TNIK drives castration-resistant prostate cancer via phosphorylating EGFR

Summary The development of castration-resistant prostate cancer (CRPC) is driven by intricate genetic and epigenetic mechanisms. Traf2- and Nck-interacting kinase (TNIK) has been reported as a serine/threonine kinase associated with tumor cell proliferation or unfavorable cancer behavior. The microarray approach revealed a substantial upregulation of TNIK expression levels, enabling us to investigate the functional behaviors of the TNIK gene in CRPC. Specifically, we discovered that AR suppresses TNIK gene transcription in LNCaP and C4-2 cells by forming a complex with H3K27me3. Following the reduction of AR levels induced by androgen deprivation therapy (ADT), TNIK is recruited to activate EGFR signaling through phosphorylation in C4-2 cells, thereby promoting CRPC progression. Our findings unveil a regulatory role of AR as a repressor for TNIK while also highlighting how TNIK activates the EGFR pathway via phosphorylation to drive CRPC progression. Consequently, targeting TNIK may represent an appealing therapeutic strategy for CRPC.


INTRODUCTION
The prevalence of CRPC continues to be the leading cause of mortality among male patients worldwide. 1,2The rapid progression, ease of transfer, and development of late castration resistance of CRPC pose challenges to treatment. 3,46][7][8][9] The identification of targets is imperative for the advancement of CRPC development. 10raf2-and Nck-interacting kinase was initially identified as germinal center kinases (GCKs) and plays a crucial role in regulating various fundamental cellular processes through phosphorylation of its downstream substrates. 11,124][15][16][17][18] For example, TNIK protein phosphorylates AKT and promotes the proliferation of gastric cancer cells. 19Furthermore, TNIK-deficient mice showed reduced expression of Myc and Cd44. 20TNIK was also shown to be involved into the NF-kB and SMAD signaling pathways. 11,17merging evidence has demonstrated the crucial involvement of epidermal growth factor receptor (EGFR) signaling pathways in prostate regulatory mechanisms during prostate tumorigenesis.Activation of the EGFR signaling pathway plays a pivotal role in promoting cancer cell survival under androgen-depleted conditions.Upregulation of EGFR signaling may be a mechanism by which PC cells escape castrationinduced cell death. 21The association between TNIK and EGFR, however, remains unestablished in existing literature.
In this study, we employed GeneArray analysis to elucidate the mechanism underlying the development of CRPC.Our findings revealed TNIK as a potential driver gene for CRPC that interacts with EGFR.Notably, our evidence demonstrated that AR directly repressed TNIK transcription through the AR-H3K27me3 complex.Upon si-AR or MDV3100 treatment, TNIK gene expression was activated, leading to its binding to the extracellular domain (ECD) of EGFR and subsequent promotion of phosphorylation.Previous studies have implicated the ECD domain of EGFR in cellular drug resistance, suggesting that the interaction between TNIK and ECD may play a crucial role in CRPC emergence. 22The activated EGFR is mainly located in the nucleus and regulates the transcription of target genes, thereby promoting the progression of CRPC.Pharmacological inhibitors of TNIK (NCB-0846) inhibited the growth of CRPC cell xenografts.Furthermore, we observed that silencing TNIK also enhances EGFR-mediated, erastin-induced ferroptosis in CRPC cells.This kinase represents a promising candidate for targeted drug therapy in CRPC and provides valuable insights for potential combination strategies involving ferroptosis-based therapies.

