Gli Activity Research Articles

The Antagonistic Action of B56-containing Protein Phosphatase 2As and Casein Kinase 2 Controls the Phosphorylation and Gli Turnover Function of Daz Interacting Protein 1

The Hedgehog (Hh) pathway is evolutionarily conserved and plays critical roles during embryonic development and adult tissue homeostasis. Defective Hh signaling has been linked to a wide range of birth defects and cancers. Hh family proteins regulate the expression of their downstream target genes through the control of proteolytic processing and the transcriptional activation function of Gli transcription factors. Although Hh-dependent regulation of Gli has been studied extensively, other Gli regulatory mechanisms remain relatively unappreciated. Here we report our identification of a novel signaling cascade that controls the stability of Gli proteins. This cascade consists of Daz interacting protein 1 (Dzip1), casein kinase 2 (CK2), and B56 containing protein phosphatase 2As (PP2As). We provide evidence that Dzip1 is involved in a novel Gli turnover pathway. We show that CK2 directly phosphorylates Dzip1 at four serine residues, Ser-664/665/706/714. B56-containing PP2As, through binding to a domain located between amino acid residue 474 and 550 of Dzip1, dephosphorylate Dzip1 on these CK2 sites. Our mutagenesis analysis further demonstrates that the unphosphorylatable form of Dzip1 is more potent in promoting Gli turnover. Consistently, we found that the stability of Gli proteins was decreased upon CK2 inhibition and increased by inhibition of B56-containing PP2As. Thus, reversible phosphorylation of Dzip1, which is controlled by the antagonistic action of CK2 and B56-containing PP2As, has an important impact on the stability of Gli transcription factors and Hh signaling.

The α Subunit of the G Protein G13 Regulates Activity of One or More Gli Transcription Factors Independently of Smoothened

Smoothened (Smo) is a seven-transmembrane (7-TM) receptor that is essential to most actions of the Hedgehog family of morphogens. We found previously that Smo couples to members of the G(i) family of heterotrimeric G proteins, which in some cases are integral although alone insufficient in the activation of Gli transcription factors through Hedgehog signaling. In response to a report that the G(12/13) family is relevant to Hedgehog signaling as well, we re-evaluated the coupling of Smo to one member of this family, G(13), and investigated the capacity of this and other G proteins to activate one or more of forms of Gli. We found no evidence that Smo couples directly to G(13). We found nonetheless that Gα(13) and to some extent Gα(q) and Gα(12) are able to effect activation of Gli(s). This capacity is realized in some cells, e.g. C3H10T1/2, MC3T3, and pancreatic cancer cells, but not all cells. The mechanism employed is distinct from that achieved through canonical Hedgehog signaling, as the activation does not involve autocrine signaling or in any other way require active Smo and does not necessarily involve enhanced transcription of Gli1. The activation by Gα(13) can be replicated through a G(q)/G(12/13)-coupled receptor, CCK(A), and is attenuated by inhibitors of p38 mitogen-activated protein kinase and Tec tyrosine kinases. We posit that G proteins, and perhaps G(13) in particular, provide access to Gli that is independent of Smo and that they thus establish a basis for control of at least some forms of Gli-mediated transcription apart from Hedgehogs.

Blocking Hedgehog Survival Signaling at the Level of the GLI Genes Induces DNA Damage and Extensive Cell Death in Human Colon Carcinoma Cells

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.

