Entosis: it's a cell-eat-cell world

In this issue, Overholtzer et al. (2007) describe a new nonapoptotic cell death pathway termed "entosis" in mammary epithelial cells that have detached from the extracellular matrix (ECM). Given that surviving detachment from the ECM is an event associated with the progression of epithelial cancers, entosis--along with apoptosis--may contribute to tumor suppression by promoting the elimination of cancer cells.     

Glycosylation defects,offset by PEPCK-M,drive entosis in breast carcinoma cells

On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from ¹³C-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies.Subject terms: Entosis, Cancer metabolism     

Competition between human cells by entosis

Human carcinomas are comprised of complex mixtures of tumor cells that are known to compete indirectly for nutrients and growth factors. Whether tumor cells could also compete directly, for example by elimination of rivals, is not known. Here we show that human cells can directly compete by a mechanism of engulfment called entosis. By entosis, cells are engulfed, or cannibalized while alive, and subsequently undergo cell death. We find that the identity of engulfing (“winner”) and engulfed (“loser”) cells is dictated by mechanical deformability controlled by RhoA and actomyosin, where tumor cells with high deformability preferentially engulf and outcompete neighboring cells with low deformability in heterogeneous populations. We further find that activated Kras and Rac signaling impart winner status to cells by downregulating contractile myosin, allowing for the internalization of neighboring cells that eventually undergo cell death. Finally, we compute the energy landscape of cell-in-cell formation, demonstrating that a mechanical differential between winner and loser cells is required for entosis to proceed. These data define a mechanism of competition in mammalian cells that occurs in human tumors.     

Cell death leaves a new TRAIL

Cell death involves numerous mechanisms that can be cross-regulated through a complex signaling network. In this issue, Bozkurt et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202010030) identify a new connection in the network: signaling from TRAIL, a canonical inducer of apoptosis, can also induce a form of cell death called entosis, which has implications for cancer progression.

