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Androgen metabolite-dependent growth of hormone receptor-positive breast cancer as a possible aromatase inhibitor-resistance mechanism

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Abstract

Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3β,17β-diol (3β-diol) generated from DHT by 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3β-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of DHT into 3β-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen metabolite-induced ER activation and growth mechanisms. Androgen metabolite-dependent growth of breast cancer cells may therefore play a role in AI-resistance.

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Abbreviations

AI:

Aromatase inhibitor

TS:

Testosterone

DHT:

Dihydrotestosterone

3β-Diol:

5α-Androstane-3β,17β-diol

HSD3B1:

3β-Hydroxysteroid dehydrogenase type 1

AKR1C3:

Aldo–keto reductase 1C3

AR:

Androgen receptor

ERα:

Estrogen receptor α

E2:

Estradiol

OHT:

4-Hydroxytamoxifen

GFP:

Green fluorescent protein

SERM:

Selective estrogen receptor modulator

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Acknowledgments

We would like to thank Takashi Suzuki (Tohoku University Department of Pathology and Histotechnology) for discussions and helpful suggestions. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare, Japan; the Program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (NIBIO); and a Grant from the Smoking Research Foundation.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standard

All experiments complied with the current laws of Japan.

Author information

Authors and Affiliations

  1. Department of Molecular and Functional Dynamics, Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan

    Toru Hanamura, Toshifumi Niwa, Sayo Nishikawa, Hiromi Konno, Tatsuyuki Gohno, Chika Tazawa & Shin-ichi Hayashi

  2. Center for Regulatory Epigenomics and Diseases, Graduate School of Medicine, Tohoku University, Sendai, Japan

    Shin-ichi Hayashi

  3. Division of Breast and Endocrine Surgery, Department of Surgery, Shinshu University School of Medicine, Nagano, Japan

    Toru Hanamura & Ken-ichi Ito

  4. Department of Pathology, Saitama Cancer Center, Saitama, Japan

    Yasuhito Kobayashi & Masafumi Kurosumi

  5. Division of Breast Surgery, Saitama Cancer Center, Saitama, Japan

    Hiroyuki Takei

  6. Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan

    Yuri Yamaguchi

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  1. Toru Hanamura
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Correspondence to Toru Hanamura.

Electronic supplementary material

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10549_2013_2595_MOESM1_ESM.ppt

Fig. S1 Establishment of E10 cells and screening of variant cell lines. a E10 cells report ER activity via GFP expression. ERE-tk-GFP-MCF-7 cells (E10 cells) were established from the human breast cancer cell line, MCF-7, by introduction of a plasmid carrying the ERE fused with ERE-GFP gene. These cells show luminescence via GFP, corresponding to ER transcription activity. b Screening of variant cell lines that show ER activity depending on androgen metabolites. We used E10 cells to establish variant cell lines. After 3 months of culture in TS-supplemented steroid-depleted medium, five clones expressing GFP (representing ER activity) were obtained. ER activities at each step of treatment (a, b, c, d, e) were assessed in a single experiment, as described in Materials and methods. E estrogen; Let letrozole; Ful fulvestrant (PPT 449 kb)

10549_2013_2595_MOESM2_ESM.ppt

Fig. S2 Expression of HSD3B1 in E10-HSD3B1 cells. Cells transfected with HSD3B1 expression vector (E10-HSD3B1) or control vector (E10-Control) were generated as described in Materials and methods. The expression level of HSD3B1 under regular growth medium was analyzed using real-time PCR. Values relative to RPL13A are shown (PPT 134 kb)

10549_2013_2595_MOESM3_ESM.ppt

Fig. S3 Interaction coculture system using insert layers. To assess the inhibitory effect of AIs in E10 and variant cell lines, we used interaction cultures, defined as cocultures where cancer cells and stromal cells are grown separately by a membrane, but are allowed to interact via soluble factors, and have the same microenvironment, such as local estrogen synthesis from androgen by aromatase in stromal cells (PPT 136 kb)

10549_2013_2595_MOESM4_ESM.ppt

Fig. S4 Induction of estrogen- or androgen-responsive genes by androgen or 3β-diol. After 3 days of incubation in steroid-depleted medium, 100 nM TS, DHT, 3β-diol or vehicle control (EtOH) was added to each cell line for 4 days before total RNA extraction. Total RNA was extracted from each cell line cultured in the indicated culture conditions, and real-time PCRs for expression of the indicated mRNA were carried out as described in Materials and methods. All PCRs were performed in duplicate, and the expression of the target gene relative to vehicle control is shown (mean ± SD) (PPT 198 kb)

10549_2013_2595_MOESM5_ESM.ppt

Fig. S5 Estrogen synthesis and androgen metabolism. In postmenopausal women, estrogens are mainly supplied by aromatase from androgens (TS and androstenedione) biosynthesized in the adrenal gland. In contrast, 5α-androstane-3β,17β-diol (3β-diol) is generated from DHT by HSD3B1 type 1 and AKR1C3. It was suggested that the metabolizing pathway from DHT to 3β-diol is up-regulated in variant cell lines (PPT 149 kb)

10549_2013_2595_MOESM6_ESM.ppt

Fig. S6 Blockage of 3β-diol-induced growth in variant cell lines by SERMs. After 3 days of culture in steroid-depleted medium, each cell line was plated in 24-well culture plates at a density of 10,000 cells/well in steroid-depleted medium. A volume of 100 nM 3β-diol or vehicle control (EtOH), with or without 1 μM OHT, 3 μM toremifene (TOR), 1 μM fulvestrant (Ful), 100 nM letrozole (Let), 100 nM anastrozole (Ana) or 100 nM exemestane (Exe), was added to each well for 4 days. Cells from each well were then harvested and counted. Values relative to the vehicle control are shown. All data are shown as mean ± SD of three independent experiments (PPT 161 kb)

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Hanamura, T., Niwa, T., Nishikawa, S. et al. Androgen metabolite-dependent growth of hormone receptor-positive breast cancer as a possible aromatase inhibitor-resistance mechanism. Breast Cancer Res Treat 139, 731–740 (2013). https://doi.org/10.1007/s10549-013-2595-x

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