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Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma

  • Preclinical study
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Abstract

Purpose

The tumor microenvironment plays pivotal roles in promotion of many malignancies. Cancer-associated fibroblasts (CAFs) have been well-known to promote proliferation, angiogenesis, and metastasis but mechanistic understanding of tumor–stroma interactions is not yet complete. Recently, estrogen synthetic enzymes were reported to be upregulated by co-culture with stromal cells in ER positive breast carcinoma (BC) but effects of co-culture on androgen metabolism have not been extensively examined. Therefore, we evaluated roles of CAFs on androgen metabolism in ER-negative AR-positive BC through co-culture with CAFs.

Methods

Concentrations of steroid hormone in supernatant of co-culture of MDA-MB-453 and primary CAFs were measured using GC–MS. Cytokines derived from CAFs were determined using Cytokine Array. Expressions of androgen synthetic enzymes were confirmed using RT-PCR and Western blotting. Correlations between CAFs and androgen synthetic enzymes were analyzed using triple-negative BC (TNBC) patient tissues by immunohistochemistry.

Results

CAFs were demonstrated to increase expressions and activities of 17βHSD2, 17βHSD5, and 5α-Reductase1. IL-6 and HGF that were selected as potential paracrine mediators using cytokine array induced 17βHSD2, 17βHSD5, and 5α-Reductase1 expression. Underlying mechanisms of IL-6 paracrine regulation of 17βHSD2 and 17βHSD5 could be partially dependent on phosphorylated STAT3, while phosphorylated ERK could be involved in HGF-mediated 5α-Reductase1 induction. α-SMA status was also demonstrated to be significantly correlated with 17βHSD2 and 17βHSD5 status in TNBC tissues, especially AR-positive cases.

Conclusions

Results of our present study suggest that both IL-6 and HGF derived from CAFs could contribute to the intratumoral androgen metabolism in ER-negative BC patients.

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Abbreviations

CAFs:

Cancer-associated fibroblasts

ER:

Estrogen receptor

AR:

Androgen receptor

PR:

Progesterone receptor

HER2:

Human epidermal growth factor receptor

GC–MS:

Gas chromatography–mass spectrometry

RT-PCR:

Reverse transcription polymerase chain reaction

TNBC:

Triple-negative breast carcinoma

17βHSD:

17β hydroxysteroid dehydrogenase type

5α-Red1:

5α-Reductase1

IL-6 (-8):

Interleukin-6 (-8)

HGF:

Hepatocyte growth factor,

STAT3:

Signal transducer and activator of transcription 3

ERK:

Extracellular signal-regulated kinase

IDC:

Invasive ductal carcinoma

α-SMA:

Α-smooth muscle actin

FBS:

Fetal bovine serum

PAI-1:

Plasminogen activator inhibitor-1

MCP-1:

Monocyte chemotactic protein-1

CM:

Conditioned medium

siRNA:

Small interfering RNA

DHT:

Dihydrotestosterone

FASN:

Fatty acid synthase

PRLR:

Prolactin receptor

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Acknowledgements

Research for this article was supported in part by JSPS KAKENHI Grant Number 15K18396. We would like to acknowledge all the members of their laboratories, whose informal input was extremely valuable. We would also like to acknowledge the support and assistance of the members of the Department of Pathology, Tohoku University School of Medicine.

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Authors and Affiliations

  1. Department of Pathology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-Ku, Sendai-shi, Miyagi, 980-8575, Japan

    Kyoko Kikuchi, Keely May McNamara, Fumiya Omata, Minako Sakurai, Yoshiaki Onodera & Hironobu Sasano

  2. Department of Disaster Obstetrics and Gynecology, International Research Institute of Disaster Science (IRIDeS), Tohoku University, 2-1, Seiryo-machi, Aoba-Ku, Sendai-shi, Miyagi, 980-8575, Japan

    Yasuhiro Miki

  3. Molecular Recognition Research Center, Korea Institute of Science and Technology, 5, Hwarang-ro 14-gil, Seoul, 02792, Korea

    Ju-Yeon Moon & Man Ho Choi

  4. Sagara Hospital, Social Medical Corporation Hakuaikai, Kagoshima, Japan

    Yoshiaki Rai, Yasuyo Ohi & Yasuaki Sagara

  5. Department of Breast and Endocrine Surgical Oncology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-Ku, Sendai-shi, Miyagi, 980-8575, Japan

    Minoru Miyashita, Takanori Ishida & Noriaki Ohuchi

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  1. Kyoko Kikuchi
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Correspondence to Keely May McNamara.

