Abstract
Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5+K19- hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5+K19- hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5+K19- hMEC differentiation. The present data validate the utility of the K5+K19- hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation.
Highlights
Molecular profiling of breast cancer revealed unexpected heterogeneity of this disease [1,2]
As for the biochemical mechanisms, it was previously reported that sustained activation of the Epidermal growth factor receptor (EGFR)-MEK-Erk pathway was required for myoepithelial differentiation of primary human mammary epithelial cells (MECs) [23]. Taking these prior observations into consideration, here we examined the effects of three EGFR ligands, epidermal growth factor (EGF), AREG and TGFα in the differentiation-promoting MEGM medium on the human telomerase reverse transcriptase (hTERT)-immortalized stem/progenitor hMEC line characterized by the presence of cytokeratin 5 and absence of cytokeratin 19 (K5+K19- hMEC)
K5+K19- hMEC cells were cultured for three weeks in MEGM medium containing 5 nM EGF and cell differentiation was examined by morphology, immunofluorescence and flow cytometry
Summary
Molecular profiling of breast cancer revealed unexpected heterogeneity of this disease [1,2]. There are technical challenges to follow developmental processes as they progress in vivo To address these issues, Band and colleagues have previously established human telomerase reverse transcriptase (hTERT)-immortalized MEC lines that can be propagated indefinitely in a stem/progenitor-like undifferentiated state but can be induced to differentiate into luminal as well as myoepithelial lineages in vitro under defined conditions [12]. Band and colleagues have previously established human telomerase reverse transcriptase (hTERT)-immortalized MEC lines that can be propagated indefinitely in a stem/progenitor-like undifferentiated state but can be induced to differentiate into luminal as well as myoepithelial lineages in vitro under defined conditions [12] Because they are amenable to complex manipulations, these models should be useful for studying mechanisms of MEC self-renewal, differentiation, transformation and cancer progression
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