Entosis: aneuploidy by invasion - Nature

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is a potent inhibitor of lysosomal cysteine peptidases12. However, Spi2A, like all clade. A serpins, encodes a signal peptide and is secreted13. So, how could ...
newS and viewS is a potent inhibitor of lysosomal cysteine peptidases12. However, Spi2A, like all clade A serpins, encodes a signal peptide and is secreted13. So, how could Spi2A be secreted and make its way back to the cytosol in sufficient quantities to block the enzymatic activities of cathepsins B and L? Interestingly, previous studies show that Spi2A can use an alternative 5ʹ translational start site to exclude the signal peptide14. This amino-terminally truncated serpin, which is also a functional peptidase inhibitor, is directed to the cytosol. Thus, an alternatively spliced Spi2A could be well positioned to serve as the cytosolic inhibitor of cathepsins B and L that is, in turn, downregulated by Stat3 during involution. However, there are several caveats to this scenario. First, it is not known whether alternative start-site utilization in Spi2A occurs frequently, if at all in mammary gland tissue. Second, there is no Spi2A orthologue in humans. Do other clade A serpins assume this role? Currently, there are no data showing that human clade A serpins inhibit cysteine peptidases or that they are targeted to the cytosol by using alternative splicing to bypass their signal peptides. Third, although the expression studies failed to detect any clade B serpins that were downregulated during involution, only a few of these paralogues are included as probes on the microarray. As the clade B repertoire in

the mouse (n = 28) is greatly expanded relative to that of humans (n = 13; ref. 6), and many of the clade B serpins (SERPINB1, -3, 4, -6 and -13) neutralize papain-like cysteine peptidases, there is a possibility that one of these serpins, and not Spi2A, is responsible for blocking mammary gland involution. Nonetheless, these findings contribute significantly to the mounting evidence that intracellular serpins capable of neutralizing lysosomal cysteine peptidases have a crucial role in blocking lysosomal-mediated cell death6. Although the leakage of lysosomal cysteine peptidases into the cytosol can augment caspase-dependent and -independent apoptosis, massive increases in LMP are likely to occur only in apoptotic cells that avoid phagocytosis and subsequently acquire a ‘necrotic’ appearance15. In contrast to apoptosis, the abrupt, widespread and conspicuous increase in LMP, visible by fluorescence microscopy, is emerging as one of the early features of necrotic cell death, regardless of the proximal signalling events2,3,6,15,16. Although most of these signalling events were considered pathologic stressors, the data from this study suggests that physiological stressors, such as hormone withdrawal, can also activate some forms of physiological necrosis. Thus, even the ‘Darth Vader’ of cell death (necrosis) can be turned to the Light side by being co-opted to perform a beneficial biological function. A

future challenge is to determine how this system can be modulated for clinical purposes. By identifying compounds that modulate key regulatory molecules (such as Stat3 and intracellular serpins) it may be possible to deactivate this powerful death pathway in myocardial cells or neurons after a myocardial infarction or stroke, respectively, or to activate the system in malignant cells. Let the Force be with you…whichever side you prefer. Competing finAnCiAl inteRests The authors declare no competing financial interests. 1. Kroemer, G. et al. Cell Death Differ. 16, 3–11 (2009). 2. Zong, W. X. & Thompson, C. B. Genes Dev. 20, 1–15 (2006). 3. Festjens, N., Vanden Berghe, T. & Vandenabeele, P. Biochim. Biophys. Acta 1757, 1371–1387 (2006). 4. Golstein, P. & Kroemer, G. Trends Biochem. Sci. 32, 37–43 (2007). 5. Boya, P. & Kroemer, G. Oncogene 27, 6434–6451 (2008). 6. Luke, C. J. et al. Cell 130, 1108–1119 (2007). 7. Kreuzaler, P. A. et al. Nat. Cell Biol. 13, 303–309 (2011). 8. Chapman, R. S. et al. Genes Dev. 13, 2604–2616 (1999). 9. Fehrenbacher, N. et al. Cancer Res. 68, 6623–6633 (2008). 10. Goulet, B. et al. Mol. Cell 14, 207–219 (2004). 11. Clarkson, R. W. et al. Breast Cancer Res. 6, R92–109 (2004). 12. Liu, N. et al. EMBO J. 22, 5313–5322 (2003). 13. Silverman, G. A. et al. J. Biol. Chem. 285, 24299– 24305 (2010). 14. Inglis, J. D., Lee, M., Davidson, D. R. & Hill, R. E. Gene 106, 213–220 (1991). 15. Berghe, T. V. et al. Cell Death Differ. 17, 922–930 (2010). 16. Yamashima, T. & Oikawa, S. Prog. Neurobiol. 89, 343– 358 (2009).

