Cyclin E Drives Human Keratinocyte Growth Into Differentiation

ORIGINAL ARTICLE Cyclin E drives human keratinocyte growth into differentiation

A Freije1,2, L Ceballos1,2, M Coisy3, L Barnes3, M Rosa1,2, E De Diego4, JM Blanchard3 and A Gandarillas1,2,3,5

Human epidermis is continuously exposed to environmental mutagenic hazard and is the most frequent target of human cancer. How the epidermis coordinates proliferation with differentiation to maintain homeostasis, even in hyperproliferative conditions, is unclear. For instance, overactivation of the proto-oncogene MYC in keratinocytes stimulates differentiation. Here we explore the cell cycle regulation as proliferating human keratinocytes commit to terminal differentiation upon loss of anchorage or overactivation of MYC. The S-phase of the cell cycle is deregulated as mitotic regulators are inhibited in the onset of differentiation. Experimental inhibition of mitotic kinase cdk1 or kinases of the mitosis spindle checkpoint Aurora B or Polo- like Kinase, triggered keratinocyte terminal differentiation. Furthermore, hyperactivation of the cell cycle by overexpressing the DNA replication regulator Cyclin E induced mitosis failure and differentiation. Inhibition of Cyclin E by shRNAs attenuated the induction of differentiation by MYC. In addition, we present evidence that Cyclin E induces DNA damage and the p53 pathway. The results provide novel clues for the mechanisms committing proliferative keratinocytes to differentiate, with implications for tissue homeostasis maintenance, HPV ampliﬁcation and tumorigenesis.

Oncogene (2012) 31, 5180--5192; doi:10.1038/onc.2012.22; published online 20 February 2012 Keywords: skin; stem cells; DNA damage; MYCER; cell size; endoreplication

INTRODUCTION able to disrupt differentiation (for example, E2F, Cyclin D1, MDM2, Epidermal homeostasis involves continuous cell renewal, expan- 11 sion and enlargement.1,2 In the basal layer of the epidermis, cdk2, cdk4; references in Zanet et al. ). Cyclin-dependent kinases cdk2 and cdk1 are key regulators of mainly quiescent, reside the epidermal stem cells. Some daughters 12 of the stem cells activate their cell cycle, undergo a short clonal S-phase and mitosis, respectively. In order to gain insight into the expansion phase of rapid proliferation and migrate into the mechanisms linking cell growth with differentiation, we have suprabasal layers. As they leave the basal layer, they cease compared the dynamics of Cyclin/cdk complexes during the proliferation, initiate terminal differentiation and increase their commitment to differentiation in normal keratinocytes or after cellular size.3,4 The mechanisms that coordinate epidermal MYC overactivation. We have inhibited cell cycle kinases, including keratinocyte cell growth and differentiation are unclear. Actively kinases of the mitosis spindle checkpoint, and explored the effects proliferating cells have a limited potential, as they are committed on differentiation. The results reveal that the cell cycle is to differentiate after four or ﬁve rounds of cell divisions by deregulated in the onset of differentiation, involving strong unknown mechanisms.1 Epidermal proliferation and differentia- accumulation of the S-phase regulator Cyclin E. Overexpression tion are controlled by cell adhesion to the basement membrane of Cyclin E in human basal keratinocytes induced a mitosis block, via integrins. Terminal differentiation can be rapidly induced by re-replication and differentiation. This response appears to be part depriving keratinocytes of cell anchorage in suspended cultures.5 of a novel keratinocyte anti-proliferative mitosis-differentiation Proto-oncogene MYC, a potent inducer of cell growth and cell checkpoint. Interestingly, MYC might promote keratinocyte differ- cycle,6 stimulates epidermal stem cell differentiation.7,8 Activation entiation by inducing Cyclin E. In turn, accumulation of Cyclin E of ectopic MYC in basal cells favours differentiation versus would trigger the mitosis checkpoints by causing DNA damage proliferation, what causes a rise of differentiation markers after and inducing the p53/p21 pathway. This mechanism might explain 4--5 days. We previously proposed that this is the time required by how actively proliferating keratinocytes commit to differentiation keratinocyte to undergo the clonal expansion phase of rapid and how the epidermis is protected from cell growth alterations. proliferation that precedes terminal differentiation.7 MYC drives differentiation in part by inhibiting cell adhesion.7,8 However, the cell cycle mechanisms by which MYC elicits this function have not RESULTS been elucidated. Cdk complexes of S-phase and mitosis during suspension-induced We have previously shown that postmitotic keratinocytes differentiation continue DNA synthesis and become polyploid during differentia- Integrins mediate cell adhesion to the basal membrane of the tion in vitro9 and in vivo,10,11 and that the process of endoreplica- epidermis and sustain keratinocyte proliferation.13 Inactivation of tion is stimulated by MYC. As for MYC, other molecules involved in integrin function in suspension rapidly triggers terminal differ- cell cycle progression caused epidermal hyperplasia, but were not entiation. Within 5 h keratinocytes irreversibly lose their capacity

1Cell Cycle, Stem Cell Fate and Cancer Laboratory. Institute for Training and Research of the Fundacio´ n Marque´s de Valdecilla (IFIMAV-FMDV), Santander, Spain; 2Molecular Biology Department of Universidad de Cantabria (UC), Santander, Spain; 3Institut de Geńe`tique Molećulaire de Montpellier (CNRS/UMPII), Montpellier, France; 4Servicio de Cirugıá Pedia´trica, Hospital Universitario Marque´s de Valdecilla (HUMV), Santander, Spain and 5Laboratoire de Dermatologie Molećulaire, UPRES EA 3754, Institut Universitaire de Recherche Clinique, Inserm ADR Languedoc-Roussillon, Montpellier, France. Correspondence: Dr A Gandarillas, Cell Cycle, Stem Cell Fate and Cancer Laboratory, Institute for Training and Research of the Fundacioń Marque´s de Valdecilla (IFIMAV-FMDV), Av. Herrera Oria s/n., Santander 39011, Spain. E-mail: ifi[email protected] Received 2 June 2011; revised 6 January 2012; accepted 8 January 2012; published online 20 February 2012 Cyclin E and keratinocyte differentiation A Freije et al 5181