TNIK was upregulated in castration-resistant prostate cancer
Previously, LNCaP cells were cultured under androgen deprivation conditions using 10% Certified FBS Charcoal Stripped and RPMI Medium 1640 culture medium.After about four months of culture and multiple passages, the CR-LNCaP cell line was obtained.Subsequently, a model of castration-resistant LNCaP tumors (CR-LNCaP) and androgen-sensitive tumors (HS-LNCaP) was established with LNCaP xenografts. 8In order to identify unrecognized molecular mechanisms of CRPC between intact and castrated mice, we quantified changes in mRNA levels of human genes in HS/CR-LNCAP tumors.We found that 1,884 genes were upregulated and 588 genes were repressed in CR-LNCaP compared with HS-LNCaP tumors (log 2 FC > 1.5, p < 0.05) (Figure 1A; Table S1).GO analysis revealed a significant enrichment of upregulated differentially expressed genes involved in mRNA processing and nuclear transport signaling pathway (Figure 1B).Among these genes, we focused on TNIK serine/threonine kinase due to its upregulated expression in CRPC associated with the EGFR signaling pathway.To validate the microarray results, we performed qPCR analysis on xenograft tumor samples from CR-LNCaP (4 castrations) and HS-LNCaP (4 uncastrations), confirming higher mRNA expression of TNIK in CR-LNCaP compared with HS-LNCaP (Figure 1C).Furthermore, we investigated the potential role of TNIK by analyzing prostatectomy samples obtained from patients with hormone-sensitive prostate cancer (HSPC) (26 cases), CRPC (29 cases), and their respective paracancerous tissues using immunohistochemistry staining for TNIK expression patterns (Figure 1D).The results showed that both cytoplasmic and nuclear compartments exhibited circumscribed expression of TNIK within the epithelial cells, with a progressively significant increase observed in CRPC samples.These findings are consistent with the data obtained from microarray analysis shown in Figures 1C and 1D, indicating increased mRNA and protein expression of TNIK in CRPC cells.To investigate the impact of TNIK on cell proliferation in CRPC, siRNA targeting TNIK was transfected into CRPC cells (C4-2, 22RV1, and PC3).Efficient knockdowns of TNIK were successfully achieved in these cell lines (Figure 2A).Subsequently, we observed a clear inhibition of proliferation in C4-2, 22RV1, and PC3 cells upon downregulation of TNIK (Figure 2B).Furthermore, the reduction of TNIK expression significantly attenuated the invasive C4-2, 22RV1, and PC3 cells as demonstrated by Matrigel assays (Figure 2C).These findings strongly support that TNIK plays a crucial role in promoting cell proliferation within CRPC.In summary, our results highlight the independent functions exerted by TNIK in androgen-independent PC cells while emphasizing its pivotal contribution to the growth and metastasis of castration-resistant tumors.

Androgen-AR signaling suppressed TNIK gene expression
The upregulated expression of TNIK in CR-LNCaP compared with HS-LNCaP tumors suggests a potential regulation of TNIK by androgens.Therefore, we investigated the impact of dihydrotestosterone (DHT) treatment (10 nM) on TNIK levels.A time-course study of DHT incubation in LNCaP cells revealed that androgen treatment suppressed the expression and phosphorylation of TNIK (Figure 3A).We observed increased TNIK protein expression and phosphorylation levels upon AR knockdown in PCa cells, whereas treatment of LNCaP cells with the AR antagonist MDV3100 also significantly upregulated TNIK mRNA and protein levels, as well as phosphorylation levels of TNIK (Figures 3B-3D).Consistent with the upregulation of TNIK detected in CR-LNCaP, we also observed that inhibition of androgen exposure led to increased nuclear abundance of TNIK through immunofluorescence imaging (Figure 3E).In our previous study, we reported a cooperative role between EZH2 and AR for YAP1 transcriptional repression. 23The sole identified methyltransferase with activity toward H3K27, EZH2, is solely responsible for all methylation of H3K27. 24Therefore, we performed co-immunoprecipitation (Co-IP) experiments to verify whether there is an interaction between AR and H3K27me3 in PCa cells.As depicted in Figure 3F, AR formed a stable complex with H3K27me3 in PCa cells.The specificity of these protein interactions was confirmed, as no visible interaction was observed in the IgG control.Furthermore, chromatin immunoprecipitation experiments demonstrated that both AR and H3K27me3 were recruited to the TNIK gene promoter; however, treatment with MDV3100 abolished the ability of AR to form a complex (Figure 3G).Treatment of PCa cells with DZNeP (EZH2 inhibitor) resulted in an increase in TNIK protein expression as well as phosphorylation levels, while interestingly downregulating AR levels as well (Figure 3H).In order to validate the role of H3K27me3 in transcriptional repression by AR, we utilized GSK-J1 (H3K27 demethylase inhibitor) to elevate the level of H3K27me3.We observed a decrease in TNIK expression when H3K27me3 levels increased, suggesting the inhibitory effect of H3K27me3 on TNIK, whereas the non-significant change in AR expression levels confirmed that EZH2-mediated regulation of AR levels is independent of its methylation function, consistent with previous studies (Figure 3I). 25 In addition, the elevation of H3K27me3 effectively counteracted the promoting effect of AR downregulation on TNIK expression (Figure 3J).Overall, these experiments demonstrated that the AR and H3K27me3 complex mediated the androgen-driven epigenetic repression of TNIK.

