Independent Progression in Prostate Cancer Via Signal Transducers and Activators of Transcription 3–Mediated Suppression of Apoptosis

Research Article

Increased Hsp27 after Androgen Ablation Facilitates Androgen- Independent Progression in Prostate Cancer via Signal Transducers and Activators of Transcription 3–Mediated Suppression of Apoptosis

Palma Rocchi,1 Eliana Beraldi,1 Susan Ettinger,1 Ladan Fazli,1 Robert L. Vessella,3 Colleen Nelson,1 and Martin Gleave1,2

1The Prostate Centre, Vancouver General Hospital; 2Division of Urology, University of British Columbia, Vancouver, British Columbia, Canada; and 3Department of Urology, University of Washington, Seattle, Washington

Abstract Unfortunately, androgen-independent (AI) progression is inevita- One strategy to improve therapies in prostate cancer involves ble, and the development of hormone-refractory disease and death targeting cytoprotective genes activated by androgen with- occurs within 2 to 3 years in most men. AI progression is a multi- drawal to delay the emergence of the androgen-independent factorial process by which cells acquire the ability to both survive (AI) phenotype. The objectives of this study were to define in the absence of androgens and proliferate using non-androgenic changes in Hsp27 levels after androgen ablation and to stimuli for mitogenesis, and involves variable combinations of evaluate the functional relevance of these changes in AI adaptive up-regulation of antiapoptotic genes, ligand-independent progression. Using a tissue microarray of 232 specimens of activation of the androgen receptor, and alternative signaling hormone-naı¨ve and post-hormone ablation–treated prostate pathways including Her2/neu, epidermal growth factor receptor, h cancer, we found that Hsp27 levels increase after androgen transforming growth factor- , and insulin-like growth factor-I ablation to become highly expressed (>4-fold, P V 0.01) in AI (1–3). Several antiapoptotic genes have been functionally linked to tumors. Hsp27 overexpression rendered LNCaP cells highly the development of hormone resistance, including Bcl-2, Bcl-xL, resistant to androgen withdrawal both in vitro and in vivo. and clusterin (4–6). The elucidation of the mechanisms that Tumor volume and serum prostate–specific antigen levels mediate AI progression, including key cytoprotective molecules, is increased 4.3- and 10-fold faster after castration when an important step towards identifying newly targeted therapeutic Hsp27 was overexpressed. Treatment of LNCaP tumor cells strategies. in vitro with Hsp27 antisense oligonucleotides (ASO) or short- Many components of survival and apoptotic pathways are interfering RNA suppressed Hsp27 levels in a dose-dependent regulated by molecular chaperones, such as heat shock proteins and sequence-specific manner increased the apoptotic sub– (Hsp). Using array analysis to compare gene expression profiles G0-G1 fraction and caspase-3 cleavage >2-fold, as well as before and after castration, we identified Hsp27 as one of the most decreased signal transducers and activators of transcription 3 highly expressed genes in AI prostate tumors (7). Hsp27 is a 27-kDa (Stat3) levels and its downstream genes, c-fos and sPLA-2. protein highly induced during the stress response to a wide variety The cytoprotection afforded by Hsp27 overexpression was of physiologic and environmental insults (8). Various roles have attenuated by Stat3 knockdown using specific Stat3 ASO. been proposed for Hsp27 to explain its cytoprotective effects during Coimmunoprecipitation and immunofluorescence confirmed cellular stress, including its role as a molecular chaperone, inhibitor that Hsp27 interacts with Stat3 and that Stat3 levels correlated of protein unfolding, direct interference with caspase activation, directly with Hsp27 levels. Hsp27 ASO treatment in athymic modulation of oxidative stress, and regulation of the cytoskeleton mice bearing LNCaP tumors significantly delayed LNCaP (9). Higher levels of Hsp27 are commonly detected in various tumor growth after castration, decreasing mean tumor cancers including breast (10), gastric (11), ovarian and endometrial volume and serum prostate–specific antigen levels by 57% (12, 13), osteosarcoma (14), glial tumors (15), and prostate (16). and 69%, respectively. These findings identify Hsp27 as a Increased Hsp27 levels in breast, endometrial, and gastric cancer modulator of Stat3-regulated apoptosis after androgen is associated with metastasis, poor prognosis, and resistance to ablation and as a potential therapeutic target in advanced chemotherapy or radiation (10, 17–19). prostate cancer. (Cancer Res 2005; 65(23): 11083-93) In prostate cancer, elevated Hsp27 expression has been linked to poor outcome, and is highly expressed in AI prostate cancer cells Introduction (7, 20–22). Bubendorf et al. (22) identified Hsp27 as an overex- Androgen withdrawal is the most effective form of systemic pressed gene in androgen-resistant CWR22 prostate xenografts. therapy for men with advanced prostate cancer, producing The objectives of this study were to define the changes in Hsp27 symptomatic and/or objective responses in >80% of patients. levels after androgen ablation and during AI progression in human prostate cancer, as well as to evaluate the functional relevance of these changes in AI prostate cancer. We chose the LNCaP tumor model, which closely mimics AI progression in humans by pro- Requests for reprints: Martin Gleave, Division of Urology, Vancouver Hospital, University of British Columbia, 27333 Heather Street, Vancouver, British Columbia, ducing prostate-specific antigen (PSA)–secreting and androgen- Canada V5Z 3J5. Phone: 604-875-5003; Fax: 604-875-5604; E-mail: gleave@ dependent tumors when injected into male immunodeficient mice, interchange.ubc.ca. I2005 American Association for Cancer Research. and develop non–androgen-regulated PSA gene expression after doi:10.1158/0008-5472.CAN-05-1840 castration as a surrogate end point of progression to AI (23). www.aacrjournals.org 11083 Cancer Res 2005; 65: (23). December 1, 2005

