Oral Oncology 50 (2014) e9–e11

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Oral Oncology

journal homepage: www.elsevier .com/locate /ora loncology

Letter to the Editor

Does entosis curb the detached cancer cellsbetter?

1368-8375/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.oraloncology.2013.11.010

Cell death can be classified according to its morphologicalappearance, enzymological criteria, functional or immunologicalcharacteristics [1]. The Nomenclature Committee on Cell Death(NCCD) proposed unified criteria for the definition of cell deathand of its different morphologies in 2005 with additions made in2009. It was stated that in the absence of apoptosis, other typesof developmental cell death can occur [2]. The one that caughtour attention was described by Overholtzer et al., 2007; a newmode of non-apoptotic cell elimination called ‘‘entosis’’ thatresembles cell cannibalism and the cell-in-cell phenotype [3].Though reports of cell-in-cell structures date back to the mid1800’s, cellular engulfment or ‘‘cell eating cell’’ has been observedin tumor cells recently. Tumor cells possibly use this strange func-tion to feed in conditions of low nutrient supply [4]. Cell-in-cellstructures can be formed homotypically, between the same celltype, or heterotypically between different cell types. The hetero-typic live cell engulfments involve leukocytes ingested into a vari-ety of host cells such as epithelial cells and fibroblasts. Homotypiclive cell engulfment are seen in malignant tumors of breast, lung,gallbladder, and gastric carcinoma, also in metastatic melanoma[5]. The various terminologies used in the literature to describecell-in-cell structures includes entosis, emperipolesis, cytophago-cytosis, and cannibalism (xeno-cannibalism) [6].

Entosis, a non-apoptotic cell death process, that occurs in hu-man tumors presumed to be provoked by loss of attachment tothe underlying matrix [7]. It describes a cell death mechanismlinked to the ‘cell-in-cell’ phenotype that is frequently exhibitedby non-phagocytic cells in clinical tumour samples, consisting ofinvasion of one live cell into another, followed by degradation ofinternalized cells by lysosomal enzymes. It is to be consideredwhen all the following conditions are satisfied [8].

1. Engulfed cells should never exit the phagosome, and should bedegraded within the lysosome (implying that entosis can beblocked by lysosomal inhibitors).

2. The ‘cell-in-cell’ phenotype should arise from homotypic inter-actions, and should not engage professional phagocytes.

3. The process should be insensitive to chemical and genetic inter-ventions that normally block caspase-dependent and indepen-dent intrinsic apoptosis.

Entosis induces a unique type of cell-in-cell, non-autophago-some dependent lysosomal death pathway [9]. LAMP1, a lysosomalmembrane protein, is found to confine around dying cells and acid-ification occurs at the earlier stages of death, suggesting a lyso-somal involvement. This is an integral part of entosis, making it

different from apoptosis [10]. The differentiating points of entosisfrom apoptosis are outlined in Table 1. In this process of entosis,the effector cells gets enveloped in a vacuole inside target cells,leading to promotion of Light Chain 3 (LC3) recruitment via trans-location onto the entotic vacuole membranes. The translocation ofLC3 depends on autophagy lipidation machinery such as autoph-agy protein 5 (Atg5), Atg7 and the lipid kinase VPS34 (vacuolarprotein sorting 34). After entotic vacuoles fuse with lysosomes oftarget cells, effector cells are deleted by target cells [9]. Also, anunbalanced myosin II dependent force associated with the adher-ens junction compaction drives one cell into another [3].

Leydon was the first to describe cannibalism as ‘‘birds eye cells’’in 1904, which are cells with one or many vacuoles, containingdegrading cells, that push the nuclei to the periphery giving it ashape of a crescent moon [11]. These cells are considered to eateverything without distinguishing between the feeding materials,with a mechanism different from typical phagocytosis [11]. Luginiet al. deduced that the driving force of the ‘‘cannibalistic vacuole’’is represented by a simple and highly efficient mechanism throughwhich any live or dead material touching the tumour cell’s externalmembrane is endocytosed, and digested through a ‘‘quicksand’’like mechanism, thus providing a constant nutrient supply to thetumour cells [12]. They comment about the mechanism involvingan acidic environment, with continuous activation of specific lyticenzymes as cathepsin B. The differentiating points of entosis fromcannibalism are outlined in Table 2 [13].

Abodief et al., in 2006 reported breast tumors with cytologicalfeatures of cell cannibalization resembling entosis but could notexplain the mechanism and significance [14]. Entosis can also betermed as ‘homotypic cannibalism’ where epithelial cells exertautonomous control over their uptake. The formation of adherensjunctions between epithelial cells, mediated by E-cadherin, fol-lowed by engulfment in a Rho-GTPase- and Rho-kinase-dependentmanner is suggestive of an internalizing cells, possibly the uptakeis controlled autonomously by an invasion-like mechanism (Figs. 1and 2) [15]. Repression of entosis, by inhibiting ROCK activity, in-creases anchorage-independent growth of cancer cells as observedby Overholtzer et al., 2007 [3]. Krajcovic et al. reported, this cell incell phenomena can disturb host cell cytokinesis resulting in theformation of a tetraploid cell containing twice the normal amountof chromosomes, and the resulting binucleate host cell gives rise toaneuploid offspring following several divisions [15].

