Natural killer cells suppress enzalutamide resistance and cell invasion in the castration resistant prostate cancer via targeting the androgen receptor splicing variant 7 (ARv7)

Accepted Manuscript Natural killer cells suppress enzalutamide resistance and cell invasion in the castration resistant prostate cancer via targeting the androgen receptor splicing variant 7 (ARv7) Shin-Jen Lin, Fu-Ju Chou, Lei Li, Chang-Yi Lin, Shuyuan Yeh, Chawnshang Chang PII:

S0304-3835(17)30217-3

DOI:

10.1016/j.canlet.2017.03.035

Reference:

CAN 13299

To appear in:

Cancer Letters

Received Date: 6 January 2017 Revised Date:

22 March 2017

Accepted Date: 23 March 2017

Please cite this article as: S.-J. Lin, F.-J. Chou, L. Li, C.-Y. Lin, S. Yeh, C. Chang, Natural killer cells suppress enzalutamide resistance and cell invasion in the castration resistant prostate cancer via targeting the androgen receptor splicing variant 7 (ARv7), Cancer Letters (2017), doi: 10.1016/ j.canlet.2017.03.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Natural killer cells suppress enzalutamide resistance and cell invasion in the castration resistant prostate cancer via

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targeting the androgen receptor splicing variant 7 (ARv7)

Shin-Jen Lin1, #, Fu-Ju Chou1, #, Lei Li1, 2, #, Chang-Yi Lin1, Shuyuan Yeh1 and

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Chawnshang Chang1, 3, *

1

George Whipple Lab for Cancer Research, Departments of Pathology, Urology, and Radiation

Oncology, and The Wilmot Cancer Center, University of Rochester Medical Center, Rochester, NY 14642, USA 2

Sex Hormone Research Center, Department of Urology, The First Affiliated Hospital of Xi'an

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Jiaotong University, Xi'an 710061, China 3

#

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Sex Hormone Research Center, China Medical University/Hospital, Taichung 404, Taiwan

Contributed equally

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*Correspondence to: Chawnshang Chang, (E-mail: [email protected])

Running title: NK cells suppress enzalutamide resistant prostate cancer

Keywords: Natural Killer cells, ARv7, Enzalutamide

ACCEPTED MANUSCRIPT Abstract Despite the success of androgen-deprivation therapy (ADT) with the newly developed anti-androgen enzalutamide (Enz, also known as MDV3100) to suppress castration resistant prostate cancer (CRPC) in extending patient survival by an extra 4.8 months, eventually patients

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die with the development of Enz resistance that may involve the induction of the androgen receptor (AR) splicing variant ARv7. Here we identify an unrecognized role of Natural Killer (NK) cells in the prostate tumor microenvironment that can be better recruited to the CRPC cells to

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suppress ARv7 expression resulting in suppressing the Enz resistant CRPC cell growth and invasion. Mechanism dissection revealed that CRPC cells compared to normal prostate epithelial

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cells could recruit more NK cells that might then lead to alterations of the microRNA-34 and microRNA-449 to suppress both ARv7 expression and ARv7-induced EZH2 expression to suppress CRPC cell invasion. Together, these results identify a new potential therapy using recruited NK cells to better suppress the Enz resistance and cell invasion in CRPC at the later

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enzalutamide resistant stage.

ACCEPTED MANUSCRIPT 1. INTRODUCTION Natural Killer (NK) cells, one type of cytotoxic lymphocytes, are critical to the innate immune system that can rapidly respond to virally infected cells and tumor development [1]. NK cells are differentiated from the common lymphoid progenitor cells that can also generate B and T

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lymphocytes [2]. NK cells are best known to differentiate and mature in the bone marrow [2]. In addition to the innate immunity, NK cells also play a role in adaptive immune response [1], which is becoming increasingly important for their roles to suppress various tumors progression [3-5].

