Calmodulin-Androgen Receptor (AR) Interaction: Calcium- Dependent, Calpain-Mediated Breakdown of AR in Lncapprostatecancer Cells

Research Article

Calmodulin-Androgen Receptor (AR) Interaction: Calcium- Dependent, Calpain-Mediated Breakdown of AR in LNCaPProstateCancer Cells

Ronald P. Pelley,1 Kannagi Chinnakannu,1 Shalini Murthy,1 Faith M. Strickland,2 Mani Menon,1 Q. Ping Dou,3 Evelyn R. Barrack,1 and G. Prem-Veer Reddy1,3

1Vattikuti Urology Institute and 2Department of Dermatology, Henry Ford Hospital; 3Karmanos Cancer Institute and Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan

Abstract Introduction Chemotherapy of prostate cancer targets androgen receptor Adenocarcinoma of the prostate is the most frequently (AR) by androgen ablation or antiandrogens, but unfortu- diagnosed cancer and second leading cause of cancer deaths in nately, it is not curative. Our attack on prostate cancer American men (1). Although androgen ablation is the most envisions the proteolytic elimination of AR, which requires a common therapy for disseminated prostate cancer, it is palliative fuller understanding of AR turnover. We showed previously in nature and most patients eventually succumb to hormone- that calmodulin (CaM) binds to AR with important con- refractory disease resistant to chemotherapy. Whether normal or sequences for AR stability and function. To examine the mutated, androgen receptor (AR) is required for growth in both involvement of Ca2+/CaM in the proteolytic breakdown of AR, androgen-sensitive and androgen-insensitive prostate cancer (2). we analyzed LNCaP cell extracts that bind to a CaM affinity Therefore, it is of paramount importance to dissect the various column for the presence of low molecular weight forms of AR ways in which AR is regulated to not simply inactivate but to (intact AR size, f114 kDa). Using an antibody directed against physically eliminate AR (3). This requires understanding the the NH2-terminal domain (ATD) of AR on Western blots, we cellular mechanisms of AR breakdown. identified f76-kDa, f50-kDa, and 34/31-kDa polypeptides in Numerous studies have examined the proteolytic destruction of eluates of CaM affinity columns, suggesting the presence of AR, and our knowledge can at present be divided into three areas. CaM-binding sites within the 31/34-kDa ATD of AR. Under First, AR is degraded to small (V10 residues) peptides by the cell-free conditions in the presence of phenylmethylsulfonyl threonine proteases of the ubiquitin-proteasome system (4–9). fluoride, AR underwent Ca2+-dependent degradation. AR Second, AR can be cleaved by serine proteases (10–16). These degradation was inhibited by N-acetyl-leu-leu-norleu, an studies established that, structurally, AR consists of at least three inhibitor of thiol proteases, suggesting the involvement of domains, the ligand-binding domain (LBD), the DNA-binding calpain. In intact cells, AR breakdown was accelerated by domain (DBD), and the NH2-terminal domain (ATD), separated by raising intracellular Ca2+ using calcimycin, and increased AR protease-sensitive linker regions. Serine proteases cleave AR to breakdown was reversed with the cell-permeable Ca2+ chelator yield a 34-kDa COOH-terminal LBD (12, 17) and the remaining bis-(O-aminophenoxy)-ethane-N,N,N¶,N¶-tetraacetic acid tetra- f76 kDa of AR comprising the ATD and DBD. Other studies (11) (acetoxymethyl)-ester. In CaM affinity chromatography stud- established that proteolysis can cleave AR into two fragments: one ies, the Ca2+-dependent protease calpain was bound to and of f50 kDa containing both the LBD and the DBD and another of eluted from the CaM-agarose column along with AR. Caspase-3, approximately the same size, presumably the ATD (13). Third, the which plays a role in AR turnover under stress conditions, did caspase family of thiol proteases is capable of cleaving AR, not bind to the CaM column and was present in the proenzyme particularly those forms of AR with expanded polyglutamine 2+ form. Similarly, AR immunoprecipitates prepared from whole- repeats (18–22). To date, the cleavage of AR by the Ca -activated, cell extracts of exponentially growing LNCaP cells contained thiol protease calpain has not been described. both calpain and calpastatin. Nuclear levels of calpain and The amount, localization, physiologic functions, and degradation calpastatin (its endogenous inhibitor) changed in a reciprocal of AR are influenced by the following factors. First, AR is transcribed, translated (23, 24), and post-translationally phosphor- fashion as synchronized LNCaP cells progressed from G1 to S phase. These reciprocal changes correlated with changes in ylated (25, 26). Androgen binds to the LBD, inducing conformational change critical to the transcription factor activity of AR as AR level, which increased in late G1 phase and decreased as S phase progressed. Taken together, these observations well as resistance to degradation (17, 27). Following androgen suggest potential involvement of AR-bound CaM in calcium- binding, AR is ready to act as a transcription factor (23), as the DBD controlled, calpain-mediated breakdown of AR in prostate binds to the androgen response element (ARE). The activity of the cancer cells. (Cancer Res 2006; 66(24): 11754-62) DNA-bound complex is governed by binding of coactivator and corepressor proteins to the LBD and the ATD. Finally, AR activity is also controlled by AR-calmodulin (CaM) interaction (28). It is the effect of AR interaction with CaM on the breakdown of AR that we

