Expression of Cyclophilin B is Associated with Malignant Progression and Regulation of Genes Implicated in the Pathogenesis of Breast Cancer
2008; Elsevier BV; Volume: 174; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080753
ISSN1525-2191
AutoresFeng Fang, Ayanna J. Flegler, Pan Du, Simon Lin, Charles V. Clevenger,
Tópico(s)Cell Adhesion Molecules Research
ResumoCyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor α, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility. Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor α, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility. Cyclophilin B (CypB) is a 21-kDa protein belonging to the cyclophilin family of peptidyl-prolyl cis-trans isomerase.1Price ER Zydowsky LD Jin MJ Baker CH McKeon FD Walsh CT Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence.Proc Natl Acad Sci USA. 1991; 88: 1903-1907Crossref PubMed Scopus (277) Google Scholar In this role, cyclophilins promote alterations in protein conformation by binding to substrates in a proline-specific manner and enabling conversion of the proline imide bond through 180° of rotation.2Fischer G Tradler T Zarnt T The mode of action of peptidyl prolyl cis/trans isomerases in vivo: binding vs. catalysis.FEBS Lett. 1998; 426: 17-20Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Cyclophilins are widely expressed in the human body and highly conserved throughout the evolution. Cyclophilins act as molecular chaperones that fold, translocate, and process newly synthesized proteins.3Bhattacharyya T Karnezis AN Murphy SP Hoang T Freeman BC Phillips B Morimoto RI Cloning and subcellular localization of human mitochondrial hsp70.J Biol Chem. 1995; 270: 1705-1710Crossref PubMed Scopus (187) Google Scholar They are also binding targets of cyclosporine A, a complex that inhibits the activation of the calcineurin/nuclear factor of activated T-cells signaling pathway, which contributes to the immunosuppressive effects of this drug.2Fischer G Tradler T Zarnt T The mode of action of peptidyl prolyl cis/trans isomerases in vivo: binding vs. catalysis.FEBS Lett. 1998; 426: 17-20Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 4Kofron JL Kuzmic P Kishore V Colon-Bonilla E Rich DH Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay.Biochemistry. 1991; 30: 6127-6134Crossref PubMed Scopus (483) Google Scholar By the reversible modification of their protein structure, cyclophilins also serve as signaling switches,5Hunter T Prolyl isomerases and nuclear function.Cell. 1998; 92: 141-143Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar regulating the activity of transforming growth factor β6Huse M Chen YG Massague J Kuriyan J Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12.Cell. 1999; 96: 425-436Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar receptor; epidermal growth factor receptor,7Lopez-Ilasaca M Schiene C Kullertz G Tradler T Fischer G Wetzker R Effects of FK506-binding protein 12 and FK506 on autophosphorylation of epidermal growth factor receptor.J Biol Chem. 1998; 273: 9430-9434Crossref PubMed Scopus (48) Google Scholar tyrosine kinases,8Brazin KN Mallis RJ Fulton DB Andreotti AH Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A.Proc Natl Acad Sci USA. 2002; 99: 1899-1904Crossref PubMed Scopus (237) Google Scholar and transcription factors such as c-Myb9Leverson JD Ness SA Point mutations in v-Myb disrupt a cyclophilin-catalyzed negative regulatory mechanism.Mol Cell. 1998; 1: 203-211Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar and interferon regulatory factor 4.10Mamane Y Sharma S Petropoulos L Lin R Hiscott J Posttranslational regulation of IRF-4 activity by the immunophilin FKBP52.Immunity. 2000; 12: 129-140Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Structurally, CypB contains a high degree of homology with other members of the cyclophilin family in its core β-barrel/isomerase region, which contains a surface hydrophobic pocket that constitutes the proline binding motif. Both the N- and C-termini of CypB differ significantly from other cyclophilin family members. Within the N-terminus, CypB contains a nuclear translocation motif (DEKKKGPKV), while an endoplasmic reticulum (ER) retention sequence (AIAKE) resides in its C-terminus. This ER-retention motif is proteolytically clipped in the ER, enabling secretion of CypB into the extracellular milieu.1Price ER Zydowsky LD Jin MJ Baker CH McKeon FD Walsh CT Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence.Proc Natl Acad Sci USA. 1991; 88: 1903-1907Crossref PubMed Scopus (277) Google Scholar CypB is present in serum and breast milk in concentrations of up to 150 ng/ml.11Allain F Boutillon C Mariller C Spik G Selective assay for CyPA and CyPB in human blood using highly specific anti-peptide antibodies.J Immunol Methods. 