The GLI genes as the molecular switch in disrupting Hedgehog signaling in colon cancer

The Hedgehog (HH) signaling pathway leads to activation of GLI, which transcriptionally regulate target genes. Regulated HH signaling activity is critical during embryogenesis while aberrantly activated HH signaling is evident in a variety of human cancers. Canonical HH signaling engages the transmembrane receptor Patched (PTCH) and the signaling intermediate Smoothened (SMO) to activate GLI1 and GLI2. In addition GLI1 and GLI2 are activated by non-canonical oncogenic signaling pathways to further drive HH-dependent survival. We have demonstrated in human colon carcinoma cells that inhibition of the RAS/RAF pathway by U0126 decreases p-ERK protein expression and also inhibits GLI-luciferase activity and GLI1 mRNA and protein levels. Of importance is the demonstration that targeting of SMO (using cyclopamine) has minimal effect on cell survival in comparison to the inhibition of GLI (using GANT61), which induced extensive cell death in 7/7 human colon carcinoma cell lines. Genetic inhibition of the function of GLI1 and GLI2 by transient transfection of the C-terminus deleted repressor GLI3R, reduced proliferation and induced cleavage of caspase-3 and cell death in HT29 cells, similar to the effects of GANT61. Mechanistically, downstream of GLI1 and GLI2 inhibition, γH2AX (a marker of DNA double strand breaks) expression was upregulated, and γH2AX nuclear foci were demonstrated in cells that expressed GLI3R. Activation of the ATM/Chk2 axis with co-localization of γH2AX and p-Chk2 nuclear foci were demonstrated following GLI1/GLI2 inhibition. GANT61 induced cellular accumulation at G1/S and early S with no further progression before cells became subG1, while cDNA microarray gene profiling demonstrated downregulation of genes involved in DNA replication, the DNA damage response, and DNA repair, mechanisms that are currently being pursued. These studies highlight the importance of targeting the GLI genes downstream of SMO for terminating HH-dependent survival, suggesting that GLI may constitute a molecular switch that determines the balance between cell survival and cell death in human colon carcinoma.

Increased hedgehog signaling in postnatal kidney results in aberrant activation of nephron developmental programs

Hedgehog (Hh) is a core signaling pathway implicated in fundamental processes during embryonic kidney development. We previously found that loss-of-function mutations in the transcription factor GLIS2, a putative vertebrate ortholog of Drosophila Ci, cause nephronophthisis type 7 in humans and mice. Kidney tubular cells in Glis2-knockout mice acquire mesenchymal phenotype, but the cellular mechanisms of this transition are unknown. Here, we demonstrate that Glis2 is a functional component of Hh signaling and is necessary to suppress this pathway in the postnatal kidney. In the epithelial compartment, Glis2 opposes Gli1 activity by binding cis-acting regulatory sequences in the 5' flanking regions of Snai1 and Wnt4, thereby inhibiting de-differentiation of tubular cells. We conclude that Glis2 is necessary to inhibit Hh signaling and to maintain the mature tubular epithelial phenotype in the adult kidney. This is the first description of a molecular mechanism that links the Hh signaling pathway to cystic kidney diseases and can open new avenues for the treatment of diverse ciliopathies.

Tcf/Lef repressors differentially regulate Shh-Gli target gene activation thresholds to generate progenitor patterning in the developing CNS

During neural tube development, Shh signaling through Gli transcription factors is necessary to establish five distinct ventral progenitor domains that give rise to unique classes of neurons and glia that arise in specific positions along the dorsoventral axis. These cells are generated from progenitors that display distinct transcription factor gene expression profiles in specific domains in the ventricular zone. However, the molecular genetic mechanisms that control the differential spatiotemporal transcriptional responses of progenitor target genes to graded Shh-Gli signaling remain unclear. The current study demonstrates a role for Tcf/Lef repressor activity in this process. We show that Tcf3 and Tcf7L2 (Tcf4) are required for proper ventral patterning and function by independently regulating two Shh-Gli target genes, Nkx2.2 and Olig2, which are initially induced in a common pool of progenitors that ultimately segregate into unique territories giving rise to distinct progeny. Genetic and functional studies in vivo show that Tcf transcriptional repressors selectively elevate the strength and duration of Gli activity necessary to induce Nkx2.2, but have no effect on Olig2, and thereby contribute to the establishment of their distinct expression domains in cooperation with graded Shh signaling. Together, our data reveal a Shh-Gli-independent transcriptional input that is required to shape the precise spatial and temporal response to extracellular morphogen signaling information during lineage segregation in the CNS.

