Amphiregulin Confers Regulatory T Cell Suppressive Function and Tumor Invasion via the EGFR/GSK-3β/Foxp3 Axis

Abstract

Previous studies mainly focused on the role of the epidermal growth factor receptor (EGFR) in tumor cells, whereas the effects of the EGFR on immune responses has not been determined. Our study shows that the EGFR signaling pathway play a role in the regulation of regulatory T cells (Treg cells) in cancer patients. The EGF-like growth factor Amphiregulin (AREG) protein was frequently up-regulated in a tissue microarray, which was associated with worse overall survival. Additionally, in sera, tissue specimens, and effusions of lung or gastric cancer patients, up-regulated AREG protein enhanced the suppressive function of Treg cells. AREG maintained the Treg cell suppressive function via the EGFR/GSK-3β/Foxp3 axis in vitro and in vivo Furthermore, inhibition of EGFR by the tyrosine kinase inhibitor gefitinib restored the activity of GSK-3β and attenuated Treg cell function. β-TrCP was involved in GSK-3β-mediated Foxp3 degradation, and mass spectrometry identified Lys³⁵⁶ as the ubiquitination site of Foxp3 by β-TrCP. These findings demonstrate the posttranslational regulation of Foxp3 expression by AREG in cancer patients through AREG/EGFR/GSK-3β signaling, which could lead to Foxp3 protein degradation in Treg cells and a potential therapeutic target for cancer treatment.

Keywords: GSK-3β; epidermal growth factor receptor (EGFR); forkhead box P3 (FOXP3); glycogen synthase kinase 3 (GSK-3); lung adenocarcinoma; phosphorylation; protein degradation; protein phosphorylation; regulatory T cells; ubiquitylation (ubiquitination).

FIGURE 1.

Clinical association of AREG expression level and Treg cell ratio with survival of cancer patients. A, AREG expression levels in serum, tissue specimens, and effusions from HC donors and LC and GC patients. B, mean frequencies of CD4⁺CD25^hiFoxp3⁺ Treg cells in CD4⁺ T cells from the indicated groups (top left, n = 7/group) and paired samples (top center and top right, n = 6/group). C, representative results of AREG immunoreactivity in tissue specimens of LC and GC patients. The arrows point to positive cells. D, Kaplan-Meier overall survival curves of AREG in LC and GC patients. *, p < 0.05.

FIGURE 2.

AREG in malignant effusions is required for maintaining Treg cell suppressive function. A, representative FACS results of isolated Treg cells (CD4⁺CD25^hi) from malignant effusions. The purity of Treg cells was >95%. B, the suppressive function of Treg cells is significantly impaired in malignant effusions. C, the proliferation inhibition ratio of CD4⁺CD25⁻ T (Teff) cells (from LC-PBMC) by CD4⁺CD25^hi T (Treg) cells from matched LC-pleural effusions at different dilutions. D, the proliferation inhibition ratio of CD4⁺CD25⁻ T (Teff) cells (from LC-PBMC) by CD4⁺CD25^hi T (Treg) cells from matched LC-pleural effusion at the optimal dilution (1:1), the LC-pleural effusion was pretreated with anti-TNF-α, anti-AREG, anti-TGF-α or anti-EGF, respectively. E, the proliferation inhibition ratio of CD4⁺CD25⁻ T cells by Treg cells (from matched LC-PBMC) in response to recombinant AREG protein showing a dose-dependent effect. Data are mean ± S.D. of five independent experiments. *, p < 0.05.

FIGURE 3.

Treg cells as well as tumor cells express EGFR. A, representative FACS analysis of EGFR expression in CD4⁺CD25^hi Treg cells derived from LC-PBMC and GC-PBMC. B, absolute EGFR expression is compared with β2 m expression and relative EGFR expression of CD4βCD25^hi Treg cells are compared with different CD4 T cells in PMBC groups. Treg cells and CD4⁺ T cells were sorted by flow cytometry. C, mRNA from B16, MFC, Lewis lung carcinoma tumor cells and control mice lung tissue were purified, and quantitative RT-PCR was performed. Relative EGFR expression of different tumor and control cells was the mean of ratio GAPDH:EGFR. Data are mean ± S.D. of five independent experiments. MFC, mouse forestomach carcinoma. *, p < 0.05.

