TSG6 promotes epithelial-mesenchymal transition and tumor-associated macrophage polarization through Smad2/3 and MAPK signaling by facilitating TSG6-CD44-TGFβR1 or EGFR complex formation

Abstract

TSG6 is highly expressed during PLK1-induced epithelial-mesenchymal transition (EMT). However, the role of TSG6 in the tumor microenvironment (TME) remains poorly understood. We investigate the function and regulatory mechanisms of TSG6 in immune plasticity within the TME of lung adenocarcinoma (LUAD). The simultaneous high expression of TSG6 and PLK1 in LUAD patients was associated with lower survival rates. TSG6 and CD44 were markedly upregulated during EMT driven by TGF-b or active PLK1 in A549 and HCC827 cells. TSG6 treatment enhanced EMT by increasing N-cadherin and phosphorylated Smad2 levels. TSG6 depletion blocked the effects, which was restored upon TSG6 retreatment. Additionally, TSG6 treatment induced polarization of THP-1 monocytes into M2d tumor-associated macrophages (TAMs). In cocultures of THP-1 monocytes with A549 cells expressing TSG6, M2d-inducing factors in A549 cells and M2d markers in THP-1 cells were upregulated. Immunoprecipitation showed that TSG6 binds CD44, enhancing CD44's interaction with TGFbR or EGFR. In TSG6-treated LUAD cells, both total CD44 and its cleaved intracellular domain increased by activating TGFβR1-Smad2/3 and MAPK-ERK1/2-AP-1 pathways. Thus, TSG6 promotes EMT and M2d-TAMs polarization by activating TGFβR1/Smad and MAPK/ERK pathway through direct interaction between CD44 and TGFβR1 or EGFR.

Keywords: CD44; TSG6; invasiveness; tumor-associated macrophages.

Figure 1

Clinical correlation between the survival rate of patients and the expression of TSG6 and PLK1 in lung adenocarcinoma (LUAD). (A) Analysis of Spearman's and Pearson's correlation coefficients between the expression of TNFAIP6 and PLK1 in LUAD patients using cBioportal. (B-C) The overall survival (OS) of LUAD patients (n = 656, Log-rank P = 1e-04) (B) and the progression-free survival (PFS) of LUAD patients (n = 115, Log-rank P = 0.03) (C) were analyzed according to the expression of TNFAIP6 and PLK1 using KM PLOTTER. High (Hi) and low (Lo) were generated by dividing patients according to their expression at the median cut-off. (D) A heatmap analysis was performed using a dataset of LUAD patients from TCGA for the expression of TNFAIP6, PLK1, epithelial-mesenchymal transition markers (EMT), and cytokines and chemokines markers in paired normal (left) and tumor tissues (right) depending on stages. (E) The ratio of increased expression of TNFAIP6, PLK1, and EMT markers (CDH2, SNAI1, and SNAI2) and (F-G) cytokine and chemokine markers (IL1A, IL6, CXCL1, CXCL8, VEGFA, and VEGFB) in tumor tissue compared to adjacent normal tissue were plotted.

Figure 2

Co-expression of TSG6 and PLK1 in TGF-β induced EMT of LUAD cells. (A) The gene expression in the GSE114761 dataset in which EMT was induced by treating the LUAD cell line with TGF-β was visualized as a heatmap (left panel), and the ratio of increased expression of each factor in the TGF-β-treated group compared to the control group was plotted (right panel). (B-D) A549 and HCC827 LUAD cells were treated with 2.5 ng/ml of TGF-β for 48 hours. (B) Immunoblotting was performed to measure the protein levels of TSG6, p-PLK1, PLK1, Vimentin, N-cadherin, E-cadherin, CD44, and GAPDH in A549 (left panel) and HCC827 (right panel) cells. The relative band intensities were analyzed and plotted in A549 (right upper panel) and HCC827 (right lower panel) cells. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (C-D) TNFAIP6, PLK1, HAS2, CD44, CDH2, VIM, and CDH1 expression was measured in in A549 (C) and HCC827 (D) cells by qRT-PCR. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3).