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This study was approved by Ethics Committee at Tohoku University School of Medicine. Informed consent was obtained from all patients.

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Supplementary material 1 (DOC 45 kb)

Supplementary material 2 (PDF 79 kb)

Supplementary Fig. 1 CAFs induced ER negative AR positive breast carcinoma cell lines proliferation, migration, and invasion. (A) Carcinoma cell lines (MDA-MB-453, MFM-223, and SUM-185-PE) were incubated with conditioned medium (CM) derived from CAFs (TH-BC24 N and TH-BC26 N) or carcinoma cells or non-CM as negative control for 96 h. Changes of cell viability were evaluated using WST-7 assay. (B) MFM-223 cells were seeded migration or invasion chamber while CAFs were seeded in 24 well plate. After 24 h, MFM-223 cells were cultured in the absence or presence of CAFs for 24 h. The migrated or invaded cells were fixed and stained with hematoxylin. Migration or invasion rate were evaluated as the average number of cells three fields (× 200) randomly selected on the lower surface on the membrane. *, p < 0.05

10549_2017_4464_MOESM3_ESM.pdf

Supplementary material 3 (PDF 138 kb)

Supplementary Fig. 2 Androgen synthetic enzymes were regulated by co-culture with CAFs in ER negative breast carcinoma cells. (A) 5α-Reductase type (5α-Red) 1 and 5α-Red2 expression levels in ER negative breast cancer cell lines (MDA-MB-453, MFM-223, and SUM-185-PE) were determined using RT-PCR. (B) RT-PCR for androgen responsive genes fatty acid synthase (FASN) and prolactin receptor (PRLR) was performed from mRNA harvested from MDA-MB-453 and MFM-223 treated with control, 10 nM dihydrotestosterone (DHT), and 10 nM DHT + androgen receptor inhibitor (Bic; Bicalutamide) 10 μM for 72 h. Worth noting is the potential for a known AR mutation to alter the androgen responsiveness of the MDA-MB-453 and MFM-223 cell lines to DHT [15] although if this is the case was not assessed in this study. (C) AR mRNA expressions in ER negative breast carcinoma cells after co-culture with CAFs were determined using RT-PCR. (D) Aromatase mRNA expression in MDA-MB-453 after co-culture with CAFs was examined using RT-PCR. *, p < 0.05

Supplementary material 4 (PDF 3412 kb)

Supplementary Fig. 3 Secretions of various cytokines derived from CAFs were determined using cytokine array. (A) Cytokines were secreted from two CAFs to their conditioned medium (CM), TH-BC24 N (upper) and TH-BC26 N (bottom). Membrane images were detected using ChemiDoc XRS + System. (B) Quantification of cytokines secreted from CAFs. Membrane images were quantified using mage Lab version 5.0. (C) mRNA levels of androgen synthetic enzymes in ER negative breast carcinomas after 72 h PAI-1, MCP-1, or IL-8 treatment were determined using RT-PCR. (D) MDA-MB-453 was incubated with IL-6 or HGF with or without testosterone and androgen receptor inhibitor (bicaltamide) in serum-free medium for 72 h. Changes of cell viability were evaluated using WST-7 assay. NC; negative control, T; testosterone, Bic; bicaltamide, *, p < 0.05

Supplementary material 5 (PDF 51 kb)

Supplementary Fig. 4 Androgen synthetic enzymes were upregulated by androgen treatment in ER negative breast carcinoma cells. RT-PCR for androgen synthetic enzymes was performed from mRNA harvested from MDA-MB-453 treated with control, 10 nM dihydrotestosterone (DHT), and 10 nM DHT + androgen receptor inhibitor (Bic; bicalutamide) 10 μM for 72 h. *, p < 0.05

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Kikuchi, K., McNamara, K.M., Miki, Y. et al. Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma. Breast Cancer Res Treat 166, 709–723 (2017). https://doi.org/10.1007/s10549-017-4464-5

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