Entosis has been noted in high-grade bladder carcinomas [5]and breast carcinomas [14], exhibiting rapid progression, increasedrecurrence, and decreased overall patient survival, thus indicatingpoor prognosis in cancer [5]. As entosis results mostly in death ofthe internalized cell, it may prove to be a novel pathway to elim-inate detached cancer cells. Novel therapeutic targets directed atthese cells should be developed, which should help curbing thesedetached tumour cells, hence controlling its progression.

Table 1Difference between apoptosis and entosis.

Apoptosis Entosis

Cell death mechanism that is aggravated by loss of attachmentto ECM

Provoked by loss of attachment to ECM, occurs independent of apoptosis

Internalization occurs by caspase activation and driven byphosphatidylserine exposure

Internalization occurs by Rho-GTPase and Rho-kinase suggestive of cell invasion, and notengulfment [3].

Dying cells are cleared by phagocytosis Internalized cells are released or undergo cell division, which highlights a further distinguishingaspect.50% of the internalized cells die

Table 2Difference between entosis and cannibalism.

Cannibalism Entosis

Effector cells Dead or live cells Live cellsTarget cells Tumor cells Tumor cellsFate of effector cells Cell death Cell death or mitosis or releaseTriggering factors Starvation UnknownEngulfment of effector cells Adherens junctions Adherens junctions; Rho-ROCK signalling pathway; myosin-

based contractile forceMolecules participating in the

processesCaveolin-1, actin, ezrin, cathepsin B, a nine transmembranesegment (TM9SF4), vimentin

LC3, Atg5, Atg7, Rho ROCK, Vps34, cadherin

Cell death pathway Lytic enzymes mediation Lysosome-mediated caspase-3 independent cell deathBiological function Nourishment of target cells Suppression of transformed growth; induction of aneuploidy

Fig. 1. Mechanism of Entosis.

Fig. 2. Photomicrograph of an entocysed cell in the tumor stroma.

e10 Letter to the Editor / Oral Oncology 50 (2014) e9–e11

References

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[3] Overholtzer M, Mailleux AA, Mouneimne G, Normand G, Schnitt SJ, King RW,et al. A nonapoptotic cell death process, entosis, that occurs by cell-in-cellinvasion. Cell 2007;131:966–79.

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[7] White E. Entosis: it’s a cell-eat-cell world. Cell 2007;131(5):840–2.[8] Galluzzi L, Vitale1 I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV,

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[9] Florey O, Kim SE, Sandoval CP, Haynes CM, Overholtzer M. Autophagymachinery mediates macroendocytic processing and entotic cell death bytargeting single membranes. Nat Cell Biol 2011;13:1335–43.

[10] Florey O, Krajcovic M, Sun Q, Overholtzer M. Entosis. Current Biology2010;20(3):R88–9.

[11] Fais S. Cannibalism: a way to feed on metastatic tumors. Cancer Lett2007;258(2):155–64.

[12] Lugini L, Matarrese P, Tinari A, Lozupone F, Federici C, Iessi E, et al.Cannibalism of live lymphocytes by human metastatic but not primarymelanoma cells. Cancer Res 2006;66(7):3629–38.

[13] He M-F, Wang S, Wang Y, Wang X-N. Modeling cell-in-cell structure into itsbiological significance. Cell Death Dis 2013;4:e630.

[14] Abodief WT, Dey P, Al-Hattab O. Cell cannibalism in ductal carcinoma ofbreast. Cytopathology 2006;17:304–5.

[15] Krajcovic M, Overholtzer M. Mechanisms of ploidy increase in human cancers:a new role for cell cannibalism. Cancer Res 2012;72(7):1596–601.

Samapika RoutrayDepartment of Oral Pathology & Microbiology, Institute of Dental

Sciences, ‘SOA’ University, Bhubaneswar 751003, Odisha, IndiaTel.: +91 9937149690.

E-mail address: drroutray.samapika@gmail.com

Akhil.A. ShankarDepartment of Oral Pathology & Microbiology, Y.M.T. Dental College,

Institutional Area, Sector-4, Kharghar, Navi Mumbai 410210, IndiaE-mail address: akhil1904@hotmail.com

Niharika SwainDepartment of Oral Pathology & Microbiology, MGM Dental College &

Hospital, Junction of NH-4 and Sion Panvel Expressway, Sector-18,Kamothe, Navi Mumbai 410209, Maharashtra, India

E-mail address: niharikadec30@gmail.com

Available online 18 December 2013