Abstract 2818: Flt-4 expression on tumor cells and its autocrine mechanism with VEGF-C/contactin-1 stimulate progression of head and neck squamous cell carcinoma.
Authors: Shigetomi Seiji , Imanishi Yorihisa , Shimoda Masayuki , Kameyama Kaori , Shibata Katsushi , Sakai Nobuya , Sakamoto Koji , Fujii Ryoichi , Habu Noboru , Ootsuka Kuninori , Ogawa Kaoru 
Published: August 2014

Background: Tumor cell-derived VEGF-C has been considered to promote lymphangiogenesis by activating Flt-4 (VEGFR-3) expressed on lymphatic endothelial cells via a paracrine mechanism in tumor microenvironment. Recent studies have shown that Flt-4 is expressed not only in endothelial cells but also selectively in certain subsets of a variety of cancer cells. In addition, the VEGF-C/Flt-4 autocrine loop on lung adenocarcinoma cells was reported to enhance cell motility and invasiveness through up-regulation of the neural cell adhesion molecule Contactin-1 (CNTN-1), although little of such mechanism is known in head and neck squamous cell carcinoma (HNSCC). Present study was aimed to examine Flt-4 expression and VEGF-C/Flt-4 autocrine loop in HNSCC cells, and clinicopathological significance of the expressions of Flt-4, VEGF-C, and CNTN-1 in the tumor specimens of the patients with HNSCC. Methods: We examined expression of Flt-4 in HNSCC cell lines using quantitative real-time PCR, RT-PCR, and immunocytochemistry. The role of the VEGF-C/Flt-4 axis in Flt-4-positive HNSCC cells in vitro was investigated by stimulating or blocking Flt-4. Immunohistochemical studies were performed on 56 oral tongue squamous cell carcinoma (TSCC) specimens. The associations of the immunohistochemical expressions of Flt-4, VEGF-C, and CNTN-1 with the clinicopathological factors were statistically analyzed. Results: Real-time PCR detected Flt-4 mRNA expression in several HNSCC cells, which was further confirmed by RT-PCR and immunocytochemistry in SAS and HO1U1. Stimulation of Flt-4 using recombinant VEGF-C up-regulated expression of CNTN-1 and VEGF-C itself in SAS, whereas blocking using neutralizing anti-Flt-4 antibody or Flt-4 inhibitor down-regulated expression of CNTN-1 in both SAS and HO1U1, and that of VEGF-C itself in SAS. Immunohistochemical analyses using TSCC specimens found significant correlation of CNTN-1 expression with both VEGF-C and Flt-4 expressions, but not between VEGF-C and Flt-4. With regard to clinicopathological factors, expressions of all the three molecular markers in tumor cells significantly correlated with lymph node (LN) metastasis, which was also correlated with T classification and lymphatic invasion in univariate analysis. Multivariate analysis elucidated that T classification (p=0.003), lymphatic invasion (p=0.024), and expression of Flt-4 in tumor cells (p=0.046) were independent predictors of LN metastasis. Conclusions: Our results suggest that VEGF-C activates FLt-4 expressed not only on lymphatic endothelial cells but also selectively on subset of HNSCC cells, resulting in VEGF-C/Flt-4 autocrine mechanism that up-regulates CNTN-1 and VEGF-C itself in tumor cells. Therefore, both paracrine and autocrine mechanisms of VEGF-C/Flt-4 may contribute to progression of HNSCC including LN metastasis.

Citation Format: Seiji Shigetomi, Yorihisa Imanishi, Masayuki Shimoda, Kaori Kameyama, Katsushi Shibata, Nobuya Sakai, Koji Sakamoto, Ryoichi Fujii, Noboru Habu, Kuninori Ootsuka, Kaoru Ogawa. Flt-4 expression on tumor cells and its autocrine mechanism with VEGF-C/contactin-1 stimulate progression of head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2818. doi:10.1158/1538-7445.AM2013-2818