Ack1-mediated Androgen Receptor Phosphorylation Modulates Radiation Resistance in Castration-resistant Prostate Cancer*

Background: The molecular mechanisms of acquisition of radioresistance in CRPC are not fully understood. Results: Ack1/AR signaling modulates ATM expression to promote radioresistance. Conclusion: Ack1/AR signaling plays a critical role in acquisition of radioresistance in CRPC by modulating the DNA damage response pathways. Significance: Ack1/AR signaling represents a new paradigm of radioresistance in CRPC that can be targeted with AIM-100. Androgen deprivation therapy has been the standard of care in prostate cancer due to its effectiveness in initial stages. However, the disease recurs, and this recurrent cancer is referred to as castration-resistant prostate cancer (CRPC). Radiotherapy is the treatment of choice; however, in addition to androgen independence, CRPC is often resistant to radiotherapy, making radioresistant CRPC an incurable disease. The molecular mechanisms by which CRPC cells acquire radioresistance are unclear. Androgen receptor (AR)-tyrosine 267 phosphorylation by Ack1 tyrosine kinase (also known as TNK2) has emerged as an important mechanism of CRPC growth. Here, we demonstrate that pTyr267-AR is recruited to the ATM (ataxia telangiectasia mutated) enhancer in an Ack1-dependent manner to up-regulate ATM expression. Mice engineered to express activated Ack1 exhibited a significant increase in pTyr267-AR and ATM levels. Furthermore, primary human CRPCs with up-regulated activated Ack1 and pTyr267-AR also exhibited significant increase in ATM expression. The Ack1 inhibitor AIM-100 not only inhibited Ack1 activity but also was able to suppress AR Tyr267 phosphorylation and its recruitment to the ATM enhancer. Notably, AIM-100 suppressed Ack1 mediated ATM expression and mitigated the growth of radioresistant CRPC tumors. Thus, our study uncovers a previously unknown mechanism of radioresistance in CRPC, which can be therapeutically reversed by a new synergistic approach that includes radiotherapy along with the suppression of Ack1/AR/ATM signaling by the Ack1 inhibitor, AIM-100.

stood. Thus, identification of gene(s) modulated by androgen independent AR, which facilitates survival of irradiated CRPC cells is crucial to provide a better understanding of the molecular pathway(s) that confer radioresistance.
Genetic integrity is monitored by components of the DNA damage response pathways, which rapidly respond to perturbations in genetic integrity to coordinate processes that pause cell cycle to allow time for repair and evade cell death (17). The ATM (ataxia telangiectasia mutated) gene product is a major player in the DNA damage and cell cycle checkpoint signaling pathways and is vital to ensure genetic stability within cells (18 -21). Although high levels of ATM expression are correlated with radioresistance, and conversely, the presence of missense mutations in the ATM gene is predictive of poor radiotherapy response and enhanced radiosensitivity (22)(23)(24), the molecular mechanisms by which cancer cells acquire increased ATM expression is not known.
To understand the molecular basis of radiation resistance of CRPC cells, we performed ChIP-on-chip analysis, which revealed the specific recruitment of pTyr 267 -AR⅐Ack1 complex to the ATM gene enhancer. ATM mRNA and consequently protein expression is modulated by the Ack1-mediated phosphorylation of AR in prostate cancer cell lines, which is antagonized by the selective Ack1 inhibitor AIM-100. Furthermore, AIM-100 suppressed growth of radioresistant CRPC xenograft tumors by decreasing ATM expression. Thus, our data reveals for the first time the molecular basis by which the oncogenic kinase Ack1 directly modulates radiation resistance of the aggressive form of prostate cancer.
Chromatin immunoprecipitation (ChIP) and ChIP-on-chip-LAPC4 (5 ϫ 10 7 cells) were either untreated or treated with EGF ligand for 45 min. Cells were treated with 1% formaldehyde for 10 min, lysed, homogenized, and centrifuged. The pellet was suspended in shearing buffer and sonicated for 25 s. The soluble chromatin was incubated overnight at 4°C with pTyr 267 -AR antibody or total AR antibody and protein G-magnetic beads. The soluble chromatin was processed in the same way without immunoprecipitation and termed input DNA. The amount of immunoprecipitated DNA was determined by real time PCR. In the second step, PCR-amplified immunoprecipitated DNAs were ligated to short oligonucleotides to the ends. The PCR reactions were assembled and labeled with biotin using the NuGen FL-Ovation Biotin V2 kit. This fragmented material was hybridized to Affymetrix Human promoter 1.0R arrays following the Affymetrix procedure. The primer sequences amplifying the pTyr 267 -AR binding region in ATM enhancer is shown in supplemental Table 4.
