Disruption of the ciliary GTPase Arl13b suppresses Sonic hedgehog overactivation and inhibits medulloblastoma formation

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and overactivation of the Sonic Hedgehog (Shh) signaling pathway, which requires the primary cilium, causes 30% of MBs. Current treatments have known negative side effects or resistance mechanisms, so new treatments are necessary. Shh signaling mutations, like those that remove Patched1 (Ptch1) or activate Smoothened (Smo), cause tumors dependent on the presence of cilia. Genetic ablation of cilia prevents these tumors by removing Gli activator, but cilia are a poor therapeutic target since they support many biological processes. A more appropriate strategy would be to identify a protein that functionally disentangles Gli activation and ciliogenesis. Our mechanistic understanding of the ciliary GTPase Arl13b predicts that it could be such a target. Arl13b mutants retain short cilia, and loss of Arl13b results in ligand-independent, constitutive, low-level pathway activation but prevents maximal signaling without disrupting Gli repressor. Here, we show that deletion of Arl13b reduced Shh signaling levels in the presence of oncogenic SmoA1, suggesting Arl13b acts downstream of known tumor resistance mechanisms. Knockdown of ARL13B in human MB cell lines and in primary mouse MB cell culture decreased proliferation. Importantly, loss of Arl13b in a Ptch1-deleted mouse model of MB inhibited tumor formation. Postnatal depletion of Arl13b does not lead to any overt phenotypes in the epidermis, liver, or cerebellum. Thus, our in vivo and in vitro studies demonstrate that disruption of Arl13b inhibits cilia-dependent oncogenic Shh overactivation.

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Soluble Heparin and Heparan Sulfate Glycosaminoglycans Interfere with Sonic Hedgehog Solubilization and Receptor Binding

Molecules ◽

10.3390/molecules24081607 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1607 ◽

Cited By ~ 3

Author(s):

Manikowski ◽

Jakobs ◽

Jboor ◽

Grobe

Keyword(s):

Heparan Sulfate ◽

Sonic Hedgehog ◽

Receptor Binding ◽

Feedback Loop ◽

Cancer Cell Growth ◽

Shh Signaling ◽

Epithelial Cancers ◽

And Function

Sonic hedgehog (Shh) signaling plays a tumor-promoting role in many epithelial cancers. Cancer cells produce soluble a Shh that signals to distant stromal cells that express the receptor Patched (Ptc). These receiving cells respond by producing other soluble factors that promote cancer cell growth, generating a positive feedback loop. To interfere with reinforced Shh signaling, we examined the potential of defined heparin and heparan sulfate (HS) polysaccharides to block Shh solubilization and Ptc receptor binding. We confirm in vitro and in vivo that proteolytic cleavage of the N-terminal Cardin–Weintraub (CW) amino acid motif is a prerequisite for Shh solubilization and function. Consistent with the established binding of soluble heparin or HS to the Shh CW target motif, both polysaccharides impaired proteolytic Shh processing and release from source cells. We also show that HS and heparin bind to, and block, another set of basic amino acids required for unimpaired Shh binding to Ptc receptors on receiving cells. Both modes of Shh activity downregulation depend more on HS size and overall charge than on specific HS sulfation modifications. We conclude that heparin oligosaccharide interference in the physiological roles of HS in Shh release and reception may be used to expand the field of investigation to pharmaceutical intervention of tumor-promoting Shh functions.

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FoxF1 is Required for Ciliogenesis and Distribution of Sonic Hedgehog Signaling Components in Cilium

Current Molecular Medicine ◽

10.2174/1566524019666190405115420 ◽

2019 ◽

Vol 19 (5) ◽

pp. 326-334

Author(s):

Lu Huang ◽

Marco Tjakra ◽

Desha Luo ◽

Lin Wen ◽

Daoxi Lei ◽

...

