RASSF8 downregulation promotes lymphangiogenesis and metastasis in esophageal squamous cell carcinoma

Abstract

Lymphatic vessels are the major routes of human esophageal squamous cell carcinoma (ESCC) metastasis. Tumor cells secrete pro-lymphangiogenic factors to induce new lymphatic vessels, promoting lymph node metastasis. In this study, we show that RAS association domain family 8 (RASSF8) expression in ESCC clinical samples was inversely correlated with lymph node metastasis and patients survival. Tumor cells with low RASSF8 expression had higher apparent migratory ability, and promoted and lymphangiogenesis both in vitro and in vivo. RASSF8 downregulation enhanced VEGF-C expression and caused subcellular redistribution of p65 in ESCC. Our results show that RASSF8 acts as a tumor suppressor in ESCC and is a potential therapeutic target for preventing lymph node metastasis.

Keywords: RASSF8; esophageal cancer; lymphangiogenesis; tumor metastasis.

Figure 1. RASSF8 is frequently downregulated in ESCCs

A., Western blotting (left) and quantitative real-time PCR (right) results showing RASSF8 levels in eight ESCC cell lines and the NE1 immortalized esophageal epithelial cell line. B., Representative images of RASSF8 immunochemical staining in ANT and tumor tissues with or without lymph node metastasis (LN+ and LN-, respectively). Bars = 200 μm (left); 50 μm (right). C., Quantitative real-time PCR analyses of RASSF8 expression in eight paired primary ESCC tissues and matched ANT tissues. D., Kaplan–Meier curves of the overall survival of 137 ESCC patients with high or low RASSF8 expression. The p-value was computed by log-rank test. E., RASSF8 protein expression was lower in LN-positive (LN+) specimens than in LN-negative (LN-) specimens (p = 0.004). F., Representative staining images of RASSF8 and the lymphatic marker LYVE-1. Arrows indicate LYVE-1–positive lymphatic vessels (left). Correlation of RASSF8 expression and lymphatic vessel density (LVD) was analyzed (right). Bars = 100 μm. IHC, immunohistochemistry.

Figure 2. RASSF8 is essential for ESCC cell motility and invasiveness

A., Western blotting (top) and quantitative real-time PCR (bottom) analysis of RASSF8 overexpression and RASSF8 knockdown in EC109 and KYSE410 cell lines. B., Representative images of wound healing assay showing the motility of RASSF8 overexpression and knockdown cells as compared with the control cells. Bars = 200 μm. C., Representative images of Transwell invasion assay showing ESCC cell invasion ability. Bars = 100 μm. *p < 0.05 (Student's t-test). NC, negative control.

Figure 3. RASSF8 downregulation increases the ability of ESCC cells to induce lymphangiogenesis in vitro

A., Representative images (left) of HLECs cultured on Matrigel-coated plates with TSNs from ESCC cells. Bars = 500 μm. The average number of tube junctions per field was calculated (right). B., Representative images (left) and quantification (right) of HLEC migration assay. Bars = 100 μm. Data shown are the mean ± SEM of three fields from three independent experiments. *p < 0.05 (Student's t-test). NC, negative control.

Figure 4. RASSF8 downregulation in ESCC cells promotes lymphangiogenesis and lymph node metastasis

A., Anti–LYVE-1 antibody immunostaining of lymphatic vessels following ESCC cell inoculation in nude mice footpads. Shown are representative images (left) and quantitative analysis of peritumor lymphatic vessels (right). Bars = 100 μm. B., Representative images (left) and volumes (right) of popliteal lymph nodes. C., Representative images of anti-cytokeratin antibody−immunostained popliteal lymph nodes from mice inoculated with ESCC cells. Bars = 50 μm. D., Ratios of metastatic to total dissected popliteal lymph nodes from mice inoculated with ESCC cells.

Figure 5. RASSF8 downregulates VEGF-C expression in ESCC cells

A., RASSF8 protein levels are inversely correlated with VEGF-C in clinical ESCC tissues (n = 23, p = 0.014, R = 0.503). Left: Images of two representative cases. Right: Correlation analyses of RASSF8 and VEGF-C protein expression in 23 human ESCC tissues. Bars = 100 μm. B., ELISA of VEGF-C protein levels in the supernatants of the indicated cells. C., Immunohistochemical staining detection of VEGF-C protein levels in footpad tumors. Bars = 100 μm. D., Western blots of phospho-AKT (p-AKT, Ser 473) and phospho-ERK (p-ERK, T202/Y204) protein levels in TSN-treated human lymphatic endothelial cells (HLECs). E., Western blot analysis of phosphor-AKT (Ser374) and phosphor-ERK (T202/Y204) protein expression in HLECs cultured in TSNs derived from RASSF8 knockdown EC109 cells treated with U0126 or LY294002. F., G., Representative images and quantifications of matrigel tube formation (Bars = 500 μm) and transwell migration (Bars = 100 μm) assays, respectively, of HLECs cultured with TSN from the indicated cell. Error bars indicate the mean±SEM of three independent experiments. *p < 0.05.

Figure 6. RASSF8 downregulation causes subcellular redistribution of p65 in ESCC cells

A., Relative expression of NF-κB luciferase reporter activities in the cells. Data represents percent change in normalized luciferase activity following RASSF8 or RASSF8-RNAi co-transfection relative to the negative control (NC) vector ± SEM from three independent experiments. *p < 0.05. B., Western blots of nuclear (nuc), cytoplasmic (cyto), and total NF-κB p65 expression in the cells. Cytoplasmic control, GAPDH; nuclear control, lamin B1. C., Quantification analysis of subcellular distribution acquired from western blotting using ImageJ software (Wayne Rashband). Error bars indicate the mean±SEM from three independent exprements. *p < 0.05.