entosis: aneuploidy by invasion Aniek Janssen and René H. medema aneuploidy is one of the most prevalent phenotypes of human tumours, but the underlying cause of this phenomenon remains highly debated. entosis, the invasion of a living cell into another cell’s cytoplasm, is now shown to perturb cytokinesis and induce the formation of aneuploid cells. Numerical chromosomal aberrations, termed aneuploidy, were suggested long ago to contribute to tumourigenesis1. However, how tumour cells acquire abnormal chromosome numbers in vivo is a persisting question in tumour biology. Aneuploidy was first induced experimentally almost a century ago with the introduction Aniek Janssen and René H. Medema are in the Department of Medical Oncology and Cancer Genomics Centre, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands. e-mail: [email protected]

of two sperm cells into a single sea urchin egg and the subsequent shaking of the dispermic egg to produce a tripolar spindle1. On page 324 of this issue, Krajcovic et al. now show that nature has adopted an even more remarkable way of producing aneuploid cells, through a process called entosis, whereby an intact cell invades a host cell’s cytoplasm2. The presence of cell-in-cell structures, in which a complete cell resides in the cytoplasm of a neighbouring host cell, is commonly observed in a variety of tumours3. Entosis is a recently described mechanism that can produce these

nature cell biology VOLUME 13 | NUMBER 3 | MARCH 2011 © 2011 Macmillan Publishers Limited. All rights reserved

cell-in-cell structures. This process is prevalent in tumour cell populations grown under nonadhesive conditions and most often results in the death of the invading cell2. Consequently, entosis was initially thought to have a tumour suppressive function, specifically killing cells growing in non-adherent, metastatic conditions2. Krajcovic et al. now provide evidence that this naturally occurring process can also contribute to tumorigenesis by inducing aneuploidy. The authors show that entosis can disturb host cell cytokinesis resulting in the formation of a tetraploid cell containing twice the 199

newS and viewS

Death of invading cell Entosis

Release of invading cell

Mitotic entry host

Diploid host cell 2N

Invading cell outside cleavage plane

Invading cell in cleavage plane

Tetraploid host cell 4N

Cell death or release of internalized cell Normal mitotic progression

Aberrant mitotic progression

Diploid progeny 2N

Aneuploid progeny 2N ± n chromosomes

Figure 1 Cytokinesis failure induced by entosis is a prelude to aneuploidy. Invasion of a live cell into a neighbouring host cell during entosis results in a cell-in-cell structure. The internalized cell can either undergo cell death or, in some cases, can be released. If the internalized cell is still in the cytoplasm on entry of the host cell into mitosis, but is positioned outside the host cell cleavage plane, division will produce two diploid daughter cells. In contrast, positioning of the invading cell within the cleavage plane of the host can perturb cytokinesis by disturbing contractile ring formation. This will produce one tetraploid host cell, with the internalized cell still present in its cytoplasm. In both cases, the internalized cell can undergo cell death or be released, but on mitotic entry of the tetraploid formerhost cell, spindle abnormalities can arise owing to the presence of extra centrosomes, which can produce aneuploid offspring.

normal amount of chromosomes (Fig. 1). This is not dissimilar to the mode of action of asbestos fibres that induce tetraploidy by sterically hindering cleavage furrow ingression of the dividing cell4. The tetraploid intermediate generated can produce aneuploid progeny in the following cell division, thus increasing the tumorigenic potential of cells5–7. Krajcovic et al. confirm this is also the case when tetraploidy is caused by entosis by showing that the resulting binucleate host cell gives rise to aneuploid offspring following several divisions8 (Fig. 1). The main contributing factor to the formation of aneuploid progeny from tetraploid cells 200

is thought to be the extra centrosomes inherited as a result of earlier cytokinesis failure, which drive aberrant mitotic spindle formation in the subsequent mitosis, resulting in chromosome segregation errors7,9. The first clue that entosis contributes to the generation of aneuploid cells came from the observation that many of the outer host cells harbouring cell-in-cell structures in breast tumour tissue were binucleated or multinucleated8. When investigating this in more detail, the authors discovered that in a high percentage of host cells the invading cells physically blocked the cleavage furrow ingression (Fig. 1). This perturbation of cytokinesis was not merely due to

the presence of any invading cell, as divisions were only aborted when the internalized cell was positioned right within the cleavage plane (Fig. 1). Intriguingly, Krajcovic et al., show that during entosis, the internalized cell does not disturb spindle morphology, but merely inhibits correct contractile ring formation. Myosin light chain 2 (MLC2) phosphorylation marks active myosin contraction, which is necessary for the correct formation of the actomyosin ring during cell division. The authors discovered that the presence of the invading cell at the onset of furrowing resulted in the asymmetric distribution of the phosphorylated form of MLC2 at the host cell cortex, so that cortical myosin phosphorylation was absent proximally to the invading cell. Exactly how myosin phosphorylation is disturbed remains unclear, but one possibility is that although the upstream kinases responsible remain active, they fail to reach the host cortex because of the presence of the internalized cell. The lack of kinase access to the cortex is supported by the fact that MLC2 instead becomes phosphorylated on the vacuolar membrane that surrounds the internalized cell. Alternatively, the presence of the internalized cell could disturb the cortical distribution of myosin itself. Distribution of myosin on the cortex is an important early step in cleavage furrow formation10 and it would be interesting to determine whether it is affected in the presence of cell-in-cell structures. In addition to cytokinesis failure, several other mechanisms, such as cell fusion and endoreduplication, have also been shown to cause tetraploidy and supernumerary centrosomes. However, to what extent these defects contribute to polyploidy in vivo remains unknown11. The work of Krajcovic et al. provides proof that cellin-cell structures can produce polyploidy in vivo as well. This is best exemplified by the identification of multinucleated host cells in sections of primary human breast tumour tissue, as well as in metastatic exudates of breast cancer patients. Furthermore, the authors find that the frequency of cell-in-cell structures correlates well with tumour grade. These observations in combination with the fact that entosis is more prevalent under non-adherent conditions, indicate that this process may provide a platform for metastatic host cells to become more tumorigenic by changing the genetic make-up of their progeny2,8. However, an equally plausible explanation for the correlation between cell-in-cell structures and tumour grade could be that cells in the high grade tumours have an increased propensity to

