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

Saito-Diaz, Kenyi; Benchabane, Hassina; Tiwari, Ajit; Tian, Ai; Li, Bin; Thompson, Jeffrey Y.; Hyde, Annastasia S.; Sawyer, Leah; Jodoin, Jeanne N.; Santos, Eduardo Alves Portela; Lee, Laura A.; Coffey, Robert J.; Beauchamp, R. Daniel; Williams, Christopher S.; Kenworthy, Anne K.; Robbins, David J.; Ahmed, Yashi; Lee, Ethan

doi:10.1016/j.devcel.2018.02.013

Cited by 78 publications

(104 citation statements)

References 56 publications

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“…While this can be explained in part by a decrease in ß-catenin (CTNNB1) mRNA expression (data not shown), the total cellular protein level of ß-catenin decreased more than its CTNNB1 mRNA, suggesting that ß-catenin protein degradation is enhanced. This is in line with findings from Saito-Diaz et al (Saito-Diaz et al, 2018), which suggest that LRP6 associates with the ß-catenin destruction complex in the absence of a Wnt ligand, partially inhibiting its activity. Thus the loss of LRP6 allows the destruction complex to be more active, decreasing overall ß-catenin protein.…”

Section: Lrp6 Modulates Wnt Signaling Levels In Apc-mutant Colon Cancersupporting

confidence: 92%

“…The loss of LRP6 affects the stability of ß-catenin due to the release of the destruction complex from the plasma membrane. (Saito-diaz et al, 2018) J. Scratch assay of SW620 Cas9 versus LRP6KO shows a significant difference in cell motility only after 48 hours, suggesting with a more dramatic Wnt inhibition, there is an increase in cell motility.…”

Section: Supplementary Materialsmentioning

confidence: 99%

See 1 more Smart Citation

Disrupting ß-catenin dependent Wnt signaling activates an invasive gene program predictive of colon cancer progression

Chen¹,

Tifrea²,

Murad³

et al. 2019

Preprint

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The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the long-standing understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we lower the levels of Wnt signaling in colon cancer via interference with two different steps in the pathway that lie upstream or downstream of the effector protein ß-catenin.We find that these Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and localized invasion in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion. We identify a set of upregulated genes common among the Wnt perturbations and find that elevated expression of these genes is strongly predictive of poor patient outcomes in early-invasive colon cancer. These genes may have clinical applications as patient biomarkers or new drug targets to be used in concert with existing therapies.

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Section: Lrp6 Modulates Wnt Signaling Levels In Apc-mutant Colon Cancersupporting

confidence: 92%

Section: Supplementary Materialsmentioning

confidence: 99%

Disrupting ß-catenin dependent Wnt signaling activates an invasive gene program predictive of colon cancer progression

Chen¹,

Tifrea²,

Murad³

et al. 2019

Preprint

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“…Wnt activation is known to induce endocytosis of the receptor complex (11,12). We confirmed that Lrp6 endocytosis was observed in SW480, a colon cancer cell line in which the absence of APC triggered, and APC reconstitution inhibited, Wnt signaling and Lrp6 endocytosis (SI Appendix, Movie S1); this is in agreement with recent results showing that APC is a major regulator of endocytosis (16,29). As expected for regulators of Lrp6 endocytosis, the Wnt antagonists Dkk1 (30) or Bighead (31) induced endocytosis of Lrp6-APEX2 into large vesicle clusters in the cytoplasm next to the nuclear bay region, where the lysosomal system is located Fig.…”

Section: Lrp6-apex2 Receptor Fusion Is Active In Wnt Signalingsupporting

confidence: 91%

Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

Colozza

Jami‐Alahmadi²,

Dsouza

et al. 2020

Preprint

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The canonical Wnt signaling pathway serves as a hub connecting diverse cellular physiological processes, such as β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many components of the ESCRT (Endocytic Sorting Components Required for Transport) machinery interacted with Lrp6 within 5 minutes of Wnt3a treatment. This supports the proposal of a central role of multivesicular endosomes in canonical Wnt signaling. Interestingly, proteomic analyses identified the Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, as being strongly enriched in the proximity of Lrp6. We provide evidence that TFG specifically co-localized with MVBs after Wnt stimulation. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling. SignificanceWnt/β-catenin signaling is a conserved pathway involved in cell differentiation and in the regulation of many other processes, including cell growth and proliferation, macropinocytosis, and cell metabolism. Endocytosis is required to regulate Wnt signaling, but the precise factors at play are still elusive. Here, we describe a biotin-dependent proximity labeling approach using ascorbate peroxidase-tagged Lrp6, a Wnt co-receptor. Proteomic analysis of biotinylatedenriched targets identified numerous multivesicular endosome proteins that were recruited to the receptor shortly after addition of Wnt protein. Additionally, we identified the protein TFG as one of the strongest interactors with Lrp6. TFG co-localized with Wnt-induced multivesicular endosomes. Xenopus embryo assays revealed that TFG is required in vivo for canonical Wnt signaling. \body

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“…Specifically, whereas intact APC inhibits ligandindependent clathrin-mediated endocytosis, APC mutation in colorectal cancer allows augmented β-catenin signaling via increased endocytic trafficking. 42 Alternatively, increased vesicle acidification in colorectal cancer has been shown to promote APC degradation via the endolysosomal pathway, leading to enhanced β-catenin signaling. 43 Nevertheless, the roles of endosome trafficking and Wnt/β-catenin signaling in oral dysplasia remain unexplored, and hence, our findings showing that increased Rab5 activity, endosomal sequestration of the destruction complex, and nuclear localization of β-catenin, contribute to understanding the relevance of this mechanism in early oral lesions.…”

Section: Discussionmentioning

confidence: 99%

Nuclear accumulation of β‐catenin is associated with endosomal sequestration of the destruction complex and increased activation of Rab5 in oral dysplasia

et al. 2020

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Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of β‐catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re‐localization of β‐catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the β‐catenin destruction complex allows nuclear accumulation of β‐catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non‐dysplastic oral keratinocytes (OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of β‐catenin and Tcf/Lef‐dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3β, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co‐localization of GSK3β, APC, and Axin, with early endosome antigen 1‐ and Rab5‐positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the β‐catenin destruction complex leads to stabilization and nuclear accumulation of β‐catenin in oral dysplasia.

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

Cited by 78 publications

References 56 publications

Disrupting ß-catenin dependent Wnt signaling activates an invasive gene program predictive of colon cancer progression

Disrupting ß-catenin dependent Wnt signaling activates an invasive gene program predictive of colon cancer progression

Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

Nuclear accumulation of β‐catenin is associated with endosomal sequestration of the destruction complex and increased activation of Rab5 in oral dysplasia

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