Oncology/endocrineEstrogen Receptor β/α Ratio Predicts Response of Pancreatic Cancer Cells to Estrogens and Phytoestrogens
Introduction
Pancreatic cancer (PaCa) is currently the fourth leading cause of cancer deaths in the United States, and carries a 5-y survival rate of only 4% [1]. Even after complete resection, only between 10 and 25% of patients are alive at 5 y [2, 3]. No systemic treatment has shown improved efficacy over that with the cytosine analogue gemcitabine alone [4], so that new and improved therapy options are desperately needed. Based on the apparent hormonal imbalance of PaCa incidence, with a male to female ratio of between 1.25–1.75 to 1 [5], strategies to investigate whether PaCa is a hormonally susceptible disease have been pursued. We have become interested in estrogenic regulation of PaCa cell growth after encountering strong antitumor effects of the phytoestrogenic supplement PC-Spes [6]. Earlier analyses had found evidence for estrogen binding within normal or neoplastic pancreatic tissue [7, 8, 9]. Numerous studies of estrogen receptor (ER) expression and treatment response of ER modulation in PaCa have generated conflicting results. In support of ER representing an important PaCa progression mediator or therapeutic target are reports of preclinical or in vitro treatment benefit of the ER modulator tamoxifen (Tam) [8, 10, 11], or clinical trials of Tam containing therapy [12, 13, 14, 15, 16, 17, 18]. Of note, only one of these clinical trials was a prospective, randomized trial with three treatment arm in 108 patients, and despite a median survival of 5.25 mo (Tam) versus 3 mo (controls), statistical significance was not achieved [14]. Nevertheless, other estrogenic agents such as 2-methoxyestradiol [19] and estradiol (E2) have shown antitumor benefits against PaCa in vivo [20, 21], and estrogens have been found to inhibit experimental pancreatic carcinogenesis [22, 23, 24]. In addition, progesterones can mediate significant apoptotic antitumor effects in PaCa cells [25].
On the other hand, several attempts to link hormonal mechanisms to PaCa progression or treatment have failed to find such support. Earlier attempts to detect ERs in PaCa tissues failed, likely due to the lack of sensitive methods [26, 27, 28]. PaCa risk was not found to be affected by the use of estrogen replacement therapy [29]. Diethylstilbestrol (DES) had no effect on pancreatic carcinogenesis in an in vivo model [30]. More importantly, Tam-based therapy failed to show any effect in a treatment model of PaCa [31] in small single-arm clinical trials [32, 33] or in two randomized clinical trials with 176 [34] or 44 patients [35].
It has become obvious that estrogenic effects on various target tissues are predominantly mediated through two ER types, ERα and ERβ. Agonistic or antagonistic effects depend on the combination of expression of these receptor subtypes and the different agonistic/antagonistic effects of various selective ER modulators (SERMs) [36]. In those studies that successfully identified the presence of ER in pancreatic tissues through traditional immunoabsorbance assays (equating to ERα detection), intratumoral levels were considered to be lower in cancer tissues than in normal pancreatic parenchyma [37, 38]. ERα and progesterone receptor levels in the PaCa cell line CaPan1 are higher during exponential growth, and low while stationary, indicating variability of ER expression at least in this cell line [39]. Interestingly, knowledge about ERβ protein expression in PaCa remains insufficient to date. On the transcriptional level, ERβ message was found to be higher in PaCa than that of (traditional) ER negative or positive breast cancer, while ERα RNA levels were low [40]. Because of the strong PC-Spes mediated G2/M cell cycle block and the resulting growth inhibition of PaCa cells [6], and due to the known increased ERβ binding and transactivation activity of phytoestrogens [41, 42], evaluating the ERβ status of PaCa and its relation to ER modulator response became of interest. We therefore examined ERβ (and ERα) protein expression in PaCa cells and investigated their function in estrogen-mediated cell proliferation in vitro.
Section snippets
Cell Culture and Reagents
Eight human PaCa cell lines from ATCC were studied, including BxPC3, MIA PaCa2, Panc-1, ASPC, CFPAC, HS766T, HTB 147, and CaPan2. Cells were cultured in RPMI medium supplemented with 10% FBS. Two breast cancer cell lines, MCF-7 (expressing a wild type ER α) and T47D (with a wild type ER β) were used as controls. ER modulators estradiol, distilbestrol (DES), 4-hydroxy tamoxifen (Tam), genistein (Gen), and coumestrol (Coum) were obtained from Sigma Chemicals (St. Louis, MO). The cytotoxic agent
ER Protein Expression
Based on Western blot analysis, measurable ERα levels were each detected in all eight PaCa cell lines. Visible bands corresponding with ERβ protein were identified in seven PaCa cell lines, but not for the cell line Panc-1 (Fig. 1). Median relative expression compared with the two positive breast cancer controls was 44% (ERα, comparison cell line MCF7) and 102% (ERβ, comparison cell line T47D). Based on densitometry analysis, the ERβ/ERα ratio was determined for each PaCa line. This ratio
Discussion
Our results show that pancreatic cancer cells express functional estrogen receptors, generally with a high ERβ to ERα ratio. This is the first report confirming ERβ protein expression in PaCa cells, lending support to previous observations of high ERβ RNA levels in PaCa [40]. In vitro proliferative responses are observed in all cell lines, and stimulation and inhibition was a dose-dependent response to all SERMs evaluated. Cell lines with higher ERβ to ERα ratio tended to show greater
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