As part of our ongoing investigations to identify key pathways Research Laboratory at the Vancouver General Hospital. Specimens were mediating AI progression, we use forced overexpression for gain-of- chosen to represent various treatment durations of androgen withdrawal function analyses and antisense oligonucleotides (ASO) or short- therapy prior to radical prostatectomy ranging from no treatment interfering RNA (siRNA) for loss-of-function analyses to evaluate (n = 35), 0 to 3 months (n = 58), 3 to 6 months (n = 52), and 6 months (n = 57). AI tumors were also identified (n = 30). Most tissues were from the functional role of castration-induced changes in gene radical prostatectomy specimens, whereas AI tissues were obtained from expression. In this study, we characterized changes in Hsp27 after transurethral resections or metastatic lesions from men with hormone- androgen ablation in LNCaP tumors and employed Hsp27 gain- refractory disease. Metastatic lesions were obtained from warm autopsy and loss-of-function approaches to determine the effects of Hsp27 specimens of 13 men who succumbed to prostate cancer. All radical on LNCaP cell survival and tumor progression after androgen prostatectomy specimens in the array were Gleason grade V4 and clinical withdrawal in LNCaP cells in vitro and in vivo. stage T1 or T2. One metastatic prostate lesion was obtained per patient from seven bone lesions, three lymph node lesions, two liver lesions, and one adrenal lesion. Materials and Methods Tissue microarray analysis. A tissue microarray analysis was Tumor cell lines. The human prostatic cancer cell line LNCaP was constructed using a Beecher microarrayer from the above paraffin- kindly provided by Dr. Leland W.K. Chung (Emery University, Atlanta, GA) embedded specimens with matching H&E slides used to identify the and was maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg, tumor and accurately obtain the core sample. Each case is represented MD) supplemented with 5% FCS. with three cores in the tissue microarray analysis. Sections were Microarray experiments. Total RNA from each tumor sample, obtained deparaffinized and rehydrated through xylene and ethanol, then before and 35 days post-castration, were compared on the same chip to transferred to the 0.02% triton for permeabilization. Slides in citrate tumor samples from intact controls. A dye-swap for each pair was done to buffer (pH = 6) were heated in the steamer for 30 minutes. After cooling account for dye bias. Three tumors from each post-castration time point for 30 minutes and washing thrice for 5 minutes in PBS, the slides were were analyzed and results were confirmed with Northern analysis. incubated in 3% BSA for a further 30 minutes. The slides were Microarrays of 13,791 (70-mer) human oligos (Operon, Huntsville, AL) successively transferred to 3% H2O2 for 10 minutes and then were printed in duplicate in 3Â SSC onto amine-coated slides (Ezray, Apogent, incubated overnight with anti-Hsp27 antibodies from Nova Castra Fisher Scientific, Ottawa, Ontario, Canada) were supplied by the Array (Newcastle, United Kingdom) at the concentration of 1:400 in 1% BSA. Facility of the Prostate Centre at Vancouver General Hospital. Slides were The next day, the primary antibody was washed extensively with PBS and printed with a BioRobotics Microgrid II (Harvard Biosciences and Genomic the LSAB+ kit (Dako, Carpinteria, CA) was used as the detection system. Solutions, Ann Arbor, MI) under 60% humidity at 22jC; then UV cross- Chromogen Nova-red (Vector Laboratories, Burlingame, CA) was applied linked using 3,000 AJ (Stratalinker, Stratagene, La Jolla, CA). Slides were for 2 minutes and counterstaining was done with H&E (Vector prehybridized in 5Â SSC, 0.1% SDS, and 0.2% bovine serum albumin (BSA; Laboratories). After ethanol rehydrating, the slides were covered with a Sigma, St. Louis, MO) at 48jC for 45 minutes, washed in deionized water, cover glass with Cytoseal, a xylene-based mounting medium (Stephen dipped in isopropanol, and dried in a centrifuge at 2,000 rpm for 2 minutes. Scientific, Riverdale, NJ). Negative control slides were processed in an Arrays were hybridized in a humid Hybaid oven (ThermoHybaid, England) identical fashion to that above, with the substitution of 1% BSA for the with reverse-transcribed fluorescently labeled (Cy3- or Cy5-dUTP; Amer- primary antiserum. Photomicrographs were taken through a Leica DMLS sham Pharmacia Biosciences Inc., Quebec, Canada) cDNA (from 20 Agof microscope coupled to a digital camera (Photometrics CoolSNAP, Roper total RNA) at 42jC for 16 hours in a hybridization buffer consisting of 50% Scientific, Inc., Glenwood, IL). formamide, 5Â SSC, 0.01% SDS, 8 Ag BSA, 25 Ag yeast tRNA, and 20 Ag Scoring of Hsp27 staining. The staining intensity of malignant tissue salmon testes DNA. Following stringent washes (1Â SSC and 0.1% SDS, was evaluated and scored by one pathologist (L. Fazli) and automated then 0.1Â SSC), fluorescent images of the slides were acquired using an quantitative image analysis was done with pro-plus image software ArrayWoRx, Microarray Scanner (Applied Precision, Seattle, WA). Signal (MediaCybernetics, San Diego, CA). Specimens were graded from 0 to +3 quality and quantity were assessed using Imagene 5.6 (BioDiscovery, San intensity representing the range from no staining to heavy staining. The Diego, CA). Data from Imagene were analyzed in GeneSpring 6.1 using a overall percentage of cancer cells showing staining (0-100%) was also per spot and per chip intensity-dependent (LOWESS) normalization indicated. All comparisons of staining intensities and percentages were (Silicon Genetics, Agilent, Palo Alto, CA) for profiling significant changes made at Â200 magnification. in gene expression. Lentiviral infection of Hsp27 into LNCaP cells. The full-length Northern blot analysis. Total RNA was isolated from cultured cell lines cDNA for human Hsp27 was subcloned into the lentiviral vector pHRV- or xenografts using the Trizol method (Invitrogen, Life Technologies, Inc., cytomegalovirus (CMV)-enhanced green fluorescent protein (EGFP) at the Burlington, Ontario, Canada). The quality and quantity of RNA was assessed BamHI and XhoI sites. Two vectors were created for study: pHRV-CMV- with an Agilent 2100 bioanalyzer (Caliper Technologies Corp., Mountain Hsp27 and pHRV-CMV (empty vector). Clone identity was verified using View, CA). Electrophoresis, hybridization, and washing conditions were restriction digest analysis and plasmid DNA sequencing. Infectious carried out as previously reported (24). Human Hsp27 cDNA probes was lentivirus was generated by cotransfection of 1.5 Â 106 293T cells with obtained from StressGen (Victoria, Canada). The density of bands for Hsp27 target plasmids with pCMVDR8.2 (carries sequence necessary for viral were normalized with 28S rRNA by densitometric analysis (Quantity One, assembly of lentivirus) and pMD.G, which expresses the vesicular stomatitis Bio-Rad Laboratories, Hercules, CA). Each assay was done in triplicate. virus envelop glycoprotein G pseudotype. The 293T cells were transfected

Human secretory nonpancreatic phospholipase A2 (sPLA2-IIA) probe was for 12 to 15 hours, after which fresh medium were added for 24 hours. After generated by reverse transcription of total RNA from LNCaP using this, the virus containing media was collected and filtered through a

Superscript II (Life Technologies) and random hexamers [p(dN)6, Roche] 0.45-Am filter. Early passage LNCaP cells (passage 30) were plated in 10 cm as primers. The cDNA generated was used as a template for PCR with the plates, and competent retrovirus was added to 30 to 40 multiplicity of following primer pairs from Operon: 5V-aaggaagccgcactcagtta-3V(sense) and infection. Cells were harvested for UV microscopy to verify green 5V-ttgcacaggtgattctgctc-3V(antisense). PCR products were cloned into pCRII- fluorescent protein expression, and Western blotting was used to verify TOPO (Invitrogen, Life Technologies), and the resulting construct was Hsp27 expression. transformed into chemically competent Escherichia coli. The density of For in vitro growth assays, LNCaP-Hsp27 and LNCaP-empty were plated bands for Hsp27 was normalized with 28S rRNA by densitometric analysis at different densities, 2.5, 10, 50, and 100 Â 104 cells into a 75 cm2 flask in (Quantity One, Bio-Rad Laboratories). RPMI 1640 supplemented with 5% FCS. The following day, cells were treated Prostate tissue specimens. Prostate tissues (n = 232) were obtained in RPMI 1640 serum-free media. After 5 weeks, cells were harvested for flow from the tissue bank in the Department of Pathology and Prostate cytometry analysis as described below.