Ack1 Transgenic and Xenograft Mice-Mice breeding and colony maintenance was performed according to Institutional Animal Care and Use Committee protocols approved by University of Florida Division of Research Integrity and Compliance. LNCaP-caAck cells (2 ϫ 10 6 ) were suspended in 100 l of PBS and 100 l of Matrigel (Discovery Labware, Bedford, MA) and injected subcutaneously into the flanks of male nude castrated mice. Mice were injected with AIM-100 (4 mg/kg of body weight per injection) on the seventh day post-injection of cells (n ϭ 8 mice for each treatment). Five injections of AIM-100 were followed on 11th, 15th, 19th, 23rd, and 27th day. Tumor volumes were measured twice weekly using calipers. Two independent experiments were performed; a representative data set is shown.
IC 50 (nM) Determination for AIM-100-Kinase assays were performed at Reaction Biology Corp. (Malvern, PA) using the "HotSpot" assay platform. In brief, substrates were freshly prepared, and required cofactors were added individually to each kinase reaction. Indicated kinases were added into the substrate solution followed by addition of AIM-100 (or staurosporine) in dimethyl sulfoxide into the kinase reaction mixture. [ 33 P]ATP (specific activity, 0.01Ci/l final concentration) was added into the reaction mixture to initiate the reaction, followed by incubation of kinase reaction for 120 min at room temperature. The reactions are spotted onto P81 ion exchange paper (Whatman no. 3698-915), and filters were washed extensively in 0.1% phosphoric acid.
Tissue Microarray (TMA) Analysis-For assessment of ATM expression in human prostate cancer, immunohistochemistry was carried out on high-density TMAs (n ϭ 250 cores) containing samples of different stages of disease as described previously (3,10). For statistical assessment of ATM expression in prostate cancer, box plots were used to summarize the intensity sampling distribution at each progression stage. The Spearman rank correlation coefficient was estimated to access the relationship between ATM levels and progression stages and groups of prostate cancer. The progression stages from Benign prostatic hyperplasia, Prostatic intraepithelial neoplasia, G6, G7, G8 -10, and CRPC were used for the correlation analysis. Analysis of variance (v) was performed to examine whether the ATM expression levels differ among different tumor stages. The Tukey-Kramer method was further performed to examine between which pairs of stages the expression levels are different. This post hoc procedure adjusts for pairwise comparisons and simultaneous inference. The Spearman rank correlation coefficient also was estimated to access the correlation between ATM levels and pTyr 267 -AR.
Immunohistochemical Staining of Xenograft Tumors-For assessment of ATM expression in xenografts, the xenograft tumors were excised, fixed, paraffin-embedded, and sectioned. Antigen retrieval was performed by heating the samples at 95°C for 30 min in 10 mmol/liter sodium citrate (pH 6.0). After blocking with universal blocking serum (DAKO Diagnostic, Mississauga, Ontario, Canada) for 30 min, the samples were then incubated with rabbit monoclonal ATM antibody (1:300 dilution; Epitomics) at 4°C overnight. The sections were incubated with biotin-labeled secondary and streptavidin-peroxidase for 30 min each (DAKO Diagnostic). The samples were developed with 3,39-diaminobenzidine substrate (Vector Laboratories, Burlington, Ontario, Canada) and counterstained with hematoxylin. The blue staining in Fig. 5C is due to hematoxylin staining. The oxidized hematoxylin colors nuclei of cells (and a few other objects, such as keratohyalin granules) blue.
Cell Cycle Analysis-LNCaP or LAPC4 cells were untreated, treated with 10 M AIM-100 alone, with 5 Gy IR, or incubated overnight with AIM-100 followed by 5 Gy IR. IR treatment was performed with an x-ray machine (XRAD160 Precision x-ray, Bradford, CT). Cells were harvested after 16 h. Cells were stored in 100 l of sodium citrate buffer. Cells were trypsinized in 450 l of trypsin solution at room temperature for 10 min, incubated in 375 l of trypsin inhibitor/RNase A (Sigma) solution for 10 min, stained with 250 l of ice-cold propidium iodide solution for 10 min, and kept in the dark. Samples were analyzed using the FACSCalibur flowcytometer, 10,000 events were collected, and cell cycle analysis was carried out using the ModFit program.