“I will not follow where the path may lead, but I will go where there is no path, and I will leave a trail” (1).Cell death, it turns out, is complicated. While it was once thought there was a single mechanism, one path that could lead to the death of metazoan cells, now up to 12 or more different paths are known. Regulated forms of necrosis (e.g., necroptosis, pyroptosis, and ferroptosis), a type of cell ingestion called entosis, and at least seven other mechanisms can, like the classical apoptosis, eliminate damaged or infected cells or remove supernumerary cells during development (2).Although some forms of cell death have unique properties that make them well suited for specific contexts, many forms appear to occur more broadly. Cell death responses to stress or infection, for example, can involve the parallel induction of numerus forms of cell death—apoptosis, forms of necrosis, and entosis—each occurring at different frequencies within a cell population (3, 4, 5). While crosstalk between some mechanisms (e.g., apoptosis, necroptosis, and pyroptosis) has been extensively studied (2), little is known about how other forms of cell death, such as entosis, are regulated within mixtures. How mixed death profiles might impact physiology is also not well understood. In this issue, Bozkurt et al. uncover an unexpected path that may reveal some important new clues: signaling from the TNF-related apoptosis-inducing ligand (TRAIL), a well-known inducer of apoptosis, can initiate entosis as well (Fig. 1; 6).Open in a separate windowFigure 1.A new TRAIL of cell death. Signaling from TRAIL through its receptors (DR4/5) is well known to induce apoptosis (left path) by a mechanism involving formation of a death-induced signaling complex involving fas-associated death domain protein (FADD) and caspase-8, and subsequent activation of caspase-8 activity that leads to cell death. Bozkurt et al. now discover that TRAIL can also induce entosis (right path) through a mechanism requiring DR4/5 and a noncatalytic function of caspase-8 (6). Whereas apoptosis is executed in a cell-autonomous manner and is largely anti-inflammatory, entosis is executed in a non-cell-autonomous manner and is competitive between cells.To examine the spatiotemporal dynamics of cell death induced by TRAIL, the authors used colon cancer cells expressing a fluorescence resonance energy transfer–based reporter of caspase activity, a hallmark of apoptosis, as well as tetramethylrhodamine methyl ester (TMRM) staining to indicate mitochondrial membrane potential, and examined cells by time-lapse microscopy. Some cells underwent apoptosis, as indicated by the induction of caspase activity and loss of mitochondrial membrane potential, but others showed different patterns, with slower kinetics of caspase activation or in some cases increased TMRM staining, an unusual observation that prompted further examination. By carefully inspecting cell morphology, the authors observed engulfment events involving whole cells within the TRAIL-treated cultures that were reminiscent of entosis, a death mechanism that results from the ingestion of live cells by their neighbors. Indeed, through further examination of the localization of cell adhesion proteins and functional requirement for the cytoskeletal regulator Rho-kinase, which mediates entotic cell ingestion, the authors showed that entosis is induced by TRAIL treatment.The induction of entosis by a canonical apoptosis-inducing ligand was surprising and presented an opportunity for the authors to identify new regulators of this unusual mechanism. By using knockout and inhibitor-based strategies, they showed that the death receptors to which TRAIL binds, called death domain–containing receptors 4 and 5 (DR4 and DR5), were required for entosis induction, and that, intriguingly, the presence of caspase-8, but not its activity, was required for entosis as well. They further demonstrated that while ingested cells underwent what is called entotic cell death, characterized by the recruitment of lysosomes and acidification of the large endocytic compartment (an activity that also accounted for the increased TMRM staining they initially observed), apoptotic factors were involved in the execution of cell death in this context. The knockout of BAX and BAK, whose protein products function at mitochondria to control apoptosis, as well as the inhibition of caspase activity, reduced the percentage of entotic cells that died and increased the percentage that escaped from their hosts and were rescued.To begin to investigate how the regulation of entosis by TRAIL might relate to pathophysiology, the authors examined colorectal cancer specimens, where entotic cell structures could be identified by histology and the expression levels of components of the TRAIL signaling pathway could also be quantified (6). Notably, correlations were identified between entotic structures and the expression of TRAIL and cellular FLICE-like inhibitory protein, a factor that binds to a TRAIL-induced signaling complex, with a trend toward caspase-8 as well, consistent with TRAIL-mediated regulation of entosis in colorectal cancer. The authors further identified a correlation between the presence of entotic structures at the invasive front of stage III colon cancer specimens and poor patient outcomes, suggesting that the induction of entosis in response to TRAIL signaling could promote the development of more aggressive disease.These findings by Bozkurt et al. uncover an important new path that leads to entosis, underscoring newfound complexity and the parallel nature of death signaling (Fig. 1). Different death mechanisms have unique properties and physiological effects (2). Apoptosis, for example, occurs mostly silently, or undetected by the immune system, a feature that is particularly well suited for death in normal tissues. Forms of regulated necrosis, on the other hand, spew intracellular contents and can alert immune responses to infection (2). Entosis may be the most unusual because it involves the ingestion and killing of individual cells by their neighbors, a form of death that is non-cell-autonomous in nature and intrinsically competitive between individual cells within a population. The findings in this study identify an important signaling node involving caspase-8 that now connects each of these multiple forms of cell death. While caspase-8 activity is required for apoptosis, here entosis is shown to be unaffected by caspase activity but curiously requires the presence of the caspase-8 protein. Noncatalytic functions for caspase-8 have been reported, including, intriguingly, regulation of the activity of the Rac-GTPase, a known regulator of entosis (7), through interaction with the PI-3-kinase scaffolding subunit p85 (8), as well as control over inflammatory signaling through formation of a complex called the “FADDosome” (9). Whether these or other reported noncatalytic functions of caspase-8 might contribute to entosis regulation will be important to uncover in future studies.The parallel induction of different death mechanisms suggests that the physiological effects of cell death, for example during cancer therapy, relate not only to the extent of death that occurs but also to the types of death and their relative proportions within a population. Treatments inducing more necrosis than apoptosis, for example, could generate more pronounced immune responses. A more predominant induction of entosis is predicted to select for cells that can ingest their neighbors, called “winners,” which have been shown to have competitive advantages and could promote the development of more aggressive disease (10). While the authors show that TRAIL receptors are required within the cells that are internalized when DR4/5 knockout cells are mixed with control cells, they also find that the winner cells exhibit lower levels of caspase activation than others in response to TRAIL, suggesting, overall, that entosis may select for cells with resistance to TRAIL-induced cell death. TRAIL treatment is known to result in the fractional killing of tumor cells (11), which may limit the efficacy of TRAIL in cancer therapy. Its newfound control over entosis raises the possibility that the fraction that survives might also be selected through this competitive mechanism.     