TNIK interacts directly with EGFR
Gene set enrichment analysis (GSEA) was conducted to investigate the involvement of activating EGFR pathways in LNCaP-CR vs. LNCaP-HS, where the differential expression profile of genes was analyzed (Table S2).Although the role of TNIK in certain cancers has been extensively documented, its role in CRPC remains less understood.To validate the regulatory function of TNIK on EGFR expression, we utilized C4-2 cell line with an active EGFR pathway.The interaction between TNIK and EGFR was examined in C4-2 cells by immunoprecipitating TNIK from lysates and probing for interaction with EGFR through western blotting (Figures 4A and 4B).As expected, direct binding between TNIK and EGFR was observed.Furthermore, using immunofluorescence imaging, we found that overexpression of TNIK led to increased abundance of nuclear-localized EGFR, whereas decreased levels of TNIK resulted in reduced nuclear import of EGFR (Figure 4C).In order to determine the binding domain between TNIK and EGFR more precisely, we generated several plasmids expressing truncated versions of Myc-tagged-EGFR (Myc-EGFR).Immunoprecipitation experiments were performed in C4-2 cells coexpressing these Myc-tag mutants along with TNIK.Results indicated that when the ECD domain was deleted from these mutants, they were not detected in the immunoprecipitates pulled down by anti-Myc immunomagnetic beads (Figure 4D).Next, we investigated the impact of TNIK overexpression or siRNA-mediated knockdown on EGFR phosphorylation and its downstream target genes.Our findings revealed that TNIK overexpression activated EGFR phosphorylation and its associated signaling pathways, including the ferroptosis-related gene NRF2.Conversely, knockdown of TNIK had the opposite effect (Figures 4E and 4F).Mutants with attenuated catalytic activity at S171 exhibited reduced EGFR phosphorylation and NRF2 upregulation compared with mutant controls (Figure 4G).
To further validate these results, we suppressed the activity of the ECD domain in EGFR using a plasmid.We observed that deletion of the ECD domain reversed the inhibitory effect of TNIK knockdown on EGFR phosphorylation and the changes in EGFR-associated NRF2 levels (Figure 4H).This further illustrates the importance of the ECD domain for TNIK to bind and function with EGFR.Notably, knockdown of TNIK decreased C4-2 cell resistance to erastin (a ferroptosis inducer).Similar to previous results, the ferroptosis sensitivity caused by TNIK knockdown was reversed when the ECD domain was deleted.(Figure 4I).In conclusion, our study highlights the essential role of TNIK in optimal EGFR phosphorylation and transcriptional activation while emphasizing its regulatory effects on ferroptosis as a potential avenue for combination ferroptosis therapies.

