Gene Expression Profiling Leads to Identification of GLI1-binding Elements in Target Genes and a Role for Multiple Downstream Pathways in GLI1-induced Cell Transformation

Abstract

The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1-and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1-and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5 regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.Important gene hierarchies, in part coding for components of signal transduction pathways, regulate growth and differentiation during development. One such pathway is the Sonic hedgehog-Patched-Gli pathway (1). SHH 1 signaling is critical to the genetic specification of fate of many tissues during early organogenesis including the central nervous system (2, 3), lung (4), prostate (5), bone (6 -8), and muscle (9). SHH signaling is mediated by the GLI family of transcription factors (10). One of these genes, GLI1, has been shown to be a transcriptional activator operating through a C-terminal VP-16-like acidic helical domain (11). GLI1 transforms cells in culture, and its expression is associated with significant human cancers including basal cell carcinoma (12), medulloblastoma (13), and sarcomas (14). Few downstream targets of GLI1 are known, which precludes a clear understanding of its action in carcinogenesis. Genetic evidence suggests that PTCH and Wnt genes are downstream targets of GLI1 (15), and biochemical evidence has established HNF-3␤ (Hepatocyte Nuclear Factor-3␤) as a target of GLI1 during development (16).Microarray technology has provided a methodology to study the expression of thousands of genes simultaneously and has been used in many important settings (17). Among these is the dissection of signal transduction pathways. To identify unique downstream targets of GLI1, we have utilized a cell transformation phenotype as a selection system for the stable integration and expression of either GLI1 or Ha-ras in RK3...

GLI1 facilitates the migration and invasion of pancreatic cancer cells through MUC5AC-mediated attenuation of E-cadherin

“…These cell lines express a high level of GLI1 upon the withdrawal of doxycycline (DOX), an analog of tetracycline, from the culture medium. Using these and their control cell lines, PANC-1 Tet/Empty clone 1 and clone 2, we found that MUC5AC but not MUC3B expression was further increased by the induction of GLI1, as evidenced by the increased expression of a known direct target of GLI1, Patched1 (PTCH) (Yoon et al, 2002) (Figure 1b and Supplementary Figures 1 and 2a). As seen in PANC-1 cells, the transient transfection of GLI1 induced MUC5AC expression in PA-TU-8902 and PA-TU-8988T cells (Figure 1c), which did not express any endogenous GLI1, GLI2 or MUC5AC (Figure 1a).…”

“…MUC5AC is a direct target of GLI1 GLI1 has been reported to control the expression of many downstream target genes, either directly or indirectly (Yoon et al, 2002;Eichberger et al, 2006;Kasper et al, 2006). To determine whether MUC5AC is a direct target of GLI1, we next generated two stable cell lines, PANC-1 GLI1ER and its control PANC-1 ER .…”

“…These clinical and experimental data highlight the importance of the downstream targets of GLI transcription factors in the development and cellular properties of PDA. Prior to this study, many attempts have been made to identify the transcriptional target genes of GLIs (Yoon et al, 2002;Eichberger et al, 2006;Kasper et al, 2006). However, whether GLIs regulate mucin expression remains unknown.…”

Expression patterns of hedgehog signaling peptides in naturally acquired equine osteochondrosis

“…Glil has been shown to alter the expression of developmentally important genes including: hedgehog target genes, BMP promoters, and genes regulating cell cycle, cell adhesion, signal transduction, and apoptosis [13,15,32,33].…”

IGF2: Epigenetic regulation and role in development and disease

“…Though putative Gli-binding sites have been identified in the mouse Igf2 promoter [72], it is not known whether these sites are functional, or if they exist in the human VEGF promoter. However, functional Gli sites have been documented in the human IGFBP-6 promoter [74]. IGFBP-6 specifically binds IGF2 and is generally thought to have anti-proliferative properties.…”