Joon Won Yoon

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

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 stableHa-ras expression. Comparison of expression profiles fromGLI1- 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- andHa-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 theGLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.

N-myc Can Substitute for Insulin-Like Growth Factor Signaling in a Mouse Model of Sonic Hedgehog–Induced Medulloblastoma

Medulloblastoma is a malignant brain tumor that arises in the cerebellum in children, presumably from granule neuron precursors (GNP). Advances in patient treatment have been hindered by a paucity of animal models that accurately reflect the molecular pathogenesis of human tumors. Aberrant activation of the Sonic hedgehog (Shh) and insulin-like growth factor (IGF) pathways is associated with human medulloblastomas. Both pathways are essential regulators of GNP proliferation during cerebellar development. In cultured GNPs, IGF signaling stabilizes the oncogenic transcription factor N-myc by inhibiting glycogen synthase kinase 3beta-dependent phosphorylation and consequent degradation of N-myc. However, determinants of Shh and IGF tumorigenicity in vivo remain unknown. Here we report a high frequency of medulloblastoma formation in mice following postnatal overexpression of Shh in cooperation with N-myc. Overexpression of N-myc, alone or in combination with IGF signaling mediators or with the Shh target Gli1, did not cause tumors. Thus, Shh has transforming functions in addition to induction of N-myc and Gli1. This tumor model will be useful for testing novel medulloblastoma therapies and providing insight into mechanisms of hedgehog-mediated transformation.

Selective down-regulation of glioma-associated oncogene 2 inhibits the proliferation of hepatocellular carcinoma cells.

The sonic hedgehog (Shh) pathway contributes to the initiation and progression of tumors with various origins when aberrantly activated. In this study, we investigated if the Shh pathway is important for the proliferation of hepatocellular carcinoma (HCC) cells and also began to identify which components of the pathway play a pivotal role in the biology of HCC. Expression levels of components in the pathway were measured, and glioma-associated oncogene (Gli) 2 levels were found to be considerably higher in human HCC lines compared with normal liver. Gli2 levels were also higher in tumor tissue from HCC patients compared with normal liver. Antisense oligonucleotides (ASO) were used to specifically down-regulate Gli2, and this led to decreased proliferation of various HCC cell lines. However, inhibition of Gli1 and Gli3 with ASOs did not decrease proliferation in most HCC cell lines and inhibitors targeting the upstream components of the pathway, including smoothened (Smo), displayed antiproliferative effects in only a subset of HCC cell lines. Moreover, in cancer cells harboring Smo mutations or unresponsive to the Smo inhibitor 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine, the Gli2 ASO was still able to inhibit proliferation. The importance of Gli2 in HCC proliferation was further confirmed by the changes in expression levels of genes, such as Bcl-2, c-Myc, and p27, following suppression of Gli2 expression. Taken together, these results suggest that, among the Gli transcription factors, Gli2 plays a predominant role in the proliferation of HCC cells and the suppression of Gli2 expression may provide a useful therapeutic option for the treatment of HCC.

Conservation of the C.elegans tra-2 3'UTR translational control.

The Caenorhabditis elegans sex‐determination gene, tra‐2, is translationally regulated by two 28 nt elements (DREs) located in the 3′UTR that bind a factor called DRF. This regulation requires the laf‐1 gene activity. We demonstrate that the nematode Caenorhabditis briggsae tra‐2 gene and the human oncogene GLI are translationally regulated by elements that are functionally equivalent to DREs. Here, we rename the DREs to TGEs (tra‐2 and GLI elements). Similarly to the C.elegans tra‐2 TGEs, the C.briggsae tra‐2 and GLI TGEs repress translation of a reporter transgene in a laf‐1 dependent manner. Furthermore, they regulate poly(A) tail length and bind DRF. We also find that the C.elegans TGEs control translation and poly(A) tail length in C.briggsae and rodent cells. Moreover, these same organisms contain a factor that specifically associates with the C.elegans TGEs. These findings are consistent with the TGE control being present in C.briggsae and rodent cells. Three lines of evidence indicate that C.briggsae tra‐2 and GLI are translationally controlled in vivo by TGEs. First, like C.elegans tra‐2 TGEs, the C.briggsae tra‐2 and GLI TGEs control translation and poly(A) tail lengths in C.briggsae and rodent cells, respectively. Second, the same factor in C.briggsae and mammalian cells that binds to the C.elegans tra‐2 TGEs binds the C.briggsae tra‐2 and GLI TGEs. Third, deletion of the GLI TGE increases GLIs ability to transform cells. These findings suggest that TGE control is conserved and regulates the expression of other mRNAs.