Figure 1. Dynamics of cdk complexes of S-phase and mitosis, during the keratinocyte commitment to suspension-induced differentiation. (a) Quantitation of the flow-cytometry analyses of primary human keratinocytes placed in suspension for the periods of time indicated. Top histogram: percentage of involucrin-expressing cells or cells with a differentiated morphology (High Scatter). Bottom histogram: percentage of cells undergoing DNA synthesis (BrdU-incorporation) in G2/M (propidium iodide, PI) or polyploid; cells were incubated with BrdU for 3 h before harvest, or for the last 12 h in the case of 24 h. Data are the average of duplicate samples and representative of four independent experiments with cells from three different individuals. Error bars are s.e.m. The plots and populations are shown in Supplementary Figure 1. (b)Expressionofthe regulators indicated in suspended keratinocytes as determined by western blotting; CA, Cyclin A; CB, Cyclin B; CE, Cyclin E; p-Rb, phosphorylated Rb. b-actin (b-act) was used a loading control. *Cyclin B (CB) was first immunoprecipitated owing to low expression. (c)Normalhuman keratinocytes were double stained for Cyclin E (CE, in green) and involucrin (Invol, in red) or Cyclin A (CA; in red) and analysed by confocal microscopy. The images show a differentiating layer. Note that Cyclin E accumulates (arrows) and that stratified cells express mitotic Cyclin A (arrowhead; see Supplementary Figures 2a--c). Scale bar, 50 mm. (d) Representative analyses of kinase activity on histone H1 associated with the regulators indicated; histogram bars shows the quantitation of the radioactivity of duplicates, small bars being s.e.m. (e) Immunoprecipitation (IP) for the regulators indicated on the top and WB for the regulators indicated on the left. PST: motive common to cdk1 and cdk2; i, a: inactive or active forms of the regulators indicated according to mobility shift due to phosphorylation. Results with cells from two different individuals.

to proliferate and commit to differentiate.5 By 24 h all cells are cdk2 or to cdk1 during the S/G2 transition, and Cyclin B/cdk1 differentiated. We placed primary keratinocytes in suspension to controls mitosis.12 The Cyclin A/cdk2 complex has been previously analyse changes in the cell cycle during the transition to shown to depend on cell anchorage,15 also in keratinocytes.16,17 differentiation. Epidermal differentiation is associated with mor- Expression of Cyclins A and B was high for the first 6 h in phological changes and with the induction of the cornified suspension but it was suppressed by 12 h (Figures 1b and 2a and precursor involucrin that can be assessed by flow cytometry Supplementary Figure 1b). Strikingly, Cyclin E followed an (Figure 1a and Supplementary Figure 1a).9,14 Cellular size and opposite pattern, and markedly accumulated from 6 to 24 h complexity (light scattering), and the expression of involucrin (Figures 1b and 2a and Supplementary Figure 1b). Similar results increased markedly after 12 h in suspension. were observed by confocal microscopy analyses of stratified Bromodeoxyuridine (5-bromo-20-deoxyuridine, BrdU) incorpora- cultures (Figure 1c and Supplementary Figure 2). Interestingly, the tion experiments showed that DNA synthesis stays active during switch from mitotic Cyclins to high Cyclin E was associated with differentiation (Figure 1a and Supplementary Figure 1a). Labelling- the increase in cell size and involucrin expression, characteristics adherent cycling cells by a BrdU pulse before suspension showed of epidermal differentiation (Figures 1a-c and 2a and Supplemen- that differentiating cells accumulated in G2/M by 12 h and tary Figures 1-3).3,4 subsequently, bypassed mitosis and became polyploid (Supple- Consistent with the changes in Cyclin expression, the expres- mentary Figure 1a). Therefore, DNA synthesis stayed active during sion of mitotic kinase cdk1 was downregulated in suspended cells differentiation beyond the time of commitment (6 h). Concomi- (Figure 1b). Although the expression of Cyclin E and cdk2 proteins tantly, we analysed the dynamics of cdk2 and cdk1 complexes. was sustained, all the kinase activities were inhibited after 6 h in Cyclin E/cdk2 complexes control DNA replication, Cyclin A binds to suspension (Figure 1d). The inhibition of Cyclin A-associated

& 2012 Macmillan Publishers Limited Oncogene (2012) 5180 --5192 Cyclin E and keratinocyte differentiation A Freije et al 5182

Figure 2. Dynamics of cdk complexes of S-phase and mitosis during MYC-promoted differentiation. (a) WB for the regulators indicated on the left of parallel samples from keratinocytes in suspension or after activation of MYCER by OHT, for the periods of time indicated. Invol bands are due to differentiation crosslinking. Regulators abbreviated as in Figure 1; broken lines: phase of rapid cell ampliﬁcation. (b) Representative analyses of kinase activity on histone H1 associated with the regulators indicated, after activation of MYCER as in a; bar histogram shows the quantitation of duplicate samples; small bars are s.e.m. (c) Expression of the cell cycle inhibitors indicated after activation of MYCER as in a;(d) Or in freshly isolated keratinocyte populations from human skin enriched for stem cells (SC), rapidly amplifying cells (TAC), or terminally differentiating cells (TDC); MIX: unsorted. Results with cells from two different individuals. Bars in c and d indicate quantitation (% of total) of the different mobility forms detected for Rb: hypophosphorylated (white; active, a), phosphorylated (grey), hyperphosphorylated (black; inactive, i). C, negative antibody control on adherent cells; E, exponentially growing HDF; M, human dermal ﬁbroblasts (HDF) blocked in mitosis by Nocodazole for 24 h.

