cell growth sequentially fixed in formalin, dehydrated, and embedded in paraffin. through AR-dependent signaling. Most importantly, we show that Then, IHC was conducted by incubating the tissue sections with depletion of KIF4A reverses enzalutamide resistance and enhances the primary antibodies including KIF4A, PSA, or Ki67 overnight at 4C. sensitivity of CRPC cells to endocrine therapy. Subsequently, after washing three times with PBS, the sections were incubated with the secondary antibody (1:200; GB23303; Servicebio, Inc.) at room temperature for 2 hours. Immunofluorescence (IF) was Materials and Methods performed as described previously (16). Cells were fixed in 4% Tissue microarray paraformaldehyde, permeated by 0.3% Triton X-100, and blocked A human prostate cancer tissue microarray (TMA) was purchased with 3% BSA for 1 hour at 37C, followed by incubation with primary from Alenabio (PR1921b). All detailed clinical information including KIF4A and AR antibodies. pathology, diagnosis, stage, Gleason scores, and PSA level is freely available on the Web (http://www.alenabio.com/public/details? ChIP assays productId¼59058&searchText¼). The following antibodies were The ChIP assay was performed according to the protocol of the used: anti-KIF4A (1:50; Proteintech) and anti-AR (1:100; sc-816; Santa ChIP Assay Kit (CST). 22Rv1 and C4-2 cells were cultured in 10-cm Cruz Biotechnology). The TMA was evaluated using by scoring the dishes. Then, the chromatin in the cells was cross-linked by adding staining intensity in a range of 0 to 3 (0 ¼ absent, 1 ¼ weak, 2 ¼ formaldehyde to a final concentration of 1% at room temperature for intermediate, 3 ¼ strong) and the percentage of positively stained cells 10 minutes, followed by washing twice with 2 mL of ice-cold PBS in a range of 0 to 4 (0 5%, 1 ¼ 5%–25%, 2 ¼ 25%–50%, 3 ¼ 50%–75%, containing protease inhibitors, lysis in ChIP lysis buffer, and sonica- 4 75%). The two scores were multiplied to obtain an immunore- tion to completely lyse nuclei. The digested, cross-linked chromatin activity score (IRS) ranging from 0 to 12. The IHC data were evaluated was diluted with ChIP buffer. A 10-mL sample of the diluted chromatin by two blinded pathologists. was removed as a 2% input sample. The remaining 500 mL of diluted chromatin was incubated with anti-AR or anti-IgG at 4C overnight Cell culture and drug treatment with rotation. After elution of chromatin, reversal of cross-links and 293T, LNCaP, C4-2, and 22Rv1 cells were purchased from ATCC. DNA purification, qRT-PCR was performed to amplify the potential 293T cells were maintained in DMEM. LNCaP, C4-2, and 22Rv1 cells AR binding sites on the KIF4A promoter region. were maintained in RPMI1640 medium. All media were supplemented with 10% FBS and 1% penicillin–streptomycin solution. All cells were Cell proliferation assay 3 cultured at 37 C in an incubator containing 5% CO2. Drug concen- A total of 3 10 cells were added to each well of a 96-well plate. The trations (unless otherwise indicated) were enzalutamide (20 mmol/L), cell proliferation rate was determined using Cell Counting Kit-8 bicalutamide (10 mmol/L), DHT (10 nmol/L), and cycloheximide (CCK-8; Dojindo) every 24 hours. Briefly, 10 mL of CCK-8 solution (CHX; 10 mmol/L). was added to each well. After 4 hours, the absorbance was measured at 450 nm. Establishment of the enzalutamide-resistant cell line C4-2-ENZ-R Immunoprecipitation and Western blot analysis The starting treatment concentration of enzalutamide in the paren- The collected cells were lysed in RIPA buffer containing protease tal C4-2 cell culture medium was 0.2 mmol/L. At this concentration, the inhibitor. The lysate was kept on ice for 30 minutes and centrifuged at

