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A Step Forward in Signaling

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Thanks to systems biology screening approaches, scientists have identified an important link in the chain when it comes to the Wnt/beta-catenin signaling pathway and cancer. Composed of a complex network of interacting proteins and known to be involved in cancer, the pathway has so far eluded small molecule therapy. The recent work, which was published in the journal Nature in October, identified a small molecule, XAV939, that can prevent the degradation of axin, a key protein in the pathway, and thereby promote beta-catenin degradation through inhibiting another protein, tankyrase. Because the pathway is abnormally regulated in many cancers, including colorectal, finding out which molecule to aim for in this intricate cellular signaling pathway could lead to targeted Wnt pathway therapies.

"The Wnt pathway has been clearly implicated in cancer, especially colon cancer," says senior author Feng Cong at the Novartis Institutes for BioMedical Research. "The major issue here is that the Wnt pathway is not clearly druggable — there are no clear druggable targets in the pathway." Other contributors to the work include Cellzome and scientists within the department of systems biology at Harvard University.

Combining chemical genetics and proteomic screening approaches with mass spectrometry, Cong's team discovered the XAV939 small molecule, which effectively deactivates the pathway, leading to reduced transcription of beta-catenin-regulated genes. In normal cells, Wnt signaling leads to accumulation of beta-catenin in the cytosol, translocation to the nucleus, and transcription of Wnt genes. When the pathway is off, beta-catenin in the cytosol is instead phosphorylated and destroyed by the beta-catenin destruction complex. In many cancers, there is overactivation of the pathway. In this effort, scientists found that XAV939 stimulates beta-catenin degradation by stabilizing axin, one component of the degradation complex.

Starting with a high-throughput, cell-based screen, they found that XAV939 inhibits Wnt signaling activity in colon cancer cells using a luciferase reporter assay. Then they collaborated with Cellzome and, using a chemical proteomics approach, discovered that their target compound inhibits two proteins that normally associate with and degrade axin, tankyrase 1 and tankyrase 2. Cong says, "We get a compound that inhibits Wnt signaling, but we do not know the cellular target of the compound. So Cellzome made a linkable version of the compound, [and] basically conjugated the compound to beads." When the beads were incubated with protein lysate, the proteins that bound were possible targets. Mass spec revealed those proteins — including the poly(ADP-ribose) polymerases PARP1, PARP2, tankyrase 1, tan-kyrase 2, and several known PARP substrates. Using siRNA to knock out TNKS1 and TNKS2, they saw that the protein levels of axin 1 and 2 increased. Loss-of-function experiments also showed that depleting TNKS1 and TNKS2 increased beta-catenin phosphorylation, decreased beta-catenin levels, and inhibited the transcription of beta-catenin target genes in colorectal cancer cells.

Axin1 and axin 2 "are well-known regulators of the Wnt pathway," Cong says. "They bind to several other proteins of the pathway and form the beta-catenin degradation complex." The concentration of axin is limiting, so if you increase the concentration of axin, you increase the concentration of the degradation complex and "you're going to inhibit Wnt signaling," he says. "Our work shows that tankyrase 1 and tankyrase 2 regulate the protein stability of axin."
As for clinical applications, Cong says this is a first step toward developing a cancer drug that targets the Wnt signaling pathway. "We have an early hit, at least," he says. "We're still trying to figure out the biology around tankyrase and evaluate [XAV939] as a cancer target."