Identification of a novel estrogen-regulated gene, EIG121, induced by hormone replacement therapy and differentially expressed in type I and type II endometrial cancer.

PURPOSE: The identification of genes and pathways that are affected by estrogenization may shed light on the mechanisms of estrogen action. Here, we describe the expression pattern of a novel estrogen-induced gene, EIG121, in distinct types of endometrial cancer. EXPERIMENTAL DESIGN: EIG121 was identified by cDNA microarray analysis of endometrial RNA from women receiving either placebo or estrogen replacement therapy. The expression level of EIG121 was then measured by real-time quantitative reverse transcription-PCR in benign, hyperplastic, and malignant endometrial samples. A polyclonal antibody was used to detect EIG121 protein by immunohistochemistry. RESULTS: In postmenopausal endometrium, estrogen replacement therapy with Premarin and synthetic estrogen sulfate conjugates induced the expression of EIG121 2- and 3-fold, respectively. In premenopausal endometrium, the expression of EIG121 was higher in the estrogen-dominated proliferative phase than the secretory phase. In endometrial complex, hyperplasia, and endometrioid adenocarcinoma, neoplastic proliferations associated with estrogen excess, the expression of EIG121 was significantly elevated (on average 3.8-fold in hyperplasias and 21-fold in grade 1 tumors). Although the level of EIG121 mRNA in grade 3 endometrioid carcinoma was still 3.5-fold of that in benign endometrium, EIG121 expression tended to decline with increasing tumor grade and disease stage. Immunohistochemistry showed faint staining of normal endometrial epithelium, but intense staining of endometrioid tumors. In sharp contrast, EIG121 expression was significantly suppressed in both uterine papillary serous carcinoma and uterine malignant mixed mullerian tumor, two tumors not associated with estrogen exposure, to <5% of the level in benign endometrium. CONCLUSIONS: Our results suggest that EIG121 is a good endometrial biomarker associated with a hyperestrogenic state and estrogen-related type I endometrial adenocarcinoma.

p53-dependent inhibition of progestin-induced VEGF expression in human breast cancer cells.

VEGF, a potent angiogenic growth factor, is up-regulated in many tumors including human breast tumors and stimulates growth of vascular networks that support tumor growth and metastasis. We previously reported that natural and synthetic progestins (P) increased VEGF mRNA and protein levels in progesterone receptor (PR) containing T47-D human breast cancer cells in a PR dependent manner, but not in PR positive ZR-75 and MCF-7, or in PR negative MDA-MB-231 cells. This indicated that factors beside PR are involved in progesterone-dependent VEGF regulation. We, therefore, tested additional tumor cell lines reported to contain PR for progestin-dependent VEGF induction. Out of nine PR-positive breast tumor cell lines, progestins induced VEGF in three cell lines that lack wild-type p53 (T47-D, BT-474, and HCC-1428) but not in cell lines that contained the wild-type p53 protein. The T47-D and BT-474 cells express mutant p53, while the p53 protein is absent HCC-1428 cells. The anti-progestin RU-486 blocked progestin-dependent induction of VEGF in T47-D and BT-474 cells but not in HCC-1428 cells. However, RU-486 partially blocked medroxyprogesterone acetate-dependent induction of VEGF in HCC-1428 cells. Estrogen receptor (ER) and PR agonists and antagonists also induce VEGF in HCC-1428 cells and this effect was partially blocked by anti-estrogen ICI-182, 780. Progestin-dependent VEGF induction was completely inhibited by PRIMA-1-activated p53 in all cell-types, but progestin-dependent transcription of a progesterone-regulated minimal promoter was only partially inhibited. PRIMA-1 induced activation of p53 in tumor cell lines was confirmed with a p53-responsive p21 reporter plasmid and by detecting increased levels of p21 proteins in cell lysates. PRIMA-1 induced p53 protein in the HCC-1428 cells while levels of mutant p53 protein in T47-D and BT-474 remained unaltered. Progestin-dependent induction of VEGF was also inhibited by stable transfection of wild-type p53 in T47-D cells. These results are consistent with the hypothesis that wild-type p53 blocks progestin-dependent induction of VEGF in breast cancer cells and this may be a novel anti-angiogenic mechanism for controlling the growth of progestin-dependent tumors.

Coordinate regulation of the production and signaling of retinoic acid by estrogen in the human endometrium.

To determine whether estrogen regulates retinoic acid (RA) production and signaling in the human endometrium as it does in the rodent uterus, we investigated the effects of estrogens on the expression of RA-metabolizing enzymes, retinoid receptors, and biomarker genes in the post- and premenopausal human endometrium. Real-time quantitative PCR revealed that retinaldehyde dehydrogenase (RALDH) 2, a critical enzyme in RA biosynthesis, was induced 4-fold by estrogen replacement therapy with either Premarin or a mixture of estrone and equilin sulfates for 3 months. Estrogen replacement therapy also increased the expression of the RA receptor RAR alpha 1.9-fold. In parallel, there was a marked increase in the expression of two RA-regulated genes, cellular retinoic acid-binding protein II and tissue transglutaminase. In the premenopausal endometrium, the levels of RALDH1, RALDH2, RAR alpha, and cellular retinoic acid-binding protein II were increased in the estrogen-dominated proliferative phase, and the transcripts for the RA catabolic enzyme retinoic acid 4-hydroxylase (CYP26A1) and tissue transglutaminase were significantly increased in the secretory phase. Our results suggest that estrogen coordinately up-regulates RA production and signaling in the human endometrium. This coordinate mechanism may play a role in the antiproliferative effects that counterbalance the estrogen-induced endometrial proliferation.

Inhibition of progesterone-induced VEGF production in human breast cancer cells by the pure antiestrogen ICI 182,780.

The 'pure' antiestrogen ICI 182,780 (Faslodex) is in clinical trials for treatment of human breast cancer. Recently, we showed that ICI 182,780 exhibits a novel antiprogestin activity in transient transfection assays in the total absence of estrogen receptors. In this work, we determined if ICI 182,780 displays antiprogestin activity for an endogenous progesterone responsive gene. For this purpose, we examined the effect of ICI 182,780 on progestin induction of a potent angiogenic growth factor, vascular endothelial growth factor (VEGF), in T47-D human breast cancer cells. ICI 182,780 blocks the progestin induction of VEGF at both the mRNA and protein levels in T47-D cells. The antihormone does not block progestin binding to the progesterone receptor (PR), nor does it enhance the down regulation of the endogenous PR in cells that occurs upon progestin exposure. These results establish that ICI 182,780, generally considered to be a highly selective antiestrogen, displays antiprogestin activity for an endogenous progestin-regulated gene. These observations raise the possibility that an antiprogestin activity of ICI 182,780 may contribute to the antitumor activity in a subset of human breast cancers similar to T47-D cells, by inhibiting angiogenesis secondary to blockade of VEGF induction.