Elsevier

Neoplasia

Volume 16, Issue 4, April 2014, Pages 365-374.e2
Neoplasia

NHERF1/EBP50 Controls Morphogenesis of 3D Colonic Glands by Stabilizing PTEN and Ezrin-Radixin-Moesin Proteins at the Apical Membrane1,2

https://doi.org/10.1016/j.neo.2014.04.004Get rights and content
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Abstract

Na+/H+ exchanger 3 regulating factor 1/ezrin-radixin-moesin (ERM)–binding phosphoprotein 50 (NHERF1/EBP50), an adaptor molecule that interacts with the ERM–neurofibromatosis type 2 family of cytoskeletal proteins through its ERM-binding region and with phosphatase and tensin homolog (PTEN) and β-catenin through its PDZ domains, has been recently implicated in the progression of various human malignancies, including colorectal cancer (CRC). We report here that NHERF1 controls gland morphogenesis, as demonstrated in three-dimensional (3D) human intestinal glands developing from a single nonpolarized cell. Starting from the early two-cell developmental stage, NHERF1 concentrates at the cellular interface in a central membrane disc that marks the apical pole delimiting the forming lumen. NHERF1 depletion leads to severe disruption of the apical-basal polarity, with formation of enlarged and distorted cell spheroids devoid of a central lumen. This characteristic and the increased number of mitoses in NHERF1-depleted spheroids, including multipolar ones, mimic high-grade dysplasia lesions observed in CRC progression. NHERF1 ERM-binding or PDZ-domain mutants fail to localize apically and impair gland formation most likely by outcompeting endogenous ligands, with the latter mutant completely aborting gland development. Examination of NHERF1 ligands showed that even if both ezrin and moesin colocalized with NHERF1 at the apical membrane, moesin but not ezrin depletion disrupted morphogenesis similarly to NHERF1. NHERF1 depletion resulted also in membrane displacement of PTEN and nuclear translocation of β-catenin, events contributing to polarity loss and increased proliferation. These findings reveal an essential role of NHERF1 in epithelial morphogenesis and polarity and validate this 3D system for modeling the molecular changes observed in CRC.

Abbreviations

3D
three-dimensional
CIP
calf intestinal phosphatase
IF
immunofluorescence
CRC
colorectal cancer
ERM
ezrin-radixin-moesin
NF2
neurofibromatosis type 2
NHERF1/EBP50
Na+/H+ exchanger 3 regulating factor 1/ERM-binding phosphoprotein 50
pHH3
phospho-histone H3
PIP2
phosphatidylinositol 4,5-bisphosphate
PIP3
phosphatidylinositol 3,4,5-trisphosphate
PM
plasma membrane

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1

This work was supported by NCI-CA107201, American Recovery and Reinvestment Act ARRA supplement, (ARRA) supplement and GS Hogan Gastrointestinal Research Fund to M.-M.G. The authors declare no conflict of interest.

2

This article refers to supplementary materials, which are designated by Figures W1 to W3 and are available online at www.neoplasia.com.