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NK cells play a critical role in tumor immuno-surveillance by directly inducing the death of tumor cells even without the surface adhesion molecules and antigenic peptides [4]. Compared

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to the cytotoxic T cells, NK cells are more critical for the tumor surveillance particularly because T cells are unable to recognize pathogens in the absence of surface antigens [2, 4, 6]. Lysis of tumor cells by NK cells is mediated by alternative receptors, including NKG2D, NKp44, NKp46, and DNAM [7]. NKG2D is a disulfide-linked homodimer that recognizes a number of ligands,

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including ULBP and MICA that are typically expressed in tumor cells [8]. Prostate cancer (PCa) is one of the most common cancers in the United States, with the highest incidence at 29% and second highest mortality at 11% in men [9]. It starts with prostate

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luminal epithelial cells malignant transformation into small clumps of cancer cells confined in normal prostate glands, named prostatic intraepithelial neoplasia (PIN). Over time these PIN

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cells spread to the stromal compartment forming the prostate tumor. The cancer cells may metastasize from the prostate to other parts of the body, particularly lymph nodes and bones [10].

The standard treatment of advanced PCa is the androgen-deprivation therapy (ADT) via surgical or chemical castration, which can shrink the primary androgen-dependent PCa, but the patients may eventually develop castration resistant PCa (CRPC) after 12-18 months of ADT [10, 11]. The newly developed antiandrogen enzalutamide (Enz, also known as MDV3100) has a better efficacy to suppress the CRPC with an increase in patients’ survival by 4.8 months [12].

ACCEPTED MANUSCRIPT However, the resistance to Enz still occurs eventually [13-16], which may involve the induction of the androgen receptor (AR) splicing variant 7 (ARv7, also known as AR3) [13]. Presently, there is no further therapeutic approach after the failure of Enz treatment, and therefore development of a new therapy to suppress the Enz resistant CRPC is needed to extend the patients survival.

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Here we found the recruited NK cells might function through suppressing the ARv7 to further suppress the CRPC cells at the Enz resistant stage. Since early studies indicated that using NK cell-based immunotherapy can efficiently delay cancer progress [17, 18], our finding using

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recruited NK cells to further suppress the Enz resistant CRPC may help us to develop a better

2. MATERIALS AND METHODS 2.1 Antibodies

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therapeutic strategy to suppress the Enz resistant CRPC.

Anti‐GAPDH (6c5), anti‐AR (N‐20), and anti-STAT5b antibodies were purchased from Santa

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Cruz Biotechnology. Anti-EZH2, anti-HIF1á, anti-c-Myc, and anti-p-Src, antibodies were purchased from Cell Signaling Technology. Anti-VEGFa antibody was purchased from Abcam.

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2.2 Cell culture

The NK cells, NK92-MI (NK92) cells (that grow as suspension culture), the PCa cell lines

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CWR22Rv1 and VCaP (both are enzalutamide resistant), and the normal prostate epithelial cell RWPE1 were obtained from the American Type Culture collection (ATCC, Rockwell, MD). NK92 cells were maintained in αMEM (GIBCO) without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate. The following components were added to the αMEM: 0.2 mM inositol; 0.1 mM 2-mercaptoethanol; 0.02 mM folic acid; 12.5% horse serum; 12.5% fetal bovine serum; and 1% Antibiotic-Antimycotic solution (Invitrogen). CWR22Rv1 and VCaP cells were maintained in RPMI 1640 media (GIBCO) supplemented with 10% fetal bovine serum and 1% Antibiotic-Antimycotic solution (Invitrogen). RWPE1 cells were

ACCEPTED MANUSCRIPT maintained in complete keratinocyte serum-free media (KSF media), supplemented with 1% penicillin/streptomycin/amphoterycin B, 50 mg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor (Life Technologies).

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2.3 Boyden chamber transwell recruitment assay

NK cells recruitment assay was performed using 24-well transwell inserts (5 µm pores) according to the manufacturer’s instructions (Corning, #3422). 105 NK cells were seeded on the

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upper chambers of transwell plates and 105 RWPE1 or CWR22Rv1 cells were seeded to the lower chambers. Each sample was assayed in triplicate and incubated for 24 h for migration. The

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migrated NK cells were counted by the automated cell counter.

2.4 MTT assay

105 NK cells were co-cultured with 5 x 105 CWR22Rv1 cells. MTT cell growth assay of

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CWR22Rv1 cells were performed after 72 h of co-culture. NK cells were removed from the culture dish and the CWR22Rv1 cell number was calculated using MTT agent.