Cancer Res 2005; 65: (23). December 1, 2005 11084 www.aacrjournals.org

Antisense oligonucleotides and short-interfering RNA sequences. (v/v distilled H2O) in a water bath at 56jCto60jC for 20 minutes. Slides Hsp27 phosphorothioate ASO targeting the human Hsp27 translation were then incubated for 10 minutes in 0.2% triton, and endogenous initiation site (5V-GGGACGCGGCGCTCGGTCAT-3V) were purchased from peroxidase quenched in 3% hydrogen peroxide for 5 minutes. Detection of Qiagen Operon (Alameda, CA). Human signal transducers and activators of cleaved apoptotic DNA fragments were done using mouse monoclonal transcription 3 (Stat3) ASO (5V-GCTCCAGCATCTGCTGCTTC-3V)was antibody MAB3299 (Chemicon International, Temecula, CA). generously provided by Dr. B. Monia at Isis Pharmaceuticals (Carlsbad, Flow cytometric analysis. Flow cytometric analysis of propidium CA). Scrambled control oligonucleotide (5V-CAGCGCTGACAACAGTTTCAT- iodide–stained nuclei was done as described previously (7). Briefly, the 3V) was purchased from Qiagen Operon. Small RNA interference siACE-RNAi LNCaP cells were plated in 75 cm2 dishes and were treated as described was purchased from Dharmacon, Inc. (Lafayette, CO). The sequence above the following day. The cells were trypsinized 2 days after ASO of Hsp27 siRNA duplex used corresponded to the human Hsp27 site treatment and analyzed for relative DNA content on a dual laser flow (5V-GUCUCAUCGGAUUUUGCAGC-3V). A scrambled siRNA duplex cytometer (Beckman Coulter Epics Elite, Beckman, Inc., Miami, FL). Each (5V-CAGCGCUGACAACAGUUUCAU-3V) was used as a control. assay was done in triplicate. Treatment of cells with antisense oligonucleotides or short- Measurement of caspase-3 cleavage. Caspase-3 cleavage was detected interfering RNA. Cells were plated at a density of 30,000 cells by 1.9 cm2 by Western blotting as described above using 40 Ag of proteins. Polyclonal and treated the day after with the indicated siRNA or ASO for 1 or 2 days, antibody (New England BioLabs, Inc., Mississauga, Ontario, Canada) was respectively. OligofectAMINE, a cationic lipid (Invitrogen, Life Technolo- used to detect full length (32-35 kDa) and large fragments of activated gies), was used to increase ASO or siRNA uptake into the cells. LNCaP cells caspase-3 (17-20 kDa) which results from cleavage after Asp175. were treated with increasing concentrations of ASO or siRNA after a Assessment of in vivo tumor growth. For in vivo studies, using a preincubation for 20 minutes with 3 mg/mL OligofectAMINE in serum-free 27-gauge needle, 106 LNCaP cells were inoculated s.c. with 0.1 mL of OPTI-MEM (Life Technologies). Four hours after the beginning of the Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ) in the flank incubation, the medium was replaced with standard culture medium region of 6- to 8-week-old male athymic nude mice (Harlan Sprague- described above. Dawley, Inc., Indianapolis, IN) under halothane anesthesia. All animal Western blot analysis. Hsp27 and caspase-3 cleavage Western blot procedures were done according to local guidelines on animal care and analysis was done as described previously (7). Stat3 and c-fos were detected with appropriate institutional certification. Tumors were measured twice by Western blotting using 40 Ag of proteins. Rabbit polyclonal antibody weekly and their volumes were calculated by the formula: length Â width (Santa Cruz Biotechnology, Inc. Delaware, CA) was used to detect total Stat3 Â depth Â 0.5236. Mice bearing tumors between 200 and 300 mm3 in (92 kDa) and chicken polyclonal antibody (Abcam, Cambridge, MA) was volume were castrated via scrotal approach and randomly assigned to a used to detect c-fos (46 kDa). treatment arm. Mice were treated beginning 1 day after castration with Immunofluorescence. LNCaP-empty and -Hsp27 cells were grown 10 mg/kg of ASO or control oligonucleotide i.p. once daily. Tumor volume on glass coverslips in RPMI plus 5% fetal bovine serum for 48 hours. Subsequently, cells were fixed with cold 3% acetone in methanol for 10 minutes at À20jC and permeabilized in 0.2% Triton in PBS. Slides were incubated in blocking solution, 3% milk in PBS for 1 hour, and simultaneously treated overnight with primary antibodies, mouse monoclonal Hsp27 (StressGen) and rabbit polyclonal Stat3 (Santa Cruz Biotechnology). Secondary fluorescent antibodies antimouse FITC and antirabbit Texas red conjugated were added for 1 hour at room temperature with three 5-minute washes (0.1% Triton in PBS). Cells examined for localization of red and green protein were mounted with fluorescent 4V,6- diamidino-2-phenylindole vectashield mounting medium (Vector Laborato- ries). Images were captured using a Zeiss Axioplan II fluorescence microscope at Â63 magnification followed by analysis with imaging software (Northern Eclipse, Empix Imaging, Inc., Mississauga, Ontario, Canada). Analysis of focal colocalization was also done with Northern Eclipse and Adobe Photoshop 5.5 software with an assignment of yellow (Y) for colocalized foci and green (G) or red (R) as non-colocalization. Immunoprecipitation. LNCaP cells were lysed in radioimmunoprecipitation assay buffer without SDS containing complete proteinase inhibitor cocktail (Roche Diagnostic, Mannheim, Germany). Lysates from LNCaP- Hsp27, LNCaP-empty, or LNCaP-Hsp27 was incubated with 5 Ag anti-Stat3 (Santa Cruz Biotechnology), anti-Hsp27 (StressGen), and anti-IgG antibodies (Santa Cruz Biotechnology). After 12 hours of incubation, 50 ALof protein A beads (Amersham Pharmacia Biosciences) were added into the reaction tubes and incubated for 2 hours. The beads were washed three times using radioimmunoprecipitation assay lysis buffer and resolved in 5Â loading buffer (MBI, Fermentas Inc., Burlington, Canada). In vitro mitogenic assay. The in vitro growth-inhibitory effects of Hsp27 ASO on LNCaP cells were assessed using the crystal violet assay as previously described (4). Cells were treated once daily with 20 nmol/L of oligonucleotide for 2 days or 1 nmol/L siRNA for 1 day. Every 24 hours, over a period of 4 days, crystal violet assays were done. Each assay was done in Figure 1. Castration-induced changes of Hsp27 mRNA expression in vivo. triplicate. LNCaP tumors were harvested from intact male athymic mice before and Immunocytochemistry for in situ apoptosis. LNCaP cells were plated 35 days after castration. A, total RNA was extracted from each tumor tissue and in 9.87 cm2 labtek (Nunc, Roskilde, Denmark) and treated with analyzed for Hsp27 and 28S levels by Northern blotting. B, quantitative analysis oligonucleotides or siRNA as described above. Two days after transfection, of Hsp27 mRNA levels after normalization to 28S ribosomal mRNA levels was done using the optical densitometer and serum PSA was collected in each cells were harvested and fixed with methanol for 10 minutes. Slides were animal before (day 0) and 35 days post-castration. *, P V 0.05; **, P V 0.01, then transferred into a coplin jar containing 50 mL of 50% formamide experiments were repeated in triplicate. www.aacrjournals.org 11085 Cancer Res 2005; 65: (23). December 1, 2005

