Overexpression Of Gli1 Research Articles

Hedgehog signaling overrides p53-mediated tumor suppression by activating Mdm2

The hedgehog (Hh) signaling pathway regulates the development of many organs in mammals, and activation of this pathway is widely observed in human cancers. Although it is known that Hh signaling activates the expression of genes involved in cell growth, the precise role of the Hh pathway in cancer development is still unclear. Here, we show that constitutively activated mutants of Smoothened (Smo), a transducer of the Hh signaling pathway, inhibit the accumulation of the tumor suppressor protein p53. This inhibition was also observed in the presence of Hh ligand or with the overexpression of the transcription factors Gli1 and Gli2, downstream effectors of Smo, indicating that this inhibition is specific for the Hh pathway. We also report that Smo mutants augment p53 binding to the E3 ubiquitin-protein ligase Mdm2 and promote p53 ubiquitination. Furthermore, Hh signaling induced the phosphorylation of human Mdm2 protein on serines 166 and 186, which are activating phosphorylation sites of Mdm2. Smo mutants enhanced the proliferation of mouse embryonic fibroblasts (MEFs) while inducing a DNA-damage response. Moreover, Smo partially inhibited p53-dependent apoptosis and cell growth inhibition in oncogene-expressing MEFs. We also found that accumulation of p53 is inhibited by Hh signaling in several human cancer cell lines. Therefore, the Hh pathway may be a powerful accelerator of oncogenesis by activating cell proliferation and inhibiting the p53-mediated anti-cancer barrier induced by oncogenic stress.

Crosstalk of hedgehog and Wnt pathways in gastric cancer

Morphogenic signals like Hedgehog (Hh) and Wnt are reported to play critical roles in the progression of gastric cancer. We aimed to assess the relationship between Hh and Wnt signaling pathways. In 58 gastric cancer specimens, Wnt pathway activation was inversely correlated with Hh pathway activation. When AGS gastric cancer cells, in which Wnt signaling was constitutively active, were used as a target cell line, Gli1 overexpression suppressed Wnt transcriptional activity, nuclear β-catenin accumulation and proliferation of AGS cells. Knock-down of β-catenin by siRNA suppressed Wnt pathway activity and proliferation of AGS cells. Our data may provide some clues for the treatment of gastric cancer associated with Wnt signaling activation.

Indian hedgehog promotes the migration of rat activated pancreatic stellate cells by increasing membrane type‐1 matrix metalloproteinase on the plasma membrane

Indian hedgehog (Ihh) is a member of hedgehog peptides family that exerts diverse effects on multiple cellular functions. Since Ihh expression is elevated in the pancreas of chronic pancreatitis patients, Ihh has been assumed to participate in the chronic pancreatic injury, especially in pancreatic fibrosis. However, its function in pancreatic fibrosis is still unknown. We thus examined Ihh effects on rat activated pancreatic stellate cells (PSCs) that play a central role in pancreatic fibrosis. Activated PSCs express both patched-1 and smoothened that are essential components of hedgehog receptor system. Ihh did not alter the PSC expression of collagen-1 or alpha-smooth muscle actin, a parameter of PSC transformation, or did not change PSC proliferation. However, Ihh enhanced PSC migration in both chemotactic and chemokinetic manners. Furthermore, Ihh increased the amount of membrane-type 1 matrix metalloproteinase (MT1-MMP) and altered its localization on the plasma membrane, which plays a stimulatory role in cellular migration. In addition, tissue inhibitor of metalloproteinase-2 (TIMP-2) attenuated Ihh-stimulated PSC migration. Since most hedgehog intracellular signals are mediated by Gli-1 transcription factor, we investigated its contribution to Ihh-enhancement of PSC migration. Ihh induced Gli-1 nuclear accumulation in PSCs, indicating that Ihh stimulates Gli-1-dependent signaling pathway in PSCs. Unexpectedly, however, adenovirus-mediated Gli-1 overexpression blocked the Ihh enhancement of both MT1-MMP localization on the plasma membrane and PSC migration. Furthermore, reduction of Gli-1 expression with RNA interference augmented Ihh-stimulated PSC migration. These data indicate that Ihh promotes PSC migration by enhancing MT1-MMP localization on the plasma membrane but is negatively regulated by Gli-1.

