Published OnlineFirst June 3, 2019; DOI: 10.1158/1541-7786.MCR-18-1154 Cancer Genes and Networks Molecular Cancer Research Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and b-Catenin– Independent Targets by Proteomics Olesja Popow1,2,3,Joao~ A. Paulo2, Michael H. Tatham4, Melanie S. Volk3, Alejandro Rojas-Fernandez5, Nicolas Loyer3, Ian P. Newton3, Jens Januschke3, Kevin M. Haigis1,6, and Inke Nathke€ 3 Abstract Adenomatous Polyposis Coli (APC) is the most frequently tion between endogenous MINK1 and APC and further mutated gene in colorectal cancer. APC negatively regulates confirmed the negative, and b-catenin–independent, regu- the Wnt signaling pathway by promoting the degradation of lation of MINK1 by APC. Increased Mink1/Msn levels were b-catenin, but the extent to which APC exerts Wnt/b-cate- also observed in mouse intestinal tissue and Drosophila nin–independent tumor-suppressive activity is unclear. To follicular cells expressing mutant Apc/APC when compared identify interaction partners and b-catenin–independent with wild-type tissue/cells. Collectively, our results highlight targets of endogenous, full-length APC, we applied label- the extent and importance of Wnt-independent APC func- free and multiplexed tandem mass tag-based mass spec- tions in epithelial biology and disease. trometry. Affinity enrichment-mass spectrometry identified more than 150 previously unidentified APC interaction Implications: The tumor-suppressive function of APC, the partners. Moreover, our global proteomic analysis revealed most frequently mutated gene in colorectal cancer, is mainly that roughly half of the protein expression changes that attributed to its role in b-catenin/Wnt signaling. Our study occur in response to APC loss are independent of b-catenin. substantially expands the list of APC interaction partners and Combining these two analyses, we identified Misshapen- reveals that approximately half of the changes in the cellular like kinase 1 (MINK1) as a putative substrate of an APC- proteome induced by loss of APC function are mediated by containing destruction complex. We validated the interac- b-catenin–independent mechanisms. Introduction and functions of APC crucial for effectively targeting APC-mutant cells. Mutations in Adenomatous Polyposis Coli (APC) are a frequent The tumor-suppressive function of APC has been mainly (>80%) and early event in the development of sporadic colorectal attributed to its role in Wnt signaling. In conjunction with Axin, cancer (1, 2). Germline mutations in APC also form the genetic APC acts as a scaffold for the b-catenin destruction complex, basis of familial adenomatous polyposis (FAP), an inherited form thereby limiting the transcription of proproliferative b-catenin of the disease, which is characterized by hundreds of colorectal target genes in the absence of Wnt ligands (4). The vast majority of polyps that progress to cancerous lesions if left untreated (3). This APC mutations result in the translation of a truncated protein and makes a comprehensive understanding of the normal interactions consequent deregulation of Wnt signaling (1, 2). Nevertheless, Wnt-independent roles of APC likely also contribute to its func- fi 1Cancer Research Institute and Department of Medicine, Beth Israel Deaconess tion as a tumor suppressor. This is exempli ed by the rare Medical Center, Boston, Massachusetts. 2Department of Cell Biology, Harvard detection of mutations in other Wnt signaling components, Medical School, Boston, Massachusetts. 3Cell and Developmental Biology, including b-catenin, in colorectal cancer (5). Although deletion School of Life Sciences, University of Dundee, Dundee, Scotland, United King- of Apc in the intestinal epithelium in mice phenocopies homo- 4 dom. Centre for Gene Regulation and Expression, School of Life Sciences, zygous truncation mutations, it leads to more rapid onset of 5 University of Dundee, Dundee, Scotland, United Kingdom. Center for Interdis- tumors despite lower levels of Wnt activation (6). It thus emerges ciplinary Studies on the Nervous System (CISNe) and Institute of Medicine, Universidad Austral de Chile, Valdivia, Chile. 6Harvard Digestive Disease Center, that loss of wild-type (WT) APC confers additional advantages to Harvard Medical School, Boston, Massachusetts. cells beyond b-catenin–mediated proliferation, but the extent of APC's Wnt-independent functions is unclear. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). A variety of proteins have been described to interact with APC in addition to b-catenin destruction complex components (7). How- Corresponding Author: Inke Nathke,€ University of Dundee, Dow Street, Dundee, ever, proteome-wide studies of APC-binding proteins are limited Tayside DD15EH, United Kingdom. Phone: 44-1382-385821; Fax: 44-1382- 385386; E-mail: [email protected] to interactome and yeast-two-hybrid experiments with overex- pressed, tagged, and/or fragments of APC (8–11). Using tagged Mol Cancer Res 2019;XX:XX–XX APC in interaction studies is problematic because the C-terminal doi: 10.1158/1541-7786.MCR-18-1154 PDZ-binding domain must remain free to interact with other Ó2019 American Association for Cancer Research. proteins (12). Similarly, the N-terminal oligomerization domains www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst June 3, 2019; DOI: 10.1158/1541-7786.MCR-18-1154 Popow et al. rely on coiled-coil formation and may be compromised by N- more comprehensive set of interacting partners of endogenous, terminal tags (13). To overcome these limitations, we used label- nontagged APC. Furthermore, we applied an untargeted global free affinity-enrichment mass spectrometry (AE-MS) to identify a approach using tandem mass tag (TMT)-based and label-free MS A B Significant in N&C-APC CoIP Untreated HeLa cells HeLa Cells treated with 12 Significant in N-APC CoIP Co-immunoprecipitation with siRNA against Significant in C-APC CoIP 10 N-APC C-APC APC β-Catenin β-Catenin 8 APC Control antibody APC Control & β-Catenin 6 Label-free LC-MS2 Label-free TMT-labeled 4 LC-MS2 LC-MS3 ) 2 Proteins interacting Proteins regulated with APC by APC -4 -2 2 4 6 8 10 12 (C-APC/ctrl. 2 -2 Log Overlap: proteins potentially regulated -4 by APC-containing complex(es) Log2 (N-APC/ctrl.) C 25 (Actin) Cytoskeleton Cell-cell contact 20 RNA Processing Other 15 10 Enrichment factor 5 0 A binding PDZ (Pfam) LSM (Pfam) Cell junction Tight junction SCARHULC complex complex Lamellipodium Cell projection Catenin complex Cell-cell junction U6 snRN Cytoskeleton organiz. Reg. of actin nucleation Cell projection organiz.Spliceosomal complex DevelopmentalReg. of cellular process process Guanylate kinase (Pfam) Lateral plasma membrane Reg. of actin cytoskeleton Reg. of organelle organiz. Cell-cell adherens junction Reg. of cytoskeleton organiz. Arp2/3 comp.-med. actinArp2/3 nuc. comp.-med.U4/U6 x U5 actin tri-snRNP nuc. complex Pos. reg. of organelle organiz. Cellular component movement Rac protein signal transduction Reg. of cellular component organiz. Reg. of Pos. reg. of cellular component organiz. Pos. reg. of Figure 1. Identification of APC-interacting and -regulated proteins. A, Experimental outline. Proteins in APC-containing complexes and changes in protein expression in response to siRNA-mediated depletion of APC and/or b-catenin were analyzed by label-free and TMT-based mass spectrometry. The overlap between the two datasets constitutes potential targets of alternative APC-containing complexes. B, Proteins significantly enriched in C- and/or N-APC co-IPs. Log2 fold change in mean LFQ intensities between N-APC co-IP versus control IP (x-axis) plotted against log2 fold change in mean LFQ intensities between C-APC co-IP versus control IP (y-axis, n ¼ 4 experimental replicates). Significance determined by two-sided t test with permutation-based FDR < 0.01 and s0 ¼ 2 used for truncation (49). C, GO, Pfam, and KEGG terms significantly enriched in the APC interactome dataset. Enrichment calculated by Fisher exact test, significance determined by Benjamini–Hochberg corrected FDR < 0.02. comp.-med., complex-mediated; nuc., nucleation; organiz., organization; pos., positive; reg., regulation. OF2 Mol Cancer Res; 2019 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst June 3, 2019; DOI: 10.1158/1541-7786.MCR-18-1154 The APC Interactome and Its b-Catenin–Independent Targets to identify proteins that are regulated by APC in their abundance. normocin. MINK1 protein expression was screened by Western These two datasets provide a unique resource for the exploration blotting analysis. Genomic DNA of MINK1 KO cells was ampli- of Wnt/b-catenin–dependent and -independent functions of APC. fied by PCR and sequenced to confirm the introduction of In addition, we could identify potential targets of APC-containing frameshift mutations. destruction complexes by combining our data on APC-interacting and APC-regulated proteins (Fig. 1A). Although no direct evi- HeLa SEC-C mNeonGreen-MINK1. The same pBabeD dence for the assembly of such complexes by APC exists, other pU6 gMINK1 vector used for the generation of MINK1 knock- components of the b-catenin destruction complex, such as GSK- out cells was used for the fusion of mNeonGreen to the N- b 3b and SCF -TrCP, are known to have many targets (14, 15). We terminus of MINK1 in HeLa SEC-C cells as described previous- thus hypothesized that APC may directly regulate the abundance ly (16). A donor vector was designed to replace the ATG start of other proteins in addition to b-catenin. codon of MINK1 with the start codon of an mNeonGreen cDNA cassette, flanked by approximately 500 bp homology arms. The donor vector was synthesized by GeneArt (Life Technologies). Materials and Methods Expression of mNeon-MINK1 in selected and expanded single- Cell culture cell clones was validated by Western blotting analysis and Colo320, HeLa, and SW480 cells were obtained from the ATCC microscopy.