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12,000 rpm for 5 minutes. Then, the supernatant was collected in two growth of advanced prostate cancer (both CSPC and CRPC) depends parts: a small amount of lysate was taken as input, and the remaining on AR signaling. In this study, by screening four independent prostate lysate was incubated with 2 mg of the corresponding antibody and cancer gene sets (TCGA, Arredouani, Grasso, and Varambally; 30 mL of agarose beads at 4C overnight. The immune complexes were refs. 18–20), three genes (KIF4A, BIRC5, ATP8A2) that correlated centrifuged at 3,000 rpm for 2 minutes. The supernatant was carefully with AR were selected on the basis of their significant differences in discarded, and the agarose beads were washed three to four times with expression in prostate cancer tissues compared with normal tissues 200 mL of lysis buffer. Finally, 40 mL of RIPA lysate and 10 mL of loading (logFC > 1.5, P < 0.05; Fig. 1A; Supplementary Fig. 1A). Furthermore, buffer were added to the beads and boiled for 10 minutes. For Western Kaplan–Meier survival analysis and log-rank tests were conducted to blotting, total proteins were separated by 10% SDS-PAGE and trans- determine whether the disease-free survival (DFS) and overall survival ferred to polyvinylidene fluoride (PVDF) membranes. The PVDF (OS) of patients were associated with KIF4A, BIRC5, and ATP8A2 membranes were blocked and then incubated with primary antibodies. expression in tumors. As shown in Fig. 1B, only patients with high The antibodies used in Western blotting were KIF4A (ab124903; levels of KIF4A mRNA expression had worse DFS (P ¼ 0.0002) and OS Abcam), AR (sc-7305; Santa Cruz Biotechnology), AR-V7 (ab198394; (P ¼ 0.033) than those with low KIF4A expression. Higher BIRC5 or Abcam), Flag (20543-1-AP; ProteinTech), MYC (60003-2-Ig; Protein- ATP8A2 expression did not appear to affect OS in prostate cancer Tech), HA (66006-2-Ig; ProteinTech), eGFP (66002-1-Ig; Protein- (Fig. 1B). A comprehensive consideration of these results led us to Tech), and b-actin (AC026; ABclonal). The proteins were visualized focus on KIF4A for further study. using ChemiDoc-XRsþ (Bio-Rad). Previous studies have shown that increased KIF4A mRNA expression is a potential prognostic factor in prostate cancer (14). KIF4A may RNA isolation and RT-PCR play a key role in prostate cancer progression. Next, to further confirm Total RNA was extracted from cells using TRIzol reagent (Invitro- the previous findings and the interaction between KIF4A and AR, we gen) and then reverse transcribed by the PrimeScript RT reagent Kit examined KIF4A and AR protein levels with IHC staining using a with gDNA Eraser (Takara). Real-time PCR was conducted using tissue array containing 160 cases of human prostate cancer and 32 Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fermentas) on adjacent normal tissue specimens. Similar to a previous result, we the ABI-7500 qRT-PCR system. The primers used for qRT-PCR are observed that KIF4A was positively correlated with AR expression in listed in Supplementary Table S1. human normal prostate tissues (r ¼ 0.37, P < 0.05) and human prostate cancer tissues (r ¼ 0.55, P < 0.0001; Fig. 1C). KIF4A protein levels were Luciferase assays significantly higher in the prostate cancer tissues than in the normal Cells were seeded in a 24-well plate at 70% confluence and tran- tissues and were related to tumor stage and PSA levels (Supplementary siently transfected with 0.8 mg of expression vector plasmids and 0.4 mg Figs. S1B–S1D). Taken together, these results suggested a functional of promoter reporter plasmids. The fluorescence intensity was mea- interaction between KIF4A and AR during prostate cancer sured after 48 hours. The luciferase activity of the gene promoter was progression. normalized to Renilla luciferase activity as an internal standard control. The plasmids of KIF4A-luc and mutated KIF4A-luc were AR transcriptionally activates KIF4A in prostate cancer designed and synthesized by Genechem. AR acts as a transcription factor to regulate the expression of its downstream target genes and promote prostate cancer progres- Tumor xenograft study sion (21). Given that KIF4A and AR are positively correlated in Three- to 4-week-old castrated male nude mice were purchased prostate cancer, there is a possibility that AR may transcriptionally from Beijing Vital River Laboratory Animal Technology Co., Ltd. regulate