Note: R.P. Pelley, K. Chinnakannu, and S. Murthy contributed equally to this work. examine herein. Requests for reprints: G. Prem-Veer Reddy, Vattikuti Urology Institute, Henry Ford Extending our observation that CaM binds to AR directly (28), Health System, One Ford Place, 2D, Detroit, MI 48202. Phone: 313-874-5991; Fax: 313- we now map the CaM-binding site(s) to the ATD of AR. We show 874-4324; E-mail: [email protected]. 2+ I2006 American Association for Cancer Research. for the first time that Ca stimulates AR breakdown and that CaM doi:10.1158/0008-5472.CAN-06-2918 bound to AR may play an important role in regulating AR levels by

Cancer Res 2006; 66: (24). December 15, 2006 11754 www.aacrjournals.org

a Sorvall RT7 centrifuge and resuspended in buffer A [0.16 mol/L sucrose,

50 mmol/L Tris-HCl (pH 7.6), 25 mmol/L KCl, 10 mmol/L MgCl2, 70 mmol/L HEPES (pH 7.6), 0.025 mmol/L CaCl2, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2 mmol/L DTT, 1 mmol/L EDTA] at a density of 2 Â 107/mL. The cell suspension was then homogenized in a top-driven Wheaton homogenizer (Wheaton Co., Wheaton, IL) until f90% of the cells were stained with trypan blue dye (usually three strokes at a rotation setting of 3). The cytosolic supernatant was then separated from the nuclear pellet by centrifugation at 4jC in a Beckman Microfuge B at 6,000 rpm for 5 minutes (Beckman Instruments, Palo Alto, CA). A volume of buffer A equal to that used for homogenization was added to the microfuge tube, and the nuclei were then subjected twice to 30 pulses of sonication with a Branson Sonifier 250 (Branson Co., Danbury, CT) set at an output control of 2 and a duty cycle of 20, with intermittent cooling on ice. The sonicated nuclear extract was cleared by centrifugation in the microfuge as described above. Protein concentration in cytosolic and nuclear extracts was assessed with the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) using the Bradford reagent in 96-well plates and read using an EL340 Micro Colorimeter (BioTek Instruments, Winooski, VT). Whole-cell extracts were prepared essentially as described by Cifuentes et al. (28). Briefly, LNCaPcells were suspended at a density of 2 Â 107/mL in buffer A and sonicated and the extracts were clarified as above. Figure 1. AR and its breakdown products in LNCaP cell lysate and UVB irradiation. UV irradiation was administered (31) using a single CaM-agarose affinity column fractions. An f1 mL volume of cell lysate of FS40 lamp (National Biological, Twinsburg, OH). FS40 lamps produce 0.5% 2 Â 107 LNCaP cells (harvested by trypsinization) was applied to 2mL bed UVC (260–280 nm), 62% UVB (280–320 nm), and 38% UVA (320–400 nm) volume columns of CaM-agarose in affinity chromatography buffer containing 2+ drop thru with a peak emission at 313 nm. The average irradiance of the source was 2mmol/L Ca and 1 mmol/L PMSF. After nonadherent material ( ) Â À4 washed through, CaM-binding materials were eluted with buffer lacking Ca2+ approximately 1.58 10 W at 17-cm distance as measured by an IL1700 but containing 10 mmol/L EGTA (EGTA Eluate). Each lane was loaded with Research Radiometer (International Light, Newburyport, MA) with a UVB-1 f20 Ag protein and respectively contained the following: crude cell lysate filter (#25628) and SED 240 detector. LNCaPcells were grown to 70% lane 1 lane 2 ( ), material not binding to CaM, drop thru ( ), and material eluted from confluence in P100 plates. Immediately before UV radiation treatment, the CaM with EGTA (lane 3). After PAGE and transfer, the blots were stained with the pAb N20 and developed with anti-rabbit conjugated antibody as medium was removed from each culture, the cells were washed once with described in Materials and Methods. Data are representative of two experiments, PBS, and fresh PBS was added. Cultures were exposed to one of two doses of each comprising four chromatographic runs. UV radiation (300 J/m2 for 40 seconds or 600 J/m2 for 80 seconds) under sterile conditions with the cover of the culture dish off. Following UV exposure, the PBS was removed and replaced with serum-containing culture engaging calpain/calpastatin in cleavage of AR under physiologic medium and cultured for an additional 12 or 24 hours. Control cultures conditions. Other pathways involving caspase-3 may also play a were identically treated but were not UV irradiated. The cells were then role in AR breakdown under stress conditions. These observations harvested by pipetting the culture medium up and down on the cells with a raise the possibility that anti-CaM drugs and calpastatin inhibitors Pasteur pipette until all cells were detached. The cells were washed once in can be used in the treatment of prostate cancer, particularly when PBS/EDTA before sonicating to prepare cell extracts as above. the disease is metastatic and hormone refractory. Treatment of cells with Ca2+ ionophore and intracellular Ca2+ chelator. Subconfluent, P150 plates of exponentially growing cultures of LNCaPcells were treated with the Ca 2+ ionophore A23187 (calcimycin) Materials and Methods or the cell-permeable calcium chelator 1,2-bis-(O-aminophenoxy)- Cell culture. LNCaPcells (obtained from the American Type Culture ethane-N,N,N¶,N¶-tetraacetic acid tetra-(acetoxymethyl)-ester (AM-BAPTA; Collection, Manassas, VA) were grown in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10 nmol/L testosterone (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FCS (Atlanta Biologicals, Table 1. Densitometric analysis of the Western blot in A Lawrenceville, GA), 2.5 mmol/L glutamine, 100 g/mL streptomycin, and Fig. 1 to determine the degree of breakdown of AR in the 100 units/mL penicillin. Cells were maintained in a humidified incubator initial sample and in the EGTA eluate j with 5% CO2 and 95% air at 37 C. PBS (pH 7.4) without magnesium or calcium (10Â concentrate), glutamine, and antibiotics were obtained from Integrated densitometric gray-scale units* Life Technologies. (% of total in lane) Synchronization of LNCaP cells by isoleucine deprivation. Isoleucine deprivation was done by our previously published method (29). The cells Form of AR Cell lysate, Drop thru EGTA eluate, were grown to 60% to 70% confluence, and then the medium was replaced n (%) n (%) with isoleucine-free RPMI 1640 supplemented with 6% dialyzed FCS (Life Technologies), 2.5 mmol/L glutamine, 100 Ag/mL streptomycin, 100 units/mL penicillin, and 10 nmol/L testosterone. Cells were maintained Intact AR 114,249 (38) 8,167 90,001 (9) f in isoleucine-free medium for 36 hours in an incubator as above. Cells were 50-kDa fragment 107,436 (36) 432,440 (43) released from isoleucine block by replacing the medium with the 31/34-kDa fragment 78,006 (26) 428,377 (43) corresponding complete medium containing 10% FCS. Preparation of nuclear and cytoplasmic fractions. LNCaPcells were *On an arbitrary scale of 0 to 256 density units above background per subjected to nuclear and cytoplasmic fractionation by slight modification of pixel determined using the Stratagene (La Jolla, CA) Eagle Eye II the method of Subramanyam et al. (30). All chemicals were obtained from system. Sigma-Aldrich (St. Louis, MO), except where otherwise noted. After harvest, cells were pelleted by centrifugation for 10 minutes at 4jC at 2,000 rpm in www.aacrjournals.org 11755 Cancer Res 2006; 66: (24). December 15, 2006

