GLI2 Knockdown Using an Antisense Oligonucleotide Induces Apoptosis and Chemosensitizes Cells to Paclitaxel in Androgen-Independ

Cancer Therapy: Preclinical

GLI2 Knockdown Using an Antisense Oligonucleotide Induces Apoptosis and Chemosensitizes Cells to Paclitaxel in Androgen-Independent Prostate Cancer Shintaro Narita,1Alan So,1, 2 Susan Ettinger,1Norihiro Hayashi,1Mototsugu Muramaki,1Ladan Fazli,1 Yo un g s o o K i m , 3 and Martin E.Gleave 1, 2

Abstract Purpose: GLI transcription factors mediate hedgehog signaling and have been implicated in several human malignancies, including prostate cancer.The objectives of this study were to characterize GLI2 expression levels in human prostate cancer cell lines and tissues to test the effect of antisense oligonucleotide (ASO) targeting GLI2 on androgen-independent (AI) prostate cancer cell lines. Experimental Design: A tissue microarray was used to characterize differences in GLI2 expression in benign prostate hyperplasia, prostate cancer treated by neoadjuvant hormonal therapy and AI prostate cancer.The effects of GLI2 ASO on PC-3 cell growth and paclitaxel chemosensitivity were assessed in vitro and in vivo.Oligonucleotide spotted microarray analysis was used to determine alteration in GLI2 coregulated genes afterASO treatment. Results: The expression of GLI2 was significantly higher in prostate cancer than in benign prostate hyperplasia, decreased after androgen ablation in a time-dependent fashion, but became highly expressed again in AI prostate cancer.GLI2 ASO treatment of PC-3 cells reduced GLI2 mRNA and protein levels in a dose-dependent manner.GLI2 knockdown increased PC-3 cell apoptotic rates and significantly decreased cell growth and modulated levels of apoptosis-related genes, such as Bcl2, Bcl-xL, and clusterin.GLI2 knockdown also changed levels of several cell cycle regulators, such as cyclin D1, p27, and PKC-D.Systematic administration of GLI2 ASO in athymic mice significantly delayed PC-3 tumor progression and enhanced paclitaxel chemosensitivity. Conclusions: These findings suggest that increased levels of GLI2 correlates with AI progression and that GLI2 may be a therapeutic target in castrate-resistant prostate cancer.

Prostate cancer is the most common cancer in men and a growth factor and signaling pathways. To significantly leading cause of death from cancer in North America (1). improve survival in men with prostate cancer, new thera- Androgen ablation and chemotherapy, the only therapies that peutic strategies designed to inhibit the emergence of this have been shown to prolong survival, only induce short-term phenotype must be developed. remissions because surviving tumor cells usually recur with The sonic hedgehog (SHH) signaling pathway is integral in an androgen-independent and chemoresistant phenotype human organ embryogenesis, and recently, activation of this with the eventual progression of disease (2–4). Development pathway in adults has been implicated in many different of advanced disease is a complex process involving up- tumor types, including skin (5), breast (6), lung (7), regulation of antiapoptotic survival genes and activated pancreatic (8), stomach (9), liver (10), and prostate cancers (11–13). SHH is one of three hedgehog ligands in humans that bind the transmembrane receptor Patched1 (Ptch1). Ptch1 constitutively inhibits a second transmembrane protein 1 Authors’ Affiliations: The Prostate Center, Vancouver General Hospital, British Smoothened (Smo). After SHH binds to Ptch1, inhibition of Columbia, Canada; 2Division of Urology, University of British Columbia,Vancouver, Canada; and 3Isis Pharmaceuticals, Carlsbad, California Smo is lost, which leads to the activation of a group of Received 11/1/07; revised 2/12/08; accepted 3/3/08. transcription factors, including GLI1, GLI2, and GLI3. GLI1 Grant support: Pilot Project Grant from Pacific Northwest Prostate Cancer Spore and GLI2 are transcriptional activators of hedgehog target Grant. genes, whereas GLI3 acts mainly as a repressor (14–16). The costs of publication of this article were defrayed in part by the payment of page Although normally dormant in adult prostate, recent evidence charges.This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. indicate that the SHH-GLI pathway is activated in human Note: Supplementary data for this article are available at Clinical Cancer Research prostate cancers when compared with normal prostate Online (http://clincancerres.aacrjournals.org/). epithelium (12, 13, 17). In situ hybridization and quantitative Requests for reprints: Martin E.Gleave,The Prostate Center, 2660 Oak Street, reverse transcription–PCR (RT-PCR) of prostatectomy speci- Vancouver, BC, V6H-3Z6.Phone: 604-874-4818; Fax: 604-875-5654; E-mail: [email protected]. mens indicate that cancers have increased expression of F 2008 American Association for Cancer Research. markers of SHH-GLI pathway activation, including SHH, doi:10.1158/1078-0432.CCR-07-4282 GLI1, and PTCH1, compared with normal epithelium.

