在流行病學研究中，大豆類食物可以預防多種的人類癌症，膀胱癌亦是其中之一。研究發現三羥異黃酮可以調節基因功能並影響細胞的生理現象。然而在三羥異黃酮調節膀胱癌抑癌基因EMP2的研究領域中，其分子機制還尚未明瞭。因此本研究首先發現在泌尿上皮細胞及組織中，正常細胞及組織的EMP2 mRNA與蛋白質表現量都比癌化細胞及組織來的高。第二，利用RNAi干擾技術 (RNA interference) 將EMP2基因表現量弱化後，明顯增強了癌化泌尿上皮細胞的細胞增生，以及在plate colony formaion中增加了癌化傾向的細胞團塊形成、另外癌化細胞移動性及侵入性能力都會提高；相反的，超量表現EMP2則會抑制這些癌化細胞的惡性表現。第三，實驗結果顯示三羥異黃酮經由聯結增強EMP2表現而抑制了細胞的增生。經由建構不同長度片段的EMP2啟動子區域表現量實驗，得知三羥異黃酮可以影響EMP2核心啟動子區域進而提高EMP2轉錄活性。最後，使用定點突變技術及染色質免疫沉澱法證明了CREB1轉錄因子可以經由直接結合EMP2啟動子區域進而正向調節EMP2基因轉錄。本研究結果顯示EMP2表現抑制了泌尿上皮癌細胞的生長、移動及侵入能力；另外首次發現在泌尿上皮癌細胞中，三羥異黃酮可以經由誘導CREB1 轉錄因子結合EMP2啟動子區域而促進EMP2轉錄活性並增強基因表現。

Epidemiologic data suggest that soy consumption may protect against cancer induction in several tissues in humans, including urothelial carcinoma. Genistein have been reported to regulate genes that are involved in several cellular events. However, the molecular mechanism of genistein -induced upregulation of epithelial membrane protein 2 (EMP2), candidate urothelial tumor suppressor, is not entirely understood. At first, we found that the mRNA and protein expression levels of EMP2 were significantly greater in the normal urothelial tissues and human urothelial cells than those in urothelial bladder carcinoma tissues and urothelial cell carcinoma-derived cell lines. Second, EMP2 knockdown via RNA interference markedly enhanced cell proliferation, colony formation, migration and invasiveness. By contrast, EMP2 overexpression suppressed these malignant behaviors. Third, we showed that genistein-induced inhibition in cell proliferation is associated with an increase in EMP2 expression. Using various deleted EMP2 promoter constructs, we defined that the EMP2 core promoter is enough to observe the genistein-induced upregulation of EMP2 transcriptional activity. Using site direct mutagenesis and chromatin immunoprecipitation assays demonstrated that cyclic-AMP response element binding protein 1 (CREB1) acts as a positive regulator of EMP2 transcription by directly binding to its promoter. These results showed EMP2 suppressed urothelial cell carcinoma-derived cell growth, motility and invasion and for the first time that genistein promoted EMP2 expression in urothelial cell carcinoma-derived cells by inducing EMP2 transcriptional activity via CREB1 binding.

Contents

Chinese abstract I
English abstract II
Tables of contents III
Chapter one: Literature review of epithelial membrane protein-2 (EMP2) gene 1
Tumor suppressor 1
Disease relevance 3
Medical therapy 4
Functional configuration 6
Activation of FAK/Src pathway 7
Trafficking of specific membrane-bound proteins 9

