WISP-1 promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-300 in human oral squamous cell carcinoma cells

Abstract

Oral squamous cell carcinoma (OSCC), which accounts for nearly 90% of head and neck cancers, is characterized by a poor prognosis and a low survival rate. Vascular endothelial growth factor-C (VEGF-C) has been implicated in lymphangiogenesis and is correlated with cancer metastasis. WNT1-inducible signaling pathway protein-1 (WISP)-1/CCN4 is an extracellular matrix-related protein that belongs to the CCN family and stimulates many biological functions. Our previous studies showed that WISP-1 plays an important role in OSCC migration and angiogenesis. However, the effect of WISP-1 on VEGF-C regulation and lymphangiogenesis in OSCC is poorly understood. Here, we showed a correlation between WISP-1 and VEGF-C in tissue specimens from patients with OSCC. To examine the lymphangiogenic effect of WISP-1, we used human lymphatic endothelial cells (LECs) to mimic lymphatic vessel formation. The results showed that conditioned media from WISP-1-treated OSCC cells promoted tube formation and cell migration in LECs. We also found that WISP-1-induced VEGF-C is mediated via the integrin αvβ3/integrin-linked kinase (ILK)/Akt signaling pathway. In addition, the expression of microRNA-300 (miR-300) was inhibited by WISP-1 via the integrin αvβ3/ILK/Akt cascade. Collectively, these results reveal the detailed mechanism by which WISP-1 promotes lymphangiogenesis via upregulation of VEGF-C expression in OSCC. Therefore, WISP-1 could serve as therapeutic target to prevent metastasis and lymphangiogenesis in OSCC.

Keywords: OSCC; VEGF-C; WISP-1; lymphangiogenesis; miR-300.

Figure 1. Clinical significance of WISP-1 and VEGF-C in specimens from patients with OSCC

mRNA expression levels of VEGF-C in specimens from patients with OSCC microarray datasets GSE3524 (A) and GSE2280 (B). Tumor specimens were immunostained (IHC) with anti-VEGF-C antibody. The staining intensity was scored 1–5. (C) IHC photographs (arrow shown VEGF-C staining). (D and E) Quantitative results and correlation between WISP-1, VEGF-C, and OSCC clinical grade.

Figure 2. WISP-1 promotes lymphangiogenesis through up-regulation of VEGF-C in OSCC cells

(A and B) Cells were incubated with WISP-1 (0–30 ng/mL) for 24 h, and VEGF-C expression was measured by qPCR and ELISA (n = 5). (C and D) SAS cells were incubated with WISP-1 (0–30 ng/mL) for 24 h, or pre-treated for 30 min with IgG control antibody or VEGF-C antibody (1 μg/mL) followed by stimulation with WISP-1 (30 ng/mL) for 24 h. The medium was collected as CM, then applied to LECs for 24 h, and capillary-like structure formation and in vitro cell migration in LECs were examined by assessment of tube formation and Transwell assay (n = 5). (E and F) Cells were incubated with the integrin αvβ3 antibody for 30 min, followed by stimulation with WISP-1 (30 ng/mL) for 24 h. VEGF-C expression was examined by qPCR and ELISA (n = 5). Data are expressed as mean ± SEM *P < 0.05 compared to control; #P < 0.05 compared to the WISP-1-treated group.

Figure 3. The ILK-dependent Akt signaling pathway is involved in WISP-1-induced VEGF-C expression

(A–D) Cells were pretreated for 30 min with KP-392 (3 μM) and an Akt inhibitor (10 μM) or transfected with ILK and Akt siRNA for 24 h, followed by stimulation with WISP-1 (30 ng/mL) for 24 h. VEGF-C expression was examined by qPCR and ELISA (n = 6). (E) SAS cells were incubated with WISP-1 for the indicated times. Cell lysates were prepared and immunoprecipitated with anti-ILK. Immunoprecipitated proteins were subjected to western blot analysis using anti-pGSK3β, anti-GSK3β, and anti-ILK. SAS cells were pretreated for 30 min with integrin αvβ3 antibody or KP-392 for 30 min and stimulated with WISP-1 for 10 min. ILK activity (F) and Akt phosphorylation (G) were examined by ILK kinase assay and western blotting (n = 4). Data are expressed as mean ± SEM *P < 0.05 compared to control; #P < 0.05 compared to the WISP-1 treated group.

Figure 4. WISP-1 promotes VEGF-C expression by down-regulating miR-300

(A and B) Cells were infected with WISP-1 shRNA for 24 h or incubated with WISP-1 (0–30 ng/mL) for 24 h, and miR-300 expression was examined by qPCR (n = 5). (C and D) Cells were transfected with an miRNA control or an miR-300 mimic for 24 h and stimulated with WISP-1 for 24 h. VEGF-C expression was examined by qPCR and ELISA (n = 5). (E and F) Medium was collected as CM, then applied to LECs for 24 h, and capillary-like structure formation and in vitro cell migration in LECs were examined by assessing tube formation and Transwell assay (n = 5). Data are expressed as mean ± SEM *P < 0.05 compared to control; #P < 0.05 compared to the WISP-1 treated group.

Figure 5. miR-300 directly represses VEGF-C expression via binding to the 3′-UTR of human VEGF-C

(A–C) Cells were pretreated for 30 min with integrin αvβ3 antibody, KP-392, and an Akt inhibitor or transfected with ILK and Akt siRNA for 24 h and stimulated with WISP-1 for 24 h. miR-300 expression was examined by qPCR (n = 6). (D) Schematic representation of the 3′-UTR of human VEGF-C containing a miR-300 binding site. (E) SAS cells were co-transfected with a miR-300 mimic or control miRNA and wt-VEGFA-3′-UTR or mt-VEGFA-3′-UTR plasmid for 24 h, and the relative luciferase/renilla activities were measured, as described in the Methods section (n = 4). (F and G) SAS cells were pretreated for 30 min with KP-392 and an Akt inhibitor or co-transfected with ILK and Akt siRNA for 24 h and stimulated with WISP-1 for 24 h. The wt-VEGFA-3′-UTR relative luciferase/renilla activities were measured as described in the Methods section (n = 4). Data are expressed as mean ± SEM *P < 0.05 compared to control; #P < 0.05 compared to the WISP-1 treated group.

Figure 6. Schema of signaling pathways involved in WISP-1-promoted VEGF-C expression and lymphangiogenesis in OSCC

WISP-1 induces VEGF-C expression in OSCC cells by inhibiting miR-300 expression through the integrin αvβ3/ILK/Akt pathway. WISP-1-induced VEGF-C production subsequently recruits LECs to the OSCC tumor microenvironment, promoting lymphangiogenesis.