Hedgehog Signaling in Development and Disease

HEDGEhog 2014

Hedgehog Signaling in Development and Disease August 4th - August 8th, 2014 University of Michigan Ann Arbor, Michigan

Conference Program, Abstracts & Information

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THANKS to our sponsors:

our organizing committee:

Monday August 4, 2014 Welcome! 4:00pm-10:00pm

Registration (BSRB Atrium)

6:00pm-7:00pm

Welcome Reception (BSRB Atrium)

7:15pm

Opening Remarks (BSRB Kahn Auditorium)

Introductory Session: A Taste of Michigan 7:30pm-8:00pm Scott Barolo (University of Michigan, USA) “Gli binding sites in Hh-regulated enhancers: Patching together some unexpected results” 8:00pm-8:15pm Brandon Carpenter (University of Michigan, USA) “The Heterotrimeric Kinesin-2 Complex Regulates GLI Protein Function” 8:15pm-8:30pm Shelby Peterson (University of Michigan, USA) “Basal cell carcinoma preferentially arises from stem cells within the hair follicle and mechanosensory epithelia”

ConferenceSchedule

DAY 1

ConferenceSchedule

DAY 2

HEDGEhog 2014

Tuesday August 5, 2014 Session 1: Hedgehog Ligand Secretion, Trafficking and Reception 9:00am-9:30am Pascal Therond (University of Nice, France) “Hedgehog Secretion and Extracellular Transport in Drosophila” 9:30am-10:00am Suzanne Eaton (Max Planck Institute, Germany) “Lipids in Hedgehog Signaling” 10:00am-10:15am Mattias Alenius (Linkoping University, Sweden) “Cilia-mediated Hedgehog Signaling in Drosophila” 10:15am-10:30am Don van Meyel (McGill University, Canada) “Ihog and Boi elicit Hedgehog signaling via Ptc but do not aid Ptc in

sequestering the Hh ligand”

10:30am-11:00am

Coffee/Tea Break (BSRB Seminar Rooms)

11:00am-11:30am Stacey Ogden (St. Jude Children’s Research Hospital, USA) “Mechanisms of Smoothened Signaling” 11:30pm-12:00pm Frederic Charron (IRCM, Canada) “The Role of Hedgehog Co-Receptors in Nervous System Development and Pathologies” 12:00pm-2:00pm

Lunch Break

Session 2: Hedgehog Signal Reception and Transduction 2:00pm-2:30pm Adrian Salic (Harvard University, USA) “Hedgehogs and Sterols” 2:30pm-3:00pm Jeremy Reiter (UCSF, USA) “The transition zone controls Hedgehog signaling and ciliary composition” 3:00pm-3:15pm Alexander Holtz (University of Michigan, USA) “Novel role for HHIP1 as a secreted antagonist of Hedgehog signaling” 3:15pm-3:30pm Ljiljana Milenkovic (Stanford University, USA) “Single-molecule imaging of Smoothened in primary cilia reveals binding events

that are modified by Hedgehog pathway agonists”

3:30pm-4:00pm


4:00pm-4:30pm Christian Siebold (Oxford University, USA) “Molecular mechanisms of Hedgehog ligand-receptor interactions” 4:30pm-4:45pm Xuecai Ge (Stanford University, USA) “Integration of Neuropilin and Hedgehog signal transduction through

control of protein kinase A”

4:45pm-5:00pm Saikat Mukhopadhyay (UT-Southwestern, USA) “Role of the G-protein-coupled receptor, Gpr161, in hedgehog signaling” 5:30pm

Bill’s Beer Garden (218 S. Ashley Street, see events page for more info)

HEDGEhog

Wednesday August 6, 2014 Session 3: Hedgehog Signal Reception and Transduction 9:00am-9:30am Rajat Rohatgi (Stanford University, USA) “Hedgehog Signaling in Primary Cilia” 9:30am-10:00am Natalia Riobo (Thomas Jefferson University, USA) “Modulation of Patched-1 pro-apoptotic activity: an itchy problem” 10:00am-10:15am James Chen (Stanford University, USA) “Arhgap36-dependent activation of Gli transcription factors” 10:15am-10:30am Jianhang Jia (University of Kentucky, USA) “Direct roles of aPKC in Drosophila Hh signaling” 10:30am-11:00am


11:00am-3:00pm

Canoe Trip/Free Time ( see events page for more info )

Session 4: Hedgehog Signaling in Development 3:30pm-4:00pm Kathryn Anderson (MSKCC, USA) “Cilia and Hedgehog signaling in the mouse embryo” 4:00pm-4:30pm James Briscoe (NIMR, UK) “Gene regulatory logic of Shh patterning of the neural tube” 4:30pm-4:45pm Susan Mackem (NIH/NCI, USA) “Does Sonic Hedgehog Function as a Morphogen in the Developing Limb?” 4:45pm-5:00pm Steven Vokes (UT-Austin, USA) “Spatiotemporal Regulation of GLI Target Genes in the Mammalian Limb Bud” 5:00pm-7:00pm

Poster Session 1 (BSRB Seminar Rooms)

2014

ConferenceSchedule

DAY 3

ConferenceSchedule

DAY 4

HEDGEhog 2014

Thursday August 7, 2014 Session 5: Hedgehog Signaling in Stem Cells, Development and Disease 9:00am-9:30am Alexandra Joyner (MSKCC, USA) “The Double Life of SHH in the Cerebellum: Developmental Scaling and Cancer” 9:30am-10:00am Anna Mae Diehl (Duke University, USA) “Hedgehog Regulation of Adult Liver Repair” 10:00am-10:15am Maike de la Roche (University of Cambridge, UK) “Hedgehog signaling controls T cell killing at the immunological synapse” 10:15am-10:30am Ronald Buckanovich (University of Michigan, USA) “Identification of a BMP/hedgehog signaling loop between ovarian

carcinoma-associated mesenchymal stem cells and ovarian tumor cells”

10:30am-11:00am


11:00am-11:30am Deborah Gumucio (University of Michigan, USA) “Mesenchymal Bmp signaling downstream from Hedgehog establishes a

Turing field that patterns intestinal villi”

11:30pm-12:00pm Thomas Willnow (Max Delbruck Center, Germany) “LRP2 Regulates SHH Signaling in the Developing Mammalian Forebrain and Eye” 12:00pm-2:00pm

Lunch Break

Session 6: Hedgehog Signaling in Disease 2:00pm-2:30pm Marina Pasca di Magliano (University of Michigan, USA) “Dosage-Dependent Regulation of Pancreatic Cancer Growth and

Angiogenesis by Hedgehog Signaling”

2:30pm-3:00pm Tony Oro (Stanford University, USA) “Hedgehog Pathway Inhibition Meets Tumor Evolution” 3:00pm-3:15pm Lalita Shevde-Samant (University of Alabama, USA) “Hedgehog Signaling and Social Networking in the Tumor Microenvironment” 3:15pm-3:30pm Junhao Mao (University of Massachusetts, USA) “Hedgehog-mediated rhabdomyosarcoma genesis” 3:30pm-4:00pm


4:00pm-4:30pm Phil Beachy (Stanford University, USA) “Organ homeostasis and malignancy” 4:30pm-5:00pm Robert Krauss (Icahn School of Medicine, USA) “Modeling Holoprosencephaly in the Mouse” 5:00pm-7:00pm