Chapter two: Tumor suppressor function of EMP2 in human urothelial bladder carcinoma 11
Introduction 11
Materials and methods 16
Cell lines 16
Antibodies 16
shRNA vectors and transfection 17
Expression vectors and transfection 18
RNA extraction, cDNA preparation and quantitative RT-PCR 19
Western immunoblotting 21
3-(4, 5-dimethylthiahiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation assay 21
5-Bromo-2-deoxyuridine (BrdU) incorporation assay 22
Wound-healing assay 23
Cell invasion assay 23
Colony formation assay 24
Cell cycle analysis and annexin V staining 24
Genistein treatment 25
Construction of EMP2 promoter reporter plasmids 25
Cells transfection, genistein or 5-aza-CdR treatments, and dual luciferase reporter assay 26
Chromatin immunoprecipitation (ChIP) assay 27
Site direct mutagenesis 28
Bisulfite modification and bisulfite sequencing 29
Statistics 30
Supplementary materials and methods 30




Contents

Results 31
Loss of EMP2 with malignant phenotype of human urothelial bladder carcinoma 31
Knockdown of EMP2 enhanced urothelial bladder carcinoma cell growth 31
Knockdown of EMP2 reduced the migratory and invasive potentials of urothelial bladder carcinoma cells 33
EMP2 overexpression enhanced urothelial bladder carcinoma cell growth, motility, invasion 33
EMP2 expression affected cell cycle distribution of urothelial bladder carcinoma cells 34
EMP2 did not enhanced apoptosis in urothelial bladder carcinoma cells 35
Genistein increased mRNA and protein levels of EMP2 in J82 cells 36
Identification of the putative EMP2 promoter region 36
Either genistein or 5-aza-CdR induced the promoter activities of the EMP2 proximal regions 37
CREB1 as a positive regulator of EMP2 expression in urothelial bladder carcinoma cells 38
Exogenous expression of CREB1 proteins induced, while knockdown CREB1 gene repressed, EMP2 mRNA and protein levels in urothelial bladder carcinoma cells 40
Promoter methylation contributed to EMP2 downregulation in urothelial bladder carcinoma cells 41
Demethylation of CRE element of the EMP2 proximal prompter by genistein in urothelial bladder carcinoma cells 41
Discussion 43
References 51
Figures and figure legends 56
Figure 1 Genistein induced EMP2 mRNA and EMP2 protein levels in human J82 UCC-derived cells. 56
Figure 2 Knockdown of EMP2 gene in RT4 cells enhanced cell viability, proliferation and migration, and invasion capacity. 57
Figure 3 Exogenous expression of the EMP2 gene in J82 cells inhibited cell viability, proliferation, migration and invasion capacity. 59
Figure 4 Exogenous expression or knockdown of EMP2 gene in UCC-derived cells altered their cell cycle distributions. 61
Figure 5 Genistein induced EMP2 mRNA and EMP2 protein levels in the J82 bladder carcinoma cells in a time-dependent manner 63
Figure 6 Either genistein or 5-aza-CdR treatment induced the promoter activity of the EMP2 gene 64
Figure 7 Mutagenesis on CREB1-binding sites in the EMP2 promoter region decreases its promoter activities in J82 and TSGH8301 cells. 66
Figure 8 Exogenous expression and knockdown of CREB1 gene, respectively, induce and repress EMP2 mRNA and protein levels in TSGH8301 and J82 cells. 68
Figure 9 Promoter methylation contributes to EMP2 downregulation in UCC-derived cells. 71



Contents

Supplementary 73
Materials and methods 73
Figure S1 Genistein induced EMP2 transcription in high-grade urothelial carcinoma-derived cells. 74
Figure S2 Exogenous expression of the EMP2 gene was not able to induce J82 cell apoptosis 75
Figure S3 Effect of on the growth of J82 cells in NOD/ SCID mice 76
Appendix 77
Table 1 Details of cell lines used 77
Table 2 Primer sequences and PCR conditions 78
Appendix Figure 1 Subcloning strategy for EMP2 79
Appendix Figure 2 Nucleotide sequence of the human proximal EMP2 promoter region (-1420 to +268 bp) 80
Appendix Figure 3 Appendix Figure 3 MethPrimer software was used to design primers around transcription start sites 81
Publications 84

Ben-Porath I, Kozak CA, Benvenisty N (1998). Chromosomal mapping of Tmp (Emp1), Xmp (Emp2), and Ymp (Emp3), genes encoding membrane proteins related to Pmp22. Genomics 49: 443-7.

