Anchorage-independent cytokinesis as part of oncogenic ...

[Cell Cycle 7:8, 984-988; 15 April 2008]; ©2008 Landes Bioscience

Perspective

Anchorage-independent cytokinesis as part of oncogenic transformation? Minna Thullberg and Staffan Strömblad Karolinska Institutet; Department of Biosciences and Nutrition; Huddinge, Sweden

Key words: anchorage, cell adhesion, cell cycle, check-point, extracellular matrix, G1-phase, integrin, ras, restriction point, signaling

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far been on the G1-phase. The G1-phase was early proposed to maintain a molecular switch called the restriction point, monitoring the balance between un-preferable and promoting signals, and passed this point the cell was committed to divide.5 Importantly, the regulation of the restriction point was shown to be disrupted in most if not all cancer cells.6 Cell cycle progression through the G1-phase is dependent on growth factors stimulating growth factor receptor tyrosine kinases, together with integrin-mediated anchorage to the extracellular matrix (ECM).7-10 These two classes of receptors coordinately stimulate the activation of the G1-phase cyclin dependent kinases (cdk) 4 and 6.11,12 Cdk4/6 in complex with D-type cyclins phosphorylate the retinoblastoma protein (pRb) and thereby initiate the preparation for DNA synthesis and continued G1- and S-phase progression.11 The induced phosphorylation of pRb coincides in time with the proposed restriction point and the pRb-pathway has therefore been suggested to be the major controller of G1-phase progression.13 However, anchorage-dependent regulation of G1-phase progression has turned out to be more complex.14 Firstly, anchorage is a prerequisite for the induction of cyclin D1 in several cell types and therefore necessary for the phosphorylation of pRb.11,12 In addition, cell adhesion to the ECM is necessary for the induction of the cyclin E-dependent kinase cdk2-activity that controls the subsequent late G1-phase progression.11 Importantly, the anchorage-control of cyclin E-cdk2 activity also persist in primary fibroblasts independent of cyclin D activity.14 The effect of anchorage on cyclin E-cdk2 activity is therefore not a consequence of the lack of previous cyclin D-induction.14 When comparing primary fibroblasts lacking both pRb and the pRb-family member p107, we further found that these cells progressed through the mid-G1-phase independent of growth factors, indicating that pRb/p107 restricts the growth factor requirement in mid-G1-phase. However, importantly, pRb/p107 null fibroblasts still needed anchorage for the progression through midand late G1-phase.14 Without anchorage, the pRb/p107 knockout fibroblasts could not induce cyclin E-dependent cdk2-activity most likely due to the insufficient downregulation of the cdk2-inhibitors p21CIP1 and p27KIP1.11,14-17 Therefore, the G1-phase promotion by growth factors, whose effects appear to be linked to the inactivation of pRb/p107, is not at all required during late G1-phase. In contrast, anchorage is a prerequisite for progression during early, mid and late G1-phase.14 In consequence, the previously suggested model of a single restriction point in the G1-phase, where all different extracellular cues are controlled for, is not applicable. The anchorage-mediated signaling during the G1-phase is initiated by integrin binding to ECM followed by integrin-clustering

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Cell anchorage to the extracellular matrix (ECM) controls the cell proliferation in all multicellular organisms and the abrogation of this control is an indicator of cellular transformation. In fact, two distinct periods of the cell cycle are subject to anchoragedependent regulation. Firstly, anchorage exerts an extensive control of the G1-phase, a control that we found to be more rigorous than for example the control by growth factors. Secondly, anchorage regulates the progression through cytokinesis. In order to achieve anchorage-independent growth a cell must circumvent these controls. To this end, we recently found that oncogenic H-RasV12 can provide sufficient signals to overcome the anchorage-dependence for cytokinesis. Together with earlier findings on G1-phase control, this demonstrates that oncogenic signaling contributes to de-regulation of anchorage-dependence during both the G1-phase and the cytokinesis. This also suggests that de-regulated cytokinesis may be part of oncogenic transformation.

Introduction

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Cancer is a genetic disease, which renders all cancers unique. Nevertheless, the evolution of cancer cells triggers genetic changes that all promote uncontrolled cell growth and thus there are several common features of cancer cells.1 One of these features is the capability of anchorage-independent growth.1 In fact, anchorage-independent growth was frequently used as an indicator of transformation in the era when oncogenes where identified in mouse cell models using cell growth in soft agar as the read-out.2,3 To achieve anchorage-independent growth, the cell has to overcome cell cycle inhibitory signaling and/or apoptosis.4 This Perspective focuses on how anchorage regulates cell cycle progression and how oncogenes may contribute to cancer transformation by promoting anchorage-independent cell proliferation.

