Abstract
Signaling cascades depend on scaffold proteins that regulate the assembly of multiprotein complexes. Missense mutations in scaffold proteins are frequent in human cancer, but their relevance and mode of action are poorly understood. Here we show that cancer point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth in vivo through a gain-of-function mechanism. The effect is conserved for both the human and Drosophila proteins. Mutated Axin forms nonamyloid nanometer-scale aggregates decorated with disordered tentacles, which 'rewire' the Axin interactome. Importantly, the tumor-suppressor activity of both the human and Drosophila Axin cancer mutants is rescued by preventing aggregation of a single nonconserved segment. Our findings establish a new paradigm for misregulation of signaling in cancer and show that targeting aggregation-prone stretches in mutated scaffolds holds attractive potential for cancer treatment.
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Acknowledgements
We thank members of the laboratories of M.M.M. and S.G.D.R. for experimental support, helpful discussions and suggestions. We thank B. Kleizen and I. Braakman (Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Utrecht University) for providing the CFTR ΔF508 construct and H. Bellen (Baylor College of Medicine), I. Duncan and D. Duncan (Washington University) for antibodies. This work was supported by the European Research Council ((ERC) starting grant 242958 to M.M.M. and ERC advanced grant 294523 to J.-P.V.); Utrecht University (High Potential Grants to M.M.M. and S.G.D.R.); Boehringer Ingelheim Fonds (PhD fellowship to E.C.v.K.); the European Union (Framework Programme (FP) 7 Marie Curie ITN 608180 'WntsApp' to M.M.M. and S.G.D.R., FP7 Marie Curie ITN-IDP 317371 'ManiFold' to S.G.D.R., FP6 Marie Curie Excellence Grant 25651 'chaperoning cascades' to S.G.D.R. and BioNMR project 261863 to R.B. and T.M.); the Netherlands Organization for Scientific Research ((NWO) VICI grant to M.M.M., Vidi career development grant to S.G.D.R. and instrumentation support for a TCI probe to R.B.); the Internationale Stichting Alzheimer Onderzoek ((ISAO) grant to S.G.D.R.); the Medical Research Council of Great Britain (grant U117584268 to J.P.V.); the European Molecular Biology Organization ((EMBO) ALTF 983-2009 to H.N.); the Uehara and Kanae Foundations (to H.N.); the Austrian Academy of Sciences (APART-fellowship to T.M.); the Bavarian Ministry of Sciences, Research and the Arts in the framework of the Bavarian Molecular Biosystems Research Network (to T.M.); and the German Research Foundation (Emmy Noether program MA 5703/1-1 to T.M.). T.Y.L. and A.J.R.H. were supported by the NWO embedded roadmap program Proteins@Work (project 184.032.201) and PRIME-XS, grant number 262067, funded by the European Union FP7. We thank the Deutsches Elektronen Synchrotron (DESY) synchrotron radiation facilities for support of the SAXS data collection and B. Demeler for providing a license for the UltraScan software package.
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Z.A., H.N., E.C.v.K., J.-P.V., S.G.D.R. and M.M.M. conceived and designed the experiments. Z.A., H.N., E.C.v.K., T.B., M.S., I.J., M.V., T.Y.L., R.C.v.S., I.K. and K.R. performed the experiments. Z.A., H.N., E.C.v.K., I.J., T.Y.L., M.V., T.M., R.B., A.J.R.H., J.-P.V., S.G.D.R. and M.M.M. analyzed the data. Z.A., J.-P.V., E.C.v.K., S.G.D.R. and M.M.M. wrote the manuscript. The other authors commented on the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Alignment of Axin RGS domains and structural consequences of mutation-induced destabilization.
(a) Alignment of Axin RGS domain amino acid sequences of Homo sapiens and other indicated species. Mutated residues found in cancer patients are indicated in red and show full, partial or no conservation. (b) CD spectra of purified wild-type (WT) and mutant RGS domains are shown at the indicated temperatures. At low temperatures all CD spectra, with the exception of RGS-L106R, exhibit typical α-helical profiles showing two peak minima at 208 and 220 nm and a peak maximum at 190 nm. For each of these mutants, loss of α-helical structure is shown at or above the unfolding temperature that were calculated from fluorescence-based thermal denaturation (Fig. 1f). CD spectra of RGS-L106R do not display α-helical content and remain unaffected by temperature change. Results represent two independent experiments. (c) 2D NMR spectra showing signals of RGS-L106R. As compared to the 2D NMR spectrum of RGS-wt (Fig. 3b), Trp side chain signals (purple squares) as well as signals from folded regions (grey squares) are lost for RGS-L106R, indicating loss of regular 3D structure. By contrast, signals from disordered termini of the protein remain in place (blue squares).
Supplementary Figure 2 Characterization of Axin L106R aggresome-like structures and analysis of SDS solubility.
(a) Quantification of the percentage of Axin-L106R expressing cells carrying an aggresome-like structure. Per field >50 cells were counted. Error bars represent SDs of 7 fields (representing >350 cells) per condition. (b) γ-tubulin staining of cells expressing Axin-wt, Axin-L106R and Axin-L106R F119R reveals that a fraction of Axin-L106R and Axin-L106R F119R (Axin-L106R (F>R) proteins accumulate in a pericentriolar structure. Scale bars 10 μm. (c) Pericentriolar accumulations of Axin-L106R do not display enrichment of HDAC6 or vimentin, in contrast to previously described aggresomes formed by mutant CFTR-ΔF508 protein29. Scale bars 10 μm. (d) Axin-L106R is fully soluble in SDS-based lysis buffer (SDS), similar to the Axin-wt (WT) protein. SDS-resistant pellet fractions (dissolved in formic acid (FA)) were devoid of Axin protein. Actin control is shown for comparison.
