Uptake transporter organic anion transporting polypeptide 1B3 contributes to the growth of estrogen-dependent breast cancer

doi:10.1016/j.jsbmb.2010.06.014

The Journal of Steroid Biochemistry and Molecular Biology

Volume 122, Issue 4, October 2010, Pages 180-185

https://doi.org/10.1016/j.jsbmb.2010.06.014 Get rights and content

Abstract

Estrone-3-sulfate is one of the most abundant estrogen precursors in postmenopausal women. We previously showed that estrone-3-sulfate transporters are present in human breast cancer-derived MCF-7 cells (J. Pharmacol. Exp. Ther. 311 (2004) 1032–1037) and that inhibition of estrone-3-sulfate uptake resulted in the suppression of cell growth (Pharm. Res. 22 (2005) 1634–1641); therefore, estrone-3-sulfate transporter should be a novel target for therapy of hormone-dependent breast cancers. The purpose of the present study is to identify the transporter(s) responsible for the uptake of estrone-3-sulfate in breast cancer cells. We obtained two subclones of MCF-7 cells with different estrone-3-sulfate uptake activities and searched for differentially expressed transporter genes by means of DNA microarray analysis. Among several candidate transporters identified, OATP1B3 was further evaluated, since the uptake characteristics of estrone-3-sulfate by MCF-7 cells seemed consistent with the transport properties of OATP1B3. The contribution of OATP1B3 to estrone-3-sulfate uptake by MCF-7 cells was examined by the relative activity factor (RAF) method, and was calculated to amount to 6%. This result suggests that OATP1B3 is one of the transporters contributing to the supply of the estrogen precursor estrone-3-sulfate to estrogen-dependent breast cancer cells.

Introduction

Breast cancer is one of the major causes of cancer death in women. Since two-thirds of breast cancers are estrogen-dependent, estrogen is an important risk factor for progression of breast tumors [1]. However, breast cancers frequently occur during the postmenopausal period, when the circulating estrogen level is low because of loss of ovarian function [2], [3]. Although the circulating level of estrogen is low, tissue concentrations of estrogen in breast cancer are significantly higher than those found in the plasma or in adjacent normal breast tissues, suggesting a specific mechanism of local biosynthesis from precursors [2]. The biologically active form of estrogen is estradiol, which is synthesized from the precursors through two main pathways; one is the aromatase pathway, in which aromatase converts androgens to estrogens, and the other is the sulfatase pathway, in which sulfatase converts estrone-3-sulfate to estrone. Both of them are currently molecular targets of endocrine therapy of breast cancer. Although estrone-3-sulfate is a biologically weak ligand of estrogen receptor, it is one of the most important forms of circulating estrogen. The plasma concentration of estrone-3-sulfate is about 5–10 times higher than that of other conjugated estrogens, and its half-life is longer than that of estradiol in postmenopausal women [4], [5], [6]. Moreover, sulfatase activity is 50–200 times higher than aromatase activity in breast cancer cells [6], [7], [8]. In addition, sulfatase activity in breast cancer cells is higher than that of normal breast cells [9]. Therefore, estrone-3-sulfate is thought to play an important role in the progression of breast cancer as a precursor of active estrogen.

Estrone-3-sulfate is highly hydrophilic, so that it cannot readily cross the plasma membrane, whereas unconjugated estrogens, such as estrone and estradiol, are lipophilic and can enter cells via simple diffusion. Thus, estrone-3-sulfate import into cells across the plasma membrane is expected to involve active transport. Indeed, we previously reported that the uptake of estrone-3-sulfate across the plasma membrane in T-47D cells and MCF-7 cells, which are estrogen-dependent breast cancer cell lines, is mediated by a specific transport mechanism [10], [11].

Accordingly, we hypothesized that the transporter responsible for this transport process could be a novel target for endocrine therapy of estrogen-dependent breast cancers. To develop this concept further, it is necessary to identify the estrone-3-sulfate uptake transporter(s) in breast cancer cells. In our previous study, mRNA expression of some organic anion transporting peptide (OATP) and organic anion transporter (OAT) molecules was detected in MCF-7 cells by RT-PCR [11]. In addition, the uptake of estrone-3-sulfate by MCF-7 cells and T-47D cells was inhibited by anionic compounds, such as bromosulfophthalein, taurocholate, cholate, and probenecid, which are substrates of OATPs or OATs. Some of these transporters may be involved in the uptake of estrone-3-sulfate in MCF-7 cells, but their contribution remains to be examined.

The purpose of this study is to identify the responsible transporter molecules among previously identified estrone-3-sulfate transporters, based on the correlation between mRNA expression levels and the uptake activity of estrone-3-sulfate in subclones of breast cancer-derived MCF-7 cells with differential transport activity.

Section snippets

Materials

[³H]Estrone-3-sulfate, ammonium salt (1702.0 Gbq/mmol) and [³H]cholecystokinin octapeptide (CCK8) (3590 Gbq/mmol) were purchased from Perkin Elmer Life and Analytical Sciences (Boston, MA) and GE Healthcare UK Ltd. (Buckinghamshire, England), respectively. MCF-7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Fetal bovine serum (FBS) was obtained from Invitrogen Life Technologies (Carlsbad, CA). All other reagents were purchased from Sigma–Aldrich (St. Louis, MO)

Microarray analysis

To identify transporters involved in the uptake of estrone-3-sulfate into MCF-7 cells, MCF-7 cells were subcloned and the uptake activity in each clone was measured. Those clones showed different uptake activities for estrone-3-sulfate (data not shown). Among them, two subclones were selected for further experiments, i.e., the one that exhibited the highest activity of estrone-3-sulfate uptake (MCF-7-High cells), and the one that exhibited the lowest activity (MCF-7-Low cells) (Fig. 1). The

Discussion

Estrone-3-sulfate is a major circulating estrogen and also participates in the progression of breast cancer cells [19]. Although estrone-3-sulfate itself is a weak ligand of the estrogen receptor, it is converted to the active estrogen, estradiol, by cytosolic sulfatase and 17β-hydroxysteroid dehydrogenase type 1. Since estrone-3-sulfate is hydrophilic, it was thought that a specific transporter would be involved in import of estrone-3-sulfate across the plasma membrane of breast cancer cells.

Acknowledgment

This study was partly supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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Uptake transporter organic anion transporting polypeptide 1B3 contributes to the growth of estrogen-dependent breast cancer

Abstract

Introduction

Section snippets

Materials

Microarray analysis

Discussion

Acknowledgment

J. Biol. Chem.

J. Steroid Biochem. Mol. Biol.

Adv. Pharmacol.

Drug Metab. Pharmacokinet.

Gastroenterology

Anal. Biochem.

Biochem. Biophys. Res. Commun.

J. Steroid Biochem.

J. Biol. Chem.

Gastroenterology

Gastroenterology

Cancer of the breast: the past decade (second of two parts)

N. Engl. J. Med.

Recent insight on the control of enzymes involved in estrogen formation and transformation in human breast cancer

J. Steroid Biochem. Mol. Biol.

In situ production of estrogens in human breast carcinoma

Breast Cancer