LRIG1 acts as a critical regulator of melanoma cell invasion, migration, and vasculogenic mimicry upon hypoxia by regulating EGFR/ERK-triggered epithelial-mesenchymal transition

Abstract

Intratumoral hypoxia is a well-known feature of solid cancers and constitutes a major contributor to cancer metastasis and poor outcomes including melanoma. Leucine-rich repeats and Ig-like domains 1 (LRIG1) participate in the aggressive progression of several tumors, where its expression is frequently decreased. In the present study, hypoxia exposure aggravated melanoma cell invasion, migration, vasculogenic mimicry (VM), and epithelial-mesenchymal transition (EMT). During this process, LRIG1 expression was also decreased. Importantly, overexpression of LRIG1 notably counteracted hypoxia-induced invasion, migration, and VM, which was further augmented after LRIG1 inhibition. Mechanism analysis corroborated that LRIG1 elevation muted hypoxia-induced EMT by suppressing E-cadherin expression and increasing N-cadherin expression. Conversely, cessation of LRIG1 further potentiated hypoxia-triggered EMT. Additionally, hypoxia stimulation activated the epidermal growth factor receptor (EGFR)/ERK pathway, which was dampened by LRIG1 up-regulation but further activated by LRIG1 inhibition. More important, blocking this pathway with its antagonist erlotinib abrogated LRIG1 suppression-induced EMT, and subsequently cell invasion, migration, and VM of melanoma cells under hypoxia. Together, these findings suggest that LRIG1 overexpression can antagonize hypoxia-evoked aggressive metastatic phenotype by suppressing cell invasion, migration, and VM via regulating EGFR/ERK-mediated EMT process. Therefore, these findings may provide a promising target for melanoma therapy.

Keywords: EGFR/ERK pathway; LRIG1; hypoxia; melanoma; metastasis; vasculogenic mimicry.

Figure 1. Hypoxia induced more aggressive phenotype in melanoma cells

(A) Cells were exposed to normoxia or hypoxia for 12 h. Then, cell invasion ability was evaluated by Transwell analysis (×200 magnification). (B) Transwell assay was performed to assess cell migration ability (×200 magnification). (C) Cells were subjected to 3D Matrigel culture for 24 h under hypoxia exposure. Then, the formation of tube-like structure was photographed under light microscope (×100 magnification). (D) Morphological changes were observed under light microscope (×200 magnification). *P<0.05 compared with normoxia group.

Figure 2. Effects of LRIG1 expression on hypoxia-augmented melanoma cell invasion and migration

(A) After exposure to hypoxia for 4, 12, 24, and 48 h, the mRNA levels of LRIG1 were detected by qRT-PCR. (B) The protein levels of LRIG1 at 24 h post incubation were analyzed by Western blotting. *P<0.05 compared with normoxia control group. (C) Cells were transfected with the recombinant plasmid of LRIG1 or empty vector, and then LRIG1 expression was determined. *P<0.05 compared with vector group. (D) After transfection with LRIG1 siRNA or si-NC, the protein expression of LRIG1 was evaluated. *P<0.05 compared with si-NC group. (E,F) Effects of LRIG1 overexpression or inhibition on hypoxia-induced melanoma cell invasion (E) and migration (F). *P<0.05 compared with control group. ^#P<0.05 compared with normoxia group.

Figure 3. LRIG1 elevation weakens, but its inhibition increased hypoxia-enhanced VM and EMT

(A) After transfection with LRIG1 plasmids or si-LRIG1, cells were then subjected to 3D Matrigel culture under hypoxia conditions for 24 h. Then, the formation of VM was observed under a light microscope. (B) The expression of VM-related marker VE-cadherin was detected by Western blotting. (C) The expression of EMT markers E-cadherin and vimentin was measured by Western blotting. *P<0.05 compared with control group. ^#P<0.05 compared with normoxia group.

Figure 4. Effects of LRIG1 on hypoxia-induced activation of the EGFR/ERK pathway

(A,B) After exposure to hypoxia for 24 h, the expression of p-EGFR, EGFR, p-ERK, and ERK was detected. *P<0.05 compared with control group. (C,D) Cells were transfected with the LRIG1 vector or si-LRIG1, prior to hypoxia exposure. Then, the activation of the EGFR/ERK pathway was evaluated. *P<0.05 compared with hypoxia group. (E) Following pretreatment with erlotinib for 2 h, the expression of E-cadherin, vimentin, and the activation of the EGFR-ERK pathway in LRIG1-silenced cells upon hypoxia was measured. (F) The effects on morphological changes were assessed under light microscope (×200 magnification). (G–I) The subsequent effects on cell invasion (G), migration (H), and VM formation (I) were also evaluated. *P<0.05 compared with hypoxia group. ^#P<0.05 compared with si-LRIG1+hypoxia group.