VEGF-C/Flt-4 axis in tumor cells contributes to the progression of oral squamous cell carcinoma via upregulating VEGF-C itself and contactin-1 in an autocrine manner

Abstract

Tumor cell-derived vascular endothelial growth factor (VEGF)-C has been primarily implicated in promoting lymphangiogenesis by activating Flt-4 (VEGFR-3) expressed on lymphatic endothelial cells via a paracrine mechanism. Flt4 has also been shown to be expressed selectively in subsets of cancer cells. However, little is known about the functional role of VEGF-C/Flt4 signaling via an autocrine mechanism, as well as the clinicopathological implication of the VEGF-C/Flt4 axis and its downstream effector molecules, in head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC). In the present study, we detected Flt-4 expression selectively in several HNSCC cell lines by quantitative PCR, and its internalization reflecting receptor activation was confirmed by immunocytochemistry in SAS and HO1U1 cells. Flt-4 stimulation upregulated the expression of contactin-1 (CNTN-1, a neural cell adhesion molecule) and VEGF-C itself in SAS cells, while Flt-4 inhibition downregulated the expression of CNTN-1 in both SAS and HO1U1 cells and that of VEGF-C itself in SAS cells. In vitro cell proliferation and migration assays using SAS cells demonstrated that both cell proliferation and migration were promoted by Flt-4 stimulation, while those were suppressed by Flt-4 inhibition. Clinicopathological factors and immunohistochemical expression of Flt-4, VEGF-C, and CNTN-1 in tumor cells were evaluated using surgical specimens from patients with tongue squamous cell carcinoma. We found a significant correlation of CNTN-1 expression with both VEGF-C and Flt-4 expression, but not between VEGF-C and Flt-4. Multivariate logistic regression analysis revealed that T classification (P = 0.003), lymphatic invasion (P = 0.024), and Flt-4 expression in tumor cells (P = 0.046) were independently predictive of neck lymph node metastasis. These results suggest that the VEGF-C/Flt-4 axis in tumor cells enhances tumor cell proliferation and migration via upregulating the expression of VEGF-C itself and CNTN-1 in an autocrine manner, thereby contributing to cancer progression of OSCC, including neck metastasis. Hence, targeting the VEGF-C/Flt-4 axis in tumor cells can be an attractive therapeutic strategy for the treatment of cancer.

Keywords: Flt-4; VEGF-C; autocrine manner; contactin-1; metastasis; migration; oral squamous cell carcinoma; proliferation; tumor cell.

Figure 1

Baseline mRNA expression of Flt-4, VEGF-C, and CNTN-1 in HNSCC cells. The mRNA expression levels of each gene in the HNSCC cell lines were assessed by quantitative real-time PCR. The relative expression levels of the genes were compared after normalization using those of GAPDH. A: Flt-4. B: VEGF-C and CNTN-1, in which the relative expression levels were calibrated by dividing each value by that of HSC-2 for the sake of convenience. C: The selective expression of Flt-4 in SAS and HO1U1 cells was further confirmed by standard PCR. D: Immunofluorescent staining of SAS and HO1U1 cells, as well as HMVEC cells, revealed the cytoplasmic localization of Flt-4, indicating the receptor internalization that reflects its activation. Nuclei were stained with Hoechst 33258. Scale bar: 10 μm.

Figure 2

Effects of Flt-4 stimulation and inhibition on the mRNA expression levels of CNTN-1 and VEGF-C in OSCC cells. Alterations in the mRNA expression of CNTN-1 and VEGF-C in OSCC cells were examined by quantitative real-time PCR using recombinant VEGF-C or VEGF-C (Cys156Ser) (selective Flt-4 agonist) as stimulation reagents and recombinant human Flt-4/Fc chimera (specific VEGF-C neutralizer) or MAZ51 (specific Flt-4 inhibitor) as inhibition reagents. In SAS cells, Flt-4 stimulation upregulated both CNTN-1 and VEGF-C expression compared to the control (A), whereas in contrast, Flt-4 inhibition downregulated both CNTN-1 and VEGF-C expression compared to the control (B). In HO1U1 cells, Flt-4 stimulation resulted in relatively less upregulation of CNTN-1 expression and no change in VEGF-C expression (C), whereas Flt-4 inhibition led to downregulation of CNTN-1 expression but no change in VEGF-C expression (D). The values represent the mean ± standard deviation. Difference between each condition and control was statistically analyzed using a two-tailed t-test: *, P < 0.05; N.S., not significant.

Figure 3

Alterations in the proliferation and migration activity of SAS cells following Flt-4 stimulation and inhibition. The effects of Flt-4 stimulation and inhibition on proliferation and migration activity were examined using recombinant VEGF-C or VEGF-C (Cys156Ser) (selective Flt-4 agonist) as stimulation reagents and recombinant human Flt-4/Fc chimera (specific VEGF-C neutralizer) or MAZ51 (specific Flt-4 inhibitor) as inhibition reagents. (A and B) An in vitro cell proliferation assay was conducted using SAS cells. The data are presented as the fold increase in the OD values of the absorbance measured at 450 nm compared with the respective controls. Flt-4 stimulation promoted proliferation activity in a dose-dependent manner (A), whereas Flt-4 inhibition suppressed proliferation activity in a dose-dependent manner (B). (C and D) An in vitro migration assay was performed using SAS cells. The results are presented as the fold increase in the number of migrated cells compared with the respective controls. To eliminate the possible influence of differences in proliferation activity, each value was normalized according to the corresponding ratio of the proliferation rate found in the same condition. Flt-4 stimulation enhanced migration activity in a dose-dependent manner (C), whereas Flt-4 inhibition attenuated migration activity in a dose-dependent manner (D). The values represent the mean ± standard deviation. Difference between each condition and control was statistically analyzed using a two-tailed t-test: *, P < 0.05; N.S., not significant.

Figure 4

Immunohistochemistry of Flt-4, VEGF-C, and CNTN-1 in TSCC specimens. In the representative cases with positive staining shown in the top panels, staining of Flt-4 (A), VEGF-C (C), and CNTN-1 (E) was observed in the cytoplasm of the tumor cells. The bottom panels show cases with negative staining of Flt-4 (B), VEGF-C (D), and CNTN-1 (F). Original magnification: ×200. Scale bar: 100 μm.

Figure 5

A schematic diagram demonstrating the VEGF-C/Flt-4 autocrine system in OSCC cells. Tumor cell-derived VEGF-C binds to Flt-4 expressed on the tumor cells, and its activation upregulates the expression levels of VEGF-C itself and CNTN-1 in an autocrine manner, thereby enhancing tumor cell proliferation and migration. This autocrine mechanism of the VEGF-C/Flt-4 axis in tumor cells likely contributes to cancer progression, including the development of lymphatic metastasis, along with the primary well-known paracrine mechanism involving lymphatic endothelial cells that leads to lymphangiogenesis.