Cancer Letters

Cancer Letters

Volume 452, 28 June 2019, Pages 213-225
Cancer Letters

Original Articles
Induction of breast cancer stem cells by M1 macrophages through Lin-28B-let-7-HMGA2 axis

https://doi.org/10.1016/j.canlet.2019.03.032Get rights and content

Highlights

  • M1 macrophages induce CSCs through EMT process from non-stem breast cancer cells in a paracrine manner.

  • Stat3/NF-κB-activated Lin-28B-let-7-HMGA2 axis regulates M1-induced EMT/CSC formation, with HMGA2 playing a central role.

  • Macrophages contribute to CSC formation and maintenance by orchestrating M1 and M2 phenotype.

Abstract

Proinflammatory macrophage (M1) is now being suggested as a potential therapeutic strategy for cancer because of its tumoricidal capacity. However, few studies have been focused directly on the effects of M1 macrophages on cancer cells. Here, we found that M1 induced a subpopulation of CD44high/CD24/low or ALDH1+ cells with CSC-like phenotypes from different types of breast cancer cells (BCCs) in a paracrine manner. Stat3/NF-κB pathways in BCCs were activated by proinflammatory cytokines, igniting Lin-28B-let-7-HMGA2 axis to induce CSC through epithelial-mesenchymal transition (EMT). Previously, we reported that Stat3-coordinated Lin-28B-let-7-HMGA2 axis initiated EMT in BCCs. Here, inhibition of Stat3/NF-κB pathways or Lin-28B-let-7-HMGA2 axis suppressed EMT/CSCs program. Notably, HMGA2 knockdown directly repressed M1-induced CSC formation and expression of Klf-4 and Nanog. Meanwhile, prolonged coculture with BCCs endowed M1 with M2 properties. M1 supernatant induced CSC from non-stem cancer cells, while M2 supernatant sustained a higher proportion of ALDH1+ cells. Our data suggest that macrophages might modulate CSC formation and maintenance by transferring between M1/M2 phenotype. Given that M1 are being considered as a promising immunotherapy tool, it is important to inhibit their CSC-inducing potential by targeting key molecules and pathways.

Introduction

Macrophages have been demonstrated to be the most abundant immune cells in solid tumors, comprising 5–40% of tumor mass. In early tumors, macrophages appear to possess “classically activated” (M1) phenotype to play proinflammatory tumoricidal roles [1]. As tumors are established, macrophages tend to be educated by tumor cells towards immunosuppressive “alternatively activated” phenotype (M2) to aid tumor progression [2]. Mounting evidence suggests that an elevated number of TAMs with M2 profile is correlated with therapy failure and poor prognosis in patients [[3], [4], [5], [6]], indicating TAM an important target in anti-tumor therapy [7,8]. Attempts have been made to increase M1/M2 ratio in tumors by inducing TAMs to switch from M2 to M1 or injecting polarized M1 into tumors [[9], [10], [11]], showing that increased M1 macrophages efficiently reduced tumor burden and malignancy in mouse models. Furthermore, modulation of TAMs is also required by other immunotherapies to succeed. Change of TAM phenotype to M1 augmented the anti-tumor efficacy of CD8+ T cells in lung and melanoma cancers in vivo [12,13]. Monoclonal antibodies targeting programmed cell death protein-1 (α-PD-1) have shown notable clinical efficacy in patients with various cancer. However, PD-1- TAMs could capture α-PD-1 from T cell surface and dramatically reduce the efficacy of α-PD-1 in activating T cells [14]. These data suggest that modulating TAMs would be a promising cancer therapy and also beneficial for other established immunotherapies. Though M2 phenotype has been considered to be the major phenotype of TAM, TAMs with a mixed M1/M2 phenotype are also present in patients of pancreatic cancer, T cell/histiocyte rich large B cell lymphoma and ovarian cancer [[15], [16], [17], [18]]. In pancreatic ductal adenocarcinoma (PDAC) patients, TAMs exhibiting M1 phenotype were detected and both pro- and anti-inflammatory macrophages contributed equally to EMT in PDAC initiation and development [15]. Very recently, it has also been revealed that polarized M1 macrophages enhances the metastasis potential of ovarian cancer cell lines via NF-κB pathway [19]. These evidences indicate that the pro-inflammatory signals of tumor-associated macrophages might function in tumor progression, so strategies using M1 macrophages in cancer treatment should be regarded with some caution and more studies should be focused on the effect of M1 macrophages on cancer cells.