GLI1 repression of ERK activity correlates with colony formation and impaired migration in human epidermal keratinocytes

Basal cell carcinoma (BCC) of the skin is a highly compact, non-metastatic epithelial tumour type that may arise from the aberrant propagation of epidermal or progenitor stem cell (SC) populations. Increased expression of GLI1 is a common feature of BCC and is linked to the induction of epidermal SC markers in immortalized N/Tert-1 keratinocytes. Here, we demonstrate that GLI1 over-expression is linked to additional SC characteristics in N/Tert-1 cells including reduced epidermal growth factor receptor (EGFR) expression and compact colony formation that is associated with repressed extracellular signal-regulated kinase (ERK) activity. Colony formation and repressed ERK activity remain evident when EGFR is increased exogenously to the basal levels in GLI1 cells revealing that ERK is additionally inhibited downstream of the receptor. Exposure to epidermal growth factor (EGF) to increase ERK activity and promote migration negates GLI1 colony formation with cells displaying an elongated, fibroblast-like morphology. However, as determined by Snail messenger RNA and E-cadherin protein expression this is not associated with epithelial-mesenchymal transition (EMT), and GLI1 actually represses induction of the EMT marker vimentin in EGF-stimulated cells. Instead, live cell imaging revealed that the elongated morphology of EGF/GLI1 keratinocytes stems from their being 'stretched' due to migrating cells displaying inefficient cell-cell detachment and impaired tail retraction. Taken together, these data suggest that GLI1 opposes EGFR signalling to maintain the epithelial phenotype. Finally, ERK activity was predominantly negative in 13/14 BCCs (superficial/nodular), indicating that GLI1 does not routinely co-operate with ERK to induce the formation of this common skin tumour.

Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function.

Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

Blockade of Hedgehog Signaling Inhibits Pancreatic Cancer Invasion and Metastases: A New Paradigm for Combination Therapy in Solid Cancers

In the context of pancreatic cancer, metastasis remains the most critical determinant of resectability, and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines, Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin, consistent with inhibition of epithelial-to-mesenchymal transition, and was mirrored by a striking reduction of in vitro invasive capacity (P < 0.0001). Conversely, Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P < 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model, cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of "primary" tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced "ALDH-high" cells by approximately 3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites, Hh inhibitors may provide a new paradigm for therapy of disseminated malignancies, particularly when used in combination with conventional antimetabolites that reduce "bulk" tumor size.

Hedgehog Signaling Promotes Medulloblastoma Survival via BclII

Activation of the Hedgehog (Hh) pathway has been identified in several cancers, including medulloblastoma, but the mechanisms by which this pathway affects tumor survival and growth are incompletely understood. We investigated whether Hedgehog might promote survival of medulloblastoma cells via up-regulation of BclII. We found that mRNA levels of the Hedgehog pathway effector Gli1 were significantly associated with BclII expression in medulloblastoma and that Gli1 and BclII are both present in regions of decreased apoptosis in nodular medulloblastoma. Transient overexpression of Gli1 and Gli2 in medulloblastoma cultures induced a BclII transcriptional reporter and increased BclII protein levels, whereas stable overexpression of Gli1 was associated with increased BclII mRNA. The Hedgehog antagonist cyclopamine blocked expression of the Hh pathway targets PTCH1 and Gli1, lowered BclII levels, and increased apoptosis in DAOY and UW228 medulloblastoma cells. Apoptotic induction caused by cyclopamine could be rescued in part by enforced expression of Gli1 or BclII. Hh pathway blockade also sensitized medulloblastoma to the effects of the proapoptotic agent lovastatin. These data demonstrate that BclII is an important mediator of Hh activity in medulloblastoma and suggest new strategies for combined chemotherapeutic regimens.

