Receptor Tyrosine Kinase EphB4 Is a Survival Factor in Breast Cancer
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.050889
ISSN1525-2191
AutoresS. Ram Kumar, J. B. Singh, Guangbin Xia, Valery Krasnoperov, Loubna Hassanieh, Eric J. Ley, Jeffrey S. Scehnet, Neil G. Kumar, Debra Hawes, Michael F. Press, Fred A. Weaver, Parkash S. Gill,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoEphB4, a member of the largest family of receptor tyrosine kinases, is normally expressed on endothelial and neuronal cells. Although aberrant expression of EphB4 has been reported in several human tumors, including breast cancer, its functional significance is not understood. We report here that EphB4 is expressed in 7 of 12 (58%) human breast cancer specimens and 4 of 4 (100%) breast tumor cell lines examined. Overexpression of EphB4 in breast cancer cells was driven by gene amplification and by the erbB family of receptors via activation of Janus tyrosine kinase-signal transducers and activators of transcription and protein kinase B. The aberrantly expressed receptor was phosphorylated by its natural ligand, EphrinB2, and signaled via the protein kinase B pathway. Targeted knockdown of EphB4 expression by small interference RNA (and antisense oligodeoxynucleotides (ODNs)) led to dose-dependent reduction in cell survival, increased apoptosis, and sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Antisense ODN-mediated EphB4 knockdown resulted in reduced tumor growth in a murine tumor xenograft model. Antisense ODN-treated tumors were 72% smaller than control tumors at 6 weeks, with an 86% reduction in proliferating cells, 15-fold increase in apoptosis, and 44% reduction in tumor microvasculature. Our data indicate that biologically active EphB4 functions as a survival factor in breast cancer and is a novel target for therapy. EphB4, a member of the largest family of receptor tyrosine kinases, is normally expressed on endothelial and neuronal cells. Although aberrant expression of EphB4 has been reported in several human tumors, including breast cancer, its functional significance is not understood. We report here that EphB4 is expressed in 7 of 12 (58%) human breast cancer specimens and 4 of 4 (100%) breast tumor cell lines examined. Overexpression of EphB4 in breast cancer cells was driven by gene amplification and by the erbB family of receptors via activation of Janus tyrosine kinase-signal transducers and activators of transcription and protein kinase B. The aberrantly expressed receptor was phosphorylated by its natural ligand, EphrinB2, and signaled via the protein kinase B pathway. Targeted knockdown of EphB4 expression by small interference RNA (and antisense oligodeoxynucleotides (ODNs)) led to dose-dependent reduction in cell survival, increased apoptosis, and sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Antisense ODN-mediated EphB4 knockdown resulted in reduced tumor growth in a murine tumor xenograft model. Antisense ODN-treated tumors were 72% smaller than control tumors at 6 weeks, with an 86% reduction in proliferating cells, 15-fold increase in apoptosis, and 44% reduction in tumor microvasculature. Our data indicate that biologically active EphB4 functions as a survival factor in breast cancer and is a novel target for therapy. Breast cancer is the most common malignancy in women in the United States, with an estimated incidence of 135 cases per 100,000 population (U.S. Cancer Statistics Working Group. United States Cancer Statistics: 1999–2001 Incidence and Mortality Web-based Report Version. Atlanta (GA): Department of Health and Human Services, Centers for Disease Control and Prevention, and National Cancer Institute; 2004. Available at: http://www.cdc.gov/cancer/npcr/uscs. Accessed March 31, 2005). A significant body of evidence links the erbB family of receptor tyrosine kinases to breast cancer and poor outcome.1Gullick WJ Srinivasan R The type 1 growth factor receptor family. 