Figure 2. Changes in Hsp27 immunostaining in human prostate cancer tissue microarray. A-i, neoplastic glands in nontreated tumors: a few foci of weakly positive cancer cells are visible but most of tumor is not immunoreactive. A-ii and iii, strong immunoreactivity of cancer cells after 3 and 6 months of androgen ablation, respectively. A-iv, sheets of uniformly and intensely reactive tumor cells characteristic of AI tumors. B, mean Hsp27 staining after androgen ablation. Specimens spotted on the tissue microarray were graded from 0 to +3 intensity, representing the range from no staining to heavy staining by visual scoring and automated quantitative image analysis by pro-plus image software. Data from 232 samples were used to calculate average F SE. All comparison of stain intensity was made at 200Â magnification. **, P V 0.01; ***, P V 0.001, indicate significance between groups. C and D, highly intensive and uniformly reactive tumors characteristic of hormone-refractory bone and liver metastases, respectively. and serum PSA measurements were done weekly. Data points for both sets Hsp27 mRNA levels in hormone-dependent LNCaP xenografts of experiments were expressed as average tumor volume F SE of the mean collected at baseline with AI tumors 35 days after castration. Log2 based on 10 determinations. plots from comparative microarray analyses of tumors at baseline F Statistical analysis. All of the results were expressed as the mean and 35 days post-castration identified Hsp27 within the top 1% of SE. Statistical analysis was done with a one-way ANOVA followed by overexpressed genes in AI tumors. Changes in Hsp27 correlated Fisher’s protected least significant difference test (StatView 512, Brain Power, Inc., Calabasas, CA). *, P V 0.05 was considered significant; **, P V highly (95%) with changes in tumor PSA mRNA levels (data not 0.01; ***, P V 0.001. shown). Northern blot analysis of tumors confirmed that Hsp27 mRNA increased by 77.6% 35 days after castration (**, P V 0.01) compared with the precastrate levels (Fig. 1A and B). Changes in Results Hsp27 mRNA expression also paralleled changes in serum PSA in Hsp27 mRNA expression increases after androgen ablation tumor-bearing mice, the main end point of AI progression in the in LNCaP xenografts. Expression profiling using 13,791 (70-mer) LNCaP model (Fig. 1B). human oligos (Operon) printed in duplicate onto amine-coated Hsp27 immunostaining increases after androgen withdrawal slides were used to quantify changes in Hsp27 expression during in human prostate cancer. To determine whether Hsp27 levels castration-induced apoptotic stress and AI progression, comparing also increase after androgen-ablation or during AI progression in

Cancer Res 2005; 65: (23). December 1, 2005 11086 www.aacrjournals.org

Table 1. Hsp27 staining in prostate cancer before and after androgen ablation

Prostate cancer No. of cases Immunohistochemistry SD SE score

Not treated 35 0.66 0.14 0.07 Neoadjuvant hormone therapy–treated (mo) <3 58 1.29 0.20 0.09 3-6 52 1.76 0.22 0.11 >6 57 1.99 0.507335 0.10 Hormone-refractory prostate specimens 16 2.08 0.25 0 Hormone-refractory metastasis Bone 6 2.16 0.6645 0.48 Pelvic lymph nodes 3 1.33 0.308 1.2 Non-osseous distant metastasis (liver, lung, adrenal) 5 1.7 0.2885 0.55

human disease, 232 prostate cancer specimens spotted on a respectively, when scored as described in Materials and Methods tissue microarray were stained for Hsp27 by immunohistochem- (Fig. 2B). AI tumor tissue from prostate and metastatic sites istry (Fig. 2; Table 1). Hsp27 protein was present in the exhibited uniform and highly positive Hsp27 staining in all cytoplasm of the epithelial cells (Fig. 2A). More specifically, specimens (Fig. 2C and D). Hsp27 staining was limited to the basal layer of the benign Overexpression of Hsp27 inhibits androgen withdrawal– prostate glands, and there was very weak or no staining in induced apoptosis and promotes androgen-independent untreated prostate cancer. Hsp27 staining increases 4-fold after tumor growth. Figure 3A verifies elevated Hsp27 protein levels in neoadjuvant hormone therapy and was significantly higher at all LNCaP cells stably expressing human Hsp27 cDNA (LNCaP-Hsp27). time points after hormone therapy compared with untreated LNCaP-Hsp27 and empty vector controls were cultured at different hormone-naı¨ve prostate cancer (***, P V 0.001). The mean densities in serum-free media for 5 weeks (see Materials and intensity of positive cells in the untreated, <3 months, 3 to 6 Methods). Flow cytometry analysis shows that apoptotic rates months, >6 months, and AI were 0.66, 1.29, 1.76, 1.99, and 2.08, (sub-G0 phase) were significantly lower (*, P V 0.05; ***, P V 0.001)

Figure 3. Effect of Hsp27 overexpression on AI LNCaP cell survival in vitro and tumor growth in vivo. A, the full-length cDNA for human Hsp27 was subcloned into the lentiviral vector pHRV-CMV-EGFP. After 1 week, protein was extracted from cultured cells and Hsp27 and glyceraldehyde-3- phosphate dehydrogenase protein levels were analyzed by Western blotting. B, LNCaP-Hsp27 and -empty vector cells were cultured at different densities in RPMI 1640 serum-free medium for 5 weeks and apoptotic rates quantified by flow cytometry. The results are expressed in percentage and the value obtained for LNCaP-mock at 2, 5.104 density of cells was chosen as control (100%). C, effects of Hsp27 overexpression on LNCaP tumor growth post-castration. Twenty nude mice were injected with LNCaP-Hsp27 or LNCaP-empty (106 cells). The mice were castrated when PSA increased >50 ng/mL. *, mean tumor volume increased significantly in LNCaP-Hsp27 compared with LNCaP-empty tumors beginning 1 week after castration. D, effects of Hsp27 overexpression on serum PSA levels post-castration. Mean serum PSA levels increased significantly faster in the LNCaP-Hsp27 group compared with LNCaP-empty group beginning 1 week after castration.

www.aacrjournals.org 11087 Cancer Res 2005; 65: (23). December 1, 2005

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research in LNCaP-Hsp27 cells than the LNCaP-empty control cells (Fig. 3B). LNCaP-empty control group (46 F 21.8 ng/mL; *, P V 0.05). LNCaP-Hsp27 cells were also more resistant to paclitaxel-induced These data imply that increased Hsp27 levels protect androgen- apoptosis compared with LNCaP-empty cells (P V 0.001, data dependent prostate cancer cells from treatment stress induced not shown). by androgen withdrawal and facilitate AI tumor progression after We next compared the rate of AI progression in LNCaP castration. tumors in vivo after castration in LNCaP-Hsp27- (n = 10) and Sequence-specific and dose-dependent inhibition of Hsp27 control-transfected (n = 10) tumors. Beginning 1 week after expression by antisense oligonucleotides and short-interfering castration, tumor volume and serum PSA levels increased more RNA. To further study the functional relevance of castration- rapidly in LNCaP-Hsp27 tumors compared with mock-transfected induced increases in Hsp27, inhibition of Hsp27 gene expression controls. At the time of sacrifice, tumor volume (Fig. 3C) was using Hsp27 ASO or siRNA was employed with the appropriate 4.3-fold higher in the LNCaP-Hsp27 group (1,912 F 330 mm3) scrambled controls. As shown in Fig. 4A and B, ASO treatment of compared with the LNCaP-empty control group (437 F 57 mm3; LNCaP cells reduced Hsp27 mRNA levels in a dose-dependent **; P V 0.01), and serum PSA (Fig. 3D) was 10-fold higher in the manner up to 73% at 50 nmol/L (**, P V 0.01), whereas Hsp27 LNCaP-Hsp27 group (466 F 114 ng/mL) compared with the mRNA expression was not significantly suppressed by scrambled