TNIK inhibitor inhibited proliferation and invasion of CRPC cell
The efficacy of the small molecule TNIK inhibitor NCB-0846 on tumor cells has been confirmed. 13,26To test the efficacy of NCB-0846 during prostate cancer treatment and in CRPC, we initially assessed the efficacy of NCB-0846 in suppressing TNIK protein expression in C4-2 and PC3 cells.
Treatment with 1 mm or 10 mm of NCB-0846 significantly reduced the levels of TNIK protein and phosphorylation after 24 h (Figure 5A).Furthermore, western blot analysis revealed a concurrent decrease in p-EGFR activity upon TNIK downregulation following inhibitor treatment in both C4-2 and PC3 cells.Notably, C4-2 and PC3 cells exhibited sensitivity to NCB-0846 treatment at a concentration of 10 mm (Figure 5B).Additionally, NCB-0846 treatment effectively inhibited cell invasion in both C4-2 and PC3 cells at a concentration of 10 mm (Figure 5C).Collectively, these findings highlight the potential therapeutic targeting of TNIK as an innovative approach for treating CRPC by modulating the EGFR signaling pathway.

Targeting TNIK suppressed CRPC tumor progression in vivo
To investigate the impact of the TNIK inhibitor on the growth of CRPC xenograft tumors, 2 3 10 6 C4-2 and PC3 cells were subcutaneously implanted in BALB/c mice.Once the tumors reached approximately 100 mm 3 in size, the mice were randomly assigned to receive either vehicle (10% DMSO in PBS) or NCB0846 (80 mg/kg of body weight) daily via oral gavage for a duration of 10 days (n = 4 mice per treatment group).While robust subcutaneous CRPC tumors formed in the DMSO-treated mice, tumor growth was noticeably smaller in the NCB-0846treated group (Figures 6A and S1A).A significant inhibition of tumor growth was observed in the NCB-0846-treated mice compared with those treated with DMSO alone (Figures 6B, 6C, S1B, and S1C).Importantly, administration of NCB-0846 did not result in any apparent toxicity, as it had no effect on body weight changes (Figures 6D and S1D).Furthermore, we found that tumors treated with NCB-0846 exhibited reduced TNIK expression levels and decreased Ki67 and p-EGFR expression (Figures 6E and S1E).Because previous articles have demonstrated that EGFR is critical for the regulation of EMT in PCa, we speculate that TNIK can also regulate the EMT process through EGFR. 27Therefore, we tested three EMT markers (b-catenin, vimentin, and E-cadherin) and found that inhibiting TINK seemed to block EMT in PCa.More importantly, in order to further explore the therapeutic potential of TNIK-targeted therapy in PCa, we detected two more bone metastasis markers (BMP6, BMP7) in PCa.Surprisingly, NCB-0846 also had inhibitory effects on PCa bone metastasis.These findings underscored the potential therapeutic value of targeting TNIK signaling to enhance sensitivity toward CRPC therapy and inhibit its progression and metastasis.

