Signal transducers and activators of transcription 3 up-regulates vascular endothelial growth factor production and tumor angiogenesis in head and neck squamous cell carcinoma

Summary

Overexpression of vascular endothelial growth factor (VEGF) is associated with angiogenic phenotypes and poor prognosis of numerous tumors, including head and neck squamous cell carcinoma (HNSCC). However, the precise mechanism that causes VEGF overexpression in HNSCC remains unknown. Since there is evidence that a transcriptional factor, signal transducers and activators of transcription 3 (Stat3), is constitutively activated in HNSCC and this activation is significantly associated with aggressive phenotypes of this disease, we investigated the roles of Stat3 activation on VEGF production and tumor angiogenesis in HNSCC both in vitro and in clinical samples. VEGF promoter assays with YCU-H891 cells demonstrated that dominant negative Stat3 significantly inhibited VEGF promoter activity in the full length (−2279 to +54) and two truncated forms of VEGF promoter luciferase-reporter construct (−1179 to 54) or (−1014 to +54), which retain the putative Stat3 responsive elements (−849 to −842). However, this was not seen in the shorter construct (−794 to +54), which lacks the putative Stat3 responsive elements. In the derivative of YCU-891 cells that stably express dominant negative Stat3 protein, cellular levels of VEGF mRNA and VEGF protein were significantly inhibited. In the 51 clinical samples obtained from the patients with tongue carcinoma, the expression levels of phosphorylated (activated) form of Stat3 protein were significantly correlated with VEGF (P < 0.05) production and intratumoral microvessel density IMVD (P < 0.01). These results strongly indicate that Stat3 directly up-regulates VEGF transcription and thereby promotes angiogenesis in HNSCC. Inhibition of Stat3 activity may provide a new anti-angiogenic therapy in HNSCC.

Introduction

Angiogenesis, a recruitment of new capillaries from existing vessels, is indispensable process for tumor development, three dimensional tumor growth, and metastasis in solid tumors,1, 2, 3 including head and neck squamous cell carcinoma (HNSCC). To date, several angiogenic factors have been identified. Among them vascular endothelial growth factor (VEGF) is one of the most prominent mitogens for the endothelial cells and the roles of VEGF in carcinogenesis are extensively investigated.2, 4 Indeed, the elevated VEGF production is associated with the higher levels of intratumoral microvessel density (IMVD) and poor prognosis of patients with several malignancies including HNSCC.5, 6, 7, 8, 9 However, the precise mechanism by which VEGF is overexpressed in HNSCC is not known.

Our previous studies and others demonstrated that a transcriptional factor, signal transducers and activators of transcription 3 (Stat3), is constitutively activated in HNSCC and this activation is significantly associated with aggressive phenotypes of this disease.10, 11, 12, 13 Recently it was demonstrated by Xi et al.¹⁴ that administration of the Stat3 decoy significantly decreased the expression level of VEGF and thereby inhibited tumor growth and angiogenesis in a xenograft model of HNSCC. In addition, recent studies provided strong evidence that Stat3 directly regulates VEGF production through a putative Stat3 responsive element (−849 to −842) on the VEGF promoter.15, 16, 17 These findings led us to hypothesize that Stat3 directly transactivates VEGF expression and thus enhances tumor angiogenesis in HNSCC. Based on this hypothesis, we presently examined the effects of Stat3 activation on VEGF production and intratumoral microvessel density (IMVD) both in vitro and in clinical samples. In our initial study, we examined the inhibitory effects of a dominant negative Stat3 construct on VEGF promoter activity and the expression level of VEGF mRNA and VEGF protein, employing a YCU-891 cell line which displays constitutive activation of Stat3 due to autocrine activation of EGFR/erbB2 by TGF-α, and produces relatively high level of VEGF protein even under normoxic condition. We then examined the expression levels of activated (phosphorylated)-Stat3, VEGF and IMVD in 51 clinical samples obtained from patients with HNSCC. Correlations of these parameters were statistically analyzed.