FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling
2015; Elsevier BV; Volume: 185; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2015.03.011
ISSN1525-2191
AutoresVictor Mak, Oscar G.W. Wong, Michelle K.Y. Siu, Esther S.Y. Wong, Wai-Yan Ng, Richard Wing-Cheuk Wong, Ka-Kui Chan, Hys Ngan, Any Cheung,
Tópico(s)Chromatin Remodeling and Cancer
ResumoHuman placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts. Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts. Gestational trophoblastic disease (GTD) is a group of heterogeneous trophoblastic lesions including hydatidiform moles (partial mole and complete mole) that carry malignant potential, as well as frankly malignant tumors such as choriocarcinoma (CCA), placental site trophoblastic tumor, and epithelioid trophoblastic tumor.1Cheung A.N. Pathology of gestational trophoblastic diseases.Best Pract Res Clin Obstet Gynaecol. 2003; 17: 849-868Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 2Shih Ie M. Gestational trophoblastic neoplasia–pathogenesis and potential therapeutic targets.Lancet Oncol. 2007; 8: 642-650Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Although they are distinct diseases, about half of CCA cases are preceded by hydatidiform mole. In fact, although most cases of hydatidiform mole may regress after suction evacuation, 8% to 30% of cases of hydatidiform mole may develop into persistent trophoblastic neoplasm that requires chemotherapy.1Cheung A.N. Pathology of gestational trophoblastic diseases.Best Pract Res Clin Obstet Gynaecol. 2003; 17: 849-868Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 3Shih I. Mazur M. Kurman R. Gestational trophoblastic disease and related lesions.in: Kurman R.J. Blaustein's Pathology of the Female Genital Tract. Springer, New York2002: 1193-1250Google Scholar In recent years, hyperactivation of proto-oncogenic cell signaling pathways, such as the p21-activated kinases, leading to aggressive outcome in GTD patients, has been reported.4Siu M.K. Yeung M.C. Zhang H. Kong D.S. Ho J.W. Ngan H.Y. Chan D.C. Cheung A.N. p21-Activated kinase-1 promotes aggressive phenotype, cell proliferation, and invasion in gestational trophoblastic disease.Am J Pathol. 2010; 176: 3015-3022Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 5Cheung A.N. Zhang H.J. Xue W.C. Siu M.K. Pathogenesis of choriocarcinoma: clinical, genetic and stem cell perspectives.Future Oncol. 2009; 5: 217-231Crossref PubMed Scopus (56) Google Scholar In addition, GTD has also been characterized by abnormal proliferation and apoptosis.6Chiu P.M. Ngan Y.S. Khoo U.S. Cheung A.N. Apoptotic activity in gestational trophoblastic disease correlates with clinical outcome: assessment by the caspase-related M30 CytoDeath antibody.Histopathology. 2001; 38: 243-249Crossref PubMed Scopus (63) Google Scholar, 7Xue W.C. Khoo U.S. Ngan H.Y. Chan K.Y. Chiu P.M. Tsao S.W. Cheung A.N. Minichromosome maintenance protein 7 expression in gestational trophoblastic disease: correlation with Ki67, PCNA and clinicopathological parameters.Histopathology. 2003; 43: 485-490Crossref PubMed Scopus (33) Google Scholar Although TP53 is commonly mutated in human malignancies, lack of TP53 mutation has been reported in hydatidiform mole and CCA.8Cheung A.N. Srivastava G. Chung L.P. Ngan H.Y. Man T.K. Liu Y.T. Chen W.Z. Collins R.J. Wong L.C. Ma H.K. Expression of the p53 gene in trophoblastic cells in hydatidiform moles and normal human placentas.J Reprod Med. 1994; 39: 223-227PubMed Google Scholar Findings from our recent studies of apoptosis-stimulating protein of p53 1 and 2, DJ-1, and phosphatase and tensin homologue suggest that these p53 regulators and mediators may have a crucial role in the pathogenesis and progression of GTD.9Mak V.C. Lee L. Siu M.K. Wong O.G. Lu X. Ngan H.Y. Wong E.S. Cheung A.N. Downregulation of ASPP1 in gestational trophoblastic disease: correlation with hypermethylation, apoptotic activity and clinical outcome.Mod Pathol. 