Estrogen Stimulates the Proliferation of Human Endometrial Cancer Cells by Stabilizing

Supplementary Information

Estrogen stimulates the proliferation of human endometrial cancer cells by stabilizing nucleophosmin/B23 (NPM/B23)

Angel Chao1*§, Chiao-Yun Lin1*, Chia-Lung Tsai1, Swei Hsueh2, Ying-Yu Lin1,

Cheng-Tao Lin1, Hung-Hsueh Chou1, Tzu-Hao Wang1,3, Chyong-Huey Lai1§, Hsin-

Shih Wang1§

1 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital and

Chang Gung University College of Medicine, Taoyuan, Taiwan

2 Department of Clinical Pathology, Chang Gung Memorial Hospital and Chang Gung

University College of Medicine, Taoyuan, Taiwan

3 Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taiwan

1 Supplementary Table 1. Characteristics of endometrial cancer patients (for immunohistochemistry)

Number Age Pathology Stage 1 52 endometrioid adenocarcinoma IIIA 2 54 endometrioid adenocarcinoma IIIA 3 49 endometrioid adenocarcinoma IB 4 61 endometrioid adenocarcinoma IA 5 46 endometrioid adenocarcinoma IA 6 44 endometrioid adenocarcinoma IB 7 62 endometrioid adenocarcinoma IB 8 64 endometrioid adenocarcinoma IB 9 67 endometrioid adenocarcinoma I1B 10 51 endometrioid adenocarcinoma IA 11 58 endometrioid adenocarcinoma IC 12 53 endometrioid adenocarcinoma IB 13 71 endometrioid adenocarcinoma IIIC 14 68 endometrioid adenocarcinoma I1A 15 53 endometrioid adenocarcinoma IA 16 60 endometrioid adenocarcinoma IIIC 17 53 endometrioid adenocarcinoma IVA 18 67 endometrioid adenocarcinoma IIIC 19 59 endometrioid adenocarcinoma IIIA 20 72 endometrioid adenocarcinoma IIIC 21 54 endometrioid adenocarcinoma IIIC 22 65 endometrioid adenocarcinoma IIIC 23 59 endometrioid adenocarcinoma IIIC 24 55 endometrioid adenocarcinoma I A

2 Supplementary Table 2. Characteristics of endometrial cancer patients (for real- time quantitative PCR analysis)

Number Age Pathology Stage 1 53 endometrioid adenocarcinoma IB 2 51 endometrioid adenocarcinoma IC 3 53 endometrioid adenocarcinoma IIIC 4 36 endometrioid adenocarcinoma IIIA 5 75 endometrioid adenocarcinoma IIIA 6 59 endometrioid adenocarcinoma IB 7 45 endometrioid adenocarcinoma II 8 44 endometrioid adenocarcinoma IB 9 46 endometrioid adenocarcinoma IA 10 61 endometrioid adenocarcinoma I A

3 Supplementary Figure 1. E2 activates NPM/B23 protein expression in an ERα- dependent manner. (a) Lysates were prepared from the MCF-7 (ERα-positive) and

MB-MDA231 (ERα-negative) human breast cancer cell lines. Western blotting was performed using ERα and β-actin antibodies. (b) MCF-7 and MDA-MB-231 cells were treated with E2 for 24 h. Western blotting was performed using NPM/B23 and β- actin antibodies. The data shown represent the mean ± standard error and were derived from three independent experiments. ＊, P < 0.05 as compared to the controls.

Supplementary Figure 2. Expression levels of the ERα and ERβ receptors and the effects of E2, an ERα-specific agonist (PPT), and an ERβ agonist (DPN) on endometrial cancer cells. (a) The human endometrial cancer cell lines (Ishikawa and

ARK1 cells) and ERα-positive breast cancer MCF7 cells were measured for ERα

5 expression. Equal amounts of protein lysate were separated by SDS-PAGE and subjected to immunoblotting with antibodies for ERα and β-actin. The titration concentrations of (b) Ishikawa cells treated with E2, an ERα-specific agonist (PPT), or an ERβ agonist (DPN) are shown. (c) ARK1 cells were treated with the ERα- specific agonist (PPT) and an ERβ agonist (DPN). The ARK1 cell viability was less than 10% when treated with 10 μM PPT; thus, the experiment was not performed at this concentration. The data shown represent the mean ± standard error and were derived from three independent experiments. ＊, P < 0.05 as compared to the controls.

6 (a) (b)

Supplementary Figure 3.

E2 induces the proliferation of human cancer cells by activating NPM/B23 in more than one clone of Ishikawa and ARK1 cells. NPM/B23 knockdown in an additional (a) Ishikawa and (b) ARK1 cell clone suppressed 1 μM E2-mediated cell proliferation, as assessed by BrdU incorporation. The data shown represent the mean

± standard error and were derived from three independent experiments. ＊, P < 0.05 as compared to the controls.