activity preceded the inhibition of Cyclin B-associated activity, binding of p21 to Cyclin E complexes peaked by 12 h (Figure 1e). which was still high by 6 h. Both became undetectable by 12 h. Reverse experiments by pulling down p21 showed consistent However, a remaining low Cyclin E-associated activity was results (Figure 1e). detectable by 12 h. Cyclin B and Cyclin E activities increased p21 was barely detectable in Cyclin E IPs after 24 h, suggesting slightly and tumour suppressor Rb and histone H3 were that most of the Cyclin E protein was free of the inhibitor hyperphosphorylated as soon as cells were placed in suspension (Figure 1e). Conversely, IP for p21 revealed a neat band of Cyclin E by trypsin treatment (0 h: Figures 1b and 2a). Inactivation of Rb by by 24 h, suggesting that most of the p21 protein was bound to phosphorylation marks cell cycle entry and total Rb is down- Cyclin E/cdk2 (Figure 1e). In summary, the transient increase of regulated during keratinocyte differentiation.16,18 Consistent with p21 appears to suffice to inhibit the mitotic Cyclin/cdk complexes previous reports,16,17 cdk inhibitors p21CIP1 (hereafter, p21) and by 6 h, but not the high levels of Cyclin E that are subsequently p27KIP1 (hereafter, p27) were upregulated in suspension produced. Consistently, immunodepletion of p21 in cell lysates (Figure 1b). However, whereas p27 reached maximum levels late removed most of the Cyclin A/cdk complex by 6 h, but left during differentiation, p21 was transiently induced by the time of significant levels of Cyclin E/cdk2 complex by 12 h (Supplementary commitment (6 h). Figure 4a). Interestingly, downregulation of Cyclin A in suspended We next analysed the dynamics of Cyclin/cdk complexes by mouse keratinocytes requires p21.17 immunoprecipitation (IP) and western blotting (WB; Figure 1e and Supplementary Figure 4a). Cyclin A bound to both cdk2 and cdk1 (Figure 1e). By 12 h, the detection of both kinases in Cyclin A IPs Cdk complexes of S-phase and mitosis during MYC-induced was much reduced and by 24 h, only the inactive form of cdk1 differentiation was barely detectable (Figure 1e). Cyclin B/cdk1 peaked by 6 h. Constitutive activation of proto-oncogene MYC drives epidermal Increasing levels of cdk2 were detected in Cyclin E IPs up to 24 h stem cells into the clonal amplification phase (referred to as transit (Figure 1e; PSTAIR is a homologous motive of the cdks; Cyclin E amplifying cells or TAC) that precedes terminal differentiation.7,8 binds to cdk2). It is worth noting that the binding of the cdk Once terminal differentiation initiates, MYC is downregulated inhibitor p21 to Cyclin B complexes peaked by 6 h whereas the (Figure 2a).19,20 Keratinocytes expressing MYCER constitute an

Oncogene (2012) 5180 --5192 & 2012 Macmillan Publishers Limited Cyclin E and keratinocyte differentiation A Freije et al 5183 inducible differentiation system in the presence of cell adhesion terminal differentiation by 48 h, as it was evident by light that is more gradual than the suspension method. An increase in scattering, involucrin expression and cell morphology (Figure 3a the proportion of terminally differentiating cells is observed over 5 and Supplementary Figures 5 and 6). days after the activation of MYCER, the time estimated for stem To further determine whether inhibition of cdk1 triggered cells to amplify before differentiation (see involucrin in terminal differentiation, we expressed specific short hairpin RNAs Figure 2a).7,9 We have examined the dynamics of cdk complexes (shRNAs) in primary keratinocytes by lentiviral infection. Two during MYC-promoted differentiation. As the induction of different shRNAs were able to inhibit the expression of differentiation by MYC is more gradual than in the suspension endogenous cdk1 as analysed by real-time reverse transcrip- system, the changes were not as marked but were consistent. A tion--polymerase chain reaction (RT--PCR; Figure 3b). The cell transient increase of the expression of Cyclins A and B was cultures showed areas of rounded cells typical of mitosis arrest observed over 2--3 days after MYCER activation (Figure 2a and (Supplementary Figure 7). Inhibition of cdk1 provoked an increase Supplementary Figure 4c). In contrast, MYC continuously induced in the proportion of cells expressing the terminal differentiation the expression of Cyclin E (Figure 2a). This is consistent with keratins K1 and K10 as measured either by RT--PCR or by previous reports showing that MYC induces Cyclin E expres- percentage of positive cells (Figure 3b). sion.21,22 The kinase activities associated with Cyclin A, Cyclin B Genotoxic agents might cause keratinocyte differentiation by and cdk2 increased over the first 2 days and dropped by 7 days triggering a mitosis checkpoint.11 Mitosis kinases Aurora B or below the initial levels (Figure 2b). Cyclin E-associated activity also Polo-like kinase 1 (Plk1), which are involved in chromosome increased and decreased to levels similar to the start of the segregation, participate in the mitotic spindle checkpoint32,33 and experiment (Figure 2b). All regulators thus reflected an early some of their inhibitors are being tested in cancer clinical activation of the cell cycle by MYC. Consistently, the hyperpho- trials.34 To explore whether the keratinocyte mitotic checkpoints sphorylated (inactive) form of Rb became predominant by 6--12 h are involved in the signal to differentiation, we inhibited and phospho-Rb Ser780 and phospho-cdk2 Y15 increased after these molecules by specific chemical inhibitors. A 48 h treatment MYCER activation (bars, Figure 2c and Supplementary Figure 4b). with Aurora B kinase inhibitor, ZM44743935 or Plk1 inhibitor, The inhibition of the kinase activities after 3 days might be due to BI253636 strongly induced an accumulation of keratinocytes in G2/ the striking transient induction of total p21 levels (Figure 2c). p21 M and polyploidy and a remarkable increase of cell size and bound to both Cyclin A and Cyclin E complexes in the presence of differentiation (Figure 4a and Supplementary Figures 5 and 6). activated MYC (Supplementary Figure 4c). In contrast, p27 was To confirm the specificity of the effects, we inhibited Aurora B in undetectable in the MYCER keratinocytes (Figure 2c). keratinocytes by transfection of specific small interference RNA Altogether, the results suggest that the transient induction of (siRNA). Transient inhibition of Aurora B produced 24 h after p21 might be a keratinocyte response to cell cycle deregulation. transfection a significant increase in the proportion of cells Expression of p21 has been previously described in the expressing the differentiation marker involucrin (Figure 4b). epidermis.23 To further study this issue, we isolated keratinocytes The induction of terminal differentiation by the mitotic from the normal human skin and selected stem cells, transit inhibitors was irreversible as cells did not recover colony growth amplifying cells and terminally differentiating cells on the basis of after being released from the 48 h treatment with Roscovitine or their adhesion capacity via integrins.14 Strong expression of p21 BI2536 (Figure 4c and Supplementary Figure 6). In contrast, the was detected in the slowly adhering population, previously colonies grew bigger after the G1/S block by the AG555 inhibitor defined as transit amplifying cells (TAC; Figure 2d). The presence (Figure 4c and Supplementary Figure 6). of p21 was again accompanied by a higher phosphorylation of Rb (Figure 2d). These results were therefore consistent in suspension- induced differentiation, MYC-induced differentiation and sponta- Overexpression of Cyclin E-GFP neously occurring differentiation. We have shown that epidermal differentiation is associated with a marked accumulation of Cyclin E in vitro (Figures 1a and 2a and Supplementary Figures 5 and 6) and in vivo.11 To test whether a Mitosis block and differentiation direct hyperactivation of the cell cycle is able to drive differentia- The results reported above suggest that keratinocyte differentiation, we constructed a retroviral green fluorescent protein (GFP) tion involves mitotic inhibition and cell cycle deregulation. To form of Cyclin E under a constitutive retroviral promoter for its functionally explore this issue, we treated normal primary expression in basal primary keratinocytes (Figures 5a and b and keratinocytes with cdk inhibitors in order to block either DNA Supplementary Figure 8a). In vitro assays showed that Cyclin E-GFP replication cdk2 or mitosis cdk1. The specificity of these inhibitors was functional in activating cdk2 (Supplementary Figure 8b and on cdk2 or cdk1 vary depending on the concentration and the cell not shown). The exogenous Cyclin E-GFP localised to the nucleus type.24 However, AG555 was identified as a cdk2 inhibitor.25,26 and was sensitive to posttranscriptional regulation, as it was Roscovitine and NG97 were selected by searching for inhibitors of absent in metaphasic cells (Supplementary Figure 9a, INSET), as Cyclin B/cdk1 and have been shown to block the mitosis described for the wild-type protein.12 Keratinocytes overexpres- machinery.27 --29 As it is not technically possible to test the activity sing Cyclin E-GFP (KCEGFP) were significantly larger and presented of the inhibitors in vivo, we tested their capacity to inhibit S-phase large or various nuclei (Figure 5b and Supplementary Figure 9a). or mitosis by analysing their effect on DNA replication and cell KCEGFP cultures widely expressed the terminal differentiation cycle.30,31 Treating primary keratinocytes with AG555 for 48 h at marker involucrin (Figure 5b). the concentration used in Figure 3a significantly inhibited Flow-cytometry analyses of unselected cultures after infection progression through S-phase, causing an accumulation of cells with Cyclin E-GFP (GFP þ /À; Figure 5c) or stably selected by in G1/S and a reduced G2/M phase. In contrast, although puromycin (KGFP/KCEGFP; Supplementary Figure 9b) showed that Roscovitine and NG97 have the capacity to inhibit both cdk1 Cyclin E caused an accumulation of cells in the G2/M phase of the and cdk2, they provoked a marked accumulation of keratinocytes cell cycle and induced a significant proportion of polyploid cells. In in G2/M and polyploidy and yet allowed DNA replication addition, overexpression of Cyclin E induced a significant increase in (Figure 3a and Supplementary Figures 5 and 6). AG555 slightly the rate of large cells (High Scatter) and involucrin expression inhibited cell size and terminal differentiation as measured by (Figure 5c). Overexpression of Cyclin E also induced an increase in light scattering, involucrin expression and cellular morphology the rate of DNA synthesis, consistent with cell cycle activation (Figure 3a and Supplementary Figures 5 and 6). In contrast, both (Figure 5d and Supplementary Figure 9c), and a decrease in Cyclin A, Roscovitine and NG97 strongly induced basal cells to undergo consistent with terminal differentiation (Figure 5d; see Figure 1a).