2011; 24: 522-532Crossref PubMed Scopus (16) Google Scholar, 10Zhang H.J. Siu M.K. Jiang L.L. Mak V.C. Ngan H.Y. Cheung A.N. Overexpression of the Parkinson disease protein DJ-1 and its regulator PTEN in gestational trophoblastic disease.Int J Gynecol Pathol. 2010; 29: 468-475Crossref PubMed Scopus (8) Google Scholar, 11Mak V.C. Lee L. Siu M.K. Wong O.G. Lu X. Ngan H.Y. Wong E.S. Cheung A.N. Downregulation of ASPP2 in choriocarcinoma contributes to increased migratory potential through Src signaling pathway activation.Carcinogenesis. 2013; 34: 2170-2177Crossref PubMed Scopus (24) Google Scholar POZ and Krüppel family proteins display essential regulatory functions in many physiological and pathological processes.12Costoya J.A. Functional analysis of the role of POK transcriptional repressors.Brief Funct Genomic Proteomic. 2007; 6: 8-18Crossref PubMed Scopus (74) Google Scholar Factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A/LRF] is a proto-oncogenic POZ and Krüppel family transcription repressor on ARF and potentially leads to the inactivation of the p53 pathway.13Maeda T. Hobbs R.M. Pandolfi P.P. The transcription factor Pokemon: a new key player in cancer pathogenesis.Cancer Res. 2005; 65: 8575-8578Crossref PubMed Scopus (90) Google Scholar, 14Maeda T. Hobbs R.M. Merghoub T. Guernah I. Zelent A. Cordon-Cardo C. Teruya-Feldstein J. Pandolfi P.P. Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.Nature. 2005; 433: 278-285Crossref PubMed Scopus (287) Google Scholar, 15Choi W.I. Jeon B.N. Yun C.O. Kim P.H. Kim S.E. Choi K.Y. Kim S.H. Hur M.W. Proto-oncogene FBI-1 represses transcription of p21CIP1 by inhibition of transcription activation by p53 and Sp1.J Biol Chem. 2009; 284: 12633-12644Crossref PubMed Scopus (65) Google Scholar FBI-1 may also play a role in the pathogenesis of GTD. Aberrant expression of FBI-1 is associated with the cellular transformation of several solid tumors. FBI-1 is overexpressed in cancers of the colon and bladder, in which the normal functions of cyclin-dependent kinase inhibitor 2A and p53 are lost.13Maeda T. Hobbs R.M. Pandolfi P.P. The transcription factor Pokemon: a new key player in cancer pathogenesis.Cancer Res. 2005; 65: 8575-8578Crossref PubMed Scopus (90) Google Scholar, 14Maeda T. Hobbs R.M. Merghoub T. Guernah I. Zelent A. Cordon-Cardo C. Teruya-Feldstein J. Pandolfi P.P. Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.Nature. 2005; 433: 278-285Crossref PubMed Scopus (287) Google Scholar In contrast, overexpression of FBI-1 has been reported in both p53 mutant and p53 wild-type lung cancer cases.16Apostolopoulou K. Pateras I.S. Evangelou K. Tsantoulis P.K. Liontos M. Kittas C. Tiniakos D.G. Kotsinas A. Cordon-Cardo C. Gorgoulis V.G. Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer.J Pathol. 2007; 213: 294-302Crossref PubMed Scopus (57) Google Scholar In ovarian cancer, we previously revealed the oncogenic role of FBI-1 and its ability to induce proliferation and cell migration through direct interaction with the promoter of membrane type 1–matrix metalloproteinase 1 in association with adverse outcome,17Jiang L. Siu M.K. Wong O.G. Tam K.F. Lam E.W. Ngan H.Y. Le X.F. Wong E.S. Chan H.Y. Cheung A.N. Overexpression of proto-oncogene FBI-1 activates membrane type 1-matrix metalloproteinase in association with adverse outcome in ovarian cancers.Mol Cancer. 