An essential role for the Gai2 protein in Smoothened-stimulated mammary epithelial cell proliferation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE70210 | GEO | 2015/09/08
SECONDARY ACCESSION(S): PRJNA287876
REPOSITORIES: GEO
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Project description:Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. Smoothened (SMO), the primary effector of Hh signaling, is expressed ectopically in human breast cancer, as well as in other cancers. Constitutive activation of SMO in mouse mammary glands leads to paracrine stimulation of proliferation, as well as hyperplasia. In canonical signaling, SMO functions via GLI transcription factor activation. However, recent data from Drosophila and mammalian cell lines indicate that SMO can function non-canonically as a G-protein coupled receptor (GPCR) by coupling to heterotrimeric G proteins, particularly those in the pertussis toxin (PTX)-sensitive G-alpha-i (Gai) class. Whether SMO functions as a GPCR in mammalian tissues in vivo is not known. Using genetically modified mouse models, we demonstrate here that SMO-induced stimulation of proliferation is PTX sensitive, and requires Gai2, but not Gai1 or Gai3. Our findings provide evidence for a non-canonical GPCR function of activated SMO in vivo, a finding that may have clinical significance given that most SMO-targeted agents were selected based largely on their ability to block canonical GLI-mediated transcription. Primary mammary epithelial cell RNA was deep-sequenced from mT-mG/SmoM2;MMTV-Cre (EGFP), mT-mG/SmoM2;MMTV-Cre (tdTomato), and mT-mG/SmoM2;+ cells to examine the effects of SmoM2 overexpression in the mammary gland.
2015-09-08 | E-GEOD-70210 | biostudies-arrayexpress
Project description:The Hedgehog (Hh) pathway is essential for tissue homeostasis, and misregulation of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase 2 (GRK2)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking vertebrate Hh signal transduction at the membrane to transcription factor activation in the nucleus. In contrast to existing models, our work shows that this aspect of Hh signaling is fundamentally similar between species. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades.
2021-04-25 | PXD019346 | Pride
Project description:The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.
2021-04-25 | PXD023893 | panorama
Project description:Mutations in Hedgehog (Hh) pathway genes, leading to constitutive activation of Smoothened (Smo), occur in sporadic medulloblastoma, the most common brain cancer in children. Antagonists of Smo induce tumor regression in mouse models of medulloblastoma and hold great promise for targeted therapy for this tumor. However, acquired resistance has emerged as one of the major challenges of targeted cancer therapy. Here, we describe novel mechanisms of acquired resistance to Smo antagonists in medulloblastoma. NVP-LDE225, a potent and selective Smo antagonist, inhibits Hh signaling and induces tumor regressions in allograft models of medulloblastoma that are driven by mutations of Patched (Ptch), a tumor suppressor in the Hh pathway. However, after long-term treatment, evidence of acquired resistance was observed. Genome-wide profiling of resistant tumors revealed distinct mechanisms to evade the inhibitory effects of Smo antagonists. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, reactivated Hh signaling and restored tumor growth. Analysis of pathway gene-expression signatures selectively deregulated in resistant tumors identified increased phosphoinosite-3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we showed that the combination of NVP-LDE225 with the dual PI3K/mTOR inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma.