HEDGEHOG PATHWAY ACTIVATION IN ACUTE INJURY AND REGENERATION IN ADULT MOUSE PANCREAS

Background & Aims: The Hedgehog (Hh) signaling pathway specifies patterns of cell growth and differentiation during embryogenesis, and is also involved in mature tissue homeostasis, particularly in response to injury. Recently, the seminal role of this pathway in carcinogenesis, particularly in tumors of endodermal derivation such as pancreatic cancer, has been elucidated. Currently, little is known of the distribution of Hh-responsive cells in the mature exocrine pancreas, or the requirement for this pathway in epithelial regeneration. In this study, we examined the status of the Hh signaling pathway during pancreatic regeneration following exocrine injury, and also assessed the effect of Hh pathway inhibition on pancreatic regeneration, using both pharmacologic and genetic approaches. Methods: Acute exocrine pancreatic injury was induced by caerulein injection in C57BL/6J mice. To identify Hh-responding cells, we utilized a reporter mouse harboring a lacZ "knock-in" in one of the Ptch alleles (Ptch-lacZ mice). A second group of mice was additionally treated by, a specific inhibitor of the Hh-pathway. To genetically inhibit the Hh pathway in all pancreatic epithelial cell types, we used PdxI-Cre/smoflox/flox mice, while Ela-Cre-ERT2/smoflox/flox mice were used for acinar cell-specific Hh inactivation. Pancreatic tissue was harvested on days 1-7 following injury, and processed for Xgal staining (as a surrogate for Ptch activation), as well as for quantitative real-time PCR and immunohistochemical analysis. Results: We observed striking activation of the Hh pathway during acute pancreatic injury and regeneration, confirmed by marked Shh and Ptch-lacZ expression, as well as overexpression of Hh target genes, including Ptch and Gli1. Global blockade of pancreatic Hh signaling in either cyclopamine-treated or PdxI-Cre/smoflox/flox mice led to impaired regeneration of exocrine pancreas and the abnormal persistence of a novel cell population expressing both nestin and pdx1. Acinar cell-specific inhibition of Hh signaling in Ela-Cre-ERT2/smoflox/flox mice resulted in an identical phenotype. Conclusion: Our studies confirm that the Hh pathway is activated in acute pancreatic exocrine injury and repair. Inhibition of the Hh pathway prolonged the process of exocrine pancreatic regeneration. Because chronic injury in both pancreas and other solid organs is often associated with future cancer risk, activation of the Hh pathway during pancreatic injury may play a role in the initiation of pancreatic tumorigenesis.

The Zinc Finger Transcription Factor Gli2 Mediates Bone Morphogenetic Protein 2 Expression in Osteoblasts in Response to Hedgehog Signaling

Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation.

Enhancement of GLI1-transcriptional activity by β-catenin in human cancer cells

The Hedgehog (Hh) signaling pathway and the Wnt signaling pathway are known to play important roles in carcinogenesis and the progression of various human malignant tumors. Although a relationship between these two pathways has recently been reported, the mechanism by which beta-catenin, one of the key molecules of the Wnt signaling pathway, influences the Hh pathway has not yet been revealed in detail. To clarify the role of beta-catenin in relation to the Hh signaling pathway, we transfected GLI1 and beta-catenin expression constructs into human malignant cells, including stomach, colon, and lung cancers, and evaluated the luciferase activity of GLI-responsive reporter constructs. While exogenous GLI1 increased the luciferase activity, exogenous beta-catenin also enhanced the activity under overexpression of GLI1. However, co-transfection with T-cell factor (TCF)-4 or lymphocyte enhancer factor (LEF)-1 did not influence the activity, indicating that the enhancement of beta-catenin in relation to the Hh signaling pathway is not TCF/LEF-dependent. Our results suggest that beta-catenin might be involved in the Hh signaling pathway via enhancement of the transcriptional activity of GLI.

Hedgehog Signaling and Bmi-1 Regulate Self-renewal of Normal and Malignant Human Mammary Stem Cells

The epithelial components of the mammary gland are thought to arise from stem cells with a capacity for self-renewal and multilineage differentiation. Furthermore, these cells and/or their immediate progeny may be targets for transformation. We have used both in vitro cultivation and a xenograft mouse model to examine the role of hedgehog signaling and Bmi-1 in regulating self-renewal of normal and malignant human mammary stem cells. We show that hedgehog signaling components PTCH1, Gli1, and Gli2 are highly expressed in normal human mammary stem/progenitor cells cultured as mammospheres and that these genes are down-regulated when cells are induced to differentiate. Activation of hedgehog signaling increases mammosphere-initiating cell number and mammosphere size, whereas inhibition of the pathway results in a reduction of these effects. These effects are mediated by the polycomb gene Bmi-1. Overexpression of Gli2 in mammosphere-initiating cells results in the production of ductal hyperplasia, and modulation of Bmi-1 expression in mammosphere-initiating cells alters mammary development in a humanized nonobese diabetic-severe combined immunodeficient mouse model. Furthermore, we show that the hedgehog signaling pathway is activated in human breast "cancer stem cells" characterized as CD44+CD24-/lowLin-. These studies support a cancer stem cell model in which the hedgehog pathway and Bmi-1 play important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells.