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Figure 3. Changes in cell cycle and differentiation after treating keratinocytes with cdk inhibitors. (a) Representative ﬂow-cytometry analyses of DNA content, DNA synthesis (BrdU incorporation), cell size and morphology (light scattering) and involucrin expression after treating keratinocytes for 48 h with the cdk inhibitors 50 mM AG555 (AG), 30 mM Roscovitine (ROSCO; Supplementary Figure 5), or 1 mM NG97 (NG). Bar histograms: quantitation of BrdU-positive cells, polyploid cells (44N) or differentiating cells (High Scatter). Numbers are from the regions shown in the histograms above, means of duplicates and representative of experiments with cells from two different individuals. The circle in the morphology histograms indicates the proliferative basal population. The involucrin positive region (p) was determined by a negative isotype antibody control (CD8). Bars are s.e.m. Ctr: control cells. (b) Inhibition of cdk1 by lentiviral infection with shRNA 2. Top histograms: expression of cdk1 or differentiation markers K1 and K10 as measured by RT--PCR in control cells (Ctr, white) or in the presence of shRNA to cdk1 (grey). Bottom histogram: percentage of keratin K1-expressing cells. Microphotographs: expression of K1 and nuclear staining by DAPI of the same ﬁeld, as indicated.

Overexpression of Cyclin E caused a reduction in the clonogenic Supplementary Figure 9d) and by assays on the capacity to form amplification capacity (Figure 6a). This reduction was not due to cornified envelops, the final product of epidermal differentiation senescence (Supplementary Figure 10) or apoptosis (no sub-G1 in (Figure 6c).37 Therefore, all the parameters analysed show that the the DNA content profiles; Figure 5c and Supplementary Figure 9b). activation of the cell cycle by Cyclin E in basal keratinocytes results Basal KCEGFP were able to attach and to proliferate but formed a in terminal differentiation. higher proportion of abortive-differentiated colonies (Figure 6a). MYC has been shown to induce Cyclin E.21,22 Activation of The increased terminal differentiation rate was further confirmed conditional MYCER significantly induced Cyclin E in keratinocytes by a higher expression of keratins K1 and 10 (Figure 6b and (Figures 2a and 6d). We questioned whether Cyclin E might

Oncogene (2012) 5180 --5192 & 2012 Macmillan Publishers Limited Cyclin E and keratinocyte differentiation A Freije et al 5185

