EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.

Journal of Biochemistry Advance Access published May 19, 2015

EDAC (Epithelial Defense Against Cancer) —Cell competition between normal and transformed epithelial cells in mammals— Mihoko Kajita and Yasuyuki Fujita* Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University Graduate School of Chemical Sciences and Engineering, Sapporo, Japan

*Yasuyuki Fujita, Division of Molecular Oncology, Institute for Genetic Medicine,

Nishi 7, Kita-ku, Sapporo, Hokkaido 060-0815, Japan. Tel: +81-11-706-5530, Fax: +81-11-706-7544, email: [email protected]

Running title: Cell competition in mammals

© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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Downloaded from http://jb.oxfordjournals.org/ at :: on June 30, 2015

Hokkaido University Graduate School of Chemical Sciences and Engineering, Kita 15,

During embryonic development or under certain pathological conditions, viable but suboptimal cells are often eliminated from the cellular society through a process termed cell competition. Cell competition was originally identified in Drosophila where cells with different properties compete for survival; ‘loser’ cells are eliminated from tissues and consequently ‘winner’ cells become dominant. Recent studies have shown that cell competition also occurs in mammals. While apoptotic cell death is the major fate for losers in Drosophila, outcompeted cells

extrusion and cellular senescence. Molecular mechanisms underlying these processes have been recently revealed. Especially, in epithelial tissues, normal cells sense and actively eliminate the neighboring transformed cells via cytoskeletal proteins by the process named Epithelial Defense Against Cancer (EDAC). Here, we introduce this newly emerging research field: cell competition in mammals.

Keywords: cell competition/mammals/apical extrusion/EDAC/Filamin

Abbreviations: Cdc42-CA, constitutive active form of Cdc42; EDAC, Epithelial defense against cancer; ESCs, embryonic stem cells; EVL, enveloping layer; HSPCs, hematopoietic and progenitor cells; iMOS, inducible random genetic mosaics; IR, ionizing radiation; JNK, c-Jun-N-terminal kinase; Lgl, Lethal giant larvae; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; SILAC, stable isotope labeling by amino acids in cell culture; T-ALL, T-cell acute lymphoblastic 2


show more variable phenotypes in mammals, such as cell death-independent apical

leukemia.


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Introduction In multicellular organisms, cells communicate with each other to form a harmonious and coordinated society. When suboptimal or harmful cells arise within tissues, those unwanted cells must be eliminated to maintain a homeostatic status. Cell competition is thought to be the mechanism that has been evolved to select and exclude such undesirable cells from the body. Cell competition was originally identified in the imaginal discs of Drosophila melanogaster over 30 years ago (1). Minute is a genetic

flies (Minute+/-) develop slowly, they are viable and able to develop normal organisms. However, when heterozygous Minute cells are neighboring to wild-type epithelial cells in imaginal wing discs, Minute cells undergo apoptosis and the neighboring wild-type cells proliferate to fill the gap, eventually develop a wing with the normal size. These data suggest that wild-type and Minute cells compete with each other for survival; Minute cells are eliminated as losers, whereas wild-type cells become dominant as winners. Such competitive interactions between the same types of cells with different properties are termed ‘cell competition’. In addition, it has been demonstrated that dMyc, a Drosophila homolog of the c-Myc proto-oncogene, is also involved in cell competition (2, 3). When dMyc-overexpressing cells are co-present with wild-type epithelial cells within wing discs, cells with higher dMyc levels become super-competitors and outcompete the neighboring wild-type epithelial cells. Moreover, deletion of tumor suppressor protein Lethal giant larvae (Lgl), Scribble, or Rab5 in a mosaic manner within imaginal discs also induces cell competition, and the mutant cells are eliminated 4


mutant that harbors mutations in ribosomal proteins. Although heterozygous Minute

by apoptosis (4-7). Thus, in Drosophila, cell competition occurs between wild-type and various types of transformed cells. On the other hand, using mammalian cell culture systems, Stoker et al. reported cell competition-like phenomena in 1960s-70s (8-10). They showed that the growth of virus-transformed fibroblast cells is inhibited when they are surrounded by a monolayer of non-transformed fibroblast cells (8). This growth inhibition of the transformed cells requires direct contact with, or close proximity to the normal cells (9, 10). Comparable phenomena have been also reported for fibroblast cells

Furthermore, Oliver et al. demonstrate that Minute mutant cells show significant growth or survival disadvantage in Minute and wild-type chimera mice (15), suggesting that cell competition-like phenotypes occur in vivo as well. In addition, recent reports have shown that various types of other mutations can also induce competitive phenotypes in mammals in vitro and in vivo. It was first thought that the difference in proliferation rates determines the winners and losers, but it has become evident that there exist other factors that affect the outcome of cell competition(16). Moreover, in Drosophila, apoptotic cell death is the major fate for losers, but in mammals outcompeted cells show other phenotypes as well (Table 1). For example, when Ras- or Src-transformed cells are surrounded by normal epithelial cells, the transformed cells are outcompeted and apically extruded from a monolayer of normal epithelial cells in a cell death-independent fashion (17-19). In addition, in a hematopoietic system, p53 is involved in cell competition, and the outcompeted cells with higher p53 activity show the senescence-like phenotype (20). 5


transformed by carcinogens (11) as well as by transfection of oncogenes (12-14).