nature cell biology VOLUME 13 | NUMBER 3 | MARCH 2011 © 2011 Macmillan Publishers Limited. All rights reserved

newS and viewS grow under non-adherent, metastatic conditions, resulting in an increased frequency of cell-in-cell structures. Thus, the extent to which entosis inhibits or enhances tumorigenesis, and which outcome is dominant in human tumour tissue, remains to be determined. Over the last decade, several mouse models have been created in which aneuploidy is induced in a variety of tissues12. All of these models involve genetic defects that produce constant gains and losses of whole chromosomes, termed chromosomal instability. There is substantial evidence that chromosomal instability can enhance tumorigenesis by contributing to loss of tumour suppressor genes, gain of oncogenes and acquisition of chemo-resistance13. However, the conclusions drawn from these models are hampered by the fact that the targeted genes could perform other functions to suppress tumour initiation independently of their role in chromosome stability. Moreover, most of the genetic defects induced in the mouse models do not reflect genetic defects present in human tumours in vivo. In contrast, entosis is an aneuploidy-inducing process that occurs naturally when tumour cells are grown under non-adherent conditions. Therefore, it would be interesting to test whether entosis itself

can enhance tumour growth. Experimentally, aneuploid cells could be produced using nonadherent growth conditions, to induce cell-incell structures and cytokinesis failure. The fate and tumorigenic capacity of these aneuploid offspring could then be further tested using cell growth assays in vitro and in vivo. Such studies would contribute to uncovering the role of entosis in tumour growth, and the significance of aneuploidy in this process. Moreover, induction of entosis and subsequent cytokinesis failure may prove a valuable tool in studying the existence of the ‘tetraploidy checkpoint’, a p53-dependent cell-cycle checkpoint thought to be activated as a result of tetraploidy after failed cytokinesis14. Obtaining substantial evidence to prove that such a cellcycle control mechanism truly exists has been hampered by the fact that all methods used so far to induce tetraploidization could also lead to indirect off-target effects that activate p53 independently of cytokinesis failure15. It is possible that such limitations may be overcome by the use of entosis, which could provide a cleaner system for tetraploidization. In summary, the work of Krajcovic et al. presents a new, interesting mechanism by which cytokinesis failure and subsequent

nature cell biology VOLUME 13 | NUMBER 3 | MARCH 2011 © 2011 Macmillan Publishers Limited. All rights reserved

aneuploidy can occur. Although the extent to which entosis contributes to aneuploidy in cancer remains unknown, it is important to stress that the findings presented here can be extrapolated to human tumour tissue, providing us with useful information on how aneuploidy can arise in vivo. Competing finAnCiAl inteRests The authors declare no competing financial interests. 1. Boveri, T. Zur Frage der Entstehung maligner tumoren (Jena, Gustav Fischer, 1914). 2. Overholtzer, M. et al. Cell 131, 966–979 (2007). 3. Overholtzer, M. & Brugge, J. S. Nat. Rev. Mol. Cell Biol. 9, 796–809 (2008). 4. Jensen, C. G., Jensen, L. C., Rieder, C. L., Cole, R. W. & Ault, J. G. Carcinogenesis 17, 2013–2021 (1996). 5. Mazumdar, M. et al. Curr. Biol. 16, 1559–1564 (2006). 6. Fujiwara, T. et al. Nature 437, 1043–1047 (2005). 7. Shi, Q. & King, R. W. Nature 437, 1038–1042 (2005). 8. Kracjovic, M. et al. Nat. Cell Biol. 13, 324–330 (2011). 9. Ganem, N. J., Godinho, S. A. & Pellman, D. Nature 460, 278–282 (2009). 10. Barr, F. A. & Gruneberg, U. Cell 131, 847–860 (2007). 11. Ganem, N. J., Storchova, Z. & Pellman, D. Curr. Opin. Genet. Dev. 17, 157–162 (2007). 12. Ricke, R. M., van Ree, J. H. & van Deursen, J. M. Trends Genet. 24, 457–466 (2008). 13. Kops, G. J., Weaver, B. A. & Cleveland, D. W. Nat. Rev. Cancer 5, 773–785 (2005). 14. Andreassen, P. R., Lohez, O. D., Lacroix, F. B. & Margolis, R. L. Mol. Biol. Cell 12, 1315–1328 (2001). 15. Ganem, N. J. & Pellman, D. Cell 131, 437–440 (2007).

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