Stat3 Promotes Metastatic Progression of Prostate Cancer
2008; Elsevier BV; Volume: 172; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.071054
ISSN1525-2191
AutoresJunaid Abdulghani, Lei Gu, Ayush Dagvadorj, Jacqueline Lutz, Benjamin E. Leiby, Gloria Bonuccelli, Michael P. Lisanti, Tobias Zellweger, Kalle Alanen, Tuomas Mirtti, Tapio Visakorpi, Lukas Bubendorf, Marja T. Nevalainen,
Tópico(s)Bone Metabolism and Diseases
ResumoThere are currently no effective therapies for metastatic prostate cancer because the molecular mechanisms that underlie the metastatic spread of primary prostate cancer are unclear. Transcription factor Stat3 is constitutively active in malignant prostate epithelium, and its activation is associated with high histological grade and advanced cancer stage. In this work, we hypothesized that Stat3 stimulates metastatic progression of prostate cancer. We show that Stat3 is active in 77% of lymph node and 67% of bone metastases of clinical human prostate cancers. Importantly, adenoviral gene delivery of wild-type Stat3 (AdWTStat3) to DU145 human prostate cancer cells increased the number of lung metastases by 33-fold in an experimental metastasis assay compared with controls. Using various methods to inhibit Stat3, we demonstrated that Stat3 promotes human prostate cancer cell migration. Stat3 induced the formation of lamellipodia in both DU145 and PC-3 cells, further supporting the concept that Stat3 promotes a migratory phenotype of human prostate cancer cells. Moreover, Stat3 caused the rearrangement of cytoplasmic actin stress fibers and microtubules in both DU145 and PC-3 cells. Finally, inhibition of the Jak2 tyrosine kinase decreased both activation of Stat3 and prostate cancer cell motility. Collectively, these data indicate that transcription factor Stat3 is involved in metastatic behavior of human prostate cancer cells and may provide a therapeutic target to prevent metastatic spread of primary prostate cancer. There are currently no effective therapies for metastatic prostate cancer because the molecular mechanisms that underlie the metastatic spread of primary prostate cancer are unclear. Transcription factor Stat3 is constitutively active in malignant prostate epithelium, and its activation is associated with high histological grade and advanced cancer stage. In this work, we hypothesized that Stat3 stimulates metastatic progression of prostate cancer. We show that Stat3 is active in 77% of lymph node and 67% of bone metastases of clinical human prostate cancers. Importantly, adenoviral gene delivery of wild-type Stat3 (AdWTStat3) to DU145 human prostate cancer cells increased the number of lung metastases by 33-fold in an experimental metastasis assay compared with controls. Using various methods to inhibit Stat3, we demonstrated that Stat3 promotes human prostate cancer cell migration. Stat3 induced the formation of lamellipodia in both DU145 and PC-3 cells, further supporting the concept that Stat3 promotes a migratory phenotype of human prostate cancer cells. Moreover, Stat3 caused the rearrangement of cytoplasmic actin stress fibers and microtubules in both DU145 and PC-3 cells. Finally, inhibition of the Jak2 tyrosine kinase decreased both activation of Stat3 and prostate cancer cell motility. Collectively, these data indicate that transcription factor Stat3 is involved in metastatic behavior of human prostate cancer cells and may provide a therapeutic target to prevent metastatic spread of primary prostate cancer. Progression of prostate cancer to metastatic disease is one of the key problems in the clinical management of prostate cancer.1Arya M Bott SR Shergill IS Ahmed HU Williamson M Patel HR The metastatic cascade in prostate cancer.Surg Oncol. 