2010-09-29 | GSE19657 | GEO
Project description:Mutations in Hedgehog (Hh) pathway genes, leading to constitutive activation of Smoothened (Smo), occur in sporadic medulloblastoma, the most common brain cancer in children. Antagonists of Smo induce tumor regression in mouse models of medulloblastoma and hold great promise for targeted therapy for this tumor. However, acquired resistance has emerged as one of the major challenges of targeted cancer therapy. Here, we describe novel mechanisms of acquired resistance to Smo antagonists in medulloblastoma. NVP-LDE225, a potent and selective Smo antagonist, inhibits Hh signaling and induces tumor regressions in allograft models of medulloblastoma that are driven by mutations of Patched (Ptch), a tumor suppressor in the Hh pathway. However, after long-term treatment, evidence of acquired resistance was observed. Genome-wide profiling of resistant tumors revealed distinct mechanisms to evade the inhibitory effects of Smo antagonists. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, reactivated Hh signaling and restored tumor growth. Analysis of pathway gene-expression signatures selectively deregulated in resistant tumors identified increased phosphoinosite-3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we showed that the combination of NVP-LDE225 with the dual PI3K/mTOR inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma. mRNA profiling: RNA was prepared from tumours from vehicle or NVP-LDE225 treated nude mice allografted with medulloblastoma tumors derived from Ptch+/-p53-/- transgenic mouse and hybridized on Affymetrix Mouse Genome 430 2.0 RNA expression array. The dosage terminology (BID & QD) reflects the dosing schedule, where BID = twice a day, QD = once a day. aCGH: DNA was prepared from tumors from vehicle or NVP-LDE225 treated nude mice allografted with medulloblastoma tumors derived from Ptch+/-p53-/- transgenic mouse and hybridized on Agilent mouse CGH 244K Array.
2010-09-29 | E-GEOD-19657 | biostudies-arrayexpress
Project description:Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor of many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling profoundly depend on interactions with other pathways such as epidermal growth factor receptor-mediated signaling which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. We demonstrate that the human medulloblastoma cell line Daoy possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic Hedgehog or Smoothened agonist induced expression of GLI1 protein and prevented processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed on the transcriptomic as well as proteomic level. Daoy cells responded to the co-treatment by downregulating GLI1, PTCH, and HHIP on the transcript level which was also seen when Amphiregulin (AREG) was used instead of EGF. The finding that EGFR signaling silences proteins acting as negative regulators of HH signaling is firstly described here as a novel crosstalk mechanism. Furthermore, combined EGFR/HH signaling maintains high GLI1 protein levels contrasting its downregulation on the transcript level. On the other hand, high level synergism was observed with respect to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile pointing to more wide-spread, yet context-dependent, interactions between HH/GLI and growth factor receptor signaling in human malignancies. To study interactions between HH- and EGF-induced signaling, time-resolved measurements were carried out over a period of 24 h at 14 different time points after stimulation by EGF with and without additional stimulation by SHH. Furthermore, as a control, cells without any stimulation by EGF and SHH (control) and cells in the presents of SHH were analyzed. Overall, three biological replicates of 60 different treatment/timepoints were analyzed yielding 180 different samples.
2014-07-31 | E-GEOD-46045 | biostudies-arrayexpress
Project description:Aberrant activation of Hedgehog pathway is responsible for initiation and maintenance of various cancers, including medulloblastoma (MB), basal cell carcinoma (BCC), and other solid and hematological tumors. Therefore, targeting Hh pathway represents promising therapeutic prospects for Hh-driven cancers. In recent years, tremendous efforts have been dedicated to the discovery of Hh pathway inhibitor. While the majority of Hh pathway inhibitors target the upstream membrane protein Smoothened (SMO). Here, we performed Next Generation Sequencing to reveal the target genes of Hh pathway by treating mouse SHH-subtype medulloblastoma cells (SmoWT) with SMO inhibitor (GDC0449) or DMSO.