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Dual Periods of Regulation of Anchorage-Dependent Cell Division Anchorage-dependent regulation of the cell cycle G1-phase. The center of attention for anchorage-dependent cell cycle control has so Correspondence to: Minna Thullberg; Karolinska Institutet; Department of Biosciences and Nutrition; SE-141 57 Huddinge, Sweden; Email: [email protected]/ Staffan Strömblad; Karolinska Institutet; Department of Biosciences and Nutrition; SE-141 57 Huddinge, Sweden; Email: [email protected] Submitted: 01/25/08; Accepted: 01/27/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5674 984

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Anchorage-dependent cell cycle control

has not been thoroughly investigated. However, integrin binding is a likely source, since integrins upon attachment are the major transducers of intracellular signals. The work by Aszodi et al., showed that integrin β1 null chondrocytes in mice were bi-nucleated and that this defect affected the bone structure.33 This demonstrates that integrin β1 is essential for cytokinesis in vivo within the tissue, at least in certain cells.33 However, the potential role of other integrins has not been thoroughly investigated. Our results show that both integrin αv and integrin β1 can support cytokinesis in fibroblasts.25 Taken together, these data also support the hypothesis that attachment is necessary for the progression through cytokinesis of most or all untransformed cells. However, the nature of attachment may not be critical, different integrins or other cell surface receptors might contribute depending on the surrounding ECM.25

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Role of Anchorage in Regulation of Cytokinesis: Tension or Signaling?

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Given that primary fibroblasts in suspension can initiate ingression of the cleavage furrow but are unable to complete the ingression,25 a central question is why the ingression is not completed. To this end, at least three alternative explanations has been proposed for the role of anchorage in cytokinesis.34 Firstly, signaling from loss of anchorage may initiate a checkpoint in cytokinesis to prevent the cells to complete division. Secondly, anchorage may be necessary for proper actin polymerization providing tension in the cleavage furrow to enable complete ingression. A third alternative is that anchorage may be necessary for re-establishment of cell-matrix adhesion complexes and cell spreading in the end of cytokinesis to provide traction forces necessary for ingression and abscission of the midbody.34,35 Our recent findings indicate that anchorage provides necessary regulatory signals or that lack of anchorage triggers a checkpoint in cytokinesis, but that tension or traction forces may not be needed for ingression during cytokinesis.25 Firstly, we found that cell spreading was not necessary for complete furrow ingression (Fig. 1A).25 In fact, we found that complete ingression in anchored fibroblasts occurred before the cells regained extensive attachment and spreading (Fig. 1A and B).25 Therefore, cell spreading and ingression of the cleavage furrow seems to be events occurring independent from each other. In addition, by studying video time-lapse of many human primary fibroblasts during cytokinesis, we found that some cells completed ingression in a cleavage plane different from the focus plane.25 In such situations, attachment of only one of the daughter cells was sufficient to promote complete ingression and division (Fig 1B).25 This also showed that the orientation of the spindle in relation to the attachment site as well as to gravity is not important for the final outcome of cytokinesis.25 Ingression of the cleavage furrow is enforced by Myosin II-driven cortical actin reorganization.34,36 Are then cells cultured without anchorage incapable of Myosin II activity or sufficient actin organization? To this end, we found that oncogenic H-RasV12 could promote cytokinesis in cells cultured without anchorage.25 We also found that Myosin II activity and proper organization of the actin were still necessary for cytokinesis also in the presence of active H-Ras V12.25 This suggests that cells in suspension have sufficient Myosin II activity and actin organization, since overexpressed HRasV12 promoted cytokinesis in these cells. H-RasV12 is a strong inducer of various intracellular signaling.37-41 The finding that

