Wnt canonical pathway activates macropinocytosis and lysosomal degradation of extracellular proteins

Abstract

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Wnt treatment markedly increased the endocytosis and degradation in lysosomes of BSA. In this study, we report that in addition to receptor-mediated endocytosis, Wnt also triggers the intake of large amounts of extracellular fluid by macropinocytosis, a nonreceptor-mediated actin-driven process. Macropinocytosis induction is rapid and independent of protein synthesis. In the presence of Wnt, large amounts of nutrient-rich packages such as proteins and glycoproteins were channeled into lysosomes after fusing with smaller receptor-mediated vesicles containing glycogen synthase kinase 3 (GSK3) and protein arginine ethyltransferase 1 (PRMT1), an enzyme required for canonical Wnt signaling. Addition of Wnt3a, as well as overexpression of Disheveled (Dvl), Frizzled (Fz8), or dominant-negative Axin induced endocytosis. Depletion of the tumor suppressors adenomatous polyposis coli (APC) or Axin dramatically increased macropinocytosis, defined by incorporation of the high molecular weight marker tetramethylrhodamine (TMR)-dextran and its blockage by the Na+/H+ exchanger ethylisopropyl amiloride (EIPA). Macropinocytosis was blocked by dominant-negative vacuolar protein sorting 4 (Vps4), indicating that the Wnt pathway is dependent on multivesicular body formation, a process called microautophagy. SW480 colorectal cancer cells displayed constitutive macropinocytosis and increased extracellular protein degradation in lysosomes, which were suppressed by restoring full-length APC. Accumulation of the transcriptional activator β-catenin in the nucleus of SW480 cells was inhibited by methyltransferase inhibition, EIPA, or the diuretic amiloride. The results indicate that Wnt signaling switches metabolism toward nutrient acquisition by engulfment of extracellular fluids and suggest possible treatments for Wnt-driven cancer progression.

Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

“…Wnt activation is known to induce endocytosis of the receptor complex (11,12). We confirmed that Lrp6 endocytosis was observed in SW480, a colon cancer cell line in which the absence of APC triggered, and APC reconstitution inhibited, Wnt signaling and Lrp6 endocytosis (SI Appendix, Movie S1); this is in agreement with recent results showing that APC is a major regulator of endocytosis (16,29). As expected for regulators of Lrp6 endocytosis, the Wnt antagonists Dkk1 (30) or Bighead (31) induced endocytosis of Lrp6-APEX2 into large vesicle clusters in the cytoplasm next to the nuclear bay region, where the lysosomal system is located Fig.…”

“…Here, we describe the results obtained with a Human Embryonic Kidney 293T (HEK293T) cell line stably expressing a chimeric Lrp6-APEX2 receptor. Our proteomic data indicate that in presence of Wnt3a, the Lrp6-interactome was enriched in components of the ESCRT machinery that forms MVBs, providing independent support to our previous findings that endocytosis is at the core of Wnt pathway intracellular regulation (11,15,16). We also discovered that Tropomyosin-receptor kinase fused gene (Trk-fused gene, TFG), a protein involved in several pathologies including cancer (26), was a highly enriched target of Wntactivated Lrp6-APEX2 and localized to endosomal vesicles.…”

“…During the 30 years since its discovery (13,14), the Wnt pathway has been the subject of intense studies, but its molecular mechanisms have not yet been fully elucidated, in particular the role of membrane trafficking in receptor activation (11,12,(15)(16)(17). To dissect the dynamics of Wnt signaling, we decided to use a protein interaction approach to identify additional regulators of the pathway.…”

ATM inhibition drives metabolic adaptation via induction of macropinocytosis

“…Next we aimed to determine the mechanism of macropinocytosis induction downstream of ATM inhibition. Towards this goal, we used pharmacological or genetic inhibition of PI3K/AKT, WNT, and AMPK, three known inducers of macropinocytosis (Commisso et al, 2013;Kim et al, 2018;Redelman-Sidi et al, 2018;Tejeda-Munoz et al). While inhibition of PI3K, AKT, or WNT had no effect ( Fig.…”

“…Macropinocytosis is an endocytic process whereby cells take up fluid, macromolecules, metabolites, and other cargo from the surrounding microenvironment (King and Kay, 2019;Palm, 2019;Zhang and Commisso, 2019). Many studies have evaluated the critical role of macropinocytosis as a nutrient scavenging mechanism in cancers under nutrientdeprived conditions (Commisso et al, 2013;Davidson et al, 2017;Hodakoski et al, 2019;Kamphorst et al, 2015;Kim et al, 2018;Redelman-Sidi et al, 2018;Tejeda-Munoz et al;Yao et al, 2019). To date, these studies have focused on cancers with high PI3K activity, such as those with mutant RAS or PTEN loss, which act upstream of the Rac1-Pak1 actin remodeling pathway to promote macropinosome formation .…”