Figure 2. Breakdown of AR is activated by Ca2+ and inhibited by ALLN. Nuclear extracts were prepared from exponentially growing LNCaP cells harvested by scraping. Aliquots were incubated at 4jC for 48 hours in the presence of varying (0–5 mmol/L) concentrations of Ca2+ and in the absence (A) or presence (B)ofthe tripeptide cysteine protease inhibitor ALLN. All reactions were terminated by addition of 35 AL of sample into 1 mL of ice-cold 5% trichloroacetic acid. PAGE gels were run with f7 Ag protein, and Western blots were developed with either rabbit pAb to the ATD of human AR (top) or antibody to actin (bottom) to ascertain the degree of protein loading as described in Materials and Methods. Data are representative of four experiments.

Molecular Probes, Eugene, OR) for 24 hours. BAPTA or control vehicle was precipitation in ice-cold 5% trichloroacetic acid and dissolved in PAGE added to cultures and incubated at 37jC. Thirty minutes later, A23187 was loading buffer. added and cells were incubated for 24 hours. The cells were harvested, as Immunoprecipitation. AR immunoprecipitation to analyze AR-inter- above under UV treatment, by pipetting the culture medium up and down to acting proteins was done by modification of the method of Cifuentes et al. detach the cells followed by washing and sonication to prepare the extracts. (28). Cell extracts were diluted 10-fold in 250 mmol/L NaCl and 50 mmol/L Western blot analysis. Samples were dissolved in PAGE loading buffer Tris (pH 7.4; dilution buffer) and incubated overnight with 4 Ag/mL of (Bio-Rad, Richmond, CA) and subjected to denaturing 10% or 12% SDS/ N20 or 441 anti-AR antibody at 4jC. The immune complexes were then PAGE and then transferred to nitrocellulose membranes. Individual adsorbed to protein A/G agarose immunoadsorbent (Pierce) by incubation membranes were probed with rabbit polyclonal antibody (pAb) against for 1 hour at ambient temperature with gentle agitation. The adsorbed the 20 NH2-terminal residues (N20) or murine monoclonal antibody (mAb) complexes were washed twice in dilution buffer by centrifugation at 4jC against residues 301 to 317 (mAb 441) of human AR (Santa Cruz and eluted with PAGE loading buffer. Mock immunoprecipitates were Biotechnology, Santa Cruz, CA). Other primary antibodies used were obtained by diluting and incubating the extracts without AR antibody. The mAb against the following: CaM (Upstate Cell Signaling Solutions, Lake mixture was then incubated with immunoadsorbent and eluted as above. Placid, NY); caspase-3 (Santa Cruz Biotechnology); the COOH-terminal domain of poly(ADP-ribose) polymerase (PARP), which reacts with both the intact 112-kDa and the clipped 85-kDa forms of PARP (Santa Cruz Results Biotechnology); A-calpain large subunit and calpastatin (Chemicon CaM-binding site in the NH2-terminal region of AR. In International, Temecula, CA); and rabbit mAb to cleaved caspase-3 (Cell LNCaPcell extracts prepared from cells harvested by trypsiniza- Signaling Technology, Beverly, MA). Immunoreactive bands were developed tion, there was a noticeable breakdown of AR to fragments with by using horseradish peroxidase–conjugated secondary antibodies and approximate molecular weights of f50 and 31/34 kDa. Like SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) intact AR, each of these AR fragments was detected on Western and visualized by using X-ray film. blots using affinity-purified pAbs raised against the most NH - CaM-agarose affinity chromatography. The CaM affinity chromatog- 2 raphy procedure was essentially as described by Cifuentes et al. (28). Briefly, terminal 20 amino acids of human AR (pAb N20, Santa Cruz nuclear (f2.5 mg protein) and cytoplasmic (f7.5 mg protein) lysates of Biotechnology; Fig. 1). Therefore, the AR fragments detected by LNCaPcells prepared as described above were applied to 2 mL CaM- pAb N20 are referred to as AR ATD fragments. Following up on agarose affinity columns (Sigma Chemical) poured in 25-cm Econocolumns our observation that AR in LNCaPcell extracts binds to CaM (28), (Bio-Rad). Nonadherent proteins were washed from the column with buffer we attempted to map the CaM-binding region on AR by testing B [20 mmol/L Tris (pH 7.4), 4 mmol/L MgCl2, 2 mmol/L CaCl2, 10 mmol/L whether the f50-kDa or 31/34-kDa AR ATD fragments retain KCl, 1 mmol/L PMSF] until A280 returned to the baseline. This nonadherent CaM-binding ability. CaM-agarose affinity chromatography of fraction was called the drop thru. Proteins retained on the column were lysate from trypsinized LNCaPcells showed strong binding of then eluted with modified buffer B [20 mmol/L Tris (pH 7.4), 4 mmol/L both the f50-kDa and 31/34-kDa AR ATD fragments to CaM. MgCl2, 10 mmol/L EGTA, 10 mmol/L KCl, 1 mmol/L PMSF] in which the Ca2+ chelator EGTA was substituted for Ca2+. All chromatography Although Ponceau S stainable protein could be detected equally procedures, including cell lysate application, washing, and elution, were well in both the ‘‘drop thru’’ and EGTA eluate fractions of a carried out without application of pressure to the gel bed other than the CaM-agarose affinity column (data not shown), the pAb N20 hydrostatic pressure of 15-cm (maximum) column of liquid above the gel. immunoreactive bands of AR and AR ATD fragments could only Protein in the drop thru and eluate fractions was concentrated by be detected in the EGTA eluate (Fig. 1, lane 2). In addition, we

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Calpain Cleavage of Androgen Receptor in LNCaP Cells observed that, whereas the pAb N20 detected all ATD fragments of AR, mouse mAb (32) raised against amino acids 301 to 317 of the AR (mAb 441, Santa Cruz Biotechnology) detected intact AR and the f50-kDa ATD fragment but not the 31/34-kDa ATD fragment (data not shown). These observations together suggest that the CaM-binding site lies within the 31/34-kDa NH2-terminal region of AR and that the 31/34-kDa fragment lacks the 301 to 317 epitope. AR cleavage is accentuated in LNCaP cell extracts subjected to CaM affinity column. Quantitative analysis of the relative distribution of pAb N20 immunoreactive bands of intact and ATD fragments of AR in cell lysates versus those enriched in the EGTA eluate of a CaM affinity column suggested that AR in LNCaPcell lysates may have undergone additional proteolytic breakdown during the course of affinity chromatography despite the presence of PMSF in the buffer. Densitometry of pAb N20 immunoreactive bands in cell lysate showed an approximate distribution of intact AR and the f50-kDa and 31/34-kDa ATD fragments to be 38%, 36%, and 26%, respectively (Table 1). By comparison, the same cell lysates, when subjected to CaM affinity chromatography, yielded EGTA eluate that contained pAb N20 immunoreactive bands of intact AR and the f50-kDa and 31/34-kDa ATD fragments with a

Figure 4. Calcium-dependent binding of AR and calpain, but not caspase-3, to CaM-agarose columns. LNCaP cells were harvested by trypsinization, extracts were subjected to CaM-agarose affinity chromatography, and the unbound flow-through protein fraction (Drop-Thru) and the protein fraction eluted with EGTA (EGTA Eluate) were subjected to Western blot analysis using antibodies to AR, A-calpain, or caspase-3 as described in Materials and Methods. Data are from one of four chromatographic runs.