Thus, cell competition now needs to be redefined as more variable cellular processes than previously envisioned. In this review, we introduce recently published cell competition studies in mammals. For cell competition in Drosophila, please refer to other excellent reviews(21-24).

Cell competition in cell culture systems In humans, more than 80% of malignant tumors arise from epithelial tissues such as

from single cells that acquire mutations in an oncogene or tumor suppressor gene. During this initial stage of carcinogenesis, the newly emerging transformed cells are supposed to interact with the neighboring normal cells. Recently, several studies using mammalian cell culture systems have revealed that interactions between transformed cells and the neighboring normal cells induce cell competition, and profoundly influence their signaling pathways and cellular behaviors, often leading to elimination of transformed cells in a variety of ways.

Apical extrusion of transformed cells Ras is one of the small GTPase superfamily that is involved in multiple cellular processes including cell proliferation, differentiation, and motility (25). Ras is frequently mutated to its active form (e.g. RasV12) in various types of human cancers, such as pancreatic and colonic carcinomas. Using a newly established cell culture system, Hogan et al. demonstrate that when RasV12-transformed cells are surrounded 6


lung, colon, cervix, and mammary glands. The majority of human cancers originate

by normal epithelial cells, the majority of RasV12 cells are apically extruded from a monolayer of normal epithelial cells in an apoptosis-independent manner (Fig. 1A) (17). Importantly, when RasV12 cells alone are cultured, apical extrusion does not occur (Fig. 1B), indicating that activation of the cell-autonomous RasV12 signaling pathway itself is not sufficient to induce apical extrusion, but that the presence of surrounding normal cells is also required. Similarly to RasV12 cells, when oncoprotein v-Src-transformed cells are surrounded by normal epithelial cells, v-Src cells are apically extruded from a

observed in the enveloping layer (EVL) of zebrafish embryos as well, suggesting that apical extrusion is an evolutionarily conserved process in vertebrates for elimination of transformed cells from the epithelium. Apical extrusions of RasV12 or v-Src cells share several common features. First, apical extrusion is induced only when the transformed cells are surrounded by normal epithelial cells. Second, the transformed cells are apically extruded in an apoptosis-independent manner. Third, the height of the transformed cells is increased when they are surrounded by normal cells. However, non-extruded RasV12 cells frequently form basal protrusions underneath the neighboring normal cells and are eventually delaminated into the underling matrix (17, 26, 27), whereas non-extruded v-Src cells do not form basal protrusions (18), indicating that distinct signaling pathways are also involved in the interaction between normal and these transformed cells. Moreover, recent studies have shown that other types of transformed cells are also apically extruded upon interaction with normal epithelial cells (Table 1). For example, expression of a constitutively active form of Cdc42 7


monolayer of normal epithelial cells (18). Apical extrusion of v-Src-expressing cells is

(Cdc42-CA) in single cells within a monolayer of normal epithelial cells induces apical extrusion of Cdc42-CA-expressing cells from the epithelium (28). In addition, in the 3D organotypic culture, ErbB2-overexpressing cells are translocated into the apical lumen of breast epithelial cysts (29). These results indicate that apical extrusion is a general mechanism of the elimination of transformed cells from epithelia.

Various changes occurring in transformed cells during apical extrusion

modulated in transformed cells during apical extrusion (Fig. 2). Hogan et al. have shown that prior to apical extrusion, RasV12 cells significantly increase cell height, accumulate intercellular F-actin, and show higher activity of myosin-II and Cdc42 (17), suggesting that the presence of the neighboring normal cells influences the cytoskeletal organization of RasV12 cells. Overexpression of a dominant negative mutant of Cdc42 or Rho kinase in RasV12 cells significantly suppresses their apical extrusion and instead promotes basal protrusion formation, indicating that activity of these signaling pathways plays an important role in these processes. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, Anton et al. have identified multiple proteins of which phosphorylation is elevated in RasV12-transformed cells that are surrounded by normal epithelial cells (30). VASP is one of those proteins, and S239 phosphorylation is significantly enhanced in RasV12 cells that interact with normal cells. When VASP is knocked down, apical extrusion of RasV12 cells is significantly 8


Recent studies have revealed that various molecules and signaling pathways are

promoted, which is rescued by expression of wild type or non-phosphorylatable mutant (S239A) of VASP, but not by that of phosphomimetic mutant of VASP (S239D), suggesting that VASP plays an inhibitory role in the apical extrusion of RasV12 cells and that phosphorylation of VASP at S239 relieves its suppressive effect. In addition, PKA is activated in RasV12 cells when they are surrounded by normal cells, thereby promoting VASP phosphorylation. Ohoka et al. demonstrate that membrane microdomains are also involved in apical

