E-cadherin/Epidermal Growth Factor Receptor Complexes Are Mechano-Switches That Regulate Force Transduction and Morphogenesis in Three Dimensional Epithelial Cultures

Thursday, October 12, 2023

8:45 AM – 9:00 AM PDT

Location: 339 (Center, Level 3)

Presenting Author(s)

Deborah Leckband, PhD

University of Illinois at Urbana-Champaign
Urbana, Illinois, United States

Co-Author(s)

YZ

Yubo Zou

University of Illinois at Urbana-Champaign
Urbana, Illinois, United States
EH

Evan Hebner

University of Illinois at Urbana-Champaign
Urbana, Illinois, United States
AR

Al-Sadiq Rehemtulla

University of Illinois at Urbana-Champaign
Urbana, Illinois, United States
JL

Jon N. Lakins

University of California San Francisco, California, United States
WV

Weaver M. Valerie

University of California San Francisco, California, United States

Introduction::
Cadherins are essential adhesion proteins that zip up intercellular junctions in all tissues, by binding cadherins on opposing membranes. They are mechanically coupled to actin through cytosolic binding partners, catenins. Cadherin complexes are also force transduction hubs¹. Increased intercellular (cadherin) tension can increase cell proliferation and alter the barrier properties of tissues such as the vascular endothelium and skin. We are investigating cadherin-medicated force transduction mechanisms and their impact on cell and tissue functions.

In epithelia, E-cadherin is the predominant intercellular adhesion protein at cell-cell junctions. Prior studies demonstrated that E-cadherin force transduction involves a mechanically-induced conformational change in the actin binding protein, alpha catenin, in cadherin complexes^2,3. The latter change potentiates cytoskeletal remodeling at stressed cell-cell adhesions. However, the alpha catenin-based force transduction mechanism does not activate signaling.

We identified a second force transduction mechanism in which E-cadherin tugging mechanically activates epidermal grown factor receptor, EGFR signaling^4,5, to increase global cell contractility, regulate local cytoskeletal remodeling, and initiate proliferation. E-cadherin is a tumor suppressor that binds EGFR and suppresses EGF-dependent proliferation, in contact-inhibited proliferation⁶. We demonstrated that tugging on E-cadherin receptors in 2D epithelial cultures overrides contact inhibition, to activate EGFR (Fig. 1A) and MAPK signaling and induce cell proliferation. We tested how E-cadherin mutations that disrupt E-cadherin/EGFR complexes impact force transduction. We further tested how the same E-cadherin mutants alter cell polarization and organization in 3D breast epithelial cultures.

Materials and Methods::
Studies used E-cadherin knockout MCF10A cells or A431D epithelial cells, which do not express E-cadherin. The cells were engineered to stably express E-cadherin mutants that disrupt E-cadherin/EGFR interactions, without altering E-cadherin adhesion. Mutations focused on the E-cadherin extracellular region, which is hypothesized to mediate E-cadherin/EGFR interactions⁷. We also tested cancer linked E-cadherin mutations that affect EGFR signaling⁸. Cell adhesion was quantified with shear flow assays. EdU uptake assays tested the impact of cadherin mutants on cell proliferation. With 2D cultures, we used magnetic twisting cytometry (MTC) to quantify the impact of E-cadherin mutants and matrix rigidity on cadherin force transduction⁴. We cultured 3D spheroids with cells expressing wild type E-cadherin or E-cadherin mutants at similar expression levels. Spheroids are cultured in Matrigel/Col 1 matrices and analyzed after 2 weeks. Matrix rigidity was altered by changing the Col 1 concentration or use of a mechanical reactor⁹.

Results, Conclusions, and Discussions::

Co-immunoprecipitation studies demonstrated that the extracellular domain of E-cadherin is required for both EGFR complex formation and E-cadherin mediated force transduction. We localized the putative binding region to E-cadherin extracellular domain 4, EC4 (Fig. 1A), which has also been implicated in the epithelial to mesenchymal transition¹⁰. Point mutations in the EC4 domain that are linked to diffuse gastric cancer and lobular breast cancer also reduce hetero-receptor complex levels.

Force transduction studies of two-dimensional epithelial cultures used magnetic twisting cytometry (MTC), which quantifies cell stiffening in response to receptor tugging (Fig. 1B). Results obtained with cells expressing different E-cadherin mutants showed that disrupting interactions with EGFR attenuated or fully abolished cadherin-mediated force transduction (Fig. 1B). This result confirmed that EGFR/E-cadherin complexes are intercellular mechano-switches and that direct E-cadherin binding to EGFR is required for force-activated signaling. We also identified key E-cadherin regions required for hetero-complex formation and force transduction.

We investigated cell polarization and cell organization in 3D breast epithelial cultures, using parental MCF10A cells and MCF10A spheroids in which E-cadherin is knocked out or rescued with E-cadherin mutants (Fig. 1C). Initial results show that merely deleting the cadherin EC4 domain impairs cell polarization and the formation of acinar structures with defined lumens in 3D cultures. These changes are not due to differences in cadherin adhesion and are attributed to the disruption of E-cadherin/EGFR interactions. We are currently investigating other E-cadherin mutants, as well as the impact of the matrix rigidity on the 3D spheroid cultures.

In conclusion, these results demonstrated that EGFR/E-cadherin complexes are required for force transduction and morphogenesis in 3D cultures. Moreover, E-cadherin mutations that disrupt binding to EGFR ablate force transduction and result in abnormal cell organization in 3D spheroids. Further studies are testing how matrix rigidity and E-cadherin force transduction cooperate to regulate intercellular adhesion and morphogenesis in 3D cultures.

Acknowledgements (Optional): :
Supported by NIH RO1 GM127554.

References (Optional): :
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