2010; 9: 318Crossref PubMed Scopus (34) Google Scholar suggesting the importance of FBI-1 in gynecological malignancies and its potential in promoting oncogenesis in diverse molecular pathways more than that formerly described. However, the tumor suppressor activity of FBI-1 has been recently reported in Pten-null prostate tumor models.18Wang G. Lunardi A. Zhang J. Chen Z. Ala U. Webster K.A. Tay Y. Gonzalez-Billalabeitia E. Egia A. Shaffer D.R. Carver B. Liu X.S. Taulli R. Kuo W.P. Nardella C. Signoretti S. Cordon-Cardo C. Gerald W.L. Pandolfi P.P. Zbtb7a suppresses prostate cancer through repression of a Sox9-dependent pathway for cellular senescence bypass and tumor invasion.Nat Genet. 2013; 45: 739-746Crossref PubMed Scopus (108) Google Scholar The oncogenic role of FBI-1 may therefore be context dependent. Interestingly, an earlier study showed that GTD tends to display high phosphatase and tensin homologue expression.10Zhang H.J. Siu M.K. Jiang L.L. Mak V.C. Ngan H.Y. Cheung A.N. Overexpression of the Parkinson disease protein DJ-1 and its regulator PTEN in gestational trophoblastic disease.Int J Gynecol Pathol. 2010; 29: 468-475Crossref PubMed Scopus (8) Google Scholar Hence, the clinical importance and molecular aspects of FBI-1 in GTD deserve an in-depth investigation. Here, the expression pattern and significance of FBI-1 were evaluated in various types of GTD. The in vitro effects of FBI-1 in relation to apoptosis and cell signaling pathways putatively involved were also investigated. The role of FBI-1 in tumor growth was further explored in vivo. For immunohistochemistry (IHC) analysis, 87 trophoblastic tissue samples were retrieved from the Department of Pathology, University of Hong Kong, Queen Mary Hospital (Hong Kong, China). These included 17 first-trimester and 9 term placentas, 18 partial and 32 complete moles, eight CCAs, and three placental site trophoblastic tumors. Among 50 hydatidiform moles, 12 developed gestational trophoblastic neoplasm (GTN) requiring chemotherapy. For real-time PCR, snap-frozen samples of 47 hydatidiform moles, 16 first-trimester placentas, and 8 term placentas were also retrieved for cDNA preparation. First-trimester and term placenta were collected after induced abortion by suction evacuation and normal delivery, respectively. The tissues of hydatidiform moles and CCAs were obtained from specimens of uterine evacuate and/or hysterectomy. The use of such trophoblastic tissues has been approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster. All tissue sections were histologically reviewed using generally accepted diagnostic criteria.3Shih I. Mazur M. Kurman R. Gestational trophoblastic disease and related lesions.in: Kurman R.J. Blaustein's Pathology of the Female Genital Tract. Springer, New York2002: 1193-1250Google Scholar, 19Cheung A. Gestational trophoblastic disease. Robboy's Pathology of the Female Reproductive Tract.in: Robboy S. Mutter G. Prat J. Bentley R. Russell P. Anderson M. Elsevier Churchill Livingstone, China2009: 881-907Google Scholar, 20Paradinas F. Elston C. Gestational trophoblastic disease. Haines and Taylor: Obstetrical and Gynaecological Pathology.in: Fox H. Wells M. Churchill Livingstone, Edinburgh2003: 1359-1430Google Scholar Twenty hydatidiform moles in this series also were previously assessed by fluorescent microsatellite genotyping after microdissection and chromosome in situ hybridization as an adjunct to histological diagnosis.21Cheung A.N. Khoo U.S. Lai C.Y. Chan K.Y. Xue W.C. Cheng D.K. Chiu P.M. Tsao S.W. Ngan H.Y. Metastatic trophoblastic disease after an initial diagnosis of partial hydatidiform mole: genotyping and chromosome in situ hybridization analysis.Cancer. 2004; 100: 1411-1417Crossref PubMed Scopus (43) Google Scholar, 22Lai C.Y. Chan K.Y. Khoo U.S. Ngan H.Y. Xue W.C. Chiu P.M. Tsao S.W. Cheung A.N. Analysis of gestational trophoblastic disease by genotyping and chromosome in situ hybridization.Mod Pathol. 