Brancolini C, Sgorbissa A, Schneider C (1998). Proteolytic processing of the adherens junctions components beta-catenin and gamma-catenin/plakoglobin during apoptosis. Cell Death Differ 5: 1042-50.

Burvall KM, Palmberg L, Larsson K (2002). The tyrosine kinase inhibitor genistein increases basal cAMP and potentiates forskolin-induced cAMP accumulation in A549 human airway epithelial cells. Mol Cell Biochem 240: 131-3.

Castillo-Martin M, Domingo-Domenech J, Karni-Schmidt O, Matos T, Cordon-Cardo C (2010). Molecular pathways of urothelial development and bladder tumorigenesis. Urol Oncol 28: 401-8.

Chen YH, Wu LC, Wu WR, Lin HJ, Lee SW, Lin CY et al (2012). Loss of epithelial membrane protein-2 expression confers an independent prognosticator in nasopharyngeal carcinoma: a cohort study. BMJ Open 2: e000900.

Comb M, Goodman HM (1990). CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. Nucleic Acids Res 18: 3975-82.

Dixon RA, Ferreira D (2002). Genistein. Phytochemistry 60: 205-11.

Dyrskjot L, Thykjaer T, Kruhoffer M, Jensen JL, Marcussen N, Hamilton-Dutoit S et al (2003). Identifying distinct classes of bladder carcinoma using microarrays. Nat Genet 33: 90-6.

Fabbretti E, Edomi P, Brancolini C, Schneider C (1995). Apoptotic phenotype induced by overexpression of wild-type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A. Genes Dev 9: 1846-56.

Fang MZ, Chen D, Sun Y, Jin Z, Christman JK, Yang CS (2005). Reversal of hypermethylation and reactivation of p16INK4a, RARbeta, and MGMT genes by genistein and other isoflavones from soy. Clin Cancer Res 11: 7033-41.

Forbes A, Wadehra M, Mareninov S, Morales S, Shimazaki K, Gordon LK et al (2007). The tetraspan protein EMP2 regulates expression of caveolin-1. J Biol Chem 282: 26542-51.

Fournier DB, Erdman JW, Jr., Gordon GB (1998). Soy, its components, and cancer prevention: a review of the in vitro, animal, and human data. Cancer Epidemiol Biomarkers Prev 7: 1055-65.

Fu M, Maresh EL, Soslow RA, Alavi M, Mah V, Zhou Q et al (2010). Epithelial membrane protein-2 is a novel therapeutic target in ovarian cancer. Clin Cancer Res 16: 3954-63.

Habeeb O, Goodglick L, Soslow RA, Rao RG, Gordon LK, Schirripa O et al (2010). Epithelial membrane protein-2 expression is an early predictor of endometrial cancer development. Cancer 116: 4718-26.

Herman JG (1999). Hypermethylation of tumor suppressor genes in cancer. Semin Cancer Biol 9: 359-67.

Iguchi-Ariga SM, Schaffner W (1989). CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev 3: 612-9.

Jetten AM, Suter U (2000). The peripheral myelin protein 22 and epithelial membrane protein family. Prog Nucleic Acid Res Mol Biol 64: 97-129.

Li Y, Che M, Bhagat S, Ellis KL, Kucuk O, Doerge DR et al (2004). Regulation of gene expression and inhibition of experimental prostate cancer bone metastasis by dietary genistein. Neoplasia 6: 354-63.

Liu D, Jiang H, Grange RW (2005). Genistein activates the 3',5'-cyclic adenosine monophosphate signaling pathway in vascular endothelial cells and protects endothelial barrier function. Endocrinology 146: 1312-20.