Poster Session 2 (BSRB Seminar Rooms)

HEDGEhog 2014

Friday August 8, 2014 Session 7: Hedgehog Pathway Therapeutics 9:00am-9:30am Charlotte Mistretta (University of Michigan, USA) “HH signaling: required for taste organs and taste sensation” 9:30am-10:00am Brandon Wainwright (University of Queensland, Australia) “Gene networks that control medulloblastoma growth in Ptc1 mutant mice” 10:00am-10:15am Hayley Sharpe (Genentech, USA) “Smoothened inhibition in the clinic” 10:15am-10:30am Michelle Muza-Moons (University of Michigan, USA) “Modulation of the Hedgehog pathway in the intestine affects basal inflammatory

cytokine secretion and acute response to inflammatory challenge”

10:30am-11:00am Coffee/Tea Break (BSRB Seminar Rooms)

11:00am-11:15am Abhinav Dey (Emory University) “Why be one dimensional? Multifunctionality of Y-box protein 1 (YB1) at the helm of Hedgehog-driven medulloblastomas”

11:15pm-11:30pm Jiang Wu (UT-Southwestern) “An Epigenetic Switch Induced by Sonic Hedgehog Signaling Regulates Gene Activation during Development and Medulloblastoma Growth” 11:30pm-11:45pm

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12:00pm Departure End of Meeting

ConferenceSchedule

DAY 5

ConferenceEvents

Tuesday August 5, 2014 Bill’s Beer Garden 5:30pm

Bill’s Beer Garden

218 S. Ashley Street (see map in next section) Join us for a drink (or two!). Bill’s Beer Garden includes Michigan craft beer and wine. Food can be brought into Bill’s Beer Garden from adjacent Mark’s Carts — a collection of eight food carts serving an eclectic mix of tasty, affordable and locally sourced food. Check your name badge for drink tickets.

Wednesday August 6, 2014 Canoe Trip 11:00am-3:00pm

Canoe Trip/Free Time

River fun for everyone! Spend the afternoon boating on the Huron River. Transportation from BSRB to the Argo Canoe Livery and then later from Gallup Canoe Livery to BSRB is planned. Check out the brochure to the right and the trip map below. More details will be announced at the meeting.

Argo Canoe Livery 1055 Longshore Dr

Gallup Canoe Livery 3000 Fuller Rd BSRB - Kahn Auditorium 109 Zina Pitcher Place, 48109

rapids

portage

flow direction

nature parks

liveries

Delhi

put-in points

Wagner

dam

Bar ton

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14

Bandemer Park

A Argo Cascades

Park

S. Division

N. Main

Tube Rental Rates Two hours

$10/tube

Tubes are a fun way to float down the Argo Cascade drops and pools and then hike back up to float down again. Our tubes have bottoms, lifejackets provided and required, shoes required (we have water shoes for purchase), one/person per tube and minimum age is 8 years old.

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Island

accessible.

Argo Canoe Livery

rive

TUBE RENTALS *Argo Only

14

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Argo Dam

Barton Park

Ba

Barton Dam

Our stillwater paddles are

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Huron

$18/boat

$18/boat

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Paddle a boat in the river where there is little or no current. Great for beginners and families, no van ride needed, just jump in a boat and off you go!

Stand Up Paddleboard 2 hours, Argo only

Rowboats 2 hours, Gallup only

$10/boat

$14/boat

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Cash, checks and credit cards accepted Deposit of drivers license or car keys Life jackets required and provided Assumption of Risk/Waiver must be signed Alcohol & Styrofoam coolers NOT allowed Maximum 3 people in a canoe unless 2 children have combined weight of 4-fold in the Gr1 subpopulation compared to the Gr1 cells. + +/-/Slfn4 induction was 48% or 78% less in the Gr1 population of the Gli1 or Gli1 mice + respectively. Although loss of Gli1 did not affect the total number of Gr1 cells, Gli1 was + required for IFNα11 Slfn4 induction. These Slfn4 cells have increased expression of nitric + oxide synthase 2 and arginase I, and inhibit Cd4 T cell proliferation. Conclusion: Hedgehog ligand over-expression accelerates Helicobacter-induced SPEM and the + + appearance of Slfn4 MDSC-like myeloid cells. Slfn4 mRNA was induced mainly in the Gr1 population in a Gli1-dependent manner. Since IFNa strongly induced Slfn4 and the induction was mitigated by loss of the Gli1 allele, our results suggest cooperative regulation of this myeloid differentiation gene by pro-inflammatory cytokines and Hedgehog signaling.

Abstracts

Lin Ding

Abstracts

Alexandre Ermilov Research Lab Specialist University of Michigan

Gli transcriptional activity is required in mature hair follicles for epithelial proliferation, survival, and hair shaft elongation Basal cell carcinomas (BCCs) are driven by uncontrolled Hh signaling which stimulates activity of Gli transcription factors. Treatment of BCC patients with Hh pathway inhibitors (HPIs) targeting SMO, a proximal effector of both oncogenic and physiologic Hh signaling, leads to tumor regression as well as hair loss. Although genetic and pharmacological studies have established that Hh signaling is required for activation of the hair growth cycle, the Hh pathway remains active in several cell populations in mature (anagen phase) hair follicles, but little is known of Hh pathway functions during this phase of the hair cycle. We have begun exploring this issue using mice carrying a doxycycline-inducible dominantnegative Gli2 allele, Gli2ΔC4, to inhibit Hh pathway-driven Gli transcriptional activity selectively in hair follicle epithelium. In keeping with previous studies using other approaches, we confirmed that Gli2ΔC4 is a potent inhibitor of hair follicle growth when induced prior to anagen initiation. We then activated Gli2ΔC4 expression between 9 and 14 days after depilation, which triggers a synchronous wave of hair follicle growth. Hh signaling activity in highly proliferative hair matrix cells, based on beta-gal staining of mice carrying a Hh pathway responsive Gli1-lacZ allele, was strongly inhibited by expression of Gli2ΔC4. This was associated with a marked reduction in the number of Ki67+ matrix cells, increased apoptosis, the precocious appearance of several differentiating cell types, and the formation of misshapen and markedly shorter hairs. The rapid alterations brought about by Gli2ΔC4 expression in anagen hair follicles argue that Hh pathway/Gli activity has critical functions required for sustained proliferation and cell survival in rapidly-growing hair matrix cells that produce the hair shaft, and that disruption of these functions in HPI-treated patients is responsible for the hair loss that develops in the majority of treated patients.