Figure 3

Expression of TSG6 in an active PLK1-driven metastatic lung cancer mouse model. (A) Representative TSG6 staining was performed using lung tissue from mice transplanted with wild-type (WT), a constitutively active T210D (TD), and a substrate-nonbinding mutant at W414F/V415A (FA) of the polo-box domain of PLK1 (left panel). The relative density of TSG6 staining was analyzed and plotted (right panel). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 8). (B) Representative CD44 staining was performed using lung tissue from mice transplanted by various versions of PLK1 (left panel), and the relative density of CD44 staining was analyzed and plotted (right panel). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 8). (C) Representative TSG6 staining was performed on the lung tissue of mice transplanted with active PLK1-treated volasertib (PLK1 inhibitor) (left panel), and the relative density of TSG6 staining was analyzed and plotted (right panel). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 8). (D) Representative CD44 staining was performed using lung tissue from mice transplanted with active PLK1-treated volasertib (PLK1 inhibitor) (left panel), and the relative density of CD44 staining was analyzed and plotted (right panel). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 8).

Figure 4

Recombinant TSG6 protein induced EMT by activation of TGF-β signaling. (A-B) A549 and HCC827 cells were treated with 200 ng/ml of rhTSG6 for 2 hours. (A) Immunoblotting was performed to measure the protein levels of TSG6, PLK1, p-PLK1^T210, E-cadherin, N-cadherin, Vimentin, CD44, p-Smad2^S465/S467, Smad2 and GAPDH in A549 (left panel) and HCC827 (right panel) cells. (B) qRT-PCR was used to evaluate CDH1, CDH2, CD44, PLK1, SMAD2, and VIM expression in A549 (right upper panel) and HCC827 (right lower panel) cells. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (C-F) TSG6 shRNA targeting the positions 530-550 (#1) or 693-713 (#2) was applied to A549 and HCC827 cells. (C) Immunoblotting was performed to measure the protein levels of TSG6, PLK1, CD44, N-cadherin, Smad2/3, p-Smad2^S465/S467, and β-actin in A549 (left panel) and HCC827 (right panel) cells. (D) qRT-PCR was used to evaluate TNFAIP6, PLK1, CD44, CDH1, VIM, SMAD2, and CDH2 expression in A549 (upper panel) and HCC827 (lower panel) cells. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (E-F) qRT-PCR was used to evaluate TNFAIP6, PLK1, CD44, CDH1, VIM, SMAD2, and CDH2 expression in A549 (E) and HCC827 (F) cells after rhTSG6 treatment. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3).

Figure 5

TSG6 induced the polarization of tumor-associated macrophages. (A) Monocyte THP-1 cells were treated with 200 ng/ml of rhTSG6 for 8 hours, and qRT-PCR was used to measure TNFAIP6, TGFB1, VEGFA (M2d macrophage markers), CD206, CD163 (M2 macrophage markers), IL12B, and iNOS (M1 macrophage markers) expression. (B-C) TSG6- overexpressing A549 cells and monocyte THP-1 cells were cocultured. (B) qRT-PCR was used to evaluate TNFAIP6, VEGFA, IL6, IL4 and IL10 expression in A549 cells. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (C) qRT-PCR was used to evaluate VEGFA, TGFB1 (M2d macrophage markers), CD206, CD163 (M2 macrophage markers), IL12B, and iNOS (M1 macrophage markers) expression in THP-1 cells. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (D-E) TSG6-depleted A549 cells and monocyte THP-1 cells were cocultured. (D) qRT-PCR was used to evaluate TNFAIP6, VEGFA, IL6, IL4 and IL10 expression in A549 cells. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (E) qRT-PCR was used to evaluate VEGFA, TGFB1, CD206, CD163, IL12B, and iNOS expression in THP-1 cells. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3).