Keyword(s):

Sonic Hedgehog ◽

Hedgehog Signaling ◽

3T3 Cells ◽

Shh Signaling ◽

Nih 3T3 ◽

Signaling Components ◽

Nih 3T3 Cells

Background: In vertebrates, cilium is crucial for Hedgehog signaling transduction. Forkhead box transcriptional factor FoxF1 is reported to be associated with Sonic Hedgehog (Shh) signaling in many cases. However, the role of FoxF1 in cilium remains unknown. Here, we showed an essential role of FoxF1 in the regulation of ciliogenesis and in the distribution of Shh signaling components in cilium. Methods: NIH/3T3 cells were serum starved for 24h to induce cilium. Meanwhile, shRNA was used to knockdown the FoxF1 expression in the cells and CRISPR/Cas9 was used to generate the FoxF1 zebrafish mutant. The mRNA and protein expression of indicated genes were detected by the qRT-PCR and western blot, respectively. Immunofluorescence staining was performed to detect the cilium and Shh components distribution. Results: FoxF1 knockdown decreased the cilium length in NIH/3T3 cells. Meanwhile, the disruption of FoxF1 function inhibited the expression of cilium-related genes and caused an abnormal distribution of Shh components in the cilium. Furthermore, homozygous FoxF1 mutants exhibited defective development of pronephric cilium in early zebrafish embryos. Conclusion: Together, our data illustrated that FoxF1 is required for ciliogenesis in vitro and in vivo and for the proper localization of Shh signaling components in cilium.

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Mediator Modulates Gli3-Dependent Sonic Hedgehog Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00443-06 ◽

2006 ◽

Vol 26 (23) ◽

pp. 8667-8682 ◽

Cited By ~ 78

Author(s):

Haiying Zhou ◽

Seokjoong Kim ◽

Shunsuke Ishii ◽

Thomas G. Boyer

Keyword(s):

Gene Transcription ◽

Sonic Hedgehog ◽

Target Gene ◽

Dominant Negative ◽

Transactivation Domain ◽

Functional Interaction ◽

Shh Signaling ◽

Target Gene Transcription

ABSTRACT The physiological and pathological manifestations of Sonic hedgehog (Shh) signaling arise from the specification of unique transcriptional programs dependent upon key nuclear effectors of the Ci/Gli family of transcription factors. However, the underlying mechanism by which Gli proteins regulate target gene transcription in the nucleus remains poorly understood. Here, we identify and characterize a physical and functional interaction between Gli3 and the MED12 subunit within the RNA polymerase II transcriptional Mediator. We show that Gli3 binds to MED12 and intact Mediator both in vitro and in vivo through a Gli3 transactivation domain (MBD; MED12/Mediator-binding domain) whose activity derives from concerted functional interactions with both Mediator and the histone acetyltransferase CBP. Analysis of MBD truncation mutants revealed an excellent correlation between the in vivo activation strength of an MBD derivative and its ability to bind MED12 and intact Mediator in vitro, indicative of a critical functional interaction between the Gli3 MBD and the MED12 interface in Mediator. Disruption of the Gli3-MED12 interaction through dominant-negative interference inhibited, while RNA interference-mediated MED12 depletion enhanced, both MBD transactivation function and Gli3 target gene induction in response to Shh signaling. We propose that activated Gli3 physically targets the MED12 interface within Mediator in order to functionally reverse Mediator-dependent suppression of Shh target gene transcription. These findings thus link MED12 to the modulation of Gli3-dependent Shh signaling and further implicate Mediator in a broad range of developmental and pathological processes driven by Shh signal transduction.

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GENE-04. THE ONCOGENIC FUNCTIONS OF YAP1-GENE FUSIONS CAN BE INHIBITED BY DISRUPTION OF YAP1-TEAD INTERACTION

Neuro-Oncology ◽

10.1093/neuonc/noz175.406 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi98-vi98

Author(s):

Frank Szulzewsky ◽

Pia Hoellerbauer ◽

Hua-Jun Wu ◽

P J Cimino ◽

Franziska Michor ◽

...

