HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk
Yingying Wang, … , Jinsong Lu, Jianhua Wang
Yingying Wang, … , Jinsong Lu, Jianhua Wang
Published September 11, 2018
Citation Information: J Clin Invest. 2018;128(12):5235-5250. https://doi.org/10.1172/JCI99974.
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Research Article Cell biology Oncology

HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk

Abstract

Breast cancer (BrCa) is the malignant tumor that most seriously threatens female health; however, the molecular mechanism underlying its progression remains unclear. Here, we found that conditional deletion of hypermethylated in cancer 1 (HIC1) in the mouse mammary gland might contribute to premalignant transformation in the early stage of tumor formation. Moreover, the chemokine (C-X-C motif) ligand 14 (CXCL14) secreted by HIC1-deleted BrCa cells bound to its cognate receptor GPR85 on mammary fibroblasts in the microenvironment and was responsible for activating these fibroblasts via the ERK1/2, Akt, and neddylation pathways, whereas the activated fibroblasts promoted BrCa progression via the induction of epithelial-mesenchymal transition (EMT) by the C-C chemokine ligand 17 (CCL17)/CC chemokine receptor 4 (CCR4) axis. Finally, we confirmed that the HIC1-CXCL14-CCL17 loop was associated with the malignant progression of BrCa. Therefore, the crosstalk between HIC1-deleted BrCa cells and mammary fibroblasts might play a critical role in BrCa development. Exploring the progression of BrCa from the perspective of microenvironment will be beneficial for identifying the potential prognostic markers of breast tumor and providing more effective treatment strategies.

Authors

Yingying Wang, Xiaoling Weng, Luoyang Wang, Mingang Hao, Yue Li, Lidan Hou, Yu Liang, Tianqi Wu, Mengfei Yao, Guowen Lin, Yiwei Jiang, Guohui Fu, Zhaoyuan Hou, Xiangjun Meng, Jinsong Lu, Jianhua Wang

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Figure 6

CXCL14-activated fibroblasts induce migration of BrCa cells via CCL17.

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CXCL14-activated fibroblasts induce migration of BrCa cells via CCL17. (...
(A) Upper panel: schematic showing the coculture of MDA-231-LM2 BrCa cells with primary NAF10, CXCL14-activated NAF10, or primary CAF10 cells in a Transwell apparatus (0.4 μm pore size) for 4 days. Lower panel: Boyden chamber assay of MDA-231-LM2 cells that were treated as above. (B) Upper panel: Human XL Cytokine Array Kits (R&D Systems) were used to measure the levels of 102 cytokines in the CM from diverse fibroblasts. Cytokines that were upregulated in the CM of CXCL14-activated NAF10 and CAF10 cells are indicated by colored boxes; they include CCL17 (red), IL-5 (green), and angiopoietin-2 (blue). Black frames indicate the positive controls, and the dashed boxes indicate the negative controls in each membrane. Lower panel: table showing the relative signal intensities of the 3 selected cytokines noted above. The signal intensities were quantified by densitometry using ImageJ software and normalized to the intensity of the internal positive controls. (C) Boyden chamber assay of MDA-231-LM2 cells plated with rhCCL17, rhIL-5, and rh angiopoietin-2 in the lower chambers at 100 ng/ml for 20 hours. (D) Boyden chamber assay of MDA-231-LM2 cells that were cocultured with primary NAF10, CXCL14-activated NAF10, or primary CAF10 cells in a Transwell apparatus for 4 days in the presence or absence of α-CCL17 (1 μg/ml) or an isotype-matched IgG control. (E) MCF7 or MDA-231-LM2 cells were treated with various concentrations (0–100 ng/ml) of rhCCL17 for 4 days, and lysates of the cells were analyzed by Western blot using antibodies against E-cadherin, N-cadherin, and GAPDH. (F) MCF7 or MDA-231-LM2 cells were treated with rhCCL17 at 100 ng/ml for the indicated times (0, 5, 15, 30, and 60 minutes). Cell lysates were analyzed by Western blot with antibodies against p-Akt (Ser 473), Akt, p-GSK-3β (Ser9), GSK-3β, and GAPDH. (G) Knockdown of CCR4 expression by siRNA-3 in MCF7 and MDA-231-LM2 cells in the presence or absence of rhCCL17 at 100 ng/ml for 4 days. Cell lysates were analyzed by Western blot with antibodies against E-cadherin, N-cadherin, p-Akt (Ser 473), Akt, and GAPDH. Data are shown as mean ± SEM. n = 3 independent experiments. ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test.