Hepatitis B virus X protein stimulates the Hedgehog–Gli activation through protein stabilization and nuclear localization of Gli1 in liver cancer cells

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver diseases, which frequently results in hepatits, cirrhosis, fibrosis, and ultimately hepatocellular carcinoma (HCC). Recent studies have shown the activation of Hedgehog signaling in HCC. Here, we provide evidences that HBV induces Gli-directed gene transactivation. HBx increases the protein stability of Gli proteins, which are key transcription factors of the Hedgehog signaling pathway, and nucleus translocation of Gli1 through direct protein interaction of HBx and Gli1. This functional synergism of Gli1 protein by HBx increases the Hedgehog activation-directed gene expression. Taken together, these results suggest that HBV infection might induce hepatocellular carcinoma by modulating post-translational activation of the hedgehog signaling components.

Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations

Uncontrolled Hedgehog (Hh) signaling leads to the development of basal cell carcinoma (BCC), the most common human cancer, but the cell of origin for BCC is unclear. While Hh pathway dysregulation is common to essentially all BCCs, there exist multiple histological subtypes, including superficial and nodular variants, raising the possibility that morphologically distinct BCCs may arise from different cellular compartments in skin. Here we have shown that induction of a major mediator of Hh signaling, GLI2 activator (GLI2ΔN), selectively in stem cells of resting hair follicles in mice, induced nodular BCC development from a small subset of cells in the lower bulge and secondary hair germ compartments. Tumorigenesis was markedly accelerated when GLI2ΔN was induced in growing hair follicles. In contrast, induction of GLI2ΔN in epidermis led to the formation of superficial BCCs. Expression of GLI2ΔN at reduced levels in mice yielded lesions resembling basaloid follicular hamartomas, which have previously been linked to low-level Hh signaling in both mice and humans. Our data show that the cell of origin, tissue context (quiescent versus growing hair follicles), and level of oncogenic signaling can determine the phenotype of Hh/Gli-driven skin tumors, with high-level signaling required for development of superficial BCC-like tumors from interfollicular epidermis and nodular BCC-like tumors from hair follicle stem cells.

GLI Activation is Required for Pancreatic Tumor Formation In Vivo

GLI Activation is Required for Pancreatic Tumor Formation In Vivo

The Requirement of GLI Transcriptional Activity in KRAS-Induced Pancreatic Neoplasia

Background and Aim: Kras mutations are detected in 90% of human pancreatic ductal adenocarcinoma (PDAC) and are believed to be the critical oncogenic event during pancreatic tumorigenesis. However, the downstream transcriptional mechanisms by Kras in pancreatic cancer still remain poorly understood. Recent In Vitro studies suggested that Gli transcriptional factors, the intracellular mediator of Hedgehog signaling, are important for Kras activity in human PDAC cell lines. However, a requirement for Gli activation in pancreatic malignancy has not been demonstrated In Vivo. Here we carried out both loss-of-function and gain-offunction genetic analyses to establish the functional significance of Gli transcriptional activation in Kras-Induced pancreatic tumorigenesis using genetically modified mouse models. Methods: We utilized a conditional Rosa26 allele of Gli3T, R26-Gli3T. Gli3T is a dominant repressor of Gli3 that blocks Gli transcriptional activation. The R26-Gli3T mice were crossed to the Ptf1a-Cre;LDL-KrasG12D mice, a mouse model that allows pancreatic-specific Kras activation and tumor formation. A cohort of Ptf1a-Cre;LDL-KrasG12D;R26-Gli3T mice was generated and monitored for pancreatic tumor development. For Gli gain of function studies, we ectopically expressed Gli1 along with oncogenic Kras in mouse pancreas to determine whether Gli1 cooperates with Kras to promote tumorigenesis. Results: Our results showed that Gli3T expression caused a severe growth defect and loss of tumorigenicity in human PDAC cell lines. More importantly, the mice carrying Gli3T along with oncogenic Kras failed to develop PanIN lesions, in contrast to the mice with Kras activation alone. Mice expressing Gli1 along with activated Kras in pancreas also showed marked acceleration of tumor formation. Furthermore, our gene expression profiling identified a distinct transcriptional program mediated by Gli proteins in pancreatic tumor cells. Conclusions: Gli transcriptional activation not only accelerates pancreatic tumorigenesis in cooperation with Kras, but is also required for Kras-induced tumorigenesis In Vivo.

