Abstract 4165: The gain-of-function truncated GLI1 (TGLI1) promotes glioblastoma angiogenesis by direct upregulation of VEGF-C and TEM7 expression

Glioblastoma (GBM) is the most frequent and deadliest brain malignancy in adults. GBM is associated with poor patient outcomes with less than five percent of patients surviving five years after diagnosis. High tumor vascularity is a hallmark of GBM and is associated with poor patient prognosis. Glioma-associated oncogene homolog 1 (GLI1) is a transcription factor that is the terminal effector of the Sonic Hedgehog (Shh) signaling pathway. Our laboratory discovered an alternatively spliced variant of GLI1, termed truncated GLI1 (TGLI1), which has an in-frame deletion of 123 nucleotides corresponding to 41 amino acids. We have demonstrated that: 1) TGLI1 is highly expressed in GBM cell lines, patient tumors, and primary culture from the Wake Forest University Brain Tumor Center of Excellence, but not in normal brain tissues; 2) TGLI1 is a gain-of-function GLI1 with a higher propensity than GLI1 to induce tumor cell migration and invasiveness; 3) TGLI1-expressing GBM xenografts have greater vascularity compared to those expressing GLI1. However, to date, the mechanisms underlying TGLI1-mediated angiogenesis in GBM are still not well understood. To address this knowledge gap, we compared TGLI1- with GLI1-expressing GBM xenografts for the expression profile using an angiogenesis PCR array consisting of 84 angiogenesis-associated genes. Our results showed that seven genes, including vascular endothelial growth factor-C (VEGF-C) and tumor endothelial marker 7 (TEM7), were expressed at higher levels in TGLI1-expressing tumors compared to those with GLI1. VEGF-C is a member of the VEGF family and binds VEGFR2/3 to promote angiogenesis and lymphangiogenesis. TEM7 is a transmembrane protein identified in the tumor endothelium with a currently unidentified function, but it may play a role in capillary morphogenesis. Results of the chromatin immunoprecipitation assay revealed that TGLI1, but not GLI1, directly bound the promoters of both VEGF-C and TEM7 genes. Conditioned medium from TGLI1-expressing GBM cells strongly induced tubule formation of brain microvascular endothelial cells and the induction was prevented when VEGF-C or TEM7 expression in the GBM cells were knocked down. Consistent with these results, we found that TGLI1 expression was positively correlated with VEGF-C, TEM7, and microvessel density in122 patient gliomas across all grades. The levels of VEGF-C and TEM7 in patient specimens were also correlated with microvessel density. Taken together, these findings further support an important role for TGLI1 in promoting GBM angiogenesis, identify VEGF-C and TEM7 as novel TGLI1 target genes and their involvement in TGLI1-mediated vascularity, and provide a new insight into the molecular underpinning of the excessive angiogenesis that is characteristic of GBM (T32CA079448 to RC; 7R01NS087169-02 to HWL). Citation Format: Richard L. Carpenter, Ivy Paw, Sherona Sirkisoon, Fei Xing, Denise Gibo, Kounosuke Watabe, Waldemar Debinski, Hui-Wen Lo. The gain-of-function truncated GLI1 (TGLI1) promotes glioblastoma angiogenesis by direct upregulation of VEGF-C and TEM7 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4165. doi:10.1158/1538-7445.AM2015-4165