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2.5 Anchorage-independent colony forming assay CWR22Rv1 and VCaP cells cultured with or without NK cells were suspended at a density of

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2.5×105 cells/ml in 0.4% Noble agar (Sigma, St. Louis, MO) containing media and seeded on top of 0.8% agarose gel in culture plates. After solidification, 2 ml of media was added on the top. Media were changed every week for one month before the pictures were taken.

2.6 Boyden chamber transwell invasion assay NK cells were co-cultured with CWR22Rv1 in the 0.4 ìm pore size transwell. After 24 h of incubation, CWR22Rv1 cells were trypsinized and were seeded on the top well of 8 µm transwells pre-coated with matrigel. Each sample was assayed in triplicate and incubated for 24

ACCEPTED MANUSCRIPT h for invasion. The CWR22Rv1 cells invaded into the matrigel were stained with toluidine blue and 5 representative areas counted.

2.7 Quantitative real-time PCR (QPCR) for gene and microRNA expression and microRNA

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inhibitors

For RNA extraction, total RNAs were isolated using Trizol reagent (Invitrogen). 1-2 µg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen).

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Quantitative PCR (QPCR) for mRNA was conducted using a Bio-Rad CFX96 system with SYBR green to determine the mRNA expression level of a gene of interest. Expression levels were

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normalized to the expression of GAPDH mRNA. The microRNAs (miRNAs) were isolated using PureLink®miRNA kit. QPCR for miRNA was conducted using a Bio-Rad CFX96 system with FAM/FITC to determine the miRNA expression level. Primers were individual miRNA primer + universal primer. Tagman FITC probe was used for fluorescence labeling. Expression levels

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were normalized with the expression of U6 RNA.

The inhibitors for miR-34c-3p and miR-449b-5p were purchased from Qiagen (cat:

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MIN0003327 and MIN0004677, respectively).

2.8 Western Blot Analysis

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CWR22Rv1 and VCaP cells were lysed in RIPA buffer (50 mM Tris–HCl/pH 7.4, 1% NP‐40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 mM okadaic acid and 1 mg/ml aprotinin, leupeptin and pepstatin). Individual samples (78 µg proteins) were prepared for electrophoresis run on 10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore). After blocking the membranes with 5% fat-free milk in TBST (50 mM Tris/pH 7.5, containing 0.15 M NaCl and 0.05% Tween‐20) for 1 h at room temperature, the membranes were incubated with appropriate dilutions (diluted in 2.5% BSA) of specific primary antibodies overnight at 4°C. After TBST washing, the blots wer e incubated with anti‐rabbit or anti‐mouse

ACCEPTED MANUSCRIPT secondary antibodies with horseradish peroxidases and conjugated for 1 h. After TBST washing, the blots were developed in ECL mixture (Thermo Fisher Scientific Inc.).

2.9 Luciferase assay

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CWR22Rv1 cells prepared as stable luciferase clone (CWR22Rv1-Luc) were lysed and detected for firefly luciferase activity by adding substrate LAR II. After 24 h, ARE activity was detected using a Dual-Luciferase Reporter Assay System (Promega). Light intensity was

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2.10 Orthotopic xenograft model

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measured using a plate reader (ARVOTM MX, Perkin Elmer, Inc., Waltham, MA, USA).

Male 6-8 weeks old nude mice were purchased from NCI. 5x105 CWR22Rv1-Luc with or without 2.5x105 NK cells were orthotopically injected into both anterior prostates in nude mice. The primary and metastatic tumors were evaluated each week and at 8 weeks endpoint using

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the non-invasive in vivo imaging system (IVIS). After sacrifice, tumors and metastases were removed to measure and count.

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2.11 Statistics

All experiments were performed three times in triplicate. The data values were presented as

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the mean±SEM. P values were calculated by unpaired Student's t test or Fisher’s exact test. P<0.05 was considered statistically significant. The mouse metastasis results were calculated using Fisher’s exact test. P values of *P<0.05; **P<0,01; ***P<0.001 were considered statistically significant.

3. RESULTS 3.1 PCa cells have better capacity than normal prostate cells to recruit NK cells To compare the recruitment of NK cells to the PCa cells vs the normal prostate cells, we

ACCEPTED MANUSCRIPT applied the Boyden chamber co-culture system [19, 20] to assay the NK cell migration with NK-92 cells on top and the CRPC CWR22Rv1 or VCaP cells (both are CRPC and resistant to Enz [13, 21]) or normal prostate epithelial cell RWPE1 cells on bottom (see diagram in Figure 1,

cells as compared to the RWPE1 cells (Figure 1, bottom panel).

