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, 108 (29), 11930-5

E-cadherin Mediates Contact Inhibition of Proliferation Through Hippo Signaling-Pathway Components

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E-cadherin Mediates Contact Inhibition of Proliferation Through Hippo Signaling-Pathway Components

Nam-Gyun Kim et al. Proc Natl Acad Sci U S A.

Abstract

Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms, and the growth of tumors is characterized by a loss of contact inhibition of proliferation. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. The modulation of the phosphorylation and nuclear localization of Yes-associated protein (YAP) by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied. However, cell-surface receptors regulating the Hippo signaling pathway in mammals are not well understood. In this study, we show that Hippo signaling pathway components are required for E-cadherin-dependent contact inhibition of proliferation. Knockdown of the Hippo signaling components or overexpression of YAP inhibits the decrease in cell proliferation caused by E-cadherin homophilic binding at the cell surface, independent of other cell-cell interactions. We also demonstrate that the E-cadherin/catenin complex functions as an upstream regulator of the Hippo signaling pathway in mammalian cells. Expression of E-cadherin in MDA-MB-231 cells restores the density-dependent regulation of YAP nuclear exclusion. Knockdown of β-catenin in densely cultured MCF10A cells, which mainly depletes E-cadherin-bound β-catenin, induces a decrease in the phosphorylation of S127 residue of YAP and its nuclear accumulation. Moreover, E-cadherin homophilic binding independent of other cell interactions is sufficient to control the subcellular localization of YAP. Therefore, Our results indicate that, in addition to its role in cell-cell adhesion, E-cadherin-mediated cell-cell contact directly regulates the Hippo signaling pathway to control cell proliferation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Hippo pathway components are required for E-cadherin–dependent contact inhibition of proliferation. At 12 to 24 h posttransfection of siRNA, cells were harvested and seeded at very low density on fibronectin-coated coverslips. Fc-hE–coated microspheres were presented to create pure E-cadherin homophilic binding. Anti-HLA or polylysine-coated microspheres were used as controls for bead binding to MCF-7 or MCF10A cells, respectively. Control and Fc-hE beads were bound for 24 h and cells were treated with 50 μM of BrdU for the last 6 h. BrdU incorporation was calculated by counting the number of positive BrdU immunofluorescence staining cells from the population of completely isolated, DAPI stained cells. (A and B) Depletion of α-catenin blocks E-cadherin–dependent contact inhibition of proliferation in SW480/E-cadherin (A) or MCF-7 (B) cells. (C and D) Depletion of Merlin (C) or NHERF (D) eliminates E-cadherin–dependent contact inhibition of proliferation in MCF-7 cells. (E and F) Depletion of β-catenin or Lats1/2 inhibits the E-cadherin bead-induced inhibition of proliferation in MCF-7 (E) and MCF10A (F) cells. Decrease of endogenous β-catenin or Lats1 protein levels by siRNA transfection in MCF-7 cells is shown in Fig. S1A. (G) Depletion of Kibra leads to the elimination of the E-cadherin bead-induced inhibition of proliferation in MCF10A cells. (H) Compared with β-catenin or Lats1/2 depletion, knockdown of Mst1/2 does not inhibit E-cadherin–dependent contact inhibition of proliferation in MCF10A cells. Decrease of endogenous Mst1/2 by siRNA transfection in MCF-7 cells is shown in Fig. S1B.
Fig. 2.
Fig. 2.
E-cadherin expression regulates cell density-dependent redistribution of YAP from nucleus to cytoplasm. (A and B) Parental MDA-MB-231 cells and stable MDA-MB-231 clones, which contain doxycycline-inducible full-length E-cadherin, E-cadherin Δβ-catenin, E-cadherin–α-catenin fusion, or E-cadherin Δp120 transgene were seeded on fibronectin-coated coverslips in 24-well plates sparsely (1 × 104 cells) or densely (2 × 105 cells). Wild- or mutant-type E-cadherin expression was induced by the treatment of 2 μg/mL of doxycycline for 2 d. Cells were cultured under sparse (A) or dense (B) conditions, and endogenous YAP was stained by anti-YAP antibody. (Magnification, 200×.) (C) Subcellular localization of YAP in A and B was quantified using Blobfinder.
Fig. 3.
Fig. 3.
Depletion of β-catenin in MCF10A cells induces the nuclear accumulation of YAP and decreases the phosphorylation of the YAP S127 residue in dense cell cultures. (A and B) MCF10A cells transfected with control, β-catenin, or Lats1/2 siRNA (positive control) were harvested and 2 × 105 cells were seeded on fibronectin-coated coverslips in 24-well plates for 2 d more. (Magnification, 400×.) Localization of endogenous YAP was identified by immunofluorescence staining (A) and quantified (B). (C) Knockdown of β-catenin or Lats1/2 decreases the phosphorylation of YAP S127 residue in densely cultured MCF10A cells.
Fig. 4.
Fig. 4.
Homophilic ligation of E-cadherin controls the localization of endogenous YAP protein. MCF10A cells transfected with control, β-catenin, or Lats1/2 siRNA were plated at very low density. (A) Cells were plated on either E-cadherin protein and fibronectin-coated coverslips (FN + Fc-hE), or fibronectin and Fc domain alone as control (FN + hFc). (B) Fc-hE coated or polylysine coated control microspheres were applied to the surface of MCF10A cells. After 24 h, endogenous YAP was stained and the nuclear-to-cytoplasmic YAP ratio (NCR) was quantified.
Fig. 5.
Fig. 5.
YAP overexpression blocks the proliferation inhibitory effect of E-cadherin. (A) MCF10A cells stably expressing Flag-YAP or Flag-YAP S127A were generated. Endogenous E-cadherin protein level and overexpression of exogenous protein was verified by Western blot. (B) Overexpression of YAP or YAP S127A inhibits the E-cadherin–dependent contact inhibition of proliferation.
Fig. 6.
Fig. 6.
A model for an E-cadherin–mediated Hippo signaling pathway. Homophilic binding of E-cadherin between two cells stimulates the Hippo signaling pathway, which control proliferation by inhibiting the activity of YAP in the nucleus. Broken arrows indicate steps unresolved by the present study (see Discussion for more details).

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