Figure 4. Sequence-specific and dose-dependent suppression of Hsp27 mRNA and protein levels by Hsp27 ASO and siRNA. A, LNCaP cells were treated daily with various concentrations of Hsp27 siRNA or ASO or a scrambled control for 1 or 2 days, respectively. One day after treatment, total RNA was extracted from cultured cells, and Hsp27 and 28S levels were analyzed by Northern blotting. Oligofectamine, cells treated with Oligofectamine only. B, quantitative analysis of Hsp27 mRNA levels after normalization to 28S rRNA levels by densitometry analysis in LNCaP cells after treatment with various concentrations of Hsp27 ASO or scrambled control. Points, means of triplicate analysis; bars, SE. *, P V 0.05; **, P V 0.01, difference from scrambled controls using Student’s t test. C, LNCaP cells were treated once with various concentrations of Hsp27 siRNA or scrambled control. Two days after treatment, total RNA was extracted from cultured cells, and Hsp27 and 28S levels were analyzed by Northern blotting as described above. D and E, LNCaP cells were treated daily with various concentrations of Hsp27 ASO or siRNA or scrambled control. Three days after the first treatment, proteins were extracted from cultured cells, and Hsp27 and vinculin protein levels were analyzed by Western blotting.

Cancer Res 2005; 65: (23). December 1, 2005 11088 www.aacrjournals.org

Figure 5. Hsp27 ASO induces apoptosis in LNCaP cells in vitro. A, LNCaP cells were treated daily with 20 nmol/L of Hsp27 ASO or scrambled control for 2 days. After an additional 24 hours of incubation, cell viability was determined daily for 4 days using the crystal violet assay. Points, means of quadruplicate analyses; bars, SE. **, P V 0.01; ***, P V 0.001, difference from scrambled control using Student’s t test. B, 2 days after 20 nmol/L Hsp27 ASO or scrambled control treatment, LNCaP cells were fixed and stained with antibody MAB3299 (Chemicon) that specifically reacts with ssDNA characteristic of apoptotic cells. C, the apoptotic index was determined after counting the number of highly positive labeled cells divided by the total cell number in the slide. The experiment was repeated in triplicate. ***, P V 0.001, significance of differences between Hsp27 ASO- and scrambled control-treated groups. D, flow cytometry was used to quantify the percentage of cells undergoing apoptosis (cells in sub–G0-G1). The experiment was repeated in triplicate. *, P V 0.05; **, P V 0.01, differ from scrambled controls using Student’s t test. E, after 48 hours of treatment with 20 nmol/L Hsp27 ASO or scrambled, cells were harvested and protein extracted for Western blotting with caspase-3 antibody that recognizes both full-length and cleaved caspase-3 fragments.

oligonucleotide. Similar dose-dependent inhibition was observed 8.9-fold after treatment with Hsp27 ASO (**, P V 0.001) in LNCaP using siRNA between 10À3 and 1 nmol/L (Fig. 4C). Significant cells compared with those treated with control oligonucleotide inhibition of Hsp27 protein levels in LNCaP cells were also (Fig. 5B and C). detected after treatment with Hsp27 ASO (Fig. 4D) and siRNA The induction of apoptosis by Hsp27 ASO was also clearly shown (Fig. 4E). using flow cytometry. After the same treatment schedule described Hsp27 antisense oligonucleotides and short-interfering RNA above, the fraction of cells undergoing apoptosis (sub–G0-G1 induce apoptosis and increase caspase-3 cleavage. Hsp27 ASO fraction) was significantly higher after treatment with Hsp27 ASO treatment significantly reduced LNCaP cell growth by 29% (**, P V 20 nmol/L (35.2% versus 18.9 %; **, P V 0.01), compared with 0.01), 31% (**, P V 0.01), 40% (**, P V 0.01), and 63% (***, P V 0.001) control oligonucleotides (Fig. 5D). 1, 2, 3, and 4 days posttreatment, respectively, compared with Hsp27 has been reported to interact with and inhibit caspase-3 scrambled controls (Fig. 5A). Similar growth suppression occurred activation (25). Using coimmunoprecipitation (data not shown), using 1 nmol/L Hsp27 siRNA (data not shown). Apoptosis, detected we confirmed that Hsp27 interacts with several regulators of by ssDNA nuclear staining (MAB 3299, Chemicon), increased the mitochondrial apoptotic pathway (bax and cytochrome c), and www.aacrjournals.org 11089 Cancer Res 2005; 65: (23). December 1, 2005

Figure 6. Effect of Hsp27 knock-down on Stat3 signaling pathway. LNCaP cells were treated daily with various concentrations of Hsp27 ASO or scrambled control for 2 days. A and B, 3 days after the first treatment, proteins were extracted from cultured cells, and Stat3, c-fos, and vinculin protein levels were analyzed by Western blotting. C, 2 days after the first treatment, total RNA was extracted from cultured cells, and sPLA2-IIA and 28S levels were analyzed by Northern blotting. Oligofectamine, cells treated with Oligofectamine only. D, LNCaP-Mock and -Hsp27 were treated with 500 nmol/L of Stat3 ASO or scrambled control for 2 days. Serum-free media and 10 nmol/L of paclitaxel were added after transfection for 24 hours and the cells were then analyzed for their growth properties by crystal violet assay (OD, 560 nmol/L). The experiment was repeated in triplicate. **, P V 0.01; *, P V 0.05, significance of differences between Stat3 ASO- and scrambled control-treated groups.