Postdoctoral Research Fellow Stanford University

Neuropilins regulate Hedgehog signal transduction by modulating cAMP production and PKA activity Hedgehog (Hh) signaling is critical for neuronal patterning, cell proliferation and differentiation during development. Mis-regulation of Hh signaling may lead to various birth defects and cancers. It is therefore crucial to understanding how Hh signal is transduced. We previously discovered that Neuropilins, proteins involved in axon guidance and angiogenesis, are positive regulators of Hh signal transduction. Our new data suggest that the Neuropilin ligand Semaphorin, but not VEGF, promotes Hh signaling by inhibiting the activity of PKA, a well-known negative regulator of Hh signaling. We then screened for proteins interacting with the cytoplasmic domain of Neuropilin, and identified PDE4D as a participant in the signaling cascade. We found that Semaphorin enhances the NeuropilinPDE4D interaction, and promotes PDE4D localization to the cytoplasmic membrane. PDE4D then degrades cAMP and reduces the overall intracellular cAMP level; consequently it inhibits PKA activity and promotes Hh signaling. These data revealed a hitherto unknown signaling pathway downstream of Neuropilins, and elucidated a novel pathway crosstalk between the Neuropilin and Hh signaling. To study Neuropilin-mediated Hh signal regulation in vivo, we examined neurogenesis in the developing cerebellum, where the proliferation of granule neuron precursors (GNPs) highly depend on Hh signaling. We blocked Semaphorin signaling by knocking out both Neuropilins from GNPs, and found significant reduction in GNP proliferation, a phenotype frequently seen in mutants with repressed Hh signal transduction. These findings underscore the essential roles of Neuropilin signaling in Hh signal transduction in vivo. In short, our findings deepened the understanding of Hh signal transduction, and revealed a novel role of Neuropilins in the regulation of cAMP production. All these discoveries potentially point to novel therapeutic targets for Neuropilin- and Hh-related diseases.

Abstracts

Xuecai Ge

Abstracts

Marina Grachtchouk

Assistant Research Scientist University of Michigan

Overriding hair follicle stem cell resistance to oncogenic Hedgehog signaling Previous results have shown that acute induction of oncogenic Hedgehog (Hh) signaling in skin leads to the development of basaloid hyperplasia affecting multiple progenitor cell populations, with the striking exclusion of follicle bulge stem cells. We re-evaluated the resistance of bulge stem cells to Hh oncogenic transformation by comparing responses to GLI2* expression driven by the Keratin 14 promoter, using K14-rtTA;tetO-GLI2* (iK14;GLI2*) mice, versus the Keratin 5 promoter, using K5-rtTA;tetO-GLI2* (iK5;GLI2*) mice. In both of these models treatment with doxycycline (doxy) leads to activation of GLI2* transgene in basal cell populations, enabling us to examine and compare responsiveness of bulge cells and other basal cells. H&E histology did not reveal significant differences in telogen skin phenotype after 2 days of transgene activation, but extensive basaloid hyperplasia was detected in the bulge compartment of K5-, but not K14-driven mouse model after 5 days of induction. In situ hybridization and immunostaining analysis demonstrated substantially higher level of transgene and Hh target genes expression in all basal layers, including the bulge in iK5;GLI2* compared to iK14;GLI2* mice. Despite the lack of a morphological response affecting bulge cells of iK14;GLI2* mice, the proliferation marker Ki67 was upregulated in this normally quiescent population in both the K14- and K5-driven mouse models. However, cell cycle progression assessment, using colchicine administration to arrest cells in mitosis, revealed pHH3+ mitotic cells in the bulge compartment of K5- but not K14-driven bitransgenic mice. The lack of basaloid hyperplasia in the bulge compartment of K14-driven GLI2* mice is associated with lower-level Hh signaling than in K5-driven mice and may be related to impaired cell cycle progression and increased apoptosis. In contrast, high-level GLI2* expression and Hh signaling in iK5;GLI2* mice overrides bulge resistance to stimulate hyperplasia, supporting the concept that even cells which are normally protected from cancer development can produce tumors in the setting of sufficiently high oncogenic signaling.

Research Associate Fox Chase Cancer Center

Determining the mechanisms of Hh release in response to feeding A healthy diet improves adult stem cell function and delays diseases such as cancer, heart disease, and neurodegeneration. Defining molecular mechanisms by which nutrients dictate stem cell behavior is a key step toward understanding the role of diet in tissue homeostasis. Here we elucidate the mechanism by which dietary cholesterol controls epithelial follicle stem cell (FSC) proliferation in the drosophila ovary. In nutrient-restricted flies, the transmembrane protein Boi sequesters Hedgehog (Hh) ligand at the surface of Hh-producing cells within the ovary, limiting FSC proliferation. Upon feeding, dietary cholesterol stimulates S6 kinase-mediated phosphorylation of the Boi cytoplasmic domain via the nuclear hormone receptor DHR96, triggering Hh release and FSC proliferation. This mechanism enables a rapid, tissue-specific response to nutritional changes, tailoring stem cell divisions and egg production to environmental conditions sufficient for progeny survival. Mass spec and genetic analysis of Boi-associated proteins in flies fed a normal or nutrientrestricted diet has identified novel proteins involved in nutrient-responsive release of Hh. Identifying the specific molecular mechanism occurring in this system will likely have important implications for studies on molecular control of stem cell function in conserved pathways in the tissues of many organisms in which the benefits of low calorie and low cholesterol diets are beginning to emerge.

Abstracts

Tiffiney Hartman

Abstracts

David Hipfner

Associate Professor IRCM

A broadly conserved G-protein-coupled receptor kinase phosphorylation mechanism controls Drosophila Smoothened activity Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a Cterminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.

Graduate Student University of Michigan

Distinct roles for secreted and cell surface associated HHIP1 in the control of Hedgehog-dependent spinal cord growth and patterning Hedgehog (HH) ligands are secreted morphogens that are essential for vertebrate and invertebrate embryogenesis. We have recently shown that three HH-binding antagonists, PTCH1, PTCH2, and HHIP1, play overlapping and essential roles to restrict HH activity in the developing mouse neural tube. However, the mechanisms employed by these structurally distinct antagonists remain unclear. In particular, little is known about the molecular and cellular mechanisms of HHIP1-mediated inhibition of HH signaling. HHIP1 was initially characterized as a transmembrane-anchored glycoprotein that binds and sequesters HH ligands. Using chicken in ovo neural tube electroporations, we find that ectopic PTCH1 and PTCH2 inhibit HH-dependent neural patterning in a cell autonomous manner. By contrast, ectopic HHIP1 uniquely causes profound non-cell autonomous growth defects in the developing chicken neural tube by limiting neural progenitor proliferation. Strikingly, HHIP1 also antagonizes HH-dependent patterning in a non-cell autonomous manner. These findings led us to hypothesize that despite its putative membrane anchor, HHIP1 is a secreted antagonist of HH signaling, representing a novel mechanism to control HH pathway function. In support of this idea, artificial tethering of HHIP1 to the cell surface abolishes its non-cell autonomous effects. Intriguingly, membrane-tethered HHIP1 inhibits HH-dependent patterning in a cell-autonomous manner without affecting tissue growth. Using biochemical approaches we demonstrate that HHIP1 is secreted independently of direct proteolytic cleavage. Instead, HHIP is retained at the cell surface through interactions between heparan sulfate and a stretch of basic amino acids in the N-terminal cysteine rich domain of HHIP1. Additionally, we show that the balance between secreted and membrane-associated HHIP1 is regulated by cell-type specific differences in heparan sulfate modifications. Overall, these data redefine HHIP1 as a secreted antagonist of vertebrate HH signaling and show that the effects of HHIP1 on growth and patterning of the neural tube are functionally separable and governed by secreted and membraneassociated HHIP1, respectively.