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top panel). The results revealed that NK-92 cells migrated better to the CWR22Rv1 and VCaP

Together, results from Figure 1 suggest that Enz resistant CRPC cells have better capacity

3.2 Recruited NK cells suppress CRPC cell growth

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than normal prostate cells to recruit the NK cells.

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To examine the impacts of increased recruitment of NK cells to the Enz resistant CRPC cells, we assayed the Enz resistant CRPC cell growth after recruitment of more NK cells. We first co-cultured NK-92 cells with CWR22Rv1 or VCaP cells in the same dish for 48 h (Figure 2a, top panel). We then removed the suspended NK cells from the culture dish for the MTT growth

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assay. Interestingly, we found the recruited NK-92 cells could result in suppressing the CWR22Rv1 and VCaP cells growth (Figure 2a, bottom panel) compared to control/Mock with no NK culture.

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Importantly, when we applied the 2nd growth assay with anchorage independent colony-formation-assay, we also observed the similar results (Figure 2b).

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Together, results from Figure 1-2 using various Enz resistant CRPC cells with 2 different growth assays conclude that NK cells can migrate more to the Enz resistant CRPC cells to suppress their growth compared to the normal prostate cells.

3.3 Recruited NK cells suppress CRPC cell invasion As most PCa patients die from tumor metastases [10], we also assayed the recruited NK cells impacts on Enz resistant CRPC cell invasion. We used Boyden chambers coated with matrigel to mimic the extracellular matrix for the cell invasion assay [20]. We first cultured

ACCEPTED MANUSCRIPT CWR22Rv1 cells with/without NK cells for 24 h, removed the NK cells, then trypsinized the CWR22Rv1 cells to plated them onto a new Boyden chamber top well for 24 h before staining the cells invaded into the matrigel with toluidine blue (Figure 3, top panel). The results showed that recruited NK cells could significantly suppress CWR22Rv1 cell invasion (Figure 3, bottom

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panels).

Together, results from Figure 3 using Enz resistant CRPC cells with invasion assay conclude that recruited NK cells can suppress Enz resistant CRPC cells invasion. These conclusions are

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clinically important since more evidences indicated that the ADT with antiandrogens, including the recently developed powerful Enz might have unwanted side effects of increasing Enz

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resistance and enhancing the PCa cell invasion [14, 15, 21-28]. These unexpected and exciting findings may therefore suggest that recruited NK cells may be applied in the future as a novel therapy to further suppress CRPC after development of the Enz resistance.

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3.4 Mechanism dissection how recruited NK cells can suppress Enz resistant CRPC cells Recent clinical studies strongly suggested that the key mechanism for the development of Enz resistance during treatment of CRPC could be due to the induction of the ARv7 as clinical

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surveys concluded that CRPC patients with higher ARv7 expression had poor response to Enz treatment [24, 27]. We were interested to see if recruited NK cells could also suppress the

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Enz-induced CRPC cell invasion via altering the ARv7 signaling. Surprisingly, we found the mRNA and protein expressions of ARv7 were reduced in CRPC CWR22Rv1 cells after co-cultured with NK cells (Figure 4a). Similar results were also obtained when we replaced CWR22Rv1 cells with VCaP cells, another Enz resistant CRPC cell line (Figure 4b). Together, results from Figure 4a-b suggest that recruited NK cells can further suppress Enz resistant CRPC cells invasion via inhibiting the ARv7 expression.

3.5 Mechanism dissection how recruited NK cells can suppress AR-v7 expression

ACCEPTED MANUSCRIPT Early studies suggested that a variety of non-coding RNAs including microRNAs (miRNAs) or long non-coding RNAs (lncRNAs) might be able to alter the CRPC progression via targeting AR [29]. We were interested to see whether those miRNAs and/or lncRNAs are the mediators for the NK cells-suppressed ARv7 (and/or AR expression). Among those miRNAs screened we found

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the expression of miRNA-34, miRNA-449 and miRNA-488 were altered after co-cultured with NK cells (Figure 4c), and were further confirmed via qPCR assay (Figure 4d). Importantly, using Interruption assays with an inhibitor of miRNA-449, we found that targeting miRNA-449 could

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partially reverse NK cells-suppressed ARv7 expression (Figure 4e) and NK cells-suppressed cells invasion (Figure 4f).