that caspase-3 cleavage increases significantly within 2 days after Stat3-regulated genes c-fos protein and sPLA2-IIa mRNA also Hsp27 ASO treatment (Fig. 5E). Increased caspase-3 cleavage after decreased after the same treatment (Fig. 6B and C). Moreover, Hsp27 ASO was also detected by immunostaining using an when a Stat3 ASO was used to specifically knock down Stat3 antibody specific for the cleaved caspase-3 fragment (data not levels in LNCaP-empty and LNCaP-Hsp27 cells, the cyto- shown). Hsp27 siRNA (1 nmol/L) resulted in similar increases in protection to in vitro androgen withdrawal or paclitaxel DNA fragmentation, sub–G0-G1 fraction, and caspase-3 cleavage treatment normally conferred by Hsp27 overexpression in (data not shown). Collectively, these data suggest that Hsp27 LNCaP-Hsp27 cells was no longer detected (Fig. 6D). Collectively, knockdown increases the rate of apoptosis by interfering with these data suggest that the increased rate of apoptosis after Hsp27-regulated inhibition of caspase-3 activation. Hsp27 knock down is in part due to attenuation of Hsp27- Hsp27 antisense oligonucleotides inhibits proliferation and mediated regulation of Stat3 activity. induces apoptosis via inhibition of signal transducers and Hsp27 colocalizes and interacts with signal transducers and activators of transcription 3 activity. In many human cancers, activators of transcription 3. To further define the role of aberrant constitutive activation of Stat3 is sufficient to induce Hsp27 in the regulation of, or interaction with, Stat3, we examined tumorigenesis through activation of proto-oncogenes like c-fos whether Hsp27 colocalizes and interacts with Stat3 using immu- and sPLA2-IIA (26–28). Stat3 enhances cell survival and nofluorescence and coimmunoprecipitation. Figure 7A illustrates oncogenesis in breast carcinoma cells (18), whereas Stat3 that Hsp27 colocalizes in the cytoplasm with Stat3 in LNCaP-empty signaling is implicated in the development of AI progression and LNCaP-Hsp27 cell lines, and that the intensity of Stat3 staining (29, 30). To determine whether androgen ablation–induced and colocalization is higher in Hsp27 transfectants relative to changes in Hsp27 could act as an upstream regulator, Stat3, mock-transfected cells. Using coimmunoprecipitation, Hsp27 LNCaP, and PC-3 cells were treated with increasing concen- coimmunoprecipitated with Stat3 in both LNCaP-mock and trations of Hsp27 ASO or control oligonucleotides. RNA and LNCaP-Hsp27 and higher levels of Stat3 were detected in Hsp27- protein were extracted 2 and 3 days after treatment, respectively, transfectants (Fig. 7B). Collectively, the results illustrated in Figs. 6 and analyzed for Stat3 expression levels and the downstream and 7 indicate that Hsp27 interacts, either directly or indirectly Stat3-regulated genes, c-fos and sPLA2-IIA. Stat3 protein levels as part of a protein complex, with Stat3, and that Stat3 levels decreased within 2 days in LNCaP (Fig. 6A) and PC-3 cells (data and transcriptional regulation of c-fos and sPLA2-IIA directly not shown) after treatment with Hsp27 ASO. In addition, the correlate with Hsp27 levels.

Cancer Res 2005; 65: (23). December 1, 2005 11090 www.aacrjournals.org

Hsp27 antisense oligonucleotides delay LNCaP tumor Discussion progression after castration in vivo. Twenty male mice bearing Androgen ablation induces apoptotic prostate cancer cell death, LNCaP tumors were castrated 6 to 8 weeks after tumor im- but despite the initially high response rates, remissions are tem- plantation and were randomly selected for treatment with Hsp27 porary because surviving tumor cells eventually recur within ASO versus scrambled control. Mean tumor volume and PSA were several years. Cellular and molecular events that mediate AI prog- similar in both groups at the beginning of the treatment. Beginning ression are complex and multifactorial, and include adaptive 1 day after castration, 10 mg/kg of oligonucleotide was diluted changes in the expression of various apoptosis-regulating genes, in PBS and administered once daily by i.p. injection for including androgen receptor, bcl-2, clusterin, IGFBP-2, and 10 weeks. As shown in Fig. 8, LNCaP tumor volume (A) and serum IGFBP-5 (1–6). PSA levels (B) decreased more rapidly in mice treated with Hsp27 Hsp27 expression is induced by various stressors such as ASO, compared with those treated with scrambled controls. chemotherapy and can inhibit cell death triggered by various All mice (n = 10) treated with castration plus Hsp27 ASO had stimuli when overexpressed (31). Hsp27 prevents formation of the a significant inhibition of AI tumor growth during the 10 weeks apoptosome and the subsequent activation of caspases through of analysis. At sacrifice, mean PSA levels were 3.2-fold higher in direct sequestration of cytochrome c released from the mitochon- the scrambled control (42.67 F 8.2 ng/mL) compared with the dria into the cytosol (32). When expressed at high levels, Hsp27 also Hsp27 ASO–treated group (13.12 F 3 ng/mL; **, P V 0.01), whereas interferes with caspase activation upstream of the mitochondria, tumor volume was 2.3-fold higher in scrambled control (506.51 F for instance, by preventing the caspase-8-triggered activation of the 106.8 mm3) compared with the Hsp27 ASO–treated group (214.27 F proapoptotic Bcl-2 family member, Bid (33). Hsp27 may also inhibit 57.45 mm3;*,P V 0.05). No side effects were observed with Hsp27 cytochrome c–mediated caspase activation by sequestering both ASO or scrambled control treatment. pro-caspase-3 and cytochrome c (34).

Figure 7. Hsp27 colocalizes and interacts with Stat3. A, LNCaP-empty and -Hsp27 cells were grown on glass coverslips on RPMI with 5% fetal bovine serum for 48 hours. Subsequently, cells were fixed and incubated with mouse monoclonal Hsp27 (green) and rabbit polyclonal Stat3 (red) antibody simultaneously. Analysis of focal colocalization was done with Northern Eclipse and Adobe Photoshop 5.5 software with an assignment of yellow (Y) for a colocalized foci and green (G) or red (R) as non-colocalization. B, LNCaP-empty and LNCaP-Hsp27 cells were harvested and cell lysate was used to immunoprecipitate Hsp27 and Stat3 using anti-Stat3 and anti-Hsp27 antibodies, respectively. After resolving immuno-complexes on 10% SDS-PAGE and transfer to polyvinylidene difluoride membrane, the membrane was probed with anti-Stat3 and anti-Hsp27 antibodies, respectively. www.aacrjournals.org 11091 Cancer Res 2005; 65: (23). December 1, 2005