Abstracts

Alexander Holtz

Abstracts

Jianhang Jia

Associate Professor University of Kentucky Markey Cancer Center

Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling but how the localization of Smo is controlled remains poorly understood. In this study, we demonstrate that atypical Protein Kinase C (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation downregulates Smo accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively-active aPKC elevates Smo accumulation and promotes Hh signaling. aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by CK1, and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is upregulated by Hh signaling in a Ci-dependent manner. Our findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity.


Hedgehog-Dependent Maintenance and Regeneration of the Mammalian Olfactory Epithelium

In the olfactory epithelium (OE), olfactory sensory neurons (OSNs), which are necessary for the sense of smell, are continuously produced by the differentiation of two populations of basal stem cells, globose basal cells (GBCs) and horizontal basal cells (HBCs). GBCs are an active cell population that frequently divides, while HBCs are a quiescent cell population that rarely divides except following severe injury to the OE. The fate of these cells during the maintenance and restoration of the OE has been studied by various groups, but the signaling pathways that mediate their activation and differentiation are not well defined. The Hedgehog (HH) pathway is a well-established regulator of cell proliferation/differentiation in developing and adult tissues. Recently, we defined the expression of several components of the HH pathway (i.e., the receptor, Patched1, the coreceptor Gas1, and the transcription factors, Gli1 and Gli2) in cells of the OE, including OSNs and HBCs. We hypothesize that HH signaling through HBCs regulates OE tissue maintenance and regeneration. To test this hypothesis we used several mouse models to investigate the effects of HH signaling on the regulation of HBC function in OE tissue maintenance and regeneration. We found that mice with HBC-specific activation of Gli2, demonstrate hyperproliferative HBCs that correlates with a reduction in neuronal-fated GBCs. In addition, the OE of these mice do not recover properly following chemical ablation compared to wildtype mice. Conversely, we found that conditional deletion of Gli2 in HBCs results in a reduction in the number of olfactory basal cells. Overall, these data suggest an important role for the HH pathway, and specifically GLI2, in the regulation of HBCs and the maintenance of the OE. To further investigate the effects of HH pathway disruption in maintenance and regeneration of the OE, wildtype mice were treated with the Smoothened inhibitor, LDE225, in addition to chemical ablation of the OE. Immunofluorescent analyses and behavioral studies revealed that the OE was able to recover without HH pathway function, but that mice displayed reduced attractant behaviors. These data suggest that GLI proteins function in a non-canonical manner during restoration of the OE, while canonical HH signaling selectively controls olfactory behavior.

Abstracts

Ariell Joiner

Abstracts

Jennifer Kong

Graduate Student University of California Los Angeles

A requirement for Notch pathway activity for the interpretation of Sonic hedgehog signaling and the assignment of neural progenitor fates Throughout the developing nervous system, neural stem and progenitor cells give rise to functionally diverse classes of neurons and glial cells in a spatially and temporally coordinated manner. In the ventral spinal cord, much of this diversity emerges though the morphogen actions of Sonic hedgehog (Shh). Interpretation of the Shh signaling gradient depends on both the amount of ligand and duration of exposure, but the mechanisms preserving this competence are not known. We demonstrate that Notch signaling plays an essential role in this process, enabling neural progenitors to accumulate sufficiently high levels of Shh pathway activity needed to direct the ventral-most cell fates. Notch activity regulates the localization of the Shh receptor Patched1, gating the translocation of the key Shh effector Smoothened into primary cilia and its downstream signaling activities. These data reveal an unexpected role for Notch in shaping the interpretation of the Shh morphogen gradient and impacting cell fate determination.

Graduate Student Max-Planck-Institute of Molecular Physiology

Novel Smo binding and non-binding Inhibitors of the Hedgehog Signaling Pathway The Hedgehog (Hh) signaling pathway plays a major role in embryonic development, stem cell homeostasis and cancer. Its dysregulation can lead to congenital defects and malignancies. Due to this immense biological and medicinal significance, potent inhibitors of the Hedgehog signaling pathway are in high demand. Since first therapy failures occurred because of acquired resistance to Smoothened (Smo)-binding compounds, especially inhibitors that do not bind to Smo are of particular importance. So far, all clinically successful inhibitors are Smo-binders. In order to identify inhibitors of the Hh signaling pathway, a high throughput screen has been performed at the Compound Management und Screening Center (COMAS) in Dortmund, employing an osteoblast differentiation assay. Out of the compounds that were found to inhibit osteoblast differentiation via inhibition of alkaline phosphatase in the low micromolar range, seven have been selected for further characterization and showed a dose-dependent reduction of Gli-mediated transcription in SHH-light II cells. The influence on the Hedgehog target gene Patched-1 (Ptch-1), that is upregulated upon pathway activation as a negative feedback-loop, was determined by quantitative PCR. Treatment of NIH/3T3 cells with the known Hedgehog pathway activator purmorphamine led to an upregulation of Ptch-1. Simultaneous treatment of these cells with purmorphamine and the hit compounds or vismodegib as a positive control for pathway inhibition led to a concentration-dependent decrease of Ptch-1 expression. In addition, five compounds could not displace bodipy-cyclopamine from the Smoothened receptor. Most likely, these compounds mediate their effect on the Hh signaling pathway via alternative targets. Future experiments will aim at elucidating the substances influence on the protein levels of Gli transcription factors and focus on the identification of their cellular targets.

Abstracts

Lea Kremer

Abstracts

Archana Kumari

Postdoctoral Fellow University of Michigan

Inhibition of hedgehog pathway by cancer drug LDE225 disrupts taste organ maintenance and taste sensation in mice

Hedgehog (HH) signaling is crucial in development and maintenance of several tissues including taste papillae and taste buds. In contrast, deregulation of the HH pathway promotes tumorigenesis, and therefore a current cancer therapeutic approach uses inhibition of HH signaling, by blocking the signaling effector Smoothened. However, the HH Pathway Inhibitor (HPI) drugs, including LDE225, result in severe taste disturbances that often lead patients to discontinue treatment. To understand HH pathway functions in regulating taste sensation, we investigated effects of LDE225 on fungiform papilla taste organs and gustatory function in adult mice. Mice received LDE225 (20mg/kg) or Vehicle gavage for 16 days. In recordings made from the chorda tympani nerve, there were reduced or no responses to various chemical taste stimuli with LDE225 treatment compared to Vehicle. On the other hand, tongue responses to mechanical touch and cold stimuli were not affected. Lingual tissue analysis demonstrated that Gli1-expressing, HHresponding cells were reduced in the fungiform papilla epithelium of LDE225 treated mice. Fungiform papillae in LDE225 treated tongues acquired a conical apex with thick layers of keratin. Taste buds were either absent (27%) or very thin with disoriented cells (66%) in these aberrant papillae. In association with taste bud cell loss, the taste bud restricted, Shh ligand was also reduced after treatment. The remaining abnormal taste bud cells were positive for the taste cell marker keratin 8 and retained innervation to papillae and taste buds, as demonstrated with neurofilament and P2X3 antibodies. These data identify a critical requirement for HH signaling in maintaining taste papilla and taste bud integrity and function, and strongly support the concept that taste disturbances in LDE225-treated patients reflect an on-target response to HH pathway blockade in human taste organs.