EdU Assay-For rapid detection of DNA synthesis in proliferating cells, we used the chemical method, which is based on the incorporation of 5-ethynyl-2Ј-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)catalyzed [3 ϩ 2] cycloaddition reaction ("click" chemistry) (25). Reagents are available as a kit from Invitrogen (catalog no. 350002). Cells were treated with IR or AIM-100 as described.
EdU was added at a final concentration of 10 M for 2 h, and cells were harvested. EdU-labeled cells were fixed with 4% paraformaldehyde, and cells were processed for measuring DNA synthesis as described in the Click-it-Edu Alexa Flour 488 Flow Cytometry Assay protocol (Invitrogen).
In Situ Labeling of DNA Strand Breaks-LAPC4 or LNCaP cells were treated with individually with AIM-100, ␥-irradiated (15 Gy) or pretreated with AIM-100, followed by ␥-irradiation, and fixed after 30 min or 5 h with 1% paraformaldehyde. DNA strand breaks were directly labeled with dUTP-FITC by using Terminal Deoxynucleotidyl transferase (Biovision catalog no. K401-60), and detected by flow cytometry as described previously (26).

pTyr 267 -AR Initiates Distinct Transcriptional Program-Our
earlier studies have demonstrated that Ack1-mediated AR Tyr 267 -phosphorylation is critical for the progression of prostate cancer to CRPC stage (2). To identify novel genes modulated by pTyr 267 -AR, we performed chromatin immunoprecipitation and tiled microarray (ChIP-on-chip) analysis. The specificity of the pTyr 267 -AR antibody for ChIP analysis has been validated extensively and reported (10). Earlier, we have demonstrated that prostate-derived LAPC4 cells expressing wild type AR exhibit robust Ack1 activation upon EGF ligand treatment, which in turn, phosphorylates AR at Tyr 267 (2). LAPC4 cells depleted of androgen and serum were either treated or untreated with EGF ligand, and ChIP was performed with pTyr 267 -AR antibodies. A false discovery rate cut-off of Ͻ0.6 yielded 219 sequences (supplemental Table 1a) mostly in the enhancer regions (supplemental Table 1b). We compared peaks identified in this pTyr 267 -AR ChIP-on-chip experiment with the published dihydrotestosterone (DHT) bound-AR ChIP-on-chip studies (27) and observed little overlap (supplemental Table 2). Only 10 peaks were found to be overlapping, which indicates that pTyr 267 -AR initiates a distinct transcriptional program than DHT-bound AR.
The signaling pathways that are associated with enriched genes that correspond to pTyr 267 -AR ChIP-on-chip binding peaks are shown in Table 1. It includes three members of the p53-dependent DNA damage signaling pathway: p300, MDM2, and ATM. pTyr 267 -AR was bound to a 364-bp fragment, located on chromosome 11, at 2414 nucleotides upstream of the ATM gene transcription start site (supplemental Fig. S1). ATM is a critical upstream regulator of the DNA damage signaling and checkpoint pathway that maintains genetic integrity and facilitates DNA repair in response to DNA damage. The direct up-regulation of ATM by tyrosine phosphorylated AR has not been reported earlier and therefore provides a novel target for combating the radioresistance of prostate cancer cells by inhibiting AR/Ack1 function in cells.
To assess whether ATM is a target for Ack1/AR signaling, LAPC4 cells were treated with EGF and equal amount of the whole cell extracts were immunoblotted with ATM, pTyr-Ack1, Ack1, CHK2, and tubulin antibodies. A significant increase in pTyr 284 -Ack1 and ATM protein levels was seen upon EGF treatment, whereas total Ack1, CHK2 and tubulin levels were unchanged (Fig. 1A).
