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.     

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.     

Meiosis and sex chromosome aneuploidy: how meiotic errors cause aneuploidy; how aneuploidy causes meiotic errors

As a group, sex chromosome aneuploidies - the 47,XXY, 47,XYY, 47,XXX and 45,X conditions - constitute the most common class of chromosome abnormality in human live-births. Considerable attention has been given to the somatic abnormalities associated with these conditions, but less is known about their meiotic phenotypes; that is, how does sex chromosome imbalance influence the meiotic process. This has become more important with the advent of assisted reproductive technologies, because individuals previously thought to be infertile can now become biological parents. Indeed, there are several recent reports of successful pregnancies involving 47,XXY fathers, and suggestions that cryopreservation of ovarian tissue might impart fertility to at least some Turner syndrome individuals. Thus, the possible consequences of sex chromosome aneuploidy on meiotic chromosome segregation need to be explored.     

Inherited aneuploidy: germline mosaicism

Germline mosaicism has been thought to be a rare cause of aneuploidy in the human population. Recent evidence from cytological and population studies suggests otherwise. Approximately 5% of young couples with a Down syndrome child show evidence of germinal mosaicism. Molecular cytogenetic analysis of oocytes has proved germinal or gonadal mosaicism for trisomies of chromosomes 13 and 21 in several studies involving both oocytes and first polar bodies. Most recently direct analysis of fetal ovarian pre-meiotic, meiotic, and stromal cells proved low level trisomy 21 mosaicism in every sample tested. Based upon this evidence, germinal or gonadal mosaicism is likely to make a significant contribution to aneuploidy in the human population, particularly where younger women are concerned.     

Whole chromosome aneuploidy: Big mutations drive adaptation by phenotypic leap

Despite its widespread existence, the adaptive role of aneuploidy (the abnormal state of having an unequal number of different chromosomes) has been a subject of debate. Cellular aneuploidy has been associated with enhanced resistance to stress, whereas on the organismal level it is detrimental to multicellular species. Certain aneuploid karyotypes are deleterious for specific environments, but karyotype diversity in a population potentiates adaptive evolution. To reconcile these paradoxical observations, this review distinguishes the role of aneuploidy in cellular versus organismal evolution. Further, it proposes a population genetics perspective to examine the behavior of aneuploidy on a populational versus individual level. By altering the copy number of a significant portion of the genome, aneuploidy introduces large phenotypic leaps that enable small cell populations to explore a wide phenotypic landscape, from which adaptive traits can be selected. The production of chromosome number variation can be further increased by stress- or mutation-induced chromosomal instability, fueling rapid cellular adaptation.     

Bacterial invasion: Force feeding by Salmonella

Recent findings have shed new light on mammalian-cell invasion by Salmonella. Using a type III secretion system, Salmonella deliver virulence factors into the host cell that directly activate signal transduction pathways, initiating cytoskeletal rearrangements and bacterial uptake by a ruffling mechanism.     

A balancing act: focus on aneuploidy

Aneuploidy has emerged as a major health concern in cancer and fertility. This issue of EMBO reports features four reviews that discuss aneuploidy and its consequences from different viewpoints, and are contextualized in this editorial.EMBO reports (2012) 13, 472; doi:10.1038/embor.2012.66Faithful chromosome segregation is crucial for the viability of cells and organisms, as evidenced by the fact that in humans only one autosomic trisomy—and no autosomic monosomies—allow survival into adulthood. Cells therefore use sophisticated mechanisms to ensure that each daughter receives an intact copy of the genome during cell division. Eukaryotic chromosomes have a specialized region known as the centromere, which recruits a complex proteinaceus structure—the kinetochore—that binds spindle microtubules to enable the separation of chromosomes during mitosis. The mitotic checkpoint and the machinery that controls kinetochore–microtubule attachment ensure correct chromosome segregation. However, several processes can lead to aneuploidy—the deviation from a haploid chromosomal number—such as defects in mitotic checkpoint proteins or sister chromatid cohesion, incorrect or hyperstabilized chromosome-spindle attachments, centrosome amplification or defects in cytokinesis.Aneuploidy is a major health concern. It is the leading cause of mental retardation and spontaneous miscarriage, and the current trend towards advanced maternal age has increased the frequency of trisomic fetuses by 71% in the past ten years [1]. Furthermore, most solid tumours and about 50% of haematopoietic cancers are aneuploid. During the past few years, the cell-cycle, cancer and fertility fields have therefore made a substantial effort to understand the causes and consequences of aneuploidy.To bring together knowledge from different viewpoints and highlight recent advances in this exciting field, this issue of EMBO reports features four reviews on aneuploidy. An article by Rolf Jessberger analyses the process of oocyte meiosis and how it becomes less accurate with age, and reviews by Holland & Cleveland, Pfau & Amon and Swanton & colleagues focus on aneuploidy in the context of cancer.An overarching theme is the importance of intact sister chromatid cohesion to ensure the fidelity of chromosome segregation. In mammalian oocytes—which remain arrested in meiosis for up to four decades in humans—cohesin is loaded onto chromosomes during development and is probably not turned over for the life of the oocyte. Progressive loss of cohesin or ‘exhaustion'' seems responsible for the dramatic increase in aneuploid eggs with age. Similarly, defects in cohesion proteins are frequently found in various types of cancer.As will become apparent in the three cancer-related reviews, it is important to distinguish between aneuploidy and chromosomal instability (CIN)—a high rate of gain or loss of chromosomes. CIN leads to aneuploidy, but stable aneuploidy can occur without CIN, which is associated with a good prognosis in cancer and occurs in normal brain and liver tissue. An outstanding question is how and whether aneuploidy and CIN predispose to tumorigenesis. Technological advances have allowed the characterization of CIN status of a variety of cancers, underscoring the prevalence of aneuploidy. However, whether aneuploidy is a driving cause of tumour formation remains unclear. Despite the extensive association of aneuploidy with tumours in vivo, extensive data from yeast, mouse and human cell culture indicate that abnormal chromosome content provides a growth disadvantage in vitro, and the presence of CIN in some tumours correlates with good prognosis: this is the so-called ‘aneuploidy paradox''.In this review series, the Cleveland, Amon and Swanton groups provide their own particular views on this paradox. CIN could endow tumour cells with extreme evolvability that is beneficial in vivo, but would be a growth disadvantage under the constant, rich conditions of cell culture. On the other hand, aneuploidy could interfere with cell proliferation—as seen in vitro—and would be selected against; further mutations or chromosomal alterations would allow cells to overcome this restriction and reveal their full tumorigenic potential. According to this view, CIN would allow cells to overcome the negative effects of aneuploidy and promote tumorigenesis below a certain threshold. However, as Swanton and colleagues discuss, the nonlinear relationship between the extent of CIN and cancer prognosis suggests that, beyond this threshold, CIN would become unfavourable owing to the accumulation of deleterious genomic alterations.An increase in genomic material is generally accompanied by an increase in the expression of proteins encoded there, leading to altered metabolic properties, imbalances in the cell proteome and proteotoxic stress due to an overloading of protein degradation pathways. These effects imply that therapeutically targetable pathways would be common in a variety of aneuploid tumour cells. Initial proof-of-principle screens show promise in this regard and, as discussed in these reviews, have led to potential drug candidates.Swanton and colleagues provide a much needed—but rare—translational perspective into the issue of aneuploidy and CIN. Their review highlights the prognostic value of CIN assessment in human tumours, evaluates the methods used to analyse CIN and provides insights into how it could be therapeutically targeted.We hope this selection of comprehensive reviews will contribute to a better understanding of the complexities of aneuploidy and its causes. The possibility of targeting this imbalanced state in cancer therapy and harnessing our increasing knowledge to alleviate fertility problems are exciting prospects. We look forward to future developments in this fast-moving field.     

KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres

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The tmRNA Website: invasion by an intron

tmRNA (also known as 10Sa RNA or SsrA) plays a central role in an unusual mode of translation, whereby a stalled ribosome switches from a problematic mRNA to a short reading frame within tmRNA during translation of a single polypeptide chain. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA Website, a collection of sequences for tmRNA and the encoded proteolysis-inducing peptide tags, alignments, careful documentation and other information; the URL is http://www.indiana.edu/~tmrna. Four pseudoknots are usually present in each tmRNA, so the database is rich with information on pseudoknot variability. Since last year it has doubled (227 tmRNA sequences as of September 2001), a sequence alignment for the tmRNA cofactor SmpB has been included, and genomic data for Clostridium botulinum has revealed a group I (subgroup IA3) intron interrupting the tmRNA T-loop.     

Setting the stage: host invasion by HIV

For more than two decades, HIV has infected millions of people worldwide each year through mucosal transmission. Our knowledge of how HIV secures a foothold at both the molecular and cellular levels has been expanded by recent investigations that have applied new technologies and used improved techniques to isolate ex vivo human tissue and generate in vitro cellular models, as well as more relevant in vivo animal challenge systems. Here, we review the current concepts of the immediate events that follow viral exposure at genital mucosal sites where most documented transmissions occur. Furthermore, we discuss the gaps in our knowledge that are relevant to future studies, which will shape strategies for effective HIV prevention.     

Polyploidy and aneuploidy induced by colcemid in Drosophila melanogaster

Colcemid was fed to Drosophila melanogaster larvae throughout most of the larval period. Surviving individuals were then mated with untreated flies, and their progeny were examined for polyploid flies or flies resulting from X-chromosome nondisjunction. A total of 251 polyploid offspring was recovered from the experimental matings, none from the control. All of the polyploids were evidently triploids, and all but one were obtained from colcemid-fed females: males produced significantly lower frequencies of triploid offspring than females. The highest average frequency of triploid offspring obtained from any treatment group was 18%. Nonrandom distributions of triploid offspring were observed among females raised identically, indicating tht polyploidization occurs mitotically, rather than meiotically, giving rise to clones of tetraploid oogonia. 9 colcemid-fed females produced exclusively triploid offspring. Colcemid also caused a significant increase in X-chromosome nondisjunction in females, though the frequencies of such offspring were at least several-fold lower than the frequencies of triploid offspring. Somatic polyploidy was apparently also indiced since patches of large cells were found on the wings of some flies raised on colcemid-containing food. Various teratological abnormalities were observed among the treated flies, including deformed or missing eyes and partially duplicated thoraxes.     

Mosaic double aneuploidy: Down syndrome and XYY

Chromosomal abnormalities are seen in nearly 1% of live born infants. We report a 5-year-old boy with the clinical features of Down syndrome, which is the most common human aneuploidy. Cytogenetic analysis showed a mosaicism for a double aneuploidy, Down syndrome and XYY. The karyotype was 47, XY,+21[19]/48, XYY,+21[6]. ish XYY (DXZ1 × 1, DYZ1 × 2). Mosaic double aneuploidies are very rare and features of only one of the aneuploidies may predominate in childhood. Cytogenetic analysis is recommended even if the typical features of a recognized aneuploidy are present so that any associated abnormality may be detected. This will enable early intervention to provide the adequate supportive care and management.     

Duplicating dangerously: linking centrosome duplication and aneuploidy

Centrosomes are far more fascinating than the first explorers of this organelle a century ago could ever have imagined. Recent evidence indicates that deregulation of centrosome duplication affects centrosome number and promotes aneuploidy, features characteristic of human tumors.