Role of GLI2 in the growth of human osteosarcoma

The Hedgehog pathway functions as an organizer in embryonic development. Aberrant activation of the Hedgehog pathway has been reported in various types of malignant tumours. The GLI2 transcription factor is a key mediator of Hedgehog pathway but its contribution to neoplasia is poorly understood. To establish the role of GLI2 in osteosarcoma, we examined its expression by real-time PCR using biopsy tissues. To examine the function of GLI2, we evaluated the growth of osteosarcoma cells and their cell cycle after GLI2 knockdown. To study the effect of GLI2 activation, we examined mesenchymal stem cell growth and the cell cycle after forced expression of GLI2. We found that GLI2 was aberrantly over-expressed in human osteosarcoma biopsy specimens. GLI2 knockdown by RNA interferences prevented osteosarcoma growth and anchorage-independent growth. Knockdown of GLI2 promoted the arrest of osteosarcoma cells in G(1) phase and was accompanied by reduced protein expression of the cell cycle accelerators cyclin D1, SKP2 and phosphorylated Rb. On the other hand, knockdown of GLI2 increased the expression of p21(cip1) . In addition, over-expression of GLI2 promoted mesenchymal stem cell proliferation and accelerated their cell cycle progression. Finally, evaluation of mouse xenograft models showed that GLI2 knockdown inhibited the growth of osteosarcoma in nude mice. Our findings suggest that inhibition of GLI2 may represent an effective therapeutic approach for patients with osteosarcoma.

Abstract 2585: Snail-p53 interaction inhibitor GN25 and its combination with Oxaliplatin targets both hedgehog signaling and K-ras pathway in pancreatic cancer

Abstract Activation of hedgehog (Hh) signaling is widely observed in human cancers including pancreatic cancer (PC). Similarly, mutations in K-Ras gene is found in >80% of PC. Constitutive activation of Hh signaling component Gli in turn enhances p53 suppressor Snail. Studies from our group have revealed that p53 is further suppressed by direct binding with oncogenic K-Ras-induced Snail. Therefore in order to target combined suppressive effects of Hh signaling and K-Ras-induced snail on p53 pathway, a specific inhibitor of the p53-Snail binding (GN25) was developed. This inhibitor can induce p53 expression and functions in K-Ras-mutated cells, with no toxicity to normal cells (Oncogene 2010). Here we show that GN25 when combined with Oxaliplatin induces synergistically (combination index CI<1) enhanced growth inhibition (MTT) and apoptosis (Histone DNA ELISA and Annexin V FITC) in multiple PC cell lines (AsPC-1, Capan-2, BxPC-3 and Colo-357) irrespective of the mutational or functional status of both p53 and K-Ras. Interestingly, we found that some of the above cell lines have hyper activation of Hh signaling that results in constitutively over activation of Gli. Western blot analysis demonstrated that GN25/oxaliplatin re-activated the p53 protein and downstream effector molecules such as pro-apoptotic Bax and cell cycle regulator p21. Further analysis showed that GN25/oxalipltin interferes with the Hh signaling and suppresses Snail. In SCID mice studies in vivo, the maximum tolerated dose of GN25 was found to be 35 mg/kg i.v. In addition, it can be given in combination with oxaliplatin at 5 mg/kg i.p twice a week for two weeks. The combination studies of GN25/oxaliplatin in multiple tumor xenografts (wt-k-Ras BxPC-3 and AsPC-1 mut-p53 k-Ras) are ongoing. So far our results suggest that GN25/oxaliplatin is a strong combination against hyper activated Hh and mutant K-Ras-initiated human pancreatic cancer irrespective of mutational status of p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2585. doi:10.1158/1538-7445.AM2011-2585