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GLI2 seems to be the major nuclear effector of hedgehog total volume of 30 ALat25jC for 10 min, followed at 37jC for 1 h and signaling (18, 19), and the role in carcinogenesis has been denaturation at 95jC for 5 min. The ABI PRISM 7900HT Sequence shown in the context of several cancers (20–22). In prostate Detection System (Applied Biosystems) was used for real-time cancer, GLI2 protein has been reported to be highly expressed monitoring of PCR amplification of complementary DNA. Comple- mentary DNA samples were mixed with the Taqman Universal PCR in prostate cancer cell lines and human prostate cancer Master Mix (Applied Biosystems). Relative quantification of gene tissues (23). expression was done as described in the manual using rRNA as an In this study, we further characterized the relationship internal standard. For GLI2 amplification, Taqman Gene Expression between GLI2 expression and human prostate cancer in men Assay reagent (Hs00257977_m1) was purchased from Applied Bio- receiving hormone therapy, as well as hormone refractory systems. For Bcl2 amplification, the primers and Taqman probes tumors. In addition, we tested the effect of antisense described previously were used (26). The thermal cycling conditions oligonucleotide (ASO)–induced GLI2 knockdown on andro- were 50jC for 2 min, 95jC for 15 s, and 60jC for 1 min. The C gen-independent (AI) prostate cancer cell lines. comparative cycle threshold ( t) method was used for relative quantification of target mRNAs. Each assay was done in triplicate. Western blot analysis. Samples containing equal amounts of Materials and Methods protein (depends on the antibody, 20-100 Ag) from lysates of cultured PC-3 cells underwent electrophoresis on a SDS-polyacrylamide gel and Tumor cell lines and reagents. The human prostate cancer cell lines were transferred to nitrocellulose filters. The filters were blocked in PC-3 and DU145 were purchased from the American Type Culture Odyssey Blocking Buffer (LI-COR Biosciences) at 4jC for 1 h and then Collection and maintained in DMEM (Invitrogen-Life Technologies, incubated overnight with a 1:5,000 dilution of antihuman GLI2 rabbit Inc.) supplemented with 5% fetal bovine serum. LNCaP and C4-2 cells polyclonal antibody (Abcam, Inc.) at 4jC and for 1 h with 1:10,000 were kindly provided by Dr. Leland W.K. Chung (Emery University) diluted antihuman Vinculin mouse monoclonal antibody (Sigma and maintained in RPMI 1640 (Invitrogen) supplemented with 5% Chemical Co.) at room temperature with gentle shaking and washed fetal bovine serum. thrice with washing buffer (PBS containing 0.1% Tween 20) for 5 min. Chemotherapeutic agents. Paclitaxel was purchased from Biolyse Filters were then incubated for 1 h with 1:5,000 diluted Alexa Fluor Pharma (St. Catharines). Dr. Helen M. Burt (Pharmaceutical Science, 680 goat anti-rabbit and anti-mouse antibodies (Invitrogen) at room University of British Columbia) generously supplied the polymeric temperature. Specific proteins were detected using ODYSSEY IR micellar paclitaxel used for in vivo studies. Stock solutions of paclitaxel imaging system (LI-COR Biosciences) after washing, as described were prepared in PBS to the required concentrations before each in vitro above. The monoclonal and polyclonal antibodies (Bcl2, Bcl-xL, Mcl-1, or in vivo experiment. clusterin, cyclin D1, cyclin E2, p27, PKC-D) were purchased from Santa Treatment of cells with GLI2 ASO. The GLI2 ASO used in this study Cruz Biotechnology for screening of the genes related with GLI2 and was modified at the 2¶ position of the sugar with a 2¶-O-(methoxyethyl) apoptosis assessment. The anti-GLI1 polyclonal antibody was pur- group on the five bases at both the 5¶ and 3¶ termini (Isis 183652). The chased from Abcam, and the anti–poly(ADP-ribose) polymerase and central 10 bases contained deoxyribose and the entire oligonucleotide anti–phosphorylated Rb antibody was obtained from Cell Signaling backbone contained phosphorothioate linkages. The sequence of GLI2 Technology. ASO used was 5¶-GTGGCGGCACTCCAGCCAGG-3¶. The control oligo- Neoadjuvant hormone therapy tissue microarray. Slides (H&E) nucleotide (control ODN) used was designed not to match any mRNA containing tissue samples from 112 radical prostatectomy specimens in the human or mouse transcriptomes and has the sequence 5¶- (collected between 1989 and 2003) were obtained from the Vancouver CCTTCCCTGAAGGTTCCTCC-3¶. Cells were plated at the density of General Hospital. Fourteen AI specimens were obtained from meta- 4,000 cell/cm2 and treated a day later for two successive days with ASO. sitatic lesions (bone, lymph node, liver, and adrenal) of the warm Oligofectamine (Invitrogen), a cationic lipid, was used as a transfection autopsy at the University of Washington. All AI patients had progressive reagent. PC-3 cells were treated with various concentrations of ASO in prostate cancer after either chemical or surgical castration, both a manner described previously (24). clinically and with rising PSA, and died of prostate cancer. Supplemen- Northern blot analysis. Total RNA was isolated from cultured PC-3, tary Table S1 shows the treatment groups. Benign and cancer sites were DU145, LNCaP, and C4-2 cells using the TRIzol Reagent (Invitrogen). identified and marked in donor paraffin blocks using matching H&E Electrophoresis, hybridization, and washing conditions were carried reference slides. Tissue microarray was constructed using a manual out, as reported previously (24, 25). Briefly, 20 Ag of total RNA from tissue microarrayer (Beecher Instruments). Each marked block was each sample were subjected to electrophoresis on 1.0% agarose- sampled thrice with a core diameter of 0.6 mm arrayed in a rectangular formaldehyde gels and transferred to nylon membranes overnight at pattern with 1 mm between the centers of each core, creating a triplicate room temperature according to standard procedures. Human GLI2 and tissue microarray layout and ordered by treatment of the patients glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary (Supplementary Table S1). The 126 patient specimens were spotted DNA probes were generated by RT-PCR from RNA of DU145 or LNCaP in triplicate to create a tissue microarray with 378 cores. The tissue cells using primer 5¶-TCAGACTATTACCACCAGATGAC-3¶ (sense) or microarray paraffin block was sectioned into 0.5-Am sections and 5¶-AGATGTAGGGTTTCTCGTTGGAGT-3¶ (antisense) for GLI2 and mounted on the positively charged slides. Using rabbit polyclonal primer 5¶-TGCTTTTAACTCTGGTAAAGT-3¶ (sense) and 5¶-ATATTTGG- antibody against GLI2 (Abcam), immunohistochemical staining was CAGGTTTTTCTAGA-3¶ (antisense) for GAPDH. The RNA blots were conducted by Ventana autostainer model Discover XT (Ventana Medical hybridized overnight with the human GLI2 probe labeled with System) with enzyme labeled biotin streptavidin system and solvent [32P]dCTP by random primer labeling. The RNA blots were exposed resistant 3,3¶-diaminobenzidine Map kit. Slides were mounted with to film after washing. After the detection of GLI2 expression, the coverslips using the xylene-based mounting medium, Cytoseal membranes were reprobed with human GAPDH to verify the integrity (Stephan Scientific). Using identical microscopic and camera settings of the RNA. [reverse transcription color-SPOT high-resolution digital camera Quantitative RT-PCR. To quantify the expression of GLI2 mRNA, (Diagnostic Instruments) mounted on an Olympus System light quantitative RT-PCR was done. The same RNAs used for Northern blot microscope (model BX51)], digital images were taken from represen- analysis were used for quantification. After treating total RNA with tative areas reflecting the overall staining to strong staining by a DNaseI (Invitrogen), total RNA (2 Ag) was reversed transcribed using pathologist (L. F.) The staining intensity was evaluated and scored by a random hexamers (Perkin-Elmer Applied Biosystems) and 20 units of pathologist (L. F.). All comparisons of staining intensities and Molony murine leukemia virus reverse transcriptase (Invitrogen) in a percentages were made at 200Â magnifications.

