Abstract
The tumour microenvironment is a key regulators of tumour progression through the secretion of growth factors that activate epithelial-mesenchymal transition (EMT). Induction of EMT is a key step for transition from a benign state to a metastatic tumour. Adipose tissue forms a bulk portion of the breast cancer microenvironment, emerging evidence indicates the potential for adipocytes to influence tumour progression through the secretion of adipokines that can induce EMT. The molecular mechanisms underlying how adipocytes enhance breast cancer progression is largely unknown. We hypothesized that paracrine signalling by adipocytes can activate EMT and results in increased migration and invasion characteristics of breast cancer cells. We found that IL-6 secreted by adipocytes induce EMT in breast cancer cells. The effect of IL-6 expression on EMT is mediated through activation of the signal transducer and activated of transcription 3 (STAT3). Blocking of IL-6 signalling in breast cancer cells and adipocytes, decreased proliferation, migration and invasion capabilities and altered the expression of genes regulating EMT. Together, our results suggest that matured human adipocytes can enhance the aggressive behaviour of breast cancer cells and induce an EMT-phenotype through paracrine IL-6/STAT3 signalling.
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Introduction
Breast cancer is the most common malignancy and a leading cause of cancer-related death in women worldwide1. Presently, treatment and management of breast cancer has significantly improved with increased understanding to the disease biology. In breast cancer, the tumour microenvironment is dominated by stromal cells such as fibroblasts, endothelial cells, immune cells and adipocytes2,3. Recent studies have focused on elucidating the mechanisms by which growth factors secreted by components of the tumour microenvironment enhance tumour progression. Epithelial-mesenchymal transition (EMT) is a key process hypothesized to be activated by tumour microenvironment that enhance tumour procession. Epithelial-mesenchymal transition (EMT) is a well-documented molecular event that enhance invasion and metastasis of breast cancer cells4,5. The EMT process in cancer cells is characterised by tumour epithelial cells undergoing molecular and genetic changes resulting in the loss of cell junctions, apical-basal polarity, acquisition of a mesenchymal phenotype with enhanced migration and invasion potential6. Epithelial cells express specific proteins such as E-cadherin’s, occludins and cytokeratin; however, during EMT, epithelial cells decrease expression of E-cadherin and increase the expression of mesenchymal phenotype specific proteins such as N-cadherin’s and vimentin6,7. Regulation of EMT is associated with aberrant expression of usually repressed transcriptional factors such as snail homolog 1 (SNAIL), twist basic helix-loop-helix transcription factor (TWIST), FOXC2, ZEB1 and ZEB28. In breast cancer, the EMT phenotype is associated with increased cell motility, invasion and enhanced metastasis9. The components of the tumour microenvironment have emerged as key contributors to tumourigenesis. Paracrine interaction between stromal and breast cancer cells have been shown to enhance the metastatic potential breast cancer cells2,3. Several signalling pathways activated in the tumour microenvironment are essential regulators of EMT7,10,11.
Adipose tissues are the most abundant tissues in the breast cancer microenvironment, initially regarded as providing support, insulation and serving as site for energy storage12,13. The potential for adipocytes to influence breast cancer cells migration and invasion, and ultimately result in metastasis has begun to emerge12,14,15. With various studies focused on determining how paracrine signalling by adipocytes enhance breast cancer progression. The secretion of hormones, growth factors and cytokines (collectively referred to as adipocytokines) by adipocytes have been hypothesized to activate various signalling pathways in the nearby tumour cells resulting in increased migration and invasion in breast cancer cells16. Among the growth factors secreted by adipocytes, transforming growth factor-beta (TGF-β) and interleukin-6 (IL-6) have been independently proven to be potent regulators of EMT in various cancer cells7,10,17,18. TGF-β through the SMADs transcription factors can induce EMT, invasion and migration in epithelial cells and breast cancer cells18,19. The pleotropic cytokine, IL-6 is highly expressed in adipose tissue and play a multifactorial role in cancer, influencing EMT, metastasis, angiogenesis, cachexia, stemness and therapeutic resistance20,21,22. Addition of synthesized IL-6 to breast cancer cells was demonstrated to induced EMT via activation of the signal transducer and activated of transcription 3 (STAT3)7,1.
Western blotting
Harvested cells were lysed in RIPA buffer (150 mM sodium chloride, 1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5 and 2 mM EDTA) (GenDEPOT, TX, USA) containing 1% protease inhibitor cocktail (GenDEPOT). 20 μg of each sample is separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK). Western blotting was performed as previously described by Liu et al.38 with primary antibodies to mouse anti-E-cadherin 1:1000 (Cell Signalling, Danvers, MA, USA), rabbit anti-Vimentin 1:500 (Abcam, Cambridge, UK), rabbit anti-Zeb1 1:500 (Sigma), rabbit anti-IL-6 1:1000 (Abcam), rabbit anti-STAT3 1:500 (phosphor Y705: Abcam), mouse anti-STAT3 1:1000 (Cell signalling), rabbit anti-α-lamin 1:1000 and goat anti-β-actin 1:5000 (Santa Cruz Biotechnology, Santa Cruz, USA). All primary antibodies were diluted in 5% Bovine Serum Albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C. Secondary antibody included goat anti-rabbit IgG-HPR 1:5000 (Santa Cruz Biotechnology), goat anti-mouse IgG-HPR 1:2000 (Santa Cruz Biotechnology) and rabbit anti-goat IgG-HPR (GenDepot, TX, USA). Protein bands were visualised using enhanced chemiluminescence reagents (Western Lighting Plus, PerkinElmer, USA).