Cancer stem cells (CSCs) are a minority subpopulation of highly tumorigenic cells in tumors, which have been identified in a wide variety of cancers [20]. It has been suggested that CSCs may arise from non-stem cancer cells (NSCCs) upon microenvironment signals [21,22]. However, the mechanisms of CSC formation still remain elusive. Ablation of macrophages in mouse mammary fat pad severely impaired the function of stem/progenitor cells in developing mammary gland and almost completely blocked tumor initiation in mouse models, suggesting a critical correlation between macrophages and CSCs [23]. M2 TAMs have been indicated to support CSC signatures [24,25]. Both M1 and M2 TAMs are observed in tumor tissues and the transient activation status is essential for macrophages to acquire M2 phenotype. Also, pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and IL-8 contributed to the establishment of CSC niche. Since CSCs are a major cause for tumor metastasis, drug resistance and relapse and M1 macrophages are being introduced into cancer treatments, it is worth investigating any potential correlation between macrophages and CSCs.

Accumulating evidence indicates that stem cell-like properties can be induced in cancer cells via epithelial-mesenchymal transition (EMT), during which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties. Previously, we reported that in breast cancer cells, Stat3-coordinated Lin-28B-let-7-HMGA2 axis effectively initiated EMT in response to inflammatory cytokine Oncostatin M (OSM) and inflammatory pathways IL-6/Stat3/Jagged-1/Notch could increase drug resistance of gastric cancer cells via EMT/CSC process [26,27].

In this study, we found that M1 macrophages contribute to the initiation of CSC phenotypic transformation in breast cancer cells. M1-associated inflammatory cytokine network triggers the expansion and self-renewal of CSCs through Lin-28B-let-7-HMGA2 axis. Our data also suggest that upon interplay with tumor cells, M1 transdifferentiate into M2 phenotype, which function in supporting CSC maintenance. This study not only sheds a new light on the potential mechanism of CSC generation, but more importantly, indicates that when M1 macrophage is utilized as a powerful tool to kill cancer cells, it is of great importance to suppress potential CSC formation and maintenance by blocking key pathways and preventing M1 from switching to M2 phenotype.

Section snippets

Cell culture

Human breast cancer cell lines MCF-7, T47D, BT474, and MDA231 were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37 °C. Human monocytic cell line THP-1 was cultured in RPMI 1640 medium supplemented with 10% FBS. Stable depletion of HMGA2 in MDA-MB-231 cells was achieved by transfection of the HMGA2 shRNA expression vector and the transfected cells were maintained in DMEM containing 10% FBS and 400 ng/ml

M1 macrophage induces CSC subpopulation in breast cancer cells

Human monocytic cell line THP-1 is widely used as a model for monocyte/macrophage differentiation. We produced polarized M1 and M2 macrophage from THP-1 and human monocytes according to established methods [28] (Supplemental Fig. 1A), which have also been used in studying the effect of polarized M1 on tumor cells both in vivo and in vitro [10,11]. THP-1 cells treated with IFN-γ/LPS firmly attached to the plates and most of the cells extended multiple elongated processes. IL-4/IL-13-induced

Discussion

Nowadays, it has been widely accepted that tumorigenesis is orchestrated by innate immunity [42]. As a major component in innate immunity and the most abundant immune cells infiltrating in solid tumor, macrophages regulate tumor initiation and progression. The phenotype of macrophage is considered to switch from proinflammatory M1 in tumorigenesis to immunosuppressive M2 since tumors are established, with its role changing from tumoricidal to pro-malignant in tumor. An increased M1/M2 ratio

Compliance with ethics guide lines

All authors declare they have no conflict of interest. This study was approved by the ethics committee of Institute of Basic Medical Sciences. All animal studies were performed in accordance with the institutional guidelines and the experiments were approved by the Animal Care and Use Committee of the institute.

Conflicts of interest

All authors read and approved the final version of the manuscript, and the authors declare no conflict interest.

Acknowledgements

Grant Support: National Key Technologies R&D Program for New Drugs (2013ZX09102056), the National High-Tech Research and Development Plan (863 Program, No. 2014AA020604), National Natural Science Foundation of China (No. 31500702, 31370825, 81272232, 81402562, 81572845, and 81401311), Beijing Natural Science Foundation (No. 7162144, 7122124, and 7132163), and China Postdoctoral Science Foundation (No. 2015T81095). The authors are grateful to all staffs who contributed to this study.

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