APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

Abstract

Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting β-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased β-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote β-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.

Keywords: APC; LRP6; Wnt signaling; caveolin; clathrin; colorectal cancer; endocytosis; β-catenin.

Figure 1. LRP6 Is Required for Wnt Signaling in APC-Deficient Cells

(A) LRP6 knockdown inhibits Wnt signaling in APC mutant CRC cells. WT KO (wild-type knockout) refers to deletion of the wild-type copy of CTNNB1. (B–D) LRP6 knockdown prevents Wnt activation upon APC loss. HEK293 STF and (C) RKO APC^KO cells were transfected with LRP6 small interfering RNA (siRNA) and APC siRNA. (D) MEF LRP6^WT and MEF LRP6^KO cells were incubated with Wnt3a or APC siRNA. R.L.U., relative light units. (E) LRP5 and LRP6 are required for Wnt activation in the absence of APC. RKO APC^KO cells were incubated with LRP5 or LRP6 siRNA. (F) LRP6 is not required for Wnt activation in the absence of Axin. MEF LRP6^WT and MEF LRP6^KO cells were incubated with Axin siRNA. Graph shows mean ± SEM. n.s., non-significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.

Figure 2. mAb7E5 Inhibits Wnt Signaling upon APC Loss

(A) mAb7E5 inhibits Wnt signaling in APC mutant CRC cells. CRC cells were incubated with mAb7E5 followed by immunoblotting. (B and C) mAb7E5 prevents accumulation of nuclear β-catenin. (B) CRC cells incubated with mAb7E5 or (C) RKO cells treated with APC siRNA and either LRP6 siRNA or mAb7E5 were immunostained. Scale bar, 40 µm. (D and E) mAb7E5 inhibits Wnt signaling upon APC loss. (D) HEK293 STF and (E) RKO STF cells were incubated with APC siRNA and mAb7E5. (F) mAb7E5 blocks Wnt pathway activation in APC-deficient cells. RKO APC^KO cells were incubated with mAb7E5 or mAb7E5^Fab followed by immunoblotting. (G) Specificity of Wnt signaling inhibition by mAb7E5 in APC-deficient cells. A non-Wnt-regulated reporter cell line, HEK293 CMV-luc, was incubated with APC siRNA and mAb7E5. Graph shows mean ± SEM. n.s., non-significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S2.

Figure 3. Loss of APC Promotes Ligand-Independent Signalosome Formation

(A) Fz and Dvl are required for ligand-mediated Wnt activation. HEK293 STF cells were transfected with Xdd1 and Fz1-ER for 24 hr and then incubated with L-cell conditioned medium (L CM) or Wnt3a conditioned medium (Wnt3a CM). (B) Dvl and Fz are required for Wnt activation in APC-deficient cells. HEK293 STF cells were transfected with APC siRNA plus Xdd1 or Fz1-ER. (C) APC depletion fails to activate the Wnt pathway in Dvl null cells. HEK293 and HEK293 Dvl TKO were transfected with APC siRNA or treated with Wnt3a CM or 30 mM LiCl. (D) GSK3 inhibition activates the Wnt pathway in Dvl null cells. HEK293 Dvl TKO cells were incubated with 2 µM CHIR. (E) Wnt signaling due to β-catenin stabilization is not dependent on Fz or Dvl. HEK293 STF cells were transfected with Xdd1 or Fz1-ER and incubated with 30 mM LiCl. (F) Similar to Wnt ligand-activated cells, LRP6 forms high molecular complexes in the absence of APC (sucrose density gradient analysis). Graphs show mean ± SEM. n.s., non-significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4. Wnt Receptor Activation in APC-Deficient Cells Is Wnt Ligand Independent

(A–D) Fz8-Fc blocks Wnt ligand-mediated signaling but not signaling in APC-deficient cells. (A) RKO STF cells were incubated with Wnt3a and Fz8-Fc at the indicated concentrations. (B) HEK293 STF cells transfected with APC siRNA and (C) RKO APC^KO cells were incubated with Fz8-Fc. (D) Wnt activation in CRC cells is not dependent on Wnt ligand. CRC cells were incubated for 48 hr with Fz8-Fc at the indicated concentrations. (E) APC-depleted cells are responsive to Wnt ligand. RKO APC^KO cells were preincubated for 30 min with Fz8-Fc followed by incubation with Wnt3a. (F) Inhibition of secreted Wnt3a by the PORCN inhibitor Wnt-C59. HEK293 STF were transfected with Wnt3a and treated with Wnt-C59 (0.3 nM). (G) Wnt pathway activation by exogenous Wnt3a is not blocked by Wnt-C59. HEK293 STF cells were preincubated for 30 min with Wnt-C59 followed by incubation with Wnt3a. (H and I) PORCN inhibition does not block Wnt activation upon loss of APC. (H) HEK293 STF cells transfected with APC siRNA and (I) RKO APC^KO cells were incubated with Wnt-C59. (J) PORCN inhibition does not block Wnt activation in CRC cells. CRC cells were incubated for 48 hr with Wnt-C59. (K) LRP6 is required for Wnt activation upon loss of APC in the absence of PORCN. PORCN^KO MEF and wild-type MEF were transfected with APC and LRP6 siRNA. Graphs show mean ± SEM. n.s., non-significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S3.