accumulated at lateral and apical membranes of RasV12 cells that are surrounded by normal cells. When lipid rafts are disrupted by chemical compounds or Cav-1-knockdown, apical extrusion of RasV12 cells is significantly suppressed, indicating a crucial role of membrane microdomains in apical extrusion of transformed cells. Furthermore, the authors have identified EPLIN (Epithelial protein lost in neoplasm), a crucial regulator of actin dynamics and cell-cell adhesions (32, 33), as a Cav-1-interacting protein and demonstrated that EPLIN acts upstream of Cav-1 in this process. EPLIN also regulates myosin-II and PKA independently of Cav-1 (Fig. 2), indicating that EPLIN is a master regulator of the apical extrusion of transformed cells. Similarly to RasV12 cells, some of the common signaling pathways are activated in Src-transformed cells surrounded by normal epithelial cells (Fig. 2). For example, EPLIN is accumulated in Src cells at the interface with normal cells, which functions upstream of Cav-1 accumulation and myosin-II activation (18, 31). In addition, focal adhesion kinase (FAK) is also activated in Src cells, and both FAK and myosin-II 9


extrusion(31). Caveolin-1 (Cav-1), a major component of caveolae, is specifically

mediate activation of mitogen-activated protein kinase (MAPK) (18). Grieve et al. show that E-cadherin cleavage by matrix metalloproteinase (MMP) is involved in apical extrusion of Cdc42-CA-expressing cells (28). MMP also plays a crucial role in the apical elimination of ErbB2-overexpressing cells in organotypic mammary acini (29). The substrate proteins of MMPs during these processes need to be clarified in future studies. Physical properties of cells also affect apical extrusion of transformed cells. Wu et al.

(19). At the steady status, cell cortical tension is significantly higher at the zonula adherens than lateral cell-cell contact sites. N-WASP accumulates at the zonula adherens and enhances apical junctional tension by stabilizing local F-actin networks. Interestingly, at the interface between normal and RasV12-transformed cells, N-WASP is re-distributed into lateral junctions, leading to increased lateral tension. Such redistribution of N-WASP provides altered patterning of intra-junctional tension, which promotes apical extrusion of RasV12-expressing cells from the epithelial monolayer.

EDAC (Epithelial Defense Against Cancer) Although expression and activity of various molecules in transformed cells are specifically regulated during apical extrusion, it was not known whether and how the neighboring normal cells play an active role in this process. By a biochemical screening, Kajita et al. show that Filamin and Vimentin are specifically modulated under the mix culture condition of normal and Src-transformed epithelial cells (34). 10


demonstrate that the defect of integrity of apico-basal tension induces apical extrusion

Immunofluorescence analyses demonstrate that both Filamin and Vimentin are strongly accumulated in the neighboring normal epithelial cells at the interface with Src- or RasV12-transformed cells (Fig. 3A and B). Knockdown of Filamin or Vimentin from the surrounding normal cells significantly suppresses apical extrusion of the transformed cells, indicating that normal epithelial cells sense the neighboring transformed cells and actively eliminate them via Filamin and Vimentin. Vimentin accumulation depends on the presence of Filamin, suggesting that Filamin acts

accumulated Vimentin filaments generate tensile forces around the neighboring transformed cells, providing physical forces for the apical extrusion. Furthermore, the Rho/Rho kinase pathway regulates Filamin accumulation, and PKC is a potential regulator for Filamin-regulated Vimentin accumulation (Fig. 2). Moreover, the authors have examined whether comparable phenomena also occur in vivo using zebrafish embryos. They have found that Filamin accumulates at the interface with v-Src-expressing cells in the zebrafish enveloping layer (Fig. 3C). In addition, knockdown of Filamin using morpholino oligonucleotides significantly suppresses apical extrusion of v-Src-expressing cells, suggesting that Filamin is an evolutionarily conserved key regulator in apical extrusion of transformed cells. The authors have further examined how Filamin accumulates at the interface between normal and transformed cells. As described above, myosin-II activity is elevated in Srcor Ras-transformed cells that are surrounded by normal cells. When myosin-II activation in the transformed cells is suppressed by expression of a dominant negative 11


upstream of Vimentin. In addition, laser ablation experiments show that the

mutant of myosin-II (MLC-AA), Filamin accumulation in the neighboring normal cells is significantly suppressed. Furthermore, analyses by atomic force microscopy show that enhanced myosin-II activity is related to increased cellular elasticity in the transformed cells. Together with the previous study showing that Filamin functions as a mechanosensor/transducer, these data support the notion that normal cells sense and respond to the physical environments modulated by myosin-II-driven forces in the neighboring transformed cells.

active role in the apical extrusion of transformed cells, implying that normal epithelial cells are endowed with anti-tumorigenic activity, which does not involve immune systems. This process is named as EDAC (Epithelial Defense Against Cancer) (34).

Apoptotic phenotype of transformed cells Lgl is a tumor suppressor protein in both Drosophila and mammals (35, 36). Tamori et al. have identified Mahjong as an evolutionarily conserved binding partner of Lgl (5). Mahjong is a cytosolic protein that contains LisH and WD40-like domains. In Drosophila, homozygous Mahjong-knockout (mahj-/-) cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Interestingly, the comparable cell competition phenotype is also observed in a mammalian cell culture system; when Mahjong-knockdown cells are surrounded by normal epithelial cells, Mahjong-knockdown cells undergo apoptosis and are eliminated from the epithelia. In addition, the c-Jun N-terminal kinase (JNK) pathway plays a vital role in 12


Collectively, these results indicate that via Filamin, normal epithelial cells play an

Mahjong-mediated cell competition in both Drosophila and mammals. This is the first report showing that apoptosis-mediated cell competition also occurs in mammals. Scribble is a crucial cell polarity regulator and functions as a tumor suppressor in both Drosophila and mammals (37, 38). In Drosophila eye discs, when homozygous scrib mutant cells are surrounded by wild-type cells, the scrib mutant cells are eliminated from the epithelium by apoptosis (6). Using a mammalian cell culture system, Norman et al. demonstrate the comparable cell competition phenomenon in mammals;

Scribble-knockdown cells undergo apoptosis and leave the epithelial monolayer (39). Apoptosis of Scribble-knockdown cells depends on activation of p38 MAPK, while the JNK pathway is involved in Scribble-mediated cell competition in Drosophila, indicating the involvement of distinct molecular mechanisms between Drosophila and mammals.