Immunofluorescence staining
Cells were seeded on cover slide placed in co-culture insert and cultured with/without adipocytes for 48 hr. Cells was rinsed in PBS and fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with appropriate primary antibodies. For double staining experiments, antibodies were diluted together and incubated with cells overnight at 4 °C. Goat Anti-Rabbit IgG (Alexa Fluor 647) and Goat Anti-Mouse IgG (Alexa Fluor 488) antibodies (Abcam) were used as secondary antibodies. Counter staining of cell nuclei was performed using DAPI (Invitrogen, Carlsbad, CA, USA). Stained cells were visualized using the ZEISS LSM 710 microscope (ZEISS, Germany). Antibodies used included mouse anti-E-cadherin 1:500 (Cell Signalling, Danvers, MA, USA), rabbit anti-Vimentin 1:500 (Abcam, Cambridge, UK), mouse anti-STAT3 (dilution 1:200) (Cell Signalling Technology) and rabbit anti-P-STAT3(Y705) (dilution 1:500) (Abcam).
Transfections and Luciferase reporter assays
MDA-MB-468 and MCF-7 breast cancer cells were seeded at 1 × 106 cells in a 100 mm dish in antibiotic free media overnight and transfected with 20 nM IL-6R siRNA from On-target Plus Smart Pool (Dharmacon, Lafayette, CO, USA) or control siRNA (Dharmacon) as previous described by Rosner et al.39 using lipofectamine RNAiMAX reagent (Invitrogen). Experiments with siRNA transfected cells were conducted 72 hrs after transfection and co-culture with human adipocytes. Differentiated human adipocytes was also transfected with 20 nM IL-6R siRNA from On-target Plus Smart Pool (Dharmacon, Lafayette, CO, USA). The transfection efficiency was determined by western blot analysis.
MDA-MB-468 and MCF-7 were transfected with STAT3 reported plasmid (Cignal Lenti STAT3 Reporter, QIAGEN, Hilden, Germany) using SureENTRY transduction reagent (Qiagen). Stable STAT3 reporting cells were selected with 400 µg/ml of puromycin (Sigma) over 10 days to generate a stable STAT3 reporter cell line for MDA-MB-468 and MCF-7. STAT3 promoter activity was determined by Promega Dual-Luciferase reporter assay system (Promega corporation, Madison, USA) and luciferase activity measured in the Tecan™ microplate-Luminometer (Tecan Group limited, Männedorf, Switzerland). The constitutively expressed non-inducible Renilla luciferase activity served as internal control for normalizing transfection efficiencies.
Adipocytes IL-6 Neutralisation
To differentiated human adipocytes in 6-well plate, 400 ug/ml IL-6 antibody (Abcam) was added to adipocytes media to neutralise IL-6 secreted by adipocytes.
Statistical analysis
Data were analysed and graphs plotted with Graphpad Prism version 6 software (GraphPad Inc.). Student’s t-test was used to compare differences between two groups and multiple analysis was performed using analysis of variance (ANOVA). Multiple analysis of groups was checked for after ANOVA using Bonferroni’s multiple comparison test. Statistical significance was defined as P < 0.05.
Change history
29 July 2020
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Acknowledgements
The authors thank Haerin Jang and Juwon Kang of the Laboratory of Translational cancer research, Yonsei University for their generous assistance. This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF 2014R1A1A1002443, 2016M3C7A1913844, NRF 2017R1D1A1B03033362 and NRF 2018R1A6A1A03023718) and the Faculty research grant of Yonsei University, Wonju College of Medicine.
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J.C. and J.G. conceived and designed experiments, Y.H.L. isolated and characterised human adipose stem cells. J.G. and J.C. performed other experiments, J.G. and J.C. analysed data. J.G. and J.C. edited and/or drafted the manuscript. J.C. and Y.H.L. supervised the study. All authors have read and approved the final version of the manuscript.
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Gyamfi, J., Lee, YH., Eom, M. et al. Interleukin-6/STAT3 signalling regulates adipocyte induced epithelial-mesenchymal transition in breast cancer cells. Sci Rep 8, 8859 (2018). https://doi.org/10.1038/s41598-018-27184-9
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DOI: https://doi.org/10.1038/s41598-018-27184-9
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