Abstract 1656: Termination of hedgehog (HH) survival signaling at the level of the GLI genes induces DNA damage and extensive cell death in human colon carcinoma cells

Abstract Temporally and spatially regulated activity of the HH signaling pathway is important for normal embryonic development and organ patterning while aberrant activation of this pathway is involved in the development of genomic instability and dysregulated proliferation in several human cancers. There is emerging evidence that HH signaling progresses during colon carcinogenesis and in metastatic disease, however little is known functionally about this signaling pathway and how it affects the survival and pathogenesis of colon cancer. Canonical HH signaling occurs via Ptch1, Smo, and activation of Gli1 and Gli2, which transcriptionally regulate HH target genes. We demonstrate that a small molecule inhibitor of Gli1 and Gli2, GANT61, in contrast to cyclopamine (Smo inhibitor), induced extensive cell death in six human colon carcinoma cell lines. Detailed mechanistic studies in HT29 cells demonstrated that GANT61 induced transient accumulation of cells at the G1/S boundary and in early S at 24 hr and 32 hr, respectively, along with upregulation of p21Cip1, cyclin E and cyclin A in G1- and S- phase cells. By 40 hr, GANT61-treated cells (but not cells treated with cyclopamine) became subG1. shRNA-mediated knockdown of p21Cip1 failed to alter the cytotoxicity of GANT61. cDNA microarray gene profiling in GANT61-treated cells identified significant changes in expression of genes involved in DNA damage signaling, DNA replication and DNA repair. In single cell assays (Comet), GANT61 induced DNA damage by 24 hr with increased Tail Moment and in particular Tail Length; within 4 hr, γH2AX foci were detected at sites of DNA breaks (confocal microscopy). Upregulation of activated forms of ATM and Chk2 but not ATR or Chk1 at 4 hr, that were sustained (24 hr), was observed by western analysis following GANT61 exposure. Further, by 4 hr, p-Chk2 foci co-localized with γH2AX foci in nuclei. Partial knockdown of GLI1 and GLI2 using shRNA partially protected HT29 cells from GANT61-induced cell death, and reduced nuclear γH2AX foci. Studies of the extrinsic and intrinsic pathways of apoptosis demonstrated increased expression of Fas and DR5 receptors, decreased expression of PDGFRα (GLI1 target which regulates Fas), and decreased Bcl-2 expression (GLI2 target) following GANT61 treatment. Expression of DNFADD (to abrogate death receptor signaling) and/or over-expression of Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued from GANT61-induced cell death. Data demonstrate the critical role of HH signaling in colon cancer cell survival, and that inhibition of this pathway at the level of the GLI genes in contrast to Smo induces 1) DNA damage, 2) transient accumulation of cells at G1/S and early S, 3) ineffective checkpoint activation, and 4) extensive cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2011-1656

Abstract 513: Stromal desmoplasia is induced by a hypoxia and HIF-1α mediated increase in sonic hedgehog formation in pancreatic cancer