density distribution of 9%, 43%, and 43%, respectively (Table 1). Thus, there was a significant decrease of intact AR and an increase of 50-kDa and 31/34-kDa ATD fragments in the EGTA eluate fraction compared with that in the cell lysate before CaM affinity chromatography. Ca2+ stimulates AR breakdown in LNCaP cell extracts. The cleavage of AR on chromatography on CaM affinity columns observed above occurred in the presence of 1 mmol/L PMSF, suggesting that AR proteolysis was caused by an enzyme that was not a serine esterase. Given that the loading buffer for applying extracts to the CaM affinity column contained 2 mmol/L Ca2+,we sought to determine if Ca2+ was activating a proteolytic factor in the cell extracts. Therefore, LNCaPnuclear extracts (prepared from cells harvested by scraping rather than trypsinization) were incubated for varying times in the presence of increasing concentrations of Ca2+ before being separated by PAGE and analyzed by Western blots using pAb N20. Figure 2A illustrates that AR proteolysis proceeded even in the presence of the low level of Ca2+ that was present in the buffer used for the preparation of nuclear extract at 4jC. Increasing Ca2+ concentration to the millimolar range caused complete breakdown of AR into lower molecular weight fragments over a 48-hour incubation at 4jC. Similar effects were seen with cytosolic extracts at 4jC and with both nuclear and cytosolic extracts at 23jC, albeit at a more rapid Figure 3. Increasing intracellular Ca2+ by ionophore increases AR breakdown, rate (data not shown). Thus, the enzyme responsible for and chelation of intracellular Ca2+ decreases AR breakdown. LNCaP cells production of ATD fragments from AR is Ca2+ dependent. were incubated for 24 hours with the ionophore calcimycin (A23187) in the 2+ presence or absence of the intracellular Ca2+ chelator AM-BAPTA. Cells were Ca -stimulated breakdown of AR in cell extracts is then harvested by scraping into medium, and experimental and control attenuated by N-acetyl-leu-leu-norleu. One of the best-charac- (whole cell) lysates were prepared at a concentration of 2 Â 107 cells/mL. PAGE terized Ca2+-activated proteases is calpain. Therefore, we tested was done, and the blots were analyzed with antibodies to AR (pAb N20), calpastatin, CaM, or actin as described in Materials and Methods. The whether the breakdown of AR into ATD fragments might be experiment was repeated twice, and representative data are shown. inhibited by the tripeptide analogue thiol protease inhibitor www.aacrjournals.org 11757 Cancer Res 2006; 66: (24). December 15, 2006

N-acetyl-leu-leu-norleu (calpain inhibitor I or ALLN). Addition of experiment, cellular integrity was unaffected as evidenced by the 20 Amol/L ALLN to the incubation system described above in equal amount of actin in equal volumes of extract (Fig. 3). Fig. 2A resulted in protection of AR from Ca2+-activated Relative to actin, A23187 calcimycin caused a substantial decrease proteolytic cleavage into ATD fragments (Fig. 2B). A similar in intact AR and AR fragments (Fig. 3, lane 3). This A23187- inhibitory effect of ALLN on Ca2+-stimulated AR breakdown was induced decrease in AR was also associated with a decrease in observed in cytosolic extracts incubated at 4jC and with both calpastatin, a natural inhibitor of calpain, and calpastatin nuclear and cytosolic extracts incubated at 23jC (data not breakdown products (Fig. 3). Pretreatment of LNCaP cells with shown). Thus, Ca2+-stimulated AR cleavage and ALLN attenuation AM-BAPTA partially ameliorated the effect of calcimycin iono- of Ca2+-stimulated cleavage suggest the involvement of calpain in phore (Fig. 3, lane 4). AR cleavage. Calpain, but not caspase-3, binds to CaM affinity columns Ca2+ ionophore stimulates and cell-permeable Ca2+ chelator along with AR. Cleavage of AR by calpain has not been previously suppresses breakdown of endogenous AR in LNCaP cells. Given reported, whereas there are numerous reports of caspase-3- that the breakdown of AR in vitro is accelerated by increasing the mediated AR breakdown (19–22). Furthermore, ALLN is not levels of Ca 2+, we investigated the effect of artificially altering specific for calpain. Therefore, we sought further evidence for intracellular levels of Ca2+. We cultured cells in the presence of the involvement of calpain in AR degradation. LNCaPcell extracts the cell-permeable Ca2+ chelator acetomethyl ester form of were subjected to CaM-agarose affinity chromatography under the BAPTA (AM-BAPTA) and/or in the presence of A23187, an conditions described in Fig. 1, a procedure found to accelerate AR ionophore that raises intracellular Ca2+ levels. Cells in this study cleavage. The drop thru and EGTA eluate fractions were then were harvested by scraping rather than trypsinization. Treatment analyzed by Western blots probed with antibodies to caspase-3, of LNCaPcells with AM-BAPTA had little effect, per se, on the A-calpain, and AR (Fig. 4). Caspase-3 failed to bind to CaM and was level of intact AR but attenuated the formation of the f50-kDa present only in the drop thru fraction (Fig. 4, lane 1). Furthermore, and 31/34-kDa fragments of AR (Fig. 3, compare lanes 1 and 2). the molecular weight of the material immunoreactive with the This is consistent with a potential role of Ca2+-dependent AR antibody to caspase-3 was consistent with the presence of caspase- cleavage. On the other hand, treatment of LNCaPcells with the 3 in its inactive form. No material was observed in these extracts A23187 calcimycin ionophore had a striking effect. Cell rounding that reacted with antibody to cleaved caspase-3 (data not shown), and detachment were observed, consistent with induction of which is the active form of the enzyme. All of the AR bound to the apoptosis, although, within the 24-hour time course of the CaM column and was eluted with EGTA (Fig. 4, lane 2). Calpain bound to the CaM-agarose column and was present in the EGTA eluate (Fig. 4, lane 2). Thus, the Ca2+-dependent binding of AR and calpain to CaM affinity columns is consistent with a potential role of calpain in AR cleavage. Calpain, calpastatin, and CaM, but not caspase-3, are in AR immunoprecipitates. The previous experiments suggested that calpain, but not caspase-3, was responsible for calcium-activated cleavage of AR to ATD fragments. Therefore, we sought evidence using immunoprecipitation that calpain is associated with AR in whole-cell extracts. Caspase-3, although present in crude lysates, did not coprecipitate when AR was immunoprecipitated with either mAb 441 or pAb N20 (Fig. 5), consistent with our conclusion that caspase-3 was not responsible for AR breakdown in our experiments. CaM, on the other hand, coprecipitated with AR in immunoprecipitates prepared with both antibodies (anti-AR mAb 441 and pAb N20), confirming our earlier report of AR binding to CaM (28). Calpain and calpastatin also coprecipitated with AR (Fig. 5). Coprecipitation of calpastatin with AR was more apparent with the mAb 441 than with pAb N20. We infer from this that the binding of calpastatin to AR may be disrupted when pAb N20 binds to the most NH2-terminal residues of AR. Thus, it seems that, in crude lysates at a concentration of Ca2+ that is physiologic for the intracellular milieu, AR, CaM, calpain, calpastatin, and Ca2+ form a complex. Changes in calpain and calpastatin levels are associated Figure 5. Calpain, calpastatin, and CaM, but not caspase-3, are associated with progression of synchronized LNCaP cells from G0-G1 to with AR immunoprecipitates. Exponentially growing LNCaP cells were harvested S phase. Given that AR, CaM, calpain, and calpastatin seem to by scraping. Whole-cell extracts (Cell Lysate) were incubated with either mAb (mAb 441) to a synthetic peptide comprising residues 301 to 317 in the middle form a complex in the living cell and given that intracellular of the ATD of AR, pAb (pAb N20) to a synthetic peptide comprising residues Ca2+ fluctuates significantly during the cell cycle (33), we 1 to 20 of the ATD of AR, or immunoprecipitation buffer lacking antibody (No Ab). The mixtures were then treated with an immunoglobulin adsorbent (protein examined the possibility that variation of AR levels throughout A/G mix), and the complexes were washed and then eluted with PAGE buffer. the cell cycle might be associated with concomitant changes in Eluates were subjected to PAGE, and Western blots were developed with the levels of calpain and calpastatin. For this, we used LNCaP antibodies to AR, calpain, calpastatin, CaM, and caspase-3 as described in Materials and Methods. The experiment was repeated thrice, and representative cells synchronized by isoleucine deprivation that progress blots are shown. synchronously from G0-G1 to S phase on release from isoleucine