AIM-100 Specifically Inhibits Ack1 Kinase Activity-To assess the role of Ack1 in pTyr 267 -AR/ATM signaling, a Ack1specific inhibitor is desirable. 4-amino-5,6-biaryl-furo[2, 3-D]pyrimidine derivative, also known as AIM-100, has been identified as a potent inhibitor of Ack1 activity in vitro (10,28). However, the kinase specificity and tumor inhibitory effect of AIM-100 was not known. To establish the specificity of AIM-100, we synthesized and determined IC 50 values by performing A, LAPC4 cells were either untreated or treated with EGF (10 ng/ml, 1 h). Equal amounts of whole cell extracts were analyzed by immunoblotting with the indicated antibodies (Ab). B, pTyr 267 -AR binding to the ATM enhancer in prostate cells. Serum and androgen-depleted LAPC4 cells were either treated (or untreated) with DHT (5 nM, 16 h) or AIM-100 (1uM, 16 h) or EGF (10 ng/ml, 45 min), and EGFϩAIM-100. ChIP of pTyr 267 -AR bound to the ATM enhancer was performed, followed by quantitative PCR (*, p ϭ 0.013). C, LAPC4 cells were both untreated or treated with AIM-100 (1 M, 16 h), and EGF was added (10 ng/ml, 1 h). Equal amounts of whole cell extracts were analyzed by immunoblotting with indicated antibodies. D, LAPC4 cells were either untreated or treated with AIM-100 (2 M, 16 h), and EGF was added (10 ng/ml, 1 h). Total RNA was extracted and analyzed by quantitative RT-PCR. Data are representative of three independent experiments (*, p ϭ 0.014). E, the schematic of two luciferase reporter construct with the pTyr 267 -AR binding site (ATM-pARE) and deletion construct lacking them (ATM-␦pARE) are shown. F, LAPC4 cells were transfected with the ATM-pARE or ATM-␦pARE luciferase reporter constructs (500 ng). Twenty-four hours after transfection, cells were treated with AIM-100 (10 M) for 16 h and followed by EGF (10 ng/ml) for 3 h, and luciferase activity was determined (*, p ϭ 0.01). G, LAPC4 cells were either transfected with control or Ack1 siRNAs followed by EGF (10 ng/ml, 45 min), AIM-100 (1 M, 16 h), or DHT treatment. ChIP analysis for pTyr 267 -AR binding to the ATM enhancer was performed, followed by quantitative PCR (*, p ϭ 0.013). H, LAPC4 cells were transfected with vector (control) FLAG-tagged AR or the mutated FLAG-tagged ARY267F constructs and were either unstimulated or stimulated with EGF ligand for 45 min. ChIP was performed using FLAG antibodies followed by real time PCR for the ATM enhancer.
kinase assays in the presence of increasing concentrations of AIM-100 or with staurosporine as control ( Table 2). It revealed that AIM-100 specifically inhibits Ack1 with an IC 50 of 21 nM but not other kinases, including all the tested PI3K subfamily (to which ATM belongs) members such as ATR (ataxia telangiectasia and Rad3-related), DNA-dependent protein kinase, and mTOR (mammalian target of rapamycin).
pTyr 267 -AR Is Recruited Specifically to ATM Enhancer-To confirm pTyr 267 -AR binding to the ATM enhancer region, LAPC4 cells were either untreated or treated with DHT or EGF (1 h) or EGF plus AIM-100. ChIP using pTyr 267 -AR antibodies followed by real time PCR for the ATM enhancer region revealed that pTyr 267 -AR was recruited specifically to the ATM enhancer upon EGF stimulation (Fig. 1B). As a negative control, we performed ChIP using pTyr 267 -AR and AR antibodies in DU145 cells that lack functional AR expression, which exhibited no binding to the ATM enhancer (data not shown). Consistent with the requirement for AR Tyr 267 phosphorylation, AIM-100 treatment that specifically inhibits Ack1-mediated AR Tyr 267 phosphorylation (Fig. 1B) inhibited AR recruitment to the ATM enhancer (Fig. 1A). To assess the effect of pTyr 267 -AR recruitment on ATM protein expression, LAPC4 cells were either untreated or pretreated with AIM-100 alone or with AIM-100, and EGF was added. EGF-treated cells exhibited significantly higher levels of pTyr-Ack and pTyr-AR, which correlated with increased ATM levels; however, upon AIM-100 treatment, significant loss of pTyr-Ack/pTyr-AR levels and a concomitant decrease in ATM protein expression was observed (Fig. 1C). In contrast, protein expression of the two neighboring genes of ATM, NPAT, and ACAT were not altered (Fig. 1C). Consistent with LAPC4 data, LNCaP prostate cancer cells too exhibited significant decrease in ATM protein expression upon AIM-100 treatment (Supplemental Fig. S2A).