DISCUSSION
In the present study, we have elucidated crucial components of the interplay between the AR and EGFR signaling pathways via TNIK.By selectively inhibiting TNIK to impede EGFR phosphorylation, we effectively suppressed the proliferation of CRPC.
The previous study reported that TNIK was frequently upregulated in high-grade ovarian cancer tumors and serous hepatocellular carcinoma. 18Moreover, TNIK hyperactivity contributed to human lung adenocarcinoma cell metastasis. 17In this study, we initially identified TNIK as a potential biomarker of CRPC by analyzing gene array files from mouse models.We observed elevated expression levels of TNIK not only in CR-LNCaP tumors in mice but also in CRPC patients compared with those with localized PCa and benign prostatic hyperplasia (BPH) (Figure 1D).These findings suggest a correlation between TNIK and aggressive behavior in cancer.
The principal findings of our study revealed that AR forms a repressive complex with H3K27me3 at the TNIK promoter, leading to the suppression of TNIK transcription (Figure 3).Consequently, ADT induces TNIK mRNA expression, which in turn regulates EGFR phosphorylation to activate the EGFR signaling pathway and contribute to CRPC growth.The transcriptional activity of AR is regulated by interacting coactivators that positively modulate receptor function.9][30] A few reports have studied AR inhibition of transcription.Some hinted to indirect mechanisms-DNA methylation or protein phosphorylation 23,31 -whereas others hinted to direct mechanisms in involving the epigenetic silencing complex. 24,32Using TNIK as a model, we discovered that AR can also suppress gene expression through hormone-induced recruitment of H3K27me3 to the AR/H3K27me3 complex, thereby directly inhibiting transcription.Consequently, ADT restores TNIK expression by disrupting the association between AR and H3K27me3 at the TNIK promoter.
EGFR signaling pathway also plays a crucial role in CRPC.Substantial evidence has accumulated, indicating that the expression of EGFR is associated with an increased risk of high-grade, advanced disease, as well as prostate-specific antigen recurrence. 32Castration of mature animals can increase the expression of EGFR protein in the prostate in a time-dependent manner. 33Our findings also suggest that castration resistance may be induced by reciprocal interaction between TNIK and the EGFR signaling pathway, with TNIK playing a major role in promoting nuclear translocation and activation of EGFR (Figure 4).Phosphorylation of EGFR is crucial for its intracellular distribution and transcriptional activity.ECD domain can recognize and bind to specific ligands, thereby facilitating the activation of EGFR.In this study, we discovered that TNIK can interact with the ECD domain on EGFR to induce phosphorylation and enhance nuclear translocation of EGFR.When the ECD domain is deleted, TNIK loses its regulatory effect on EGFR and EGFR-associated ferroptosis sensitivity.It has been reported that inhibition of TNIK reduces cell viability in ERG-positive PCa cells DU145 and 22RV1. 24Our current data further demonstrate that TNIK has the ability to phosphorylate EGFR and promote transcriptional activation of its target genes.The use of TNIK inhibitors has shown promising inhibitory effects in CRPC cells and tumors (Figures 5 and 6).
Interestingly, TNIK also plays a regulatory role in ferroptosis.Ferroptosis is primarily caused by an imbalance between the generation and degradation of intracellular lipid reactive oxygen species (ROS), which is closely associated with cellular metabolic activity.In comparison to normal cells, tumor cells exhibit heightened metabolic rates and consequently face an increased susceptibility to ferroptosis.Previous studies have demonstrated that TNIK regulates glucose and lipid homeostasis in both Drosophila and mice. 34The knockout of TNIK has been shown to enhance glucose and lipid metabolism; however, there is currently no research linking TNIK to ferroptosis.Given its role as a crucial regulatory factor in CRPC, the upregulation of TNIK expression may serve as a cellular self-protective mechanism against ferroptosis.Similarly, AR also plays a significant protective role in PCa cells by inhibiting lipid peroxidation and preventing ferroptosis. 