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triggering signal transduction.18 This signal transduction promotes actin cytoskeletal remodeling to induce cell spreading and actin stress fiber formation.19,20 The actin remodeling is considered to further initiate signaling which controls cell cycle progression.21-23 Integrin binding and importantly, the subsequent cell spreading, are necessary for the induction of cyclin D1.21 The treatment of cells with actin disrupting drugs will equally to having them in suspension induce a G1-phase arrest caused by inhibition of cyclin D1 synthesis.21,22,24 Loss of anchorage thus induces actin remodeling, which triggers signals to stop the G1-phase progression. In addition, integrin-mediated attachment may control cell cycle G1-phase progression by other routes of signaling, such as through integrin-mediated activation of the small GTPases Cdc42 and Rac1 leading to degradation of the cdk2-inhibitors p21CIP1 and p27KIP1.15 Anchorage-dependent regulation of cytokinesis. As explained above, anchorage exerts an extensive control of the G1-phase, indicating a more significant role of anchorage than growth factors in restricting cell proliferation. Therefore, while growth factors exert no effect on any other cell cycle phases,5 anchorage might regulate additional cell cycle phases. However, human primary fibroblasts progress perfectly through the S- and G2-phases without anchorage.25 Nevertheless, it may be indicated from visual inspection that anchoring proteins and cytoskeleton remodeling might regulate some of the stages during mitosis. Onset of mitosis coincide with cellular de-adhesion and actin cytoskeleton remodeling inducing cell rounding that is prominent during prometaphaseanaphase (Fig. 1A).26 Then, the cell regains full attachment and spread when the ingression of the cleavage furrow is complete (Fig. 1B).25,26 Whether lack of anchorage influences early stages of mitosis is still controversial but it appears likely that too strong attachment might influence cell rounding and progression through prometaphase until telophase. Two recent studies focusing on the function of integrin β1 during mitosis suggest that it is important for progression through this period.27,28 Toyoshima et al., described differences in spindle orientation when inhibiting integrin β1, but did not thoroughly investigate how this affected progression through mitosis and cytokinesis.27 In addition, Reverte et al., indicated that an integrin β1-Y783 mutant affecting integrin-mediated intracellular signaling, induced an impaired centrosome duplication, abnormal tubulin orientation and prevented proper progression through mitosis in transfected CHO cells.28 In contrary, we found that human primary fibroblasts progressed through mitosis until cytokinesis when cultured without anchorage, and that the tubulin spindles were still formed and sister chromatids separated in suspension.25 Further, the central spindle was formed and contained tubulin, Aurora B and Rho A similar to the anchored cells , and also the midbody was formed in primary human fibroblasts cultured in suspension.25 Importantly, lack of anchorage appears to play a role during cytokinesis, since several studies published almost 30 years ago described the formation of bi-nucleated cells caused by culture without proper substratum.29-32 However, it was unclear why these cells become bi-nuclear. We investigated how far into cytokinesis primary human fibroblasts could proceed without anchorage and found that primary human fibroblasts in suspension could form and initiate ingression of the cleavage furrow, but could not complete the ingression nor the final cleavage.25 The nature of attachment necessary for cytokinesis www.landesbioscience.com

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Figure 1. Human fibroblasts round up in mitosis and spread after ingression of the cleavage furrow. Representative differential interference contrast (DIC) images of human fibroblasts progressing through mitosis. Normal cells lose attachment during mitosis and are rounded from the prometaphase until cleavage furrow ingression.25,26 (A) Image sequence of a human immortal fibroblast (BJT) proceeding from the G2-phase to mitosis, the dividing cell is indicated by arrows. (B) Image sequence of a primary human fibroblast (NHDF) progressing through mitosis with one of the daughter cells not attached. Upper with the focus plane set at the attached cell at the bottom. Lower with the focus plane set on the unanchored daughter cell. Arrows indicate the cleavage furrow. The daughter cell attached only to its anchored sister cell during ingression and then attached and spread onto the substratum after the ingression was completed. Cells maintain cell-cell contacts after the ingression.25,26 Bar = 10 μm.

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H-RasV12 promotes anchorage-independent cytokinesis further indicates that specific signaling controls anchorage-dependence in cytokinesis, rather than tension or traction forces.25 While HRasV12 was unable to overcome Myosin II inhibition, inhibition of Rho kinase that blocked cytokinesis in normal fibroblasts was overcome by H-RasV12 expression.25 This shows that H-Ras signaling overcomes certain specific cytokinesis inhibitory signals but not others. This information may be useful to elucidate in more detail how anchorage-dependent cytokinesis is controlled by signaling. Taken together, the fact that anchorage regulates several points of the cell cycle indicates its vast importance in cell proliferation control. Although the anchorage control appears to be more rigorous, most studies of cell cycle regulation have so far have been focused on growth factors. There may be good reasons for the strict and extensive anchorage-dependent control of cell proliferation, since the integrin-mediated contact with the ECM is the major way to convey information into the cell about its local environment. Cell proliferation within an inappropriate location may be disastrous, leading to developmental abnormalities or cancer growth. Therefore, 986

it appears important to further improve our understanding of anchorage-dependent cell cycle regulation.