Postdoctoral Fellow Institute of Molecular Biology, Academia Sinica

PIG-V mutations in Hh morphogen formation and signaling responses, and as a model for hyperphosphatasia mental retardation (HPMR) syndrome Hedgehog (Hh) morphogen patterns tissues via interaction with extracellular matrix heparin sulfate proteoglycans (HSPGs). Dally-like protein (Dlp), one of HSPGs with glycosylphosphatidylinositol (GPI) modification plays as co-receptor for endocytosis of Hhreceptor complex. Here, we show the functions of GPI modification on Hh response and morphogen formation by studying the mutants for phosphatidylinositol glycan anchor biosynthesis class V (PIG-V) that encodes GPI mannosyltransferase II, an enzyme involved in the synthesis of GPI core. In PIG-V mutants, Dlp modification, processing and localization are affected. Genetic mosaic analyses show that PIG-V is required cellautonomously for Hh signaling in short and intermediate ranges, consistent with the role of Dlp as a co-receptor in Hh signaling. However, long ranges of Hh signaling are unaffected in PIG-V clones. In contrast to clonal assays, long and intermediate ranges of Hh signaling are enhanced in PIG-V hypomorphic mutants, as revealed by the increased expression levels and domains of Cubitus interruptus (Ci) and Patched (Ptc). Consistent with the effects on long-range Hh signaling, anchorless forms of Dlp are increased in the larval hemolymph, and Hh puncta are increased even in the far anterior region of wing discs. Additionally, we show that knockdown of PIG-V in wing discs causes growth defect and that can be rescued by the co-expression of secreted and membrane-tethered forms of Dlp. Thus, we propose that dual functions of Dlp on Hh spreading and responses are dependent on the different states of GPI modification. Here we define the anchorless Dlp, an unidentified carrier for the long range transportation of Hh, whereas the membraneanchored Dlp acts as co-receptor required for Hh responses. Finally, we suggest that the increased activity of alkaline phosphatase, one of GPI anchored proteins in hemolymph, and the expended Hh gradient range in wing discs in PIG-V mutants can provide a disease model to study the etiology of hyperphosphatasia mental retardation (HPMR) syndrome, a congenital disease with PIG-V mutations that are associated with increased alkaline phosphatase activity in circulation and hypertelorism, a mild Hh gain-of-function phenotype in facial development.

Abstracts

Yi-Nan Lee

Abstracts

Libo Li

Research Lab Specialist University of Michigan

Taste organ maintenance is dependent on continuous Hh/Gli signaling activity We have shown that deregulated Hedgehog (Hh) signaling, which acts through the Gli family of transcription factors, alters papilla and taste bud maintenance. However, the role of Hh/Gli signaling in the adult taste system is not well studied. To learn whether acute loss of Gli function can affect fungiform papilla and taste bud integrity, we generated mice with a doxycycline-inducible Gli2 mutant, Gli2deltaC4, in K5 expressing cells throughout lingual basal cells in fungiform papilla and interpapilla epithelium. Tongues were collected at 1-2, 5 or 10 days after expressing the Gli2deltaC4 transgene. By 5 days the effects of blocking Gli function were profound. Hh/Gli signaling was reduced in fungiform papilla epithelium, assessed by analysis of Hh target gene expression. Greater than 50% of papillae had an atypical shape with a heavily keratinized apex. Of these atypical papillae, about 40% did not have intact taste buds. Some remnants of taste buds were thin with disoriented, keratin 8 positive cells. Neurofilament-positive expression in the papilla connective tissue core indicates retained innervation and suggests that effects on taste buds reflect a requirement for Gli function in epithelium and not an indirect effect due to nerve disruption and consequent taste bud loss. After 10 days of transgene expression 98% of fungiform papillae were aberrant and had no taste buds, and cell proliferation was substantially reduced in the papilla basal cells. Qualitatively similar changes were detected in the circumvallate papilla on posterior tongue, which typically contains a few hundred taste buds. The rapid alterations brought about by disruption of Gli function in adult tongue epithelium point to a strict requirement for continuous Hh/Gli signaling in the maintenance of lingual epithelium and taste organs.

Graduate Student Emory University

The inositol phosphatase Inpp5e regulates Shh signaling Inositol polyphosphate-5-phosphatase (Inpp5e) is a phosphatase that can hydrolyze the 5phosphate of PIP2 and PIP3, membrane phospholipids regulating important cellular processes such as vesicle trafficking, calcium signaling, and cell proliferation. In the mouse, Inpp5e deletion leads to abnormal embryonic development and perinatal lethality. In a forward genetic screen for genes that control mammalian neural tube patterning, we identified a novel allele of Inpp5e (M2). This allele results in a D511G amino acid substitution within the phosphatase domain. Homozygous Inpp5eM2 mice display developmental defects and die at approximately E14.5. We detected dorsal expansion of ventral spinal cell fates (p2, pMN, and p3) at E9.5 and E10.5 in Inpp5eM2 embryos. Shh mRNA expression expanded dorsally, but the protein appeared normal at E10.5. Surprisingly, by E12.5 the neural patterning looks fairly normal, indicating that a partial recovery takes place between E10.5 and E12.5. Together, these data suggest that Inpp5e regulates Shh signaling. Many of the Inpp5e-dependent phenotypes are reminiscent of mutations in Arl13b, a small ciliary GTPase. In fact, mutations in either gene can cause the ciliopathy Joubert syndrome. Additionally, Arl13b is required for Inpp5e enrichment in cilia, and the two proteins are in a common protein complex. In terms of Shh signaling, the Inpp5eM2. phenotype recapitulates a subset of Arl13b null phenotypes. Arl13b ventral patterning defects are more severe than in Inpp5eM2. At a cellular level, Smoothened (Smo) is normally enriched in cilia only upon Shh pathway stimulation. In the absence of Arl13b, Smo is found in cilia regardless of Shh stimulation and is in puncta, indicating defects in both ligand-dependent entry of Smo into the cilium and in Smo distribution within the cilium. In Inpp5eM2, however, we only observe misdistribution of Smo. These findings imply that Inpp5e regulates Shh signaling as an effector of Arl13b.

Abstracts

Chao Lin

Abstracts

David Lorberbaum Graduate Student University of Michigan

Patching together the transcriptional response of a Hedgehog target gene with multiple enhancers and low affinity binding sites Surprisingly few cell signaling pathways are responsible for generating the cellular diversity required for all organisms to survive. One of these pathways is known as the Hedgehog (Hh) signaling pathway and is essential for patterning multiple tissues during development and is equally important for maintaining homeostasis in adult tissues. Regardless of context, the immediate output of Hh signaling is the activation and repression of target genes, all of which are directly regulated by the transcription factor Cubitus interruptus (Ci). Conserved in vertebrates as the Gli family, Ci functions through binding to enhancers, or cis-regulatory regions of DNA, to regulate levels of target gene transcription. Of the known Hh target genes, one of the best studied is patched (ptc), which encodes the Hh pathway receptor and is therefore activated in all Hh-responding cells in both flies and vertebrates. Previous work identified a ptc enhancer and demonstrated that it requires three perfect Ci binding sites to activate ptc transcription in the wing, a context that requires Hh for proper patterning. Our lab has shown that this enhancer, despite its ability to respond in the wing, is not sufficient to respond to Hh signaling in all relevant contexts, including other development stages and in adults. To understand how ptc and other Hh target genes respond in all tissues, we have characterized several temporally and spatially regulated Hh responsive enhancers, many of which do not require perfect Ci binding sites. Most often, these Hh responsive enhancers require non-perfect, low affinity sites to activate transcription. These data suggest that the transcriptional regulation of ptc is highly context dependent and requires inputs from several different enhancers. This work also provides new information that better define how Hh might regulate other target genes at the transcriptional level.