35hen AR is present, in order to sustain the normal metabolic activities necessary for cell growth, AR exerts an inhibitory influence on TNIK.During treatment, the reduction in AR levels triggers the activation of TNIK as a secondary defense mechanism against ferroptosis while concurrently promoting CRPC progression.The collaborative action of AR and TNIK ensures cellular metabolism homeostasis in PCa.NCB-0846, as a TNIK inhibitor, has shown potential therapeutic effects in a variety of tumors.As a regulatory component of the transcription complex composed of b-catenin and T cell factor 4 (TCF4), TNIK can regulate the Wnt signaling pathway related to stem cell activity.In colorectal cancer, where the Wnt signaling pathway is the most prominent, NCB-0846 has been shown to bind to TNIK in an inactive structure, thereby inhibiting the activation of the Wnt pathway and blocking intestinal tumorigenesis and tumorigenic activity. 20In addition, the combination of NCB-0846 and ABT-263 (BCL-X) inhibitor also showed synergistic inhibition of colorectal tumors in the KRAS mutant xenograft model. 36The inhibitory effect of NCB-0846 on Wnt target genes also shows good tumor suppressor effects in synovial sarcoma, inducing rapid apoptosis of synovial sarcoma cells. 37Similar to colorectal cancer and synovial sarcoma, the Wnt pathway is very important for the occurrence and development of PCa, and KRAS mutations also occur in PCa.The use or combination treatment of NCB-0846 in colorectal cancer and synovial sarcoma can provide a reference for the treatment of PCa.More importantly, NCB-0846 exhibits an inhibitory effect on lung cancer cell metastasis, which is mediated through the epithelial to mesenchymal transition (EMT) mechanism.NCB-0846 blocks the activation of Smad signaling and the induction of EMT by downregulating the expression of transforming growth factor beta (TGF-b) receptor type I. 38 In PCa, the metastasis and drug resistance caused by EMT have always been problems that trouble people.Interestingly, NCB-0846 can disrupt the transport and secretion of type I procollagen and inhibit the production of matrix proteins to regulate liver fibrosis. 39In PCa, matrix proteins can induce tumor cell metastasis and adhesion to bone cells. 39Although previous studies have demonstrated that NCB-0846 has a good therapeutic effect in ERG-positive PCa, whether it has the effect of regulating PCa EMT and bone metastasis has not been verified. 13Here, our findings demonstrate that AR functions as a transcriptional repressor in TNIK by binding H3K27me3 complex.When AR levels decrease, TNIK phosphorylated EGFR and activating EGFR pathway to escape from castration treatment and promoted CRPC progression.By inhibiting the TNIK/EGFR axis, NCB-0846 showed inhibitory effects on CRPC tumor cells both in vivo and in vitro and blocked EMT of tumor cells as well as bone metastasis.Through current research, we have reason to believe that NCB-0846 has great potential for the treatment of PCa.These results provide the possibility for the combined treatment of PCa with AR and TNIK, which may be the key to inhibiting the progression of CRPC and provide a reference for the launch of ferroptosis treatment.
prior to target labeling using the RiboMinusTM Human Transcriptome Isolation Kit (Invitrogen, CA, USA).Subsequently, cDNA synthesis was performed according to the manufacturer's instructions using the GeneChipâ WT Sense Target Labeling and Control Reagents Kit (Affymetrix, Inc., CA, USA).The resulting sense cDNA was fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidinic endonuclease 1), followed by biotin labeling with TdT (terminal deoxynucleotidyl transferase) using a GeneChipâ WT Terminal Labeling Kit (Affymetrix, Inc., CA, USA).Once the biotin-labeled sense target DNA was prepared, it underwent hybridization to a gene chip known as The GeneChipâ Human Exon 1.0 ST array.Hybridization involved incubating 5 mg of biotinylated target with a GeneChipâ Hybridization Wash and Stain Kit and a GeneChipâ Fluidics Station 450 (Affymetrix, Inc., CA, USA).Finally, the arrays were scanned using a GeneChipâ Scanner 3000 7G(Affymetrix，Inc., CA，USA), and raw data were extracted from scanned images for analysis utilizing Agilent Technologies'GeneSpring GX software version11.5.