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Together, results from Figure 4a-f suggest the recruited NK cells may function through altering the miRNA-449 to suppress the ARv7 signals, which may then result in suppressing the Enz resistant CRPC cells invasion.

growth and invasion

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3.6 Mechanism dissection how recruited NK cells can suppress ARv7-mediated CRPC cell

Finally, we asked how NK-suppressed ARv7 could inhibit CRPC cells invasion via screening

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several reported PCa metastasis-related genes in CWR22Rv1 cells with vs without NK cells co-culture. The results revealed that 11 of 14 metastasis genes had mRNA expression

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suppressed in CWR22Rv1 cells after co-cultured with NK cells (Figure 5a). We then confirmed these suppressed metastasis-related genes at the protein level with Western Blot analysis, and found that the protein expressions of C-Myc, p-Src, EZH2, STAT5b, and ARv7 were significantly suppressed (Figure 5b). We then focused on EZH2 as early studies suggested that EZH2 could promote PCa cells invasion[30]. We applied the interruption approaches via overexpressing the ARv7-cDNA or EZH2-cDNA in CWR22Rv1 cells co-cultured with NK cells (Figure 5c) and results revealed that these interruptions partially reversed the recruited NK cells-suppressed cell growth (Figure 5d)

ACCEPTED MANUSCRIPT and invasion (Figure 5e). Together, results from Fig. 5a-e suggest that recruited NK cells may function through altering the ARv7-mediated EZH2 signals to suppress the Enz resistant CRPC cells growth and invasion.

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3.7 In vivo mouse model to prove new therapy to suppress Enz resistant CRPC All above in vitro studies from Enz resistant CRPC CWR22Rv1 and VCaP cells compared to normal RWPE1 cells suggest we may be able to use the recruited NK cells as a potential novel

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therapy to further suppress the CRPC after development of Enz resistance. We then applied the in vivo mouse model to prove our in vitro cell line data. We first stably transfected the luciferase

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plasmid into CWR22Rv1 cells (CWR22Rv1-Luc) and verified its activity (Figure 6a). We then orthotopically xenografted these CWR22Rv1-Luc cells with or without NK cells into nude mice prostates and monitored the tumor growth by weekly IVIS imaging. The results showed that co-injecting CWR22Rv1-Luc plus NK cells could reduce the primary tumor sizes compared to

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injecting CWR22Rv1-Luc cells only (Figure 6b). We then sacrificed the mice and results revealed that the metastatic tumors were reduced in size and numbers in the mice injected with NK plus CWR22Rv1-Luc cells compared to the mice injected only with CWR22Rv1-Luc cells, in

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which we found the metastasis in duodenum, bone, diaphragm, and lymph node (Figure 6c). Together, results from Figure 6 showed the promising therapeutic effect of NK cells to

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suppress CRPC metastasis.

4. DISCUSSION

The recent clinical observation shows CRPC patients with higher ARv7 expression respond poorly to the Enz treatment and ARv7 expression increased in patients treated with Enz [27], suggesting ARv7 may play a key role in the development of Enz resistance. Since ARv7 is generated by splicing the full length AR, which results in loss of the androgen-binding-domain [24], that renders the ARv7 able to be self-transactivated in the castration androgen environment

ACCEPTED MANUSCRIPT so that Enz is no longer able to suppress its mediated cells growth [25]. Another AR splicing variant, ARv12, also known as ARv567es, where exon 5, 6, and 7 are deleted, was also found in the CRPC [31]. ARv12 acts as a constitutively active receptor that can increase the expression and activity of full-length AR [32]. Patients with higher expression of

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ARv7 or ARv12 have a significantly shorter survival than other CRPC patients [33], with little response to the AR antagonist Enz and the androgen biosynthesis inhibitor Abiraterone to further suppress the CRPC [13, 34].

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Importantly, in addition to induce the Enz resistance, recent preclinical studies also indicated that Enz might have 2 other unwanted side effects of increasing the neuroendocrine

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differentiation [35, 36] as well as increasing the cell invasion [14, 15, 37-42]. Mechanism studies indicated that ADT with antiandrogens might increase PCa cell invasion via altering multiple signal pathways, including TGFβ1/Smad/-MMP9 [15], BRD4 [37], SOX2 [38], STAT3/CCL2 [14], lncRNA-HOTAIR/AR/MMP9 [39], FGF11/miRNA541/AR/MMP9 [40], and Hif/CCL5/CXCR4 [41,

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42].