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In vitro cell growth assays. Cell growth of PC-3 cells was assessed cells were plated in 10-cm2 dishes and, on the day after, were treated as using crystal violet assay, as described previously (27). Cells were described above. The cells were trypsinized 2 d after ASO treatment, plated in 12-well plates, allowed to attach 24 h, and treated once daily fixed in 70% ethanol overnight at 4jC, then incubated with 1 Ag/mL with various concentrations of ASO for 2 d. The absorbance was RNase (Sigma) for 30 min at 37jC before stained with 5 Ag/mL determined with a microculture plate reader (Becton Dickinson propidium iodide (Sigma) for 1 h at room temperature. The stained Labware) at 560 nm. Absorbance values were normalized to the cells were analyzed for relative DNA content on a dual laser flow values obtained from the vehicle-treated cells to determine the cytometer (Beckman Coulter Epics Elite; Beckman, Inc.). Each assay was percentage of survival. To assess the combination of GLI2 ASO and done in triplicate. paclitaxel in vitro, paclitaxel was added at various concentrations Microarray procedure, data processing, and analysis. Total RNA from (0-10 nmol/L) after GLI2 ASO treatment. Cells treated with this the PC-3 cell line was obtained 2 d after transfection with GLI2 combination treatment were cultured 2 or 4 d after transfection, and ASO and control ASO (250 nmol/L). Three independent transfections then the crystal violet assay was carried out. were done to minimize experimental variation. Total RNA from Flow cytometric analysis. Flow cytometric analysis of propidium each treatment sample (GLI2 ASO, 250 nmol/L) was compared with iodide–stained nuclei was done, as described previously (28). In brief, its control (250 nmol/L) on the same microarray. A dye swap for