LAPC4 cells treated with EGF or AIM-100 followed by quantitative RT-PCR revealed up-regulated ATM mRNA levels upon EGF treatment while AIM-100 treatment abrogated this increase (Fig. 1D). ATM mRNA up-regulation was further confirmed in LNCaP cells stably expressing activated Ack1 or caAck (1, 2); AIM-100 treatment resulted in a significant decrease in ATM mRNA levels (supplemental Fig. S3A). In contrast, DHT-treated LAPC4 cells did not reveal any changes in ATM mRNA levels (supplemental Fig. S3B). The AR-responsive gene PSA was used as control for DHT-mediated stimulation. To determine whether pTyr 267 -AR binding to the ATM enhancer affects the neighboring genes of ATM, relative mRNA levels of ACAT, C11ORF65, KDELC2, and NPAT were determined. Real time quantitative RT-PCR revealed that RNA levels of all four neighboring genes were not altered (supplemental Fig. S2B). Taken together, these data indicate Ack1 dependent pTyr 267 -AR recruitment specifically to the ATM enhancer, which can be suppressed by AIM-100.
Characterization of Tyr 267 -phosphorylated AR Recruitment to ATM Enhancer-The ATM gene is large, spanning ϳ150 kb of genomic DNA and encodes a mRNA of ϳ9.4 kb and a protein of 3056 amino acids or 350 kDa (29). With such a large RNA molecule, a robust increase in mRNA levels may not be apparent; however, the modest increase in mRNA levels translated into significant increase in the protein levels (Fig. 1C). A similar modest increase in ATM mRNA levels has been reported in the literature (30). We observed that the pTyr 267 -AR is recruited to the 364-bp ATM enhancer sequence, which contains two ARElike half-sites, TGTTCT (supplemental Figs. S1 and S5A). To further validate the pTyr 267 -AR recruitment to the ATM enhancer, luciferase reporter constructs containing 364-bp ATM enhancer regions (ATM-pARE) or lacking it (ATM-␦pARE) were constructed (Fig. 1E). EGF treatment of LAPC4or heregulin-treated LNCaP cells transfected with ATM-pARE exhibited significant increase in luciferase activity, which was inhibited by AIM-100 ( Fig. 1F and supplemental Fig. S4A). In contrast, DHT-treated ATM-pARE exhibited minimal luciferase activity (supplemental Fig. S5B). As a positive control, LAPC4 cells were transfected with AR-responsive reporter construct ARR2PB, which exhibited high luciferase activity upon DHT treatment (supplemental Fig. S5B). These data indicate that AR recruitment to the ATM enhancer is dependent on AR phosphorylation at Tyr 267 .
To evaluate further the underlying mechanism of pTyr-AR binding to the ATM enhancer, we assessed the role of Ack1. Earlier, we have demonstrated that Ack1 specifically phosphorylates AR and the pTyr-Ack1⅐pTyr-AR complex translocates to the nucleus (2). It led us to hypothesize that Ack1 may be recruited to the ATM enhancer as a component of the pTyr 267 -AR transcriptional complex. LAPC4 and LNCaP cells were transfected with control or Ack1 siRNAs followed by ligand and/or AIM-100 treatment. ChIP with the pTyr 267 -AR antibodies followed by real time PCR of the ATM enhancer revealed that Ack1 knockdown resulted in significant decrease in pTyr 267 -AR binding to the ATM enhancer ( Fig. 1G and supplemental Fig. S4B). Moreover, knockdown of Ack1 lead to sig-

Ack1/AR Signaling Regulates Radiation Resistance
nificant decrease in ATM levels (supplemental Fig. S6A). Collectively, these data reveal that Ack1⅐pTyr-AR complex is recruited to the ATM enhancer and Ack1 is required for pTyr-AR mediated optimal transcriptional up-regulation of ATM gene expression. AR Phosphorylation at Tyr 267 Promotes Binding to ATM Enhancer and Activates Transcription-To determine whether tyrosine 267 phosphorylation of the androgen receptor is required for binding to the ATM enhancer site, vector (control), FLAG-tagged AR, or the mutant FLAG-tagged ARY267F constructs were transfected into LAPC4 prostate cell lines. These cells were either untreated or stimulated with EGF after serum starvation. ChIP was performed using FLAG antibodies followed by real time PCR for the ATM enhancer. AR bound to the ATM enhancer upon EGF stimulation. In contrast, ARY267F mutant was compromised significantly in its ability to bind to the ATM enhancer (Fig. 1H).
