Canonical Hedgehog Signaling Research Articles

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.

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.

Canonical hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular cells

Hedgehog (Hh) signaling is critical to the patterning and development of a variety of organ systems, and both ligand-dependent and ligand-independent Hh pathway activation are known to promote tumorigenesis. Recent studies have shown that in tumors promoted by Hh ligands, activation occurs within the stromal microenvironment. Testing whether ligand-driven Hh signaling promotes tumor angiogenesis, we found that Hh antagonism reduced the vascular density of Hh-producing LS180 and SW480 xenografts. In addition, ectopic expression of sonic hedgehog in low-Hh-expressing DLD-1 xenografts increased tumor vascular density, augmented angiogenesis, and was associated with canonical Hh signaling within perivascular tumor stromal cells. To better understand the molecular mechanisms underlying Hh-mediated tumor angiogenesis, we established an Hh-sensitive angiogenesis coculture assay and found that fibroblast cell lines derived from a variety of human tissues were Hh responsive and promoted angiogenesis in vitro through a secreted paracrine signal(s). Affymetrix array analyses of cultured fibroblasts identified VEGF-A, hepatocyte growth factor, and PDGF-C as candidate secreted proangiogenic factors induced by Hh stimulation. Expression studies of xenografts and angiogenesis assays using combinations of Hh and VEGF-A inhibitors showed that it is primarily Hh-induced VEGF-A that promotes angiogenesis in vitro and augments tumor-derived VEGF to promote angiogenesis in vivo.

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

Linking Stem Cells to Thyroid Cancer

Recent progress in tumor cell biology and genetics provides increasing evidence that a specific subpopulation of tumor cells plays a crucial role in tumor initiation, invasive growth, and metastasis in several malignancies (for review, see Ref. 1). These cells, designated cancer stem cells (CSCs) or tumor-initiating cells, have the ability of asymmetric cell division, i.e. they generate a self-copy (selfrenewal) and give rise to progenitor cells that progressively differentiate into multiple lineages of the heterogeneous tumor cell mass (2). When transplanted into immunodeficient, i.e. NOD/SCID, mice, CSCs regenerate a phenocopy of the cancer of origin. Indeed, CSCs have been detected in several solid tumors, in leukemia, and in cancer cell lines (2). CSCs may arise through acquisition of mutations or epigenetic changes from adult stem cells or their committed progenitor cells. Adult stem cells, which are capable of self-renewal and differentiation throughout the organism’s lifetime, are characterized by their pluripotency and undifferentiated state. The CSC model (hierarchy model) argues that the striking morphological and functional heterogeneity, which is a hallmark of most tumors, is sustained by a biologically distinct subpopulation of CSCs (3). However, not all malignancies follow the CSC model of heterogeneity. Thus, an alternative model has been put forward to explain this heterogeneity. The stochastic model for tumor initiation predicts that a tumor is primarily biologically homogenous and acquires heterogeneity by randomly occurring secondary events that alter proliferation, differentiation, and survival of individual cells. All tumor cells are equally sensitive to intrinsic (e.g. levels of transcription factors, signaling pathways) or extrinsic factors (e.g. host factors, microenvironment, immune response) that affect single cells and thus may generate heterogeneity (4). Support for the CSC concept of carcinogenesis comes from findings that several signaling pathways that are known to regulate adult stem cells are altered in malignancies, including the canonical Wnt pathway, Hedgehog, and Notch signaling pathways (5–7). Furthermore, there are close relationships between gene expression pattern in embryonic stem cells and some malignancies (1).

TGF-β Promotion of Gli2-Induced Expression of Parathyroid Hormone-Related Protein, an Important Osteolytic Factor in Bone Metastasis, Is Independent of Canonical Hedgehog Signaling

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.

TGF-β promotion of GLI2 induced pthrp expression is independent of canonical hedgehog signaling

TGF-β promotion of GLI2 induced pthrp expression is independent of canonical hedgehog signaling

Activation of Erk by sonic hedgehog independent of canonical hedgehog signalling

Hedgehog (Hh) signalling is mediated through the Patched-1 (Ptch1) receptor. Hh-binding to Ptch1 blocks the inhibitory effects of Ptch1 on the activity of the transmembrane protein, Smoothened (Smo), resulting induction of target genes by the Gli-family of transcription factors. We demonstrate here that Hh-binding to Ptch1 stimulates activation of Erk1/2. This activation is insensitive to the small molecule Smo antagonists and occurs in a cell line that does not express Smo. Specifically, the C-terminus of Ptch1 harbours motifs encoding Class I and II SH3-binding sites. SH3-domain binding activity was verified using GST-c-src SH3, -Grb2 SH3 and -p85β SH3 fusion-proteins. Ectopically expressed Grb2 or p85β could also be co-immunoprecipitated with the Ptch1 C-terminus. Addition of Shh to serum-starved human mammary epithelial cells and Shh Light II fibroblasts stimulated phosphorylation of Erk1/2. Erk1/2 activation was observed in cells where Smo activity had been inhibited using cyclopamine and in the breast epithelial cell line, MCF10A, that does not express Smo. These data reveal novel binding activities for the C-terminal region of Ptch1 and define a signalling pathway stimulated by the Hh-ligands operating independently of pathways requiring Smo.

14-P012 Interactions between canonical Wnt pathway and Hedgehog signalling in retinal stem/precursor cells

14-P012 Interactions between canonical Wnt pathway and Hedgehog signalling in retinal stem/precursor cells