Figure 5. LRP6 Activation and β-Catenin Accumulation Occur Rapidly upon Loss of APC

(A) Degradation complex inhibition does not lead to LRP6 phosphorylation. HEK293 STF cells were incubated with Wnt3a or 30 mM LiCl. (B and C) Inhibition of Wnt-mediated transcription does not block LRP6 activation. (B) HEK293 STF cells were transfected with APC siRNA and dnTCF4. β-catenin levels were quantified and the average of five independent replicates are plotted in (C). (D) Schematics of APC structure and truncations tested. (E–G) β-catenin and Axin binding domains of APC are sufficient to rescue loss-of-APC phenotype. (E) HEK293 STF treated with APC siRNA, (F) RKO APC^KO, and (G) CRC cells were transfected with Myc-APC^T followed by TOPflash analysis and immunoblotting. (H and I) Loss of APC correlates with β-catenin and phospho-LRP6 accumulation independently of secreted ligands. (H) RKO APC^KO TIR1 cells were transfected with Myc-APC^T or AID-Myc-mCherry-APC^T followed by incubation with auxin. (I) RKO APC^KO TIR1 cells were transfected with AID-Myc-mCherry-APC^T followed by incubation with BFA and auxin. AID, auxin-inducible degron; mCh, mCherry; Aux, auxin. Scale bar, 10 µm. (J) Downregulation of APC2 by two distinct siRNAs. (K) Loss of APC2 promotes Wnt pathway but not receptor activation. HEK293 cells were incubated with APC or APC2 siRNA. (L) APC2 is required for β-catenin degradation complex in APC-null cells. RKO APC^KO cells were incubated with APC2 siRNA and mAb7E5. Graphs show mean ± SEM. n.s., non-significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6. Endocytosis Is Required for Wnt Signaling in APC-Deficient Cells

(A–D) Inhibition of endocytosis induced by temperature shift prevents β-catenin accumulation and LRP6 phosphorylation. RKO cells (treated with Wnt3a, 30 mM LiCl, or 2 µM CHIR), and RKO APC^KO cells were incubated at 4°C (A and B) and shifted back to 37°C (C and D). The average of three independent replicates were plotted. Representative blots are shown in Figures S4A–S4D. (E) Dynamin is required for Wnt signaling in APC-depleted cells. HEK293 STF cells were incubated with Wnt3a or APC siRNA and transfected with HA-Dynamin^K44A. Graphs show mean ± SEM. n.s., non-significant; **p < 0.01. (F and G) LRP6 is internalized in Wnt3a-treated and APC-deficient cells. RPE cells were transfected with LRP6-eYFP and incubated with (F) Wnt3a or 30mMLiCl or (G) APC siRNA and mAb7E5. Cells (>100 per condition) demonstrating membrane or internalized LRP6 were quantified. Scale bars, 20 µm. See also Figure S4.

Figure 7. Clathrin-Mediated Endocytosis Is Required for Wnt Signaling in APC-Deficient Cells

(A–D) Ligand-dependent and -independent Wnt activation requires distinct endocytic pathways. (A) RKO cells incubated with Wnt3a or (B) RKO APC^KO cells were incubated with the indicated concentrations of Pitstop-2 or nystatin. (C) RKO cells incubated with Wnt3a were transfected with caveolin-1 or clathrin siRNA. (D) RKO APC^KO cells were transfected with clathrin or caveolin-1 siRNA. (E–G) APC partially colocalizes with clathrin but not caveolin-1. (E and F) RPE cells were incubated with or without Wnt3a, fixed, and stained for APC and clathrin or caveolin. Scale bar, 10 µm. (G) Intensity correlation analysis of APC-clathrin/caveolin-1 signal. (H) APC co-immunoprecipitates with clathrin and its adaptor protein, AP2. Graphs show mean ± SEM. n.s., non-significant; **p < 0.01; ***p < 0.001. See also Figure S5.

Figure 8. Arrow and Dishevelled Are Required for the Consequences of Apc1 Loss in Drosophila ISCs Independently of Wnt Ligand

(A–H and Q) In comparison with controls (A–D and Q), Apc1 mutation leads to increased progenitor cell number (E, F, H, and Q) and cell polarity defects (G). Intestinal progenitor cells are marked with esg>GFP (green), cell membranes with Armadillo/β-catenin (Arm, magenta), nuclei with DAPI (blue), and enteroendocrine cells with Prospero (Pros, magenta). (I–L and Q) RNAi-mediated Arrow knockdown in Apc1 mutants leads to significant reduction of progenitor cell number (I, J, L, and Q) and rescue of cell polarity defects (K). (M–P and Q) RNAi-mediated Dsh knockdown in Apc1 mutants leads to significant reduction of progenitor cell number (M, N, P, and Q). (A, E, I, and M) Lower magnification view (scale bars, 50 µm). (B–D, F–H, J–L, and N–P) Higher magnification view (scale bar, 10 µm). (R) RNAi-mediated knockdown of Wg or Wls in Apc1 mutants does not reduce progenitor cell number. Graphs show mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001.