Keyword(s):

Hippo Pathway ◽

Point Mutations ◽

Tumor Formation ◽

In Vitro Assays ◽

Gene Fusions ◽

Genetic Ablation ◽

Molecular Features ◽

Cancer Types

Abstract Supratentorial ependymoma can be sub-stratified into clinically relevant subtypes characterized by distinct molecular features. The subtype defined by high YAP1 activity harbored two distinct YAP1 gene fusions, YAP1-MAMLD1 and YAP1-FAM118B. In addition, YAP1 gene fusions have been detected in several other cancer types, including Epithelioid Hemangioendothelioma and Endocervical Adenocarcinoma. YAP1 is a key transcriptional co-activator and proto-oncogene that is negatively regulated by the Hippo pathway. Here, we show that both YAP1-MAMLD1 and YAP1-FAM118B, as well as additional YAP1 fusion genes found in other cancer types, are potent oncogenic drivers that cause tumor formation in the brain and the hindlimb in mice upon overexpression by somatic cell gene transfer. Using different in vitro assays, including Luciferase, RNA-, and ChIP Seq, we show that both the N-terminal YAP1 part and the C-terminal fusion partners exert activity. We can show that the YAP1 activity still relies on the binding to TEAD transcription factors, whereas the C terminal activity does not. Furthermore, the different fusion proteins have become independent from negative Hippo pathway signaling by constitutive nuclear localization and protection from degradation. In addition, by introducing point mutations and truncations to block the YAP1 and the MAMLD1 function we can show that the activity of both halves contributes to the oncogenic function of YAP1-MAMLD1. Using in vitro and in vivo assays we can show that pharmacological and genetic ablation of YAP-TEAD interaction diminishes the oncogenic potential of the fusions, indicating that this might be a potential therapeutic approach for these tumors in the future.

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Cancer Stem Cells: Powerful Targets to Improve Current Anticancer Therapeutics

Stem Cells International ◽

10.1155/2019/9618065 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15 ◽

Cited By ~ 11

Author(s):

Rayana L. Bighetti-Trevisan ◽

Lucas O. Sousa ◽

Rogerio M. Castilho ◽

Luciana O. Almeida

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Cancer Cells ◽

Resistance Mechanisms ◽

In Vivo Studies ◽

Tumor Resistance ◽

Therapeutic Implications ◽

Anticancer Therapeutics

A frequent observation in several malignancies is the development of resistance to therapy that results in frequent tumor relapse and metastasis. Much of the tumor resistance phenotype comes from its heterogeneity that halts the ability of therapeutic agents to eliminate all cancer cells effectively. Tumor heterogeneity is, in part, controlled by cancer stem cells (CSC). CSC may be considered the reservoir of cancer cells as they exhibit properties of self-renewal and plasticity and the capability of reestablishing a heterogeneous tumor cell population. The endowed resistance mechanisms of CSC are mainly attributed to several factors including cellular quiescence, accumulation of ABC transporters, disruption of apoptosis, epigenetic reprogramming, and metabolism. There is a current need to develop new therapeutic drugs capable of targeting CSC to overcome tumor resistance. Emerging in vitro and in vivo studies strongly support the potential benefits of combination therapies capable of targeting cancer stem cell-targeting agents. Clinical trials are still underway to address the pharmacokinetics, safety, and efficacy of combination treatment. This review will address the main characteristics, therapeutic implications, and perspectives of targeting CSC to improve current anticancer therapeutics.

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Sonic Hedgehog Signaling Drives Proliferation of Synoviocytes in Rheumatoid Arthritis: A Possible Novel Therapeutic Target

Journal of Immunology Research ◽

10.1155/2014/401903 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 10

Author(s):

Mingxia Wang ◽

Shangling Zhu ◽

Weixiang Peng ◽

Qiuxia Li ◽

Zhaoxia Li ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Sonic Hedgehog ◽

Therapeutic Target ◽

Hedgehog Signaling ◽

Specific Inhibitor ◽

Sonic Hedgehog Signaling ◽

Shh Signaling ◽

Novel Therapeutic

Sonic hedgehog (Shh) signaling controls many aspects of human development, regulates cell growth and differentiation in adult tissues, and is activated in a number of malignancies. Rheumatoid arthritis (RA) is characterized by chronic synovitis and pannus formation associated with activation of fibroblast-like synoviocytes (FLS). We investigated whether Shh signaling plays a role in the proliferation of FLS in RA. Expression of Shh signaling related components (Shh, Ptch1, Smo, and Gli1) in RA synovial tissues was examined by immunohistochemistry (IHC) and in FLS by IHC, immunofluorescence (IF), quantitative RT-PCR, and western blotting. Expression of Shh, Smo, and Gli1 in RA synovial tissue was higher than that in control tissue (P<0.05). Cyclopamine (a specific inhibitor of Shh signaling) decreased mRNA expression of Shh, Ptch1, Smo, and Gli1 in cultured RA FLS, Shh, and Smo protein expression, and significantly decreased FLS proliferation. Flow cytometry analysis suggested that cyclopamine treatment resulted in cell cycle arrest of FLS in G1phase. Our data show that Shh signaling is activated in synovium of RA patientsin vivoand in cultured FLS form RA patientsin vitro, suggesting a role in the proliferation of FLS in RA. It may therefore be a novel therapeutic target in RA.