2021-07-08 | GSE171689 | GEO
Project description:Recent fate mapping studies concluded that EMT is not required for metastasis of carcinomas. Here we challenge this conclusion by showing that it failed to account for possible crosstalk between EMT and non-EMT cells that promotes dissemination of non-EMT cells. In breast cancer models, EMT cells induce increased metastasis of weakly metastatic, non-EMT tumor cells in a paracrine manner in part by non-cell autonomous activation of the GLI transcription factor. Treatment with GANT61, a GLI1/2 inhibitor, but not with IPI-926, a Smoothened inhibitor, blocks this effect, and inhibits growth in PDX models. In human breast tumors, the EMT-TFs strongly correlate with activated Hedgehog/GLI signaling, but not with the Hh ligands. Our findings indicate that EMT contributes to metastasis in part via non-cell autonomous effects that activate the Hh pathway. While all Hh inhibitors may act against tumors with canonical Hh/GLI signaling, only GLI inhibitors would act against EMT-induced GLI activation. In recent years, immunohistochemical analyses and multiplex high-throughput single cell sequencing of human tumor cells have shown that tumors are composed of diverse cell subpopulations containing different driver mutations, gene and protein expression profiles, growth rates, and responses to chemotherapeutics. Such heterogeneity is exacerbated by cellular plasticity, where some cells may undergo oncogenic epithelial-to-mesenchymal transition (EMT), resulting in loss of cell-cell adhesion and polarity, reduced epithelial and elevated mesenchymal protein expression, increased migration and invasion, and enhanced dissemination from the primary tumor. Because metastases in patients appear epithelial, the reverse process, mesenchymal-to-epithelial transition (MET), may occur to allow tumor cell colonization in secondary metastatic sites, establishing cellular plasticity as an important aspect of tumor progression. However, the role of EMT in carcinoma metastasis is controversial. Recent lineage tracing studies argue against the requirement of EMT for metastasis, as reporter-tagged cells that underwent a previous EMT were not found at the secondary site. However, these studies did not address the potential cooperation between EMT and non-EMT cells during the metastatic process, as EMT cancer cells may enable non-EMT cells to gain access to the secondary site, leading to macrometastatic growth. Thus, metastasis can be influenced by intratumoral heterogeneity: where a small proportion of primary tumor cells that have undergone an EMT may influence neighboring, non-EMT tumor cells. Twist1, Snail1, and Six1 are EMT-inducing transcription factors (EMT-TFs) that have all been associated with breast cancer metastasis. All three EMT-TFs regulate critical developmental processes such as cell survival, migration and invasion; in part by influencing EMT. In addition, they are downregulated post-embryogenesis, but re-expressed in various cancers where they cell autonomously induce EMT, resulting in increased percentages of tumor initiating cells (TICs) and enhanced metastasis. In carcinomas, Twist1 and Snail1 transcriptionally repress E-Cadherin (E- Cad) and upregulate mesenchymal genes. Similarly, Six1 overexpression induces EMT by regulating E-Cad localization and altering other EMT markers. During development and cancer, EMT-TFs act in concert with several signaling networks including TGFβ, Wnt, and Hedghog (Hh). The Hedgehog (Hh) signaling pathway is a prominent regulator of embryonic development, where Hh ligands function as morphogens to control numerous developmental processes. Interestingly, in Drosophila eye development, hh is a direct target of sine oculis (the homolog of Six1) and Six1 regulates Hh/GLI signaling during lung development and in fibroblasts. Additionally, Twist1 and Hh/GLI signaling are intimately linked during development, and recently Twist1 and Snail1 were associated with the Hh pathway in TICs. In mammals, canonical activation of Hh/GLI signaling involves binding of one the Hh ligands such as(either, Desert (DHH), Indian (IHH), or Sonic Hedgehog (SHH) to Patched-1 (PTCH1) or Patched-2 (PTCH2) receptors, relieving the inhibitory activity of PTCH on Smoothened (SMO). When inhibition is relieved, levels of the transcriptional activator forms of one or more GLI transcription factors (GLI1, 2, or 3) increase in the nucleus, resulting in activation of Hh target genes 12. Non-canonical activation of the GLI TFs can occur in a Hh- or SMO-independent