Figure 4. Changes in cell cycle and differentiation after inhibiting keratinocyte mitosis kinases of the spindle checkpoint. (a) Representative flow-cytometry analyses of DNA content, DNA synthesis, morphology and involucrin expression as in Figure 3a, after treating keratinocytes for 48 h with inhibitors of Aurora B kinase (AURB), ZM447439 (ZM; 2 mM) or Polo-like kinase 1, BI2536 (BI; 100 nM ; Supplementary Figure 5). Bar histograms and errors as in Figure 3a. Representative of experiments with cells from two different individuals. (b) Effects 24 h after transfection with siRNA to downregulate Aurora B Kinase (siAURB). Bar histograms: expression of AURB as measured by RT--PCR, or percentage of involucrin positive cells by immunofluorescence and cell counting. Ctr: non-targeting control siRNA. Bars are s.e.m. Microphotographs show representative images of immunofluorescent staining for involucrin (Invol; red) and DAPI (nuclear DNA, blue). Scale bar: 50 mm. (c) representative samples of clonogenicity capacity of keratinocytes released after a 48 h treatment with the kinase inhibitors indicated. Data are from experiments of duplicate samples with cells from two different individuals. Ctr, control; ROSCO, Roscovitine. contribute to the differentiation effect produced by MYC. To this histone H2ax (P-gH2ax), an early marker of DNA repair.38 As end, we transduced MYC in keratinocytes in combination with summarised in Figures 7a and b, KCEGFP strikingly accumulated shRNAs to Cyclin E. As shown in Figures 6e and f, inhibition of DNA damage as compared with control keratinocytes over- Cyclin E by shRNAs attenuated the increase of keratinocyte expressing GFP only. Interestingly, ectopic Cyclin E induced the differentiation markers induced by MYC. expression of the p53/p21 pathway (Figure 7c). The p53 pathway is induced in response to DNA damage and triggers mitotic checkpoints.39 Cyclin E and DNA damage We next tested whether an accumulation of DNA damage We have shown that triggering the spindle mitosis checkpoint might trigger the mitosis block and terminal differentiation effects causes in keratinocytes mitotic failure and differentiation (Figure 4 observed in KCEGFP cells. To this aim, we treated keratinocytes and Supplementary Figures 5 and 6). This effect is also provoked with doxorubicin (DOXO), a known DNA-damaging agent.40 DOXO by genotoxic agents such as bleomycin or a topoisomerase II treatment strongly induced DNA damage by 24 h (P-gH2ax; inhibitor.11 We investigated whether overexpression of Cyclin E Figure 8a), the p53/p21 pathway (Figure 8b) and a mitosis arrest caused DNA damage by measuring the phosphorylation of (Figure 8c). As a result, keratinocytes were rapidly induced to

& 2012 Macmillan Publishers Limited Oncogene (2012) 5180 --5192 Cyclin E and keratinocyte differentiation A Freije et al 5186

Oncogene (2012) 5180 --5192 & 2012 Macmillan Publishers Limited Cyclin E and keratinocyte differentiation A Freije et al 5187 undergo terminal differentiation (Figure 8c), resembling the to the basement membrane and keratinocyte proliferation.1 We effects observed after a direct mitosis block (Figures 3a and 4a). here have shown that primary keratinocytes continue DNA Interestingly, inhibition of the mitotic checkpoint component synthesis after loss of anchorage, as they commit to terminal Aurora B kinase by ZM447439 did not induce p53 as signiﬁcantly differentiation.9 While differentiation progressed, keratinocytes (ZM; Figure 8b), consistent with the model that p53 acts upstream accumulated in G2/M and became polyploid. Consistently, we of the mitosis block (Figure 8d). have found that keratinocytes continue DNA replication in the differentiating layers of the human epidermis.11 Although the activities of cell cycle kinases cdk2 (S phase) and DISCUSSION cdk1 (mitosis) were both inhibited in suspended keratinocytes, The cell cycle deregulates prior to differentiation a remaining low-intensity Cyclin E-associated activity persisted It is unclear how keratinocytes coordinate cell multiplication with during differentiation. The decrease of the kinase activities in postmitotic differentiation. Integrins maintain both cell adhesion suspended keratinocytes was likely elicited by cell cycle inhibitors

Figure 6. Effects of ectopic Cyclin E on keratinocyte growth and differentiation. (a) Clonogenic capacity of control KGFP or KCEGFP. 2000 cells of either total (upper dish) or basal (lower dishes) populations were plated per well (see Materials and methods). Bar histograms represent the percentage of actively growing colonies (grey) versus the percentage of abortive, differentiated colonies (white). Dotted line indicates the proportion of total colonies produced per thousand cells plated Â 10. Representative of triplicate samples from experiments with cells from two different individuals. (b) mRNA expression of differentiation markers K1, K10 or involucrin as measured by RT--PCR in KGFP (white bars) or KCEGFP (grey bars) cells cultured in low calcium medium. (c) Quantitation of the production rate of corniﬁed envelopes by control KGFP (white bars) or KCEGFP (grey bars). (d) Immunoﬂuorescence for Cyclin E after activation of MYCER by OHT for 4 days. (e) Detection of MYCGFP, Cyclin E (CE) or involucrin by RT--PCR in primary keratinocytes, uninfected or infected with MYCGFP and CE-shRNA1 or CE-shRNA2 as indicated. (f) Microphotographs: staining of the differentiation marker K1 in cells overexpressing MYCGFP in the absence or presence of CE- shRNA1; scale bars, 50 mm. bar histogram: quantitations of the percentage of K1-expressing cells are data from the two independent experiments.

Figure 5. Overexpression of Cyclin E-GFP in basal primary keratinocytes. (a) Detection of the Cyclin E-GFP fusion protein (arrowhead) by WB with antibodies to Cyclin E (CE) or to GFP, as indicated on the left in keratinocytes infected with the GFP-only vector (KGFP) or in keratinocytes infected with the Cyclin E-GFP construct (KCEGFP). (b) Upper panels: microphotographs of phase contrast of the cells indicated. Note that CEGFP keratinocytes are significantly larger and they have large or various nuclei. Bottom panels: Immunofluorescence for CE (in green) and involucrin (Invol, in red); DAPI in blue. Bars: 100 mm. Ctr: uninfected keratinocytes. (c) Representative flow-cytometry analyses for light scattering (SSC-A) versus GFP (upper-left dot plot, GFP positive cells in green), involucrin versus GFP (upper-right dot plot, GFP positive cells in green), DNA content (propidium iodide), morphology (Light Scatter) or involucrin as indicated, in GFP expressing (GFP þ ) or non-expressing (GFP-) keratinocytes. Bar histograms show the quantitations of the percentage of cells in G2/M, polyploidy, with differentiated morphological features (large and complex cells; High Scatter) or positive for involucrin (Invol), as indicated. Regions and controls as in Figure 3a. (d) Expression of Cyclin E and Cyclin A as measured by RT--PCR (CE, CA) and percentage of positive cells for DNA replication, as measured by BrdU-incorporation as in Figure 3a; KGFP (white); KCEGFP (grey). Numbers are means of duplicate samples, representative of experiments with cells from two different individuals. Small bars are s.e.m.