KIF4A expression. To explore this possibility, LNCaP cells Approximately 5 106 C4-2-ENZ-R cells with knockdown of KIF4A were treated with DHT for 12 hours. As shown in Fig. 2A,KIF4A or control cells suspended in 100 mL of serum-free medium were protein levels were elevated in LNCaP cells upon androgen treat- injected subcutaneously into the armpit on the right side of the mice. ment. To confirm that this effect of androgen occurred through Tumor volumes (V) were measured every 5 days based on measure- AR, we directly modulated the levels of AR in PC3 and C4-2 cells. ments of length (L) and width (W) and calculated as V ¼ (L W2)/2. As expected, the changes in KIF4A protein levels were in accor- When the tumors reached 100 to 200 mm3, the mice were randomized dance with the change in AR when we overexpressed AR in equally, treated with vehicle or enzalutamide (10 mg/kg) twice per PC3 cells and C4-2 cells or downregulated AR in C4-2 cells week, and sacrificed approximately 40 days later. (Fig. 2B–D). These results indicated that KIF4A is elevated by AR activation. Statistical analysis In the nucleus, AR binds to specific DNA sequences termed All statistical analyses were performed using Prism 5.0 (GraphPad) androgen response elements (AREs) in the promoter regions of target and SPSS 22.0 (IBM Corporation). All in vitro experiments wererepeated genes, such as prostate-specific antigen (PSA) and transmembrane three times. All data are presented as the mean SD. Survival infor- protease serine 2 (TMPRSS2; refs. 22, 23). Next, we identified a mation was verified by Kaplan–Meier analysis and compared using the canonical ARE 531 base pairs upstream of the KIF4A transcription log-rank test. A two-tailed unpaired Student t test was used to determine start site (Fig. 2E). We then cloned luciferase reporter constructs into a the P values, which were considered significant at less than 0.05. pGL3 plasmid containing the putative ARE (ARE-luc) or its mutant (AREmut-luc; Fig. 2E). As expected, ectopic AR expression significantly activated ARE-luc activity with or without DHT treatment. Results However, no activation of AREmut-luc activity was observed upon The expression level of KIF4A is positively related to AR in overexpression of AR (Fig. 2F). To further verify the binding sites on prostate cancer the promoter region, we analyzed and designed primers covering AR is a ligand-activated transcription factor that plays critical roles different sequences of interest in the promoter of KIF4A, which were in normal prostate development and prostate tumorigenesis (17). The denoted 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 cancer datasets. Bottom, In four independent prostate cancer databases, the KIF4A/AR correlation was determined by Pearson correlation. B (ﬁrst column), Kaplan–Meier DFS curves of patients with prostate cancer based on BIRC5, KIF4A, and ATP8A2 expression. (second column) Kaplan–Meier OS curves of patients with prostate cancer based on BIRC5, KIF4A, and ATP8A2 expression. C, A prostate tumor microarray containing 160 prostate cancer tissues and 32 adjacent normal tissues was stained with anti-KIF4A and AR antibodies. Spearman rank correlation analysis identiﬁed positive correlations between KIF4A and AR in the prostate cancer tumor samples and adjacent normal samples.

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Figure 2. AR directly activates KIF4A transcription. A, Immunoblot detection of KIF4A levels after 48 hours of DHT treatment in LNCaP cells. B, Immunoblot detection of KIF4A levels 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 the KIF4A promoter and its mutant (AREmut). The þ1 denotes the transcription initiation site. F, Luciferase assays performed using the control pGL3, ARE, and AREmut constructs 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 human KIF4A promoter. P1-6 show the regions of the KIF4A promoter detected by the paired primers. ChIP analysis of AR binding at the P1, P2, P3, P4, P5, and P6 loci in C4-2 cells (H) and 22RV1 cells (I)(, P < 0.05; , P < 0.01, , P < 0.001).