2004; 17: 40-48Crossref PubMed Scopus (53) Google Scholar GTN was diagnosed if there was a plateau in human chorionic gonadotropin level for 4 weeks or a further rise in human chorionic gonadotropin for three consecutive weeks after evacuation. For in vitro studies, two CCA cell lines (JAR and JEG-3; ATCC, Manassas, VA) and a normal extravillous trophoblast cell line (TEV-1)23Feng H.C. Choy M.Y. Deng W. Wong H.L. Lau W.M. Cheung A.N. Ngan H.Y. Tsao S.W. Establishment and characterization of a human first-trimester extravillous trophoblast cell line (TEV-1).J Soc Gynecol Investig. 2005; 12: e21-e32Crossref PubMed Scopus (57) Google Scholar were cultured in minimum essential Eagle's medium supplemented with 10% fetal bovine serum, and 100 U/mL penicillin and streptomycin. IHC analysis was performed on deparaffinized sections 5 μm thick using the EnVision+ Dual Link System (K4061; Dako, Carpinteria, CA)24Chan H.Y. Siu M.K. Zhang H.J. Wong E.S. Ngan H.Y. Chan K.Y. Cheung A.N. Activated Stat3 expression in gestational trophoblastic disease: correlation with clinicopathological parameters and apoptotic indices.Histopathology. 2008; 53: 139-146Crossref PubMed Scopus (25) Google Scholar after antigen retrieval by microwave heating in 0.01 mol/L citrate buffer (pH 6.0) for 10 minutes. Sections were incubated in peroxidase blocking solution (1% hydrogen peroxide solution) for 5 minutes and washed in phosphate-buffered saline (pH 7.4). A polyclonal rabbit anti-human FBI-1 (Pokémon) antibody (catalog no. Ab36606; Abcam, Cambridge, UK) was applied in 1:600 dilution and incubated overnight at room temperature. Freshly prepared 3,3′-diaminobenzidine tetrahydrochloride was applied as chromogen, and sections were counterstained with hematoxylin. Negative controls were prepared by replacing the primary antibody with phosphate-buffered saline. A known positive control from a normal first-trimester placenta was used.24Chan H.Y. Siu M.K. Zhang H.J. Wong E.S. Ngan H.Y. Chan K.Y. Cheung A.N. Activated Stat3 expression in gestational trophoblastic disease: correlation with clinicopathological parameters and apoptotic indices.Histopathology. 2008; 53: 139-146Crossref PubMed Scopus (25) Google Scholar, 25Siu M.K. Wong E.S. Chan H.Y. Ngan H.Y. Chan K.Y. Cheung A.N. Overexpression of NANOG in gestational trophoblastic diseases: effect on apoptosis, cell invasion, and clinical outcome.Am J Pathol. 2008; 173: 1165-1172Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Trophoblast subpopulations in each case were evaluated separately. The percentage of positively stained cells was scored according to the following criteria: 0, no positive staining; 1, 0.1% to 25.0% of cells were positive; 2, 25.1% to 50.0% of cells were positive; 3, 50.1% to 75.0% of cells were positive; and 4, 75.1% to 100% of cells were positive. Signal-intensity score was assigned according to the following criteria: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, strong staining. IHC score, ranging from 0 to 12, was calculated by multiplying the signal score (0 to 3) and the percentage positive score (0 to 4). Photomicrographs were taken with the assistance of Spectrum software version 11.1.1.765 (Aperio, Vista, CA). Real-time quantitative PCR (qPCR) was performed according to previously published procedures.4Siu M.K. Yeung M.C. Zhang H. Kong D.S. Ho J.W. Ngan H.Y. Chan D.C. Cheung A.N. p21-Activated kinase-1 promotes aggressive phenotype, cell proliferation, and invasion in gestational trophoblastic disease.Am J Pathol. 