& 2012 Macmillan Publishers Limited Oncogene (2012) 5180 --5192 Cyclin E and keratinocyte differentiation A Freije et al 5188

Figure 7. Effects of ectopic Cyclin E on DNA damage and the expression of the p53 pathway. (a) Expression of the DNA repair marker P-gH2ax as assessed by immunoﬂuorescence in stably selected KGFP or KCEGFP cells; bars: 100 mm. (b) Percentage of positive cells for P-gH2ax scored in: i--- the GFP-negative or -positive populations within the same cultures 48 or 72 h after infection with CEGFP; or ii--- in cells stably expressing GFP or CEGFP (ST); Ctr: uninfected cells. (c) WB for expression of the regulators indicated on the left in uninfected, KGFP or KCEGFP cells. UI, uninfected keratinocytes. b-actin (b-act) was used as the loading control.

such as p21 or p27. Induction of p21 during keratinocyte been reported to inhibit p21 expression and to induce Cyclin B.46--48 differentiation has been reported previously.16,17,23,41 In our However, our results are consistent with the model that MYC experiments, p21 was transiently induced in suspended keratino- function depends on the cell type and the cell context.10,49,50 cytes and bound Cyclin A/cdk1 and Cyclin B/cdk1 at the time of Interestingly, induction of p21 has also been found in MYCER- the commitment to differentiation (6 h), whereas the binding to induced senescence in the absence of cdk2.51 The upregulation of cdk2 peaked later on. Interestingly, p21 is required for the p21 in keratinocytes was concomitant with the cell cycle downregulation of Cyclin A in mouse keratinocytes.17 Although activation by MYC and it was also found in the rapid proliferative p21 inhibits cdk2 complexes, it is able to bind and inactivate the population isolated from human basal epidermis. mitosis complex Cyclin B/cdk1 and yet allow DNA replica- Activation of MYC in keratinocytes induced expression of tion.39,42,43 The induction of p21 in suspended keratinocytes Cyclin E, consistent with previous reports in other cell types.21,22 might not be sufficient to completely inhibit the large amounts of The regulation of Cyclins during suspension-induced keratinocyte Cyclin E/cdk2 produced, but it might be sufficient to inhibit the differentiation is consistent with what we have observed decreased amounts of Cyclin A and Cyclin B. Consistently, the in suprabasal epidermis11 and with the modified cycle that occurs levels of cdk2 required to drive DNA replication appear to be in endoreplication.52 --54 Some of these changes have also lower than the levels of cdk1 required to trigger mitosis.44,45 The been observed in keratinocytes harbouring HPV molecules.55 low activity of Cyclin E/cdk2 might thus sustain keratinocyte DNA Postmitotic DNA replication allows a large increase in cell re-replication once differentiation has initiated. Within the same size in endoreplicating systems, including plant epidermis (for lines, cdk inhibitors Roscovitine and NG97 blocked mitosis and yet example, Zanet et al.,10 Schellmann and Hulskamp54 and allowed DNA replication. references therein). The switch from proliferative Cyclins to high Overactivation of MYC in keratinocytes induced cell cycle Cyclin E observed in our experiments correlated with the changes consistent with the suspension system. The changes were induction of the differentiation marker involucrin and might more marked in suspension-induced differentiation, as it is a rapid explain the cell size increase of differentiating keratinocytes.3,4 process that includes complete lack of cell adhesion. In contrast, This in turn reconciles the function of MYC in promoting ectopic activation of MYC is thought to drive keratinocytes into epidermal differentiation with its well-established function in the clonal amplification phase of rapid proliferation that precedes driving cellular growth. terminal differentiation.7,8 Activation of MYCER in keratinocytes All the cell cycle regulators analysed increased early after loss of for 2 days caused hyperphosphorylation of Rb and a transient anchorage or MYC activation as keratinocytes committed to induction of the Cyclins, all changes that mark cell cycle entry. differentiation. This included Cyclin/cdk activities and phosphor- Subsequently, ectopic MYC induced p21 and downregulation of ylation of Rb. In addition, we and others have shown that p53 mitotic Cyclins A and B. These changes are intriguing, as MYC has antagonist MDM2 is upregulated during keratinocyte differentia-

Oncogene (2012) 5180 --5192 & 2012 Macmillan Publishers Limited Cyclin E and keratinocyte differentiation A Freije et al 5189

Figure 8. Effects of DNA damage on primary keratinocytes. (a) Immunofluorescence for the DNA repair marker P-gH2ax in control (DMSO treated) keratinocytes, or keratinocytes treated with doxorubicin (DOXO) for 24 h; scale bar, 50 mm. (b) WB for expression of the regulators indicated on the right side in keratinocytes treated with DMSO, DOXO, or the Aurora B kinase ZM447439 (ZM) for 48 h. GAPDH (GDH) was used as the loading control. (c) Representative flow-cytometry analyses of DNA content, involucrin expression and morphology after treating keratinocytes with DOXO for 48 h; top bar histograms: quantitations of the percentage of involucrin-positive cells, morphologically differentiated cells (High Scatter) and G2/M cells; regions and controls as in Figure 3a. Numbers are means of duplicates, representative of experiments with cells from two different individuals. Bars are s.e.m.; botton bar histograms: expression of mRNA of differentiation markers filaggrin and involucrin as determined by RT--PCR after a 24 h treatment with DMSO (Ctr, white bars); or with DOXO (grey bars). (d) Model for the sequence of events following a hyperactivation of the keratinocyte cell cycle: cell cycle deregulation leads to accumulation of DNA damage and induction of the p53/pathway; the mitosis checkpoints block mitosis and trigger terminal differentiation.

tion (Dazard et al.41 and references therein). Altogether, the results differentiation. This striking rapid response was not observed show that there is a loss of inhibition in the S-phase of the cell when keratinocytes were treated with inhibitors of G1/S, AG555 cycle as keratinocytes commit to differentiation and block mitosis (herein) or hydroxyurea.9 In contrast, the clonogenic potential of (Figure 8d). This response to lack of anchorage might account for keratinocytes augmented once they were released from a G1 the loss of proliferative potential as keratinocytes lose adherence arrest, induced by the cdk inhibitor AG555. This inhibitor slightly to the basal membrane via integrins.5 reduced cell size and involucrin expression. Along the same lines, overexpression of p21 in keratinocytes slowed down the cell cycle and inhibited differentiation.16,23 Consistently, inhibition of Cyclin A mitosis-differentiation checkpoint E by shRNAs, in our experiments, attenuated the differentiation The inhibition of mitosis after loss of cell anchorage or MYC effect caused by MYC. The results altogether suggest that activation in keratinocytes might be a response to counterbalance acceleration of the cell cycle supports and promotes keratinocyte cell cycle hyperactivation. In our experiments, a block of mitosis by differentiation (Figure 8d). The model is consistent with the notion inhibiting cdk1 or components of the spindle checkpoint Aurora B that epidermal stem cells are mainly quiescent, and that their kinase or Polo-like kinase, or by causing genotoxic insult by activation already initiates the differentiation programme. The doxorubicin (herein), Bleomycin or topoisomerase II inhibitor ICRF- model is in turn consistent with the fact that MYC promotes 193,11 triggered a rapid increase of the keratinocyte cell size and keratinocyte differentiation.