Abstract Pancreatic tumors are notoriously hypoxic and characterized by a dense fibro-inflammatory stromal reaction (desmoplasia). Hedgehog (Hh) signaling is a major contributor to tumor desmoplasia in pancreatic and other cancers, resulting in decreased tumor blood flow, decreased drug delivery and resistance to therapy. We hypothesized that the hypoxia inducible factor-1α (HIF-1α) transcription factor, a major component of the cell's response to hypoxia is an important mediator of the mechanism inducing desmoplasia in pancreatic cancer. We observed that a hypoxic environment (1% O2 for 24 hr) increased the expression of sonic Hh (SHH) mRNA and secreted SHH protein, in both MiaPaCa-2 and Panc-1 pancreatic cancer cells, and that this was blocked by siRNA targeting HIF-1α. NIH-3T3 fibroblasts transfected with a GLi1-firefly luciferase reporter exposed to conditioned media from hypoxic MiaPaCa-2 and Panc-1, or co-cultures of the Panc-1 and the fibroblasts showed an increase in GLi activity. siRNA to sHH transfected into Panc-1 tumor cells blocked the fibroblast GLi1 response. NIH-3T3 fibroblasts alone did not show an increase in Hh components or Hh signaling upon exposure to hypoxia. MiaPaCa-2 cells transfected with a GLi1-firefly luciferase reporter showed no increase in GLi1 activity upon exposure to hypoxia suggesting there is no autocrine stimulation of the cancer cells by the SHH they secrete. Fibroblasts co-cultured with the pancreatic cancer cells showed increased expression of two markers of desmoplasia, collagen-1 and fibronectin under hypoxic conditions that was blocked by siRNA to HIF-1α. Thus, increased pancreatic cancer cell SHH formation in response to hypoxia and mediated by HIF-1α, stimulates Hh signaling which increases fibroblast GLi1 transcriptional activity without activating Hh signaling in the cancer cell itself. We also compared levels of nuclear HIF-1α and SHH by immunohistochemistry in patient pancreatic cancer samples and found a significant correlation between tumor HIF-1α and stromal SHH (p< 0.01). Furthermore, both tumor HIF-1α and stromal SHH correlated with decreased patient survival. The findings suggest that tumor hypoxia and increased HIF-1α in pancreatic cancer which is likely a consequence of the desmoplasia, is also a cause of the desmoplasia. Breaking the cycle of hypoxia, HIF-1α, and desmoplasia, could offer a new therapy for pancreatic cancer. Supported by CA109552-05 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 513. doi:10.1158/1538-7445.AM2011-513

Abstract 5343: Aberrant activation of hedgehog signaling confers a poor prognosis in embryonal and fusion gene negative alveolar rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and comprises two major histological subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). The hedgehog (Hh) pathway has recently been implied in tumor formation and progression of various cancers. In the current study we investigated whether Hh pathway activation presents a general feature also of RMS and whether it bears prognostic impact. Here, we report that marker genes of active Hh signaling, i.e. Patched1 (Ptch1), Gli1, Gli3 and Myf5, are elevated in two distinct cohorts of RMS patients with a total number of 235 primary samples. Interestingly, all these marker genes of active Hh signaling were expressed at significantly higher levels in specific subtypes of RMS, i.e. ERMS and fusion gene negative ARMS, compared to fusion gene positive ARMS. Consistently, Gli1 expression correlates with Ptch1 expression in ERMS and fusion gene negative ARMS, but not in fusion gene positive ARMS. In addition, expression levels of MyoD1 are significantly lower in ERMS and fusion gene negative ARMS, pointing to an inverse association of Hh activation and early muscle differentiation in primary RMS samples. In support of this notion, the addition of Shh inhibits muscle differentiation in parallel experiments in muscle satellite cells and myoblasts. Moreover, we identify Myf5 is as a novel excellent class predictor for RMS by receiver operating characteristic (ROC) analysis. Most importantly, high expression of Ptch1 or low MyoD1 expression significantly correlate with reduced cumulative survival in fusion gene negative RMS (P< 0.004) underscoring the clinical relevance of these findings. By identifying aberrant Hh activation in specific subgroups of RMS, our study has important implications for the development of molecular targeted therapies, such as small molecule Hh inhibitors, in RMS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5343. doi:10.1158/1538-7445.AM2011-5343

