TNIK swaths AR to WNT pathway and drives Castration-Resistant Prostate Cancer CURRENT STATUS: POSTED

Background: The development of CRPCa was driven by complex genetic and epigenetic mechanisms that remained poorly understood. TNIK (Traf2 and Nck-interacting kinase) has been reported to be a serine/threonine kinase and associated with tumor cell proliferation or unfavorable cancer behavior. The present study was conducted to investigate the TNIK gene expressions in CRPCa. Methods: Using a microarray approach, we identified higher expression of TNIK in CRPCa. The interaction between AR and H3K27me3 upon TNIK depression was determined through molecular and cell biological methods. Co-immunoprecipitations assays were Performed to confirm that TNIK interacted and phosphorylated with β-catenin in CRPCa cell. Results: Specifically we found AR repressed TNIK gene transcription via forming complex with H3K27me3. TNIK was recruited to promote transcription of Wnt target genes in a β-catenin-dependent manner in C4-2 cells. In vitro binding showed that TNIK directly band and phosphorylated β-catenin. Depletion or mutant of TNIK kinase abrogated β-catenin transcription, highlighting the essential function of TNIK kinase activity in Wnt target gene activation. Conclusions: Our findings revealed a regulatory role of AR in TNIK repressor, TNIK interacted with β-catenin and phosphorylated activaing Wnt pathway to promote CRPCa progression. TNIK may present an attractive candidate for drug targeting in CRPCa.

Co-Immunoprecipitation and Western blotting.
LNCaP and C4-2 cells were harvested and lysed in lysis buffer (150 mM KCl,75 mM Hepes, pH 7.5, 1.5 mM EGTA, 1.5 mM MgCl2, 10% glycerol, and 0.075% NP-40 supplemented with protease inhibitor cocktail[Roche,USA]. Extract proteins were precleared using a mixture of protein A-Sepharose (CL-4B; GE Healthcare) and antibody for overnight hr at 4˚C. Immunoprecipitates were washed with lysis buffer and resuspended in sample buffer, boiled and analyzed by SDS-PAGE. Individual samples (40 µg of protein) were separated on 8% SDS polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, MA). Membranes were blocked in a PBS-Tween 20 solution with 5% fat-free milk for 1 h at room temperature, and then the membranes were incubated with appropriate dilutions of specific primary AR or TNIK antibodies overnight at 4 °C. After washing, the blots were incubated with HRP conjugated anti-rabbit or anti-mouse IgG for 1 h. The blots were developed in ECL mixture(Vector Laboratories, Burlingame, CA) and visualized by Imager.

Chromatin Immunoprecipitation
LNCaP cells were grown in 1640 (Invitrogen) supplemented with 10% charcoal-stripped FBS (CSF, HyClone, USA) for 12 h. DNA cross-linking was performed by adding 1% formaldehyde into the cell cultures at room temperature for 10 min, and glycine was then added (0.125 M final concentration) for 5 min to stop the cross-linking reaction. Cells were lysed with a lysis buffer with protease inhibitors and sonicated to shear genomic DNA to lengths between 200 and 1000 bp. One-tenth of the cell lysate was used for input control, and the rest was used for immunoprecipitation using AR or H3K27me3 antibody. After collecting the immunoprecipitates using protein G-agarose columns, protein-DNA complexes were eluted and heated at 65 °C to reverse the cross-linking. After digestion with proteinase K, DNA fragments were purified using spin columns and analyzed using PCR for 35 cycles in a sequence of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. Specific primer sets were designed to amplify a target sequence within the human TNIK promoter as table 2. PCR products were electrophoresed in a 1.5% agarose gel with ethidium bromide and visualized under ultraviolet light.
Whole-transcript expression array and microarray image processing One µg/µl of total RNA was used as a starting material for making total RNA/Poly-A RNA controls, and was mixed with a GeneChip®Eukaryotic Poly-A Control Kit (Affymetrix, Inc., CA, USA). The majority of the rRNA was removed from the total RNA samples prior to target labeling, so as to increase sensitivity by RiboMinusTM Human Transcriptome Isolation Kit (Invitrogen, CA, USA); cDNA was synthesized using the GeneChip® WT Sense Target Labeling and Control Reagents Kit, as per the manufacturer's instructions (Affymetrix, Inc., CA, USA). The sense cDNA was then fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidinic endonuclease 1), and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using a GeneChip® WT Terminal Labeling Kit.
(Affymetrix, Inc., CA, USA). After the biotin-labeled sense target DNA was prepared, the sample was ready to hybridize to gene chip (The GeneChip® Human Exon 1.0 ST array). Hybridization was performed using 5 µg of biotinylated target, which was incubated with a GeneChip® Hybridization, Wash and Stain Kit and a GeneChip® Fluidics Station 450 (Affymetrix, Inc., CA, USA). The arrays were scanned using a GeneChip® Scanner 3000 7G (Affymetrix, Inc., CA, USA). Raw data were extracted from the scanned images and analyzed with GeneSpring GX software version 11.5 (Agilent Technologies, CA, USA).