Induction of entosis by epithelial cadherin expression

Cell engulfment typically targets dead or dying cells for clearance from metazoan tissues. However, recent evidence demonstrates that live cells can also be targeted and that engulfment can cause cell death. Entosis is one mechanism proposed to mediate the engulfment and killing of live tumor cells by their neighbors, an activity often referred to as cell cannibalism. Here we report that the expression of exogenous epithelial cadherin proteins (E- or P-cadherin) in human breast tumor cells lacking endogenous expression of epithelial cadherins induces entosis and inhibits transformed growth. Entosis induced by cadherin expression is associated with the polarized distribution of Rho and Rho-kinase (ROCK) activity within entotic cells, which is dependent on p190A RhoGAP activity. ROCK inhibition or downregulation of p190A RhoGAP expression reduces entosis and increases the transformed growth of epithelial cadherin-expressing tumor cells. These data define new cell systems for the study of entosis, and identify entosis as a mechanism of cell cannibalism that is induced by the establishment of epithelial adhesion and inhibits transformed growth.     

Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling

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.Normal cells undergo cell death and/or growth arrest in the absence of attachment to extracellular matrix (ECM) or upon contact with abnormal or ectopic ECM, which constitutes a physiologically important defense mechanism in multicellular organisms for preventing re-adhesion of detached cells to foreign matrices and their dysplastic growth in inappropriate sites.^{1, 2} On the other hand, the process of cancer metastasis demands that cancer cells circumvent such cell death/growth arrest. This is true even for incipient tumors, where outgrowth and displacement of cells from their original location in a mass result in loss of adequate contact of cells with innate ECM. Cells that disseminate through foreign stroma experience more deviant conditions, and upon reaching the parenchyma of distant organs need to adapt to the non-permissive matrix in the foreign tissue. To survive through this process, cancer cells acquire resistance to cell death/growth arrest induced in the absence of appropriate adhesion to ECM. Therefore, the eradication of cancer cells in ectopic environments requires an understanding of their resistance to anchorage dependence for growth and survival based on responsiveness of their normal counterparts.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} however, 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, particularly given the current limited knowledge in this respect.In the present study, we characterized anchorage loss-induced cell death in MECs using primary human MECs immortalized with hTERT (^TertHMEC).^{17, 18} The established cells are potential stem/progenitors of mammary epithelial cells¹⁸ and show a partial differentiation toward to the luminal phenotype in the culture system developed by Stampfer et al (http://hmec.lbl.gov/mreview.htm). Unlike previous observations based on MCF-10 A cells, the detached ^TertHMECs were found to have an apparent defect in the execution of apoptosis and instead, underwent non-apoptotic cell death through simultaneous entosis, cornification, and necrotic processes. 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.     

TRAIL signaling promotes entosis in colorectal cancer

Entosis is a form of nonphagocytic cell-in-cell (CIC) interaction where a living cell enters into another. Tumors show evidence of entosis; however, factors controlling entosis remain to be elucidated. Here, we find that besides inducing apoptosis, TRAIL signaling is a potent activator of entosis in colon cancer cells. Initiation of both apoptosis and entosis requires TRAIL receptors DR4 and DR5; however, induction of apoptosis and entosis diverges at caspase-8 as its structural presence is sufficient for induction of entosis but not apoptosis. Although apoptosis and entosis are morphologically and biochemically distinct, knockout of Bax and Bak, or inhibition of caspases, also inhibits entotic cell death and promotes survival and release of inner cells. Analysis of colorectal cancer tumors reveals a significant association between TRAIL signaling and CIC structures. Finally, the presence of CIC structures in the invasive front regions of colorectal tumors shows a strong correlation with adverse patient prognosis.     