Cell competition in vivo Recent studies have shown that cell competition also occurs in vivo in mammals and plays a pivotal role in various physiological and pathological processes.

Physiological relevance of cell competition BMP signaling suppresses differentiation and sustains self-renewal and pluripotency of mouse embryonic stem cells (ESCs) (40). Bmpr1a encodes the main type I BMP receptor. Sancho et al. show that in mosaic Bmpr1a-/- embryos, mutant cells are 13


when Scribble-knockdown cells are surrounded by normal epithelial cells,

eliminated by apoptosis at the epiblast stage (41). In addition, using ESC culture systems, they demonstrate that when wild-type and Bmpr1a-/- ESCs are co-cultured, cell competition occurs between them, and Bmpr1a-/- ESCs are outcompeted by apoptosis. Secreted factors rather than direct cell-cell interactions are required for this process. Furthermore, tetraploid ESCs or autophagy-defective (Atg5-/-) ESCs are also eliminated in the presence of wild-type ESCs in vitro and in vivo. Interestingly, in all these cell competition processes, loser mutant ESCs show the lower levels of c-Myc expression

elimination of the neighboring wild-type cells. Moreover, in the mouse epiblast, ESCs spontaneously expressing low levels of c-Myc are preferentially eliminated by apoptosis. Claveria et al. also show that c-Myc-driven cell competition occurs in mouse embryos (42). To induce mosaic expression in the epiblast, the authors have successfully established the inducible random genetic mosaics (iMOS) system. In this system, a combination of two sequence variants of loxP (that can be combined with homotypic but not with heterotypic sequences) allows to generate random mosaics expressing two distinct reporter genes. Using this system, mosaic Myc-expressing embryos are established where the Myc level of one cell population is higher than the other population. The authors demonstrate that cells with lower Myc levels are eliminated by apoptosis, and instead cells with higher Myc levels proliferate to fill the vacant spaces and become dominant. Consequently, the final size and development of the embryo remain normal. For the elimination of cells with lower Myc levels, direct 14


than winner wild-type ESCs. Indeed, c-Myc overexpression is sufficient to induce the

contacts or short-range interactions with the cells with higher Myc levels are required. Furthermore, at the early embryonic stage (up to E6.75), endogenous differences in Myc levels induce cell competition and that cells with higher Myc levels become "winners" and are selected for further development. Collectively, these results indicate that c-Myc is a key regulator in the elimination of the defective ESCs. A recent report show that Myc-driven cell competition can also occur in cardiomyocytes of the mouse heart (43). In this study, Campo et al. have examined the

developing heart of embryos, the population of Myc-overexpressing cardiomyocytes expands and becomes dominant, whereas the neighboring wild-type cells are eliminated by apoptotic cell death. Accordingly, this cell replacement process is phenotypically silent with normal heart anatomy and function. Furthermore, cell competition-like phenotype is also observed in the adult heart where Myc-overexpressing cells replace wild-type cells. Intriguingly, apoptotic cell death is not involved in this replacement, instead an autophagic cell death marker Beclin is elevated in the "loser" wild-type cells neighboring "winner" Myc-overexpressing cells, though it remains unknown whether autophagic cell death is involved in this process. Thus, Myc-driven cell competition is governed, at least partially, by a distinct mechanism between embryonic and adult hearts.

Pathological relevance of cell competition The liver is well known of its ability to actively regenerate after massive injury (44). 15


phenotype of mosaic Myc-overexpression in the heart using the iMOS system. In the

Oertel et al. show that cell competition occurs in the liver by transplanting fetal rat liver stem/progenitor cells into an adult liver (45). After two-thirds partial hepatectomy of the adult host liver, freshly isolated rat fetal liver cells, which are rapidly proliferating but not yet commit to differentiate, are infused through the portal vein, and the progeny of the transplanted cells are monitored. Transplanted fetal cells are morphologically and functionally indistinguishable from the host hepatocytes, but actively proliferate in the host liver, and the repopulation percentage increases over time. Importantly,

cells, but also on the apoptotic cell death of the adjacent host hepatocytes, suggesting that cell competition-based liver reconstitution occurs. Interestingly, liver repopulation of the transplanted cells becomes much higher in older rats than in younger ones (46). This context-dependent cell competition may be affected by relative difference in proliferative activity. The transcription factor p53 has been most extensively studied in its function to mediate tumor suppression (47-49). Expression of p53 is induced upon various stress responses including irradiation-mediated DNA damage. To investigate the involvement of p53 in cell competition in mammals, Bondar and Medzhitov have utilized bone marrow chimeras with different levels of p53 activity (20). The authors demonstrate that a low dose of ionizing radiation (IR) stimulates cell competition and that hematopoietic stem and progenitor cells (HSPCs) with low activity of p53 outcompete HSPCs with high activity of p53. Furthermore, using the inducible genetic mouse model expressing an oncogenic dominant negative mutant of p53 R172H (mp53) in a mosaic manner, the 16


