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Breast Cancer Research
Published in: Breast Cancer Research 1/2015

Open Access 01-12-2015 | Research article

A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation

Authors: Angela Burleigh, Steven McKinney, Jazmine Brimhall, Damian Yap, Peter Eirew, Steven Poon, Viola Ng, Adrian Wan, Leah Prentice, Lois Annab, J Carl Barrett, Carlos Caldas, Connie Eaves, Samuel Aparicio

Published in: Breast Cancer Research | Issue 1/2015

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Abstract

Introduction

The extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes.

Methods

We developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance.

Results

We identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein–coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype–independent overall survival differences.

Conclusion

Diverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.
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Metadata
Title
A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation
Authors
Angela Burleigh
Steven McKinney
Jazmine Brimhall
Damian Yap
Peter Eirew
Steven Poon
Viola Ng
Adrian Wan
Leah Prentice
Lois Annab
J Carl Barrett
Carlos Caldas
Connie Eaves
Samuel Aparicio
Publication date
01-12-2015
Publisher
BioMed Central
Published in
Breast Cancer Research / Issue 1/2015
Electronic ISSN: 1465-542X
DOI
https://doi.org/10.1186/s13058-014-0510-y

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