Figure 6

TAM polarization was induced in active PLK1-driven metastatic lung cancer of a mouse model. (A) Representative CD68 (pan-macrophage marker) staining was performed on lung tissue of mice transplanted with various versions of PLK1 (left panel), and the relative density of CD68 staining was analyzed and plotted (right panel). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 8). (B) Representative CD163 (M2 macrophage marker) staining was performed on the lung tissue of mice transplanted with various versions of PLK1 (left panel), and the relative density of CD163 staining was analyzed and plotted (right panel). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 8). (C) Representative CD68 staining was performed on the lung tissue of mice transplanted with cells containing active PLK1 treated with volasertib (PLK1 inhibitor) (left panel), and the relative density of CD68 staining was analyzed and plotted (right panel). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 8). (D) Representative CD163 staining was performed using lung tissue from mice transplanted with cells containing active PLK1-treated volasertib (PLK1 inhibitor) (left panel), and the relative density of CD163 staining was analyzed and plotted (right panel). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 8).

Figure 7

MAPK/ERK and TGFβR1/Smad signaling are the main pathways for TSG6-mediated EMT and TAM polarization. (A) A549 and HCC827 cells were treated with 200 ng/ml of rhTSG6 for 2 hours. Immunoblotting was performed to measure the protein levels of CD44, cleaved CD44 (CD44-ICD), MMP9, p-ERK1/2, ERK1/2, c-Jun, c-Fos, IL-6, and GAPDH in A549 (left panel) and HCC827 (right panel) cells. (B-C) A549 cells were treated with 10 μM trametinib (MEK1/2 inhibitor) for 48 hours and treated with 200 ng/ml of rhTSG6 for 2 hours. (B) Immunoblot was performed to measure the protein levels of TSG6, p-ERK1/2, ERK1/2, vimentin, IL-6, CD44, CD44-ICD, and GAPDH, and the relative band intensity values were analyzed in A549 cells (Sup Fig. S4). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (C) qRT-PCR was used to evaluate TNFAIP6, VEGFA, IL6, CD44, VIM, CDH1, and CDH2 expression in A549 cells. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (D-F) A549 cells were treated with 30 μM SB431542 (TGFβR1 inhibitor) for 48 hours and with 200 ng/ml of rhTSG6 for 2 hours. (D) Immunoblot was performed to measure the protein levels of TSG6, p-Smad2, Smad2/3, vimentin, IL-6, CD44, CD44-ICD, and GAPDH (D), and the relative band intensity values were analyzed in A549 cells (E). *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3). (F) Expression of TNFAIP6, VEGFA, IL6, CD44, VIM, CDH1, and CDH2 in A549 cells was measured by qRT-PCR. *p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3).

Figure 8

TSG6 facilitates the interaction between CD44 and TGFβR or EGFR for the expression of genes involved in EMT and TAM polarization. (A) A549 cells were treated with 200 ng/ml of rhTSG6 for 2 hours. Immunoprecipitation and immunoblotting were used to measure the protein levels of CD44, CD44-ICD, TGFβR1, EGFR, and GAPDH in A549 cells. (B) Fractionation of A549 cells treated with rhTSG6 was used to observe the levels of nuclear and cytosolic fraction. Immunoblotting was used to measure the protein levels of CD44, CD44-ICD, c-Jun, c-Fos, Smad2/3, Histone H1 (nuclear loading control), and GAPDH (cytosolic loading control). (C) Nuclear fraction of A549 cells treated with 200 ng/ml of rhTSG6 for 2 hours was used for immunoprecipitation and immunoblotting analysis. Immunoblot analyses were performed using anti-CD44, anti-c-Fos, and anti-c-Jun antibodies. (D-F) ChIP assays for AP-1 (complex of c-Jun and c-Fos) binding to the promoters of VEGFA (D), IL6 (E), and TNFAIP6 (F) in rhTSG6-treated A549 cells. (G-I) ChIP assays for Smad2/3 binding to the promoters of CD274 (G), SNAI1 (H), and SNAI2 (I) in rhTSG6-treated A549 cells. Assays were performed on chromatin fragments using antibody to c-Jun (D-F) or Smad2/3 (G-I) and normalized to pre-immune normal IgG. Immunoprecipitated fractions were assayed by qRT-PCR to determine the binding to each promoter. Data are presented as mean ± SD of three independent experiments (significantly different from the experimental control). *p < 0.05; **p < 0.01; ***p < 0.001; (n = 3).