by the activation of Stat3. For example, androgen-independent DU145 prostate cancer cell lines constitutively express activated Stat3 as a result of autocrine production of IL-6, whereas androgen- sensitive LNCaP cells increase Stat3 activity following paracrine- mediated IL-6 stimulation via Janus-activated kinase (37, 38). Increased Stat3 signaling has been linked to AI progression, whereas constitutively active Stat3 signaling can antagonize androgen deprivation–induced prostate cell death (39, 40). IL-6-induced proliferation of prostate cancer cell lines is associated with the activation of Stat3, whereas inhibition of IL-6 activation of Stat3 inhibited cell growth and induction of apoptosis (35, 37). In this study, we found that Hsp27 colocalizes with Stat3 and that Stat3 protein levels correlate directly with changes in Hsp27. Our findings suggest that increased apoptotic rates after Hsp27 silencing is mediated, in part, via reduced Stat3 protein levels and Stat3-regulated genes c-fos and sPLA2-IIA. Collectively, these findings link increased Hsp27 levels with the hormone-refractory phenotype and indicate that inhibition of Hsp27 up-regulation after androgen withdrawal can inhibit AI progression. Hsps have attracted attention as new therapeutic targets for cancer, especially since the discovery and character- ization of geldanamycin as an inhibitor of Hsp90 (41), and the targeting of the clusterin gene (6, 42, 43), whose product has small Hsp-like function. We are targeting cell survival or antiapoptotic genes up-regulated after androgen withdrawal as one strategy to delay tumor progression (44), and recently reported results of a proof-of-concept phase I trial demonstrating Figure 8. Effect of Hsp27 ASO treatment on LNCaP tumor growth in vivo after that OGX-011, a second-generation ASO targeting clusterin, is castration. A and B, 20 male mice bearing LNCaP tumors were randomly well-tolerated and potently inhibits clusterin expression in selected for treatment with Hsp27 ASO or scrambled control oligonucleotides. Castration was done when tumors reached a mean of 260 mm3. Hsp27 ASO or prostate cancers (43). Phosphorothioate ASOs are chemically scrambled controls were injected (10 mg/kg/mice) daily, starting 1 day after modified stretches of ssDNA complementary to mRNA regions of castration, for 10 weeks. Tumor volume (A) and serum PSA levels (B) were a target gene, thereby inhibiting gene expression and providing a measured once weekly. Points, mean tumor volume in each experimental group containing 10 mice; bars, SE. *, P V 0.05; **, P V 0.01, differ from scrambled useful tool for in vitro and in vivo functional genomics, and can control and by Student’s t test. also serve as preclinical proof-of-principle to support the clinical development of a drug candidate (44, 45). Although siRNA offers an alternative and more potent method of sequence-specific posttranscriptional gene silencing, few chemical modifications The role of Hsp27 in prostate cancer progression has not have been identified that support systemic in vivo activity been well-defined, and only recent studies report its association (46, 47). In this report, suppression of Hsp27 using ASO after with hormone-refractory disease (7, 22). In this study, we castration provides in vivo proof-of-principle that inhibition of identified Hsp27 as highly overexpressed in AI LNCaP tumors castration-induced increases in Hsp27 can delay AI progression. using a gene microarray of 13,791 genes, and confirmed in tissue LNCaP tumor volume and serum PSA decreased more rapidly microarrays that Hsp27 levels increased significantly in human after castration and in mice treated with Hsp27 ASO. Clinical disease after neoadjuvant hormone treatment to become trials with a second-generation ASO targeting Hsp27 (OGX-427) uniformly and highly expressed in AI tumors. The highly uniform are planned to begin early in 2006. expression of Hsp27 in metastatic hormone-refractory lesions In summary, the results of this study support the hypothesis that obtained from rapid autopsy specimens further underscores the increased Hsp27 after androgen ablation is an adaptive response association of Hsp27 with the lethal component of this disease. induced by castration to enhance cell survival, and thereby We extend these observations into functional data by showing promote AI tumor growth. Hsp27 silencing using ASO and siRNA that increased Hsp27 levels following androgen withdrawal technology alters Stat3 signaling, enhances caspase-3 activation confers an antiapoptotic survival advantage to enhance the AI and apoptosis, and offers a treatment strategy to delay progression growth of LNCaP tumors. Importantly, Hsp27 knockdown using of prostate cancer after androgen withdrawal. ASO or siRNA increased active cleaved caspase-3 and apoptotic rates, supporting the negative regulation of caspase-3 activation by Hsp27 (25). Acknowledgments In this report, we identify a mechanism by which castration- Received 5/26/2005; revised 8/9/2005; accepted 9/23/2005. induced changes in Hsp27 expression serves as an upstream reg- Grant support: Terry Fox Foundation of the National Cancer Institute of Canada, and the NIH Pacific Northwest Prostate Specialized Programs of Research Excellence. ulator of Stat3 activity. Considerable evidence suggests that The costs of publication of this article were defrayed in part by the payment of page activated Stat3 functions as an oncogenic protein in many cancers, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. including prostate cancer (35, 36). The AI phenotype induced by We thank Virginia Yago, Mary Bowden, and Eleyna Gomez for their excellent interleukin-6 (IL-6) in several prostate cancer cells is accompanied technical assistance in animal experimentation.