significant increase in AR recruitment to the P5 region compared with Depletion of KIF4A decreases AR protein levels and inhibits the the IgG control in C4-2 and 22Rv1 cells (Fig. 2H–I). Furthermore, AR AR signaling pathway recruitment to the P5 region was significantly increased upon DHT To explore the role of KIF4A in prostate cancer, we knocked down treatment compared with the IgG control in castration-sensitive KIF4A in C4-2 cells using KIF4A-specific shRNA and observed that LNCaP cells (Supplementary Fig. S2A). These results demonstrated the shRNA decreased the expression levels of AR (Fig. 3A). Further- that AR directly activates KIF4A transcription in prostate cancer. more, the transcript levels of PSA and TMPRSS2, AR canonical target

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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 after KIF4A 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 cells were immunoprecipitated with anti-AR antibodies and blotted with the indicated antibodies. D, Immunoblot detection of AR and AR-V7 protein levels in 22RV1 cells after KIF4A knockdown. E, Real-time qPCR analysis of AR-V7 in 22Rv1 cells upon KIF4A knockdown. F, Whole-cell lysates of 22Rv1 cells were immunoprecipitated with anti-KIF4A antibodies and blotted with the indicated antibodies. 293 cells were transiently transfected with Flag-KIF4A and eGFP-AR for 2 days, and whole-cell lysates were immunoprecipitated with anti-Flag (G) or eGFP (H) antibodies and blotted with the indicated antibodies. I, 293 cells were transiently transfected with Flag-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 of AR-V7. Next, the potential role of KIF4A in AR and AR-V7 ubiqui- AR was not downregulated by KIF4A knockdown (Fig. 3B). AR-Vs tination was examined. In 293T cells, CHIP-dependent ubiquitination lacking the ligand-binding domain, such as AR-V7, have been impli- of AR was enhanced by the overexpression of CHIP protein (Fig. 4I), cated in the pathogenesis of CRPC and in mediating resistance to new but KIF4A expression rescued CHIP-mediated AR ubiquitination endocrine therapies that target the androgen axis (5). Next, we (Fig. 4I). Indeed, the amount of pulled down CHIP protein was knocked down KIFA in 22Rv1 cells, another CRPC cell line expressing significantly decreased by KIF4A expression (Fig. 4I). Similarly, high levels of AR-V7. As shown in Fig. 3D, the expression levels of AR AR-V7 ubiquitination was inhibited by KIF4A expression, and the and AR-V7 were also decreased by KIF4A knockdown in 22Rv1 cells. binding of CHIP protein to AR-V7 was also reduced (Fig. 4J). However, the mRNA levels of AR and AR-V7 were not decreased Meanwhile, CHIP-dependent ubiquitination of AR and AR-V7 was (Fig. 3E). Because the loss of KIF4A decreased AR and AR-V7 protein decreased by the deletion of endogenous CHIP protein in 293T cells. levels and inhibited AR signaling without downregulation of AR and And additional KIF4A expression further reduced the ubiquitination AR-V7 transcript levels, we hypothesized that KIF4A regulates the AR level of AR and AR-V7 (Supplementary Figs. S5A and S5B). Taken and AR-V7 proteins and their functions through a posttranslational together, these results suggest that KIF4A inhibits CHIP-mediated modification. To test this possibility, we first performed immunopre- ubiquitination of AR and AR-V7 by blocking the interaction between cipitation experiments. AR was immunoprecipitated from C4-2 cell AR or AR-V7 and CHIP. lysates with an anti-AR antibody and analyzed for KIF4A binding by Western blot analysis. The results showed that endogenous KIF4A KIF4A knockdown reverts endocrine therapy–resistant CRPC coimmunoprecipitated with AR (Fig. 3C). KIF4A interacted with progression in vitro AR-V7 proteins in 22Rv1 cells (Fig. 3F; Supplementary Fig. S2B). To Given that KIF4A interacts with AR and inhibits its degradation, we prove their direct interaction, Flag-KIF4A and eGFP-AR or HA-AR- assumed that the inhibition of cell growth by silencing KIF4A is V7 were coexpressed in 293T cells. Flag-KIF4A was immunoprecipi- dependent on AR. To further investigate this possibility, we transfected tated from cell lysates with an anti-Flag antibody, and eGFP-AR was AR plasmids in LNCaP and C4-2 cells in which KIF4A was silenced. pulled down and detected by Western blot analysis (Fig. 3G). When we The results showed that the inhibition of cell proliferation induced by used an anti-eGFP antibody to precipitate eGFP-AR, Flag-KIF4A was KIF4A knockdown was reversed by transient overexpression of AR in also observed in the immunoprecipitate by using an anti-Flag