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

Wnt/β-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 β-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.

Wang Z ，Tacchelly-Benites O ，Yang E ，Thorne CA ，Nojima H ，Lee E ，Ahmed Y ... -  《-》

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein β-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the β-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate β-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases β-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.

Croy HE ，Fuller CN ，Giannotti J ，Robinson P ，Foley AVA ，Yamulla RJ ，Cosgriff S ，Greaves BD ，von Kleeck RA ，An HH ，Powers CM ，Tran JK ，Tocker AM ，Jacob KD ，Davis BK ，Roberts DM ... -  《-》

A Context-Dependent Role for the RNF146 Ubiquitin Ligase in Wingless/Wnt Signaling in Drosophila.

Aberrant activation of the Wnt signal transduction pathway triggers the development of colorectal cancer. The ADP-ribose polymerase Tankyrase (TNKS) mediates proteolysis of Axin-a negative regulator of Wnt signaling-and provides a promising therapeutic target for Wnt-driven diseases. Proteolysis of TNKS substrates is mediated through their ubiquitination by the poly-ADP-ribose (pADPr)-dependent RING-domain E3 ubiquitin ligase RNF146/Iduna. Like TNKS, RNF146 promotes Axin proteolysis and Wnt pathway activation in some cultured cell lines, but in contrast with TNKS, RNF146 is dispensable for Axin degradation in colorectal carcinoma cells. Thus, the contexts in which RNF146 is essential for TNKS-mediated Axin destabilization and Wnt signaling remain uncertain. Herein, we tested the requirement for RNF146 in TNKS-mediated Axin proteolysis and Wnt pathway activation in a range of in vivo settings. Using null mutants in Drosophila, we provide genetic and biochemical evidence that Rnf146 and Tnks function in the same proteolysis pathway in vivo Furthermore, like Tnks, Drosophila Rnf146 promotes Wingless signaling in multiple developmental contexts by buffering Axin levels to ensure they remain below the threshold at which Wingless signaling is inhibited. However, in contrast with Tnks, Rnf146 is dispensable for Wingless target gene activation and the Wingless-dependent control of intestinal stem cell proliferation in the adult midgut during homeostasis. Together, these findings demonstrate that the requirement for Rnf146 in Tnks-mediated Axin proteolysis and Wingless pathway activation is dependent on physiological context, and suggest that, in some cell types, functionally redundant pADPr-dependent E3 ligases or other compensatory mechanisms promote the Tnks-dependent proteolysis of Axin in both mammalian and Drosophila cells.

Wang Z ，Tacchelly-Benites O ，Noble GP ，Johnson MK ，Gagné JP ，Poirier GG ，Ahmed Y ... -  《-》

Dual Roles for Membrane Association of Drosophila Axin in Wnt Signaling.

Wang Z ，Tacchelly-Benites O ，Yang E ，Ahmed Y ... -  《PLoS Genetics》

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

Wnt/β-catenin signaling controls intestinal stem cell (ISC) proliferation, and is aberrantly activated in colorectal cancer. Inhibitors of the ADP-ribose polymerase Tankyrase (Tnks) have become lead therapeutic candidates for Wnt-driven cancers, following the recent discovery that Tnks targets Axin, a negative regulator of Wnt signaling, for proteolysis. Initial reports indicated that Tnks is important for Wnt pathway activation in cultured human cell lines. However, the requirement for Tnks in physiological settings has been less clear, as subsequent studies in mice, fish and flies suggested that Tnks was either entirely dispensable for Wnt-dependent processes in vivo, or alternatively, had tissue-specific roles. Here, using null alleles, we demonstrate that the regulation of Axin by the highly conserved Drosophila Tnks homolog is essential for the control of ISC proliferation. Furthermore, in the adult intestine, where activity of the Wingless pathway is graded and peaks at each compartmental boundary, Tnks is dispensable for signaling in regions where pathway activity is high, but essential where pathway activity is relatively low. Finally, as observed previously for Wingless pathway components, Tnks activity in absorptive enterocytes controls the proliferation of neighboring ISCs non-autonomously by regulating JAK/STAT signaling. These findings reveal the requirement for Tnks in the control of ISC proliferation and suggest an essential role in the amplification of Wnt signaling, with relevance for development, homeostasis and cancer.

Wang Z ，Tian A ，Benchabane H ，Tacchelly-Benites O ，Yang E ，Nojima H ，Ahmed Y ... -  《-》