Barry M. Gumbiner, PhD
Cadherin-mediated contact inhibition of growth and regulation of the Hippo signaling pathway
Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms. It is highly regulated, since growth can occur in rapidly developing tissues despite cell contact, 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 the YAP transcriptional regulator by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied, but less is know about cell surface receptors regulating the Hippo signaling pathway. We discovered that the Hippo signaling pathway mediates E-cadherin–dependent contact inhibition of proliferation. E-cadherin stimulates Hippo signaling and YAP nuclear exclusion via several membrane associated proteins, and we are actively investigating the molecular mechanism linking E-cadherin engagement and the hippo pathway. Knowledge of this mechanism is being used to determine the specific role of cadherin-hippo-mediated signaling in the control of cell proliferation and tumor growth in vivo using genetic manipulations of animal models of mammary tumors.
Contact inhibition of growth can be antagonized by mitogenic growth factor signaling. We report an important mechanism for this antagonism, the inhibition of Hippo pathway signaling by mitogenic growth factors. Epidermal growth factor (EGF) treatment cells triggers the rapid translocation of YAP into the nucleus and YAP dephosphorylation, both of which depend on Lats, the terminal kinase in the Hippo pathway. A small molecule inhibitor screen revealed that EGF signaling inhibits the Hippo pathway through activation of PI3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), but independent of AKT activity. This pathway is not unique to EGF signaling because the PI3K-PDK1 pathway also mediates YAP nuclear translocation downstream of lysophosphatidic acid and serum as a result of constitutive oncogenic activation of PI3K. PDK1 associates with the core Hippo pathway-kinase complex, which dissociates in response to EGF signaling, leading to inactivation of Lats, dephosphorylation of YAP, and YAP nuclear accumulation. These findings show that an important activity of mitogenic signaling pathways is to inactivate the growth-inhibitory Hippo pathway and provide a mechanism for antagonism between contact inhibition and growth factor action. We are currently investigating the detailed molecular mechanism underlying this regulation, which will also be used to determine its role of in the control of cell proliferation and tumor growth in vivo using genetic manipulations of animal models of mammary tumors.
Regulation of cadherin-mediated adhesion in tissue morphogenesis, cancer, and inflammation.
We discovered many years ago that cadherin adhesive function can be regulated of at the cell surface to mediate dynamic changes in cell interactions in tissues, including morphogenetic cell movements during embryonic development. New findings from my laboratory provide important new insights into the mechanisms of E-cadherin regulation in epithelia as well as novel approaches to manipulate cadherin activity experimentally. We discovered a mechanism that controls cadherin conformation and adhesive activity at the cell surface independent of any changes in levels of expression or amounts of associated catenins. We are able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. Both types of mAbs recognize specific conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Moreover, we discovered a role for p120-catenin phosphorylation and microtubules in the control of E-cadherin activity state. Activation induces p120-catenin dephosphorylation, and phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane.
We hypothesize that regulation of the adhesive homophilic bond itself is a key event in cell junction regulation, allowing the catenin-associated cytoskeleton to pull the junctions apart once the adhesive bond is broken, and that this mechanism plays a role in morphogenetic cell movements, barrier regulation in both epithelia and endothelia during inflammatory processes, and cancer metastasis. We are working to understand in greater depth the mechanisms by which cadherins are regulated at the cell surface, including: the nature of the changes in the properties of the homophilic adhesive bond; the cytoplasmic mechanisms regulating p120-catenin phosphorylation and the effectors through which p120-catenin phosphorylation controls adhesion activity; the role of E-cadherin activity state in the regulation of tumor metastasis and epithelial barrier function during inflammatory processes in both cell culture and animal models.
Kim, N.G., Koh, E.J., and Gumbiner, B.M. (2011) E-cadherin mediates contact inhibition of cell proliferation through Hippo signaling pathway components. Proc. Natl. Acad. Sci.., 108: 11930-11935.
Fan, R., Kim, NG, and Gumbiner, B.M. (2013) Regulation of Hippo Pathway by Mitogenic Growth Factors via PI3-Kinase and Phosphoinositide-Dependent Kinase-1. Proc. Natl. Acad. Sci, 110(7): 2569-2574.
Petrova, Y.I., M.M. Spano, and B.M. Gumbiner, (2012) Conformational epitopes at cadherin calcium- binding sites and p120-catenin phosphorylation regulate cell adhesion. Molec. Biol. Cell. 23:2092-2108.
Gumbiner, B.M. (2005) Regulation of cadherin-mediated adhesion in morphogenesis. Nature Reviews Molecular Cell Biology, 6, 622-634.
Park, KS, and Gumbiner, B.M. (2012) Cadherin-6B stimulates an epithelial mesenchymal transition and the delamination of cells from the neural ectoderm via LIMK/cofilin mediated non-canonical BMP receptor signaling. Devel Biol., 366, 232-243.
Kim, N.G., Xu, C., and Gumbiner,, B.M. (2009) Identification of targets of the Wnt pathway destruction complex in addition to β-catenin. Proc. Natl. Acad. Sci., 106:5165-5170.
Chen, XJ and Gumbiner, B.M. (2006) Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity. J. Cell Biol., 174:301-313.