2006; 15: 117-128Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar This is because there are currently no effective therapies for metastatic prostate cancer, and metastatic prostate cancer is the lethal form of the disease. Identification of the molecular changes that lead to formation of distant metastasis is critical for improvement of therapeutic interventions for metastatic prostate cancer and for development of strategies to prevent primary prostate cancer from metastasizing. Transcription factor Stat3 has been implicated in the promotion of growth and progression of prostate cancer. Stat3, which is both a cytoplasmic signaling molecule and a nuclear transcription factor, belongs to the seven-member Stat gene family of transcription factors.2Ihle JN The Stat family in cytokine signaling.Curr Opin Cell Biol. 2001; 13: 211-217Crossref PubMed Scopus (600) Google Scholar Stat3 becomes active by phosphorylation of a specific tyrosine residue in the carboxy-terminal domain by a tyrosine kinase (pY705).3Levy DE Darnell Jr, JE Stats: transcriptional control and biological impact.Nat Rev Mol Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2544) Google Scholar Activation of Stat3 is supplemented by phosphorylation of a specific serine residue (S727).4Wen Z Zhong Z Darnell Jr, JE Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation.Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1762) Google Scholar After phosphorylation, Stat3 homodimerizes and translocates to the nucleus where it binds to specific Stat3 response elements of target gene promoters to regulate transcription.3Levy DE Darnell Jr, JE Stats: transcriptional control and biological impact.Nat Rev Mol Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2544) Google Scholar Transcription factor Stat3 is constitutively active in clinical human prostate cancer,5Mora LB Buettner R Seigne J Diaz J Ahmad N Garcia R Bowman T Falcone R Fairclough R Cantor A Muro-Cacho C Livingston S Karras J Pow-Sang J Jove R Constitutive activation of Stat3 in human prostate tumors and cell lines: direct inhibition of Stat3 signaling induces apoptosis of prostate cancer cells.Cancer Res. 2002; 62: 6659-6666PubMed Google Scholar, 6Campbell CL Jiang Z Savarese DM Savarese TM Increased expression of the interleukin-11 receptor and evidence of STAT3 activation in prostate carcinoma.Am J Pathol. 2001; 158: 25-32Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 7Huang HF Murphy TF Shu P Barton AB Barton BE Stable expression of constitutively-activated STAT3 in benign prostatic epithelial cells changes their phenotype to that resembling malignant cells.Mol Cancer. 2005; 4: 2-8Crossref PubMed Scopus (49) Google Scholar, 8Dhir R Ni Z Lou W DeMiguel F Grandis JR Gao AC Stat3 activation in prostatic carcinomas.Prostate. 2002; 51: 241-246Crossref PubMed Scopus (128) Google Scholar, 9Horinaga M Okita H Nakashima J Kanao K Sakamoto M Murai M Clinical and pathologic significance of activation of signal transducer and activator of transcription 3 in prostate cancer.Urology. 2005; 66: 671-675Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar and activation of Stat3 has been associated with advanced stage of prostate cancer.5Mora LB Buettner R Seigne J Diaz J Ahmad N Garcia R Bowman T Falcone R Fairclough R Cantor A Muro-Cacho C Livingston S Karras J Pow-Sang J Jove R Constitutive activation of Stat3 in human prostate tumors and cell lines: direct inhibition of Stat3 signaling induces apoptosis of prostate cancer cells.Cancer Res. 2002; 62: 6659-6666PubMed Google Scholar, 9Horinaga M Okita H Nakashima J Kanao K Sakamoto M Murai M Clinical and pathologic significance of activation of signal transducer and activator of transcription 3 in prostate cancer.Urology. 2005; 66: 671-675Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Moreover, several reports implicate Stat3 in promotion of prostate cancer cell proliferation and inhibition of apoptosis.5Mora LB Buettner R Seigne J Diaz J Ahmad N Garcia R Bowman T Falcone R Fairclough R Cantor A Muro-Cacho C Livingston S Karras J Pow-Sang J Jove R Constitutive activation of Stat3 in human prostate tumors and cell lines: direct inhibition of Stat3 signaling induces apoptosis of prostate cancer cells.Cancer Res. 2002; 62: 6659-6666PubMed Google Scholar, 10Lee SO Lou W Hou M de Miguel F Gerber L Gao AC Interleukin-6 promotes androgen-independent growth in LNCaP human prostate cancer cells.Clin Cancer Res. 2003; 9: 370-376PubMed Google Scholar, 11Barton BE Karras JG Murphy TF Barton A Huang H Signal transducer and activator of transcription (STAT3) activation in prostate cancer: direct STAT3 inhibition induces apoptosis in prostate cancer lines.Mol Cancer Ther. 2004; 3: 11-20Crossref PubMed Scopus (15) Google Scholar Recent studies have linked Stat3 to metastatic progression of several different cancer types. These include lung, skin, liver, ovarian, kidney, and colon cancer.12Dauer DJ Ferraro B Song L Yu B Mora L Buettner R Enkemann S Jove R Haura EB Stat3 regulates genes common to both wound healing and cancer.Oncogene. 