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Analysis of human breast cancer specimens reveals that EphB4 expression correlates with histological grade and stage and is associated with DNA aneuploidy.27Wu Q Suo Z Risberg B Karlsson MG Villman K Nesland JM Expression of Ephb2 and Ephb4 in breast carcinoma.Pathol Oncol Res. 2004; 10: 26-33Crossref PubMed Scopus (102) Google Scholar Transgenic expression of EphB4 in mouse mammary epithelium does not initiate transformation. 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Paradoxically, these authors also reported that stimulation of tumor cell EphB4 (with EphrinB2) results in a loss of tumor cell viability, suggesting that the proangiogenic effects of tumor cell EphB4 can mask its direct antisurvival effects. We sought to characterize the biological significance of EphB4 in breast cancer. We show here that EphB4 is expressed in a majority of human breast cancer specimens and breast cancer cell lines that we analyzed. EphB4 expression is regulated by signaling via members of the erbB family of receptors and by amplification of the EPHB4 gene locus. Knocking down EphB4 expression in breast tumor cell lines with small interference RNA (siRNA) (and antisense oligonucleotides) results in loss of cell viability, activation of caspase-8, and induction of apoptosis by sen-sitizing cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Finally, EphB4 knockdown with specific antisense oligonucleotides in a murine breast cancer xenograft model results in significantly smaller tumors, induction of apoptosis, and reduction in tumor vascularity. EphB4 is thus a critical survival factor in breast cancer cells and is a novel therapeutic target in breast cancer. Media and fetal bovine serum were from Invitrogen (Carlsbad, CA). Antibodies against EGFR, EphrinB2 (P20), and CD31 (M20) were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-mouse EphB4 was from R&D Systems, Inc. (Minneapolis, MN), anti-protein kinase B (Akt) and anti-p-Akt were from Cell Signaling Technology (Beverly, MA), and anti-human Fc was from Jackson Laboratories (Bar Harbor, ME). Monoclonal antibody against phosphotyrosine (clone 4G10) was from Upstate (Lake Placid, NY), anti-Ki-67 was from DAKO (Carpentaria, CA), anti-Bcl-2 and anti-Bcl-xL were from Calbiochem (San Diego, CA), anti-Mcl-1 was from BD Pharmingen (San Diego, CA), anti-FLIP was from Alexis Corp. (San Diego, CA), and anti-actin was from Sigma-Aldrich (St Louis, MO). Monoclonal EphB4 antibodies used in immunoprecipitation, immunofluorescence, immunohistochemistry, and immunoblotting and monoclonal EphrinB2 antibody used in immunohistochemistry were kindly provided by VasGene Therapeutics, Inc. (Los Angeles, CA). AG1478, AG490, PP2, SB203580, and SH5 were from Calbiochem, wortmannin and PD 98059 were from Sigma-Aldrich, EGF and EphrinB2/Fc chimeric protein were from R&D Systems, Inc., and human IgG Fc fragment was from Jackson Laboratories. Soluble human TRAIL was obtained from Santa Cruz Biotechnology. The MACSelect 4.1 transfected cell selection kit was from Miltenyi Biotec (Auburn, CA). The cell lines MCF-7, ZR75, T47D, SKBR3, and A549 were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Roswell Park Memorial Institute (RPMI)-1640 media containing 10% fetal bovine serum, 5 mmol/L l-glutamine, and penicillin/streptomycin. Primary normal breast epithelial cells harvested from normal human breast tissue obtained at the time of breast reduction surgery were cultured in the above medium. Human umbilical vein endothelial cells were obtained from Clonetics (San Diego, CA) and cultured as described before.30Masood R Xia G Smith DL Scalia P Still JG Tulpule A Gill PS Ephrin B2 expression in Kaposi sarcoma is induced by human herpesvirus type 8: phenotype switch from venous to arterial endothelium.Blood. 2005; 105: 1310-1318Crossref PubMed Scopus (55) Google Scholar Parent mouse fibroblast cells (3T3) stably transfected with either the empty vector or HER-2/neu were maintained in 10% Dulbecco's modified Eagle's medium supplemented as above. Human normal breast and breast cancer specimens were obtained under Institutional Review Board-approved protocols. Sections (5 μm) of fresh frozen human breast tumor tissues were fixed in 4% paraformaldehyde and washed in phosphate-buffered saline. Sections (5 μm) of formalin-fixed, paraffin-embedded tissues were deparafinized and hydrated. Antigen epitope retrieval was performed by heating slides in 10 mmol/L sodium citrate buffer (pH 8.5) at 80°C for 20 minutes. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 in phosphate-buffered saline for 10 minutes, followed by blocking of nonspecific sites with SuperBlock blocking buffer (Pierce, Rockford, IL) for 1 hour both at room temperature. Sections were incubated with primary antibody overnight at 4°C and, after three washes in phosphate-buffered saline, with appropriate secondary antibody for 1 hour at room temperature. Antibody binding was localized with ABC staining kit form Vector Laboratories (Burlingame, CA) according to the manufacturer's instructions, and peroxidase activity was detected using 3,3′-diamino benzidine substrate solution (Vector). Sections were counterstained with Harris hematoxylin for 45 s, dehydrated, and mounted in xylene. For immunofluorescence, secondary antibody incubation was followed by incubation for 30 minutes at room temperature with 2 μg/ml fluorescein isothiocyanate-labeled avidin (Vector). After three washes in phosphate-buffered saline, sections were counterstained with 4′,6-diamidino-2-phenylindole and mounted in immunofluorescence medium (Vector). Routine negative controls included deletion of primary and secondary antibody, premixing primary antibody with blocking peptide and substitution of normal IgG isotope for primary antibody. When using mouse anti-human Ki-67 antibody, an MOM kit (Vector) was used to block nonspecific binding to mouse tissue. For in vivo tumor specimens, the number of cells staining positively was counted by a blinded observer in five random high power fields. Cell lysates were prepared using Cell Lysis Buffer (GeneHunter, Basgvukke, TN) supplemented with protease inhibitor cocktail (Pierce). Total protein was determined using the DC reagent system (Bio-Rad, Hercules, CA). Typically, 20 μg of whole cell lysate was run on 4 to 20% Tris-glycine gradient gel (Invitrogen). The samples were electrotransferred to polyvinylidene difluoride membrane (Bio-Rad), and nonspecific binding was blocked in TBST (Tris-buffered saline with 0.1% Tween-20) buffer containing 5% nonfat milk (Bio-Rad). Membranes were first probed with primary antibody for 2 hours, washed, and probed with the secondary antibody for 1 hour, and developed. Membranes were then stripped with Restore Western Blot stripping buffer (Pierce) and reprobed with β-actin to confirm equivalent loading and transfer of protein. Signal was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). Bands were scanned and signal intensity was normalized to β-actin using ImageJ software (version 1.32j; National Institutes of Health, Bethesda, MD). Gene amplification was analyzed by quantitative polymerase chain reaction (PCR). DNA was extracted from peripheral blood monocytes of a normal donor and the breast cancer cell lines using the Blood and Cell Culture DNA Midi kit from Qiagen (Valencia, CA) according to the manufacturer's instructions. Quantitative PCR was performed on 50 ng of DNA on the MX3000P real-time PCR system (Stratagene, La Jolla, CA) using SYBR Green I Brilliant Mastermix according to the manufacturer's instructions with thermal profile of 95°C for 10 minutes followed by 40 cycles of 95°C for 30 seconds, 60°C for 1 minute, and then 72°C for 1 minute. The primers were EphB4-forward, 5′-TCC TGC AAG GAG ACC TTC AC-3′; EphB4-reverse, 5′-CAG AGG CCT CGC AAC TAC AT-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-forward, 5′-GAG GGG TGA TGT GGG GAG TA-3′; GAPDH-reverse, 5′-GAG CTT CCC GTT CAG CTC AG-3′; β-actin-forward, 5′-GTC TTC CCC TCC ATC GTG-3′; and β-actin-reverse, 5′-ACA CGC AGC TCA TTG TAG-3′. The amplification signal for EphB4 was normalized to GAPDH, and the gene copy number was normalized to healthy donor peripheral blood monocytes. Amplification of β-actin was also examined as an additional control. For assessing urokinase-type plasminogen activator (uPA) mRNA levels, total mRNA was extracted from cultured cells using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX). First strand cDNA was synthesized from 5 μg of total RNA using SuperScript III (Invitrogen). Quantitative