A novel mutation of the PTCH1 gene activates the Shh/Gli signaling pathway in a Chinese family with nevoid basal cell carcinoma syndrome

Objective: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by a predisposition to neoplasms and developmental abnormalities. Mutation of the PTCH1 gene, which is considered to be responsible for NBCCS, was investigated in a Chinese NBCCS family in this study. Methods: Genomic DNA was isolated from blood samples of all eight living individuals in this family. Mutation analysis of PTCH1 was done by amplified polymerase chain reaction and direct sequencing. Biophysical predictions of the altered protein were made using various bioinformatics tools. To determine the action of the mutated protein, the expression of Gli1 and Gli2 was investigated by immunohistochemistry. Results: A novel PTCH1 mutation at 897G → A in exon4 was identified in all four affected members. This mutation was not found in any unaffected members of this family or in 100 unrelated healthy Chinese people. The mutation causes amino acid replacement 237E → K in the first large extracellular loop of the PTCH1 protein which is required for Sonic Hedgehog (Shh) binding. This mutation changes the protein’s biochemical properties and protein activity, resulting in subsequent activation of transcription factors, Gli1 and Gli2, in the Shh/Gli signaling pathway. Immunohistochemistry showed overexpression of Gli1 and Gli2 in the keratocystic odontogenic tumor (KCOT) tissues.

The Binding of Hepatitis B Virus X Protein to Glioma-Associated Oncogene Homologue 1 and its Biological Characterization In vitro

Hepatitis B virus (HBV) X protein (HBx) is a 17-kDa transcriptional coactivator that plays a significant role in the regulation of genes involved in inflammation and cell survival. It has been known to be involved in the development of liver cancer and alteration of the cellular HBx level may influence the pathogenesis of HBV-induced liver diseases. The transcription factor GLI1, a member of the glioma-associated oncogene homologue (GLI) subfamily of Krüppel-like zinc finger proteins is involved in signal transduction within the hedgehog (Hh) signaling pathway, which is involved in the development of many human malignancies. GLI activation is important for cell proliferation and anti-apoptosis in various cancers. To investigate whether the transcriptional coactivator HBx binds to the zinc finger transcription factor GLI1, recombinant HBx and GLI1 were isolated. Expression and purification of the HBx and GLI1 proteins were successfully performed in Escherichia coli. The binding of HBx to GLI1 was detected by surface plasmon resonance spectroscopy (BIAcore), fluorescence measurement, and a His-tagged pull-down experiment. After measuring the fluorescence emission spectra of purified HBx and GLI1, it was found that the interaction of these proteins is accompanied by significant conformational changes in one or both. This study provides important clues for the structural identification of signal transduction pathways involving the HBx and GLI1 proteins.

Conditional ablation of Pten in osteoprogenitors stimulates FGF signaling

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a direct antagonist of phosphatidylinositol 3 kinase. Pten is a well recognized tumor suppressor and is one of the most commonly mutated genes in human malignancies. More recent studies of development and stem cell behavior have shown that PTEN regulates the growth and differentiation of progenitor cells. Significantly, PTEN is found in osteoprogenitor cells that give rise to bone-forming osteoblasts; however, the role of PTEN in bone development is incompletely understood. To define how PTEN functions in osteoprogenitors during bone development, we conditionally deleted Pten in mice using the cre-deleter strain Dermo1cre, which targets undifferentiated mesenchyme destined to form bone. Deletion of Pten in osteoprogenitor cells led to increased numbers of osteoblasts and expanded bone matrix. Significantly, osteoblast development and synthesis of osteoid in the nascent bone collar was uncoupled from the usual tight linkage to chondrocyte differentiation in the epiphyseal growth plate. The expansion of osteoblasts and osteoprogenitors was found to be due to augmented FGF signaling as evidenced by (1) increased expression of FGF18, a potent osteoblast mitogen, and (2) decreased expression of SPRY2, a repressor of FGF signaling. The differentiation of osteoblasts was autonomous from the growth plate chondrocytes and was correlated with an increase in the protein levels of GLI2, a transcription factor that is a major mediator of hedgehog signaling. We provide evidence that increased GLI2 activity is also a consequence of increased FGF signaling through downstream events requiring mitogen-activated protein kinases. To test whether FGF signaling is required for the effects of Pten deletion, we deleted one allele of fibroblast growth factor receptor 2 (FGFR2). Significantly, deletion of FGFR2 caused a partial rescue of the Pten-null phenotype. This study identifies activated FGF signaling as the major mediator of Pten deletion in osteoprogenitors.