Cell death in intervertebral disc degeneration

Degeneration of intervertebral disc (IVD) is mainly a chronic process of excessive destruction of the extracellular matrix (ECM), and also is thought to be the primary cause of low back pain. Presently, however, the underlying mechanism of IVD degeneration is still not elucidated. Cellular loss from cell death has been believed to contribute to the degradation of ECM and plays an important role in the process of IVD degeneration, but the mechanisms of cell death in degenerated IVD remain unclear. Apoptosis, a very important type of IVD cell death, has been considered to play a crucial role in the process of degeneration. Autophagy, a non-apoptosis death type of programmed cell death, has been considered extensively involved in many pathological and physiological processes, including the degenerative diseases. Thus, the research on cell death in IVD degeneration has become a new focus recently. In this review, by analyzing the available literature pertaining to cell death in IVD and discussing the inducing factors of IVD degeneration, NP cells and ECM in IVD degeneration, apoptotic signal transduction pathways involved in IVD cell death, the relationship of cell death with IVD degeneration and potential therapeutic strategy for IVD degeneration by regulating cell death, we conclude that different stimuli induce cell death in IVD via various signal transduction pathways, and that cell death may play a key role in the degenerative process of IVD. Regulation of cell death could be a potential and attractive therapeutic strategy for IVD degeneration.     

Programmed cell death in intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is largely a process of destruction and failure of the extracellular matrix (ECM), and symptomatic IVD degeneration is thought to be one of the leading causes of morbidity or life quality deterioration in the elderly. To date, however, the mechanism of IVD degeneration is still not fully understood. Cellular loss from cell death in the process of IVD degeneration has long been confirmed and considered to contribute to ECM degradation, but the causes and the manners of IVD cell death remain unclear. Programmed cell death (PCD) is executed by an active cellular process and is extensively involved in many physiological and pathological processes, including embryonic development and human degenerative diseases. Thus, the relationship between PCD and IVD degeneration has become a new research focus of interest in recent years. By reviewing the available literature concentrated on PCD in IVD and discussing the methodology of detecting PCD in IVD cells, its inducing factors, the relationship of cell death to ECM degradation, and the potential therapy for IVD degeneration by modulation of PCD, we conclude that IVD cells undergo PCD via different signal transduction pathways in response to different stimuli, that PCD may play a role in the process of IVD degeneration, and that modulation of PCD might be a potential therapeutic strategy for IVD degeneration.     

Integrin–ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. We recently identified Diedel, a secreted protein/cytokine, as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. Here, we show that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, we uncovered a resilience mechanism of epithelial tissues, mediated by Integrin–ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, we found that resilient epithelial cells, enriched for Diedel–Integrin–ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin–ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin–ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.

A key feature of the resilience mechanisms that underlie the control of epithelial tissue damage is the balance between cell death and survival. This study shows that the anti-oxidant enzyme catalase can relocate to membranes in order to promote the resilience of the Drosophila midgut epithelium, synergizing with integrin-ECM interactions to prevent the spread of cell death.     

