CRB3 and the FERM protein EPB41L4B regulate proliferation of mammary epithelial cells through the release of amphiregulin.

Stephanie J Walker,Ben L Margolis,Laura M Selfors,Joan S Brugge,Sang-Chul Nam

doi:10.1371/journal.pone.0207470

Stephanie J Walker, Ben L Margolis + Show 3 more

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https://doi.org/10.1371/journal.pone.0207470

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Abstract

Numerous observations have suggested a connection between the maintenance of cell polarity and control of cell proliferation; however, the mechanisms underlying these connections remain poorly understood. Here we found that ectopic expression of CRB3, which was previously shown to restore tight junctions and membrane polarity in MCF-10A cells, induced a hyperproliferative phenotype, with significantly enlarged acini in basement membrane culture, similar to structures induced by expression of proliferative oncogenes such as cyclinD1. We found that CRB3-induced proliferation is epidermal growth factor (EGF)-independent and occurs through a mechanism that involves secretion of the EGF-family ligand, amphiregulin (AREG). The increase in AREG secretion is associated with an increase in the number and size of both early and late endosomes. Both the proliferative and endocytic phenotypes associated with CRB3 expression require the FERM-binding domain (FBD) but not the PDZ-binding domain of CRB3, arguing that this proliferative phenotype is independent of the PDZ-dependent polarity signaling by CRB3. We identified the FBD-containing protein, EPB41L4B, as an essential mediator of CRB3-driven proliferation and observed that the CRB3-dependent changes in endocytic trafficking were also dependent on EPB41L4B. Taken together, these data reveal a previously uncharacterized role for CRB3 in regulating proliferation in mammalian cells that is associated with changes in the endocytic trafficking machinery.

Highlights

Glandular epithelial cells, such as those in the mammary gland, are organized into secretory structures with an epithelial monolayer that surrounds a hollow lumen and distinct morphological features, such as specialized cell–cell contacts and a polarized distribution of organelles and membrane proteins
The enlarged CRB3-expressing acini maintained a normal axis of polarity as evidenced by the orientation of the Golgi apparatus toward the apical surface of the acini (Fig 1A), demonstrating that the polarized architecture of MCF-10A acini was not disrupted in the larger structures induced by CRB3 expression
The enlarged CRB3-expressing acini displayed the hallmarks of MCF10A hyperproliferative structures, a phenotype observed in response to expression of oncogenes that allow escape from proliferative suppression, such as cyclinD1 and human papilloma virus E7 [2]

Summary

Introduction

Glandular epithelial cells, such as those in the mammary gland, are organized into secretory structures with an epithelial monolayer that surrounds a hollow lumen and distinct morphological features, such as specialized cell–cell contacts and a polarized distribution of organelles and membrane proteins. A common feature of cancers of epithelial origin is the disruption of normal cell polarity and a loss of the higher order organization of the epithelial tissue. Role of CRB3 and EPB41L4B in mammary epithelial cell proliferation observed in carcinomas. In addition to their known functions in the establishment and maintenance of cell polarity, many of these proteins regulate other processes relevant to tumorigenesis, such as proliferation and apoptosis [1]. The interplay between cell proliferation and epithelial architecture in this system is complex Proliferative oncogenes, such as cyclinD1, induce hyperproliferation of the outer cells leading to enlarged structures [2]. Whether disruption of epithelial architecture plays a causative role in cancer progression or if this loss of organization is a secondary phenotype caused by the transformed state has emerged as an important topic of current research in the field

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