FIGURE 4.

The AREG/EGFR pathway promotes tumor metastasis, Treg cell ratio, and suppressive function. A, representative results of the mouse tumor metastasis model in response to gefitinib or anti-AREG administration, as shown by pulmonary bioluminescence imaging. B, the mean lung photon flux in the mouse model of tumor metastasis in response to gefitinib or anti-AREG administration. C, the mean frequencies of Treg (Foxp3⁺CD4⁺) cells from LC-PBMC in response to gefitinib or anti-AREG administration. D, the proliferation inhibition ratio of CD4⁺CD25⁻ T cells by Treg cells (from matched LC-PBMC) in response to gefitinib or anti-AREG administration. Data are mean ± S.D. of five independent experiments. *, p < 0.05.

FIGURE 5.

AREG modulates the GSK-3β/Foxp3 axis in Treg cells. A, interaction of GSK-3β and Foxp3 in CD4⁺CD25^hi Treg cells. IP, immunoprecipitation. B, GSK-3β siRNA reduced phosphorylated Foxp3 (p270 and p274). Right panel, semiquantitative analysis of Ser²⁷⁰ and Ser²⁷⁴ phosphorylation of Foxp3 and protein expression of FLAG-Foxp3 and GSK-3β. C, measurement of Foxp3 protein half-life in control or GSK-3β siRNA knockdown CD4⁺CD25^hi Treg cells. The half-life of Foxp3 was significantly increased in GSK-3β knockdown CD4⁺CD25^hi Treg cells. CHX, cycloheximide. D and E, expression levels of GSK-3β, p-GSK-3β, and Foxp3 in CD4⁺CD25^hi Treg cells in response to recombinant AREG (100 ng/ml). AREG significantly increased phosphorylation of GSK-3β, but not total GSK-3β protein level, in CD4⁺CD25^hi Treg cells (isolated from LC- and GC-PBMC). F and G, AREG significantly increased the Foxp3 protein level, but not the Foxp3 mRNA level, in CD4⁺CD25^hi Treg cells (isolated from LC- and GC-PBMC). H, the AREG-stimulated Foxp3 protein level in CD4⁺CD25^hi Treg cells was decreased in response to gefitinib or anti-AREG administration. Data are mean ± S.D. of five independent experiments. *, p < 0.05.

FIGURE 6.

A, blockage of EGFR with gefitinib-inhibited Foxp3 expression in CD4⁺CD25^hi Treg cells derived from LC-PBMC. B, blockage of EGFR with gefitinib-inhibited GSK-3β phosphorylation in HCC827 and PC-9 cells.

FIGURE 7.

β-TrCP is involved in GSK-3β-mediated Foxp3 degradation. A, blockage of GSK-3β with siRNA decreased Foxp3 ubiquitination in HEK293T cells. IP, immunoprecipitation. B, interaction of β-TrCP with Foxp3 in HeLa and HEK293T cells. C, β-TrCP increased Foxp3 ubiquitination in the presence of GSK-3β in HEK293T cells. Ub, ubiquitin.

FIGURE 8.

The ubiquitination site of Foxp3 is Lys³⁵⁶. A, mass spectrometry identifies Lys³⁵⁶ as the ubiquitination site of Foxp3 by β-TrCP in HEK293T cells. B, the Foxp3 mutant (Lys³⁵⁶) cannot be ubiquitinated in the presence of GSK-3β. IP, immunoprecipitation.

FIGURE 9.

Schematic of the possible regulation of AREG on the EGFR/GSK-3β/Foxp3 axis.