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Calpain Cleavage of Androgen Receptor in LNCaP Cells block (29). In these cells, AR levels fluctuated during the cell reveal activated caspase-3 in exponentially growing or synchro- cycle from a low at G0-G1 (0–4 hours after release from nized LNCaPcells. Therefore, we exposed LNCaPcells to doses of isoleucine block) to a maximum just as S phase begins (12–16 UV radiation known to trigger programmed cell death in cultured hours after release from isoleucine block; Fig. 6). These changes cancer cells and examined the effect on activation of caspase-3 and were more pronounced in nuclear extracts (Fig. 6B, top) than in AR. Twelve hours after UVB exposure, LNCaPcells showed a mild cytosolic extracts (Fig. 6A, top). In both compartments, AR ATD degree of rounding up but little detachment from their substratum; fragments generally tended to increase as the cell cycle by 24 hours after exposure, the apoptotic pathway was activated progressed. as evidenced by rounding and detachment of f20% of the cells at The levels of calpain and calpastatin also varied during the cell the higher UV dose (data not shown). There was significant, UV cycle. Cytosolic calpain levels were highest early in the cell cycle, dose-dependent cleavage of PARP (Fig. 7A), a classic marker of decreased in late G1, and increased as S phase approached programmed cell death. Although UV treatment did not affect (Fig. 6A). Cytosolic calpastatin levels roughly mirrored calpain the level of intact caspase-3 (Fig. 7B, top), it led to the appearance levels (Fig. 6A). The pattern of increasing cleavage of AR to ATD of cleaved caspase-3 fragments. This confirms that caspase-3 is fragments seems to correlate to the increasing levels of calpain. present only in the inactive form in untreated, exponentially Nuclear calpain was lowest during early G1 (Fig. 6B) and increased growing, or synchronized LNCaPcells but that it can be generated as the cell cycle progressed, correlating with the progressive under specific conditions. increase in AR ATD fragments. Calpastatin levels, on the other Although there was no change in the actin level, the level of AR hand, remained relatively steady throughout the cell cycle, was dramatically reduced by 24 hours after UVB irradiation at a 2 although the level of the two highest molecular weight forms dose of 600 J/m (Fig. 7A). However, this decrease in AR was not fluctuated. accompanied by an increase in ATD fragments of AR. Rather, In summary, there seems to be a correlation, more pronounced compared with unexposed controls or low-dose UV at 24 hours, the in the nucleus, between increases in calpain expression during 31/34-kDa ATD fragments were less prominent in cells undergoing progression through the cell cycle and the appearance of AR ATD programmed cell death. fragments. This suggests that calpain might be responsible for AR cleavage as cells enter S phase. Although calpastatin is present in Discussion both nuclear and cytosolic extracts, greater variation is seen in Our studies are the first to show that Ca2+ stimulates AR cytosolic levels of calpastatin, suggesting different effects in the breakdown in vitro and in vivo and that CaM bound to AR two compartments. seems to play a role in calpain-mediated cleavage of AR in UV light activates caspase-3, triggers apoptosis in LNCaP prostate cancer cells. When AR binds to CaM in the presence of cells, and causes decreased AR levels but does not generate 2 mmol/L Ca2+, AR breakdown is accelerated despite the ATD fragments. Caspase-3 has been implicated in the proteolytic presence of PMSF (Fig. 1). AR, calpain, and its inhibitor cleavage of AR, but the experiments described above failed to calpastatin all bind to CaM in a Ca2+-dependent manner and