Senior Investigator Cancer and Developmental Biology Laboratory, National Cancer Institute

Does Sonic hedgehog function as a morphogen in the developing limb? How Shh functions as a morphogen to regulate dorsoventral cell fates in neural tube has been intensely investigated and is becoming well understood. However, the role of Shh and the network of downstream targets that cooperate to regulate patterning in the developing limb is far less clear. Different approaches have yielded disparate models of how Shh functions as a limb morphogen. Whereas pharmacologic work in chick and some genetic analysis in mouse support a requirement for sustained Shh activity integrated over time to specify different digit identities, experiments exploiting timed genetic deletion of Shh in mouse suggest only a very transient requirement in patterning. To directly test if Shh function is needed over an extended time for digit patterning, we restored cell survival in Shh mutant embryos supporting only a very transient burst of Shh activity that still produce a Shh null limb phenotype. Cell survival enforced by removing the function of pro-apoptotic Bax/Bak genes rescues the formation of 3-5 normally patterned digits in these otherwise phenotypically Shh null embryos. However, the early transient Shh pulse is essential for normal patterning; rescue of cell survival in Shh germline-null embryos fails to alter the null limb phenotype. Our experiments reveal distinct and separable Shh roles in regulating patterning and growth, and point to two different classes of Hedgehog targets requiring either transient or continuous signaling for their sustained activation. Downstream target genes regulating digit patterning would be predicted to fall in the former class requiring only transient exposure to Shh signals for their stable activation, suggesting that Shh functions more as a trigger than true morphogen in the limb. Identification of the targets belonging to these classes is currently under investigation. In parallel studies, we are also genetically mapping cells that produce and/or respond to Shh in the limb over time. Our preliminary results suggest that, even at early times, cells producing Shh do not respond to the signal, but that the Shh producing cell population changes over time. These results are difficult to reconcile with integrative morphogen function, and seem to be more compatible with a transient-trigger role.

Abstracts

Susan Mackem

Abstracts

Shigeru Makino

Research and Development Student RIKEN BioResource Center

Missense mutant mice of Sufu demarcate its regulatory actions on Gli2 and Gli3 Hedgehog signaling is transduced by two primary transcription factors, Gli2 and Gli3. Recent studies using a Sufu KO mouse have indicated that Sufu is involved in multiple steps for regulation both of Gli2 and Gli3 but each regulatory mechanism specific to Gli2 and/or Gli3 remains to be elucidated. In this study, we established allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that the Thr396-residue of Sufu played a key role in differential regulation specific to Gli3. SufuT396I/T396I embryos exhibited polydactyly and ventralized neural tube, indicative of overactivation of Hh signaling. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and proteolytically processed Gli3 (Gli3REP) were seen in vivo as well as in vitro. SufuT396I also qualitatively reduced the processing of Gli3. In contrast, SufuT396I exhibited any mutational effects on Gli2 regulation. Taken together, this study showed that Sufu regulated Gli3, but not Gli2, through a mechanism dependent on the Thr396 residue of Sufu, demonstrating that Sufu plays a central role in demarcating its regulatory actions of Gli2 and Gli3.

Postdoctoral Fellow Emory University

YAP: A Master Metabolic Regulator In Medulloblastoma Downstream of mitogenic Sonic hedgehog (Shh) signaling, Yes-Associated Protein (YAP) can drive proliferation in Cerebellar Granule Neural Progenitor (CGNP) cells. CGNPs are the proposed cells of origin of SHH medulloblastomas. They are a neural progenitor type whose developmental expansion requires signaling by Shh, a secreted ligand produced by the neighboring Purkinje neurons. Approximately 30% of human medulloblastomas bear an activated Sonic hedgehog pathway gene expression signature. Ectopic expression of YAP promotes highly aggressive Shh-driven medulloblastoma growth and radio-resistance (Fernandez et al., 2009). Medulloblastoma being the most common solid malignancy of childhood and a leading cause of pediatric mortality, the current standard of care results in about 60% “cure” rate. But the survivors are beset with long-term side effects, including cognitive impairment, seizures, premature aging, and susceptibility to cancer. Moreover, recurrence and metastasis are lethal. Therefore, identification of novel modes of molecular targeted therapies is critical for the improved quality of life for survivors and reduced incidence of recurrence and metastasis. Recently, there has been renewed interest in how altered metabolic patterns in tumors could be exploited for therapeutic purposes. Deregulating the metabolic machinery for aberrant energy utilization is one of the hallmarks of a proliferating cancer cell. Previously, our lab made the novel observation that Shh mitogenic/oncogenic signaling is tightly coupled to the reprogramming of mitochondrial bioenergetics: Shh inhibits fatty acid oxidation (FAO) while driving increased fatty acid synthesis (FAS), an early step of lipogenesis. The production of citrate, an essential component for fatty acid synthesis, occurs inside the mitochondrion via the Tri-carboxylic acid cycle (TCA). We analyzed the effect of Shh treatment and ectopic YAP expression on CGNPs and found that YAP increases levels of fatty acid synthase (FASN) and ATP citrate lyase (ACLY), while YAP knock-down in Shh-treated CGNPs resulted in reduced levels of these enzymes. Moreover, we also observed a surprising decrease in mitochondrial membrane potential. This prompted us to further analyze the ultrastructure of mitochondria using Transmission Electron Microscopy. Shh-treated or ectopic YAP-expressing mitochondria presented a swollen morphology, along with an expanded matrix space and deformed cristae structure, typical of morphologically aberrant mitochondria. Being dynamic structures, mitochondria undergo constant fusion and fission events, which contribute to their biogenesis. Analysis of genes regulating cristae shape and fission and fusion events in mitochondria under the influence of Shh and YAP will reveal the links between respiratory growth and mitochondrial dysfunction. Aberrant morphology and decrease in membrane potential also indicate a possible decrease in electron transport chain (ETC) and the Tri-carboxylic acid cycle (TCA), which take place within the mitochondria. Analysis of the different substrates of the metabolic pathways, especially succinate, which is the connecting link between TCA and ETC and the enzymes catalyzing different reactions, can unveil the mechanism behind mitochondrial dysfunction. Our lab has also demonstrated that Shh-treated CGNPs are highly glycolytic. Increased glycolysis results in high acidity in a cell, which leads to fragmented mitochondria (Alirol and Martinou, 2006). It would thus be imperative to explore the links between a high glycolytic rate in Shh-treated or ectopic YAP-expressing CGNPs and consequent aberrant mitochondrial morphology. Our study thus implicates YAP-regulated metabolic pathways and enzymes as potential targets for novel medulloblastoma therapies that may reduce or eliminate the requirement for high dose radiation.