manner via secreted factors like TGF-β19. Importantly, autocrine and paracrine Hh-mediated crosstalk between tumor cells and the tumor microenvironment 20 results in increased proliferation, stem cell self-renewal and metastasis in various cancers 21. In basal cell carcinoma (BCC) and medulloblastoma, activated Hh signaling is often due to mutations in pathway components such as PTCH and SMO, while in other tumor types including breast, mutations are not observed at high frequency. Instead there is evidence for Hh ligand-dependent pathway hyperactivity. Because numerous studies link Hh signaling to progression in multiple tumor types, derivatives of cyclopamine (e.g. GDC-0449 and IPI926), a naturally occurring plant-derived steroidal alkaloid which targets SMO, are in clinical trials for select patients with BCC and medulloblastoma with promising efficacy. However, these inhibitors have not shown promise in breast cancer despite evidence for activation of this pathway. Herein, we demonstrate that EMT-TFs Twist1, Snail1 and Six1 influence neighboring carcinoma cells in a non-cell autonomous manner, by increasing EMT features and aggressive properties of cells not expressing these TFs. Six1 is a key mediator of the non-cell autonomous effects downstream of Twist1 and Snail1, and can induce metastasis of non-Six1, non-EMT cells. All three EMT-TFs function non-cell autonomously by activation of GLI-mediated transcription in non-EMT cells, but employ different mechanisms of pathway activation, some of which are Hh ligand and SMO-independent. We find that pharmacological inhibition of GLI, but not SMO, in the non-EMT cells efficiently inhibits the non-cell autonomous phenotypes imparted by all three EMT-TFs. Importantly, we demonstrate that in selected patient derived breast cancer xenograft (PDX) models endogenously expressing high levels of these EMT-TFs (but not Hh ligands), GANT61 (a GLI1/2 inhibitor) inhibits tumor growth whereas IPI926 (a SMO inhibitor) did not. Our data suggest that upstream SMO inhibitors may not prove efficacious in tumors where a proportion of cells activate GLI-TF via EMT-TFs, and instead argue that inhibitors directly targeting GLI may be more effective.
2017-04-13 | GSE97726 | GEO
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is a highly malignant pediatric leukemia, where few therapeutic options are available for patients which relapse. The evolutionarily conserved Hedgehog (Hh) pathway plays a crucial role in patterning and organogenesis during early development, in adult tissue maintenance and repairing functions. Once Hh ligands bind to PTCH1/2 on target cells, the inhibition of Patched proteins for Smo is relieved. Smo translocates to the primary cilium and leads to the breakdown of a protein complex formed by Suppressor of Fused (SUFU) and GLI proteins. GLI transcription factors (GLI1-3) are released and translocate to the nucleus, initiating transcription of Hh target genes. GLI2 and GLI3 have transcriptional activation and repression properties, while GLI1 is a strong positive regulator of Hh transcriptional targets. In T-ALL rare hedgehog pathway mutations have been observed and approximately 20% of T-ALL cases present with ectopic expression of Hh pathway ligands and GLI1. However, the function of Hh signaling and in particular GLI transcription factors in T-ALL initiation and/or tumor progression is not well-known. RNA sequencing was performed in the CUTLL1 T-ALL cell line after knocking out GLI1 gene using CRISPR/Cas9 system.
2020-07-03 | GSE153718 | GEO
Project description:In the bone marrow B cell development occurs in initimate and essential association with stromal cells. Known effects of stromal cells on B cell development have been shown to be mediated through direct contact and by soluble factors produced by stromal cells. Our findings suggest that cell-nonautonomous Hh signaling may play an important role in B cell lymphopoiesis. We therefore interrogated OP9 stromal cells for Hh-dependent transcripts that may confer B lymphopoietic identity. We generated OP9 stromal cells deficient in Hh signaling through targeting the non-redundant, pathway-obligate GPCR Smoothened. OP9 cells were transduced with RNAi pLKO.1-Puro lentiviral particles expressing a scrambled control shRNA (NT) or shRNA targeting Smoothened (Smo-kd). RNA from stable NT and Smo-kd OP9 cells were isolated in triplicate and global changes in transcripts were analyzed using the Affymetrix Mouse Exon 1.0 ST gene chip.
2012-07-11 | E-GEOD-34421 | biostudies-arrayexpress