Figure 6. AR cleavage is associated with changes in calpain and calpastatin levels during the progression of synchronized LNCaP cells from G0-G1 to S phase. LNCaP cells were synchronized by isoleucine deprivation as described in Materials and Methods. Following release from isoleucine block, cells were harvested (trypsinized) either at various times corresponding to key phases of the cell cycle (0 hour, G0-G1; 4–8 hours, early G1; 12–16 hours, late G1; and 20–24 hours, S phase) or from exponentially growing cultures (Expo). Cytosol extracts (A) and nuclear extracts (B) were prepared, and PAGE was done as described in Materials and Methods. Blots were analyzed with antibodies to AR, calpain, calpastatin, and actin. The study was repeated twice, and representative data are shown. www.aacrjournals.org 11759 Cancer Res 2006; 66: (24). December 15, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research are eluted with the calcium chelator EGTA (Fig. 4). Similarly, AR (Fig. 8B). This model is also consistent with our earlier immunoprecipitates contain CaM, calpain, and calpastatin observation that the anti-CaM drug N-(6-aminohexyl)-5-chloro-1- (Fig. 5). In vitro,Ca2+ activates AR breakdown in both nuclear naphthalenesulfonamide, which can alter CaM conformation, and cytosolic extracts, and the inhibitor of thiol proteases, ALLN, induces AR degradation in LNCaPcells (28). prevents Ca2+-stimulated AR breakdown (Fig. 2). Based on AR Ca2+, as a second messenger, enables cells to walk a tightrope breakdown fragment sizes and AR antibody epitope locations, between life and death. In exponentially growing rat prostate cancer 2+ we propose a model of AR cleavage by calpain (see Fig. 8A). cells, increasing intracellular Ca shunts cells into G0-G1 phase and We suggest that calpain cleaves AR at protease-sensitive sites induces the growth arrest gene gadd153 (39). Thapsigargin (39–41), between the LBD and DBD (yielding an f75-kDa ATD h-lapachone (42), and ionomycin (40, 43) all induce striking fragment), between the ATD and DBD (yielding an f50-kDa increases in intracellular Ca2+ in prostate cancer cells and trigger ATD fragment), and approximately in the middle of the ATD programmed cell death. This process is accompanied by growth (yielding a 31/34-kDa NH2-terminal fragment; Fig. 8A). arrest (39) and down-regulation of AR expression (40). Our studies Proteins that bind to Ca2+ and/or are activated by Ca2+-binding show that, both in cultured LNCaPcells and in cell-free extracts, proteins, such as CaM, act as decoders of the Ca2+ signal. In vitro, increasing Ca2+ allowed AR degradation whereas decreasing Ca2+ Ca2+ is known to change calpain conformation, effecting its (by chelation either in vitro with EDTA or intracellularly with autocatalytic cleavage and activation (34). Calpain heavy and light AM-BAPTA) attenuated AR breakdown (Figs. 2 and 3). Thus, an chains both have CaM homology domains (35, 36). Calpastatin increase in intracellular Ca2+ both suppresses the expression and binds to these CaM homology domains and inactivates calpain stimulates the breakdown of proteins necessary for proliferation (37). Interestingly, virtually all proteins that bind to CaM are also and viability of prostate cancer cells. substrates for calpain (38). Calpain, calpastatin, and CaM exist in That AR is a substrate for calpain is not surprising. Estrogen a complex with AR (Fig. 5), and Ca2+ stimulates AR degradation receptor (44) and progesterone receptor (45) are cleaved by what we (Fig. 3) in LNCaPcells. As depicted in Fig. 8 B, these observations now know to be calpain. In LNCaPcells, cleavage of h-catenin, a key raise the intriguing possibility that CaM, bound to AR, recruits molecule in the Wnt signaling pathway that controls AR expression, is calpain and calpastatin to cleavage sites on AR. At low Ca2+, mediated by calpain (46). In this study, we observed that AR levels CaM-bound calpain is enzymatically inactive. However, at high inversely correlated with calpain levels during the cell cycle in LNCaP Ca2+, the conformation of the calpain-CaM-calpastatin complex is cells; when AR levels were increasing, calpain levels were low in early altered in such a way that calpain is released from the inhibitory G1 phase, whereas as AR levels were beginning to decline, calpain effect of calpastatin, thereby allowing calpain to cleave AR levels were at a maximum in S phase (Fig. 6).

