LRIG1 inhibits hypoxia-induced vasculogenic mimicry formation via suppression of the EGFR/PI3K/AKT pathway and epithelial-to-mesenchymal transition in human glioma SHG-44 cells

Abstract

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a pan-negative regulator of the epidermal growth factor receptor (EGFR) signaling pathway. The aim of this study was to investigate the underlying mechanism of LRIG1 in the regulation of vasculogenic mimicry (VM) formation in glioma cells. We constructed an enhanced green fluorescent protein plasmid (pEGFP) system, pEGFP-C1-LRIG1, for overexpression of LRIG1, and transfected it into human glioma cell line SHG-44. Under hypoxic conditions induced by CoCl2, we investigated the effects of LRIG1 overexpression on VM formation and VM-dependent malignant behaviors including migration, invasion, and proliferation. Additionally, we explored the effects of LRIG1 on the expression levels of major components of the EGFR/PI3K/AKT pathway as well as E-cadherin and vimentin. We found that LRIG1 overexpression is able to inhibit hypoxia-induced VM formation, migration, invasion, and proliferation. Furthermore, LRIG1 overexpression counteracts hypoxia-induced increase in the expression of phosphorylated EGFR (pEGFR), PI3K (pPI3K), and AKT (pAKT) and reverts hypoxia-induced alteration in E-cadherin and vimentin expression levels. In LRIG1 knockdown SHG-44 cells, however, hypoxia-induced VM formation and alteration in E-cadherin and vimentin expression levels were exacerbated. These results suggest that the inhibitory effects of LRIG1 are most likely mediated by suppression of the EGFR/PI3K/AKT pathway and epithelial-mesenchymal transition (EMT) process. Our findings provide compelling evidence implicating LRIG1 in glioma pathophysiology, suggesting that gene therapy using LRIG1 may serve as a treatment for this disease.

Fig. 1

LRIG1 expression in SHG-44 cells transfected with pEGFP-C1-LRIG1. a LRIG1 expression in the transfected cells detected by fluorescence microscopy 24 h after transfection. b Western blot analysis of LRIG1 expression. The results were normalized to the untransfected group and presented as means ± SD (N = 4). ***P < 0.001, ANOVA followed by Tukey’s post hoc test. Bar = 25 μm

Fig. 2

LRIG1 inhibits hypoxia-induced VM formation after 24-h treatment with CoCl₂. For quantitative analysis, ten nonoverlapping fields were selected from each culture well and the experiment was performed in quadruplicate. Data are presented as means ± SEM. ***P < 0.001 vs. the normoxia group, ###P < 0.001 vs. the mock group, ANOVA followed by Tukey’s post hoc test. Bar = 20 μm

Fig. 3

LRIG1 inhibits hypoxia-induced migration after 12-h treatment with CoCl₂. For quantitative analysis, ten nonoverlapping fields were selected from each culture well and the experiment was performed in quadruplicate. Data are presented as means ± SEM. ***P < 0.001 vs. the normoxia group, ###P < 0.001 vs. the mock group, ANOVA followed by Tukey’s post hoc test. Bar = 10 μm

Fig. 4

LRIG1 inhibits hypoxia-induced invasion after 12-h treatment with CoCl₂. For quantitative analysis, ten nonoverlapping fields were selected from each culture well and the experiment was performed in quadruplicate. Data are presented as means ± SEM. ***P < 0.001 vs. the normoxia group, ###P < 0.001 vs. the mock group, ANOVA followed by Tukey’s post hoc test. Bar = 10 μm

Fig. 5

LRIG1 inhibits hypoxia-induced proliferation after 24-h treatment with CoCl₂, as measured by MTT assay. Data are expressed as means ± SEM for three independent determinations. ***P < 0.001 vs. the normoxia group, ###P < 0.001 vs. the mock group, ANOVA followed by Tukey’s post hoc test

Fig. 6

Hypoxia reduces LRIG1 expression in SHG-44 cells (a) and LRIG1 counteracts hypoxia-induced increases in the expression of pEGFR, pPI3K, and pAKT (b), as detected by Western blot. For quantitative analysis, data are shown as means ± SD (N = 4). *P < 0.05 and **P < 0.01 vs. the normoxia group, #P < 0.05 and ##P < 0.01 vs. the mock group, ANOVA followed by Tukey’s post hoc test

Fig. 7

LRIG1 reverts hypoxia-induced alteration in E-cadherin and vimentin expression levels, as detected by Western blot. For quantitative analysis, data are shown as means ± SD (N = 4). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the normoxia group, #P < 0.05 and ###P < 0.001 vs. the mock group, ANOVA followed by Tukey’s post hoc test

Fig. 8

Knockdown of LRIG1 by siRNA aggravates the effects of hypoxia on VM formation and EMT. a Western blot analysis of LRIG1 expression in LRIG1 knockdown SHG-44 cells. The results were normalized to the untransfected group and presented as means ± SD (N = 4). ***P < 0.001. b Knockdown of LRIG1 exacerbates hypoxia-induced VM formation after 24-h treatment with CoCl₂. For quantitative analysis, ten nonoverlapping fields were selected from each culture well and the experiment was performed in quadruplicate. Data are presented as means ± SEM. ***P < 0.001 vs. the normoxia group, #P < 0.05 vs. the mock group. Bar = 20 μm. c Knockdown of LRIG1 aggravates hypoxia-induced alteration in E-cadherin and vimentin expression levels, as detected by Western blot. For quantitative analysis, data are shown as means ± SD (N = 4). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the normoxia group, ##P < 0.01 vs. the mock group. All statistical analyses were performed using ANOVA followed by Tukey’s post hoc test

Fig. 9

Hypothetical scheme of the proposed mechanism for the inhibitory effects of LRIG1