antibody LNCaP-stable cell lines (Fig. 5A). Similarly, overexpression of AR (Fig. 3H). Furthermore, HA-AR-V7 was pulled down by exogenous abolished the inhibition of cell proliferation induced by downregula- KIF4A (Fig. 3I). Moreover, IF assays confirmed that KIF4A and AR tion of KIF4A in C4-2 cells (Fig. 5B). Also, there was no growth had obvious colocalization (Fig. 3J–K). inhibitory effect upon KIF4A knockdown in AR-negative cells, PC3 and DU145 (Supplementary Figs. S6A and S6B). These results dem- KIF4A stabilizes the AR and AR-V7 proteins via competitive onstrated that AR plays a critical role in KIF4A-regulated prostate inhibition of CHIP-mediated ubiquitination cancer cell growth. On the basis of the above results, we expected that KIF4A controls Bicalutamide, a pharmaceutical drug commonly used as an anti- the function of AR and AR-V7 by an additional mechanism that may androgen therapy to treat recurrent prostate cancer, is a competitive involve regulation of AR and AR-V7 expression at the posttransla- inhibitor of AR. Enzalutamide, a novel AR signaling inhibitor, blocks tional level. To test this hypothesis, we constructed the enzalutamide- the growth of CRPC in cellular model systems and was shown in a resistant cell line C4-2-ENZ-R (Supplementary Fig. S3). We knocked clinical study to increase survival in patients with metastatic CRPC. down KIF4A expression by using shRNA in C4-2-ENZ-R cells and The rapid development of therapy resistance in patients with prostate treated cells with CHX to block de novo protein synthesis. We found cancer receiving bicalutamide and enzalutamide treatment is becom- that downregulation of KIF4A significantly accelerated the degrada- ing a major clinical challenge (30, 31). The sustained expression of tion of AR (Fig. 4A). In addition, the half-life of AR and AR-V7 protein AR and AR-V7 is a hallmark of endocrine therapy resistance in was shortened upon KIF4A knockdown (Fig. 4C; Supplementary prostate cancer. To further investigate whether KIF4A contributes Fig. S4A). Similarly, we overexpressed KIF4A in 22Rv1 cells and to bicalutamide and enzalutamide resistance in prostate cancer cells, treated cells with CHX for different durations. The results showed we examined the sensitivity of prostate cancer cells to these drugs that overexpression of KIF4A reduced the rate of degradation of under different conditions through CCK8 assays. As shown in Fig. 5C, AR-V7 and AR and prolonged the half-life of the AR-V7 and AR we observed that knockdown of KIF4A enhanced inhibition of growth proteins (Fig. 4B and D; Supplementary Fig. S4B). In addition, KIF4A by bicalutamide in castration-sensitive LNCaP cells. Similarly, down- overexpression decreased the ubiquitination levels of endogenously regulation of KIF4A increased inhibition of growth by enzalutamide expressed AR and AR-V7 in C4-2-ENZ-R and 22Rv1 cells (Fig. 4E in castration-resistant C4-2 cells and enzalutamide-resistant C4-2- and F), suggesting that KIF4A contributes to AR and AR-V7 stability ENZ-R cells (Fig. 5D–E). Overexpression of KIF4A alleviated the by blocking its ubiquitination. inhibition of growth by enzalutamide in C4-2 cells (Fig. 5F). Fur- Several studies have reported that AR levels are regulated by the thermore, we have established the stably expressed additional AR cells ubiquitin-proteasome degradation pathway (24–27). Several ubiquitin named PC3-AR. As shown in Supplementary Fig. S6E, we observed E3 ligases have also been implicated in AR-V7 degradation (28, 29). It that knockdown of KIF4A enhanced inhibition of growth by Enza- appears that only CHIP E3 ligase interacts with both the AR and AR- lutamide in AR-positive PC3-AR cells. But knockdown of KIF4A did V7 proteins. This finding led us to expect that CHIP is involved in not enhance inhibition of growth by Enzalutamide in AR-negative cells KIF4A depletion-induced AR and AR-V7 degradation. First, the (Supplementary Figs. S6C and S6D). Finally, Enzalutamide did not effects of KIF4A on CHIP-mediated degradation were examined. In inhibit the growth of C4-2-ENZ-R cells, and overexpression of KIF4A 293T cells, the coexpression of KIF4A and CHIP resulted in significant had same effect (Supplementary Fig. S6F). These results indicated that increases in the protein levels of AR and AR-V7 compared with inhibition of KIF4A potentiates the effects of enzalutamide and overexpression of CHIP alone (Fig. 4G and H). This result suggested bicalutamide in prostate cancer cells. KIF4A knockdown reverts that KIF4A might affect CHIP-mediated ubiquitination of AR and endocrine therapy-resistant CRPC progression.

