Targeting the VEGF-C/VEGFR3 axis suppresses Slug-mediated cancer metastasis and stemness via inhibition of KRAS/YAP1 signaling

Abstract

Vascular endothelial growth factor-C (VEGF-C) has been implicated in epithelial-mesenchymal transition (EMT) processes and various human cancers, including skin cancer. Skin cancer is an aggressive human malignancy with increasing incidence worldwide; however, the underlying mechanisms involved in VEGF-C-induced skin cancer stemness and metastasis remain unclear. Here, we report that VEGF-C enhances skin cancer migration, invasion and stemness through Slug up-regulation. Oncomine database analysis indicated that the KRAS/MAPK (mitogen-activated protein kinases) pathway and YAP1 (yes-associated protein 1) expression are positively correlated with metastatic skin cancer. We show that VEGF-C triggers the activation of KRAS/MAPK signaling to increase YAP1 and downstream Slug expression, which are suppressed by an anti-VEGFR3 (VEGF receptor 3) peptide, a specific peptide targeting VEGFR3. The VEGF-C-induced migration, invasion and stemness of skin cancer cells are also abrogated by the anti-VEGFR3 peptide. Based on these data, we reveal the role of the VEGF-C/VEGFR3-mediated KRAS/MAPK-YAP1/Slug pathway in skin cancer progression and propose that the VEGF-C/VEGFR3 axis is a promising target for the anti-VEGFR3 peptide.

Keywords: VEGF-C; YAP1; cancer stemness; metastasis; skin cancer.

Figure 1. VEGF-C increases the migration and invasion abilities of skin cancer cells

a. BCC and A2058 cells were incubated with 200 ng/ml of VEGF-C for the indicated time points, and phosphorylated VEGFR3 and VEGFR3 were detected by Western blot analysis. Tubulin was used as a loading control. b. The mRNA expression of VEGF-C was analyzed by real-time qRT-PCR. c and d. A transwell assay was performed to examine cell migration and invasion abilities. Cell motility and phosphorylation of VEGFR3 was inhibited by knockdown of VEGF-C and recovered by treatment with rhVEGF-C by transwell assay and Western blot analysis, respectively. e. Oncomine database analysis showed elevated VEGF-C expression in metastatic melanoma tissues. f. Expression of EMT-inducing transcription factors (Slug, ZEB1, Twist and Snail) was determined by Western blot analysis. g. The effects of Slug expression on skin cancer cell motility were determined by transwell assay. Western blot analysis was used to confirm the expression of the indicated proteins. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.001, ***P < 0.0001 (Student's t test).

Figure 2. VEGF-C enhances the cancer stemness properties in skin cancer

a-g. QRT-PCR analysis of the SOX2, OCT4, KLF4, NANOG, CD133, CD34 and CD44 mRNA expression in indicated cells with rhVEGF-C (200 ng/ml) treatment. h. ALDH activity of the indicated cells was measured by flow cytometry. i-q. Expression of VEGF-C (i) and Slug (j) was detected, and Slug expression restores the expression of CSC markers (SOX2 (k), OCT4 (l), KLF4 (m), NANOG (n), CD133 (o), CD34 (p) and CD44 (q)) in indicated cells by QRT-PCR analysis. r. Slug expression recovers ALDH activity in indicated cells by flow cytometry.

Figure 3. VEGF-C increases Slug expression, cell migration, invasion and stemness through YAP1

a. (Upper panel) Western blot analysis of Slug and YAP1 in indicated cells. Tubulin was used as a loading control. (Lower panel) QRT-PCR analysis of VEGF-C expression in indicated cells. b. A transwell assay demonstrated the migration and invasion abilities of indicated cells. c and d. QRT-PCR analysis of SOX2 and OCT4 mRNA expression in indicated cells. e. ALDH activity of indicated cells was determined by flow cytometry. f-h. The Oncomine database was used to analyze the correlation of VEGF-C, YAP1 and Slug with survival rate in skin cancer patients. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.001, ***P < 0.0001 (Student's t test).

Figure 4. YAP1 is regulated by VEGF-C via the RAS/MAPK pathway

a. Western blot analysis of indicated proteins in VEGF-C-knockdown cells with or without KRAS^G12V overexpression. b-d. The Oncomine dataset analysis showed a positive correlation of VEGF-C with KRAS (b), MAPK1 (c) and YAP1 (d) in skin cancer tissues.

Figure 5. Anti-VEGFR3 peptide represses VEGF-C-induced signaling, cell mobility and cancer stemness in skin cancer

a. A transwell assaydemonstrated the migration and invasion abilities in BCC cells pre-incubated with rhVEGF-C followed by anti-VEGFR3 (anti-R3) or control (Ctrl) peptide treatment. b. Western blot analysis of indicated proteins in rhVEGF-C-pre-incubated cells treated with anti-R3 or Ctrl peptide. c. qRT-PCR analysisof SOX2 and OCT4 mRNA expression in indicated cells. d. ALDH activity in indicated cells was analyzed by flow cytometry. e. A transwell assay showed migration and invasion abilities in indicated cells. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.001, ***P < 0.0001 (Student's t test).

Figure 6. A schematic model of the VEGF-C/VEGFR3-mediated KRAS/MAPK-YAP1/Slug pathway in skin cancer progression

In skin cancer cells, VEGF-C/VEGFR3 signaling activates the KRAS/MAPK axis to up-regulate YAP1 and Slug expression, further enhancing cell mobility and cancer stemness. Anti-VEGFR3 peptide functions as a repressor and could be a therapeutic strategy.