Immunohistochemistry
The tissue sections were deparaffinized in xylene and rehydrated with graded alcohol.Antigen retrieval was performed under pressure for 5 minutes in citrate buffer (pH adjusted to 6.0).Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 minutes, followed by blocking with 1.5% horse serum.Primary antibody incubation was carried out overnight at 4 C in a humidified chamber.After applying Poly-HRP anti-rabbit IgG (30 min), secondary antibody detection was performed using the Ultraview DAB detection kit (Zhongshan Co., China).All immunostained sections were evaluated under a Zeiss microscope (3200).At least ten high-power fields around each malignant gland were assessed and scored.

MTT assay
After 48 hours of transfection, a total of 2.0 3 103 cells per well were seeded in 96-well plates and subjected to treatment with either DMSO or erastin at a temperature of 37 C for durations of 24, 48, 72, and 96 hours.Subsequently, each well was supplemented with 30 ml of MTT solution and incubated at a temperature of 37 C for a period specified by the experiment protocol (indicated time).Following this incubation period, the MTT solution was aspirated and replaced with an addition of dimethyl sulfoxide (DMSO) measuring up to a volume of150 ml per well in order to dissolve the formazan crystals.Finally, absorbance measurements were taken at a wavelength of 490 nm using a microplate reader.

Determination of lipid peroxidation
After 48 hours of transfection, a total of 5.0 3 106 cells were collected and transferred into a centrifuge tube.Then, 1 ml of extract solution was added to the tube followed by cell lysis and subsequent centrifugation at 4 C for 10 minutes.The resulting supernatant was carefully collected and mixed with the specified reagent (Jining Shiye, JN24889) according to the provided instructions.After thorough mixing, the mixture was heated at 90 C for 40 minutes and then subjected to another round of centrifugation to obtain a final volume of 1 ml supernatant.Finally, the absorbance at 490 nm was measured using a microplate reader.

QUANTIFICATION AND STATISTICAL ANALYSIS
The data were presented as mean G standard deviation.Student's t-test was used to compare two samples, and p values of 0.05 or less were considered statistically significant.Tumor weight was analyzed using GraphPad Prism software.All experiments in our study were repeated at least three times.Statistical significance is indicated with *P <.05, **P < .01,***P < .005.

Figure 1 .
Figure 1.TNIK was upregulated in castration-resistant prostate cancer (A) Heatmap of the genome-wide transcript profile between (CR-LNCaP) castration tumors and (HS-LNCaP) uncastration tumors.(B) GO analysis of transcript profile.(C) qPCR detects mRNA expression of TNIK in (CR-LNCaP) castration tumors and (HS-LNCaP) uncastration tumors (p < 0.05).Data are represented as mean G SD. (D) Clinical specimens of benign prostate hyperplasia (BPH), hormone native, and CRPC were analyzed by IHC for TNIK expression.Scale bars: 200 mm.H-score of TNIK immunoreactivity in HSPC, CRPC, and their paracancerous tissues.Data were presented as median (interquartile range).Data are represented as mean G SD.The hash symbol indicates significantly different from Para (HSPC) group (p < 0.05); an asterisk indicates significantly different from Para (CRPC) group (p < 0.05).

Figure 3 .Figure 4 .
Figure 3. Continued (D) The levels of TNIK protein were measured in LNCaP cells treated for 24 h with the AR antagonist MDV3100 (enzalutamide, 100 nM).(E) Coimmunofluorescence (co-IF) analysis of TNIK and AR proteins in LNCaP treated with MDV3100.Scale bars: 50 mm.(F) Immunoprecipitation of AR or H3K27me3 in LNCaP and C4-2 cells followed by immunoblot analysis of H3K27me3 or AR.IgG represents a control antibody used for IPs.(G) ChIP-PCR analysis of H3K27me3 and AR binding to the TNIK gene promoter in LNCaP cells treated with MDV3100.ChIP-PCR primer in TableS3.Data are shown as mean G SD. ***p < 0.005.(H and I) LNCaP and C4-2 cells were treated with DZNep (0, 5, 10 mm) and GSKJ1 (0, 5, 10 mm) for 8 h, WB analysis of TNIK protein expression.(J) Western blot showed that treatment of the indicated LNCaP or C4-2 cells with GSK-J1 could reverse the effects of AR knockdown on TNIK.

Figure 4 .
Figure 4. Continued (G) Expression of a TNIK kinase mutant (D171A) reduces phosphorylation of EGFR.(H) The levels of p-EGFR and NRF2 protein were measured in C4-2 cells after knocking down the ECD domain.(I) Levels of cell death in C4-2 cells treated with 4 mm erastin (up).Levels of lipid peroxidation in C4-2 cells following the same treatment (down).Data are shown as mean G SD. ***p < 0.005.