Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-α and MMP-9

doi:10.1016/j.canlet.2018.08.025

Cancer Letters

Volume 437, 28 November 2018, Pages 25-34

https://doi.org/10.1016/j.canlet.2018.08.025 Get rights and content

Highlights

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TAMs induced invasion in MCF10A cells non-neoplastic human breast epithelial cells.
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ERO1-α was increased in MCF10A cells co-cultured with TAM.
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ERO1-α was crucial for the TAMs-induced invasive phenotype and MMP-9 upregulation.
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CCL2 was secreted from TAMs co-cultured with MCF10A cells.
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CCL2 induced ERO1-α, resulting MMP-9 upregulation and invasiveness in MCF10A cells.

Abstract

Tumor-associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non-neoplastic MCF10A human breast epithelial cells using an indirect co-culture system. Co-culture with TAMs induced epithelial-to-mesenchymal transition, invasive phenotype, and MMP-9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1-α was increased in MCF10A cells co-cultured with TAMs compared to that in mono-cultured cells. ERO1-α was crucial for TAMs-induced invasive phenotype and MMP-9 upregulation involving transcription factors c-fos and c-Jun. Cytokine array analysis showed that levels of interleukin (IL)-6, C-X-C motif ligand (CXCL)1, C-C motif ligand (CCL)2, growth-regulated protein (GRO), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in conditioned media of co-cultured cells. Among these cytokines increased in conditioned media of co-cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1-α, MMP-9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non-neoplastic breast cells by co-culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer.

Introduction

Breast cancer is one of the most common causes of cancer mortality in women [1]. The progression of breast cancer is affected by the microenvironment surrounding the cancer [2]. Tumor microenvironment may contribute to local recurrence of breast cancer after surgery by affecting phenotypic shift of tumor-adjacent non-neoplastic breast cells toward pre-cancerous cells [3]. It has been shown that the tumor microenvironment increases tumor growth, angiogenesis, cancer cell infiltration, and cancer metastasis [4]. The tumor microenvironment is mainly composed of macrophages, immune cells, and fibroblasts. M2 type macrophages, commonly refer to as tumor-associated macrophages (TAMs), are associated with tumor malignancies [5,6]. The presence of large numbers of TAMs around the tumor is associated with poor prognosis and survival of the patient [7,8].

TAMs play essential roles in tumor development and metastasis of breast cancer [7,9,10]. They produce tumor cell recruitment factors such as matrix degrading enzymes (MMP-2, MMP-9, uPA, and cathepsin) and CCL-18, thus promoting invasion and metastasis of breast tumors [7,11,12]. Furthermore, TAMs are closely associated with blood vessels. They promote the intravasation of tumor cells into circulation, causing distant metastasis [7,10,13]. Interaction of cancer cells with TAMs has been previously investigated. TAMs co-cultured with cancer cells can promote tumor cell invasion [14], epithelial-to-mesenchymal transition (EMT) in cholangiocarcinoma cells and solid tumors [15,16] as well as breast cancer cells [17].

After surgical removal of breast cancer, local recurrence can occur presumably due to shift of tumor-adjacent histologically normal breast epithelial cells toward more aggressive phenotypes [3]. Tumor microenvironment may contribute to the phenotypic shift of tumor-adjacent non-neoplastic breast cells to pre-cancerous cells. Although the crosstalk between breast cancer cells and TAMs has been reported, information on the effect of TAMs on non-neoplastic breast epithelial cells is limited.

Therefore, the objective of the present study was to investigate the interaction between TAMs and tumor-adjacent non-neoplastic breast epithelial cells using an indirect co-culture system. We showed that TAMs induced EMT and invasive/migratory phenotypes in MCF10A human breast epithelial cells. We further demonstrated that TAMs secreted C-C chemokine ligand (CCL)2 when they were co-cultured with MCF10A cells. CCL2 then induced endoplasmic reticulum oxidoreductase (ERO)1-α, leading to upregulation of MMP-9 and invasive phenotype of MCF10A cells. This study provides molecular mechanisms on phenotypic conversion of non-neoplastic breast epithelial cells to a more aggressive phenotype by TAMs in tumor microenvironment.