Loss of 4.1N in epithelial ovarian cancer results in EMT and matrix-detached cell death resistance

Epithelial ovarian cancer(EOC)is one of the leading causes of death from gynecologic cancers and peritoneal dissemination is the major cause of death in patients with EOC.Although the loss of 4.1N is associated with increased risk of malignancy,its association with EOC remains unclear.To explore the underlying mechanism of the loss of 4.1N in constitutive activation of epithelial-mesenchymal transition(EMT)and matrixdetached cell death resistance,we investigated samples from 268 formalin-fixed EOC tissues and performed various in vitro and in vivo assays.We report that the loss of 4.1N correlated with progress in clinical stage,as well as poor survival in EOC patients.The loss of 4.1N induces EMT in adherent EOC cells and its expression inhibits anoikis resistance and EMT by directly binding and accelerating the degradation of 14-3-3 in suspension EOC cells.Furthermore,the loss of 4.1N could increase the rate of entosis,which aggravates cell death resistance in suspension EOC cells.Moreover,xenograft tumors in nude mice also show that the loss of 4.1N can aggravate peritoneal dissemination of EOC cells.Single-agent and combination therapy with a ROCK inhibitor and a 14-3-3 antagonist can reduce tumor spread to varying degrees.Our results not only define the vital role of 4.1N loss in inducing EMT,anoikis resistance,and entosis-induced cell death resistance in EOC,but also suggest that individual or combined application of 4.1N,14-3-3 antagonists,and entosis inhibitors may be a promising therapeutic approach for the treatment of EOC.     

A Highlights from MBoC Selection: mTOR regulates phagosome and entotic vacuole fission

Macroendocytic vacuoles formed by phagocytosis, or the live-cell engulfment program entosis, undergo sequential steps of maturation, leading to the fusion of lysosomes that digest internalized cargo. After cargo digestion, nutrients must be exported to the cytosol, and vacuole membranes must be processed by mechanisms that remain poorly defined. Here we find that phagosomes and entotic vacuoles undergo a late maturation step characterized by fission, which redistributes vacuolar contents into lysosomal networks. Vacuole fission is regulated by the serine/threonine protein kinase mammalian target of rapamycin complex 1 (mTORC1), which localizes to vacuole membranes surrounding engulfed cells. Degrading engulfed cells supply engulfing cells with amino acids that are used in translation, and rescue cell survival and mTORC1 activity in starved macrophages and tumor cells. These data identify a late stage of phagocytosis and entosis that involves processing of large vacuoles by mTOR-regulated membrane fission.     

Regulation of myosin activation during cell-cell contact formation by Par3-Lgl antagonism: entosis without matrix detachment

Cell-cell contact formation following cadherin engagement requires actomyosin contraction along the periphery of cell-cell contact. The molecular mechanisms that regulate myosin activation during this process are not clear. In this paper, we show that two polarity proteins, partitioning defective 3 homologue (Par3) and mammalian homologues of Drosophila Lethal (2) Giant Larvae (Lgl1/2), antagonize each other in modulating myosin II activation during cell-cell contact formation in Madin-Darby canine kidney cells. While overexpression of Lgl1/2 or depletion of endogenous Par3 leads to enhanced myosin II activation, knockdown of Lgl1/2 does the opposite. Intriguingly, altering the counteraction between Par3 and Lgl1/2 induces cell-cell internalization during early cell-cell contact formation, which involves active invasion of the lateral cell-cell contact underneath the apical-junctional complexes and requires activation of the Rho-Rho-associated, coiled-coil containing protein kinase (ROCK)-myosin pathway. This is followed by predominantly nonapoptotic cell-in-cell death of the internalized cells and frequent aneuploidy of the host cells. Such effects are reminiscent of entosis, a recently described process observed when mammary gland epithelial cells were cultured in suspension. We propose that entosis could occur without matrix detachment and that overactivation of myosin or unbalanced myosin activation between contacting cells may be the driving force for entosis in epithelial cells.     

Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response

Apoptosis serves as a protective mechanism by eliminating damaged cells through programmed cell death. After apoptotic cells pass critical checkpoints, including mitochondrial fragmentation, executioner caspase activation, and DNA damage, it is assumed that cell death inevitably follows. However, this assumption has not been tested directly. Here we report an unexpected reversal of late-stage apoptosis in primary liver and heart cells, macrophages, NIH 3T3 fibroblasts, cervical cancer HeLa cells, and brain cells. After exposure to an inducer of apoptosis, cells exhibited multiple morphological and biochemical hallmarks of late-stage apoptosis, including mitochondrial fragmentation, caspase-3 activation, and DNA damage. Surprisingly, the vast majority of dying cells arrested the apoptotic process and recovered when the inducer was washed away. Of importance, some cells acquired permanent genetic changes and underwent oncogenic transformation at a higher frequency than controls. Global gene expression analysis identified a molecular signature of the reversal process. We propose that reversal of apoptosis is an unanticipated mechanism to rescue cells from crisis and propose to name this mechanism "anastasis" (Greek for "rising to life"). Whereas carcinogenesis represents a harmful side effect, potential benefits of anastasis could include preservation of cells that are difficult to replace and stress-induced genetic diversity.     

Entosis,a key player in cancer cell competition

Cell-in-cell structures, also referred to as ''entosis'', are frequently found in human malignancies, although their prognostic impact remains to be defined. Two articles recently published in Cell Research report the stimulation of entosis by one prominent oncogene, Kras, as well as by one class of tumor suppressors, namely epithelial cadherins E and P, illustrating the complex regulation of this biological process.A number of different terms have been used to describe live cell engulfments giving rise to cell-in-cell structures (CICS): entosis, emperipolesis, cannibalism and phagocytosis. Heterotypic live cell engulfment usually involves the ingestion of leukocytes by non-leukocytes (such as epithelial cells or fibroblasts). Homotypic live cell engulfment (among cells of the same type) mostly occurs in cancers, probably reflecting major alterations in cellular physiology that are associated with oncogenesis and tumor progression.CICS can be visualized by conventional hematoxylin-eosin staining and have been described to occur in many different human cancers¹. CICS produced as the result of entosis exhibit β-catenin localization patterns that are indicative of a cell junction-mediated mechanism of engulfment, and this polarized distribution of β-catenin can be taken advantage of to visualize CICS in vivo, in tumors¹. However, the prognostic impact of CICS is highly context-dependent. Thus, CICS are particularly frequent in high-grade, aggressive breast cancer with dismal prognosis². CICS are only found in castration-resistant, not in androgen-dependent, prostate cancer and hence correlate with poor prognosis in this particular malignancy³. In contrast, in pancreas adenocarcinomas, high levels of CICS correlate with a lower incidence of metastases⁴. These findings point to a complex role of CICS in cancer biology.Two papers by Sun et al.^5,6 recently published in Cell Research characterized one particular mechanism of homotypic live cell engulfment termed entosis. The first paper of this series⁵ provides evidence that one of the most prominent oncogenes, activated Kras, can stimulate entosis, while the second paper⁶ demonstrates that a prominent tumor suppressor, epithelial cadherin (E-cadherin), can increase entosis as well.Cancers are highly complex mixtures of cells in which the malignant population is genetically and epigenetically heterogeneous, reflecting a history of clonal selection. One particular type of competition among distinct cells may consist in the engulfment of one cell (the ''loser'') by another (the ''winner''), as demonstrated by Sun et al. in several cell culture models, as well as in human cancers that were xenografted into immunodeficient mice⁵. Importantly, co-culture of non-transformed cells with their malignant counterparts systematically leads to engulfment of the former by the latter, suggesting that oncogenic transformation is coupled to the ''winner'' status⁵. Indeed, competition by entosis leads to the physical elimination of the ''loser'' cells, which usually succumb to non-apoptotic cell death as soon as the phagosome enveloping the engulfed cell is decorated with LC3 and then fuses with lysosomes^1,7. What is then the difference between ''loser'' and ''winner'' cells? Sun et al.