E-cadherin Suppression Accelerates Squamous Cell Carcinoma Progression in Three-Dimensional, Human Tissue Constructs

Abstract

We studied the link between loss of E-cadherin–mediated adhesion and acquisition of malignant properties in three-dimensional, human tissue constructs that mimicked the initial stages of squamous cell cancer progression. Suppression of E-cadherin expression in early-stage, skin-derived tumor cells (HaCaT-II-4) was induced by cytoplasmic sequestration of β-catenin upon stable expression of a dominant-negative E-cadherin fusion protein (H-2K^d-Ecad). In monolayer cultures, expression of H-2K^d-Ecad resulted in decreased levels of E-cadherin, redistribution of β-catenin to the cytoplasm, and complete loss of intercellular adhesion when compared with control II-4 cells. This was accompanied by a 7-fold decrease in β-catenin–mediated transcription and a 12-fold increase in cell migration. In three-dimensional constructs, E-cadherin–deficient tissues showed disruption of architecture, loss of adherens junctional proteins from cell contacts, and focal tumor cell invasion. Invasion was linked to activation of matrix metalloproteinase (MMP)–mediated degradation of basement membrane in H-2K^d-Ecad–expressing tissue constructs that was blocked by MMP inhibition (GM6001). Quantitative reverse transcription–PCR showed a 2.5-fold increase in MMP-2 and an 8-fold increase in MMP-9 in cells expressing the H-2K^d-Ecad fusion protein when compared with controls, and gel zymography showed increased MMP protein levels. Following surface transplantation of three-dimensional tissues, suppression of E-cadherin expression greatly accelerated tumorigenesis in vivo by inducing a switch to high-grade carcinomas that resulted in a 5-fold increase in tumor size after 4 weeks. Suppression of E-cadherin expression and loss of its function fundamentally modified squamous cell carcinoma progression by activating a highly invasive, aggressive tumor phenotype, whereas maintenance of E-cadherin prevented invasion in vitro and limited tumor progression in vivo.