Cancers arise when cells evade homeostatic mechanisms that restrain their growth. A variety of homeostatic mechanisms, such as apoptosis, proliferation arrest, and senescence, maintain appropriate cell numbers in multicellular organisms and limit the formation of tumors. Acting in concert with these programs is autophagy. Autophagy is a regulated cellular degradative pathway, which may be tumor suppressive in some contexts by promoting cell death or inhibiting genomic instability, but may also promote tumor growth by enhancing cell survival. Recently we described a new cellular mechanism called entosis that, like autophagy, may influence human cancers in complex, context-dependent ways.

Entosis underlies the formation of cell-in-cell structures, where live cells are engulfed inside of their neighbors. These cell structures are reported in a variety of human cancers, but their significance is unknown. The majority of engulfed cells undergo cell death, suggesting that entosis could be a novel mechanism of tumor suppression. Paradoxically, cell-in-cell structures also promote the development of aneuploidy by disrupting cell division. Aneuploidy is thought to drive tumor progression and is a hallmark of aggressive cancers. Thus, like autophagy, entosis has two potential influences on human tumors – tumor suppression or tumor promotion.

Detachment of cells from extracellular matrix is one major mechanism that induces entosis. Within secretory glands such as the mammary gland, epithelial cells are normally organized into a single polarized layer that is adherent to a basement membrane and surrounds a hollow lumen. In early-stage tumors, such as ductal carinoma in-situ (DCIS), cells detach from the basement membrane, and populate the luminal space inside the mammary duct. As epithelial cells require attachment to matrix for survival, a common gain-of-function for breast tumor cells is the ability to survive in the absence of anchorage to the basement membrane. Indeed, one hallmark of many cancer types is the ability of cells to grow in an anchorage-independent fashion. In breast tumors, cell-in-cell structures are found in DCIS tumors, and also late-stage invasive tumors, in matrix-deprived regions. In cultured cells, cell-in-cell structures are prevalent under anchorage-independent conditions, such as growth in soft agar, where entosis may act as a barrier to transformed growth. Interestingly, although cell-in-cell structures are found in human tumors, most tumor cell lines do not exhibit a high rate of entosis compared to non-tumor cells, suggesting that this program is generally suppressed in most cancers.

Cell-in-cell formation occurs by an unusual mechanism involving the formation of adherens junctions between host cells and their targets, followed by engulfment that is driven by Rho-dependent contractile force in internalizing cells, which invade into their hosts. Tumor cells likely do not exhibit entosis owing to compromised cell adhesion, as many breast tumor cell lines, for example, do not express epithelial (E- and P-) cadherins, or α-catenin. We have shown previously that epithelial cadherins, as well as contractile force mediated by Rho, are required for entosis in epithelial cell lines. Here we demonstrate that restoration of epithelial adhesion in tumor cells, by re-expression of E- or P-cadherin, is sufficient to activate entosis, and entotic cell death. As restoration of epithelial adhesion is known to block tumor formation, entosis may be a new mechanism whereby cell adhesion can inhibit transformed growth.

Because entotic cells are engulfed alive, they actually exhibit multiple fates. Whereas the majority undergo a nonapoptotic form of cell death, a smaller percentage are able to survive for extended periods inside of their hosts, and some manage to escape from host cells altogether, emerging unharmed. Cell death involves conversion of the entotic vacuole into a lysosomal compartment, but the cellular pathways that control cell fate are not known. Here we demonstrate that the fate of entotic cells is controlled by the autophagy pathway. Autophagy proteins are normally involved in the formation of double-membrane autophagosomes that mediate bulk cytoplasmic and organelle degradation in cells under conditions of starvation. In epithelial cell lines and tumor cells with restored cell adhesion, we find that autophagy proteins modify entotic vacuole membranes, by a sequence of events including PtdIns(3)P formation and recruitment of Light Chain 3 (LC3). LC3 is lipidated at the entotic vacuole membrane in a manner dependent on core autophagy machinery including ATG5 and ATG7, but not on the mTor-regulated ULK-ATG13-FIP200 complex that is required for autophagy. LC3 lipidation facilitates lysosome fusion to the entotic vacuole, leading to necrotic-like death and degradation of internalized cells, which are murdered by their hosts. Importantly, disruption of autophagy machinery within host cells rescues internalized cells from death, demonstrating a non-cell-autonomous killing activity. Conversely, depletion of autophagic proteins within internalized cells induces apoptosis, due to a cell-autonomous requirement for autophagy in the nutrient deprived environment of the host cell vacuole. The complete rescue of cells from entotic cell death therefore requires the simultaneous inhibition of autophagy and apoptosis, a situation commonly found in human tumors. Accordingly, suppression of entotic cell death promotes the anchorage independent growth of cells in soft agar, a model of tumorgenicity.

Together, these data define a non-cell-autonomous mechanism of cell death, resembling a cell murder, involving autophagy machinery. As entosis is controlled by epithelial adhesion and autophagy, it represents a novel mechanism of cell death that may suppress tumor growth by using known tumor suppressive machinery. Alternatively, while entosis may suppress tumor growth by eliminating detached cells, it is also a strong inducer of aneuploidy, and this feature could be positively selected in some cancers. Our data define a novel cellular mechanism, entosis, that like autophagy, may have multiple, even opposing, influences on tumor growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY09-03. doi:10.1158/1538-7445.AM2011-SY09-03