& 2012 Macmillan Publishers Limited Oncogene (2012) 5180 --5192 Cyclin E and keratinocyte differentiation A Freije et al 5190 An uninhibited cell cycle might be a part of the rapid It is tempting to speculate that within the epidermis, a proliferation phase that precedes keratinocyte terminal differen- threshold of S-phase regulators, such as Cyclin E during the cell tiation. As a response, a keratinocyte mitosis checkpoint might cycle hyperactivation that precedes terminal differentiation, might trigger differentiation. This checkpoint might contribute to limit the number of keratinocyte divisions and thus control maintain homeostasis and be part of a tissue self-defence epidermal cell fate as a clock. mechanism in the case of genotoxic stress or oncogenic activations, similar to the role that apoptosis or senescence has in other systems.56, 57 These processes have been found to share MATERIALS AND METHODS some mechanisms.58 However, MYC has pleiotropic effects in cell Cell culture, plasmids and antibodies metabolism and growth.6 To challenge the model and to test Primary keratinocytes were isolated from neonatal human foreskin and whether a direct activation of the cell cycle would trigger a cultured in the presence of a mouse fibroblast feeder layer (inactivated by 2 þ differentiation response, we overexpressed Cyclin E in basal a 2 h treatment with 4 mg/ml mitomycin C), 10% serum and 1.2 mM Ca , keratinocytes. as described.62 Low passages (1--5) of keratinocytes from four different individuals were utilised. Terminal differentiation was induced by suspending disaggregated keratinocytes in culture medium supplemented Cyclin E makes the link with methyl cellulose (Sigma-Aldrich, Inc., St Louis, MO, USA) as Cyclin E accumulates during keratinocyte differentiation both described.5,41 For some studies keratinocytes were transferred to low- 11 in vitro (herein) and in human epidermis. Overexpression of calcium (o0.1 mM) Defined Keratinocyte-Serum-Free-Medium (Invitrogen, Cyclin E-GFP induced terminal differentiation of proliferative Carlsbad, CA, USA), following the manufacturer’s instructions: (i) siRNA keratinocytes although the exogenous fusion protein was transfections (small interfering RNA); (ii) shRNA (small hairpin RNA) degraded at mitosis as the natural protein.12 Despite the increase lentiviral infections; and (iii) quantitation of the expression of keratins K1 in DNA replication index upon ectopic Cyclin E, the clonogenic and K10 by immunofluorescence or RT--PCR. capacity diminished and cells accumulated in G2/M and Activation of conditional MYCER63 in primary keratinocytes was polyploidy owing to mitosis failure. The loss of proliferative achieved by addition of 100 nM 4-hydroxytamoxifen (OHT; variant Z, capacity caused by ectopic Cyclin E overexpression was not Research Biochemicals International, Natick, MA, USA) to the culture associated with senescence or apoptosis (see also Gandarillas58) medium for the lengths of time indicated. and it was more marked than the inhibition caused by over- Stem cells (SC), ‘transit amplifying cells’ (TAC) and terminally differentia- activation of MYC.7 Interestingly, sustained Cyclin E expression tion cells (TDC), were freshly isolated from human foreskin and sorted on resulted in accumulation of DNA damage and induction of p53/ basis of their adhesion capacity on collagen IV, as described.14,41 p21, a pathway known to halt the cell cycle in G2/M in response to Primary keratinocytes were treated for 24 or 48 h with cdk inhibitors or 39 DNA damage (Figure 8d). Interestingly, p53 also increased after mitosis kinase inhibitors: 50 mM of AG555 (ICN Biochemicals, Aurora, OH, activation of MYCER in keratinocytes and in the rapid-proliferative USA), 30 mM of Roscovitine (Sigma-Aldrich, Inc.) and 1 mM of NG97 (kind gift 41 fraction of basal human epidermis. of J Meijer) in DMSO; 2 mM of the Aurora B kinase inhibitor ZM447439 Cyclin E is involved in the activation of DNA replication origins (Tocris Bioscience, Bristol, UK) or 100 nM of the Polo-like kinase inhibitor and it must be downregulated for cells to undergo cell cycle BI2536 (Axon MedChem BV, Netherlands) in DMSO. For DNA damage 12 arrest. Premature Cyclin E expression accelerates cell cycle entry. analyses cells were treated for 24 or 48 h with 0.5 mM of DOXO (Sigma- In addition, oncogenes might promote genomic instability by Aldrich, Inc.) in DMSO. Parallel control cultures were always subjected to causing DNA damage.57 Recent reports have also concluded the DMSO vehicle. Optimal concentrations were tested for each inhibitor. that acceleration of the cell cycle by Cyclin E can cause For clonogenicity assays, 5000 or 10 000 of total or basal keratinocytes DNA damage.59,60 Interestingly, in our experiments, causing DNA were plated per T6 well triplicates. Basal cells were counted on basis of damage in keratinocytes with genotoxic agents such as doxor- their size (o18 mm). After 12--15 days, the cultures were coloured with ubicin induced p53 and terminal differentiation. As discussed Rhodanile blue as described.10 above, we have shown that a direct block of mitosis rapidly results Keratinocytes were subjected to retroviral infection with pBabe in terminal differentiation. By causing DNA damage, accumulation constructs as described.7 pBabe-GFP-CyclinE was constructed to express of Cyclin E might trigger the keratinocyte mitosis checkpoints and Cyclin E-GFP fusion protein in basal primary keratinocytes from pBabe-li- in turn, terminal differentiation (Figure 8d). This would create a GFP and pBabe-CyclinE (kind gifts from Pierre Roux and Bruno Amati) as link between rapid cell amplification and the commitment to described in Supplementary Figure 8. The studies were carried out either terminal differentiation, thus constituting an automatic compen- by analysing unselected pools 2 --5 days after infections or by stably satory mechanism to maintain homeostasis. This model would selecting GFP or CEGFP expressing keratinocytes by 1 mg/ml puromycin. contribute to explaining how the epidermis maintains tissue The production of cornified envelopes was measured as described in structure in benign hyperproliferative situations. Additional Sun and Green.37 alterations breaking the mitosis block response might be required The following antibodies were used: anti-Cyclin A2 (CY-A1, Sigma- to promote carcinogenesis. Aldrich, Inc.; immunofluorescence, IF and western blotting, WB), anti-Cyclin The differentiation effect induced by deregulated Cyclin E does A2 antibody (H432, Santa Cruz Biotechnology, Santa Cruz, CA, USA; IP), not only have implications for the mechanisms protecting the anti-Cyclin B1 (GNS1, Santa Cruz Biotechnology; IF, IP and WB), anti-Cyclin epidermis against hyperproliferative alterations. For long, a cell- E1 (C19, Santa Cruz Biotechnology; IF, IP and WB), anti-Cyclin E1 (HE12, autonomous clock-like mechanism has been proposed as one of Santa Cruz Biotechnology; IF and WB), anti-BrdU (BD Biosciences, San Jose, the possible models determining stem cell fate. The levels of CA, USA; flow cytometry, FC and IF), anti-cdk1 (p34cdc2, Sigma-Aldrich, Cyclin E drive cell fate in Drosophila oogenesis.53 A crossroad Inc.; WB), anti-cdk2 (M2, Santa Cruz Biotechnology; WB), anti-phospho-cdk2 between mitosis, differentiation and DNA endoreplication is (Y15, Abcam, Cambridge, UK; WB), anti-GFP (Invitrogen; FC, IF and WB), important in a variety of cell systems, including the plant anti-ER (Santa Cruz Biotechnology; WB), anti-phospho-Histone H2AX epidermis or human megakaryocytes.52 --54 In myeloid leukaemia (Ser139, JBW301, Millipore, Bedford, MA, USA; IF), anti-phospho-H3 (Ser- cells, Cyclin E drives endoreplication61 and Myc and p21 cooperate 10, Santa Cruz Biotechnology; WB), anti-involucrin (SY5, Sigma-Aldrich, Inc.; to induce megakaryocityc differentation and polyploidy.50 Onco- FC, IF and WB), anti-K1 (Covance, Vienna, VA, USA; IF), anti-MYC (Santa Cruz gene-induced accumulation of DNA damage by replication stress Biotechnology; WB), anti-p21CIP (C19, Santa Cruz Biotechnology; Depletion might trigger a similar response leading to apoptosis or assay and WB), anti-p21CIP (CP74, Sigma-Aldrich, Inc.; WB), anti-p27 senescence. The mechanisms we report here might therefore (F8, Santa Cruz Biotechnology; WB), anti-p53 (FL-393, Santa Cruz have a more global role in homeostasis beyond the epidermis. Biotechnology; WB), anti-PSTAIR (pstair, Sigma-Aldrich, Inc.; WB), anti-Rb