Abstracts

Anshu Malhotra

Abstracts

Laura Mariani Graduate Student Emory University

Role of the cilia protein Arl13b in neuronal non-canonical Shh signaling Canonical Shh signaling occurs at the primary cilium. Mutations in cilia genes cause abnormal Shh signaling and a class of human diseases called ciliopathies. The cilia gene Arl13b is linked to the ciliopathy Joubert Syndrome (JS). Loss of Arl13b is embryonic lethal and causes defects in cilia structure, misregulated localization of Smoothened, and abnormal neural tube patterning. JS patients have truncating or point mutations in Arl13b (or other cilia genes) but still show neurodevelopmental symptoms and signs of misregulated canonical Shh signaling (e.g., polydactyly). Non-canonical Shh signaling refers to downstream effects of Shh that require Shh receptors and effectors but do not require cilia or transcription. These effects include cytoskeletal rearrangements important for axon pathfinding and cell migration. In addition to canonical Shh defects, JS patients have abnormal axon guidance and heterotopias consistent with dysfunctional neuron migration, but the relationship between these phenotypes and the Shh pathway has not been examined. We hypothesize that 1) mutations in Arl13b also affect non-canonical Shh signaling, and 2) the disruption of cilia-related genes important for noncanonical Shh signaling contributes to ciliopathy phenotypes. To test our hypotheses, we deleted Arl13b in cell types that are known to exhibit noncanonical Shh signaling. We saw impaired Shh-dependent migration in Arl13b null fibroblasts in vitro and brain phenotypes consistent with JS in vivo. We tested the effect of Arl13b mutations on canonical and non-canonical Shh signaling and saw that JS-causing alleles fail to rescue the Arl13b null phenotype. Surprisingly, a mutation that prevents cilia localization of Arl13b also fails to rescue Shh-dependent migration in fibroblasts. Therefore, while the cilium itself is dispensable for non-canonical Shh signaling, ciliary proteins still affect the pathway, and non-canonical Shh defects may contribute to human ciliopathies.


Hedgehog Co-receptors in Pancreatic Tumor Growth Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. The Hedgehog (Hh) signaling pathway is upregulated in PDAC. 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is active in early PanIN lesions and persists as the cancer progresses. Interestingly, Hh ligands are secreted by the tumor cells and function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor growth. PDAC is characterized by hypovascularity and a highly desmoplastic stroma. The downstream effects of Hh activation in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdon), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Gas1, Boc, and Cdon are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by costains with α-SMA and vimentin. During cancer progression, expression of all three coreceptors is prevalent throughout the desmoplastic stroma. Functional studies on Gas1-/;Boc-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wildtype MEFs, indicating that these co-receptors are important for Hh signal transduction. Despite a reduced Hh-response, we found that Gas1-/-;Boc-/- MEFs promoted the growth of significantly larger, more vascularized tumors than their wildtype counterparts. A more physiologically relevant experiment with pancreatic fibroblasts and primary pancreatic tumor cells yielded the same counterintuitive result: removal of Gas1 and Boc from fibroblasts decreases Hh-response, and yet tumorpromoting potential was significantly increased. Moreover, chick chorioallantoic membrane (CAM) assays with tumor cells and fibroblasts revealed that Gas1-/-;Boc-/- MEFs stimulated blood vessel growth. We repeated these experiments with MEFs in which all three co-receptors were removed. Gas1-/-;Boc-/-;Cdon-/- MEFs displayed a more significantly abrogated Hh-response than both their Gas1-/-;Boc-/- and wildtype counterparts. However subcutaneous co-injection and CAM experiments revealed that these MEFs did not promote tumor growth, behaving similarly to Smo-/- MEFs in previously published findings. These results indicate the importance of Hh dosage in pancreatic cancer, which has important clinical implications. In particular, we found that intermediate levels of Hh signaling, such as that resulting from pharmaceutical inhibition, resulted in a marked angiogenesis response. This finding may partially account for the recent failure of Hh inhibition in a pancreatic cancer clinical trial.

Abstracts

Esha Mathew

Abstracts

Victor Maximov Postdoctoral Fellow Emory University

Roles of cholesterol and fatty acid transport proteins in Shhdependent medulloblastomas Medulloblastomas (MBs) are the most common solid malignant pediatric brain tumors and associated with highest mortality rate among pediatric cancers. The current treatment method combines surgical resection with chemo- and radiotherapies, resulting in an approximately 70% 5-year survival rate. However, survivors are usually left with life-long side effects such as hormonal dysfunction and intellectual impairment that greatly affect quality of life. Medulloblastomas originate in the developing cerebellum as a result of aberrant activity of critical developmental pathways, such as WNT, Notch or Shh (Sonic hedgehog) and they can be divided into 4 subtypes according to unique gene expression patterns, genomic abnormalities, and histological traits. The Shh subclass of MB can be modeled in vitro by activation of Shh pathway with Shh ligand in cerebellar granule precursor cells (CGNPs) extracted from murine neonatal cerebella. A novel approach for medulloblastoma treatment may be to target lipid transport and metabolism. We have previously demonstrated that exaggerated de novo lipid synthesis and accumulation of neutral lipids is a hallmark of highly proliferative, aggressive mouse Shh-driven medulloblastomas. To gain further insight into how lipid regulation may contribute to the biology of medulloblastoma, and to the normal role for mitogenic Shh signaling in the developing cerebellum, we carried out a mass spectrometry analysis, comparing Shh MB to adjacent non-tumor cerebellum to determine the intra-tumoral lipid composition. Here, we show that Shh mouse MB feature high levels of cholesteryl esters (CE), the form in which cholesterol is stored and transported outside of cells. CE accumulation is driven by the activity of Sterol O-acyltransferase 1 (SOAT1). SOAT1 protein levels are increased in these tumors, as are those of the fatty acid transporters CD36 and FATP4. Primary CGNPs cultures induced to proliferate by Shh also have an increased level of SOAT1 protein, suggesting that these cultures are a good model for investigation of lipid metabolism pathways downstream of activated Shh signaling. Inhibition of SOAT1 causes death in mouse and human medulloblastoma cell lines derived from Shh tumors, suggesting that accumulation of intracellular free cholesterol is cytotoxic to the tumor cells. At the same time, SOAT1 inhibitors did not significantly affect cells that do not have an increased cholesterol ester synthesis. Based on these findings, we hope to develop a novel method for medulloblastoma treatment.

Graduate Student Johns Hopkins University

Expression, purification and biophysical characterization of Patched and Smoothened Patched (Ptch) is twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In the absence of Hh, Ptch represses Smoothened (Smo), a seven-pass transmembrane protein that is involved in the transduction of the Hedgehog signal. The purpose of our study is to biophysically characterize near full length mouse Ptch and Smo proteins. We have expressed Ptch in Sf9 cells and biophysically characterized purified, detergent solubilized Ptch proteins using size-exclusion chromatography, circular dichroism and thermal denaturation assays. Additionally, we have expressed Smo in Sf9 cells, including the extracellular cysteine rich domain (CRD), and characterized purified, detergent solubilized Smo proteins using size-exclusion chromatography and thermal denaturation assays in the presence of a panel of Smo modulators.