1995; 178: 113-120Crossref PubMed Scopus (53) Google Scholar, 12Mariller C Allain F Kouach M Spik G Evidence that human milk isolated cyclophilin B corresponds to a truncated form.Biochim Biophys Acta. 1996; 1293: 31-38Crossref PubMed Scopus (17) Google Scholar Interestingly, when examined biochemically, the majority of CypB can be found to reside in the cell nucleus,13Le Hir M Su Q Weber L Woerly G Granelli-Piperno A Ryffel B In situ detection of cyclosporin A: evidence for nuclear localization of cyclosporine and cyclophilins.Lab Invest. 1995; 73: 727-733PubMed Google Scholar with lower quantities in the ER compartment.14Price ER Jin M Lim D Pati S Walsh CT McKeon FD Cyclophilin B trafficking through the secretory pathway is altered by binding of cyclosporin A.Proc Natl Acad Sci USA. 1994; 91: 3931-3935Crossref PubMed Scopus (105) Google Scholar Within the ER, CypB could be found in association with nascent precursor proteins complexed with protein components of the ER protein translocation apparatus.15Klappa P Freedman RB Zimmermann R Protein disulphide isomerase and a lumenal cyclophilin-type peptidyl prolyl cis-trans isomerase are in transient contact with secretory proteins during late stages of translocation.Eur J Biochem. 1995; 232: 755-764Crossref PubMed Scopus (60) Google Scholar The larger biological function of CypB has remained enigmatic. Studies have indicated that CD147 may serve as a cell surface receptor for CypB, as activation of this receptor by CypB results in calcium transport and mitogen-activated protein kinase activation16Yurchenko V O'Connor M Dai WW Guo H Toole B Sherry B Bukrinsky M CD147 is a signaling receptor for cyclophilin B.Biochem Biophys Res Commun. 2001; 288: 786-788Crossref PubMed Scopus (119) Google Scholar, 17Melchior A Denys A Deligny A Mazurier J Allain F Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.Exp Cell Res. 2008; 314: 616-628Crossref PubMed Scopus (34) Google Scholar, 18Pakula R Melchior A Denys A Vanpouille C Mazurier J Allain F Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis.Glycobiology. 2007; 17: 492-503Crossref PubMed Scopus (74) Google Scholar; as such CypB has been considered by some to be a cytokine. CypB is also required during the replication of the hepatitis C virus genome, providing a new therapeutic opportunity in the treatment of this disease.19Fernandes F Poole DS Hoover S Middleton R Andrei AC Gerstner J Striker R Sensitivity of hepatitis C virus to cyclosporine A depends on nonstructural proteins NS5A and NS5B.Hepatology. 2007; 46: 1026-1033Crossref PubMed Scopus (105) Google Scholar, 20Heitman J Cullen BR Cyclophilin B escorts the hepatitis C virus RNA polymerase: a viral achilles heel?.Mol Cell. 2005; 19: 145-146Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar However, in addition to these actions, CypB also functions within the nucleus at the level of transcriptional regulation. CypB facilitates the transcriptional activity of Stat5 by inducing the release of the repressor PIAS3,21Rycyzyn MA Reilly SC O'Malley K Clevenger CV Role of cyclophilin B in prolactin signal transduction and nuclear retrotranslocation.Mol Endocrinol. 2000; 14: 1175-1186Crossref PubMed Scopus (65) Google Scholar, 22Rycyzyn MA Clevenger CV The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer.Proc Natl Acad Sci USA. 2002; 99: 6790-6795Crossref PubMed Scopus (124) Google Scholar resulting in significantly enhanced Stat5-mediated gene expression. Given the association of CypB with other intranuclear transcription factors (Clevenger lab, unpublished data), these findings suggest that CypB could serve to coordinate global networks of gene expression. To examine the effects of CypB on gene expression and global cellular function in human breast cancer, microarray analysis was performed on T47D breast cancer stable transduced cells that overexpressed CypB small interfering siRNA, resulting in an effective knockdown of CypB (si-CypB) or non-specific control of siRNA for luciferase knockdown (si-Luc). These data, validated by subsequent real-time PCR analysis, revealed