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Astrocytic Sonic Hedgehog Alleviates Intracerebral Hemorrhagic Brain Injury via Modulation of Blood-Brain Barrier Integrity

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2020.575690 ◽

2020 ◽

Vol 14 ◽

Author(s):

Gebeili Xing ◽

Tianman Zhao ◽

Xiyue Zhang ◽

He Li ◽

Xiuping Li ◽

...

Keyword(s):

Brain Injury ◽

Signaling Pathway ◽

Blood Brain Barrier ◽

Sonic Hedgehog ◽

Critical Role ◽

Brain Barrier ◽

Shh Signaling ◽

Blood Brain

Background: Intracerebral hemorrhage (ICH) is a fatal subtype of stroke that lacks effective therapy. Blood-brain barrier (BBB) damage is a hallmark of ICH-induced brain injury that leads to edema formation, leukocytes infiltration, influx of blood components into the perihematomal (PHE) region, and eventually brain injury. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted molecules that contribute to the association between these cells. Sonic hedgehog (SHH) derived from astrocytes promotes the maturity and integrity of the BBB by upregulating tight junctions (TJs) in brain capillary endothelial cells (ECs). However, the effect of SHH on BBB in ICH has not been investigated.Methods: Cyclopamine (CYC) is a potent, selective inhibitor that specifically blocks the SHH signaling pathway. Here, we used pharmacological inhibitions (CYC and its derivatives) to determine a critical role of the SHH signaling pathway in promoting BBB integrity after ICH by mechanisms of regulating the TJ proteins in vivo and in vitro.Results: The expression of astrocytic SHH was upregulated in mouse brains after ICH. Compared with the vehicle-treated group, inhibition of the SHH signaling pathway with CYC and its derivatives treatments aggravated neurological function deficits, brain edema, hematoma volume, and BBB impairment by downregulating TJs in ECs through the SHH-Gli-1 axis in vivo and in vitro.Conclusions: SHH signaling pathway at the level of the BBB provides a barrier-promoting effect, suggesting that the SHH signaling pathway may function as a potential therapeutic target for restoring BBB function in ICH.

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The Tumor Suppressor MTUS1/ATIP1 Modulates Tumor Promotion in Glioma: Association with Epigenetics and DNA Repair

Cancers ◽

10.3390/cancers13061245 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1245

Author(s):

Nikhil Ranjan ◽

Vimal Pandey ◽

Manas Kumar Panigrahi ◽

Lukas Klumpp ◽

Ulrike Naumann ◽

...

Keyword(s):

High Grade Glioma ◽

Therapy Resistance ◽

Resistance Mechanisms ◽

Tumor Resistance ◽

Tumor Irradiation ◽

Damage Repair ◽

Low Passage ◽

Novel Therapeutic Strategies

Glioblastoma (GBM) is a highly aggressive brain tumor. Resistance mechanisms in GBM present an array of challenges to understand its biology and to develop novel therapeutic strategies. We investigated the role of a TSG, MTUS1/ATIP1 in glioma. Glioma specimen, cells and low passage GBM sphere cultures (GSC) were analyzed for MTUS1/ATIP1 expression at the RNA and protein level. Methylation analyses were done by bisulfite sequencing (BSS). The consequence of chemotherapy and irradiation on ATIP1 expression and the influence of different cellular ATIP1 levels on survival was examined in vitro and in vivo. MTUS1/ATIP1 was downregulated in high-grade glioma (HGG), GSC and GBM cells and hypermethylation at the ATIP1 promoter region seems to be at least partially responsible for this downregulation. ATIP1 overexpression significantly reduced glioma progression by mitigating cell motility, proliferation and facilitate cell death. In glioma-bearing mice, elevated MTUS1/ATIP1 expression prolonged their survival. Chemotherapy, as well as irradiation, recovered ATIP1 expression both in vitro and in vivo. Surprisingly, ATIP1 overexpression increased irradiation-induced DNA-damage repair, resulting in radio-resistance. Our findings indicate that MTUS1/ATIP1 serves as TSG-regulating gliomagenesis, progression and therapy resistance. In HGG, higher MTUS1/ATIP1 expression might interfere with tumor irradiation therapy.