To determine the effect on ATM transcriptional activation, luciferase assay was performed. HEK293 cells that have undetectable levels of endogenous AR expression were transfected with AR or ARY267F mutant and luciferase reporter constructs containing the ATM enhancer region, ATM-pARE (shown in Fig. 1E). 24 h post-transfection, cells were treated with AIM-100 overnight. 48 h post-transfection, cells were treated with EGF ligand, and luciferase activity was measured. A significant increase in luciferase activity was seen in AR-transfected cells, in contrast, ARY267F mutant expressing cells exhibited minimal luciferase activity (supplemental Fig. S6B). Taken together, these results suggested that AR phosphorylation at Tyr 267 promotes recruitment to the ATM enhancer and activates ATM transcription.
Probasin-Ack1 Transgenic Mice Display AR Tyr 267 Phosphorylation and ATM Up-regulation-To determine whether upregulation of ATM expression by pTyr-Ack1/pTyr-AR signaling occurs in vivo, we developed a transgenic mouse model in which Myc-tagged activated Ack1 was driven by a probasin promoter (PB-Ack) (3). These mice display a significant increase in Ack1 Tyr 284 phosphorylation and developed mouse prostatic intraepithelial neoplasia by 44 weeks. A few older mice developed adenocarcinoma in dorsal lobe of prostate by ϳ50 weeks (3). The prostate lysates of PB-Ack transgenic mice were immunoblotted with pTyr 267 -AR and ATM antibodies. In contrast to wild type littermates (WT), transgenic or transgenic mice display significant increase in AR Tyr 267 phosphorylation and ATM up-regulation (Fig. 2, A, top two panels, and B). Immunohistochemical staining of PINs revealed an increase in pTyr267-AR and the ATM protein staining in transgenic mice compared with the prostates of WT mice (Fig. 2C).

Up-regulation of ATM Expression Correlates with Human Prostate Cancer Progression to Castration Resistance-To
investigate the pTyr 267 -AR mediated up-regulation of the ATM levels in primary human CRPCs, we performed ATM immunohistochemical analysis of TMA of clinically annotated prostate tumor samples (n ϭ 250; for CRPC samples, see supplemental Table 3A). Generation of prostate TMA was described previously (10). A significant increase in the expression of ATM was observed when prostate cancers from progressive stages were examined (Fig. 3A), which was positively correlated with the severity of disease progression (r ϭ 0.44, p Ͻ 0.0001; Fig. 3B). Analysis of variance results indicated that ATM expression differed significantly among progression stages and CRPC (p Ͻ 0.0001). The results from all pair wise comparison using the Tukey-Kramer method for ATM staining between different groups are summarized in supplemental Table 3b. Previously, we observed increased pTyr-Ack1/pTyr-AR levels in CRPC tumors (2,3,10). Upon comparison, a significant positive correlation between the levels of pTyr 267 -AR and ATM expression was observed in the prostate TMA (Spearman rank correlation coefficient r ϭ 0.26, p Ͻ 0.0001) suggesting that prostate tumors expressing high levels of pTyr 267 -AR are likely to have up-regulated ATM levels (Fig. 3C).
Ack1 Inhibitor, AIM-100, Overrides IR-induced G 2 /M Checkpoint-ATM, a DNA damage sensor, rapidly activated by double strand breaks (DSBs) caused by ionizing radiation, activates signaling cascades to inhibit cell cycle progression and promote DNA repair processes (17,31,32). ATM is autophosphorylated at serine 1981 upon DSB induction (19) and in turn phosphorylates key checkpoint effectors such as p53 in response to DNA damage (17,33). We tested the effect of AIM-

FIGURE 2. PB-Ack1 transgenic mice display increased pTyr 267 -AR and ATM protein expression.
A, 45-week-old PB-Ack1 transgenic (TG) mice with PIN and WT mice prostate lysates were subjected to immunoblotting using ATM monoclonal antibody (Genetex, ATM2C1) and actin antibodies (top and fourth panel, respectively). Lysates were also immunoprecipitated using anti-pTyr antibodies followed by immunoblotting with pTyr 267 -AR antibodies (second panel) and anti-Myc antibodies followed by immunoblotting with Ack1 antibodies (third panel). B, ATM expression in transgenic and WT mice (n ϭ 5, each) was determined using immunoblotting followed by quantification by SCION Image software. A significant increase in ATM levels in transgenic mice was seen compared with the WT mice (*, p ϭ 0.016). C, hematoxylin and eosin (H&E) and immunohistochemical staining of WT and transgenic mice prostates. Histological appearance of the prostate lateral lobe from a WT mouse and corresponding lobe from age-matched transgenic mouse with PIN are shown (H&E staining, the first two panels). A significant increase in pTyr 267 -AR (middle panels) and ATM (last panels) protein levels is observed in prostates of transgenic mice.