Luciferase assays.
For the dual luciferase assay, C4-2 cells were plated in triplicate into 12-well plates and cotransfected with 1 µg of the reporter construct and 15 pmol of β-catenin luciferase together with TNIK wt or D171A of plasmids by using transfection reagent (Roche). Transfected cells were cultured and 24 h later, the supernatants were collected for luciferase assay using Dual Luminescence assay kit  Table 1). GO analysis showed that the up-regulated differentially expressed genes were significantly enriched involved in mRNA processing and nuclear transport signaling pathway (Fig. 1B). We focused on TNIK serine/threonine kinase because of up-regulated expression of the gene in PCa is associated with Wnt signaling pathway. To verify microarray, We analyzed samples obtained from mice xenografts tumor with CR-LNCaP (4castrasion) and HS-LNCaP (4 uncastrasion) for expression of TNIK by QPCR (Fig. 1C). The mRNA expression of TNIK was higher in CR-LNCaP compared with HS-LNCaP.
To investigate a potential role of TNIK, we analyzed prostatectomy samples obtained from patients with benign prostate hyperplasia (BPH 5cases), Hormone native(10cases) and CRPCa (7cases) for expression of TNIK by IHC (Fig. 1D). The expression of both TNIK was circumscribed to cell nuclear of the epithelial compartment, with TNIK showing a pattern of progressively, significantly increased expression in CRPCa. These data, as shown in Fig. 1C,D and in good agreement with the Microarray data analysis, CRPCa cells exhibit increased mRNA and protein expression of TNIK.
Results showed that TNIK had an effect on cell proliferation, significantly inhibited cell growth depended on knockdown TNIK and TNIK should be enough to induce efficient proliferation. These data indicated that TNIK was a potent inhibitor for CRPC cell proliferation. In conclusion, TNIK had independent functions in androgen independent prostate cancer cells that may contribute to the growth and spread of castration resistant tumors.
Androgen-AR signaling suppressed TNIK gene expression Up-regulated expression of TNIK in CR-LNCaP vs HS-LNCaP tumors suggested that TNIK may be regulated by androgens. Therefore, we studied how dihydrotestosterone (DHT) (10 nM) treatment effects on the levels of TNIK. A time-course study of DHT incubation LNCaP cells showed that androgen treatment repressed the expression of TNIK (Fig. 3A). We found increased TNIK protein expression levels following AR knockdown in PCa cells (Fig. 3B) while treatment of LNCaP cells with AR antagonist MDV3100 also upregulated TNIK mRNA and protein levels and phosphorylation levels of TNIK significantly (Fig. 3C,D). In line with the upregulation of TNIK detected in CR-LNCaP, we also observed that inhibited androgen exposure increased the abundance of TNIK in cell nucleus with immunofluorescence imaging (Fig. 3E). We previously reported EZH2 and AR can cooperate for YAP1 transcriptional repression [23]. while EZH2 was the only identified methyltransferase with activity toward H3K27 and was responsible for all H3K27 methylation [24]. Therefore, we hypothesized that AR may recruit the H3K27me3 to repress TNIK expression in PCa cells. To verify this hypothesis, we first investigated the interaction between AR and H3K27me3 in LNCaP cells by co-immunoprecipitation (Co-IP) experiments. As shown in Fig. 3F, the AR formed a stable complex with H3K27me3 with each other in LNCaP cells. The interactions between the two proteins were specific because not any visible interaction was showed in IgG control. Moreover, AR and H3K27me3 were recruited to the TNIK gene promoter, but the ability of AR to form a complex was abolished by treatment with MDV3100 by Chromatin immunoprecipitation experiments (Fig. 3G). Furthermore, DZNep treatment with an EZH2 inhibitor also lead to a significant increase in the expression of TNIK (Fig. 3H). Likewise, the H3K27 demethylase activator GSK-J1 increased H3K27me3 levels but decreased TNIK levels. Overall, these experiments demonstrated that AR and H3K27me3 complex mediated the androgen-driven epigenetic repression of the TNIK.