De-Regulation of Anchorage-Dependent Cell Division During Oncogenic Transformation If anchorage is necessary for progression through cytokinesis, then anchorage-independent growth must be a consequence of deregulation of both the G1-phase and the cytokinesis. Indeed, many cancer cells can proliferate in an anchorage-independent manner and it has been clear for a long time that these cells have a de-regulated G1-phase that can be achieved by oncogenic signaling.42-48 However, it remainedunclear if oncogenic signaling may also de-regulate anchorage-dependence in cytokinesis. To address this question, we used a set of human BJ fibroblasts derivatives from Hahn et al., who created transformed anchorage-independent cells with defined genetic elements.49 Immortalized BJ fibroblasts expressing the simian virus 40 large T antigen (SV40LT) became binuclear in suspension.25 When these cells in addition overexpressed oncogenic H-Ras V12 they divided in suspension, these cells were also

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a tumor to de-regulate the control for anchorage? Alternatively, is the anchorage-independence firstly needed at the later stage of tumor dissemination? In fact, it is likely that anchorage-independent growth is necessary for tumor dissemination, given that this process involves cell migration from its normal environment, including release from the cells’ normal ECM to a less defined microenvironment and spread within blood or lymph vessels without any attachment. The integrin β1 null chondrocytes becomes binuclear in vivo, showing that also cells surrounded by ECM in vivo might get binuclear if the attachment is impossible, not strong enough, or mediated by other receptors than those required.33 Each cell type may need their own specific set of integrin to ECM contacts to get proper attachment during proliferation and if the ECM content or integrin expression changes, the cell can no longer divide. For example, mammary epithelial cells must have a proper coordination between growth factor signals and the type of ECM in their microenvironment to be able to proliferate, and the coordination of the ECM composition and growth factors finely regulates the balance between cell proliferation, differentiation, and apoptosis.53,54 De-regulation of such a fine balance may make tumor cells sturdier and select cells that are able to handle those changes in the microenvironment that occur within a tumor and that a disseminating tumor cell is exposed to. The gain of anchorage-independence of tumor cells then appears as an efficient way to de-regulate this balance. Finally, given the need for anchorage not only for G1-phase progression, but also for cytokinesis, unraveling of the detailed molecular mechanisms of anchorage-dependence and anchorageindependence for cytokinesis may add to the list of potential targets for development of novel tumor therapy.

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anchorage-independent in soft agar.25,49 This implicates that H-RasV12 can provide sufficient signals to overcome the anchoragedependent checkpoint in cytokinesis, and thus that oncogenic signaling is capable not only to deregulate anchorage-dependence of the G1-phase, but also to de-regulate cytokinesis. A remaining question is whether binuclear cells can continue to progress in the cell cycle or will arrest in the G1-phase. Work by Uetake and Sluder indicated that binuclear cells continue in the cell cycle and replicate their DNA and divide.50 With this perspective, a cell in suspension would continue to go on in the cell cycle and in the end become multinuclear and supposedly giant. However, Andreassen et al., on the other hand suggest that binuclear cells induce a tetraploidy checkpoint and a subsequent arrest in the G1phase in a p53-dependent manner.51,52 However, regardless whether the cells in suspension end up as binuclear and arrest in the G1phase or continue to be giant cells they will not be able to proliferate further and will be harmless from a cancer point of view. The discoveries described above implicates that oncogenic H-Ras may contribute to transformation during at least two periods in the cell cycle. Firstly, H-Ras promotes G1-phase progression by inducing uncontrolled cyclin D-dependent kinase activity and secondly, it promotes anchorage-independent cytokinesis. This also implicates that oncogenic transformation may need to include de-regulation of cytokinesis, and a distinct possibility that other oncogenic signaling than H-Ras may also promote anchorage-independent cytokinesis. However, future research will have to reveal if this hypothesis is correct.

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Figure 2. De-regulation of anchorage-dependent cell division by oncogenic signaling. Primary human fibroblasts are sensitive to lack of anchorage during cell cycle progression while this control is abolished in many cancer cells.1 In comparison with growth factors that are necessary during early and mid G1-phase, where pRb is inactivated by phosphorylation,13,55 anchorage is necessary during a longer period in the G1-phase as well as during cytokinesis (C).14,25 However, S- and G2-phases progress independent of anchorage.25 Signals from Oncogenic H-Ras V12 are capable of promoting anchorageindependent growth.37,42 An important part of this function might be the promotion of anchorage-independent cytokinesis.25 Oncogenic H-Ras V12 also promotes anchorage-independent G1-phase progression, possibly through de-regulation of the pRb pathway and through promoting degradation of p21CIP1 and p27KIP1.44,56-58 Prophase (P); Prometaphase (PM); Metaphase (M); Anaphase (A); Cytokinesis (C).

Outlook Anchorage-independent growth has long been used as an indicator of oncogenic transformation and an indicator of cancer cell deregulated growth.1-3 However, a number of fundamental questions remain. For example, how is the anchorage-independent phenotype helping the cancer to develop? Is it actually necessary for a cell within www.landesbioscience.com

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We thank Professor William Hahn for the BJT fibroblasts and derivatives. This work was supported by grants to S.S. from The Swedish Cancer Society and The Swedish Research Council and to M.T. from The Swedish Society of Medicine. S.S. holds a senior scientist position from The Swedish Research Council.

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