Oncogene (2012) 5180 --5192 & 2012 Macmillan Publishers Limited Cyclin E and keratinocyte differentiation A Freije et al 5191 (C15, Santa Cruz Biotechnology; WB), anti-phospho-Rb (ser780, Cell ACKNOWLEDGEMENTS Signalling Technology, Beverly, MA, USA; WB), anti-b-actin (I-19, Santa We are especially grateful to Jean-Claude Rossi and Javier Leoń for their strong Cruz Biotechnology WB), anti-GAPDH (FL-335, Santa Cruz Biotechnology; support. We thank Bruno Amati, Pierre Roux and Jonathon Pines for providing DNA WB) and anti-CD8 ( Sigma-Aldrich, Inc; FC). constructs, J. Leoń for reagents, Anne Blangy, Vjeko Dulic, Ve´ronique Baldin and Thierry Lorca for technical advice, Daniel Fisher for helpful suggestions, Kathy Brooks and Marıá Aramburu for technical support at flow cytometry, J. Leon and Philippe Flow cytometry Pasero for critical reading of the manuscript, and Anna Solinı´s and Diana Solinı´sfor After removal of the mouse feeder layer, 106 trypsinized keratinocytes reviewing the English writing. Human skin biopsies were provided with consent by St were fixed, stained for BrdU and DNA (propidium iodide), for GFP or for Jean and Hoˆpital La Peronye (Montpellier, France) and by the Chirurgical Paediatric involucrin, and analysed by flow cytometry as described in Supplementary Service of Hospital Marque´s de Valdecilla (EdD; HMDV, Santander, Spain). MC was a recipient of a predoctoral fellowship from ARC (France). AG belongs to the INSERM Data. All antibody stainings were controlled by the use of isotype negative (France) and the FMDV-IFIMAV (Spain) and was financially supported by the INSERM immunoglobulins (mouse anti-cd8 or rabbit serum). In order to gate out and the ISCIII (Spain). AF and LC were employed by FMDV-IFIMAV and supported by cell aggregates, the area of the fluorescent pulse of propidium iodide (DNA the ISCIII, MR was recipient of a postdoctoral contract by the FMDV-IFIMAV. This work content; PE-A) was plotted versus the width of the fluorescent pulse (PE-W; was funded by ARC (AG; France), La Ligue contre le cancer (JMB; France), and Instituto see Supplementary Figure 11). de Salud Carlos III (AG; ISCIII-Programa Regiones Emergentes and Fondo de Investigaciones Sanitarias PI080890; Spain). Immunofluorescence Keratinocytes were grown on glass coverslips and fixed and stained as REFERENCES described in Supplementary Data. 1 Watt FM, Lo Celso C, Silva-Vargas V. Epidermal stem cells: an update. Curr Opin Genet Dev 2006; 16: 518 --524. Whole-cell extracts and WB 2 Fuchs E, Horsley V. More than one way to skin. Genes Dev 2008; 22: 976 --985. 3 Banks-Schlegel S, Green H. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2012) 5180 --5192 & 2012 Macmillan Publishers Limited