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Figure 4. KIF4A stabilizes the AR and AR-V7 proteins via competitive inhibition of CHIP-mediated ubiquitination. A, C4-2-ENZ-R cells were transiently transfected with shNC or sh-KIF4A-1 or sh-KIF4A-2 and then treated with 10 mmol/L CHX, and total cell lysates were collected at 0, 4, 8, and 12 hours after treatment and subjected to Western blot analysis. B, 22Rv1 cells were transiently transfected with vector or Flag-KIF4A and then treated with 10 mmol/L CHX, and total cell lysates were collected at 0, 4, 8, and 12 hours after treatment and subjected to Western blot analysis. C, The half-life of AR was calculated for C4-2-ENZ-R cells. D, The half-life of AR-V7 was calculated for 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, and blotted with the indicated antibodies. F, 22Rv1 cells were transiently transfected with Flag-KIF4A for 2 days. Total cell lysates were collected, immunoprecipitated with 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 for 2 days, and whole-cell lysates were collected and subjected to Western blot analysis. H, 293T cells were cotransfected with eGFP-AR with or without Flag-KIF4A and MYC-CHIP for 2 days, and whole-cell lysates were collected and subjected to Western blot analysis. I, 293T cells were cotransfected with HA-AR-V7 with or without Flag-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, 293T cells 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 eGFP antibody 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 AR overexpression. Cells were analyzed for cell proliferation by the CCK8 assay. C, LNCaP cells with stable KIF4A knockdown were treated with 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/L enzalutamide, respectively. Cell proliferation was determined by the CCK8 assay. F, C4-2 cells were transiently transfected with Flag-KIF4A and then treated with 20 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 CRPC genes. A binding site at 13 kb upstream of the TMPRSS2 transcription progression in vivo start site is necessary for AR regulation of the TMPRSS2 gene (38). A Our previous studies showed that KIF4A knockdown effectively group confirmed that the upstream sequence of the PSA promoter reverts endocrine therapy resistance in CRPC cells. To further confirm (539-320 bp) is necessary for androgen regulation. AR regulates the this function of KIF4A, we transplanted C4-2-ENZ-R cells with stable expression of PSA by interacting with the 50-AGAACAgcaAGTGCT- knockdown of KIF4A into castrated male SCID mice. The mice were 30 sequence (39). Here, we identified a half ARE site in the promoter then treated with enzalutamide (10 mg/kg) twice a week. As shown region of the KIF4A gene. AR ChIP analysis revealed that KIF4A might in Fig. 6A–C, there was no effect of enzalutamide treatment alone on be upregulated directly via AR binding to the KIF4A promoter. KIF4A the tumor growth and tumor weight of the mice bearing tumors expression is enhanced by AR upon androgen treatment. These facts without KIF4A knockdown. By contrast, KIF4A knockdown induced strongly indicate that KIF4A is a direct AR downstream target that is inhibition of tumor growth and tumor weight by enzalutamide upregulated by androgens. treatment in C4-2-ENZ-R tumors, indicating that KIF4A knockdown Infinite proliferation of cancer cells is a hallmark of progression of can restore enzalutamide treatment sensitivity. The knockdown effi- prostate cancer (40). Previous studies have showed that KIF4A is ciency of KIF4A was detected by qRT-PCR. The mRNA level of KIF4A involved in regulating the proliferation of cancer cells (11). Our data was significantly downregulated upon stable KIF4A knockdown revealed that KIF4A depletion inhibits prostate cancer cell prolifer- (Fig. 6D). Similarly, the protein level of KIF4A was verified in tumor ation. More importantly, the regulation of prostate cancer cell prolif- xenografts by IHC assays (Fig. 6F). Next, we determined the expres- eration by KIF4A is dependent on AR. These results reveal a novel sion of the AR target genes PSA and TMPRSS2 in tumor tissues. mode of AR-dependent signaling that is involved in regulating cell Similar results were observed. As shown in Fig. 6E, there was no effect biological behavior. The importance of the involvement of KIF4A in of enzalutamide treatment alone on PSA and TMPRSS2 expression in oncogenic regulation was reinforced by our finding that KIF4A bound mice bearing tumors without KIF4A knockdown. By contrast, KIF4A with AR/AR-V7 and inhibited their ubiquitination and degradation. knockdown induced inhibition of PSA and TMPRSS2 expression by The E3 ligase CHIP forms a complex with AR/AR-V7 and participates enzalutamide treatment in C4-2-ENZ-R tumors (Fig. 6E). Finally, in protein ubiquitination. KIF4A blocked CHIP and AR/AR-V7 IHC staining of tissue