2005; 24: 3397-3408Crossref PubMed Scopus (317) Google Scholar, 13Xie TX Huang FJ Aldape KD Kang SH Liu M Gershenwald JE Xie K Sawaya R Huang S Activation of stat3 in human melanoma promotes brain metastasis.Cancer Res. 2006; 66: 3188-3196Crossref PubMed Scopus (342) Google Scholar, 14Li WC Ye SL Sun RX Liu YK Tang ZY Kim Y Karras JG Zhang H Inhibition of growth and metastasis of human hepatocellular carcinoma by antisense oligonucleotide targeting signal transducer and activator of transcription 3.Clin Cancer Res. 2006; 12: 7140-7148Crossref PubMed Scopus (131) Google Scholar, 15Silver DL Naora H Liu J Cheng W Montell DJ Activated signal transducer and activator of transcription (STAT) 3: localization in focal adhesions and function in ovarian cancer cell motility.Cancer Res. 2004; 64: 3550-3558Crossref PubMed Scopus (227) Google Scholar, 16Horiguchi A Oya M Shimada T Uchida A Marumo K Murai M Activation of signal transducer and activator of transcription 3 in renal cell carcinoma: a study of incidence and its association with pathological features and clinical outcome.J Urol. 2002; 168: 762-765Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Kusaba T Nakayama T Yamazumi K Yakata Y Yoshizaki A Nagayasu T Sekine I Expression of p-STAT3 in human colorectal adenocarcinoma and adenoma; correlation with clinicopathological factors.J Clin Pathol. 2005; 58: 833-838Crossref PubMed Scopus (148) Google Scholar Contribution of Stat3 to metastatic progression of these cancers occurs through a variety of molecular mechanisms. Stat3 was associated with a migratory phenotype of lung cancer cells12Dauer DJ Ferraro B Song L Yu B Mora L Buettner R Enkemann S Jove R Haura EB Stat3 regulates genes common to both wound healing and cancer.Oncogene. 2005; 24: 3397-3408Crossref PubMed Scopus (317) Google Scholar while promoting angiogenesis of melanoma and hepatocellular cancer in animal tumor models.13Xie TX Huang FJ Aldape KD Kang SH Liu M Gershenwald JE Xie K Sawaya R Huang S Activation of stat3 in human melanoma promotes brain metastasis.Cancer Res. 2006; 66: 3188-3196Crossref PubMed Scopus (342) Google Scholar, 14Li WC Ye SL Sun RX Liu YK Tang ZY Kim Y Karras JG Zhang H Inhibition of growth and metastasis of human hepatocellular carcinoma by antisense oligonucleotide targeting signal transducer and activator of transcription 3.Clin Cancer Res. 2006; 12: 7140-7148Crossref PubMed Scopus (131) Google Scholar In ovarian cancer, Stat3 was suggested to increase cell motility and invasion through effects on cell adhesion and cytoskeleton.15Silver DL Naora H Liu J Cheng W Montell DJ Activated signal transducer and activator of transcription (STAT) 3: localization in focal adhesions and function in ovarian cancer cell motility.Cancer Res. 2004; 64: 3550-3558Crossref PubMed Scopus (227) Google Scholar Moreover, a number of studies using mouse embryo fibroblasts as the model system established Stat3 as a component of RhoGTPase-signaling cascade and an effector of cell migration via regulation of actin cytoskeleton.18Aznar S Valeron PF del Rincon SV Perez LF Perona R Lacal JC Simultaneous tyrosine and serine phosphorylation of STAT3 transcription factor is involved in Rho A GTPase oncogenic transformation.Mol Biol Cell. 2001; 12: 3282-3294Crossref PubMed Scopus (92) Google Scholar, 19Debidda M Wang L Zang H Poli V Zheng Y A role of STAT3 in Rho GTPase-regulated cell migration and proliferation.J Biol Chem. 2005; 280: 17275-17285Crossref PubMed Scopus (118) Google Scholar, 20Faruqi TR Gomez D Bustelo XR Bar-Sagi D Reich NC Rac1 mediates STAT3 activation by autocrine IL-6.Proc Natl Acad Sci USA. 2001; 98: 9014-9019Crossref PubMed Scopus (132) Google Scholar, 21Pelletier S Duhamel F Coulombe P Popoff MR Meloche S Rho family GTPases are required for activation of Jak/STAT signaling by G protein-coupled receptors.Mol Cell Biol. 2003; 23: 1316-1333Crossref PubMed Scopus (131) Google Scholar, 22Hall A Rho GTPases and the actin cytoskeleton.Science. 