significant alterations in the expression of genes related to cell proliferation, cell migration, and hormone response in the si-CypB cells. When coupled with data obtained from in vitro functional analysis of these cells, as well as the measurement of CypB levels in matched normal, malignant, and metastatic human breast tissues, our findings indicate that CypB action may significantly contribute to the pathogenesis of human breast cancer. The estrogen receptor α (ERα) positive human breast cancer cell line T47D (ATCC, Manassas, VA) and HEK293FT (Invitrogen, Carlsbad, CA) were maintained in Dulbecco's Modified Eagle Medium (Hyclone) supplemented with 10% fetal bovine serum, 50 μg/ml penicillin, and 50 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. The vectors used here include: pGL410-SV40 (The promoterless pGL4.10 vector was digested with BglII/HindIII, and inserted with SV40 minimal promoter digested from pGL2-promoter vector with BglII/HindIII), pGL2-promoter vector, pGL4.10 and renilla luciferase reporter pGL4.73 (The latter three vectors from Promega, Madison WI). Antibodies used in this study are: anti-α-Tubulin (Invitrogen, 32-2500), anti-CypB (Invitrogen, 37-0600), and Alexa 488 fluor-conjugated second antibody (Invitrogen, A11029). SiRNA targeted to the 3′-end region of the CypB coding sequence (termed si-CypB) and the luciferase gene (termed si-Luc) in the pHIV-7/Puro vectors were used to generate stable pools of transduced cells.23Robida JM Nelson HB Liu Z Tang H Characterization of hepatitis C virus subgenomic replicon resistance to cyclosporine in vitro.J Virol. 2007; 81: 5829-5840Crossref PubMed Scopus (81) Google Scholar These vectors were generously provided by Dr. Hengli Tang (Florida State University). HEK293FT cells were transfected with 3 μg pHIV-7/Puro vector and 9 μg of Viral Power Packaging (Invitrogen) using Lipofectamine 2000 (Invitrogen). Virus was harvested post-transfection, and filtered through a 0.45 μm filter to remove cellular debris. Two ml of the lentiviral supernatants were used to transduce T47D cells (50% confluency in T75 flask) in the presence of 7 μg/ml of polybrene (Invitrogen). After 24 hours following transduction, the transduced cells were selected with 1 μg/ml puromycin for 10 days. The resultant stable pools maintained in 1 μg/ml puromycin were then used for the outlined studies. For independent validation of the effects of siRNA knockdown of CypB, a siRNA against the central region of the CypB coding sequence (cat# D-001136-01-05, Dharmacon, Lafayette, CO) was transfected in T47D cells in parallel with the controls si-glyceraldehyde-3-phosphate dehydrogenase (cat# AM4623 from Ambion), and si-control RNA (cat# D-001210-01 from Dharmacon) using RNAiMAX (Invitrogen). After 48 hours, cells were harvested for Western blot or RT-PCR (Reverse transcription polymerese chain reaction). Microarray analysis was conducted with an Illumina Human Ref-6 version 2 Expression Chip (Illumina, San Diego, CA). Three independent T47D cultures were used for RNA isolation with RNeasy plus mini kit (Qiagen, Valencia, CA). RNA quality was checked by Agilent Bioanalyzer (Santa Clara, CA). An Ambion labeling kit was used for labeling cDNA followed by hybridization to Illumina chips. Scan data were extracted by Illumina Beadstudio and subsequently analyzed using Bioconductor lumi package.24Du P Kibbe WA Lin SM lumi: a pipeline for processing Illumina microarray.Bioinformatics. 2008; 24: 1547-1548Crossref PubMed Scopus (1613) Google Scholar The data were first variance stabilization transformed25Lin SM Du P Huber W Kibbe WA Model-based variance-stabilizing transformation for Illumina microarray data.Nucleic Acids Res. 2008; 36: e11Crossref PubMed Scopus (399) Google Scholar and then normalized by a quantile normalization method. To reduce false positives, probes with measurement value below the background level (detection P value <0.01) in all hybridizations were filtered out; 17,901 probes were kept for subsequent statistical analysis. Genes with significant differential expression levels were identified using analysis of variance with Bayes adjustment of the variance implemented in Bioconductor limma package.26Smyth GK Linear models and empirical bayes methods for assessing differential expression in microarray experiments.Stat Appl Genet Mol Biol. 2004; 3: Article3Crossref PubMed Scopus (9092) Google Scholar To control the effects of multiple