Contribution of Estrone Sulfate to Cell Proliferation in Aromatase Inhibitor (AI) -Resistant, Hormone Receptor-Positive Breast Cancer

<div><p>Aromatase inhibitors (AIs) effectively treat hormone receptor-positive postmenopausal breast cancer, but some patients do not respond to treatment or experience recurrence. Mechanisms of AI resistance include ligand-independent activation of the estrogen receptor (ER) and signaling via other growth factor receptors; however, these do not account for all forms of resistance. Here we present an alternative mechanism of AI resistance. We ectopically expressed aromatase in MCF-7 cells expressing green fluorescent protein as an index of ER activity. Aromatase-overexpressing MCF-7 cells were cultured in estrogen-depleted medium supplemented with testosterone and the AI, letrozole, to establish letrozole-resistant (LR) cell lines. Compared with parental cells, LR cells had higher mRNA levels of steroid sulfatase (STS), which converts estrone sulfate (E1S) to estrone, and the organic anion transporter peptides (OATPs), which mediate the uptake of E1S into cells. LR cells proliferated more in E1S-supplemented medium than did parental cells, and LR proliferation was effectively inhibited by an STS inhibitor in combination with letrozole and by ER-targeting drugs. Analysis of ER-positive primary breast cancer tissues showed a significant correlation between the increases in the mRNA levels of STS and the OATPs in the LR cell lines, which supports the validity of this AI-resistant model. This is the first study to demonstrate the contribution of STS and OATPs in E1S metabolism to the proliferation of AI-resistant breast cancer cells. We suggest that E1S metabolism represents a new target in AI-resistant breast cancer treatment.</p></div

Comparison of steroid sulfatase (STS) mRNA and organic anion transporter peptides (OATPs) mRNA expression in primary breast cancer tissues.

<p>Values were normalized to that of β-actin and converted to base 2 logarithms. R indicates the correlation index.</p

The contribution of estrone sulfate (E1S) to the proliferation of letrozole (Let)-resistant (LR) cells.

<p><b>A)</b> Relative mRNA expression of steroid sulfatase (STS) in LR cell lines and parental cells. Let letrozole, HSD17B1 17β hydroxyl steroid dehydrogenase type 1, EST estrone sulfotransferase, HSD17B2 17β hydroxyl steroid dehydrogenase type 2. <b>B)</b> Expression of organic anion transporter peptides (OATPs) mRNA. In Fig 2A and 2B, values are normalized to that of the RPL13A gene. <b>C)</b> Proliferation of LR and E10arom cell lines treated with E1S. <b>D)</b> E1S consumption by LR cells in E1S-containing medium measured via enzyme immunoassay. <b>E)</b> E1S consumption per cell. Consumption was measured by dividing the number of E1S molecules (determined via enzyme immunoassay) by the number of cells (obtained in proliferation assays of cells receiving 1μM E1S). Error bars show standard deviation. * p < 0.05. ** p < 0.01.</p

Effects of a steroid sulfatase (STS) inhibitor and estrogen receptor (ER) inhibitors on ER activity and proliferation in letrozole (Let)-resistant (LR) cell lines.

<p><b>A)</b> Relative ER activity in LR cell lines and parental cells treated with or without the STS inhibitor STX64. <b>B)</b> The relative proliferation of LR and parental cells treated with testosterone (TS) in the presence or absence of Let, STX64 or both. <b>C)</b> Relative cell proliferation with or without 4-OH tamoxifen (tam) and fulvestrant (ful). Error bars show standard deviation. * p < 0.05. ** p < 0.01.</p

Putative model of steroid metabolism in letrozole-resistant (LR) cell lines.

<p>The map shows our model of steroid metabolism in LR cell lines. We suggest that LR cell lines acquire resistance to aromatase inhibitors via augmentation of STS activity. EST estrone sulfotransferase, STS steroid sulfatase, HSD17B 17β hydroxyl steroid dehydrogenase, HSD3B1 3β hydroxyl steroid dehydrogenase type 1, SRD5A1 steroid 5α-reductase type 1, AKR1C3 aldo-keto reductase 1C3. HSD17B1 1, 2, and 5 refer to three different forms of this enzyme.</p

Establishment of letrozole-resistant (LR) cell lines.

<p><b>A)</b> Schematic representation of the establishment of E10arom cells from E10 cells. Estrogen receptor (ER) activity was visually monitored in cells treated with testosterone (TS) or TS plus letrozole (Let) via green fluorescent protein (GFP) expression. The green color indicates GFP expression. Comparison of the three images shows that E10arom cells express more GFP when treated with TS alone than with TS plus Let or when untreated (control). E estrogen, A androgen, CMV cytomegalovirus promoter, AROM aromatase gene, BSD blasticidin gene, ERE estrogen response element, EtOH ethanol. <b>B)</b> Eight candidate ER-expressing, Let-resistant cell lines. These cell lines are circled in black. <b>C)</b> ER activity in the LR cell lines was measured using a luciferase reporter driven by the estrogen response element. <b>D)</b> Cell number was determined in LR cultures receiving 100 nM Let and the indicated concentrations of TS. The vertical axis indicates cell number relative to vehicle-treated controls. <b>E)</b> mRNA expression of ERs, ER target genes including cyclin D1, bcl-2 and progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) was determined via real-time PCR. Values are normalized to that of the RPL13A gene. Error bars indicate standard deviation. * p < 0.05. ** p < 0.01.</p