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New Approach to Cancer Therapy Targets the Hedgehog Signaling Pathway

 

By: Carol A. Rouzer, VICB Communications
Published:  April 13, 2015

 

 

A high-throughput screen identifies an inhibitor of zebrafish development that targets an important pathway for cancer cell growth and survival.

 

The Hedgehog signaling pathway is a complex network that plays a critical role in embryonic development, stem cell biology, and tissue homeostasis. Aberrant Hedgehog signaling is a characteristic of some forms of cancer, leading to the hypothesis that drugs targeting members of this pathway might be effective chemotherapeutic agents. However, vismodegib, the only Hedgehog-directed drug that is currently approved for treatment of cancer, has produced disappointing results in the clinic. This has led to the quest for more efficacious Hedgehog pathway inhibitors. Now, Vanderbilt Institute of Chemical Biology member Chaz Hong reports eggmanone, a new Hedgehog pathway inhibitor that suppresses signaling by blocking phosphodiesterase 4 [C. H. Williams, et al. (2015) Cell Rep., published online April 7, DOI: 10.1016/j.celrep.2015.03.001].

 

The Hedgehog pathway (Figure 1) was first discovered through studies of mutations in Drosophila melanogaster. These mutations produced developmental abnormalities that resulted in fly embryos with hair-like bristles. The resemblance of the bristles to those of a Hedgehog led to the pathway’s name. In mammals, three soluble ligands, Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh) play various roles during the development of different tissues. All three of these ligands bind to Patched, a transmembrane receptor that represses the pathway by blocking the activity of the G protein-coupled receptor Smoothened. Binding of a Hedgehog ligand to Patched releases Smoothened, allowing it to translocate to the primary cilium of the cell. There, Smoothened acts to facilitate the processing of the Gli family of transcription factors which, in mammals, comprise three members, Gli1, Gli2, and Gli3. The Gli proteins exist in a full-length form that promotes transcription of target genes and a shortened form that represses transcription. Smoothened facilitates translocation of full-length Gli proteins to the nucleus and suppresses proteolysis to the repressor form. The result is an increase in the transcription of genes targeted by the Hedgehog pathway (Figure 1).

 

 

 


Figure 1.
A simplified outline of the Hedgehog signaling pathway. Binding of Sonic Hedgehog (Shh) to Patched releases Smoothened from repression by Patched. Smoothened translocates to the primary cilium where it mediates release of Gli transcription factors from Sufu. The free Gli proteins can be phosphorylated by cAMP-dependent protein kinase A (PKA), which is bound to A kinase anchoring protein (AKAP) at the base of the cilium. This phosphorylation inhibits translocation of the full-length (GliFL) transcription factors to the nucleus and/or promotes proteolysis of the transcription factors to a repressive form (GliR). The result is a reduction of Gli-modulated transcription and a suppression of Hedgehog-associated signaling. Hydrolysis of cAMP by phosphodiesterase 4 (PDE4) leads to inactivation of PKA and a reduction in Gli phosphorylation. This results in an increase in Gli-modulated transcription and Hedgehog-associated signaling. Reproduced from C. H. Williams, et al. (2015) Cell Rep., published online April 7, DOI: 10.1016/j.celrep.2015.03.001 under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

 

The importance of Hedgehog signaling in development led the Hong laboratory to use a high-throughput zebrafish embryo screen to search for compounds that produce defects characteristic of those found in Hedgehog-null embryos. Among these are a ventral tail curvature, small eyes, absent pectoral fins, enlarged and rounded somites, and impaired slow muscle formation (Figure 2). Screening of a 30,000 member library yielded a series of small molecules with similar structures that produced the characteristic Hedgehog-null phenotype. The investigators chose a representative member of this series eggmanone (Figure 3) for further studies.