repopulation is based not only on the greater proliferative activity of transplanted fetal

authors also show that after IR, the percentage of mp53-expressing HSPCs substantially increases. There is no significant difference in total HSPC numbers between wild-type and mosaic mice after IR, suggesting that mp53-expressing cells replace wild-type cells, rather than simply expand. Interestingly, the classical p53-mediated DNA damage response does not contribute to the cell competition, and apoptosis is not involved in this process. Instead, expression of p16INK4a is higher in the outcompeted wild-type cells than in mp53 cells in the irradiated mosaic mice, suggesting that the senescence-like

in a hematopoietic system, cellular stress can induce p53-mediated cell competition. The selection of cells with lower p53 activity may be required for the maintenance of homeostasis, but this selective advantage would result in tumor initiation. Although several lines of evidence suggest that cell competition is related to suppression of tumor initiation, it has been unclear whether the lack of competition indeed initiates tumor formation. In the thymus, bone-marrow-derived T-cell progenitors constantly replace thymus-resident T-cell progenitors (50). Martin et al. first show that this replacement of thymus-resident progenitors is, at least partially, due to competition for the survival factor interleukin-7 (IL-7), which can activate the expression of pro-survival protein Bcl-2 (51). The authors then demonstrate that the defect of cell competition between bone-marrow-derived T-cell progenitors and thymus-resident ones causes T-cell acute lymphoblastic leukemia (T-ALL)-like phenotype. In the absence of bone-marrow progenitors (under no cell competition), the thymus-resident precursors self-renew and produce T-cells to compensate this loss, 17


phenotype is induced in the outcompeted HSPCs. Collectively, these data indicate that

eventually leading to genetic transformations and formation of tumors that share many aspects with human T-ALL. Thus, fresh replacement of bone-marrow progenitors via cell competition prevents tumorigenesis. This study illustrates the example that cell competition is a tumor suppressor mechanism.

Final remarks Recent studies have revealed that cell competition is an evolutionally conserved

elimination of suboptimal or harmful cells under various physiological and pathological conditions. However, molecular mechanisms whereby cells recognize and respond to the differences from each other still remain largely unknown. Especially, it is not evident whether there exist universal cell competition regulators that play a general role in cell competition events. Identification of such cell competition-markers will uncover more cell competition-mediated phenomena or diseases, which would profoundly advance this promising new research field. It has now become clear that transformed cells are often eliminated from a society of normal cells via cell competition; in other words, normal cells are equipped with anti-tumor activity that does not involve immune systems. Thus, the cell competition research is highly related to the events occurring at the initial stage of carcinogenesis where newly emerging transformed cells are surrounded by normal cells. Further elucidation of the molecular mechanisms of cell competition will potentially lead to a novel type of cancer preventive treatment; enhancing the attacking force of normal cells 18


phenomenon occurring in flies and mammals, which is involved in the selection and

against transformed cells or attenuating the defensive force of transformed cells, thereby promoting eradication of transformed cells from epithelia.


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Acknowledgements This work is supported by Funding Program for Grant-in-Aid for Scientific Research on Innovative Areas, Grant-in-Aid for Scientific Research (A), and Strategic Japanese-Swiss Cooperative Program. MK is supported by Ono Cancer Research Fund, Akiyama Life Science Foundation, and Grant-in-Aid for Young Scientists (B). YF is also supported by the Takeda Science Foundation.


Conflict of Interest None declared

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References 1.

Morata, G., and Ripoll, P. (1975) Minutes: mutants of drosophila autonomously affecting cell division rate. Dev. Biol. 42, 211-221

2.

Moreno, E., and Basler, K. (2004) dMyc transforms cells into super-competitors. Cell 117, 117-129

3.

de la Cova, C., Abril, M., Bellosta, P., Gallant, P., and Johnston, L.A. (2004) Drosophila myc regulates organ size by inducing cell competition. Cell 117, 107-116

4.

Grzeschik, N.A., Amin, N., Secombe, J., Brumby, A.M., and Richardson, H.E. separable in Drosophila lgl mutant clones in the developing eye. Dev. Biol. 311, 106-123

5.

Tamori, Y., Bialucha, C.U., Tian, A.G., Kajita, M., Huang, Y.C., Norman, M., Harrison, N., Poulton, J., Ivanovitch, K., Disch, L., Liu, T., Deng, W.M., and Fujita, Y. (2010) Involvement of Lgl and Mahjong/VprBP in cell competition. PLoS Biol. 8, e1000422

6.

Brumby, A.M., and Richardson, H.E. (2003) scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila. EMBO J. 22, 5769-5779

7.

Ballesteros-Arias, L., Saavedra, V., and Morata, G. (2014) Cell competition may function either as tumour-suppressing or as tumour-stimulating factor in Drosophila. Oncogene 33, 4377-4384

8.

Stoker, M. (1964) Regulation of Growth and Orientation in Hamster Cells Transformed by Polyoma Virus. Virology 24, 165-174

9.