Unique and complimentary activities of the Gli transcription factors in Hedgehog signaling

The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. Aberrations in Hedgehog signaling seriously affect vertebrate development. Postnatally, Hedgehog signaling has been postulated to play a pivotal role in healing and repair processes and inappropriate pathway activation has been implicated in several types of cancers. To better understand both the upstream regulation of the Gli transcription factors, as well as their unique and combinatorial roles in regulating the expression of Hedgehog target genes, we have characterized embryonic fibroblasts (MEFs) from Gli mutant mice. Stimulation of wild-type MEFs by Sonic Hedgehog (Shh) peptide elicited unique profiles of induction of Hedgehog target genes Gli1, Ptc1, and Hip1. Gli2 loss-of-function was associated with diminished Shh-induced target gene expression, while Gli3 loss-of-function was associated with increased basal and Shh-induced target gene expression. The loss of Gli1 alone had no effect on target gene induction but did diminish Shh-induced target gene expression when combined with the loss of Gli2 or Gli3. Additionally, overexpression of Gli1 induced target gene expression in Gli2 −/− 3 −/− MEFs, while Shh stimulation did not. Using MEFs expressing only Gli2 or Gli3, we found that both cyclopamine and the PKA activator forskolin inhibited target gene induction mediated by Gli2 and Gli3. These results demonstrate that Gli2 and Gli3 share common regulatory mechanisms and modulate Hedgehog target gene expression directly and independently while also regulating Gli1 expression, which in specific contexts, coordinately contributes to target gene activation.

Overlapping and distinct transcriptional regulator properties of the GLI1 and GLI2 oncogenes

The GLI transcription factors mediate the hedgehog signal in development and carcinogenesis. Basal cell carcinoma can be caused by overexpression of either GLI1 or GLI2. Though GLI1 and GLI2 have identical or very similar DNA binding specificities, some of their activities are overlapping, some are clearly distinct. We analyzed target gene specificities of GLI1 and constitutively active GLI2 (GLI2ΔN) by global expression profiling in an inducible, well-characterized HaCaT keratinocyte expression system. Four hundred fifty-six genes up- or downregulated at least twofold were identified. GLI target gene profiles correlated well with the biological activities of these transcription factors in hair follicles and basal cell carcinoma. Upregulation of largely overlapping sets of target genes was effected by both factors, repression occurred predominantly in response to GLI2. Also, significant quantitative differences in response to GLI1 and GLI2ΔN were found for a small number of activated genes. Since we have not detected a putative processed GLI2 repressor, these results point to specific but indirect target gene repression by GLI2ΔN via preferential activation of one or more negative regulators.

Deregulation of the hedgehog signalling pathway: a possible role for the PTCH and SUFU genes in human rhabdomyoma and rhabdomyosarcoma development

The naevoid basal cell carcinoma syndrome (NBCCS) is caused by mutations in the hedgehog receptor PTCH gene. It is characterized by developmental defects and a predisposition to the development of certain tumours, such as basal cell carcinoma, medulloblastoma and meningioma, and potentially fetal rhabdomyomas and embryonal rhabdomyosarcomas. This study aimed to analyse PTCH status in an NBCCS patient with fetal rhabdomyoma and to investigate whether deregulation of hedgehog signalling, as shown by altered expression of hedgehog pathway components and/or genetic imbalances, is a general finding in sporadic rhabdomyomas and rhabdomyosarcomas. The NBCCS patient had a novel PTCH germ-line mutation, 1370insT, and developed a fetal rhabdomyoma that harboured a 30 bp in-frame deletion in the second allele resulting in homozygous inactivation of PTCH. Sporadic rhabdomyomas and rhabdomyosarcomas showed overexpression of PTCH (43/43) and GLI1 (41/43) mRNA, as determined by in situ hybridization, indicating ongoing active hedgehog signalling. Immunohistochemical staining revealed a subgroup of fetal rhabdomyomas and embryonal rhabdomyosarcomas (12/34) lacking PTCH immunoreactivity. Four of nine informative fetal rhabdomyomas and embryonal rhabdomyosarcomas showed loss of heterozygosity (LOH) in the PTCH region with two of these (one fetal rhabdomyoma and one embryonal rhabdomyosarcoma) also showing LOH in the SUFU region. These findings suggest that haploinsufficiency for the two tumour suppressor genes PTCH and SUFU, which are both active in the same signalling pathway, may be important for tumour development. Based on our results we propose that the pathogenesis of rhabdomyoblastic tumours, particularly fetal rhabdomyomas and embryonal rhabdomyosarcomas, involves deregulation of the hedgehog signalling pathway.