TNIK interacts directly withβ-catenin
Gene set enrichment analysis (GSEA) was performed involvement of activating Wnt pathways where the profile of genes differentially expressed in LNCaP-CR vs LNCaP-HS (Table 1). While the TNIK activating wnt through TCF4 in the growth of colorectal cancer has been well documented[8], less is known about the role of TNIK in CRPCa. To confirm the function of TNIK in regulation of the Wnt target gene expression, we used the C4-2 cell line in which the Wnt pathway is present. We examined the interaction between TNIK and β-catenin in C4-2 cells. TNIK was immunoprecipitated from C4-2 lysates and probed for interaction with β-catenin by western blotting (Fig. 4A,B). As expected, β-catenin also displayed direct binding to TNIK. Next, we examined the effect of over TNIK or siRNA-mediated knockdown of TNIK on β-catenin phosphorylation, we fund over expressed TNIK active β-catenin phosphorylation at Ser675 and knockdown of TNIK attenuated β-catenin phosphorylation (Fig. 4C). We also observed that over expressed TNIK increased the abundance of TNIK and β-catenin in cell nucleus with immunofluorescence imaging (Fig. 4D). Mutant of TNIK catalytic activity at S171 resulted in specific suppression of β-catenin -dependent TOPFlash activity (Fig. 4E). QPCR data showed knockdown TNIK with siRNA down-mediated β-catenin downstream genes transcriptional activation (Fig. 4F). We concluded that TNIK was required for optimal β-catenin phosphorylation and transcriptional activation.

TNIK Inhibitor Inhibited proliferation and Invasion of CRPC Cell
The novel small molecule TNIK inhibitor NCB-0846 was confirmed effective on tumor cell[25-26]. We first analyzed the ability of NCB-0846 to suppress TNIK protein expression in C4-2 and PC3 cells. NCB-0846 robustly decreased TNIK protein level after 24 h of treatment with 1 µm or 10 µm (Fig. 5A). A decreased activity of β-catenin (Ser675) in cells treated with inhibitor following TNIK downregulation were also observed in C4-2 and PC3 cells with WB (Fig. 5A). C4-2 and PC3 cells were found to be sensitive to the NCB-0846 treatment with 10 µm (Fig. 5B). NCB-0846 treatment also inhibited cell invasion of C4-2 and PC3 cells in 10 µm (Fig. 5C). Overall, these experiments suggested TNIK as a novel potential therapeutic target in CRPCa.