2010; 176: 3015-3022Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Total RNA (2.5 μg) extracted by TRIzol reagent (Invitrogen/Life Technologies Inc., Rockville, MD) was subjected to first-strand cDNA synthesis using the SuperScript Reverse Transcriptase system with oligo-dT primers (Invitrogen, Carlsbad, CA). qPCR was performed on the ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Unless specified otherwise, the cDNA of GAPDH amplified as housekeeping control for each sample. Primers used are listed in Table 1. The 2−ΔΔCT method was used for calculating the relative changes in gene expression determined from qPCR experiments. For in vitro studies, experiments were performed in duplicate and repeated at least twice.Table 1Primers Used for Real-Time Quantitative PCRPrimer nameSequencesFBI-1F: 5′-TCTGCGAGAAGGTCATCC-3′R: 5′-CGTAGTTGTGGGCAAAGG-3′GAPDHF: 5′-TCCATGACAACTTTGGTATCGCG-3′R: 5′-ACAGTCTTCTGGGTGGCAGTG-3′18SF: 5′-GTAACCCGTTGAACCCCATT-3′R: 5′-CCATCCAATCGGTAGTAGCG-3′FasF: 5′-CTCCAAGGGATTGGAATTGA-3′R: 5′-GACAAAGCCACCCCAAGTTA-3′FasLF: 5′-TCAATGAAACTGGGCTGTACTTT-3′R: 5′-AGAGTTCCTCATGTAGACCTTGT-3′TRAF2F: 5′-CTGACTTGGAGCAGAAGG-3′R: 5′-GCCGTTCAGGTAGATACG-3′TRADDF: 5′-TTCTGCGGCTATTGCTGA-3′R: 5′-TGAAACTGTAAGGGCTGG-3′Caspase-8F: 5′-CCGCAAAGGAAGCAAGAAC-3′R: 5′-AATTCTGATCTGCTCACTTCTTCTG-3′Caspase-10F: 5′-CAGAAGGCATTGACTCAGAGAACT-3′R: 5′-GATACGACTCGGCTTCCTTGT-3′Bcl-xF: 5′-GCAGGTATTGGTGAGTCGGATCGC-3′R: 5′-CACAAAAGTATCCCAGCCGCCG-3′Bcl-2F: 5′-GGTGGTGGAGGAACTCTTCA-3′R: 5′-ACCTACCCAGCCTCCGTTAT-3′SurvivinF: 5′-ATTCGTCCGGTTGCGCTTTCC-3′R: 5′-CACGGCGCACTTTCTTCGCAG-3′BAKF: 5′-GAACAGGAGGCTGAAGGGGT-3′R: 5′-TCAGGCCATGCTGGTAGACG-3′BAXF: 5′-TGCAGAGGATGATTGCTGAC-3′R: 5′-GGAGGAAGTCCAGTGTCCAG-3′Caspase-9F: 5′-TTCCCAGGTTTTGTCTCCTG-3′R: 5′-GGGACTGCAGGTCTTCAGAG-3′PARPF: 5′-AGGCTGCTTTGTCAAGAA-3′R: 5′-CTTGCTGCTTGTTGAAGAT-3′Caspase-7F: 5′-GGAGAAAGCTCATGGCTGTGT-3′R: 5′-TCCCCTTGGCTGTGTTTTG-3′Caspase-3F: 5′-CTGGACTGTGGCATTGAGACA-3′R: 5′-AGTCGGCCTCCACTGGTATTT-3′p110αF: 5′-GTATGTCTATCCTCCAAATGTGAG-3′R: 5′-CACAGTCATGGTTGATTTTCAGAG-3′p85αF: 5′-AGGTCGCCTAGCATACCTCA-3′R: 5′-AGAGCTGGCTGCTGAGAATC-3′p85βF: 5′-CGAGACCAGTACCTCGTGTG-3′R: 5′-TAATCCCCAGCCACTCGTT-3′PTENF: 5′-TGACAGCCATCATCAAAGAGA-3′R: 5′-TGCTTTGAATCCAAAAACCTT-3′BAK, Bcl-2 homologous antagonist killer; BAX, Bcl-2-associated X protein; Bcl, B-cell lymphoma; F, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly–ADP ribose polymerase; PTEN, phosphatase and tensin homologue; R, reverse; TRADD, tumor necrosis factor receptor type 1–associated death domain protein; TRAF, tumor necrosis factor receptor–associated factor. Open table in a new tab BAK, Bcl-2 homologous antagonist killer; BAX, Bcl-2-associated X protein; Bcl, B-cell lymphoma; F, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly–ADP ribose polymerase; PTEN, phosphatase and tensin homologue; R, reverse; TRADD, tumor necrosis factor receptor type 1–associated death domain protein; TRAF, tumor necrosis factor receptor–associated factor. JEG-3 and JAR cultures were transfected with FBI-1 construct17Jiang L. Siu M.K. Wong O.G. Tam K.F. Lam E.W. Ngan H.Y. Le X.F. Wong E.S. Chan H.Y. Cheung A.N. Overexpression of proto-oncogene FBI-1 activates membrane type 1-matrix metalloproteinase in association with adverse outcome in ovarian cancers.Mol Cancer. 2010; 9: 318Crossref PubMed Scopus (34) Google Scholar using Lipofectamine 2000 (Invitrogen). The pEGFP-C3 vector (Clontech Laboratories, Inc, Mountain View, CA) was used as a control. To knock down FBI-1 in the CCA cell line, Silencer select–predesigned siRNA of FBI-1 (siZBTB7A) (catalog no. 4427037; ID s27988) and nontargeting negative control 2 (scrambled) siRNA (catalog no. 43900846) were used (Ambion, Austin, TX).4Siu M.K. Yeung M.C. Zhang H. Kong D.S. Ho J.W. Ngan H.Y. Chan D.C. Cheung A.N. p21-Activated kinase-1 promotes aggressive phenotype, cell proliferation, and invasion in gestational trophoblastic disease.Am J Pathol. 2010; 176: 3015-3022Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 26Siu M.K. Wong E.S. Kong D.S. Chan H.Y. Jiang L. Wong O.G. Lam E.W. Chan K.K. Ngan H.Y. Le X.F. Cheung A.N. Stem cell transcription factor NANOG controls cell migration and invasion via dysregulation of E-cadherin and FoxJ1 and contributes to adverse clinical outcome in ovarian cancers.Oncogene. 