Sonic Hedgehog-responsive Genes in the Fetal Prostate

The Hedgehog (Hh) signaling pathway plays an important role in prostate development and appears to play an equally important role in promoting growth of advanced prostate cancer. During prostate development, epithelial cells in the urogenital sinus (UGS) express Sonic Hedgehog (Shh) and secrete Shh peptide. The secreted Hh peptide acts on adjacent mesenchymal cells to activate the Hh signal transduction pathway and elicit paracrine effects on epithelial proliferation and differentiation. To identify mesenchymal targets of Shh signaling, we performed microarray analysis on a Shh-responsive, immortalized urogential sinus mesenchymal cell line. We found 68 genes that were up-regulated by Shh and 21 genes that were down-regulated. Eighteen of those were selected for further study with Ptc1 and Gli1 serving as reference controls. We found 10 of 18 were also Hh-regulated in primary UGS mesenchymal cells and 13 of 18 in the cultured UGS. Seven of 18 exhibited Shh-regulated expression in both assays (Igfbp-6, Igfbp-3, Fbn2, Ntrk3, Agpt4, Dmp1, and Mmp13). Three of the 18 genes contained putative Gli binding motifs that bound Gli1 peptide in electrophoretic mobility shift assays. With the exception of Tiam1, target gene expression generally showed no differences in the concentration dependence of ligand-induced expression, but we observed strikingly different responses to direct pathway activation by transfection with activated Smo, Gli1, and Gli2.

GLI1 Localization in the Germinal Epithelial Cells Alternates Between Cytoplasm and Nucleus: Upregulation in Transgenic Mice Blocks Spermatogenesis in Pachytene

Abstract The zinc finger transcription factor GLI1 is the mediator of signaling by members of the Hedgehog (Hh) family. Male mice in which Desert hedgehog (Dhh), an Hh homologue expressed in Sertoli cells of the testis, was knocked out are sterile, suggesting that the Dhh/GLI1 pathway plays a role in spermatogenesis. Using an antiserum raised against human GLI1, we found that during the first round of spermatogenesis, GLI1 expression is initially cytoplasmic, then shifts to the nuclei of Sertoli and germ cells, and finally shifts back to the cytoplasm. In the adult mouse testis, GLI1 expression localized to the nuclei of germ cells, beginning with pachytene cells and persisting through round spermatids. Localization of GLI1 in elongating spermatids shifted from the nucleus to the cytoplasm and became associated with microtubules. We also examined a line of transgenic mice that overexpressed human GLI1. Male mice in this line were sterile. Spermatogenesis was blocked at the pachytene stage, and a subset of the morphologically indistinguishable pachytene cells underwent apoptosis. Patched-2, which is a Dhh receptor, and Fused, another component of the signal transduction pathway, are expressed in Leydig cells and in primary and secondary spermatocytes. Expression of GLI1 in the same cell types as Patched-2 and Fused and the disruption of spermatogenesis by GLI1 overexpression suggest that GLI1 is the mediator of the Dhh signal in the testis, and that it may be a regulator of spermatogenesis.

Nodal and Bone morphogenetic protein 5 interact in murine mesoderm formation and implantation

Summary: Mice mutant for the TGF‐β family member, nodal, lack mesoderm and die between E8.5 and E9.5. The short ear‐lethal (sel ) mutation, a deletion that eliminates Bmp‐5, causes a strikingly similar gastrulation defect. Here we analyze sel;nodal compound mutants and find a dosage effect. Embryos homozygous for one mutation show distinct gastrulation stage defects that depend on whether they are heterozygous or homozygous for the other mutation. Embryos mutant for nodal or sel;nodal compound mutants fail to execute an antigenic shift indicative of mesoderm differentiation and ectoderm cells are shunted into an apoptotic pathway. Furthermore, we find a novel phenotype in sel;nodal double mutant litters, in which two to four genetically different embryos are contained within the same deciduum. Both the gastrulation and implantation phenotypes can also arise in short ear‐viable (sev ) and sev;nodal mutant mice. These data indicate that loss of Bmp‐5 may underlie the sel gastrulation phenotype and suggest that nodal and Bmp‐5 interact during murine mesoderm formation. Our data also reveal an unsuspected role for Bmp‐5 in implantation and the decidual response in the mouse. genesis 28:1–14, 2000.