Section snippets

Cell culture and reagents

MCF10A human breast epithelial cells were maintained as previously described [18]. THP-1 human monocytic cells were kindly provided by Dr. Keon Wook Kang (Seoul National University, Seoul, Korea) and cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum and 100 unit/ml penicillin-streptomycin. All cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂.

M2 differentiation

THP-1 cells (1 × 10⁶ cells/well) were seeded into the upper insert of 6-well transwell plate (0.4-μm pore size,

Co-culture with TAMs induces EMT and invasive/migratory phenotypes in MCF10A cells

Differentiation of human monocytic THP-1 cells into TAMs was induced by treatment with phorbol 12-myristate 13-acetate (PMA) [27]. Expression of CD204, a TAM marker [28], was markedly increased by PMA, indicating that THP-1 cells were differentiated into TAMs. However, levels of CD68A, a classical marker for macrophages and monocytes [29], were not altered (Fig. 1A). To investigate the effect of TAMs on breast cells, we established an indirect co-culture system of MCF10A human breast epithelial

Discussion

Since recurrent or metastatic breast cancers often exhibit poor prognosis [3], it is important to develop efficient therapeutic strategies against recurrent breast cancer after surgery. The present study aimed to elucidate molecular mechanisms underlying the acquisition of aggressive phenotypes in tumor-adjacent non-neoplastic breast epithelial cells.

Tumor microenvironment has been recognized as a promising therapeutic target for breast cancer [42]. Several recent papers suggest that

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

The present study was supported by the National Research Foundation of Korea (No. 2013R1A2A2A04013379, No. 2015M3A9B6074045, and No. 2016R1A6A1A03007648).

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¹: These authors equally contributed to this study.

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Original ArticlesTumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-α and MMP-9

Highlights

Abstract

Introduction

Section snippets

Cell culture and reagents

M2 differentiation

Co-culture with TAMs induces EMT and invasive/migratory phenotypes in MCF10A cells

Discussion

Conflicts of interest

Acknowledgements

Cell

Canc. Cell

Canc. Cell

Biomol. Ther (Seoul)

J. Invest. Dermatol.

J. Thorac. Oncol.

Int. Immunopharm.

Cell. Signal.

Canc. Lett.

Cancer statistics, 2016, CA

Cancer J. Clin.

Role of the tumor microenvironment in breast cancer

Pathobiology

Towards a personalized surgical margin for breast conserving surgery-Implications of field cancerization in local recurrence

J. Surg. Oncol.

Tumor microenvironment: the role of the tumor stroma in cancer

J. Cell. Biochem.

Diverse macrophages polarization in tumor microenvironment

Arch Pharm. Res. (Seoul)

Distinct role of macrophages in different tumor microenvironments

Canc. Res.

Tumour-associated macrophages correlate with poor prognosis in myxoid liposarcoma and promote cell motility and invasion via the HB-EGF-EGFR-PI3K/Akt pathways

Br. J. Canc.

Targeting tumor-associated macrophages as a novel strategy against breast cancer

J. Clin. Invest.

Macrophages define the invasive microenvironment in breast cancer

J. Leukoc. Biol.

IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion

Genes Dev.

Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors

Canc. Res.

Macrophages induce invasiveness of epithelial cancer cells via NF-kappa B and JNK

J. Immunol.

Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors

BMC Canc.

Cytokines released from activated human macrophages induce epithelial mesenchymal transition markers of cholangiocarcinoma cells

Asian Pac. J. Cancer Prev. APJCP

H-ras, but not N-ras, induces an invasive phenotype in human breast epithelial cells: a role for MMP-2 in the H-ras-induced invasive phenotype

Int. J. Canc.

Human endoplasmic reticulum oxidoreductin 1-α is a novel predictor for poor prognosis of breast cancer

Canc. Sci.

Using the theory of coevolution to predict protein-protein interactions in non-small cell lung cancer

Chin. J. Canc.

SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway

Sci. Rep.

Guanylate binding protein 1 inhibits osteogenic differentiation of human mesenchymal stromal cells derived from bone marrow

Sci. Rep.

Original Articles
Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-α and MMP-9