Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase

Abstract

Wnt/b-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multiprotein "destruction complex," assembled by the scaffold protein Axin, targets the key transcriptional activator b-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. Here, we use an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. We find that for nearly all Wingless-driven developmental processes, a three-to fourfold increase in Axin is insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, we find that both the tumor suppressor adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and we define separable domains in Axin that are important for APC-or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation.

Axin proteolysis by Iduna is required for the regulation of stem cell proliferation and intestinal homeostasis in Drosophila

“…Although this may seem somewhat surprising, it is consistent with inactivation of Tnks in Drosophila, which also causes no overt abnormalities (Feng et al, 2014;Wang et al, 2016a and2016b;Yang et al, 2016). Like for Iduna, Tnks mutants have no obvious effects on wing development and the expression of Wingless target genes in larval wing discs, despite the fact that Axin levels are increased (Feng et al, 2014;Wang et al, 2016a and2016b;Yang et al, 2016). Our interpretation of these findings is that most tissues can tolerate relative modest (2-3fold) changes of Axin.…”

“…The Wingless pathway is also involved in adult tissue self-renewal in Drosophila (Lin et al, 2008). Genetic depletion of proteins in the Wingless pathway, such as Tcf, arr, dsh and pygo, leads to inhibition of Wingless signaling activation which in turn causes over-proliferation of stem cells in the Drosophila midgut (Kramp et al, 2002;Wang et al, 2016a and2016b;Tian et al, 2016). However, inactivation of Wnt signaling in the small intestine of mice decreases the proliferative potential of stem cells (Fevr et al, 2007;Korinek et al, 1998).…”

“…The principal components of this complex are adenomatous polyposis coli (APC), mice are overall normal; however, double knock-out of Tnks1/2 causes early embryonic lethality, which indicates their redundancy in mouse development (Hsiao et al, 2006;Chiang et al, 2008). On the other hand, inactivation of the single Drosophila Tnks gene produces viable flies that have slightly increased Axin levels but no overt defects (Wang et al, 2016a and2016b;Feng et al, 2014;Yang et al, 2016;Tian et al, 2016). Therefore, the exact physiological function of TNKS and Iduna-mediated regulation of Wnt-signaling remains to be determined.…”

Dual Roles for Membrane Association of Drosophila Axin in Wnt Signaling

“…Fas III staining confirmed that the morphology of cells in Apc2 or Apc1 clones was the same as the neighboring wild-type cells (Fig 2M and 2R), indicating that the decreased Axin localization to puncta at the cell periphery was not a secondary consequence of disrupted cell morphology. Importantly, previous genetic and biochemical studies had demonstrated that endogenous Axin is not destabilized as a consequence of Apc loss [16]; therefore, the decreased localization of Axin in peripheral puncta is not secondary to decreased Axin levels in Apc mutant cells. Based on these results, we conclude that Apc promotes Axin localization to puncta at the cell periphery in the unstimulated state.…”

The ADP-ribose polymerase Tankyrase regulates adult intestinal stem cell proliferation during homeostasis in Drosophila

“…Moreover, the efficacy of Tnks inhibitors could reflect an essential function for Tnks in the amplification of pathway activity in Wnt-driven cancers. Depletion of Tnks has no obvious effects on wing development or the expression of Wingless target genes in larval wing discs unless Axin levels are raised above the endogenous level (Feng et al, 2014;Wang et al, 2016). The threshold at which Axin levels inhibit Wingless signaling in wing discs is between 3-and 9-fold above the endogenous Axin level ) and more than 3-fold in embryos .…”