testing and reduce false positives, the gene selection of differentially expressed genes is based on: the false discovery rate (FDR) adjusted P value <0.05, fold-change ≥1.5 (up or down). The identified genes were used for the outlined studies (see Supplemental Table S1 available at ). Microarray data were deposited in the Gene Expression Omnibus database with accession number GSM324466 (GEO database, ). Ingenuity Pathway Analysis (IPA) was used to identify gene networks according to biological functions and/or diseases in the Ingenuity Pathways Knowledge Base (Ingenuity Systems, Redwood City, CA). The transcripts with known gene identifiers (HUGO gene symbols) and expression levels (fold change ≥1.5 up or down, P < 0.01, FDR <0.05) were filtered and then entered into the Ingenuity Pathways Knowledge Base IPA 4.0 (see Supplemental Table S1 available at for gene list). Each gene identifier mapped in the Ingenuity Pathways Knowledge Base was termed as a focus gene, which was overlaid into a global molecular network established from the information in the Ingenuity Pathways Knowledge Base. Each network contained a maximum of 35 focus genes. Genes were represented as nodes with different shapes and colors (see figure legends), and biological relationships were represented by edges (different lines). All edges were supported by at least one reference as interaction or action.27Ganter B Giroux CN Emerging applications of network and pathway analysis in drug discovery and development.Curr Opin Drug Discov Devel. 2008; 11: 86-94PubMed Google Scholar The nodes (genes) and the edges were described in the figures and figure legends. Two micrograms of RNA was used for cDNA synthesis in 20 μl reaction volume using Superscript III first strand synthesis kit (Invitrogen). Four μl of cDNA (2.5 ng/μl related to RNA concentration), 1 μl primers (2 μmol/L each), and 5 μl of 2× Power SYBR Mastermix (Applied Biosystems, Foster city, CA) were used for real-time PCR in 10 μl reaction volume performed in 384-well plate. Real-time PCR was conducted on ABI 7900HT Thermocycler (Applied Biosystems). All real-time PCR reactions were run in triplicate. Data were normalized to 18S rRNA, and fold change was represented as 2−ΔΔCt [2−([Ct target-Ct 18S]siCypB – [Ct target-Ct 18S]siLuc)]. Primers for real-time PCR are listed in Table 1.Table 1Primers Used for Real-Time PCRGene namePrimer sequencesStartEndRNA accession number18S rRNAolg200 5′-CCCCATGAACGAGGGAATT-3′16261686NR_003286olg201 5′-GGGACTTAATCAACGCAAGCTT-3′c-Mybolg80 5′-GCCAATTATCTCCCGAATCGAA-3′337507NM_005375olg81 5′-ACATTGTTTTCCAATTCTCCCCT-3′ERαolg331 5′-GGAAGCTACTGTTTGCTCCTAACTTG-3′15611635NM_000125olg332 5′-AGATCTCCACCATGCCCTCTAC-3′GHrolg337 5′-CCAGGCCTAAAGACAAATTCTTCT-3′164236NM_000163olg338 5′-AAAAAGTCTCTCGCTCAGGTGAA-3′PRolg345 5′-TCAGTGGGCAGATGCTGTATTT-3′37663862NM_000926olg346 5′-GCCACATGGTAAGGCATAATGA-3′S100A4olg323 5′-CAAGCTCAACAAGTCAGAACTAAAGG-3′150241NM_002961olg324 5′-GCTTCTGGAAAGCAGCTTCATC-3′S100A6olg347 5′-CTCACCATTGGCTCGAAGCT-3′438492NM_014624olg348 5′-AGTCTTCCATCAGCCTTGCAA-3′STMN3olg349 5′-TGCTGTCGCTCATCTGCTCC-3′132209NM_015894olg350 5′-TCACCTCCATGTCCCCGTACT-3′Primers were obtained using Primer Express 3.1 software, from Primerbank or from published sequences (STMN361Bieche I Maucuer A Laurendeau I Lachkar S Spano AJ Frankfurter A Levy P Manceau V Sobel A Vidaud M Curmi PA Expression of stathmin family genes in human tissues: non-neural-restricted expression for SCLIP.Genomics. 2003; 81: 400-410Crossref PubMed Scopus (48) Google Scholar, S100A662Ebihara T Endo R Kikuta H Ishiguro N Ma X Shimazu M Otoguro T Kobayashi K Differential gene expression of S100 protein family in leukocytes from patients with Kawasaki disease.Eur J Pediatr. 2005; 164: 427-431Crossref PubMed Scopus (35) Google Scholar). Most primer pairs span two exons, except the intronless 18S rRNA. Primers were tested by dissociation curve analysis to assure only a single amplicon. The base pairs that the forward primer starts and reverse primer ends are based on mRNA sequence. All primers are located inside the coding sequence. Open table in a new tab Primers were obtained using Primer Express 3.1 software, from Primerbank or from published sequences (STMN361Bieche I Maucuer A Laurendeau I Lachkar S Spano AJ Frankfurter A Levy P Manceau V Sobel A Vidaud M Curmi PA Expression of stathmin family genes in human tissues: non-neural-restricted expression for SCLIP.Genomics. 