 

 


Figure 2.
Examples of developmental abnormalities resulting from inhibition of Hedgehog signaling in zebrafish embryos. (Left) A normal embryo showing a straight ventral tail, large eyes, and pectoral fins (arrows). (Right) An embryo treated with eggmanone, showing a ventral curvature, small eyes, and the absence of pectoral fins (asterisks). Reproduced from C. H. Williams, et al. (2015) Cell Rep., published online April 7, DOI: 10.1016/j.celrep.2015.03.001 under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

 

 

                

Figure 3. Structure of eggmanone. Reproduced from C. H. Williams, et al. (2015) Cell Rep., published online April 7, DOI: 10.1016/j.celrep.2015.03.001 under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

 

 

It is well established that Hedgehog signaling increases the expression of Patched. Consistently, the Hong lab researchers found that treatment of zebrafish embryos with eggmanone resulted in low levels of Patched in areas, such as the pectoral fin fields, where it is normally found. They further evaluated the effects of eggmanone on Hedgehog-dependent gene expression in Shh-Light2 cells, which stably express a luciferase gene coupled to a Gli-responsive promoter. They found that the increase in luciferase expression observed in response to Shh treatment was inhibited by eggmanone in a concentration-dependent manner. The effects of eggmanone were reversed by overexpression of the Gli2 transcription factor. These results confirmed that eggmanone inhibits the Hedgehog signaling pathway.

 

Eggmanone inhibited luciferase expression in Shh-Light 2 cells responding to purmorphamine, a direct agonist of Smoothened. Eggmanone did not compete with BODIPY-labeled cyclopamine, a Smoothened antagonist, for binding to Smoothened. Eggmanone was able to inhibit constitutive Hedgehog signaling in Sufu-/- cells. Sufu is a Gli transcription factor-binding protein that suppresses Gli-mediated transcription in the absence of activated Smoothened (Figure 1). Together, these results indicated that eggmanone acts downstream of Smoothened and its effects on Sufu.

 

Having ruled out the possibility that eggmanone inhibits Hedgehog signaling by interacting with Smoothened, the investigators focused on the later steps of the pathway, which occur at the primary cilium. Treatment of mouse embryo fibroblasts with the Smoothened agonist SAG resulted in transport of Gli2 to the proximal tip of the primary cilium. This was not blocked by eggmanone. However, eggmanone prevented SAG-dependent transport of Gli2 to the nucleus and suppression of Gli3 cleavage to its repressor form. These data indicated that eggmanone works upstream of Gli processing.

 

Phosphorylation of Gli transcription factors by protein kinase A suppresses the transport of full-length transcription factors to the nucleus and promotes their proteolysis to repressor forms. These effects are similar to those observed in the presence of eggmanone, leading the investigators to hypothesize that the compound acts at the level of protein kinase A. Consistent with this hypothesis, they found that eggmanone treatment of SAG-stimulated NIH 3T3 cells resulted in activation of protein kinase A at the basal body of the primary cilium (Figure 4). Since activation of protein kinase A depends on the presence of cAMP, this finding suggested that eggmanone might act by stimulating cAMP production or inhibiting its degradation. Indeed, eggmanone exhibited a strong and selective inhibition of phosphodiesterase 4D3, a cAMP hydrolytic enzyme. Furthermore, overexpression of this enzyme increased Hedgehog signaling and partially reversed the effects of eggmanone in NIH 3T3 cells while a nonspecific inhibitor of phosphodiesterases reduced Hedgehog signaling.

 

 


Figure 4.
Eggmanone treatment results in activation of protein kinase A at the base of the primary cilium. NIH 3T3 cells are stained by immunofluroescence for the cilia marker Arl13B (green), and for the phosphorylated active form of protein kinase A (red). In untreated cells (left) and cells treated with the Smoothened activator SAG alone (center), there is no association between the two markers. However, cells treated with SAG in the presence of eggmanone demonstrate a bright red fluorescence in the vicinity of the green cilia marker, indicating active protein kinase A in the vicinity of the cilium. The white box in each case shows a magnified view of a representative cilium. Reproduced from C. H. Williams, et al. (2015) Cell Rep., published online April 7, DOI: 10.1016/j.celrep.2015.03.001 under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

 

 

Together the results demonstrate that eggmanone suppresses Hedgehog signaling. Its mechanism of action appears to be inhibition of phosphodiesterase 4D3, resulting in an accumulation of cAMP, and subsequent activation of protein kinase A. Prior attempts to modulate the Hedgehog pathway have focused primarily on Smoothened, and the clinically approved vismodegib is a Smoothened inhibitor. However, resistance of cancer cells to Smoothened inhibitors, either through mutation of the Smoothened binding site, or mutation of proteins downstream of Smoothened, has impeded the clinical success of these compounds. The discovery of a Hedgehog pathway inhibitor with a novel mechanism of action opens new possibilities for therapeutic interventions targeting this pervasive signaling pathway.

 

 

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