2013; 32: 3500-3509Crossref PubMed Scopus (120) Google Scholar Transfection with 5 nmol/L FBI-1–specific/nontargeting siRNA in siLentFect Lipid Reagent (Bio-Rad Laboratories, Hercules, CA) was performed. Total protein lysate was extracted with sodium dodecyl sulfate lysis buffer containing proteinase inhibitors.4Siu M.K. Yeung M.C. Zhang H. Kong D.S. Ho J.W. Ngan H.Y. Chan D.C. Cheung A.N. p21-Activated kinase-1 promotes aggressive phenotype, cell proliferation, and invasion in gestational trophoblastic disease.Am J Pathol. 2010; 176: 3015-3022Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Protein concentration was determined by detergent-compatible protein assay (Bio-Rad). Twenty micrograms of protein was resolved by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with corresponding antibodies. The primary antibodies used for Western blot analysis are listed in Table 2.Table 2Primary Antibodies Used for Immunoblot AnalysisTarget proteinAnimal sourceCatalog #Working solutionVendorFBI-1RabbitAb366061:1000Abcam Inc. (Cambridge, UK)ActinRabbitA50601:1000Sigma (St. Louis, MO)GFPMousesc-99961:1500Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)Akt TotalMouse92721:1000Cell Signaling (Beverly, MA)Phospho-Akt (Ser473)Mouse92711:1000Cell Signalingp85βMouseab283561:1000Abcam Inc.GFP, green fluorescent protein. Open table in a new tab GFP, green fluorescent protein. To determine the impact of FBI-1 on cell mobility, CCA cells transiently transfected with FBI-1 and corresponding control were counted and equally plated on the upper compartments of 24-well, 8-μm pore size Transwell chambers (BD Biosciences, San Jose, CA).4Siu M.K. Yeung M.C. Zhang H. Kong D.S. Ho J.W. Ngan H.Y. Chan D.C. Cheung A.N. p21-Activated kinase-1 promotes aggressive phenotype, cell proliferation, and invasion in gestational trophoblastic disease.Am J Pathol. 2010; 176: 3015-3022Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Cell migration and invasion assays were performed with self-coated gelatin and Matrigel, respectively. After 24 hours, the migrated and invaded cells on the lower surface of the membrane were fixed and stained with methanol-mixed crystal violet. For phosphoinositide 3-kinase (PI3K)/Akt pathway study, assays were performed in the presence of 20 μmol/L LY294002.27Liu P. Xu B. Li J. Lu H. LY294002 inhibits leukemia cell invasion and migration through early growth response gene 1 induction independent of phosphatidylinositol 3-kinase-Akt pathway.Biochem Biophys Res Commun. 2008; 377: 187-190Crossref PubMed Scopus (27) Google Scholar FBI-1– or pEGFP-C3–transfected cells were treated with 20 μmol/L LY294002 for 3 hours before cell plating for both migration and invasion assays. Assays were then performed in the presence of the same concentration of inhibitor in the Transwell chamber. Dimethyl sulfoxide was used as control. Each experiment was repeated at least twice. Assays were performed in duplicate wells, and the number of migrated or invaded cells in each well was counted in three randomly selected microscopic fields. For in vivo studies of tumorigenicity in CCA cells, human FBI-1–specific shRNA (SureSilencing shRNA constructs) and control shRNA vector (pGeneClip puromycin vector) were purchased from SuperArray (SABiosciences Corporation, Frederick, MD) and transfected into JAR cells under puromycin selection, as published previously.17Jiang L. Siu M.K. Wong O.G. Tam K.F. Lam E.W. Ngan H.Y. Le X.F. Wong E.S. Chan H.Y. Cheung A.N. Overexpression of proto-oncogene FBI-1 activates membrane type 1-matrix metalloproteinase in association with adverse outcome in ovarian cancers.Mol Cancer. 