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Supplementary Figure 1 Schematic illustration of the project. Endometrioid carcinomas. Immunomagnetical purification. Endometrial epithelial cells. +E2 +TAM. Microarray. 21 Up-regulated genes 14 Down-regulated genes. Overexpression. RNAi. Gain of function.
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Supplementary Figure 1 Schematic illustration of the project Endometrioid carcinomas Immunomagnetical purification Endometrial epithelial cells +E2 +TAM Microarray 21 Up-regulated genes 14 Down-regulated genes Overexpression RNAi Gain of function Loss of function PAX2 FACS Cell proliferation Nude mice Tumor growth Promoter analysis; ER recruitment; etc ER downstream target MSP; Bisulfite sequencing Hypomethylated in cancer ChIP; WB Mechanism of PAX2 Hypomethylation Hypomethylation-linked PAX2 Re-activation Is Associated with Tamoxifen-stimulated Endometrial Carcinogenesis
Some E2 Genes Unique TAM Genes Full Regulation Partial Regulation Full Regulation Supplementary Figure 2 Genomic action of tamoxifen E2 97 TAM 114 35 Table 1. Genes Regulated by both Estrogen and Tamoxifen in Endometrial Carcinomas Up-regulated genes Down-regulated genes NF I /B (nuclear factor I/B) RPIP8 (Rap2 interacting protein 8) EKLF (erythroid Kruppel-like factor) EREG1 (estrogen responsive element- associated Gene 1) MYL7 (myosin, light polypeptide 7) Cadherin-8 NTAK (neural- and thymus-derived activator for the ErbB kinase) Nebulette DLC-1 (deleted in liver cancer-1) PKC (protein kinase C) alpha TRIP9 (thyroid receptor interactor) Unknown (gb=AL109681) SH-PTP3 protein-tyrosine phosphatase Flt3 (Fms-like tyrosine kinase-3) ligand Phosphodiesterase I alpha MEA6 (meningioma-expressed antigen 6) Myelin proteolipid protein PAX2 (paired-box protein 2) IGF II(insulin-like growth factor II) Unknown (gb=AL049390) Carboxylesterase hKID (human kidney water channel) Heme oxygenase 1 (HO-1, EC 1.14.99.3) GABA-B R1a receptor OPCML (opioid binding protein/cell adhesion molecule-like) Cyclin F AILIM (activation-inducible lymphocyte immunomediatory molecule) ENaC (amiloride-sensitive epithelial sodium channel) beta subunit Myogenic determining factor 3 (MYOD1) Potassium channel homolog (KCNQ3) Unknown (gb=3413939) Unknown (gb=3882154) Retinoic Acid Receptor, Beta, Isoform 1 PRGP2 (proline-rich Gla protein 2) BK-2 (Bradykinin receptor) Experimentally-supported genomic view of tamoxifen action. See text for detailed explanation. Table 1 shows a list of genes that are up- and down-regulated by oestrogen and tamoxifen in cEECs. The complete lists of the genes and the other information on microarray experiments can be seen in http://medicine1.bjmu.edu.cn/department/ biochemistry/MicroarryData/index.htmor in Gene Expression Omnibus site with accession number: GSE3013.
Supplementary Figure 3 The growth stimulation of rat uteri by oestrogen and tamoxifen a Wet weight (g) LE (mM) C E2 TAM C E2 TAM b PCNA Ki-67 C C E2 E2 TAM TAM N N c Relative levels of mRNA PAX2 PKCa EKLF RARb1 Cyclin F rKID a: The wet weight of rat uteri under the treatment of oestrogen and tamoxifen. The ovariectomized adult female CD rats were housed for 14 days before treatment of 48 hours with 2.5 μg 17β-estradiol (E2)/rat (n = 5)/day, 500 μg tamoxifen (TAM)/rat (n = 5)/day, or vehicle only (C) (n=5). The uteri were weighed (left panel). b: Immunochemical staining for PCNA and Ki-67 in rat uteri. Uterine samples from above-treated rats were immersion-fixed in 4% formaldehyde at 4°C for 12 hours, stored at 4°C in 70% ethanol and embedded in paraffin for immunochemical staining for PCNA and Ki-67. c: Gene regulation by E2 and TAM in rat uteri. Uterine samples from above-treated rats were used for total RNA extraction, and the expression of mRNA was measured by real time RT PCR.
Supplementary Figure 4 PAX2-mediated endometrial cancer cell proliferation a aa 1 16 143 256 278 330 415 PAX2 Paired box Homeodomain Activation/Repression PAX2D b Increase in percentage of cells in S/G2/M phases PAX2 (ng) 0 100 100 500 PAX2D (ng) 0 0 500 100 c PAX2 PAX2D PAX2 (ng) 0 100 100 500 PAX2D (ng) 0 0 500 100 a: Construction of a PAX2 mutant with an activation/repression domain deletion. b: FACS analysis of ECC-1 cell proliferation under the treatment of E2 and tamoxifen. The PAX2 mutant had a dominant negative effect which could be rescued by wild type PAX2. c: Western blotting analysis of PAX2 and the PAX2 mutant expression.
Supplementary Figure 5 The effect of hKID, cyclin F, or RARb1 on the growth of endometrial carcinoma cells. a Increase in percentage of cells in S/G2/M phases - E2 TAM b No infection Vector RARb1 No infection Vector Cyclin F No infection Vector RARb1 No infection Vector hKID b-gal RARb1 hKID Cyclin F b-actin b-actin b-actin c Increase in percentage of cells in S/G2/M phases - E2 TAM V hKID Cyclin F RARb1 No RNAi RNAi d No RNAi Vector Cyclin F No RNAi Vector RARb1 No RNAi Vector RARb1 No RNAi Vector hKID b-gal RARb1 hKID Cyclin F b-actin b-actin b-actin
Supplementary Figure 6 Real time PCR quantification of ChIP DNAs a b Relative levels of DNA content Relative levels of DNA content - E2 TAM - E2 TAM - E2 TAM - E2 TAM cEECs nEECs cEECs nEECs PAX2 promoter EREG1 promoter c Relative levels of DNA content - E2 TAM - E2 TAM cEECs nEECs PAX2 promoter Real time PCR measurements of the PAX2 and EREG1 promoter fragments immunoprecipitated by antibodies against ERa, ERb, MeCP2, mSin3A, or HDAC1. The primer sequences for PAX2 promoter: forward: 5’- CGCCACCTCGGACATC-3’, reverse: 5’-CCAAGTGCTGGCGAGTTG-3’, and probe sequence: 5’-CCGGGATTGCTACTTCTCTGCCA-3’. The primer sequences for EREG1 promoter: forward: 5’-CCACTAACCGCGGGTATTAGG-3’, reverse: 5’-CCAGAGGGACCAAGTTCACTAAGA-3’, and probe sequence: 5’-TCGATGACCCCTCCGATTCCGTC-3’.
