The Role of Estrogen-Targeted Candidate Genes in Regulating Proliferation and Migration in Endometrial Cells

Estrogens induce cell proliferation through both nuclear estrogen receptors (ER) or extra-nuclear GPR30/GPER1 receptor. In general, classic ER takes a longer time to induce cell proliferation (24 hours or longer) whereas intracellular signals through GPR30/GPER1 takes only a few minutes (5-60 min) and frequently co-regulated by other growth factors. The endometrium is a target tissue of estrogens. Through estrogen receptor (ER), estrogens stimulate proliferation of the endometrial cell. In addition, estrogens and antiestrogens may activate other growth factor receptors such as IGF-1R and EGFR which in turn stimulate the phosphrylation of down-stream kinases, leading to cell proliferation. Recently, we have successfully established immortalized human endometrial cells by infection of endometrial cells with retrovirus containing human telomerase reverse transcriptase (hTERT) (國科會計劃 NSC96-2314-B-182-016). In the past few years, we demonstrated that phosphorylation of focal adhesion kinase (FAK) was involved in cell migration induced by estradiol and tamoxifen (an anti-estrogen) through the transmembrane ER (GPR30) even in endometrial cancer cell lines without nuclear ERα. Additionally, we also validated that the signaling between GPR30 and phosphorylated FAK was mediated by the EGFR/PI3K/ERK pathway. In the mean time, we reported that NPM/B23 was required for estrogen-induced endometrial proliferation, and the increase in NPM/B23 was ERα-dependent. In endometrial cancer cells, estrogen increased NPM/B23 protein levels by repressing its ubiquitination and subsequently stabilizing the protein, instead of increasing the gene expression of NPM/B23. This novel characterization of NPM/B23 in estrogen-mediated cell proliferation may extend our understanding of the tumorigenesis of steroid hormone-related cancers. To further explore the regulation of cell proliferation and migration by the estrogen-target genes, we used cDNA microarray to screen the significantly up-regulated genes in Ishikawa cells (with or without knockdown by siESR1). We also downloaded microarray data of 54 patients of endometrial cancer from the public domain (The Cancer Genome Atlas, TCGA)[Nature 2013 May 2; 497(7447):67-73]. Following analysis of data from both groups and verification the gene expression by using real-time Q-PCR, we found SAT1 (SSAT), PPM1H, NR4A2 (NURR1) and GALNT4 as the most significant estrogen-targeted candidate genes (see the part of Preliminary Results). In the next three years, we are going to perform the following studies using immortalized endometrial cells, endometrial cancer cell lines, and freshly cultured endometrial cells and endometrial cancer cells from clinical specimens. Part I (the first year)： The study of regulation on gene expression of SAT1 (SSAT), PPM1H, NR4A2 (NURR1) and GALNT4 as well as the signal pathways involved in the cell proliferation and migration by estrogen through nuclear ER. The aim of the first year of this study is to testify 1. if estrogen may induce both the gene expression and the protein production of SAT1 (SSAT), PPM1H, NR4A2 (NURR1) and GALNT4 as well as the subsequent effects on cell proliferation and migration? 2. if addition of ER inhibitor (ICI182,780) may abolish both the gene expression and the protein production of SAT1 (SSAT), PPM1H, NR4A2 (NURR1) and GALNT4 as well as the subsequent effects on cell proliferation and migration? 3. if estrogen-induced cell proliferation and migration may diminish after knock-down of SAT1 (SSAT), PPM1H, NR4A2 (NURR1) and GALNT4 as well as HSF1(heat shock factor 1) and NFkB by siRNA? 4. if estrogen-induced cell proliferation may be through signal pathways of c-Src and c-fos? 5. if estrogen-induced cell migration may be through the activation of FAK and MMP-2? Part II (the second year)： The study of cross-talk of PI3K, ERK and Akt in activation of SAT1 (SSAT), PPM1H, NR4A2 (NURR1) and GALNT4 signal pathways as well as the subsequent cell migration mediated by estrogen and antiestrogen via GPR30/GPER1 and EGFR. The aim of the second year of this study is to testify 1. if estrogen and antiestrogen enhance the phosphrylation of PI3K, ERK and Akt in EGFR signal pathway? 2. if estrogen and antiestrogen-induced phosphrylation of FAK and activation of MMP-2 may diminish by addition of inhibitors for PI3K, ERK and Akt…? 3. if estrogen and antiestrogen-induced cell migration may diminish by addition of inhibitors for PI3K, ERK and Akt…? Part III (the third year)： The study of repressive effects modulated by progestins on SAT1 (SSAT), PPM1H, NR4A2 (NURR1) and GALNT4 signal pathways as well as the subsequent cell proliferation and migration induced by estrogen. The aim of the third year of this study is to testify 1. if progestins may inhibit both the gene expression and the protein production of SAT1 (SSAT), PPM1H, NR4A2 (NURR1) and GALNT4 as well as the subsequent effects on estrogen-induced cell proliferation and migration? 2. if progestins may reduce estrogen-induced cell proliferation through signal pathways of c-Src and c-fos? 3. if progestins may reduce estrogen-induced cell migration through the activation of FAK and MMP-2?

Project ID:PC10401-0768
External Project ID:MOST103-2314-B182-058-MY2