Noncanonical regulation of the Hedgehog mediator GLI1 by c-MYC in Burkitt lymphoma.

Although Hedgehog signaling plays a major role in GLI1 transcription, there is now evidence suggesting that other pathways/genes, such as c-MYC, may also regulate GLI1 expression. We initiated studies in Burkitt lymphoma cells, which constitutively express c-MYC due to a chromosomal translocation, to determine whether Hedgehog or c-MYC regulates GLI1 expression. We show that all Burkitt lymphoma cell lines tested express GLI1, PTCH1, and SMO and that five of six Burkitt lymphomas express GLI1. Exposure to Sonic or Indian Hedgehog or cyclopamine (SMO inhibitor) does not modulate GLI1 expression, cell proliferation, or apoptosis in most Burkitt lymphoma cell lines. Sequence analysis of PTCH1, SMO, and SuFu failed to show mutations that might explain the lack of Hedgehog responsiveness, and we did not detect primary cilia, which may contribute to it. We show that c-MYC interacts with the 5′-regulatory region of GLI1, using chromatin immunoprecipitation (ChIP) assay, and E-box–dependent transcriptional activation of GLI1 by c-MYC in NIH3T3 and HeLa cells. The c-MYC small-molecule inhibitor 10058-F4 downregulates GLI1 mRNA and protein and reduces the viability of Burkitt lymphoma cells. Inhibition of GLI1 by GANT61 increases apoptosis and reduces viability of some Burkitt lymphoma cells. Collectively, our data provide evidence that c-MYC directly regulates GLI1 and support an antiapoptotic role for GLI1 in Burkitt lymphoma. Burkitt lymphoma cells do not seem to be Hedgehog responsive. These findings suggest a mechanism for resistance to SMO inhibitors and have implications for using SMO inhibitors to treat human cancers. Mol Cancer Res; 11(6); 604–15. ©2013 AACR.

Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis

Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2. Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or FOXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2. These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip. Summary: The sonic hedgehog pathway controls expression of Foxf2 in mouse cranial neural crest mesenchyme, promoting proliferation and upper lip closure. Human FOXF2 polymorphisms are associated with cleft lip.

P53 Modulates The Activity Of The GLI1 Oncogene Through Interactions With The Shared Coactivator TAF9

The GLI1 oncogene and p53 tumor suppressor gene function in an inhibitory loop that controls stem cell and tumor cell numbers. Since GLI1 and p53 both interact with the coactivator TATA Binding Protein Associated Factor 9 (TAF9), we hypothesized that competition between these transcription factors for TAF9 in cancer cells may contribute to the inhibitory loop and directly affect GLI1 function and cellular phenotype. We showed that TAF9 interacts with the oncogenic GLI family members GLI1 and GLI2 but not GLI3 in cell-free pull-down assays and with GLI1 in rhabdomyosarcoma and osteosarcoma cell lines. Removal of the TAF9-binding acidic alpha helical transactivation domain of GLI1 produced a significant reduction in the ability of GLI1 to transform cells. We then introduced a point mutation into GLI1 (L1052I) that eliminates TAF9 binding and a point mutation into GLI3 (I1510L) that establishes binding. Wild-type and mutant GLI proteins that bind TAF9 showed enhanced transactivating and cell transforming activity compared with those that did not. Therefore, GLI-TAF9 binding appears important for oncogenic activity. We then determined whether wild-type p53 down-regulates GLI function by sequestering TAF9. We showed that p53 binds TAF9 with greater affinity than does GLI1 and that co-expression of p53 with GLI1 or GLI2 down-regulated GLI-induced transactivation, which could be abrogated using mutant forms of GLI1 or p53. This suggests that p53 sequesters TAF9 from GLI1, which may contribute to inhibition of GLI1 activity by p53 and potentially impact therapeutic success of agents targeting GLI-TAF9 interactions in cancer.