Cancer Res 2005; 65: (23). December 1, 2005 11092 www.aacrjournals.org

References 16. Cornford PA, Dodson AR, Parsons KF, et al. Heat 33. Concannon CG, Orrenius S, Samali A. Hsp27 inhibits shock protein expression independently predicts clin- cytochrome c-mediated caspase activation by seques- 1. Chen CD, Welsbie DS, Tran C, et al. Molecular ical outcome in prostate cancer. Cancer Res 2000;60: tering both pro-caspase-3 and cytochrome c. Gene Expr determinants of resistance to antiandrogen therapy. 7099–105. 2000;9:195–201. Nat Med 2004;10:33–9. 17. Garrido C, Ottavi P, Fromentin A, et al. Hsp27 as a 34. Paul C, Manero F, Gonin S, Kretz-Remy C, Virot S, 2. Rocchi P, Muracciole X, Fina F, et al. Molecular analysis mediator of confluence-dependent resistance to cell Arrigo AP. Hsp27 as a negative regulator of cytochrome integrating different pathways associated with androgen- death induced by anticancer drugs. Cancer Res 1997;57: C release. Mol Cell Biol 2000;22:816–34. independent progression in LuCaP 23.1 xenograft. 2661–7. 35. Mora LB, Buettner R, Seigne J, et al. Constitutive Oncogene 2004;23:9111–9. 18. Song H, Ethier SP, Dziubinski ML, Lin J. Stat3 activation of Stat3 in human prostate tumors and cell 3. Kiyama S, Morrison K, Zellweger T, et al. Castration- modulates heat shock 27 kDa protein expression in lines: direct inhibition of Stat3 signaling induces induced increases in insulin-like growth factor-binding breast epithelial cells. Biochem Biophys Res Commun apoptosis of prostate cancer cells. Cancer Res 2000;62: protein 2 promotes proliferation of androgen-independent 2004;314:143–50. 6659–66. human prostate LNCaP tumors. Cancer Res 2003;63: 19. Vargas-Roig LM, Gago FE, Tello O, Aznar JC, Ciocca 36. Calo V, Migliavacca M, Bazan V, et al. STAT proteins: 3575–84. DR. Heat shock protein expression and drug resistance from normal control of cellular events to tumorigenesis. 4. Gleave M, Tolcher A, Miyake H, et al. Progression to in breast cancer patients treated with induction J Cell Physiol 2003;197:157–68. androgen independence is delayed by adjuvant treat- chemotherapy. Int J Cancer 1998;79:468–75. 37. Barton BE, Karras JG, Murphy TF, Barton A, Huang ment with antisense Bcl-2 oligodeoxynucleotides after 20. Thomas SA, Brown IL, Hollins GW, et al. Detection HF. Signal transducer and activator of transcription 3 castration in the LNCaP prostate tumor model. Clin and distribution of heat shock proteins 27 and 90 in (STAT3) activation in prostate cancer: direct STAT3 Cancer Res 1999;5:2891–8. human benign and malignant prostatic tissue. Br J Urol inhibition induces apoptosis in prostate cancer lines. 5. Miyake H, Monia BP, Gleave ME. Inhibition of 1996;77:367–72. Mol Cancer Ther 2004;3:11–20. progression to androgen-independence by combined 21. Gibbons NB, Watson RW, Coffey RN, Brady HP, 38. Jia L, Choong CS, Ricciardelli C, Kim J, Tilley WD, adjuvant treatment with antisense BCL-xL and antisense Fitzpatrick JM. Heat-shock proteins inhibit induction of Coetzee GA. Androgen receptor signaling: mechanism of Bcl-2 oligonucleotides plus taxol after castration in the prostate cancer cell apoptosis. Prostate 2000;45:58–65. interleukin-6 inhibition. Cancer Res 2004;64:2619–26. Shionogi tumor model. Int J Cancer 2000;86:855–62. 22. Bubendorf L, Kolmer M, Kononen J, et al. Hormone 39. Lee SO, Lou W, Hou M, de Miguel F, Gerber L, Gao 6. Miyake H, Rennie P, Nelson C, Gleave ME. Testoster- therapy failure in human prostate cancer: analysis by AC. Interleukin-6 promotes androgen-independent one-repressed prostate message-2 (TRPM-2) is an complementary DNA and tissue microarrays. J Natl growth in LNCaP human prostate cancer cells. Clin antiapoptotic gene that confers resistance to androgen Cancer Inst 1999;91:1758–64. Cancer Res 2003;9:370–6. ablation in prostate cancer xenograft models. Cancer 23. Gleave ME, Hsieh JT, Wu HC, von Eschenbach AC, 40. Lee SO, Lou W, Johnson CS, Trump DL, Gao AC. Res 2000;60:170–6. Chung LW. Serum prostate specific antigen levels in Interleukin-6 protects LNCaP cells from apoptosis 7. Rocchi P, So A, Kojima S, et al. Heat shock protein 27 mice bearing human prostate LNCaP tumors are induced by androgen deprivation through the Stat3 increases after androgen ablation and plays a cytopro- determined by tumor volume and endocrine and growth pathway. Prostate 2004;60:178–86. tective role in hormone-refractory prostate cancer. factors. Cancer Res 1992;52:1598–605. 41. Workman P. Altered states: selectively drugging Cancer Res 2004;64:6595–602. 24. Rocchi P, Boudouresque F, Zamora AJ, et al. the Hsp90 cancer chaperone. Trends Mol Med 2004; 8. Concannon CG, Gorman AM, Samali A. On the role of Expression of adrenomedullin and peptide amidation 10:47–51. Hsp27 in regulating apoptosis. Apoptosis 2003;8:61–70. activity in human prostate cancer and in human 42. Zellweger T, Miyake H, Monia B, Cooper S, Gleave M. 9. Parcellier A, Gurbuxani S, Schmitt E, Solary E, Garrido prostate cancer cell lines. Cancer Res 2001;61:1196–206. Antitumor activity of antisense clusterin oligonucleo- C. Heat shock proteins, cellular chaperones that 25. Pandey P, Nakazawa A, Ito Y, Datta R, Kharbanda S, tides is improved in vitro and in vivo by incorporation of modulate mitochondrial cell death pathways. Biochem Kufe D. Requirement for caspase activation in mono- 2V-O-(2-methoxy)ethyl chemistry. J Pharmacol Exp Ther Biophys Res Commun 2003;304:505–12. cytic differentiation of myeloid leukemia cells. Onco- 2001;298:934–40. 10. Conroy SE, Latchman DS. Do heat shock proteins gene 2000;19:3941–7. 43. Chi K, Eisenhauer E, Fazli L, et al. A phase I have a role in breast cancer? Br J Cancer 1996;74:717–21. 26. Bromberg J, Wrzeszczynska M, Devgan G, et al. Stat3 pharmacokinetic (PK) and pharmacodynamic (PD) 11. Kapranos N, Kominea A, Konstantinopoulos PA, et al. as an oncogene. Cell 1999;98:295–303. study of OGX-011, a 2Vmethoxyethyl phosphorothioate Expression of the 27-kDa heat shock protein (HSP27) in 27. Peilot H, Rosengren B, Bondjers G, Hurt-Camejo E. antisense to clusterin, in patients with prostate cancer gastric carcinomas and adjacent normal, metaplastic, Interferon-g induces secretory group IIA phospholipase prior to radical prostatectomy. J Natl Cancer Inst 2005;7: and dysplastic gastric mucosa, and its prognostic A2 in human arterial smooth muscle cells. J Biol Chem 1287–96. significance. J Cancer Res Clin Oncol 2002;128:426–32. 2000;275:22895–904. 44. Gleave ME, Zellweger T, Chi K, et al. Targeting anti- 12. Arts HJ, Hollema H, Lemstra W, et al. Heat-shock- 28. Yang E, Lerner L, Besser D, Darnell JE. Independent apoptotic genes upregulated by androgen withdrawal protein-27 (hsp27) expression in ovarian carcinoma: and cooperative activation of chromosomal c-fos using antisense oligonucleotides to enhance androgen- relation in response to chemotherapy and prognosis. Int promoter by Stat3. J Biol Chem 2003;278:15794–99. and chemo-sensitivity in prostate cancer. Invest New J Cancer 1999;84:234–8. 29. Ni Z, Lou W, Leman ES, Gao AC. Inhibition of Drugs 2002;20:145–58. 13. Wataba K, Saito T, Fukunaka K, Ashihara K, constitutively activated Stat3 signaling pathway sup- 45. Monia BP, Sasmor H, Johnston JF, et al. Sequence- Nishimura M, Kudo R. Over-expression of heat shock presses growth of prostate cancer cells. Cancer Res 2000; specific antitumor activity of a phosphorothioate proteins in carcinogenic endometrium. Int J Cancer 60:1225–8. oligodeoxyribonucleotide targeted to human C-raf 2001;91:448–56. 30. Gao B, Shen X, Kunos G, et al. Constitutive activation kinase supports an antisense mechanism of action 14. Ciocca DR, Oesterreich S, Chamness GC, McGuire of Jak-Stat3 signaling by BRCA1 in human prostate in vivo. Proc Natl Acad Sci U S A 1996;93:15481–4. WL, Fuqua SA. Biological and clinical implications of cancer cells. FEBS Lett 2001;488:179–84. 46. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, heat shock protein 27,000 (Hsp27) [review]. J Natl 31. Garrido C, Schmitt E, Cande C, Vahsen N, Parcellier Mello CC. Potent and specific genetic interference by Cancer Inst 1993;85:1558–70. A, Kroemer G. HSP27 and HSP70: potentially oncogenic double-stranded RNA in Caenorhabditis elegans. Nature 15. Zhang R, Tremblay TL, McDermid A, Thibault P, apoptosis inhibitors. Cell Cycle 2003;2:579–84. 1998;391:806–11. Stanimirovic D. Identification of differentially expressed 32. Bruey JM, Paul C, Fromentin A, et al. Differential 47. Soutschek J. Akinc A, Bramlage B, et al. Therapeutic proteins in human glioblastoma cell lines and tumors. regulation of HSP27 oligomerization in tumor cells silencing of an endogenous gene by systemic adminis- Glia 2003;42:194–208. grown in vitro and in vivo. Oncogene 2000;19:4855–63. tration of modified siRNAs. Nature 2004;432:173–8.

www.aacrjournals.org 11093 Cancer Res 2005; 65: (23). December 1, 2005

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Increased Hsp27 after Androgen Ablation Facilitates Androgen-Independent Progression in Prostate Cancer via Signal Transducers and Activators of Transcription 3− Mediated Suppression of Apoptosis

Palma Rocchi, Eliana Beraldi, Susan Ettinger, et al.

Cancer Res 2005;65:11083-11093.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/65/23/11083

Cited articles This article cites 46 articles, 16 of which you can access for free at: http://cancerres.aacrjournals.org/content/65/23/11083.full#ref-list-1

Citing articles This article has been cited by 25 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/65/23/11083.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/65/23/11083. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.