Fig. 1. Expression of GLI2 in prostate cancer cell lines and human prostate tissues. A, total cellular RNA was extracted from parental prostate cancer lines, and GLI2 and GAPDH mRNA expression was analyzed by Northern blotting. B, quantitative analysis of GLI2 mRNA levels was analyzed by quantitative RT-PCR and average GLI2 mRNA levels were normalized to levels of rRNA.Asterisks indicate significant difference between PC-3 and other prostate cancer cell lines (*, P V 0.05). C, GLI2 immunostaining in a human prostate tissue microarray.Lower magnification ( a-f), higher magnification (g-l). a and g, benign prostate hyperplasia (BPH ); b and h, neoplastic glands in untreated tumors (0-NHT); c, i and d, j, prostate cancer after NHT (<3M and 3-6M, respectively), illustrating decreased immunoreactivity; e and k, illustrating strong immunoreactivity after 6 mo after NHT; f and l, hormone refractory prostate cancer showing highly intensive and uniformly immunostaining. D, data from 126 samples were calculated.Columns, mean; bars, SE.Mean GLI2 staining in benign prostate hyperplasia, NHT, and AI tissues.Specimens were graded from 0 to +3 repres enting the range from no staining to heavy staining by visual scoring.Immunoreactivity of GLI2 is significantly higher in both untreated NHTand AI group compared wit h benign prostate hyperplasia (#, P V 0.05). After short-term androgen ablation (<6 mo), GLI2 staining is significantly reduced (*, P V 0.05; **, P V 0.01), but increases again after longer-duration androgen ablation (>6mo).

www.aacrjournals.org 5771 Clin Cancer Res 2008;14(18) September 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical each pair was done to account for dye bias. Microarrays of 21,000 the neoadjuvant hormone therapy (NHT) tissue array, GLI2 (70-mer) human oligonucleotides representing 21,000 genes (Operon expression initially decreases in prostate cancers after androgen Â Technologies) printed in duplicate in 3 SSC onto aminosilane- ablation when compared with untreated tumor (P V 0.01); coatedslides (ERIE C28) were supplied by the Array Facility of however, GLI2 expression increases again after prolonged The Prostate Center at Vancouver General Hospital. Arrays were hormone therapy (i.e., >6 months). Compared with benign hybridized with 3DNA DNA Dendrimer Probes generated from 10 Ag prostate tissues (i.e., benign prostate hyperplasia group; total RNA according to manufactures’ directions (Genisphere). Briefly, P V reverse transcription incorporated a specific sequence present on the 0.05), GLI2 expression was significantly higher in un- 5¶ end of the reverse transcription primer supplied with the kit. treated NHT and AI tumors. Moreover, because AI tumors The complementary DNA was hybridized to the array overnight at exhibited higher intensity staining of GLI2 than other groups, 42jC. After stringent washes, the fluorescent 3DNA reagent, which targeted suppression would be most appropriate in this dis- includes a ‘‘capture sequence’’ complementary to the sequence at ease state. the 5¶ end of the reverse transcription primer, was hybridized to Sequence-specific, dose-dependent inhibition of GLI2 by ASO in the complementary DNA (47jC for 2-3 h). After further washes, the PC3 cells. GLI2 ASO significantly suppressed GLI2 expression arrays were immediately scanned on a ScanArrayExpress Microarray in a dose-dependent and sequence-specific fashion at both Scanner (PerkinElmer). Signal quality and quantity were assessed mRNA and protein levels (Fig. 2). As shown in Fig. 2A and B, using Imagene 5.6 (BioDiscovery). Data from Imagene were analyzed treatment of PC-3 cells with GLI2 ASO significantly reduced in GeneSpring 7.2 (Silicon Genetics) for profiling changes in gene expression. Analyses done in GeneSpring include background cor- GLI2 mRNA levels by up to 89% in a dose-dependent manner rection, LOWESS normalization and hierarchical clustering using standard correlation. Differentially expressed genes were identified by two-sample t tests, and P values were adjusted for multiple comparisons using the false discovery rate method of Benjamini and Hochberg. The ingenuity pathway analysis (Mountain View, CA) was used to examine functional and network associations between differentially expressed genes. Significances for biological functions compared with the whole ingenuity pathway knowledge base as a reference set. Assessment of in vivo tumor growth. Approximately 2 Â 106 PC-3 cells were inoculated s.c. in flank region of 6-wk-old to 8-wk-old athymic nude mice (Harlan Sprague-Dawley, Inc.) via a 27-gauge needle under isoflurane anesthesia. When PC-3 tumors reach 200 mm3 (usually 3-4 wk after injection), mice were randomly selected for treatment with GLI2 ASO alone, control ODN alone, GLI2 ASO plus paclitaxel, and control ODN plus paclitaxel. Each experimental group consisted in 9 or 10 mice. After randomization, 15 mg/kg GLI2 or control ODN was injected i.p. thrice a week for 6 wk for ASO monotherapy and for 9 wk in the ASO plus paclitaxel groups. A total of 0.5 mg micellar paclitaxel was given i.v. thrice per week from days 7 to 14 and from days 21 to 28. Tumor volume measurements were done once weekly and calculated by the formula: length Â width Â depth Â 0.5236 (29). Data points were expressed as average tumor volume levels F SE. All animal procedures were done according to the guidelines of the Canadian Council on Animal Care and with appropriate institutional certification. Statistical analysis. All statistical analyses were done using Micro- soft Excel with Statcel 2 software. All results were expressed as the mean F SE and significance of differences were by Student’s t test or ANOVA repeated measurement. P V 0.05 (*, #), P V 0.01 (**, ##), and P V 0.001 (***, ###) were considered significant.

