Probes for INS

Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.

Postnatal liver development is characterized by hepatocyte growth, proliferation and functional maturation. Notably, canonical Wnt signaling in hepatocytes has been identified as an important regulator of final adult liver size and metabolic liver zonation. The cellular origin of Wnt ligands responsible for homeostatic liver/body weight ratio remained unclear, which was also attributable to a lack of suitable endothelial Cre driver mice. To comprehensively analyze the effects of hepatic angiocrine Wnt signaling on liver development and metabolic functions, we used endothelial subtype-specific Stab2-Cre driver mice to delete Wls from hepatic endothelial cells (HEC). The resultant Stab2-Cretg/wt;Wlsfl/fl (Wls-HECKO) mice were viable but showed a significantly reduced liver/body weight ratio. Specifically, ablation of angiocrine Wnt signaling impaired metabolic zonation in the liver, as shown by loss of pericentral, β-catenin-dependent target genes such as Glutamine Synthase (Glul), RhBg, Axin2 and CYP2E1 as well as by extended expression of periportal genes such as Arginase 1 (Arg1). Furthermore, endothelial subtype-specific expression of a c-terminally YFP-tagged Wls fusion protein in Wls-HECKO mice (Stab2-Cretg/wt ;Wlsfl/fl;Rosa26:Wls-YFPfl/wt [Wls-rescue]) restored metabolic liver zonation. Interestingly, lipid metabolism was altered in Wls-HECKO miceexhibiting significantly reduced plasma cholesterol levels, while maintaining normal plasma triglyceride and blood glucose concentrations. On the contrary, zonal expression of Endomucin, LYVE1 and other markers of HEC heterogeneity were not altered in Wls-HECKO livers.

The heterogeneity of cellular states in cancer has been linked to drug resistance, cancer progression and the presence of cancer cells with properties of normal tissue stem cells. Secreted Wnt signals maintain stem cells in various epithelial tissues, including in lung development and regeneration. Here we show that mouse and human lung adenocarcinomas display hierarchical features with two distinct subpopulations, one with high Wnt signalling activity and another forming a niche that provides the Wnt ligand. The Wnt responder cells showed increased tumour propagation ability, suggesting that these cells have features of normal tissue stem cells. Genetic perturbation of Wnt production or signalling suppressed tumour progression. Small-molecule inhibitors targeting essential posttranslational modification of Wnt reduced tumour growth and markedly decreased the proliferative potential of lung cancer cells, leading to improved survival of tumour-bearing mice. These results indicate that strategies for disrupting pathways that maintain stem-like and niche cell phenotypes can translate into effective anti-cancer therapies.