2003; 81: 400-410Crossref PubMed Scopus (48) Google Scholar, S100A662Ebihara T Endo R Kikuta H Ishiguro N Ma X Shimazu M Otoguro T Kobayashi K Differential gene expression of S100 protein family in leukocytes from patients with Kawasaki disease.Eur J Pediatr. 2005; 164: 427-431Crossref PubMed Scopus (35) Google Scholar). Most primer pairs span two exons, except the intronless 18S rRNA. Primers were tested by dissociation curve analysis to assure only a single amplicon. The base pairs that the forward primer starts and reverse primer ends are based on mRNA sequence. All primers are located inside the coding sequence. A dual luciferase assay was conducted according to Fang et al.28Fang F Antico G Zheng J Clevenger CV Quantification of PRL/Stat5 Signaling with a Novel pGL4-CISH Reporter.BMC Biotechnol. 2008; 8: 11Crossref PubMed Scopus (32) Google Scholar The firefly luciferase reporter pGL410-SV40 (500 ng/well) and renilla luciferase control vector pGL4.73 (5 ng/well) were transiently transfected into 2 × 105 cells in 24-well plate using Lipofectamine LTX (Invitrogen). Following transfection, cells were cultured in growth medium for 24 hours before luminescence assay. Data are represented as RLU (relative light unit, ratio of luciferase/renilla). Fifty microgram of lysates from T47D cells were subjected to 10% SDS-polyacrylamide electrophoresis gel, transferred onto polyvinylidene difluoride membrane and immunoblotted with the antibodies (1 μg/ml) described above. Images were captured using a Fujifilm LAS-3000 image analyzer (Fuji, Japan).28Fang F Antico G Zheng J Clevenger CV Quantification of PRL/Stat5 Signaling with a Novel pGL4-CISH Reporter.BMC Biotechnol. 2008; 8: 11Crossref PubMed Scopus (32) Google Scholar Cell motility was assayed using Boyden chambers (12 μmol/L, 12-well polycarbonate membrane transwell, Corning), as previously described.29Miller SL Antico G Raghunath PN Tomaszewski JE Clevenger CV Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization and motility of breast cancer cells.Oncogene. 2007; 26: 4668-4678Crossref PubMed Scopus (61) Google Scholar To enhance motility non-occlusively, the lower surface of Boyden chamber inserts were coated with 200 μl Collagen I (25 ng/ul, 5 μg total in 1×PBS). T47D cells were arrested in defined (fetal bovine serum-free) medium for 24 hours, then detached with Versene (Invitrogen); 2 × 105 detached cells were resuspended in the 500 μl of defined medium and added into the upper Boyden chamber. One ml of defined medium with 10% fetal bovine serum, or 17-β-estradiol (E2, 10−7M)30Malek D Gust R Kleuser B 17-Beta-estradiol inhibits transforming-growth-factor-beta-induced MCF-7 cell migration by Smad3-repression.Eur J Pharmacol. 2006; 534: 39-47Crossref PubMed Scopus (28) Google Scholar was placed in the lower chambers. Cells were cultured for 20 hours and the migrated cells were counted under microscope for five fields per insert with triplicate inserts in each experiment. T47D cells (4 × 104 cells) were plated in 96-well plate and cultured in the growth medium for 24 hours. Each well was pulsed with 0.5 μCi of [3H] thymidine (48 Ci/mmol; Amersham Pharmacia Biotech) for 4 hours. Cells were harvested onto the membrane with Filtermate Harvester (Perkin Elmer, Waltham, MA) before analysis with a MicroBeta TriLux Scintillation Counter (Perkin Elmer). Breast tissues were obtained from the Tissue Core of the Northwestern University Breast Cancer Program in the form of a tissue microarray (TMA). All tissues were stripped of all patient identifiers before use, and thus were anonymous to the investigators. The TMA examined included matched normal adjacent, invasive ductal carcinoma, and lymph node metastasis from 11 patients. In addition, unmatched ductal carcinoma in situ (DCIS) specimens from nine patients were also present on the TMA. The presence of tumor and integrity of each tissue was confirmed by H&E staining. Before analysis by both immunofluorescent (IF) and immunohistochemical (IHC) approaches, the TMA was incubated at 65°C for 10 minutes, before deparaffinization in xylene and rehydration through graded ethanol, followed by antigen retrieval in citrate buffer (Zymed 00-5001, pH = 6.0) at 95°C for 20 minutes. The slides were blocked in the blocking buffer (2.5% bovine serum albumin and 0.1% Triton X-100) for 10 minutes and incubated with CypB antibody (1:10 dilution for IF; 1:20 dilution for IHC) overnight at 4°C. Antigen-antibody complexes were detected for IF by incubation with an Alexa 488 fluor-conjugated second antibody (1:100 dilution) for one hour. For IHC, detection of antibody-antigen complex used an HRP-conjugated secondary antibody, followed by diaminobenzidine labeling, as previously described.31McHale K Tomaszewski JE Puthiyaveettil R Livolsi VA Clevenger CV Altered expression of prolactin receptor-associated signaling proteins in human breast carcinoma.Mod Pathol. 