Ack1/AR Signaling Regulates Radiation Resistance
100 on the DNA repair and checkpoint functions of ATM in response to IR-induced DNA damage. IRϩAIM-100-treated cells exhibited loss of ATM Ser 1981 phosphorylation and loss of ATM substrate phosphorylation, p53 at Ser 15 (Fig. 4A), suggesting that by inhibiting ATM expression, AIM-100 suppresses ATM autoactivation and ATM-mediated activation of other cell cycle effectors. Furthermore, cell cycle analysis was performed on cells that were irradiated in the presence and absence of the Ack inhibitor. The majority of the untreated cells were in the G 1 /S phase of the cell cycle; however, upon IR treatment, a substantial increase in the number of cells in G 2 phase (84%) was observed, indicative of G 2 /M arrest. When cells were treated with both AIM-100 and IR, the proportion of cells in G 2 phase was decreased significantly (84 to 38%), indicating the loss of G 2 arrest (Fig. 4B).
To assess whether IRϩAIM-100-treated cells had unrepaired DSBs, DSB quantitation assay was performed as described previously (26). IR treatment alone caused DSBs, which were repaired in 5 h, as seen by significant decrease in FITC-positive cells (Fig. 4, C and D). In contrast, the number of cells with DSBs remained high even after 5 h of post-IR incubation in IRϩAIM-100-treated cells, suggesting compromised DNA repair ability of these cells that failed to arrest due to the loss of G 2 arrest (Fig. 4, C and D). As a control, the cells were pretreated with ATM inhibitor KU-55933 followed by IR (34). Irradiated prostate cancer cells treated with the ATM inhibitor also showed a similar decrease in the number of cells in G 2 as seen with AIM-100 (Fig. 4E). Thus, similar outcomes in both the inhibitor treatments suggest that AIM-100 inhibits IR induced ATM G 2 /M checkpoint activity.
Inhibition of Ack1/pTyr-AR Radiosensitizes CRPC Tumor Growth-Activation of the DNA damage checkpoints in response to DSBs is known to promote radioresistance (35,36). To assess whether preferential up-regulation of ATM expression by pTyr 267 -AR may confer radioresistance, we used LNCaP cells that are stably transfected with activated Ack1 (LNCaP-caAck cells) (1). LNCaP-caAck cells exhibit AR Tyr 267 -phosphorylation and promote castration-resistant growth of xenograft tumors (2). To assess the radiosensitivity, clonogenic assay was performed, which revealed that in contrast to LNCaP cells, LNCaP-caAck cells formed a significantly higher number of colonies at high doses of radiation ( Fig. 5A  and supplemental Fig. S7A). Unirradiated or irradiated LNCaP-caAck cells were injected in castrated nude mice. One week after injection when palpable tumors were noticed, AIM-100 was injected, and tumor growth was monitored. Unirradiated LNCaP-caAck cells formed robust xenograft tumors, which showed ϳ50% decrease upon AIM-100 treatment (Fig. 5B). Notably, the irradiated LNCaP-caAck cells too formed tumors; however, their growth was significantly suppressed on injection with AIM-100 (Fig. 5B). We assessed the body weight of the dimethyl sulfoxide and AIM-100-injected castrated nude mice during the course of the experiment and did not observe any morbidity or statistically significant differences in the body weight of the two groups (supplemental Fig. S7B). The CRPC xenografts were excised and stained with the ATM antibodies, revealing high levels of ATM in dimethyl sulfoxide-injected xenografts before and after irradiation; however, upon treat-ment with AIM-100, ATM levels were significantly down-regulated (Fig. 5C). Furthermore, a significant necrosis in AIM-100-treated mice xenografts was observed (H&E stained lower panel, Fig. 5C), which explains the significant decrease in the tumor volume that was seen in Fig. 5B.