Results

GLI2 expression is associated with hormone responsiveness and androgen-independent progression. The expression levels of GLI2 mRNA in four prostate cancer cell lines were assessed by both Northern blot and quantitative RT-PCR analysis (Fig. 1A and B). GLI2 mRNA expression was significantly higher in the Fig. 2. Sequence-specific and dose-dependent suppression of GLI2 mRNA and protein expression levels by GLI2 ASO in PC-3 cells. A, PC-3 cells were treated with androgen receptor–negative AI cell lines PC-3 and DU145 10 to 250 nmol/L GLI2 ASO or control ASO for 2 d.One day after treatment, total compared with androgen receptor–positive, androgen-sensitive cellular RNA was extracted, and GLI2 and GAPDH mRNA expression was analyzed P V by Northern blotting.‘‘Control’’cells are treated with oligofectamine only. B, cell lines LNCaP and C4-2 ( 0.05, respectively). quantitative analysis of GLI2 mRNA levels was analyzed by quantitative RT-PCR; GLI2 staining in human prostate tissues was evaluated in the GLI2 mRNA levels were normalized to levels of rRNA and expressed here as mean tissue microarrays (Fig. 1C and D; Supplementary Table S1). F SE.***, P V 0.001differ from control (oligofectamine only) by Student’s t test. C, PC-3 cells were treated with indicated concentrations of GLI2 ASO and control GLI2 expression, when present, was predominantly nuclear ASO for 2 d.Two days after treatment, cellular proteins were extracted from cultured with weaker cytoplasmic expression in the epithelial cells. In cells, and GLI2, GLI1, and vinculin protein levels were analyzed by Western blotting.

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Fig. 3. Effect GLI2 ASO alone or in combination with paclitaxel on PC-3 cell growth in vitro. A, PC-3 cells were treated daily with indicated concentration of GLI2 ASO or control ODN for 2 d.After 72 h of treatment, cell viability was determined by crystal violet assay. B, 2 and 4 d after GLI2 ASO treatment, cells were incubated with different concentrations of paclitaxel.Cell viability was determined 2 or 4 d after treatment.*, P V 0.05; **, P V 0.01;***, P V 0.001differ from control by Student’s t test. C and D, GLI2 knockdown induces apoptosis. C, flow cytometry after treatment with GLI2 ASO.PC-3 cells were treated with 250 nmol/L GLI2 ASO or control daily for 2 d, treated F10 nmol/L paclitaxel, and then harvested for flow cytometry analysis.Plot of mean sub G 0-G1fraction among four separate flow samples (GLI2 ASO monotherapy, control monotherapy, GLI2 ASO plus paclitaxel, and control plus paclitaxel).**, P V 0.01and ***, P V 0.001differ from control ASO by Student’s t test. D, poly(ADP-ribose) polymerase (PARP ) expression after treatment with GLI2 ASO.PC-3 cells were treated daily with 250 nmol/L GLI2 ASO or control ODN for 2 d, and protein was extracted for detection of poly(ADP-ribose) polymerase.Poly(ADP-ribose) polymerase antibody was used forWestern blotting, and vinculin was used as an interna l control.

(P V 0.001), with similar dose-dependent knockdown of GLI2 GLI2 expression was not modulated by GLI1 ASO in both was observed at the protein level (Fig. 2C). The protein mRNA and protein level (data not shown). These results are expression level of GLI1 was reduced in PC-3 cells treated with consistent with the previous report indicated that GLI1 was GLI2 ASO in a dose-dependent manner (Fig. 2C). Conversely, direct target of GLI2 (30).

Table 1. Top 10 functional categories involved in GLI2 down-regulation

Function No. genes in class/no. genes measured Significance* Cell death and survival 53c/172b 4.07e-05 to 2.96e-02 Reproductive system disease 23/172 4.07e-05 to 2.96e-02 Cancer 38/172 5.59e-05 to 2.96e-02 Cellular growth and proliferation 64/172 6.74e-05 to 3.04e-02 Cell cycle 22/172 4.49e-04 to 2.88e-02 DNA replication, recombination, and repair 13/172 4.49e-04 to 2.61e-02 Gene expression 44/172 7.45e-04 to 2.7e-02 Cellular development 10/172 7.73e-04to 2.48e-02 Cell morphology 21/172 1.47e-03 to 2.91e-02 Cellular assembly and organization 15/172 1.47e-03 to 2.45e-02

*Calculated and analyzed by ingenuity pathway analysis. P < 0.05 is considered significant for association of genes. cNumber of genes found to be significantly changed (increased or decreased) as a function of Gli2 ASO in the corresponding function class. bNumber of genes in dataset file mapping for functional analysis.