1998; 279: 509-514Crossref PubMed Scopus (5242) Google Scholar In addition, Stat3 was linked to cell migration via regulation of microtubules by interaction with stathmin protein.23Ng DC Lin BH Lim CP Huang G Zhang T Poli V Cao X Stat3 regulates microtubules by antagonizing the depolymerization activity of stathmin.J Cell Biol. 2006; 172: 245-257Crossref PubMed Scopus (221) Google Scholar In colon and renal cancer, active Stat3 expression was associated with tumor invasion and poor clinical outcome in patients.16Horiguchi A Oya M Shimada T Uchida A Marumo K Murai M Activation of signal transducer and activator of transcription 3 in renal cell carcinoma: a study of incidence and its association with pathological features and clinical outcome.J Urol. 2002; 168: 762-765Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Kusaba T Nakayama T Yamazumi K Yakata Y Yoshizaki A Nagayasu T Sekine I Expression of p-STAT3 in human colorectal adenocarcinoma and adenoma; correlation with clinicopathological factors.J Clin Pathol. 2005; 58: 833-838Crossref PubMed Scopus (148) Google Scholar Based on these findings, we formed the hypothesis that Stat3 contributes to the progression of prostate cancer to advanced disease by promoting metastatic spread of human prostate cancer cells. Here, we show that Stat3 induces metastatic behavior of human prostate cancer cells in vitro and in vivo. First, using activation-specific Stat3 antibody we show that phosphorylated Stat3 localized in the nucleus and is activated in 77% of lymph node and 67% of bone metastases of clinical human prostate cancer. Gene delivery of wild-type Stat3 by adenovirus (AdWTStat3) to DU145 and PC-3 cells promoted migration of the cells as shown by wound filling and Boyden chamber assays. Moreover, Stat3 promoted formation of lamellipodia in both DU145 and PC-3 cells, which was accompanied by a phenotypic change in the cytoplasmic arrangement of actin stress fibers and microtubules. Importantly, Stat3 induced a 33-fold increase in colonization of DU145 cells to the lungs of nude athymic mice. Stat3 activation in human prostate cancer cells did not involve RhoGTPases but was rather mediated by Jak2 tyrosine kinase. In summary, Stat3 promotes metastatic behavior of human prostate cancer cells and may provide a therapeutic target protein to prevent metastatic progression of primary human prostate cancer. The recurrent human prostate cancer specimens (n = 188) were obtained from the Tampere University Hospital, Tampere, Finland (n = 76)24Saramäki O Willi N Bratt O Gasser TC Koivisto P Nupponen NN Bubendorf L Visakorpi T Amplification of EIF3S3 gene is associated with advanced stage in prostate cancer.Am J Pathol. 2001; 159: 2089-2094Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar and from the Institute for Pathology, University of Basel, Basel, Switzerland (n = 112).25Zellweger T Ninck C Bloch M Mirlacher M Koivisto PA Helin HJ Mihatsch MJ Gasser TC Bubendorf L Expression patterns of potential therapeutic targets in prostate cancer.Int J Cancer. 2005; 113: 619-628Crossref PubMed Scopus (142) Google Scholar All samples were transurethral resections from local recurrences. Of the 188 patients, 121 had received androgen ablation therapy (orchiectomy, n = 76; luteinizing hormone-releasing hormone, n = 19; estrogen, n = 1; anti-androgen, n = 2; orchiectomy + estrogen, n = 2; maximal androgen blockade, n = 21), whereas the rest (n = 67) had received no hormonal treatment. Paraffin-embedded prostate cancer metastases were obtained from the Turku University Hospital, Turku, Finland (n = 95) (lymph node, n = 44; to bone, n = 1; to other organs, n = 50) and from Georgetown University, Washington, DC (lymph node, n = 22; to bone, n = 14) (approved by the Thomas Jefferson University Institutional Review Board). Adenoviruses carrying human wild-type Stat3 (AdWTStat3), transcriptionally inactive Stat3 (AdDNStat3) (C-terminally truncated at amino acid 715), wild-type Jak2 (AdWtJak2), and a kinase-domain deleted dominant-negative Jak2 (AdDNJak2) were a gift from Dr. Hallgeir Rui at Thomas Jefferson University.26Sultan AS Xie J LeBaron MJ Ealley EL Nevalainen MT Rui H Stat5 promotes homotypic adhesion and inhibits invasive characteristics of human breast cancer cells.Oncogene. 