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Sonic hedgehog signaling is required during the appearance of spinal cord oligodendrocyte precursors

Development ◽

10.1242/dev.126.11.2419 ◽

1999 ◽

Vol 126 (11) ◽

pp. 2419-2429 ◽

Cited By ~ 8

Author(s):

D.M. Orentas ◽

J.E. Hayes ◽

K.L. Dyer ◽

R.H. Miller

Keyword(s):

Spinal Cord ◽

Sonic Hedgehog ◽

Ventricular Zone ◽

Developmental Period ◽

Dorsal Spinal Cord ◽

Shh Signaling ◽

Oligodendrocyte Precursors ◽

Dorsal Spinal

Spinal cord oligodendrocyte precursors arise in the ventral ventricular zone as a result of local signals. Ectopic oligodendrocyte precursors can be induced by sonic hedgehog (Shh) in explants of chick dorsal spinal cord over an extended developmental period. The role of Shh during normal oligodendrocyte development is, however, unclear. Here we demonstrate that Shh is localized to the ventral spinal cord immediately prior to, and during the appearance of oligodendrocyte precursors. Continued expression of Shh is required for the appearance of spinal cord oligodendrocyte precursors as neutralization of Shh signaling both in vivo and in vitro during a defined developmental period blocked their emergence. The inhibition of oligodendrocyte precursor emergence in the absence of Shh signaling was not the result of inhibiting precursor cell proliferation, and the neutralization of Shh signaling after the emergence of oligodendrocyte precursors had no effect on the appearance of additional cells or their subsequent differentiation. Similar concentrations of Shh induce motor neurons and oligodendrocytes in dorsal spinal cord explants. However, in explants from early embryos the motor neuron lineage is preferentially expanded while in explants from older embryos the oligodendrocyte lineage is preferentially expanded.

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Multifaceted functions of Rab23 on primary cilium and Hedgehog signaling-mediated granule cell proliferation

10.1101/2020.08.01.231985 ◽

2020 ◽

Author(s):

CHH Hor ◽

WY Leong ◽

ELK Goh

Keyword(s):

Sonic Hedgehog ◽

Primary Cilium ◽

Granule Cell ◽

Hedgehog Signaling ◽

Cell Precursor ◽

Shh Signaling ◽

Shh Pathway ◽

Granule Cell Precursor

AbstractSonic Hedgehog (Shh) signaling from the primary cilium drives cerebellar granule cell precursor (GCP) proliferation. Mutations of hedgehog (Hh) pathway repressors could cause medulloblastoma, the most prevalent and malignant childhood brain tumor that arises from aberrant GCP proliferation. We demonstrate that brain-specific knockout of a Shh pathway repressor Rab23 in mice caused mis-patterning of cerebellar folia and elevated GCP proliferation during early development, but with no prevalent occurrence of medulloblastoma at adult stage. Strikingly, Rab23-depleted GCPs exhibited up-regulated basal level of Shh pathway activities despite reduced ciliation, and were desensitized against stimulations by Shh and Smoothened (Smo) agonist in primary GCP culture. These results illustrate dual functions of Rab23 in repressing the basal level of Shh signaling, while facilitating Shh signal transduction via Shh/Smo on primary cilium. Collectively, our findings unravel instrumental roles of Rab23 in GCP proliferation and ciliogenesis. Rab23’s potentiation of Shh signaling pathway through the primary cilium and Smo, suggests a potential new therapeutic for Smo/primary cilium-driven medulloblastoma.Author SummaryC.H.H conceived, designed, lead, and performed all in vitro and in vivo experiments, analyzed data and wrote the manuscript. W.Y performed QPCR experiments and primary GCP cultures and analyzed data. E.L.G conceived and directed the study.

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