New Tool in the Fight Against Colon Cancer
By: Carol A. Rouzer, VICB Communications
Published: November 1, 2010
The Ethan Lee lab’s discovery that the anti-helminthic drug pyrvinium blocks Wnt pathway signaling is a promising new lead for developing drugs against colon cancers that often depend on this pathway.
β-Catenin is a multi-functional protein specialized for protein-protein interactions. It plays a critical role in forming the adherens junctions, that help “glue” cells together in epithelial layers. It also plays a role as a transcription factor, regulating the expression of genes during development. Considering the importance of β-catenein’s diverse functions, it is not a surprise that levels of this protein are tightly regulated. Under basal conditions, β-catenin that is not involved in adherens junction formation is bound in a destruction complex with other proteins, including Axin, glycogen synthase kinase-3 (GSK-3), adenomatous polyposis coli (APC) and casein kinase-1α (CK1α). The close association of the proteins in this complex allows CK1α to phosphorylate β-catenin, making it susceptible to further phosphorylation by GSK-3. These phosphorylations mark β-catenin to be degraded by the proteasome system, thus keeping its levels in the cell cytosol low.
Figure 1. How Wnt signaling regulates β-catenin-dependent transcription control. (Left) Normally β-cateinin is bound in a complex containing Axin, APC, and GSK-3β. Close association with GSK-3β leads to phosphorylation of β-catenin, which marks it for degradation. (Right) When Wnt binds to LRP5 and/or LRP6 and Frizzled, the complex activates Disheveled (Dsh), leading to repression of GSK-3β. As a result β-catenin is not phosphorylated and degraded, but can accumulate in the cytosol and then move to the nucleus, where it works in concert with LEF/TCF, BCL9, and pygopus to regulate gene transcription.
When β-catenin is needed to stimulate gene transcription, it must be freed from the destruction complex. This occurs through the Wnt signaling pathway initiated when the Wnt protein binds to its receptor Frizzled. Activated Frizzled works together with Disheveled and LRP5 and/or LRP6 (low density lipoprotein receptor-related protein 5 and/or 6) to down-regulate GSK-3, with a concomitant reduction in β-catenin phosphorylation and degradation. β-catenin accumulates in the cytosol and is transported to the nucleus where it works together with LEF (lymphoid enhancer factor) and/or TCF (T-cell factor), BCL9 (B-cell CLL/lymphoma 9), and Pygopus to regulate the transcription of proteins that contain TCF/LEF binding sites in their promotors (Figure 1).
The importance of β-catenin to normal epithelial cell function became apparent upon the discovery that abnormalities in Wnt signaling that lead to increased levels of β-catenin-mediated gene transcription occur in over 80% of sporadic colon cancers. This led VICB investigator Ethan Lee and his colleagues to search for modulators of Wnt signaling. [C. A. Thorne et al. (2010) Nature Chem. Biol., published online October 3, DOI: 10.1038/nchembio.453].
To aid in their quest, the Lee lab developed a high-throughput screen for modulators of Wnt signaling. The screen used extracts of Xenopus laevis eggs, which contain all of the components necessary to reproduce Wnt signaling. The investigators added Renilla reniformis luciferase-tagged Axin and firefly luciferase-tagged β-catenin plus a soluble form of LRP6 (LRP6ICD) to serve as a pathway stimulator. They then measured the luminescence produced by each luciferase to monitor the levels of the tagged proteins. As a stimulator of Wnt signaling, LRP6ICD led to an increase in β-catenin and a decrease in Axin levels. Compounds that blocked these changes were designated Wnt pathway inhibitors, while those that augmented the changes were Wnt pathway stimulators. A total of 20 such compounds were found during screening of a 2160 compound library. Of these, the most potent Wnt signaling inhibitor was the pamoate salt of pyrvinium, a known anti-helminthic drug (Figure 2).
Figure 2. Structure of pyrvinium.
The Lee lab quickly confirmed that pyrvinium blocked Wnt signaling in intact human embryonic kidney (HEK) 293 cells. They went on to demonstrate that pyrvinium inhibits Wnt pathway-mediated developmental processes in Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans. These findings confirmed pyrvinium’s efficacy in intact organisms of multiple species.
These exciting initial findings led to the next obvious question - how does pyrvinium work? In vitro studies using a complex of GSK3, Axin, CK1α, and β-catenin showed that pyrvinium could stimulate β-catenin phosphorylation. Further investigations revealed that pyrvinium binds directly to CK1α and stimulates its activity. Since CK1α-mediated phosphorylation of β-catenin increases phosphorylation by GSK-3, activation of this kinase could explain pyrvinium’s effects in the in vitro assay. CK1α-mediated phosphorylation of Axin also increased in the presence of pyrvinium. Further evidence that CK1α is the target of pyrvinium came from studies of pyrvinium analogs. Compounds that blocked Wnt signaling also augmented CK1α activity and vice versa. In addition, shRNA-mediated suppression of CK1α expression in Jurkat cells led to insensitivity to pyrvinium’s effects on Wnt pathway signaling.
Figure 3. Mechanism of pyrvinium action. Pyrvinium activates CK1α, leading to up-regulation of Axin and down-regulation of β-catenin. CK1α also down-regulates Pygopus. Together the effects lead to a blockade of Wnt pathway signaling.
Having identified CK1α as the target of pyrvinium, the Lee lab quickly hypothesized that pyrvinium blocked Wnt signaling by increasing phosphorylation, and hence degradation, of β-catenin (Figure 3). Therefore, they were puzzled when they found that pyrvinium could effectively block Wnt signaling even in a cell line that expresses a nondegradable β-catenin protein. This led them to turn to studies of β-catenin’s partners in nuclear transcription regulation, which revealed that pyrvinium induces a reduction of Pygopus levels. This effect did not occur in cells not expressing CK1α, and the finding that CK1α and Pygopus coimmunoprecipitate suggests that, once again, CK1α may be the target that mediates pyrvinium’s effects. Since Pygopus is required for β-catenin-mediated gene transcription regulation, its reduction in the presence of pyrvinium explains how the compound can block Wnt pathway signaling, even in the presence of a nondegradable form of β-catenin.
Together the findings suggest that pyrvinium activates CK1α, leading to increased degradation of both β-catenin and Pygopus, thereby blocking Wnt signaling at two different levels of the pathway (Figure 3). In light of these results, the Lee lab wasted no time in testing pyrvinium’s ability to block the growth of colon cancer cells. They found that, indeed, pyrvinium is highly effective, but only in cells that depend on heightened Wnt signaling. The pervasive role of Wnt pathway derangements in colon cancer suggests that pyrvinium represents an exciting lead compound for new therapeutic agents against this all too common disease.