Supplementary Figure 3 Excess Axin WT suppresses Axin L106R–induced β-catenin–mediated transcription.
Wnt luciferase reporter activity in HEK293T cells co-expressing Axin-L106R with increasing doses of Axin-wt protein. Axin-L106R induces basal β-catenin mediated transcription. Co-expression of increasing amounts of Axin-wt protein levels suppresses Axin-L106R induced β-catenin mediated transcription. Graph shows average (bars) and range (diamonds) of luc activity in duplicate cell cultures.
Supplementary Figure 4 Analysis of Axin L106R F119R’s structural stability and dose-response effects on tumor-suppressor activity in cells.
(a) Fluorescence-based thermal denaturation of RGS-L106R F119R at 340 nm emission. Tu, unfolding temperature. (b) Introduction of the F119R mutation into Axin-wt does not affect tumour suppressor activity. Graph shows average (bars) and range (diamonds) of luc activity in duplicate cell cultures. (c) Introduction of the F119R mutation into Axin-L106R rescues tumour suppressor activity. Wnt luciferase reporter activity was measured in HEK293T cells expressing increasing concentrations (6.25, 12.5, 25, 50, 100 and 200 ng of transfected DNA, respectively) of the indicated Axin variants. Graph shows average (bars) and range (diamonds) of luc activity in duplicate cell cultures. (d) Western blot indicating protein levels of Axin-wt (WT), Axin-L106R, rescue variants Axin-L106R F119R (F>R), Axin-L106R W118R F119R (WF>RR), and Axin-ΔRGS upon transfection of HEK293T cells with 100 ng of plasmid DNA. Ctr, control.
Supplementary Figure 5 STRING v10 analysis of lost and gained binders of Axin-variant interactomes.
(a) Interactome (grey dots) of Axin-wt (red dot) is shown. Shown interactors displayed a confidence score of >0.900 and interaction with at least one other partner in the network. Binding partners are clustered (grey circles) based on previously described protein-protein interactions and on shared activity in the regulation of cellular processes as revealed by GOTERM analysis. Loss of binding (blue dots) is indicated for (b) Axin-ΔRGS, (c) Axin-L106R and (d) Axin-L106R F119R. (e) Gained interactions (green dots) of Axin-L106R are shown, based on comparison with the Axin-wt interactome in (a). Partners that were trapped by Axin-L106R and set free by Axin-L106R F119R are indicated (yellow dots).
Supplementary Figure 6 Ectopic wingless signalling induced by Axin cancer-mutant expression in Drosophila wing discs is rescued by aggregon mutation.
Related to Figure 6. (a) Schematic presentation of gene targeting strategy to generate Axin knock-in flies. (b) Confirmation of genetic modification of Axin knock-out mutant flies. (c) Quantification of the average clone size of the P compartment occupied by GFP-positive tissue in different genotype (n = 5). Error bars represent SDs. *** p<0.0001. (d) Excess Wingless (Wnt) signalling in DAxin-V72R clones is rescued by aggregon mutation. Larvae were grown at 29°C. In GFP-negative cells, the indicated DAxin variants were knocked in the endogenous axn locus. Expression of the Wg target genes senseless (Sens) and distalless (Dll) was analysed using immunostaining. DAxin-V72R-expressing clones overgrow the posterior tissue (arrowheads) and show ectopic expression of both Sens and Dll. Wg target gene expression by the V72R cancer mutant is rescued by introduction of the secondary aggregon mutation I169R L170R. Boundary between anterior and posterior tissue in wing discs is marked by a dotted line. Scale bar 100 µm (e) Mutation of the region homologous to the human RGS aggregon does not rescue DAxin-V72R suppressor activity. In shown mosaic posterior compartments, the V72R Y86R F87R DAxin variant (homologue of human Axin suppressor mutant L106R F119R W118R) was knocked in the endogenous axn locus in GFP-negative cells. Mutant clones overgrow the posterior tissue at all temperatures tested.
Supplementary Figure 7 Aggregon mutation restores the ability of Drosophila Axin V72R to assemble in punctate cytosolic complexes.
(a) Confocal microscopy analysis of human Axin (hAxin)-wt, hAxin-L106R and hAxin-L106R F119R in HEK293T cells. Scale bars 10 μm. (b) Confocal microscopy analysis of Drosophila Axin (DAxin)-wt, DAxin-V72R, DAxin-V72R F87R (homologue of human aggregon suppressor), DAxin-V72R I169R L170R (Drosophila aggregon suppressor) in HEK293T cells. Note that the Drosophila aggregation suppressor mutation (I169R L170R) reverts the protein into puncta while the potential suppressor mutation at the position of the human aggregon (F87R) fails to do so. Scale bars 10 μm. Schematic RGS domain structures indicate the location of cancer (red) and aggregon (green) mutations.
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Supplementary Table 1
Analysis of interactomes of Axin variants (XLSX 318 kb)
Supplementary Data Set 1
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Anvarian, Z., Nojima, H., van Kappel, E. et al. Axin cancer mutants form nanoaggregates to rewire the Wnt signaling network. Nat Struct Mol Biol 23, 324–332 (2016). https://doi.org/10.1038/nsmb.3191
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DOI: https://doi.org/10.1038/nsmb.3191
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