Abstract
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin–α-catenin chimera that functions as a β-catenin–independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.
Highlights
We showed that Discosoma sp. red fluorescent protein (DsRed)-tagged cadherin cytoplasmic domains expressed in MDCK cells interact with β -catenin, reduce the β -catenin levels associated with endogenous E-cadherin, and inhibit the cell surface localization of endogenous E-cadherin, leading to morphological changes, including the inhibition of desmosome and tight junction formation and a reduction in the mechanical integrity of the epithelial cell sheets[25]
The construct was expressed under the control of a Tetrepressible transactivator in an MDCK cell clone T23 expressing the tet repressor (DNCT+ cells)
Immunoblot analysis of DNCT+ T23 cells cultured for 2 d with or without doxycycline showed that DNCT was detected in protein extracts from cells cultured without Dox, but was significantly reduced (~10%) in extracts from cells cultured with Dox (Fig. 1b)
Summary
YAP is involved in contact inhibition, as its phosphorylation and nuclear localization are regulated by cell density through the Hippo signaling pathway[18,19,20]. It was shown that E-cadherin, via the Hippo signaling pathway, directly mediates contact inhibition of proliferation by controlling YAP subcellular localization in human MCF10A mammary epithelial cells and MDA-MB-231 cells[23]. Detailed analysis revealed that expression of the cadherin cytoplasmic domain caused a loss in the contact inhibition of proliferation and conferred resistance to anoikis, while inhibiting β -catenin and YAP/TAZ signaling. This indicated that these signaling pathways did not mediate DNCT functions. Since Merlin is a critical regulator of contact-dependent inhibition of proliferation and its membrane localization is required for its growth-suppressing function, Merlin relocation may have played a role in DNCT activity
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