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GLI2 knockdown inhibits PC-3 cell growth and enhances clusterin, was up-regulated in GLI2 ASO-treated cells, whereas chemosensitivity in vitro. The effect of GLI2 ASO monotherapy levels of Mcl-1, an antiapoptotic Bcl-2 family member, was not and its effects on paclitaxel chemosensitivity in vitro were altered among three groups. investigated. PC-3 cells were treated for 2 days with 10 to Figure 4B showed the modulation of genes correlated with 250 nmol/L GLI2 ASO or control ODN and growth rates cell cycle by GLI2 knockdown. Genes regulating cell cycle were examined. Figure 3A shows significant reduction (***, enable cancer cells to evade antiproliferative signals (36), and P V 0.001) of PC-3 cell growth 3 days after treatment with hedgehog pathway and GLIs can interact with cell cycle GLI2 ASO alone compared with control in a dose-dependent machinery at various time points (34) by promoting D-type manner. To assess chemosensitivity after GLI2 ASO treatment, and E-type cyclins, thereby opposing P21-induced and PC-3 cells were incubated with different concentrations of P27-induced cell cycle arrest (37, 38). Compared with paclitaxel (0-10 nmol/L) and growth rates were examined daily oligofectamine only and control ODN groups, levels of for 2 or 4 days using crystal violet assay. Figure 3B reveals that hyperphospholylated Rb and cyclin D1 decreased in PC-3 cell 250 nmol/L ASO increases paclitaxel sensitivity compared with lysates treated by GLI2 ASO, whereas protein levels of p27 were control plus paclitaxel treatment (*, P V 0.05; **, P V 0.01). up-regulated. P21, whose mRNA level significantly increased in The combination index values of GLI2 ASO in combination GLI2 ASO group in microarray data, did not express detectable with paclitaxel using CalcuSyn software at IC50 was 0.59, which protein levels using Western blot (data not shown), where its indicates that the combination of GLI2 ASO and paclitaxel had basal level has been reported to be very low in PC-3 cell (39). a synergistic cytotoxic effect on PC-3 (31, 32). Interestingly, expression levels of PKC-D were significantly GLI2 ASO treatment induces apoptosis in PC-3 cells in vitro. lower in PC-3 treated by GLI2 ASO in both mRNA and protein The induction of apoptosis by GLI2 ASO has been assessed by flow cytometry. The fraction of cells undergoing apoptosis (sub G1-G0 fraction) was significantly higher after the treatment of PC-3 cells with 250 nmol/L GLI2 ASO for 2 days, compared with control ODN (20% versus 0.8%;***, P V 0.001; Fig. 3C). Combination treatment using GLI2 ASO plus paclitaxel significantly increased sub G0-G1 fraction over that of paclitaxel plus control or ASO monotherapy (**, P V 0.01; 25.4% versus 11.7% and 25.4% versus 20.0%, respectively). The percentage of cells in G1 and G2-M phase decreased after GLI2 ASO treatment, whereas an accumulation of cells at S phase was observed (data not shown). Furthermore, cells treated with GLI2 ASO expressed higher amounts of cleaved poly(ADP- ribose) polymerase products compared with cells treated with oligofectamine only and control (Fig. 3D). Gene expression profile associated with GLI2 knockdown. Gene expression analysis was done with microarrays of 21,000 (70-mer) human oligonucleotides (Operon) to screen the genes involved in GLI2 ASO. In determining the potential downstream targets of GLI2, we identified 142 up-regulated genes and 180 down-regulated genes between PC-3 cells treated by GLI2 ASO or control ODN (Supplementary Tables S2 and S3). The identified genes were uploaded into the ingenuity pathway analysis knowledge database, which enables the discovery, visualization, and exploration of molecular interac- tion networks in gene expression data and was used to assess high-level function categories and networks (Table 1 and Supplementary Table S4). Supporting previous studies investi- gating GLI downstream targets (33, 34), cell death (apoptosis) and survival, cancer, cellular proliferation, and cell cycle were identified as high-level function categories in our ingenuity pathway analysis results. We focused on differentially expressed genes involved in apoptosis, cell survival, and cell cycle and did confirmatory Western blots to detect potential direct and indirect GLI2 targets. Figure 4A illustrates several changes in levels of several genes Fig. 4. Confirmatory Western blot of select genes altered by GLI2 knockdown. linked with apoptosis and cell survival after GLI2 knockdown. A, PC-3 cells were treated with 250 nmol/L GLI2 ASO, control ODN, or Bcl-2 level decreased in PC-3 cell lysates treated with GLI2 ASO, oligofectamine only (control).After 48 h of incubation, cells were harvested and protein was extracted for detection of Bcl-2, Bcl-xL, Mcl-1, and clusterin.Vinculin which is consistent with previous reports that identified GLI2 was used as an internal control. B, PC-3 cells were treated with 250 nmol/L GLI2 interactions with the promoter of Bcl2 (30, 35). In addition ASO or control ODN for 2 d, and cell lysate was extracted in a time-dependent manner (24, 48, and 72 h after transfection) for detection of cyclin D1, cyclin E2, Bcl-xL was significantly down-regulated in PC-3 cell treated p27, phosphorylated Rb, PKC-D.Control (oligofectamine only) lysate was extracted by GLI2 ASO. The stress-induced cytoprotective chaperone, at 24 h after transfection.Vinculin was used as an internal control.