Our finding that better recruited NK cells could target the ARv7 to suppress the Enz resistance and cell invasion in the Enz resistant CRPC cells is clinically significant since NK

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therapy has been demonstrated in the clinical trials to suppress various tumors including metastatic melanoma, metastatic renal cell carcinoma, or poor-prognosis AML [43]. These

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therapies may involve the activation of endogenous NK cells, including NK cells in allogeneic clinical stem-cell transplantation, NK cell based donor lymphocyte infusions, or NK-cells adoptive immunotherapy [4]. Allogeneic stem cell transplantation involves the transfer of hematopoietic stem cells from unrelated or sibling donors to patients [4]. Alternatively, applying the autologous transfer of NK cells back into the patients is also an option and can eliminate the auto-immune response [4]. Importantly, treating with some cytokines (including IL-2, IL-12, IL-15, IL-18, IL-21 and type I IFNs) to enhance endogenous NK cells activities has also been used successfully to improve the NK therapy efficacy [4].

ACCEPTED MANUSCRIPT NK cells play an important role in tumor immunosurveillance by secreting perforin and granzymes to directly induce the death of tumor cells. It has been reported that CRPC cells can avoid NK immune response by secreting the NKG2D ligand-expressing PCa-derived exosomes [44]. That is probably why CRPC patients’ NK cells fail to suppress the tumor. Although these

with cytokines discussed above to boost the NK cell activity.

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exosomes selectively downregulated the NKG2D expression in NK cells, we may be able to treat

In summary, results from the current studies showing NK cells can effectively suppress Enz

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resistant CRPC cell growth and invasion may provide a potential new therapy with better efficacy

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to overcome the Enz-induced Enz resistance and cell invasion and extend patients’ survival.

Acknowledgment: These works were supported by NIH CA156700/DK73414 grants as well as grant (DOH102-TD-B-111-004) from Taiwan Ministry of Health and Welfare Clinical Trial and

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FIGURE LEGENDS Figure 1. Prostate cancer cells increase NK cells recruitment: NK cells recruitment were measured by the Boyden chamber assay. Transwells with 5 µm pore membranes were used to

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assay the NK cells migration to the RWPE1, CWR22Rv1, or VCaP cells (upper panel). 105 NK-92, RWPE1, CWR22Rv1, or VCaP cells were seeded in the chambers as shown in the figure. Migrated cells were counted by the automated cells counter 24 h after co-culture. The

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bottom panel shows the migrated NK cells number. The experiments were performed three times in triplicate. The statistic analyses were performed using Student t test. Each bar represents the

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mean ± sem. ** P<0.01; *** P<0.001.

Figure 2. NK cells decrease prostate cancer cells growth: (a) 2.5 x 105 NK cells were co-cultured with 5 x 105 CWR22Rv1 or VCaP cells (top panel). MTT cell growth assay of

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CWR22Rv1 cells were performed after 48 h of co-culture compared to PCa cells cultured alone (control). The quantification data was shown in the bottom panel. The experiments were performed three times in triplicate. The statistic analyses were performed using Student t test.

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Each bar represents the mean ± sem. * P<0.05. (b) Anchorage independent cell growth was assayed in both CWR22Rv1 and VCaP cells. 2.5 x 105 CWR22Rv1 or VCaP cells with or without

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1.25 x 105 NK cells co-culture were seeded in 0.4% of agarose gel. The cell colony pictures were taken after 1 month of incubation. The quantification data was shown in the right panels. The experiments were performed three times in triplicate. The statistic analyses were performed using Student t test. Each bar represents the mean ± sem. * P<0.05; *** P<0.001.

Figure 3. NK cells decrease CWR22Rv1 invasion: CWR22Rv1 cells were cultured with/without NK cells in the 0.4 µm pore membrane transwell. After 24 h of incubation, NK cells were removed, CWR22Rv1 cells were trypsinized and 1x105 of trypsinized cells were seeded on

ACCEPTED MANUSCRIPT the top well of 8 µm transwells pre-coated with matrigel (top panel). The invaded CWR22Rv1 cells were stained after 24 h with toluidine blue. The bottom panels show the migrated CWR22Rv1 cells staining. Control indicates CWR22Rv1 cells cultured alone (bottom left panel).