Bertram, J.S. (1979) Modulation of cellular interactions between C3H/10T1/2 cells and their transformed counterparts by phosphodiesterase inhibitors. Cancer Res. 39, 3502-3508

10.

Stoker, M.G., Shearer, M., and O'Neill, C. (1966) Growth inhibition of polyoma-transformed cells by contact with static normal fibroblasts. J. Cell Sci. 1, 297-310

11.

Borek, C., and Sachs, L. (1966) The Difference in Contact Inhibition of Cell

21


(2007) Abnormalities in cell proliferation and apico-basal cell polarity are

Replication between Normal Cells and Cells Transformed by Different Carcinogens. Proc. Natl. Acad. Sci. USA 56, 1705-1711 12.

Alexander, D.B., Ichikawa, H., Bechberger, J.F., Valiunas, V., Ohki, M., Naus, C.C., Kunimoto, T., Tsuda, H., Miller, W.T., and Goldberg, G.S. (2004) Normal cells control the growth of neighboring transformed cells independent of gap junctional communication and SRC activity. Cancer Res. 64, 1347-1358

13.

Bignami, M., Rosa, S., La Rocca, S.A., Falcone, G., and Tato, F. (1988) Differential influence of adjacent normal cells on the proliferation of mammalian cells transformed by the viral oncogenes myc, ras and src. Oncogene. 2, 509-514 Martin, W., Zempel, G., Hulser, D., and Willecke, K. (1991) Growth inhibition of oncogene-transformed rat fibroblasts by cocultured normal cells: relevance of metabolic cooperation mediated by gap junctions. Cancer Res. 51, 5348-5351

15.

Oliver, E.R., Saunders, T.L., Tarle, S.A., and Glaser, T. (2004) Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute. Development 131, 3907-3920

16.

Tamori, Y., and Deng, W.M. (2013) Tissue repair through cell competition and compensatory cellular hypertrophy in postmitotic epithelia. Dev. Cell 25, 350-363

17.

Hogan, C., Dupre-Crochet, S., Norman, M., Kajita, M., Zimmermann, C., Pelling, A.E., Piddini, E., Baena-Lopez, L.A., Vincent, J.P., Itoh, Y., Hosoya, H., Pichaud, F., and Fujita, Y. (2009) Characterization of the interface between normal and transformed epithelial cells. Nat. Cell Biol. 11, 460-467

18.

Kajita, M., Hogan, C., Harris, A.R., Dupre-Crochet, S., Itasaki, N., Kawakami, K., Charras, G., Tada, M., and Fujita, Y. (2010) Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Src-transformed cells. J. Cell Sci. 123, 171-180

19.

Wu, S.K., Gomez, G.A., Michael, M., Verma, S., Cox, H.L., Lefevre, J.G., Parton, R.G., Hamilton, N.A., Neufeld, Z., and Yap, A.S. (2014) Cortical F-actin stabilization generates apical-lateral patterns of junctional contractility that integrate cells into epithelia. Nat. Cell Biol. 16, 167-178

20.

Bondar, T., and Medzhitov, R. (2010) p53-mediated hematopoietic stem and

22


14.

progenitor cell competition. Cell Stem Cell 6, 309-322 21.

Vincent, J.P., Fletcher, A.G., and Baena-Lopez, L.A. (2013) Mechanisms and mechanics of cell competition in epithelia. Nat. Rev. Mol. Cell Biol. 14, 581-591

22.

Amoyel, M., and Bach, E.A. (2014) Cell competition: how to eliminate your neighbours. Development 141, 988-1000

23.

Moreno, E., and Rhiner, C. (2014) Darwin's multicellularity: from neurotrophic theories and cell competition to fitness fingerprints. Curr. Opin. Cell Biol. 31, 16-22

24.

de Beco, S., Ziosi, M., and Johnston, L.A. (2012) New frontiers in cell competition. Dev Dyn. 241, 831-841 Karnoub, A.E., and Weinberg, R.A. (2008) Ras oncogenes: split personalities. Nat. Rev. Mol. Cell Biol. 9, 517-531

26.

Slattum, G., Gu, Y., Sabbadini, R., and Rosenblatt, J. (2014) Autophagy in oncogenic K-Ras promotes basal extrusion of epithelial cells by degrading S1P. Curr. Biol. 24, 19-28

27.

Liu, J.S., Farlow, J.T., Paulson, A.K., Labarge, M.A., and Gartner, Z.J. (2012) Programmed cell-to-cell variability in Ras activity triggers emergent behaviors during mammary epithelial morphogenesis. Cell Rep. 2, 1461-1470

28.

Grieve, A.G., and Rabouille, C. (2014) Extracellular cleavage of E-cadherin promotes epithelial cell extrusion. J. Cell Sci. 127, 3331-3346

29.

Leung, C.T., and Brugge, J.S. (2012) Outgrowth of single oncogene-expressing cells from suppressive epithelial environments. Nature. 482, 410-413

30.

Anton, K.A., Sinclair, J., Ohoka, A., Kajita, M., Ishikawa, S., Benz, P.M., Renne, T., Balda, M., Jorgensen, C., Matter, K., and Fujita, Y. (2014) PKA-regulated VASP phosphorylation promotes extrusion of transformed cells from the epithelium. J. Cell Sci. 127, 3425-3433

31.

