Abstract
Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT (TertHMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached TertHMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.
Highlights
Anoikis is a particular type of apoptosis that is induced by inadequate or inappropriate cell–extracellular matrix (ECM) interactions, and is the best-characterized phenotype induced by loss of anchorage in anchorage-dependent epithelial cells.[2,3] On the other hand, detachment of cells from ECM has been observed to induce a variety of cell death phenotypes that are distinct from the typical anoikis; these include entosis, autophagy, and squamous transdifferentiation.[4,5,6,7,8] The emerging diversity of cell death phenotypes necessitates extension of the study of adhesion-dependent cell death beyond classical anoikis
Experiments in mice that were genetically deficient for caspasedependent cell death mechanisms revealed the requirement of caspase-dependent cell death for efficient luminal clearing but not the eventual formation of a lumen, suggesting that caspase-independent or non-apoptotic cell death programs function as an alternative mechanism for luminal clearing in the absence of apoptosis.[8]
The apoptosis program is tightly coupled with growth factor signaling; EGFR expression is downregulated upon loss of adhesion, resulting in upregulation of the Bcl-2 protein Bim.[12]
Summary
Anoikis is a particular type of apoptosis that is induced by inadequate or inappropriate cell–ECM interactions, and is the best-characterized phenotype induced by loss of anchorage in anchorage-dependent epithelial cells.[2,3] On the other hand, detachment of cells from ECM has been observed to induce a variety of cell death phenotypes that are distinct from the typical anoikis; these include entosis, autophagy, and squamous transdifferentiation.[4,5,6,7,8] The emerging diversity of cell death phenotypes necessitates extension of the study of adhesion-dependent cell death beyond classical anoikis. A considerable number of studies have suggested that anoikis is the predominant cell death phenotype induced in mammary epithelial cells (MECs) upon anchorage loss;[9,10,11,12,13] many of these studies employed rodent cells or the human cell line MCF-10 A, which has been characterized as being predominantly myoepithelial or classified into basal B subtype.[14,15,16] Given that the majority of malignant breast cancers exhibit the luminal characteristics, a phenotype based on a normal counterpart or a correspondent luminal subtype of human MECs needs to be defined, given the current limited knowledge in this respect. The roles of focal adhesion kinase (FAK) and its atypical signaling mediated by TRAIL (tumor necrosis factor (TNF)related apoptosis-inducing ligand) in this process have been highlighted
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