Activation of Wnt signaling triggers macropinocytosis and drives many tumors. We now report that the exogenous addition of the second messenger lipid sn-1,2 DAG to the culture medium rapidly induces macropinocytosis. This is accompanied by potentiation of the effects of added Wnt3a recombinant protein or the glycogen synthase kinase 3 (GSK3) inhibitor lithium chloride (LiCl, which mimics Wnt signaling) in luciferase transcriptional reporter assays. In a colorectal carcinoma cell line in which mutation of adenomatous polyposis coli (APC) causes constitutive Wnt signaling, DAG addition increased levels of nuclear β-catenin, and this increase was partially inhibited by an inhibitor of macropinocytosis. DAG also expanded multivesicular bodies marked by the tetraspan protein CD63. In an in vivo situation, microinjection of DAG induced Wnt-like twinned body axes when co-injected with small amounts of LiCl into Xenopus embryos. These results suggest that the DAG second messenger plays a role in Wnt-driven cancer progression.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.