ACTIVATION OF THE HEDGEHOG-PATCHED-SIGNALING PATHWAY IN PANCREATIC ADENOCARCINOMAS

Activation of the hedgehog signal transduction pathway, triggered by hedgehog binding to the transmembrane receptor PTCH or by mutations in the PTCH gene, plays an important role in the development of various tumors including cutaneous basal cell carcinomas and cerebellar medulloblastomas. Recent findings indicate that digestive tract tumors including pancreatic adenocarcinomas also display increased hedgehog signaling activity due to an abnormal expression of the secreted hedgehog ligand sonic hedgehog (Shh). In cell lines derived from pancreatic adenocarcinomas this pathway activity can be reverted by cyclopamine, a specific antagonist of the pathway resulting in apoptosis and reduction of cell proliferation. To investigate if alterations of PTCH may also be involved in the pathogenesis of pancreatic adenocarcinomas we screened 26 tumors for mutations using single-strand conformation polymorphism analysis (SSCP) of all coding exons and direct sequencing. We uncovered single nucleotide polymorphisms (SNPs) in exon 2, 11, 12 and 22 of the PTCH gene but no somatic mutations. Furthermore, the expression levels of known hedgehog target genes were determined using the competitive RT-PCR method. A total of 21 pancreatic adenocarcinoma biopsies and 3 pancreatic cancer cell lines were analyzed. In the majority of pancreatic cancer we found significant overexpression of the target genes GLI-1 and PTCH compared to the corresponding normal pancreatic tissue. The findings indicate that the hedgehog signaling pathway is frequently activated in pancreatic adenocarcinomas.

Hedgehog signaling is activated in subsets of esophageal cancers

The hedgehog pathway plays a critical role in the development of the foregut. However, the role of the hedgehog pathway in primary esophageal cancers is not well studied. Here, we report that elevated expression of hedgehog target genes occurs in 14 of 22 primary esophageal cancers. The hedgehog signaling activation is not associated with tumor subtypes, stages, or differentiation. While the sonic hedgehog (Shh) transcript is localized to the tumor tissue, expression of Gli1 and PTCH1 is observed both in the tumor and in the stroma. We discovered that 4 esophageal squamous cell carcinomas, which overexpress Shh, have genomic amplification of the Shh gene. Treatment of esophageal cancer cells with smoothened antagonist, KAAD-cyclopamine, or the neutralizing antibodies of Shh reduces cell growth and induces apoptosis. Overexpression of Gli1 under the CMV promoter renders these cells resistant to the treatments. Thus, our results indicate that elevated expression of Shh and its target genes is quite common in esophageal cancers. Our data also indicate that downregulation of Gli1 expression may be an important mechanism by which KAAD-cyclopamine inhibits growth and induces apoptosis in esophageal cancer cells.