PCR was then performed as above. Optimized reactions for uPA were 150 nmol/L each of the forward primer, 5′-ACT-GGC-TTG-AAG-ATC-ACC-AG-3′, and reverse primer, 5′-CCC-TCT-CAC-AGC-TCA-TGT-CT-3′, with a thermal profile of 95°C for 10 minutes followed by 40 cycles of 95°C for 30 seconds, 57°C for 1 minute, and then 72°C for 1 minute. The specificity of the amplification was confirmed by the presence of a single dissociation peak. All reactions were performed in triplicate and with no reverse transcriptase and no template as negative controls. EphrinB2/Fc or Fc fragment alone was clustered by incubating with 1:1000 dilution anti-human Fc antibody for 1 hour at 4°C. MCF-7 cells grown to 80% confluence were serum-starved overnight and treated for a specified time with 1 μg/ml clustered EphrinB2/Fc or Fc alone. Protein lysates were prepared, and 100 μg of each lysate was used for immunoprecipitation of EphB4 using 2 μg/ml anti-EphB4 monoclonal antibody overnight at 4°C. Antigen-antibody complexes were absorbed on 10 μl of protein A/G-Sepharose (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at 4°C. Pellets were immunoblotted with antiphosphotyrosine antibody to detect phosphorylation status. Efficiency of immunoprecipitation was examined on duplicate membranes with EphB4-specific monoclonal antibody. MCF-7 cells were seeded in 48-well plates at a density of 1 × 104 cells/well in a total volume of 500 μl. Medium was changed after cells were attached, and triplicate samples were treated as described in Results. Cell viability was assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described previously.31Masood R Kundra A Zhu S Xia G Scalia P Smith DL Gill PS 2003. Malignant mesothelioma growth inhibition by agents that target the VEGF and VEGF-C autocrine loops.Int J Cancer. 2003; 104: 603-610Crossref PubMed Scopus (140) Google Scholar For studying sensitivity of cells to TRAIL-induced apoptosis, appropriate concentration of ligand was added to the supernatant 16 hours before MTT assay. Various EphB4-specific anti-sense phosphorothioate-modified oligodeoxynucleotides (ODNs) and siRNA were synthesized from Qiagen (Valencia, CA). The most active antisense ODN and siRNA that knock down EphB4 expression in the transiently transfected 293T cell line were chosen (data not shown). The antisense ODN used, AS-10, spanned nucleotides 1980 to 1999 with a sequence 5′-ATG GAG GCC TCG CTC AGA AA-3′. To eliminate cytokine responses, the cytosine at the CpG site was methylated (AS-10M) without any loss in EphB4 knockdown efficiency (data not shown). Scrambled ODNs containing random nucleotide sequence and a similar CpG site, 5′-TAC CTG AAG GTC AGG CGA AC-3′, was used as control. siRNA 465 corresponding to the sequences 5′-GGU GAA UGU CAA GAC GCU GUU-3′ and 3′-UUC CAC UUA CAG UUC UGC GAC-5′ was used for RNA interference. Control siRNA was generated by mutating three bases in this sequence to effectively abrogate EphB4 knockdown. This mutated siRNA (siRNAΔ) had the sequences 5′-AGU UAA UAU CAA GAC GCU GUU-3′ and 3′-UUU CAA UUA UAG UUC UGC GAC-5′. Additionally, siRNA directed against green fluorescent protein with sequences 5′-CGC UGA CCC UGA AGU UCA TUU-3′ and 3′-UUG CGA CUG GGA CUU CAA GUA-5′ was also used as a negative control. Cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, transfection complexes containing 5 μl of Lipofectamine per 250 μl of reduced serum Opti-MEM medium and appropriate concentration siRNA diluted in same medium were added to antibiotic-free culture medium. Cells were cultured in this medium for 6 hours, after which they were returned to the original culture medium. All experiments were performed 48 hours after transfection. For sorting transfected cells, the MACSelect 4.1 transfected cell selection kit was used as per the manufacturer's instructions. In brief, cells were cotransfected with expression vector containing the plasmid of interest and pMACS 4.1 plasmid. After 36 hours, cells were harvested with 5 mmol/L ethylenediamine tetraacetic acid and incubated with MACSelect 4 Microbeads for 15 minutes at 4°C. The cell suspension was then passed via an MS+ column in a magnetic field. After three washes, the