2008; 21: 565-571Crossref PubMed Scopus (59) Google Scholar Visual scoring of the IF-labeled images was performed as described previously31McHale K Tomaszewski JE Puthiyaveettil R Livolsi VA Clevenger CV Altered expression of prolactin receptor-associated signaling proteins in human breast carcinoma.Mod Pathol. 2008; 21: 565-571Crossref PubMed Scopus (59) Google Scholar with modification. Given that virtually 100% of the breast epithelial demonstrated some level of anti-CypB IF labeling, scoring was restricted to assessment of label intensity on a 0 to 3 scale (0 = absent, 1 = dim, 2 = bright, 3 = very bright). Comparable semiquantitative results were noted with specimens labeled by anti-CypB IHC labeling (not shown). All experiments described here were performed at least three times. Statistical analysis was performed on GraphPad Prism 4 (GraphPad Software, La Jolla, CA), and specified in the figure legends. The results are shown as the means with error bars depicting ± SEM. To assess the effects of CypB expression on global gene expression in breast cancer, two T47D stable cell pools (transfected with the si-Luc siRNA expression vector targeting the luciferase gene as a non-human target control, and the si-CypB siRNA expression vector targeting the CypB gene) were generated. Western blot results showed that the parental T47D cells (also called wild-type, WT) and si-Luc cells had a similar level of CypB expression, while the CypB protein level was barely detected in the si-CypB cells (Figure 1A). The stable knockdown of CypB mRNA was also confirmed by real-time PCR (data not shown) and microarray analysis (see Supplemental Table S1 available at ). A luciferase assay confirmed that firefly luciferase expression was decreased in the control si-Luc T47D cells when co-transfected with a luciferase expression construct (Figure 1B). To corroborate the findings obtained with the siRNA knockdown pools, a CypB siRNA targeting a different region of CypB was transiently transfected with parallel controls into T47D cells (Figure 1A); these cells (demonstrating a 70% reduction in CypB levels) were used to independently validate the effects of the CypB knockdown in stable pools to exclude off-target effects (see Figure 2).Figure 2si-RNA-mediated knockdown of CypB decreases the expression of two motility-relevant gene products and the motility of T47D cells. A and B: Real-time PCR validated that STMN3 (A) and S100A4 (B) expression was down-regulated in T47D pools stably or transiently transfected with one of two non-homologous siRNA. The y axis label “Fold change” is defined in the materials and methods section; “Stable” refers to the cell pools stably transfected with the si-CypB-pHIV-7/puro construct; “Transient” refers to cells transfected directly with a CypB siRNA targeting a differing coding sequence than that used in the stable transfectants. C: Cell motility assay using Boyden chamber assay revealed a significant reduction in T47D motility in the si-CypB cells in both resting and fetal bovine serum-stimulated conditions. Cellular migration was quantitated by phase-contrast microscopy. Magnification = original ×100. Statistical analysis was performed using analysis of variance (ANOVA).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To explore the effect of CypB knockdown on global gene expression, cDNA microarray analysis was conducted with si-CypB and the control si-Luc stable transduced cell pools. To assess the statistical significance of these data, a Bayesian-moderated t-test was used to take advantage of the large number of genes being analyzed simultaneously to yield more reliable variance estimates. To reduce false positives, genes above-background in at least one experiment were used for subsequent analysis. Stringent criteria (fold change ≥1.5 up or down, P < 0.01, FDR <0.05) were used to filter differentially expressed genes. Two-dimensional hierarchical clustering was ap
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