Sonic Hedgehog Signaling Protects Human Hepatocellular Carcinoma Cells Against Ionizing Radiation in an Autocrine Manner

Sonic hedgehog (Shh) signaling is critical to embryogenesis and resistance to chemotherapy. We aimed to examine the role of Shh signaling in the response to radiation of human hepatocellular carcinoma (HCC) cells. Response to ionizing radiation therapy (RT) was evaluated by clonogenic assay. Quantitative RT-polymerase chain reaction for patched-1 (PTCH-1) expression was performed. Cytosolic accumulation of Shh and nuclear translocation of Gli-1 were assessed by immunofluorescence. Gli-1 knockdown was done by RNA interference (RNAi). Immunoprecipitation was performed to detect Shh ligand in conditioned medium. Immunofluorescent stain for γ-H2AX was used as an index of DNA double strand breaks (DSB). Expression of proteins related to DNA damage repair was assessed by Western blotting. We found that Shh ligand could protect human HCC HA22T and Sk-Hep1 cells against RT. In HA22T cells, Shh ligand activated the Shh signaling with upregulation of Shh, PTCH-1, and Gli-1 expression. The nuclear translocation of Gli-1 further supports the activation of Gli-1. The radioprotection by Shh ligand was partly blocked by Shh antibody neutralization and was abolished by Gli-1 RNAi, suggesting a critical role of Shh signaling in radiation resistance. Furthermore, we noted that soluble factors secreted into conditioned medium, either constitutively or responding to radiation, by HA22T or Sk-Hep1 cells protected subsequent culturing cells against RT. Immunoprecipitation shows the presence of Shh peptide in conditioned medium. Intriguingly, antibody neutralization of Shh ligand or knockdown of Gli-1 reversed the radioprotective effect of conditioned medium. Furthermore, Shh ligand reduced the RT-induced phosphorylation of checkpoint kinase 1 and impaired the repair of DNA DSB. Activation of Shh signaling protects HCC cells against ionizing radiation in an autocrine manner. Impairment of DNA damage repair might involve mechanism of Shh-induced radioresistance. Targeting Shh signaling pathway may be a novel strategy to enhance the radioresponse of human HCC cells.

Don't hedge your bets: Hedgehog signaling as a central mediator of endochondral bone development and cartilage diseases

Cell differentiation and patterning are vital processes in the development of the appendicular skeleton. The hedgehog (Hh) signaling pathway plays a central role in regulating the anterior-posterior axis of the distal limb as well as the length of endochondral bones. Ligand-induced Hh signaling inhibits the processing of the Gli transcription factors from activator to repressor isoforms. In the growth plate, Indian hedgehog inhibits Gli processing, resulting in accumulation of Gli activators that induce chondrocyte maturation and hypertrophic differentiation. Parathyroid hormone-like hormone promote and Gli processing to repressor forms, thus regulating the rate of hypertrophic differentiation. In cartilage diseases such as osteoarthritis and cartilage tumors, there is a recapitulation of developmental processes that involve increased Hh signaling. Studies have shown that pharmacological inhibitors of Hh signaling can attenuate the progression osteoarthritis and cartilage tumor growth. Thus, Hh blockade can serve as a potential therapy for the treatment of various cartilage diseases.