2010; 9: 318Crossref PubMed Scopus (34) Google Scholar The 1 × 106 sh-FBI-1– or vector-transfected CCA cells were inoculated s.c. (six mice per group) into BALB/c female nude mice. All s.c. tumor diameters were measured perpendicularly on days 7, 9, 11, and 16, and volumes were calculated. Mice were sacrificed 18 days after cell injection. All visible tumors were dissected and the total tumor weight was determined. The animal studies were preformed according to the Animals (Control of Experiments) Ordinance (Hong Kong, China) and the institute's guidance on animal experiments. Statistical analysis on nonparametric unpaired t-tests for continuous data was performed by SPSS version 15.1 (IBM SPSS Statistics, IBM Corporation, Armonk, NY). P < 0.05 was considered statistically significant. IHC studies showed that immunoreactivity of FBI-1 could be detected in the nuclei of cytotrophoblasts, syncytiotrophoblasts, and villous intermediate trophoblasts (Figure 1, A–F). Placental site trophoblastic tumor, another malignant type of GTD derived from extravillous implantation site–intermediate trophoblasts, also demonstrated strong nuclear FBI-1 immunoreactivity (Figure 1F). At the cytotrophoblasts, first-trimester placenta and complete mole samples showed significantly greater FBI-1 immunostaining compared with that of term placenta (P = 0.016 and P = 0.003, respectively). At the syncytiotrophoblasts, first-trimester placenta, partial mole, complete mole, and CCA samples showed significantly greater FBI-1 immunostaining compared with that of term placenta (P < 0.001, P = 0.003, P < 0.001, and P = 0.006, respectively). Moreover, FBI-1 immunoscores were significantly greater in complete mole compared with partial mole in cytotrophoblasts (P = 0.002), syncytiotrophoblasts (P = 0.005), and intermediate trophoblasts (P = 0.004). In hydatidiform mole samples, although FBI immunoscores were numerically greater in cytotrophoblasts and syncytiotrophoblasts in 12 cases that subsequently progressed to GTN compared with those in 20 cases that spontaneously regressed, statistical significance was not reached. The expression profile of FBI-1 at mRNA was also examined by qPCR analysis. In normal placenta, first-trimester and term samples had similar FBI-1 mRNA expression. FBI-1 mRNA expression was significantly increased in hydatidiform mole tissue (Figure 1G) compared with that in first-trimester placenta (P = 0.015). Overexpression of FBI-1 mRNA was also observed in the CCA cell lines JAR and JEG-3 but not in an immortalized, untransformed TEV-1 sample (Figure 1H). JEG-3 and JAR cell lines were transiently transfected with FBI-1 and assessed for cell mobility and invasiveness in Transwell assay. FBI-1 overexpression was significantly associated with the promotion of cell migration and invasion in both CCA cell lines (all P < 0.05). On cell migration assay, increases in cell motility of about 1.5- and 1.8-fold were observed in JAR and JEG-3, respectively (Figure 2). On the other hand, cell invasiveness increased by threefold subsequent to overexpression of FBI-1 in both cell lines (Figure 2). Akt activation stimulates cell survival, metabolism, and migration through its ability to phosphorylate its downstream targets.28Cantley L.C. The phosphoinositide 3-kinase pathway.Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4653) Google Scholar, 29Yuan T.L. Cantley L.C. PI3K pathway alterations in cancer: variations on a theme.Oncogene. 2008; 27: 5497-5510Crossref PubMed Scopus (1478) Google Scholar Here we investigated the potential relationship between FBI-1 and Akt activation in JAR and JEG-3. The level of a phosphorylated form of Akt at Ser 473 (pAkt-S473) was used as the readout of Akt activation on Western blot. Strikingly, Akt was activate
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