Liu D, Zhen W, Yang Z, Carter JD, Si H, Reynolds KA (2006). Genistein acutely stimulates insulin secretion in pancreatic beta-cells through a cAMP-dependent protein kinase pathway. Diabetes 55: 1043-50.

Mancini DN, Rodenhiser DI, Ainsworth PJ, O'Malley FP, Singh SM, Xing W et al (1998). CpG methylation within the 5' regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site. Oncogene 16: 1161-9.

Messing E, Gee JR, Saltzstein DR, Kim K, diSant'Agnese A, Kolesar J et al (2012). A phase 2 cancer chemoprevention biomarker trial of isoflavone G-2535 (genistein) in presurgical bladder cancer patients. Cancer Prev Res (Phila) 5: 621-30.

Morales SA, Mareninov S, Coulam P, Wadehra M, Goodglick L, Braun J et al (2009a). Functional consequences of interactions between FAK and epithelial membrane protein 2 (EMP2). Invest Ophthalmol Vis Sci 50: 4949-56.

Morales SA, Mareninov S, Wadehra M, Zhang L, Goodglick L, Braun J et al (2009b). FAK activation and the role of epithelial membrane protein 2 (EMP2) in collagen gel contraction. Invest Ophthalmol Vis Sci 50: 462-9.

Obermayr E, Sanchez-Cabo F, Tea MK, Singer CF, Krainer M, Fischer MB et al (2010). Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients. BMC Cancer 10: 666.

Palvimo JJ, Eisenberg LM, Janne OA (1991). Protein-DNA interactions in the cAMP responsive promoter region of the murine ornithine decarboxylase gene. Nucleic Acids Res 19: 3921-7.

Rao A, Coan A, Welsh JE, Barclay WW, Koumenis C, Cramer SD (2004). Vitamin D receptor and p21/WAF1 are targets of genistein and 1,25-dihydroxyvitamin D3 in human prostate cancer cells. Cancer Res 64: 2143-7.

Safe S, Abdelrahim M (2005). Sp transcription factor family and its role in cancer. Eur J Cancer 41: 2438-48.

Sandelin A, Wasserman WW, Lenhard B (2004). ConSite: web-based prediction of regulatory elements using cross-species comparison. Nucleic Acids Res 32: W249-52.

Sarvari M, Szego EM, Barabas K, Javor A, Toth S, Kovacs Z et al (2009). Genistein induces phosphorylation of cAMP response element-binding protein in neonatal hypothalamus in vivo. J Neuroendocrinol 21: 1024-8.

Satake N, Imanishi M, Keto Y, Yamada H, Ishikawa M, Shibata S (2000). Genistein potentiates the relaxation induced by beta1- and beta2-adrenoceptor activation in rat aortic rings. J Cardiovasc Pharmacol 35: 227-33.

Shell SA, Fix C, Olejniczak D, Gram-Humphrey N, Walker WH (2002). Regulation of cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) expression by Sp1 in the mammalian testis. Biol Reprod 66: 659-66.

Shimazaki K, Chan AM, Moniz RJ, Wadehra M, Nagy A, Coulam CP et al (2009). Blockade of epithelial membrane protein 2 (EMP2) abrogates infection of Chlamydia muridarum murine genital infection model. FEMS Immunol Med Microbiol 55: 240-9.

Shimazaki K, Lepin EJ, Wei B, Nagy AK, Coulam CP, Mareninov S et al (2008). Diabodies targeting epithelial membrane protein 2 reduce tumorigenicity of human endometrial cancer cell lines. Clin Cancer Res 14: 7367-77.

Shimazaki K, Wadehra M, Forbes A, Chan AM, Goodglick L, Kelly KA et al (2007). Epithelial membrane protein 2 modulates infectivity of Chlamydia muridarum (MoPn). Microbes Infect 9: 1003-10.

Siegel R, Naishadham D, Jemal A (2012). Cancer statistics, 2012. CA Cancer J Clin 62: 10-29.