Frequent activation of the hedgehog pathway in advanced gastric adenocarcinomas

The hedgehog pathway plays a critical role in the development of the foregut. Recent studies indicate that the hedgehog pathway activation occurs in the stomach and other gastrointestinal cancers. However, the association of hedgehog pathway activation with tumor stage, differentiation and tumor subtype is not well documented. Here, we report our findings that the elevated expression of hedgehog target genes human patched gene 1 (PTCH1) or Gli1 occurs in 63 of the 99 primary gastric cancers. Activation of the hedgehog pathway is associated with poorly differentiated and more aggressive tumors. The sonic hedgehog (Shh) transcript is localized to the cancer tissue, whereas expression of Gli1 and PTCH1 is observed both in the cancer and in the surrounding stroma. Treatment of gastric cancer cells with KAAD-cyclopamine, a hedgehog signaling inhibitor, decreases expression of Gli1 and PTCH1, resulting in cell growth inhibition and apoptosis. Overexpression of Gli1 under the control of the cytomegalovirus (CMV) promoter renders these cells resistant to cyclopamine-induced apoptosis. Thus, our analysis of in vivo tissues indicates that the hedgehog pathway is frequently activated in advanced gastric adenocarcinomas; our in vitro studies suggest that hedgehog signaling contributes to gastric cancer cell growth. These data predict that targeted inhibition of the hedgehog pathway may be effective in the prevention and treatment of advanced gastric adenocarcinomas.

Disruption of Meox or Gli activity ablates skeletal myogenesis in P19 cells.

Gli2 and Meox1 are transcription factors that are expressed in the developing somite and play roles in the commitment of cells to the skeletal muscle lineage. To further define their roles in regulating myogenesis, the function of wild type and dominant-negative forms of Gli2 and Meox1 were examined in the context of differentiating P19 stem cells. We found that Gli2 overexpression up-regulated transcript levels of Meox1 and, conversely, Meox1 overexpression resulted in the upregulation of Gli2 transcripts. Furthermore, dominant-negative forms of either Meox1 or Gli2 disrupted the ability of P19 cells to commit to the muscle lineage and to properly express either Gli2 or Meox1, respectively. Finally, Pax3 transcripts were induced by Gli2 overexpression and lost in the presence of either mutants Meox1 or Gli2. Taken together, these results support the existence of a regulatory loop between Gli2, Meox1, and Pax3 that is essential for specification of mesodermal cells into the muscle lineage.

Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas

Overexpression of the polycomb group gene Bmi1 promotes cell proliferation and induces leukaemia through repression of Cdkn2a (also known as ink4a/Arf) tumour suppressors. Conversely, loss of Bmi1 leads to haematological defects and severe progressive neurological abnormalities in which de-repression of the ink4a/Arf locus is critically implicated. Here, we show that Bmi1 is strongly expressed in proliferating cerebellar precursor cells in mice and humans. Using Bmi1-null mice we demonstrate a crucial role for Bmi1 in clonal expansion of granule cell precursors both in vivo and in vitro. Deregulated proliferation of these progenitor cells, by activation of the sonic hedgehog (Shh) pathway, leads to medulloblastoma development. We also demonstrate linked overexpression of BMI1 and patched (PTCH), suggestive of SHH pathway activation, in a substantial fraction of primary human medulloblastomas. Together with the rapid induction of Bmi1 expression on addition of Shh or on overexpression of the Shh target Gli1 in cerebellar granule cell cultures, these findings implicate BMI1 overexpression as an alternative or additive mechanism in the pathogenesis of medulloblastomas, and highlight a role for Bmi1-containing polycomb complexes in proliferation of cerebellar precursor cells.

Expression of the Human Poliovirus Receptor/CD155 Gene Is Activated by Sonic Hedgehog

The human poliovirus receptor/CD155 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. The ectodomain of CD155 mediates cell attachment to the extracellular matrix molecule vitronectin, while its intracellular domain interacts with the dynein light chain Tctex-1. CD155 is a primate-restricted gene that is expressed during development in mesenchymal tissues and ventrally derived structures within the CNS. Its function in adults is as yet unknown, but significantly, CD155 is aberrantly expressed in neuroectodermal tumors. We show that the expression of CD155 mRNA is up-regulated when human Ntera2 cells are treated with purified Sonic hedgehog (Shh) protein. Reporter gene expression driven by the CD155 core promoter is activated by Shh in transient co-transfection assays. Analysis of the CD155 core promoter indicates that an intact GLI binding site is required for Shh activation. In addition, overexpression of Gli1 or Gli3 potently activates reporter gene expression driven by the CD155 core promoter. These data identify the CD155 gene as a transcriptional target of Shh, a finding that has significance for the normal function of CD155 during development and the expression of CD155 in neuroectodermal tumors.