2005; 24: 746-760Crossref PubMed Scopus (162) Google Scholar Viral stocks were expanded in large-scale cultures, purified by double cesium chloride gradient centrifugation, and titered side-by-side by a standard plaque assay method in QBI-HEK-293A cells (Qbiogene, Carlsbad, CA) per the manufacturer's instructions. For adenoviral gene delivery, DU145 and PC-3 cells were infected with AdDNStat3, AdWTStat3, AdWTJak2, or AdDNJak2 at multiplicity of infection (m.o.i.) of 10 for 90 minutes, after which RPMI 1640 containing 10% fetal bovine serum (FBS) was added. Slides containing deparaffinized formalin-fixed prostate cancer sections were microwaved in a pressure-cooker with antigen retrieval solution AR-10 (BioGenex Laboratories, San Ramon, CA). Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide, and nonspecific binding of immunoglobulin was minimized by preincubation in 10% normal goat serum for 2 hours at room temperature. Anti-phosphotyrosine-Stat3 (Y705) polyclonal antibody (pAb) (Cell Signaling, Danvers, MA) was used at a concentration of 1:100. Antigen-antibody complexes were detected using a biotinylated goat anti-rabbit secondary antibody (BioGenex Laboratories) followed by streptavidin-horseradish-peroxidase complex using 3,3(prime)-diaminobenzidine as chromogen and Mayer hematoxylin as counterstain. Individual prostate tumor samples were scored (M.T.N. and J.A.) for active nuclear Stat3 levels on a scale from 0 to 3, where 0 is undetectable and 1 represented positive immunostaining. For immunoprecipitation, DU145, LNCaP, PC-3, and CWR22Rv1 prostate cancer cells were lysed in lysis buffer [10 mmol/L Tris-HCl (pH 7.6), 5 mmol/L ethylenediaminetetraacetic acid, 50 mmol/L NaCl, 30 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 1% Triton X-100, 1 mmol/L phenylmethyl sulfonyl fluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin]. The cell lysates were immunoprecipitated for 2 hours with anti-Stat3 pAb (a gift from Dr. Robert Kirken, University of Texas, El Paso). Antibodies were captured by incubation for 2 hours with protein A-Sepharose beads (Pharmacia Biotech, Piscataway, NJ). The primary antibodies were used at the following concentrations: anti-phosphotyrosine Stat3 pAb (Y705, 1:1000; Cell Signaling), anti-phosphoserine Stat3 pAb (1:1000, Cell Signaling), anti-Stat3 mAb (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Jak2 pAb (1:1000; Upstate Biotechnology, Lake Placid, NY), anti-actin pAb (1:3000; Sigma, St. Louis, MO) and detected by horseradish peroxidase-conjugated secondary antibodies in conjunction with enhanced chemiluminescence substrate mixture (Amersham, Piscataway, NJ) and exposed to film. RhoA inhibitors (555550 and Y27632) and a Rac1 inhibitor (553502) were purchased from Calbiochem (San Diego, CA). DU145 (1.5 × 106 cells/well) or PC-3 (1.25 × 106 cells/well) cells were mock-infected or infected with AdWTStat3, AdDNStat3, AdWTJak2, or AdDNJak2 at a m.o.i. of 10. Identical scratches were made in parallel wells 24 hours after infection using a 1000-μl plastic pipette tip. The cells were fixed by 0.5% crystal violet solution (Sigma) at 0, 24, 48, and 72 hours. The size of wound was measured and the percentage of the cells that had migrated was calculated. DU145 and PC-3 cells were mock-infected or infected with AdLacZ, AdWTStat3, AdDNStat3, AdWTJak2, or AdDNJak2 at a m.o.i. of 10. Cells were harvested 24 hours after infection and single-cell suspension of 2.5 × 104 cells was added to the upper chamber of a motility chamber system (8.0 μm pore size; BD BioSciences, San Jose, CA) (modified Boyden chamber assay). FBS (5%) in RPMI media was used as chemoattractant in the lower chamber. The cells were incubated for 24 hours, and the cells that traversed the membrane pores to the lower membrane were fixed, stained with the Diff Quik staining kit (Dade Behring Inc., Newark, DE) and counted in four different quadrants. Each experiment was done in triplicate and error bars represent the mean ± SE. Human Stat3 and control siRNA were from Dharmacon (Lafayette, CO). DU145 cells at 40 to 50% confluence were transfected with Stat3 or scrambled control siRNA (100 pmol/well of a 3-cm well) using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 24, 48, or 