Ohoka, A., Kajita, M., Ikenouchi, J., Yako, Y., Kitamoto, S., Kon, S., Ikegawa, M., Shimada, T., Ishikawa, S., and Fujita, Y. (2015) EPLIN is a crucial regulator for extrusion of RasV12-transformed cells. J. Cell Sci. 128, 781-789

32.

Abe, K., and Takeichi, M. (2008) EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin belt. Proc. Natl. Acad. Sci. USA 105, 13-19

23


25.

33.

Maul, R.S., Song, Y., Amann, K.J., Gerbin, S.C., Pollard, T.D., and Chang, D.D. (2003) EPLIN regulates actin dynamics by cross-linking and stabilizing filaments. J. Cell Biol. 160, 399-407

34.

Kajita, M., Sugimura, K., Ohoka, A., Burden, J., Suganuma, H., Ikegawa, M., Shimada, T., Kitamura, T., Shindoh, M., Ishikawa, S., Yamamoto, S., Saitoh, S., Yako, Y., Takahashi, R., Okajima, T., Kikuta, J., Maijima, Y., Ishii, M., Tada, M., and Fujita, Y. (2014) Filamin acts as a key regulator in epithelial defence against transformed cells. Nat. Commun. 5, 4428

35.

Mechler, B.M., McGinnis, W., and Gehring, W.J. (1985) Molecular cloning of lethal(2)giant larvae, a recessive oncogene of Drosophila melanogaster. EMBO J.

36.

Klezovitch, O., Fernandez, T.E., Tapscott, S.J., and Vasioukhin, V. (2004) Loss of cell polarity causes severe brain dysplasia in Lgl1 knockout mice. Genes & Dev. 18, 559-571

37.

Bilder, D., Li, M., and Perrimon, N. (2000) Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113-116

38.

Zhan, L., Rosenberg, A., Bergami, K.C., Yu, M., Xuan, Z., Jaffe, A.B., Allred, C., and Muthuswamy, S.K. (2008) Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma. Cell 135, 865-878

39.

Norman, M., Wisniewska, K.A., Lawrenson, K., Garcia-Miranda, P., Tada, M., Kajita, M., Mano, H., Ishikawa, S., Ikegawa, M., Shimada, T., and Fujita, Y. (2012) Loss of Scribble causes cell competition in mammalian cells. J. Cell Sci. 125, 59-66

40.

Ying, Q.L., Nichols, J., Chambers, I., and Smith, A. (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281-292

41.

Sancho, M., Di-Gregorio, A., George, N., Pozzi, S., Sanchez, J.M., Pernaute, B., and Rodriguez, T.A. (2013) Competitive interactions eliminate unfit embryonic stem cells at the onset of differentiation. Dev. Cell 26, 19-30

42.

Claveria, C., Giovinazzo, G., Sierra, R., and Torres, M. (2013) Myc-driven endogenous cell competition in the early mammalian embryo. Nature 500, 39-44

24


4, 1551-1557

43.

Villa del Campo, C., Claveria, C., Sierra, R., and Torres, M. (2014) Cell competition promotes phenotypically silent cardiomyocyte replacement in the mammalian heart. Cell Rep. 8, 1741-1751

44.

Michalopoulos, G.K., and DeFrances, M.C. (1997) Liver regeneration. Science 276, 60-66

45.

Oertel, M., Menthena, A., Dabeva, M.D., and Shafritz, D.A. (2006) Cell competition leads to a high level of normal liver reconstitution by transplanted fetal liver stem/progenitor cells. Gastroenterology 130, 507-520; quiz 590

46.

Menthena, A., Koehler, C.I., Sandhu, J.S., Yovchev, M.I., Hurston, E., Shafritz, D.A., and Oertel, M. (2011) Activin A, p15INK4b signaling, and cell Gastroenterology 140, 1009-1020

47.

Lowe, S.W., Cepero, E., and Evan, G. (2004) Intrinsic tumour suppression. Nature 432, 307-315

48.

Vogelstein, B., Lane, D., and Levine, A.J. (2000) Surfing the p53 network. Nature 408, 307-310

49.

Vousden, K.H., and Lane, D.P. (2007) p53 in health and disease. Nat. Rev. Mol. Cell Biol. 8, 275-283

50.

Frey, J.R., Ernst, B., Surh, C.D., and Sprent, J. (1992) Thymus-grafted SCID mice show transient thymopoiesis and limited depletion of V beta 11+ T cells. J. Exp. Med. 175, 1067-1071

51.

Martins, V.C., Busch, K., Juraeva, D., Blum, C., Ludwig, C., Rasche, V., Lasitschka, F., Mastitsky, S.E., Brors, B., Hielscher, T., Fehling, H.J., and Rodewald, H.R. (2014) Cell competition is a tumour suppressor mechanism in the thymus. Nature 509, 465-470

25


competition promote stem/progenitor cell repopulation of livers in aging rats.

Figure Legends Table 1 Interaction between normal and transformed cells in vertebrates. ND; not determined. Fig. 1

Apical extrusion of Ras-transformed cells surrounded by normal cells.