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Fig. 5. Effect of GLI2 ASO treatment on PC-3 xenograft growth in vivo. A, GLI2 ASO monotherapy in vivo.When tumor volume was f200 mm3, GLI2 ASO treatment was started. After 1wk of an induction daily dose of 15 mg/kg/mouse GLI2 ASO or control ODN given i.p., mice were injected with the same dose thrice per week. Tumors were measured weekly. B, when tumor volume reached f500 mm3, mice were treated for 1wk with 15 mg/kg once daily GLI2 ASO or control ODN, followed by two cycles of 0.5 kg/mg i.v. paclitaxel treatment on days 7 to 14 and 21to 28. ASO or control ODN was continued at 15 mg/kg thrice a week. Points, mean tumor volume in each experimental group containing10 mice; bars, SE.*, P V 0.05; **, P V 0.01;***, P V 0.001 (Student’s t test).##, P V 0.01;###, P V 0.001 (ANOVA repeated measurement). C, total RNAs were extracted from the xenografts treated with GLI2 ASO or control ODN at 6 wk.GLI2 ( left)andBcl2(right) mRNA expression levels were determined by quantitative RT-PCR.Columns, average values from five animals for each group; bars, SE.*, P V 0.05 differ from control ODN by Student’s t test. levels. PKC-D is expressed at higher levels in prostate cancer volume by >74% from days 21 to 63 after initiation of compared with benign prostate hyperplasia tissue (40) and treatment (Fig. 5B; *, P V 0.05; **, P V 0.01, by Student’s t test; reported to enhance the expression of G1 cyclin and p21 in ##, P V 0.05 by ANOVA repeat measurement). To investigate MCF-7 adenocarcinoma cell line (41). These results indicate that the target reduction and the effect to downstream target Bcl2 in GLI2 ASO modulates levels of several cell cycle genes, as well as PC-3 xenografts treated with GLI2 ASO or control ODN, genes correlated with G1-S phase to induce cell cycle arrest. quantitative RT-PCR was done. The mRNA expressions of both GLI2 ASO treatment inhibits PC-3 tumor progression and GLI2andBcl2inGLI2ASO-treatedtumorsweresigni- enhances chemotherapy in vivo. We next evaluated the effect ficantly down-regulated compared with that of control ODN of GLI2 ASO treatment on the growth of PC-3 tumors in vivo (*, P V 0.05; Fig. 5C). (Fig. 5). Male nude mice bearing PC-3 tumors (200 mm3) were randomly selected and treated with 15 mg/kg GLI2 ASO or Discussion control thrice a week by i.p. injection. From days 7 to 14 and 21 to 28, 0.5 mg of micellar paclitaxel was given i.v. thrice a Recent evidence suggests that the SHH-GLI pathway is week for the combination treatment. Mean PC-3 tumor volume activated in human prostate cancers (12, 13, 17). Expression at baseline was similar in all groups. Figure 5A shows that GLI2 of SHH, GLI1, and PTCH, which are known markers of SHH- ASO monotherapy significantly reduced PC-3 tumor volume GLI pathway activation, is increased in cancer compared with by >64% from days 7 to 42 (*, P V 0.05; **, P V 0.01; and normal prostate epithelium (12). Furthermore, expression of ***, P V 0.001, by Student’s t test; ###, P V 0.001 by ANOVA SUFU, a negative regulator of the pathway, is inversely repeat measurement). Moreover, treatment with GLI2 ASO, correlated with prostate cancer aggressiveness and tumor compared with scramble control, significantly increased che- progression (13). Accordingly, activation of this pathway has mosensitivity to paclitaxel in vivo, reducing mean PC-3 tumor been observed in prostate cancer metastases. Thus, the