72 hours, the cells were harvested for Western blotting, cell viability assays, or microtubule immunocytochemistry. DU145 and PC-3 cells were infected with AdWTStat3 or AdDNStat3 at a m.o.i. of 10. Twenty-four hours after adenoviral infection, the cells were seeded on Matrigel (total protein, 8.5 mg/ml; endotoxin, 1.0 U/ml) (BD Biosciences) that was spread on glass coverslips. After incubation for 96 hours, the cells were observed for morphological alterations and photographed (Nikon Stereoscope, Chuoku, Japan). Photographs of wound margins from wound filling assay of DU145 cells were analyzed for lamellipodia. The areas of lamellipodia along the wound margins were measured using the Metamorph program (Molecular Devices, Sunnyvale, CA). The surface areas of lamellipodia were calculated per cell and plotted on a graph. DU145 (3 × 104 cells) and PC-3 (1.5 × 104) cells grown on cover glasses were mock-infected or infected with AdWTStat3, AdDNStat3, AdWTJak2, or AdDNJak2 at a m.o.i. of 10. Forty-eight hours after adenoviral expression of Stat3 and Jak2 proteins, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 minutes followed by permeabilization with 0.1% Triton X-100 in 4% paraformaldehyde in phosphate-buffered saline for 15 minutes. The cells were incubated with rhodamine-conjugated phalloidin (Invitrogen, Eugene, OR) for 1 hour. For immunofluorescence cytochemistry of tubulin, 4% paraformaldehyde and 0.1% Triton X-100 were made in PHEM buffer (60 mM PIPES, 27.5 mM HEPES, 10 mM EGTA, 8 mM MgSO4, pH 7), and the cells were stained with an antibody against tubulin (clone TU-01; Invitrogen) diluted (1:100) in blocking buffer, which was detected with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Invitrogen). The nuclei were stained with mounting media containing 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and examined under a LSM510 META confocal laser scanning microscope with an ×63 objective (Zeiss, Thornwood, NY). Castrated male athymic mice were purchased from Taconic (Germantown, NY) and cared for according to the institutional guidelines. DU145 cells were infected with a m.o.i. of 10 of AdLacZ, AdWTStat3, and AdDNStat3. After 24 hours, 1 × 106 cells were suspended in 0.2 ml of phosphate-buffered saline and injected into the lateral tail vein using a 27-gauge needle. The mice were sacrificed 8 weeks after inoculation, and the lungs were perfused with 1.5 ml of 15% India ink dye in 3.7% formalin. Lungs were then removed, rinsed in water for 15 seconds, and bleached in Fekete's solution (70% ethanol, 3.7% formaldehyde, 0.75 mol/L glacial acetic acid). Lung surfaces were photographed and scored. Differences in groups with respect to number of migrated cells and wound size were assessed using two-sample t-tests or analysis of variance as appropriate. Pair-wise comparisons were performed if the overall analysis of variance test for differences in means was significant. P values for these comparisons were adjusted using Tukey's procedure. As the first step to test the hypothesis that Stat3 promotes metastatic behavior of human prostate cancer cells, we determined how frequently Stat3 is activated in clinical prostate cancer metastases (n = 131) and recurrent human prostate cancers (n = 181). Stat3 activation was analyzed by immunohistochemical detection of paraffin-embedded tissue sections. In addition to active Stat3 immunostaining, prostate cancer metastases sections were immunostained for PSA to verify the location of prostate cancer cells within the metastases-containing tissues. Parallel sections were stained with normal rabbit serum as a negative control (data not shown). Representative prostate cancer metastases to bone and lymph nodes with positive immunoreaction for active Stat3 and PSA are presented in Figure 1, A and B. A positive immunoreaction for active Stat3 was detected in 64% (84 of 131) of prostate cancer metastases (Table 1). In prostate cancer metastases to regional lymph nodes, an intense immunoreaction for active Stat3 was detected in 77% (51 of 66) of the specimens, whereas Stat3 was activated in 67% (10 of 15) of the bone metastases. Moreover, Stat3 was active in 56% (28 of 50) of prostate cancer metastases to distant organs other than bone. To further investigate