Fluorescently labeled MDCK-pTR GFP-RasV12 cells (CMTPX: red fluorescent dye) are mixed with normal MDCK cells (A) or MDCK-pTR GFP-RasV12 cells (B). Images are extracted from a representative time-lapse analysis. Scale bars; 20 m. Red arrows indicate fluorescently labeled RasV12 cells. Modified from Hogan et al., 2009 (17). Molecular mechanisms of the interaction between normal and transformed

cells. At the interface between normal and transformed cells, various signaling pathways are modulated in both cells. Fig. 3

Filamin accumulation in the neighboring normal cells at the interface with

transformed cells. (A, B) Immunofluorescence images of Filamin (red) in the mix culture of normal and transformed MDCK cells. (A) MDCK cells expressing temperature-sensitive v-Src are stained with CMFDA (green fluorescent dye) and mixed with normal MDCK cells. (B) MDCK-pTR GFP-RasV12 cells are mixed with normal MDCK cells. (C) Confocal images of a GFP-filamin-expressing zebrafish embryo expressing v-Src in a mosaic manner. Embryos from the zebrafish line harboring EVL-specific GFP-filamin A are injected with Cherry-v-Src-expressing vector at the one-cell stage. Arrows indicate the Filamin accumulation (A, B) or GFP-filamin accumulation (C) in the neighboring normal cells at the interface with transformed cells. The area in the white box is shown at higher magnification in the lower panel (A, B). White dashed lines in xy panels denote the cross-sections represented in xz panels (C). Scale bars; 10 m. Modified from Kajita et al., 2014 (34).

26


Fig. 2

signaling pathways involved

phenomena (references)

In vitro

In transformed cells

Ras

Apical extrusion or basal protrusion formation of Ras-transformed cells (17, 19, 30, 31, 34)

Src

Apical extrusion of Src-transformed cells (18, 34)

Mahjong

MAPK, Myosin-II, Cdc42, ROCK, N-WASP, Caveolin-1, EPLIN


mutations

In neighboring normal cells

Filamin

MAPK, Myosin-II, FAK

Filamin, Vimentin

Apoptosis of Mahjong-knockdown cells (5)

JNK

ND

Scribble

Apoptosis of Scribble-knockdown cells (39)

p38 MAPK

Cdc42

Apical extrusion of constitutively active Cdc42-expressing cells (28)

ErbB2

Translocation and clonal expansion of ErbB2overexpressing cells (3D culture) (29)

MAPK, MMPs

ND ND

MAPK, MT1-MMP

ND

ND

Filamin

ND

ND

ND

ND

In vivo Zebrafish

Src

Mice

p53 Myc

Apical extrusion of Src-transformed cells (18, 34) Senescence-like phenotype of cells with the higher p53 level (20) Apoptosis of cells with lower Myc levels (4143)

Table 1 Kajita et al.

A

MDCK : RasV12 = 100 : 1

CMTPX

1h

4h

7h

13 h

20 h

24 h

24 h

10’

1h

4h

7h GFP-RasV12

13 h

20 h

24 h

24 h

GFP-RasV12

B RasV12 only CMTPX

Figure 1 Kajita et al.


10’

transformed cell neighboring normal cell

EPLIN

FAK

Myo-II

PKA

FLN PKCε

MAPK

p-VASP

Vim

Rho K

?

observed at the interface between normal and:

Apical extrusion

Ras- or Src-transformed cells Ras-transformed cells Src-transformed cells



Rho

Cav-1

A MDCK:ts-Src=50:1

B

C

MDCK:Ras=50:1

Enveloping layer

xy

xz GFP-filamin/Src

Filamin/Ras



Filamin/Src

Regulation of protein degradation in normal and transformed human bronchial epithelial cells in culture.

Functional expression of VIP receptors in normal, immortalized and transformed mammary epithelial cells.

Conditionally reprogrammed normal and transformed mouse mammary epithelial cells display a progenitor-cell-like phenotype.

Oncogene alterations in in vitro transformed rat tracheal epithelial cells.

Oscillations: a key event in transformed renal epithelial cells.

ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

Temperature-sensitive mutants of chemically transformed epithelial cells.

Interactions between epithelial and mesenchymal cells in intrahepatic peribiliary glands in normal and hepatolithiatic livers.

A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC).

B cell stimulatory factor-II is expressed and released by normal and transformed human bronchial epithelial cells.

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells.

Breast cancer stem cells transition between epithelial and mesenchymal states reflective of their normal counterparts.

Phenotypic plasticity in normal breast derived epithelial cells.

Establishment of tight junctions between epithelial cells.

Simian virus 40-transformed human ovarian surface epithelial cells escape normal growth controls but retain morphogenetic responses to extracellular matrix.

Pathology of spontaneous and oncogene transformed rat liver epithelial cells and derived tumours in nude mice.

Effect of magnesium on the growth and cell cycle of transformed and non-transformed epithelial rat liver cells in vitro.

Proteolytic enzymes in normal and transformed cells.

Drug resistance in cultured rat liver epithelial cells spontaneously and chemically transformed.

Silencing KRAS overexpression in arsenic-transformed prostate epithelial and stem cells partially mitigates malignant phenotype.

Production of epithelial and mesenchymal tumours with rat liver cells transformed in vitro.

Cell fusion between gastric epithelial cells and mesenchymal stem cells results in epithelial-to-mesenchymal transition and malignant transformation.

Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells.

Ionizing radiation response of primary normal human lens epithelial cells.