⁵ propose that one cardinal feature of ''winners'' is a high degree of mechanic deformability, as demonstrated by biophysical experiments and computer simulations. This is a highly provocative finding because human tumors are known to be more mechanically heterogeneous than normal tissues and that tumor progression is increased with an elevated mechanic deformability of the cancer cells. This reduction in cell stiffness may hence not only increase the metastatic potential of tumor cells⁸, but may also reflect an increased entotic activity⁵.Transfection-enforced expression of active KrasV12 was sufficient to confer winner status onto non-tumorigenic cells, correlating with an increase in mechanic deformability⁵. This effect of KrasV12 relied on Rac1, as demonstrated by the facts that knockdown of Rac1 suppressed the ''winner'' status conferred by KrasV12, expression of constitutively active Rac1 induced a ''winner'' phenotype and dominant-negative Rac1N17 imparted a ''loser'' status⁵. However, at this point it remains to be explored whether other pathways downstream of Kras such as the phosphatidylinositide 3-kinases (PI3K)/protein kinase B (PKB, best known as AKT)/mechanistic target of rapamycin (mTOR) pathway may contribute to ''winner'' status. Inhibition of mTOR interferes with degradation of engulfed cells⁹, suggesting that activation of the PI3K/AKT/mTOR axis might favor the manifestation of the ''winner'' phenotype as well. Similarly, it remains an open question as to whether other oncogenes than Kras may regulate entosis as well.Breast cancers cells engineered to express epithelal E- or P-cadherins (but not mesenchymal-type cadherins, such as N-cadherin and cadherin-11) re-establish epithelial junctions and engulf and kill non-transfected parental cells in transformed growth assays⁶. The induction of entosis by epithelial E- or P-cadherins is associated to the polarized distribution of RhoA and contractile actomyosin dependent on the p190A Rho-GTPase-Activating Protein (p190A RhoGAP) that is recruited to epithelial junctions⁶. Inhibition of RhoA by overexpression of RhoA-N19 or p190A RhoGAP was sufficient to impart winner status to cells mixed with controls, whereas overexpression of RhoA, ROCKI, or ROCKII had the opposite effect and hence created ''loser'' cells⁵. It has been known that Rho-GTPase and Rho-kinase are not required in engulfing cells but are required in internalizing cells¹, underscoring the idea that ''loser'' cells are not just passive ''victims'' of a cannibalistic attack but somehow contribute to their fatal fate. The ''loser'' status was accompanied by the ROCK-dependent accumulation of actomyosin⁶, and computer simulations suggest that actomyosin contractility within ''loser'' cells constitutes a critical driving force of entosis⁵. The levels of phosphorylated myosin light chain 2 at Ser19 (pMLC2), a readout of contractile myosin downstream of ROCKI/II, were also increased in ''loser'' cells as compared to ''winners''⁶. RhoA, ROCKI/II, MLC2, actin and myosins all accumulated at particularly high levels in ''losers'' at the cell cortex oriented away from cell-cell adhesions⁶.The aforementioned data support a dual implication of entosis in carcinogenesis (Figure 1). On one hand, entosis carried out by ''winner'' cells may constitute a competitive advantage of aggressive tumor cells, perhaps allowing the ''winners'' to retrieve amino acids and other building blocks for anabolic reaction from their cannibalistic activity⁹ or increasing their genomic instability subsequent to mitotic aberrations^2,10. In this context, pharmacological suppression of entosis by Y27632, a ROCKI/II inhibitor, abolished the competitive advantage of transformed cells over their non-transformed siblings in mixed culture experiments⁵. On the other hand, stimulation of entosis by re-expression of epithelal E- or P-cadherins reduced the clonogenic potential of breast cancer cells. In this context, Y27632 facilitated tumor cell growth in vitro⁶. These observations underscore the need of exploring the detailed mechanisms through which entosis may repress or favor oncogenesis and tumor progression.Open in a separate windowFigure 1A dual role for entosis in cancer. (A) Entosis as a pro-tumorigenic process. (B) Entosis as a tumor-suppressive mechanism.     

Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes

Autophagy normally involves the formation of double-membrane autophagosomes that mediate bulk cytoplasmic and organelle degradation. Here we report the modification of single-membrane vacuoles in cells by autophagy proteins. LC3 (Light chain 3) a component of autophagosomes, is recruited to single-membrane entotic vacuoles, macropinosomes and phagosomes harbouring apoptotic cells, in a manner dependent on the lipidation machinery including ATG5 and ATG7, and the class III phosphatidylinositol-3-kinase VPS34. These downstream components of the autophagy machinery, but not the upstream mammalian Tor (mTor)-regulated ULK-ATG13-FIP200 complex, facilitate lysosome fusion to single membranes and the degradation of internalized cargo. For entosis, a live-cell-engulfment program, the autophagy-protein-dependent fusion of lysosomes to vacuolar membranes leads to the death of internalized cells. As pathogen-containing phagosomes can be targeted in a similar manner, the death of epithelial cells by this mechanism mimics pathogen destruction. These data demonstrate that proteins of the autophagy pathway can target single-membrane vacuoles in cells in the absence of pathogenic organisms.     

The regulation of cancer cell death and metabolism by extracellular matrix attachment

The metastasis of cancer cells to distant sites is responsible for the vast majority of cancer mortalities yet the molecular mechanisms underlying this extraordinarily complicated process have yet to be sufficiently elucidated. Recently, it has become clear that cancer cells need to inhibit anoikis, a cell death program induced by loss of attachment to the extracellular matrix (ECM), in order to successfully metastasize. These studies have motivated additional research into the relationship between ECM-detachment and cell viability, much of which reveals integral connections between ECM-detachment and cell metabolism. This review serves to thoroughly discuss the signaling pathways and metabolic changes that are induced by ECM-detachment. In addition, the molecular mechanisms by which cancer cells can alter signaling and metabolism to survive in the absence of ECM-attachment will be highlighted. Furthermore, cell death mechanisms that have been observed or implicated in cells detached from the ECM will also be examined. In aggregate, the studies discussed in this review reveal that ECM-detachment can regulate cancer cell metabolism in a variety of distinct cell types and suggest that interfering with metabolism in ECM-detached cells may be a novel and effective chemotherapeutic approach to selectively inhibit tumor progression.     

Cancer cell cannibalism: Multiple triggers emerge for entosis

Entosis is a form of epithelial cell engulfment and cannibalism prevalent in human cancer. Until recently, the only known trigger for entosis was loss of attachment to the extracellular matrix, as often occurs in the tumour microenvironment. However, two new studies now reveal that entosis can also occur among adherent epithelial cells, induced by mitosis or glucose starvation. Together, these findings point to the intriguing notion that certain hallmark properties of cancer cells, including anchorage independence, aberrant proliferation and metabolic stress, can converge on the induction of cell cannibalism, a phenomenon so frequently observed in tumours. In this review, we explore the molecular, cellular and biophysical mechanisms underlying entosis and discuss the impact of cell cannibalism on tumour biology.     

Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end-tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II-mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs.     

Cell migration—The role of integrin glycosylation

Background

Cell migration is an essential process in organ homeostasis, in inflammation, and also in metastasis, the main cause of death from cancer. The extracellular matrix (ECM) serves as the molecular scaffold for cell adhesion and migration; in the first phase of migration, adhesion of cells to the ECM is critical. Engagement of integrin receptors with ECM ligands gives rise to the formation of complex multiprotein structures which link the ECM to the cytoplasmic actin skeleton. Both ECM proteins and the adhesion receptors are glycoproteins, and it is well accepted that N-glycans modulate their conformation and activity, thereby affecting cell–ECM interactions. Likely targets for glycosylation are the integrins, whose ability to form functional dimers depends upon the presence of N-linked oligosaccharides. Cell migratory behavior may depend on the level of expression of adhesion proteins, and their N-glycosylation that affect receptor-ligand binding.

Scope of review

The mechanism underlying the effect of integrin glycosylation on migration is still unknown, but results gained from integrins with artificial or mutated N-glycosylation sites provide evidence that integrin function can be regulated by changes in glycosylation.

General significance

A better understanding of the molecular mechanism of cell migration processes could lead to novel diagnostic and therapeutic approaches and applications. For this, the proteins and oligosaccharides involved in these events need to be characterized.