sections of tumors from the four groups was complex formation, leading to AR/AR-V7 protein stabilization. Intro- performed to evaluate the protein expression of KIF4A, PSA, and Ki67. ducing KIF4A protein restored CHIP-mediated AR/AR-V7 degrada- The representative images show that the combination of KIF4A tion. Our findings on complex formation by KIF4A and CHIP/AR or knockdown and enzalutamide usage resulted in weaker staining CHIP/AR-V7 are critical as this mechanism may represent a general intensity of PSA and Ki67 compared with the KIF4A knockdown- ubiquitin–proteasome mechanism for the regulation of AR/AR-V7 only group, indicating that deletion of KIF4A enhanced the inhibition protein stability that may be involved in endocrine therapy resistance of AR activity and proliferation ability by enzalutamide in C4-2-ENZ- in prostate cancer progression. R tumors (Fig. 6G; Supplementary Fig. S7A and S7B). Taken together, The upregulation of AR protein is a hallmark of CRPC and seems to these results suggested that KIF4A knockdown effectively reversed be an adaptive response to ADT (41). An increase in AR protein levels endocrine therapy resistance in CRPC and that KIF4A is a novel is observed in most refractory cases (41). Several mechanisms account therapeutic target for CRPC (Fig. 6H). for increased AR levels because some factors confer stability of AR in CRPC or, importantly, in therapy-resistant prostate cancer. There is evidence that AR proteins are degraded by the ubiquitin–proteasome Discussion system (42). A number of E3 ligases have been implicated in AR The KIF proteins are involved in many essential cellular biological regulation by protein degradation. RNF6, an E3 ligase, is involved in functions, including mitosis and transport of intracellular vesicles and regulating the AR protein and induces AR ubiquitination to increase organelles (32). Increasing evidence indicates that KIF members AR transcriptional activity (43). Speckle-type POZ protein (SPOP) is a participate in the genesis and development of human cancers (33–36). ubiquitin E3 ligase that regulates AR protein stability (26). Mutations KIF4A, a member of the KIF family, has been reported to be abnor- in SPOP cause failure of SPOP to interact with AR, leading to mally expressed and to play a critical role in the progression of various stabilization of AR in prostate cancer cells. Other E3 ligases that solid cancers (9–13). However, the expression and function of KIF4A regulate ubiquitination of AR include Siah2, PIAS1, MDM2, SKP2, in prostate cancer have not been fully researched. and CHIP (24, 44–47). Apart from E3 ligases, some proteins affect AR In this study, we demonstrated that KIF4A was upregulated in stability. Deleted in breast cancer 1 (DBC1), for instance, binds and prostate cancer tissues compared with paired normal tissues. More- stabilizes AR (48). PC-1 reduces AR stability by enhancing the over, elevated KIF4A expression was significantly correlated with interaction between AR and CHIP, resulting in degradation of AR several clinicopathologic parameters, such as tumor stage and PSA by proteasomes (49). BMI1, a polycomb group protein (PcG), stabi- levels. In addition, high KIF4A expression was associated with poorer lizes AR by competitive inhibition of MDM2 and thereby decreases OS and DFS of patients with prostate cancer, which was partially proteasome degradation (50). Our present data suggest that KIF4A consistent with a previous study in prostate cancer (14). These findings stabilizes AR by physical interaction with AR and inhibiting the CHIP revealed that KIF4A plays an essential role in the progression of binding activity required for degradation. We hypothesize that binding prostate cancer and could act as a potential clinical prognostic KIF4A could mask the CHIP binding site on AR. However, the indicator for patients with prostate cancer. mechanism by which KIF4A stabilizes AR needs to be further clarified. The AR-mediated androgen signaling pathway plays an important In summary, our results further reveal the regulatory networks con- role in the development of prostate cancer (22). Upon androgen ferring AR protein stability. binding, AR dissociates from heat shock proteins and translocates to AR-V7, a major splice variant of AR, is constitutively expressed in the nucleus. The AR dimer then binds to AREs in the promoter regions refractory prostate cancer and can drive prostate cancer progression of androgen-dependent genes, thereby activating/inhibiting their even under enzalutamide treatment (51). AR-V7 mRNA is generated transcription (37). PSA and TMPRSS2 are two canonical AR target from 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 and B, Nude mice bearing C4-2-ENZ-R with stable KIF4A knockdown xenografts were treated with vector control or enzalutamide (10 mg/kg p.o.) for approximately 7 weeks (n ¼ 4). Tumor volumes were measured every 5 days. Data are shown as the 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 tumors were determined by qRT-PCR. IHC detection of the expression of KIF4A (F) and Ki67 (G) in each group was performed. H, Schematic representation of the KIF4A and AR/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 and In conclusion, the present data provide a strong theoretical basis for perform its functions in prostate cancer cells. However, little is known clinical KIF4A targeting either alone or in combination with ADT about the regulatory mechanism of the expression and stability of AR- drugs, such as bicalutamide or enzalutamide, to treat CRPC over- V7 protein. Protein phosphatase-1 (PP-1) and AKT kinase can govern expressing AR/AR-V7 and to improve enzalutamide treatment in AR-V7 phosphorylation status. PP-1 can phosphorylate AR-V7 at prostate cancer. Targeting of the KIF4A/AR axis could be used to serine 213, preventing MDM2-mediated AR-V7 protein degradation reverse endocrine therapy resistance in CRPC. by the ubiquitin–proteasome pathway (28). BMI1 can also stabilize AR-V7 by physical interaction at the N-terminus of AR and thereby Disclosure of Potential Conflicts of Interest inhibit MDM2-mediated degradation (50). A recent study demon- No potential conflicts of interest were disclosed. strated that DBC1 could stabilize AR-V7 by facilitating the DNA- ’ binding activity of AR-V7 and inhibiting CHIP-mediated ubiquitina- Authors Contributions tion and degradation of AR-V7 by competing with CHIP for AR-V7 Conception and design: Q. Cao, X. Zhang Development of methodology: Q. Cao, C. Wang, X. Zhang binding (53). Our functional studies show that KIF4A binding to AR- Acquisition of data (provided animals, acquired and managed patients, provided V7 acts as a positive regulator for AR-V7 stability and functions by facilities, etc.): Q. Cao, H. Ruan, C. Wang, X. Yang, K. Wang, G. Cheng, T. Xu, inhibiting the E3 ligase activity of CHIP. As a consequence, down- W. Xiao, Z. Xiong, D. Zhou, X. Zhang regulation of KIF4A decreases AR-V7 protein levels. In addition, CHIP Analysis and interpretation of data (e.g., statistical analysis, biostatistics, can block the interaction of the AR/V7 co-chaperone protein, HSP70, computational analysis): K. Chen, Q. Cao, Z. Song, H. Ruan, C. Wang, M. Yang with AR-V7 in enzalutamide- and abiraterone-resistant prostate Writing, review, and/or revision of the manuscript: K. Chen, Q. Cao, H. Ruan, H. Yang, X. Zhang cancer cells, thus leading to AR-V7 protein degradation (54). This Administrative, technical, or material support (i.e., reporting or organizing data, regulatory pathway provides an in-depth understanding of the AR-V7 constructing databases): Q. Cao, Z. Song, L. Bao, D. Liu, X. Zhang regulatory network. Study supervision: Q. Cao, H. Yang, X. Zhang In the past few years, much effort has been made to improve ADT. The development of novel anti-androgenic drugs such as abiraterone Acknowledgments and enzalutamide has resulted in promising effects (55, 56). However, This work was supported by grant from National Natural Sciences Foundation of resistance to these drugs, especially enzalutamide, occurs, and models China (No. 81672524, No. 81672528, No. 31741032, No. 81874090), Hubei Provincial Natural Sciences Foundation of China (2018CFA038), Foundation of Health and have been developed to investigate the underlying mechanisms. Family Planning Commission of Hubei Province in China (WJ2017M124), Inde- Herein, we investigated the potential of KIF4A inhibition to improve pendent Innovation Foundation of Huazhong University of Science and Technology current prostate cancer treatment strategies. Functional experiments (2016YXZD052, No. 118530309) and Clinical Research Physician Program of Tongji showed that KIF4A knockdown increases the inhibition of the growth Medical College, Huazhong University of Science and Technology (No. 5001530015). of prostate cancer cells by ADT drugs, including bicalutamide and enzalutamide. Most importantly, using our enzalutamide-resistant cell The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance model, the in vivo study revealed that targeting KIF4A significantly with 18 U.S.C. Section 1734 solely to indicate this fact. inhibits CRPC tumor growth, and the use of enzalutamide in combination with KIF4A knockdown further attenuated tumor growth Received February 2, 2019; revised May 25, 2019; accepted November 26, 2019; compared with a single treatment. published first December 3, 2019.

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Targeting the KIF4A/AR Axis to Reverse Endocrine Therapy Resistance in Castration-resistant Prostate Cancer

Qi Cao, Zhengshuai Song, Hailong Ruan, et al.

Clin Cancer Res Published OnlineFirst December 3, 2019.

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