Targeting TNIK Suppressed CRPCa Tumor Growth in vivo.
To investigate the effect of TNIK inhibitor on CRPCa xenograft tumor growth, 2 × 10 6 C4-2 cells were implanted subcutaneously in Balb/c mice. When the tumors reached approximately 100 mm 3 in size, the mice were randomized and administered daily by oral gavage either with vehicle (10% DMSO in PBS) or NCB0846 (80 mg/kg of body weight) for 10 days (n = 4 mice for each treatment). Although DMSO-treated mice formed robust subcutaneous CRPCa tumors, tumor growth was observed smaller in the NCB-0846-treated group (Fig. 6A). A significant tumor growth inhibited was noticed in NCB-0846-treated mice compared with the DMSO-treated mice (Fig. 6B). We observed that Tumors treated by NCB-0846 expressed reduced levels of TNIK in parallel to diminished cell proliferation, and decreased expression of Ki67 and β-catenin S675 markers (Fig. 6C). Together, these results highlighted the potential of targeting the TNIK signaling to sensitize to CRPCa therapy.

Discussion
In the current study, we unveiled key elements of the crosstalk between the AR and the WNT signaling pathways through TNIK and targeted TNIK inhibiting β-catenin phosphorylation, blocked the growth of CRPCa.
The previous study was reported TNIK were frequently upregulated in high-grade tumors ovarian cancer and serous hepatocellular carcinoma [14]. Moreover, TNIK hyperactivity contributes to Human Lung Adenocarcinoma cell metastasis [13]. In this study, we firstly identified TNIK as a candidate biomarker of CRPCa from gene array files using mouse models. We observed not only TNIK higher expression in mice CR-LNCaP tumors, but also TNIK was higher in CRPCa than localized PCa and BPH in patient (Fig. 1D). It suggested us TNIK was relation to cancer aggressive behavior.
The principal findings of our study were that AR assembled a repressive complex with H3K27me3 at the TNIK promoter to suppress TNIK transcription (Fig. 3). Consequently, androgen deprivation therapy could induce TNIK mRNA expression which, in turn, regulated β-catenin phosphorylation to switch on WNT/β-catenin signaling pathway and contributed to castration resistant prostate cancer growth. The transcriptional activity of AR was regulated by interacting coactivators that positively modulated receptor function. Conversely, AR inhibited target gene expression by "corepressors" such as Alien, SMRT, NCoR [27][28][29]. A few reports have studied AR inhibition of transcription. Some hinted to indirect mechanisms-DNA methylation or protein phosphorylation [23,30] while others hinted to direct mechanisms in involving the epigenetic silencing complex [24,31]. Using TNIK as a model, we found that AR can also inhibited gene expression by hormone induced recruitment of H3K27me3 to the AR/ H3K27me3 complex to directly inhibite transcription. Consequently, ADT restored TNIK expression due to the loss of AR/ H3K27me3 association to the TNIK promoter.
WNT/β-catenin signaling pathway also plays an important role in CRPCa. Evidence has accumulated that Approximately 24% of metastatic tumors from CRPCa patients were reported to be positive for nuclear localization of β-catenin [32]. Sequencing of CRPCa tumors has revealed more significant genomic alterations in multiple components of the Wnt pathway than in hormone treatment naïve prostate cancer [33][34]. Our results also suggested that castration resistance may be induced reciprocal interaction between TNIK and the WNT/β-catenin signaling pathway, and TNIK was a major inducer of the expression, nuclear translocation, and activation of β-catenin (Fig. 3C). Phosphorylation cascades that were dependent and independent of Wnt signaling played a critical role on β-catenin stability, intracellular distribution and transcriptional activity. Targeting β-catenin in N-terminal domain facilitated proteosomal degradation, whereas β-catenin non-canonical phosphorylation at Ser675 promoted its nuclear translocation and transcriptional activity, and promoted its interaction with transcriptional co-activators, including TCF4 [35][36]. In this study, we found that upregulation of TNIK band and phosphorylated β-catenin at Ser675, promoted β-catenin nuclear translocation and transcription activation. It was reported that TNIK inhibitor decreased DU145 and 22RV1 cell viability in ERG positive PCa cells [24]. Our current data further revealed that TNIK band to β-catenin, resulting in phosphorylation of β-catenin and target genes transcription activation and target TNIK reduced the proliferation and invasion of CRPC cells (Fig. 5,6).

Conclusions
Here, our mechanisms have shown that AR functions as a transcriptional repressor in TNIK by binding