column was removed from the field, and selected cells were eluted in culture medium. Selection efficiency was confirmed by fluorescence-activate cell sorting analysis of sorted cells with fluorescent EphB4 monoclonal antibody (data not shown). Chemotaxis was assessed using a modified Boyden chamber assay. For invasion studies, MCF-7 cells were appropriately treated, and 0.5 × 105 cells were transferred into 8μ Matrigel-precoated inserts (BD Bioscience, Palo Alto, CA). The inserts were placed in companion wells containing 700 μl of RPMI supplemented with 5% fetal bovine serum and 5 μg/ml fibronectin as a chemoattractant. Following 12 hours of incubation, the inserts were removed, and noninvading cells on the upper surface were scraped with a cotton swab. The cells on the lower surface of the membrane were fixed in 100% methanol for 15 minutes, air dried, and stained with Giemsa stain for 2 minutes. The cells were counted in five individual high power fields, and the percentage of reduction was calculated with respect to mutated siRNA or scrambled ODN. Assays were performed in triplicate for each treatment group. For migration studies, the same experiment was performed for 8 hours, except that the inserts were not coated with Matrigel. MCF-7 cells were cultured until 80% confluent. Cells were transfected with EphB4 siRNA or mutated siRNA (50 nmol/L) and cultured for 24 hours. Conditioned medium from equal cell numbers was collected, concentrated, and electrophoresed through a 10% Zymogram gel (Criterion Zymogram; Bio-Rad). The gel was washed three times over 1 hour with 2.5% Triton X-100 to remove the SDS and incubated for 24 hours at 37°C in collagenase buffer containing 50 mmol/L Tris, 200 mmol/L NaCl, and 10 mmol/L CaCl2, pH 7.5. Gelatinolytic activity was visualized by staining the gel in 0.5% Coomassie Blue. Apoptosis was studied in vitro using the Cell Death Detection ELISAplus Kit (Roche Applied Science, Piscataway, NJ) according to the manufacturer's instructions. Briefly, MCF-7 cells were cultured in 24-well plates to 80% confluence and treated with test compounds at various concentrations. Cells were harvested at 36 hours, and 1 × 104 cells from each well were incubated in 200 μl of lysis buffer. Nuclei were pelleted by centrifugation, and 20 μl of supernatant containing the mono- or oligonucleosomes was incubated with anti-histone-biotin and anti-DNA-peroxidase in streptavidin-coated 96-well plate for 2 hours at room temperature. Color was developed with peroxide substrate, and absorbance at 405 nm was read in a microplate reader (Molecular Devices, Sunnyvale, CA). Apoptosis was detected in deparafinized sections of animal tumors by terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL) assay using an in situ cell death detection kit (Roche Applied Science) according to manufacturer's instructions. Caspase activity was measured by colorimetric assay (R&D Systems, Inc.) according to the manufacturer's instructions. Briefly, MCF-7 cells appropriately treated were lysed in lysis buffer and clarified by centrifugation. The clarified lysate was incubated with reaction buffer and the appropriate colorimetric substrate at 37°C for 2 hours. Color development was quantified by measuring absorbance at 405 nm. Routine controls included deletion of cell lysate and substrate. MCF-7 cells were propagated, collected by trypsin digestion, and resuspended in serum-free medium. 5 × 106 cells were injected in the area of the right breast tissue in 10- to 12-week-old female Balb/C athymic mice. Mice were implanted with 1.7 mg of 17β-estradiol pellets as an exogenous estrogen source. Tumor size was measured every other day, and volume was estimated as 0.52 × a × b2, where a and b are the largest and smallest lengths of the palpable tumor. On day 4 after cell implantation, tumor volumes were calculated to ensure uniformity in size, and animals were randomly divided into three groups (n = 6 mice per group). Each group was administered daily intraperitoneal injection AS-10M or scrambled ODN at a dose of 10 mg/kg or vehicle alone (sterile normal saline, pH 7.4). Animals were sacri
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