Abstracts

Jacqueline McCabe

Abstracts

Don van Meyel

Associate Professor McGill University

Ihog and Boi elicit Hedgehog signaling via Ptc but do not aid Ptc in sequestering the Hh ligand Hedgehog (Hh) proteins are secreted molecules essential for tissue development in vertebrates and invertebrates. Hh reception via the 12-pass transmembrane protein Patched (Ptc) elicits intracellular signaling through Smoothened (Smo). Binding of Hh to Ptc is also thought to sequester Hh and thereby limit its spatial range of activity. In Drosophila, Interference hedgehog (Ihog) and Brother of Ihog (Boi) are two conserved and redundant transmembrane proteins that are essential for Hh pathway activation. How Ihog and Boi activate signaling in response to Hh remains unknown; each can bind both Hh and Ptc and so it has been proposed that they are essential for both Hh reception and sequestration. Using genetic epistasis we demonstrate that Ihog and Boi act upstream or at the level of Ptc to allow Hh signal transduction. In the Drosophila developing wing model we found that it is through Hh pathway activation that Ihog and Boi maintain the boundary between the anterior and posterior compartments. We dissociated the contributions of Ptc from those of Ihog and Boi and surprisingly found that cells expressing Ptc can retain and sequester the Hh ligand without Ihog and Boi, and that Ihog and Boi cannot do so without Ptc. Thus, Ptc is involved in both Hh pathway activation and sequestration, reinforcing its central role in Hh binding in vivo. Ihog and Boi are essential for pathway activation but not sequestration, allowing Hh to inhibit Ptc and thereby relieve its repression of Smo.

Senior Scientist Stanford University

Single-molecule imaging of Smoothened in primary cilia reveals binding events that are modified by Hedgehog pathway agonists In vertebrate cells, early steps of Hedgehog (Hh) signal transduction take place in primary cilia. The transmembrane protein Smoothened (Smo) accumulates in cilia when cells are treated with Hh or Smo agonist (SAG). The mechanism for this selective enrichment is poorly understood. We used single-molecule imaging and tracking in live cells to analyze Smo protein movement in the membrane of primary cilia. Obtained tracks, often tens of seconds long, have 30 nm localization precision and 10 ms time resolution. A detailed analysis of these trajectories revealed three distinct modes of Smo motion in cilia: predominant diffusion, rare directional movement, and distinct confinement. The diffusion rates and the frequency and velocities of the directional movement did not change in the presence of SAG. The confinement events, most likely representing binding to yet unknown cilia components, were predominantly observed at the base and at the tip of cilia. Binding of Smo at the base of cilia was shorter and less frequent in cells treated with SAG than in control, unstimulated cells. The observed parameters will be used to propose a model to help explain Smo accumulation in cilia.

Abstracts

Ljiljana Milenkovic

Abstracts

Hiromi Mizuochi Clinical Fellow Chiba University

Hedgehog signaling is synergistically enhanced by nutritional deprivation and ligands stimulation in human fibroblasts of Gorlin syndrome. Background: Hedgehog signaling is a pivotal developmental pathway, comprised of Hedgehog, PTCH1, SMO, and GLI proteins. PTCH1 mutations can cause Gorlin syndrome (GS), characterized by developmental defects such as palmar pits, rib abnormality, and falx calcification, and tumorigenesis such as medulloblastoma, ovarian fibroma, and cardiac fibroma. Although hedgehog pathway has been well investigated in drosophila and mice, little is known about precise roles in human cells. Materials and methods: To investigate the mechanism regulating hedgehog signal transduction in human diseases, we investigated human fibroblasts derived from GS patients already identified PTCH1 mutations, and normal adult fibroblasts as a control. We stimulated fibroblasts with recombinant human sonic hedgehog with serum deprivation, and then we observed expression of primary cilia on fibroblasts with immunostaining and measured the expression of mRNA of transcription factors of hedgehog pathway by quantitative-PCR. We also investigated hedgehog pathway related genes using mRNA plates. Result: Expression of Gli1 mRNA in all GS derived fibroblasts is significantly elevated especially with nutrient deprivation, compared to those of normal controls. In presence of sonic hedgehog, the expression of Gli1 mRNA is also elevated especially in GS cells. On hedgehog pathway investigation, we found enhanced several mRNA expressions such as Wnt signaling and morphogenic molecules. Conclusion: Since responses for hedgehog stimulation with nutritional deprivation are well activated in GS fibroblasts upon activation of hedgehog-related genes, ciliogenesis may have a key role for homeostasis and morphogenesis in PTCH1 deficient human cells.

Assistant Professor UT Southwestern Medical Center

Role of the GPCR Gpr161 in hedgehog signaling Long ignored as vestigial, primary cilia have now been established as sensory antennae in most vertebrate cells. Although, mechanisms driving formation of cilia have been extensively studied, the principles underlying signaling are largely unknown. The primary cilium is fundamentally important during developmental sonic hedgehog (Shh) signaling in vertebrates by acting as a compartment for dynamic localization of pathway components, and finally, regulating the bifunctional Gli transcription factors. The cAMP activated protein kinase A (PKA) triggers partial proteolytic processing that generates Gli repressors in a cilia-dependent manner; however, pathways that promote PKA activation, and the exact function of cilia is unclear. We previously showed that the tubby family proteins Tulp3 and Tubby, serve as an anchor between ciliary IFT-A complex and phosphatidylinositol 4,5bisphosphate-rich membrane compartment in gating GPCRs to the cilia (Genes & Dev. 2010. 24: 2180-2193). We have recently identified an orphan ciliary GPCR, Gpr161 that acts as a negative regulator of Shh signaling during neural tube development by mediating the generation of Gli repressor via cAMP signaling. Active Shh signaling also results in removal of this receptor from the primary cilia (Cell 2013. 152: 210-223). Thus, Gpr161 is a candidate GPCR for activating PKA by generating a ciliary cAMP gradient, while also being regulated by Shh signaling in a positive feedback circuit. We are currently utilizing proteomic, cell biological and reverse genetic approaches to study distinct aspects of Gpr161 function, such as signaling, ciliary localization, and removal from cilia, and mechanistically investigating signaling in the context of intact cilia by uncoupling these functions. Dissecting mechanisms governing the regulation and signaling of Gpr161 in both cellular and organismal contexts, and extending these emerging principles to ciliary signaling, will reveal important insights into the role of cilia as signaling compartments.

Abstracts

Saikat Mukhopadhyay

Abstracts

Michelle Muza-Moons Research Fellow University of Michigan

Modulation of the Hedgehog pathway in the intestine affects basal inflammatory cytokine secretion and acute response to inflammatory challenge In the gastrointestinal tract, Hedgehog (Hh) ligands secreted from the epithelium act on targets in the underlying stroma; we have shown that these cellular targets include subsets of myeloid cells and that loss of one allele of Gli1 sensitizes mice to chemically 1 induced colitis . Here, we show that after infection with the bacterial pathogen Citrobacter +/lacZ rodentium, an extracellular microbe that causes colitis, Gli1 mice exhibited upregulation of IL-4 and IL-6, suggesting that Hh plays a role in inflammatory response to diverse challenges. Since Hh inhibitors are now being tested clinically as therapies for Hh-mediated cancers such as advanced basal cell carcinoma, we further tested whether administration of chemical inhibitors would also sensitize mice to DSS challenge. C57Bl6 mice were treated with LDE-225, an inhibitor very similar to Vismodegib that has been used in humans. After 16 days of treatment, LDE treated mice had deeper crypts (solvent 77.3 ± 2.1mm vs LDE 91.8 ± 2.0mm p