ATM, Primary Target of pTyr-Ack1/pTyr-AR Signaling-To validate that ATM is the main target of the action of AIM-100, we performed the "rescue" experiment. We observed that irradiated LNCaP-caAck xenografts injected with AIM-100 exhibited minimal tumor growth (Fig. 5B), suggesting that AIM-100- LNCaP cells were untreated or treated with IR (5 Gy) or IR and AIM-100 or KU55933 (10 M, 24 h). Cells were stained with propidium iodide, and DNA content was measured. The experiment was performed in triplicate, and a representative data set is shown. mediated suppression of ATM expression resulted in a failure to repair of DSBs and thus the susceptibility to irradiation. We hypothesized that expression of ATM from promoter that is not regulated by pTyr 267 -AR might allow irradiated LNCaP-caAck xenografts to grow in the presence of AIM-100. LNCaP-caAck cells were transfected with FLAG-tagged kinase-dead (KD)-ATM or wild type (WT)-ATM expressing constructs that are regulated by CMV promoter (Fig. 6A). Cells were irradiated and injected in castrated mice, and later, the mice were injected with AIM-100. The KD-ATM-expressing cells exhibited minimal tumor growth; however, Wt-ATM expressing cells exhibited significant tumor growth (Fig. 6B), indicating that AIM-100-mediated inhibition of Ack1/pTyr-AR/ATM signaling is mainly responsible for the radiosensitization of xenografts.

DISCUSSION
Our studies collectively demonstrate a novel mechanism in which Ack-induced phosphorylation of AR up-regulates the expression of ATM in prostate cancer cells. ATM is a critical regulator of the DNA damage checkpoint response pathways, which ensures genetic integrity and cell survival. Checkpoint genes ensure genetic integrity by arresting the cell cycle to facilitate repair. In the absence of repair, these cells can undergo apoptosis or cell senescence or subvert checkpoints to have pathological consequences by resuming the cell cycle progression upon checkpoint termination (37). Thus, up-regulation and activation of the DNA damage checkpoint protein, ATM, in irradiated prostate cancer cells is likely to increase the DNA repair capacity and facilitate recovery of cells, underlying tumor radioresistance (38). Consistent with our observation, previous studies demonstrate that the glioma stems cells display preferential activation of the DNA damage checkpoint response pathways resulting in increased radioresistance in malignant brain cancer (35).
Radiation therapy is the primary treatment of choice for localized prostate cancer. However, resistance to ionizing radi- ation remains a major obstacle in CRPC therapy. Therefore, inhibitors of the DNA damage checkpoint pathway such as ATM, CHK1, and CHK2 inhibitors, that radiosensitize tumor cells and potentiate cytotoxicity are promising chemotherapeutic options (34,39,40). However, because they can also compromise repair of damaged DNA of the neighboring normal tissues their use is limited. Thus, inhibitors targeting the signaling pathways that confer radioresistance in specific tissues such as the prostate are urgently needed. Currently, limited therapeutic options are available for men with CRPC, and thus the treatment of CRPC remains a challenging proposition as most of the agents have been modestly effective against the CRPC (41). Our data has uncovered a new signaling mechanism that indicates ATM up-regulation due to the activation of Ack1/AR signaling in CRPC. Cells expressing activated Ack1 are significantly radioresistant as seen in clonogenic survival assays and in their ability to form xenograft tumors even after irradiation (Fig. 5, A and B). Treatment of the radioresistant CRPC cells with AIM-100 suppressed ATM expression, which was reflected in a failure of the CRPC cells to repair the DSBs upon irradiation (Figs. 4 and 5). Consistent with these data, combination of AIM-100 treatment with irradiation resulted in radiosensitization of the CRPC cells, leading to suppression of castration resistant xenograft tumors in mice (Fig. 5B). Thus, Ack1 inhibitors are likely to selectively radiosensitize prostate cancer tissues that are dependent on AR for their growth without affecting other tissues that lack AR expression. In summary, our work identifies a signaling nexus consisting of three key players ATM, a DNA damage-dependent kinase, Ack1, a growth factor regulated tyrosine kinase, and steroid receptor AR that play a crucial role in radioresistance of hormonally insensitive prostate cancer. The cross-talk between them defines a previously unknown mechanism by which metastatic prostate cancer cells survive IR-induced killing. Consequently, inhibition of the signaling by synergistic approach that includes both radiotherapy and chemotherapy with selective Ack1 inhibitors opens up a novel therapeutic option for CRPC patients.