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NK indicates CWR22Rv1 cells co-cultured with NK cells (bottom right panel).

Figure 4. Recruited NK cells reduces ARv7 expression: (a) In CWR22Rv1 cells after 48 h culture with/without NK cells, the protein level (left) and mRNA (right) levels of ARv7 were both

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significantly decreased. (b) In VCaP cells after 48 h culture with/without NK cells, the protein level of ARv7 was significantly decreased. (c) Screen for the miRNA candidates that can

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down-regulate AR expression in PCa CWR22Rv1 (22RV) cells. (d) Confirmation of the screened miRNA candidates via QPCR. (e) CWR22Rv1 (22RV) cells Western blot to show AR and ARv7 protein (Pt) level when cultured with/without NK cells and treated with miRNA inhibitors (Qiagen). (f) CWR22Rv1 cell invasion assay with different conditions as shown on the figure. Bottom right

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panel showed the quantification result. The statistic analyses were performed using Student t test. Each bar represents the mean ± sem. * P<0.05; ** P<0.01; *** P<0.001.

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Figure 5. PCa-NK co-culture reduces AR as well as metastasis-related genes mRNA expression: (a) CWR22Rv1 cells with or without NK cells co-culture were collected to extract

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RNA. The cDNA was converted from the extracted RNA and the quantitative PCR (QPCR) was performed using the metastasis-related genes primers. (b) Western blot to confirm the metastasis-related genes expression. (c) Western blot to confirm the ARv7 over-expression (top panel) and the EZH2 overexpression (bottom panel). (d) Cell viability assay of CWR22Rv1 with ARv7 (AR3) or EZH2 overexpression by MTT assay. (e) Cell invasion assay of CWR22Rv1 with ARv7 or EZH2 overexpression. The statistic analyses were performed using Student t test. Each bar represents the mean ± sem. ** P<0.01; *** P<0.001.

ACCEPTED MANUSCRIPT Figure 6. Xenograft model of CWR22Rv1-Luc cells co-injected with NK cells (a) Luciferase activity was detected on the CWR22Rv1 stably transfected with luciferase. (b) 5x105 CWR22Rv1-Luc cells with or without 2.5x105 NK cells were orthotopically injected into both anterior prostates in nude mice at 8 weeks of age. IVIS images were taken weekly for 5 weeks

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after inoculation. (c) The mice were sacrificed at 13 weeks old and the primary and metastatic tumors were measured and counted (left panel). The right panel shows the statistical results of metastases numbers between CWR22Rv1-Luc cells versus CWR22Rv1-Luc cells co-injected

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with NK cells. The statistical results were calculated using Fisher’s exact test. * P<0.05;

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***P<0.001.

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ACCEPTED MANUSCRIPT Highlights The recent clinical observation showing CRPC patients with higher ARv7 expression respond poorly for the Enzalutamide treatment and ARv7 expression increased in patients treated with Enzalutamide, suggesting ARv7 may play key role for the development of Enzalutamide resistance prostate cancer (ERPC). Since ARv7 was generated by splice full length AR that resulted in lost of androgen-binding-domain,

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and therefore allowed ARv7 to be self-transactivation in the castration androgen environment and enzalutamide can no longer suppress its mediated cell growth. Importantly, other studies also found the unwanted side effects showing while enzalutamide could suppress effectively the PCa cell growth, it might enhance the PCa cell invasion, as well as promote the development of neuronendocrine (NE) cells

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within PCa. Therefore development a new and better therapy to overcome the enzalutamide resistance is needed to extend PCa patients’ survival rate. Here we

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unexpectedly found that recruited NK cells could effectively target ARv7 protein expression and suppress ARv7-mediated ERPC progression. This is exciting since NK therapy has been studied in the clinical trial to suppress various tumors including metastatic melanoma, metastatic renal cell carcinoma, or poor-prognosis AML. In summary, results from our studies showing NK cells can effectively suppress ARv7 in ERPC may provide a potential new therapy with better efficacy to overcome the

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enzalutamide resistance and extend patients’ survival.