Oncology/endocrine
Estrogen Receptor β/α Ratio Predicts Response of Pancreatic Cancer Cells to Estrogens and Phytoestrogens

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Background

Reports on hormone receptor expression of pancreatic cancer (PaCa) cells and treatment responses to antihormonal therapy are conflicting. We examined estrogen receptor (ER) expression in PaCa cells and investigated its function in estrogen-mediated cell proliferation.

Methods

Protein levels of ERα and ERβ in 8 human PaCa lines were detected by Western blot analysis. Cell proliferation was measured by sulforhodamine B analysis. ER modulators included diethylstilbestrol (DES), estradiol (E2), 4-hydroxytamoxifen (Tam), genistein (Gen), and Coumestrol (Coum).

Results

ERα levels were detected in all eight, and ERβ in seven cell lines. ERβ/ERα ratio ranged from 0.4 to 111 (median: 6.4, >5 in seven lines). Median maximal growth stimulation (in %, observed at 20 to 200 nM) was 19 (DES), 39 (E2), 20 (Tam), 22 (Gen), and −9 (Coum); median maximal inhibition (at 40 to 60 μM) was 59 (DES), 36 (E2), 25 (Tam), 43 (Gen), and 50 (Coum). The extent of E2 and Gen stimulatory effects correlated with the ERβ/ERα ratio (Kendall’s τ: 0.714, P = 0.024), but not ERα or ERβ levels alone. Only Coum-induced inhibition correlated with the ERβ/ERα ratio (P = 0.006) and with ERα expression (r = 0.753, P = 0.03). Gemcitabine-induced PaCa cytotoxicity (at IC40) was significantly reduced by E2, Gen, and Coum.

Conclusions

PaCa proliferation in vitro is highly estrogen sensitive, and in contrast to other reports, ERs are frequently expressed. In 7/8 cell lines, ERβ expression outweighs ERα expression. The impact of the ERβ/ERα ratio on estrogen-mediated growth stimulation and reduced cytotoxicity at physiological concentrations may have clinical implications on PaCa therapy.

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

References (73)

  • I. Leav et al.

    Comparative studies of the estrogen receptors β and α and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma

    Am J Pathol

    (2001)
  • Y. Omoto et al.

    Expression, function, and clinical implications of the estrogen receptor β in human lung cancers

    Biochem Biophys Res Commun

    (2001)
  • P.S. Jones et al.

    Activation of transcription by estrogen receptor α and β is cell type- and promoter-dependent

    J Biol Chem

    (1999)
  • B.S. Katzenellenbogen et al.

    Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology

    J Steroid Biochem Mol Biol

    (2000)
  • B.D. Lyn-Cook et al.

    The effects of phytoestrogens on human pancreatic tumor cells in vitro

    Cancer Lett

    (1999)
  • A. Jemal et al.

    Cancer statistics, 2006

    CA-Cancer J Clin

    (2006)
  • Y. Fong et al.

    Long-term survival is superior after resection for cancer in high-volume centers

    Ann Surg

    (2005)
  • H.A. Burris et al.

    Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial

    J Clin Oncol

    (1997)
  • A. Andren-Sandberg et al.

    Other risk factors for pancreatic cancer: hormonal aspects

    Ann Oncol

    (1999)
  • B. Greenway et al.

    Oestrogen receptor proteins in malignant and fetal pancreas

    Br Med J (Clin Res Ed)

    (1981)
  • A. Pousette et al.

    Purification and partial characterization of a 17 β-estradiol-binding macromolecule in the human pancreas

    Cancer Res

    (1982)
  • C. Benz et al.

    Endocrine-responsive pancreatic carcinoma: Steroid binding and cytotoxicity studies in human tumor cell lines

    Cancer Res

    (1986)
  • G.J. Poston et al.

    Effect of somatostatin and tamoxifen on the growth of human pancreatic cancers in nude mice

    Pancreas

    (1990)
  • R.S. Selvan et al.

    Growth modulatory effects of some 6-methylenic steroids on human and hamster pancreatic adenocarcinoma cells in vitro

    Drug Des Discov

    (1992)
  • N.O. Theve et al.

    Adenocarcinoma of the pancreas—a hormone sensitive tumor?A preliminary report on Nolvadex treatment

    Clin Oncol

    (1983)
  • J.J. Keating et al.

    A prospective randomized controlled trial of tamoxifen and cyproterone acetate in pancreatic carcinoma

    Br J Cancer

    (1989)
  • A. Wong et al.

    Survival benefit of tamoxifen therapy in adenocarcinoma of pancreasA case-control study

    Cancer

    (1993)
  • L. Rosenberg et al.

    Low dose octreotide and tamoxifen in the treatment of adenocarcinoma of the pancreas

    Cancer

    (1995)
  • T. Horimi et al.

    The beneficial effect of tamoxifen therapy in patients with resected adenocarcinoma of the pancreas

    Hepatogastroenterology

    (1996)
  • F.A. Wenger et al.

    Hormone therapy of postoperative recurrent pancreatic carcinoma with octreotide and tamoxifen

    Chirurg

    (1999)
  • G. Schumacher et al.

    Potent antitumor activity of 2-methoxyestradiol in human pancreatic cancer cell lines

    Clin Cancer Res

    (1999)
  • C. Sumi et al.

    Inhibition of a transplantable pancreatic carcinoma by castration and estradiol administration in rats

    Cancer Res

    (1989)
  • M. Kuramoto et al.

    Tissue-type plasminogen activator predicts endocrine responsiveness of human pancreatic carcinoma cells

    Cancer

    (1995)
  • E.F. Lhoste et al.

    Effect of castration and hormone replacement on azaserine-induced pancreatic carcinogenesis in male and female Fischer rats

    Carcinogenesis

    (1987)
  • C. Sumi et al.

    Inhibitory effects of estrogen and castration on the early stage of pancreatic carcinogenesis in Fischer rats treated with azaserine

    Cancer Res

    (1989)
  • D.S. Longnecker et al.

    Effects of sex steroid hormones on pancreatic cancer in the rat

    Int J Pancreatol

    (1990)
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