Stat3 activation in advanced prostate cancer, we assessed Stat3 activation in recurrent human prostate tumors. Significant activation of Stat3 was detected in 86% (162 of 188) of recurrent human prostate cancer specimens (Table 1). Of these 188 patients, 121 had been treated with androgen deprivation before the recurrence occurred (see Materials and Methods section). Stat3 was constitutively active in 96 of the 121 recurrent prostate cancers (79%) treated with hormone therapy (Table 1). In summary, our results indicate that Stat3 is constitutively active in the majority of distant prostate cancer metastases and in recurrent hormone-refractory prostate cancer.Table 1Stat3 Activation in Prostate Cancer Metastasis and in Recurrent Hormone-Refractory Prostate CancersNumber of patients%Prostate cancer metastasis131100 Stat3 activation, negative4736 Stat3 activation, positive8464Prostate cancer to regional lymph nodes66100 Stat3 activation, negative1523 Stat3 activation, positive5177Prostate cancer to bone15100 Stat3 activation, negative533 Stat3 activation, positive1067Prostate cancer to other organs50100 Stat3 activation, negative2244 Stat3 activation, positive2856Recurrent prostate cancers188100 Stat3 activation, negative2614 Stat3 activation, positive16286Recurrent prostate cancers treated with hormone therapy121100 Stat3 activation, negative2521 Stat3 activation, positive9679 Open table in a new tab Given that Stat3 is constitutively active in the majority of clinical human prostate cancer metastases (Table 1), we aimed to determine whether Stat3 is involved in the regulation of metastatic behavior of prostate cancer cells in vitro. The metastatic process is a sequential cascade of multiple cellular events involving invasion of the cells into extracellular matrix, migration of the cells, changes in homo- and heterotypic adhesion, as well as changes in angiogenesis. We chose first to focus on establishing the effects of Stat3 on prostate cancer cell migration because previous studies have suggested that Stat3 proteins may affect cell motility in other cancer types.12Dauer DJ Ferraro B Song L Yu B Mora L Buettner R Enkemann S Jove R Haura EB Stat3 regulates genes common to both wound healing and cancer.Oncogene. 2005; 24: 3397-3408Crossref PubMed Scopus (317) Google Scholar, 15Silver DL Naora H Liu J Cheng W Montell DJ Activated signal transducer and activator of transcription (STAT) 3: localization in focal adhesions and function in ovarian cancer cell motility.Cancer Res. 2004; 64: 3550-3558Crossref PubMed Scopus (227) Google Scholar, 19Debidda M Wang L Zang H Poli V Zheng Y A role of STAT3 in Rho GTPase-regulated cell migration and proliferation.J Biol Chem. 2005; 280: 17275-17285Crossref PubMed Scopus (118) Google Scholar, 23Ng DC Lin BH Lim CP Huang G Zhang T Poli V Cao X Stat3 regulates microtubules by antagonizing the depolymerization activity of stathmin.J Cell Biol. 2006; 172: 245-257Crossref PubMed Scopus (221) Google Scholar DU145 and PC-3 cells were selected as the experimental models because both cell lines are androgen-independent with high metastatic potential in in vivo studies.27Nemeth JA Harb JF Barroso Jr, U He Z Grignon DJ Cher ML Severe combined immunodeficient-hu model of human prostate cancer metastasis to human bone.Cancer Res. 1999; 59: 1987-1993PubMed Google Scholar, 28Timár J Raso E Dome B Li L Grignon D Nie D Honn KV Hagmann W Expression, subcellular localization and putative function of platelet-type 12-lipoxygenase in human prostate cancer cell lines of different metastatic potential.Int J Cancer. 2000; 87: 37-43Crossref PubMed Scopus (72) Google Scholar, 29Pulukuri SM Gondi CS Lakka SS Jutla A Estes N Gujrati M Rao JS RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo.J Biol Chem. 2005; 280: 36529-36540Crossref PubMed Scopus (245) Google Scholar, 30Muramaki M Miyake H Hara I Kamidono S Synergistic inhibition of tumor growth and metastasis by combined treatment with TNP-470 and docetaxel in a human prostate cancer PC-3 model.Int J Oncol. 2005; 26: 623-628PubMed Google Scholar, 31Knox JD Mack CF Powell WC Bowden GT Nagle RB Prostate tumor cell invasion: a comparison of orthotopic and ectopic models.Invasion Metastasis.
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