Su S, Lai M, Yeh T, Chow N (2001). Overexpression of HER-2/neu enhances the sensitivity of human bladder cancer cells to urinary isoflavones. Eur J Cancer 37: 1413-8.

Su SJ, Yeh TM, Lei HY, Chow NH (2000). The potential of soybean foods as a chemoprevention approach for human urinary tract cancer. Clin Cancer Res 6: 230-6.

Taylor V, Suter U (1996). Epithelial membrane protein-2 and epithelial membrane protein-3: two novel members of the peripheral myelin protein 22 gene family. Gene 175: 115-20.

Theodorescu D, Laderoute KR, Calaoagan JM, Guilding KM (1998). Inhibition of human bladder cancer cell motility by genistein is dependent on epidermal growth factor receptor but not p21ras gene expression. Int J Cancer 78: 775-82.

Vlieghe D, Sandelin A, De Bleser PJ, Vleminckx K, Wasserman WW, van Roy F et al (2006). A new generation of JASPAR, the open-access repository for transcription factor binding site profiles. Nucleic Acids Res 34: D95-7.

Wadehra M, Dayal M, Mainigi M, Ord T, Iyer R, Braun J et al (2006a). Knockdown of the tetraspan protein epithelial membrane protein-2 inhibits implantation in the mouse. Dev Biol 292: 430-41.

Wadehra M, Forbes A, Pushkarna N, Goodglick L, Gordon LK, Williams CJ et al (2005). Epithelial membrane protein-2 regulates surface expression of alphavbeta3 integrin in the endometrium. Dev Biol 287: 336-45.

Wadehra M, Goodglick L, Braun J (2004). The tetraspan protein EMP2 modulates the surface expression of caveolins and glycosylphosphatidyl inositol-linked proteins. Mol Biol Cell 15: 2073-83.

Wadehra M, Iyer R, Goodglick L, Braun J (2002). The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion. J Biol Chem 277: 41094-100.

Wadehra M, Mainigi M, Morales SA, Rao RG, Gordon LK, Williams CJ et al (2008). Steroid hormone regulation of EMP2 expression and localization in the endometrium. Reprod Biol Endocrinol 6: 15.

Wadehra M, Natarajan S, Seligson DB, Williams CJ, Hummer AJ, Hedvat C et al (2006b). Expression of epithelial membrane protein-2 is associated with endometrial adenocarcinoma of unfavorable outcome. Cancer 107: 90-8.

Wadehra M, Su H, Gordon LK, Goodglick L, Braun J (2003a). The tetraspan protein EMP2 increases surface expression of class I major histocompatibility complex proteins and susceptibility to CTL-mediated cell death. Clin Immunol 107: 129-36.

Wadehra M, Sulur GG, Braun J, Gordon LK, Goodglick L (2003b). Epithelial membrane protein-2 is expressed in discrete anatomical regions of the eye. Exp Mol Pathol 74: 106-12.

Wang CX, Wadehra M, Fisk BC, Goodglick L, Braun J (2001). Epithelial membrane protein 2, a 4-transmembrane protein that suppresses B-cell lymphoma tumorigenicity. Blood 97: 3890-5.

Wang J, Eltoum IE, Lamartiniere CA (2004). Genistein alters growth factor signaling in transgenic prostate model (TRAMP). Mol Cell Endocrinol 219: 171-80.

Wilson HL, Wilson SA, Surprenant A, North RA (2002). Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus. J Biol Chem 277: 34017-23.

Xu G, Flynn J, Glickman JF, Reich NO (1995). Purification and stabilization of mouse DNA methyltransferase. Biochem Biophys Res Commun 207: 544-51.

Yan C, Han R (1997). Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein. Invasion Metastasis 17: 189-98.

Zhou JR, Mukherjee P, Gugger ET, Tanaka T, Blackburn GL, Clinton SK (1998). Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis. Cancer Res 58: 5231-8.