[Google Scholar]Nusse R, and Clevers H (2017)

[Google Scholar]Nusse R, and Clevers H (2017). by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity. TH5487 Graphical Abstract In Brief Albrecht et al. show that macropinocytosis is TH5487 triggered by GSK3 inhibition or mutation of the tumor suppressor Axin1. Wnt-regulated macropinocytosis leads to acidification and FGD4 activation of catabolism in lysosomes, increasing the utilization of extracellular macromolecules to fuel cell growth. These findings have implications for novel therapies in cancer. INTRODUCTION It is currently emerging that canonical Wnt signaling affects a panoply of cell biological responses. Classically, Wnt growth factors lead to the stimulation of the transcriptional coactivator -catenin through the inhibition of a destruction complex containing glycogen synthase kinase 3 (GSK3), casein kinase 1 (CK1), and the adaptor proteins axin and adenomatous polyposis coli (APC) (MacDonald et al., 2009; Nusse and Clevers, 2017). Axin and APC are major tumor suppressors and their loss-of-function, which leads to increased nuclear -catenin, lies at the heart of cancers such as hepatocellular and colorectal carcinomas (Galluzzi et al., 2019; Nusse and Clevers, 2017). GSK3 has many protein substrates in addition to -catenin, and up to 20% of the human proteome contains three or more consecutive GSK3 consensus motifs (S/T-XXX-S/T) that form phosphodegrons (Taelman et al., 2010). The inhibition of GSK3 by Wnt signaling leads to the stabilization of many proteins in a phenomenon known as Wnt-stabilization of proteins (Wnt-STOP) (Acebron et al., 2014; Koch et al., 2015). Since Wnt signaling is maximal at the G2/M phase of the cell cycle, Wnt-STOP mediates an increase of cell size in preparation for cell division (Acebron et al., 2014). An important yet incompletely understood intersection exists between Wnt signaling and endosomal trafficking. When Wnt ligands bind to their co-receptors low-density lipoprotein (LDL)-receptor-related protein 6 (LRP6) and Frizzled (Fz), the destruction complex binds to the receptor complex and is endocytosed into signalosomes (Bilic et al., 2007). These are then trafficked into multivesicular bodies (MVBs) in late endosomes, causing the sequestration of GSK3 and axin from the cytosol into membrane-bounded organelles via microautophagy mediated by the ESCRT (endosomal sorting complexes required for transport) machinery (Taelman et al., 2010; Vinyoles et al., 2014; Colozza et al., 2020). To these effects of Wnt on cell physiology, one must now add the regulation of macropinocytosis. Cells can incorporate extracellular liquid-phase macromolecules by pinocytosis (Greek, in embryos. GSK3 requires the destruction complex to phosphorylate -catenin. To test the role of the destruction complex, we developed a system using Axin1 mutant hepatocellular carcinoma (HCC) cells (called Alexander cells, also known as PLC/PRF/5 cells), in which a mutation causes skipping of exon 4 containing the GSK3-binding sites (Satoh et al., 2000). We found that these HCC cells displayed constitutive actin-driven macropinocytosis, which could be suppressed by the reconstitution of full-length Axin1 at physiological levels. Together with SW480 colorectal carcinoma cells (CRCs) reconstituted with APC (Faux et al., 2004), Alexander HCC cells Axin1 provide a useful model to study the effect of the function of individual Wnt tumor suppressor proteins in cancer cells. From a metabolic standpoint, TH5487 Wnt-induced macropinocytosis caused a rapid increase in glucose, lactate, and some intracellular amino acid pools. Wnt3a treatment or Axin1 mutation resulted in a striking increase in lysosomal catabolism, marked by increased cathepsin D and -glucosidase enzyme activity. Similarly, GSK3 inhibition led to strikingly elevated endolysosomal activity that was blocked by macropinocytosis inhibitors, indicating that macropinocytosis drives lysosomal catabolism. The results suggest that GSK3 inhibition stimulates acute changes in macropinocytosis and lysosomal activity. RESULTS The Damage Complex Represses Macropinocytosis GSK3 activity is definitely advertised from the adaptor protein and tumor suppressor Axin1, which brings GSK3 in contact with substrates such as -catenin (Ikeda et al., 1998). To investigate the effect of loss of practical Axin1 on macropinocytosis, we used the Alexander HCC cell collection (Alexander et. al., 1976; Satoh et. al., 2000) that lacks full-length Axin1 and expresses a defective form of endogenous Axin1, in which exon 4 is definitely skipped, resulting in a shorter protein lacking the GSK3-binding website. This mutant cell collection provides an elegant system to study the relevance of the GSK3-Axin1 axis in damage complex activity. To generate stable reconstituted cell lines, Axin1 (FLAG-tagged).