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SHH-GLI pathway seems to be associated with advanced shown to be increased in hormone refractory prostate cancer. prostate cancer and tumor progression (42). Interestingly, Regl and colleagues suggested that the oncogenic activity of forced overexpression of SHH in LNCaP cells also activates its GLI2 may rely on at least three functions: stimulation of distal activator GLI and dramatically accelerates growth of cell proliferation, repression of epidermal differentiation, and tumor both in vitro and in vivo (11). Furthermore, disruption of induction of prosurvival signals related with apoptosis (34, 35). the SHH-GLI pathway seems to inhibit prostate cancer cell They also identified several GLI2 targets, such as GLI1, Bcl2, growth. Upstream inhibition of the SHH pathway with either and cell cycle genes (cyclin D1, E2F1, inactivation of Rb). The anti-SHH antibodies or SMO inhibitors, such as cyclopamine, cell cycle inhibitors, P21 and p27, have also been reported can down-regulate GLI1 and inhibit proliferation and invasive- as targets of GLI2 (10, 38). Our microarray data confirms that ness of prostate cancer cell lines in vitro and in vivo (43). GLI2 knockdown induces changes in mRNA levels of diverse Accordingly, GLI transcription factors seem to be essential gene sets associated with survival, cancer, cellular proliferation, downstream effectors of the SHH pathway and may therefore and cell cycle, indicating that apoptosis and cell cycle control be the most suitable targets to suppress the SHH pathway. may be the critical pathways modulated by GLI2. Furthermore, In contrast to GLI1, which was originally identified as an whereas we confirmed the downstream target genes shown by oncogene (44), there is less evidence to implicate GLI2 in previous reports, such as Bcl2, GLI1, cyclin D1, and p27, we human malignancies. However, Sheng and colleagues showed also detected Bcl-xL and PKC-D as new candidate targets that mice overexpressing GLI2 developed a variety of skin modulated by GLI2. tumors, and GLI2 mRNA seemed to be up-regulated in many Whereas a survival advantage of docetaxel-based chemother- human skin tumor tissues (20). Our analysis of prostate cancer apy has been shown in men with hormone refractory prostate cell lines shows that GLI2 expression is higher in androgen cancer, these improved effects were modest, underscoring receptor–negative androgen-insensitive cell lines, such as PC-3 the need for additional therapeutic reagent (3, 4). Previous and DU-145, compared with androgen receptor–positive, studies reported that Bcl2 and Bcl-xL confers resistance to androgen-sensitive cell lines (Fig. 1). In human, GLI2 taxane chemotherapy in several prostate cancer models (26, 29, expression was significantly higher in untreated and AI prostate 46–48). In this current study, GLI2 knockdown was associated cancer tissues, decreasing temporarily after androgen ablation with decreased levels of Bcl2, increased apoptosis, and therapy and increasing again after durations longer than paclitaxel chemosensitization both in vitro and in vivo. Our 6 months of hormone therapy. These results suggest that current hypothesis is that synergy may be achieved through the GLI2 may correlate with development of castrate-resistant possible modulation of Bcl2-related antiapoptotic that seems to prostate cancer progression. Our functional studies showed also be down-regulated after ASO transfection. Furthermore, GLI2 knockdown induced apoptosis and reduced tumor increased clusterin levels after GLI2 knockdown is consistent progression rates in the AI PC-3 cell line. with clusterin’s role as a stress-induced cytoprotective chaper- ASO are one strategy to specifically silence gene expression one that inhibits apoptosis after a diverse array of anticancer that specially hybridize with complementary mRNA region to treatments. OGX-011, a second generation ASO inhibitor of inhibit target gene expression in a sequence-specific manner clusterin, is currently in clinical trials of prostate, breast, and and important loss-of-function tools for in vitro and in vivo lung cancer, and future preclinical studies examining effects study (45). The GLI2 ASO (Isis 183652) used in this study of combined suppression GLI2 and clusterin are under way incorporated a 2¶-O-methoxyethyl gapmer modified backbone. (49, 50). The GLI2 ASO used in this study has a potential to be These advanced chemistry ASO possess favorable physico- developed as a monotherapy or in combined multimodality chemical, biochemical, and pharmacokinetic properties, with regimens with chemotherapy. enhanced RNA affinity and reduced nonspecific immune In summary, the present study suggests that GLI2 is an stimulation (45). Most importantly, improved resistance attractive therapeutic target in prostate cancer by affecting the against nuclease-mediated metabolism, resulting in significant- expression of many genes that regulate apoptosis and cell cycle. ly improved tissue half-life in vivo, enables a longer duration of We show that GLI2 knockdown induces apoptosis, inhibits action and a more intermittent dosing schedule. GLI2 levels cancer growth, and chemosensitizes cells to chemotherapy were suppressed by 89% and associated with decreased cell in vitro and in vivo. These results provide preclinical proof of growth in vitro and tumor growth in vivo. This study is the first principle for the use of this novel therapeutic in castrate- to show the effect of GLI2 ASO therapy in prostate cancer cells resistant prostate cancer. either in vivo or in vitro. We showed that GLI2 expression was increased in patients with hormone refractory prostate cancer. As well, our results Disclosure of Potential Conflicts of Interest showing the effects of GLI2 down-regulation imply that GLI2 Y.Kim owns stock in ISIS Pharmaceuticals. overexpression may confer a growth advantage in this setting. Although further validation is warranted to investigate the Acknowledgments mechanisms of GLI2 overexpression and the role of SHH in AI prostate cancer, the effect of GLI2 may be related to its We thank Shannon Sinnemann, Jenny Baybik, Eliana Beraldi, Virginia Yago, and relationship with other antiapoptotic proteins that have been Mary Bowden for their excellent technical assistance.

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