Figure 7. Exponentially growing LNCaP cells were exposed to either 300 or 600 J/m2 of UV radiation; cells were scraped into medium 12or 24hours later, and whole-cell extracts were prepared and subjected to PAGE. A, Western blots were incubated with antibody to PARP to determine if the apoptotic process was initiated (top) and with the pAb N20 to the ATD of AR (middle) or antibody to actin to assess uniformity of loading (bottom). B, blots were incubated with antibody to intact caspase-3 (top) or antibody to cleaved caspase-3 (bottom). Results are from a single experiment.

Cancer Res 2006; 66: (24). December 15, 2006 11760 www.aacrjournals.org

Figure 8. Model of AR cleavage by calpain. A, structure of AR showing the location of pAb N20 and mAb 441 epitopes (amino acids 1–20 and 301–317, respectively) inferred calpain cleavage sites (arrows) that yield 76-kDa, f50-kDa, and 31/34-kDa fragments, all three of which bind to CaM in a Ca2+-dependent manner. aa, amino acid; Q, polymorphic glutamine repeat (48); P, proline repeat; G, polymorphic glycine repeat (48); H, hinge region; AF, activation function; FQNLF motif (amino acids 23–27; ref. 49). B, model of interactions between AR, CaM, calpain, and calpastatin and the role of Ca2+ in calpain-mediated cleavage of AR. At low concentrations, calpain, calpastatin, and CaM bind to the AR presumably at CaM-binding sites on the AR; calpain is enzymatically inactive under these conditions. At elevated Ca2+ concentrations, calpain becomes activated (change in conformation), calpastatin dissociates [or is cleaved by activated calpain (37, 50)], and calpain cleaves AR. This model suggests that CaM-binding sites on the AR are at calpain cleavage sites.

Caspase-3 does not seem to be the enzyme that cleaves AR under studies (7, 9) have indicated that the primary effect of proteasome physiologic conditions. AR with expanded polyglutamine repeats inhibitors in prostate cancer cells is to alter the Akt pathway, has been implicated in neurodegenerative disease wherein AR is thereby altering transcription and translation of AR. On the other cleaved between the DBD and LBD into a toxic, ‘‘truncated’’ ATD- hand, the proteasome is critical for AR transactivating activity [i.e., DBD moiety (18). Caspase-3 is suggested to be the enzyme AR binding to the ARE (6, 8)]. This study adds to the complexity by responsible for the cleavage of this pathologic AR (19–22). Based on implicating a proteolytic enzyme, calpain, novel to AR degradation these observations of AR in neurodegenerative diseases, caspase-3 but well established for cleaving key regulatory molecules. is implicated to cleave AR in prostate cancer cells (15, 23). However, Certainly, breaking down AR by a Ca2+-dependent, CaM-interact- we found that, under physiologic conditions, caspase-3 is not ing, calpastatin-regulated mechanism is consistent with our present in its activated form in exponentially growing LNCaPcells concept of the importance of protein-protein interactions during and caspase-3 is not part of the AR-CaM complex. Furthermore, the cell cycle. The localization of CaM-binding site(s) to the ATD of activation of caspase-3 following irradiation of LNCaPcells with AR focuses our attention on a region of AR ripe for targeted therapy UVB radiation is not associated with cleavage of AR into 76-kDa, development (47). We envision a critical regulatory role for a CaM- 50-kDa, or 31/34-kDa ATD fragments. Thus, caspase-3 could play a AR-calpastatin-calpain complex in the proliferation of prostate role in AR breakdown either directly or indirectly only under stress cancer cells and as targets of opportunity in the development of conditions; under normal growth conditions, it is an unlikely agents for treating prostate cancer. candidate for AR degradation during the cell cycle. The degradation of AR is complex and multifaceted, and Acknowledgments different proteolytic systems seem to be involved in transcription Received 8/9/2006; revised 10/2/2006; accepted 10/10/2006. of genes regulated by AR, degradation of damaged AR, progress Grant support: NIH grant RO1-DK57864 and Department of Defense grant through the cell cycle, and programmed cell death. Although initial W81XWH-05-1-0071. The costs of publication of this article were defrayed in part by the payment of page studies with proteasome inhibitors suggested that the proteasomal charges. This article must therefore be hereby marked advertisement in accordance pathway was a major route for degradation of AR (5), subsequent with 18 U.S.C. Section 1734 solely to indicate this fact. www.aacrjournals.org 11761 Cancer Res 2006; 66: (24). December 15, 2006

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Calmodulin-Androgen Receptor (AR) Interaction: Calcium-Dependent, Calpain-Mediated Breakdown of AR in LNCaP Prostate Cancer Cells

Ronald P. Pelley, Kannagi Chinnakannu, Shalini Murthy, et al.

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