In Vivo Overexpression of Emi1 Promotes Chromosome Instability and Tumorigenesis

ORIGINAL ARTICLE In vivo overexpression of Emi1 promotes chromosome instability and tumorigenesis

S Vaidyanathan1,3, K Cato1,3, L Tang1,2, S Pavey1, NK Haass1, BG Gabrielli1 and PHG Duijf1

Cell cycle genes are often aberrantly expressed in cancer, but how their misexpression drives tumorigenesis mostly remains unclear. From S phase to early mitosis, EMI1 (also known as FBXO5) inhibits the anaphase-promoting complex/cyclosome, which controls cell cycle progression through the sequential degradation of various substrates. By analyzing 7403 human tumor samples, we find that EMI1 overexpression is widespread in solid tumors but not in blood cancers. In solid cancers, EMI1 overexpression is a strong prognostic marker for poor patient outcome. To investigate causality, we generated a transgenic mouse model in which we overexpressed Emi1. Emi1-overexpressing animals develop a wide variety of solid tumors, in particular adenomas and carcinomas with inflammation and lymphocyte infiltration, but not blood cancers. These tumors are significantly larger and more penetrant, abundant, proliferative and metastatic than control tumors. In addition, they are highly aneuploid with tumor cells frequently being in early mitosis and showing mitotic abnormalities, including lagging and incorrectly segregating chromosomes. We further demonstrate in vitro that even though EMI1 overexpression may cause mitotic arrest and cell death, it also promotes chromosome instability (CIN) following delayed chromosome alignment and anaphase onset. In human solid tumors, EMI1 is co-expressed with many markers for CIN and EMI1 overexpression is a stronger marker for CIN than most well-established ones. The fact that Emi1 overexpression promotes CIN and the formation of solid cancers in vivo indicates that Emi1 overexpression actively drives solid tumorigenesis. These novel mechanistic insights have important clinical implications.

Oncogene (2016) 35, 5446–5455; doi:10.1038/onc.2016.94; published online 11 April 2016

INTRODUCTION human cancers are aneuploid indicate that CIN is an important 9–14 Cell cycle progression is regulated by the oscillation in expression driver of tumor evolution. and activity of various proteins, such as cyclins and cyclin- A number of studies have focused on unraveling the cell dependent kinases.1 Proteasome-mediated protein degradation is biological and in vivo function of Emi1 using loss-of-function 5,15–20 critical in this process. The anaphase-promoting complex/cyclosome and genetic ablation experiments, respectively. This has (APC/C) catalyzes the sequential proteolysis of substrates from late provided invaluable new insights. For instance, it has become 2 clear that Emi1-mediated stabilization of APC/C is required for G2 to early G1 phases of the cell cycle. At different stages, Cdc20 mitotic progression during embryogenesis and prevents DNA and Cdh1 act as co-factors required for APC/C activity. Several 16–18 APC/C inhibitors, such as Mad2 and Emi1 (early mitotic inhibitor 1, re-replication and polyploidy. However, it has remained also known as Fbxo5), compete with the APC/C for Cdc20 and Cdh1 unclear how Emi1 misexpression might be involved in the biology binding, thereby stabilizing APC/C substrates.2 In addition, of cancer development in vivo. fi Emi1 potently inhibits APC/C by blocking its catalytic site, by Here, we nd that EMI1 is overexpressed in the vast majority competitively preventing APC/C substrates from binding to APC/C of human solid tumors, but not blood cancers, and that this is co-receptors and by actively suppressing substrate mono- and a strong marker for poor clinical outcome. To mimic this polyubiquitination.3–5 Emi1 is the principal inhibitor of APC/C expression status in human cancers, we generated an inducible fi activity from S phase to early mitosis, whereas Mad2 fulfills this Emi1 overexpression mouse model. We nd that in vivo over- role later during mitosis.6 expression of Emi1 promotes CIN and the formation of a wide Defective cell cycle regulation is a hallmark of cancer. Consis- variety of aneuploid solid tumors due to delayed chromosome tently, APC/C substrates and regulators are often aberrantly alignment followed by chromosome missegregation. This indi- expressed in tumors.7,8 During mitosis, the mitotic checkpoint cates that Emi1 overexpression is not a cancer-associated tightly regulates APC/C activity. It prevents precocious sister bystander but actively promotes CIN and tumorigenesis in vivo. chromatid separation by ensuring that the APC/C does not prematurely target securin for degradation, thereby preventing RESULTS chromosome missegregation. Thus, the aberrant expression of mitotic APC/C regulators can lead to chromosome instability (CIN), EMI1 is overexpressed in a broad range of human cancers which may fuel cancer progression. The observations that CIN can We assessed EMI1 mRNA expression in human cancers using 115 promote tumorigenesis in mice and that at least two-thirds of previously published unique expression analyses.21 Together, EMI1

1University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Australia and 2School of Basic Medical Sciences, Fudan University, Shanghai, China. Correspondence: Dr PHG Duijf, University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, 37 Kent Street, Brisbane, Queensland 4102, Australia. E-mail: [email protected] 3These authors contributed equally to this work. Received 30 April 2015; revised 25 January 2016; accepted 19 February 2016; published online 11 April 2016 Emi1 overexpression promotes CIN and tumorigenesis S Vaidyanathan et al 5447

Figure 1. EMI1 overexpression is a marker for poor prognosis in human solid but not blood cancers. (a) Fractions of 115 independent studies reporting under- and overexpression of EMI1 mRNA in cancer. Fractions are shown for all cancers and separately for solid and blood cancers. P value: Fisher’s exact test. (b) Box plot showing EMI1 expression levels in normal brain and glioblastoma (see also Supplementary Data set 1 for details). P value: t-test. (c) EMI1 expression levels in breast cancer per subtype in the TCGA data set.22 Bottom P values compared with normal breast tissue. Top P values between groups: one-way analysis of variance Tukey’s multiple comparisons test. ^P valueo2.2 10 − 16. (d) Box plot showing reduced EMI1 expression in chronic lymphocytic leukemia (CLL) compared with normal control peripheral blood mononuclear cells (PBMCs; see Supplementary Data set 1 for details). P value: t-test. (e) Immunohistochemistry for EMI1 on five types of human solid cancers comparing matched normal control tissue to tumor tissue. Insets show enlargements of areas in the dashed boxes. For extended data, see Supplementary Figure 1. Scale bar, 100 μm. (f) Quantification of extended EMI1 immunohistochemistry analysis using the multiplicative IHC quickscore as previously described.45 See also Supplementary Figure 1. P values above bars compared with normal, other comparisons as indicated by horizontal bars. All P values: t-test. (g) Western blot analysis on primary normal control and tumor breast tissue of various breast cancer subtypes (left) and quantification of actin-normalized EMI1 levels (right). Numbers above bars indicate fold increase compared with normal control expression level. P values: t-test. (h and i) Survival curves for patients with high (red) and low (green) EMI1 mRNA expression in breast and lung cancer, respectively. P values: log-rank test. See also Supplementary Table 2. expression levels in 7403 tumor samples were compared with Data set 1). Thus, solid and blood cancers not only show those in 1467 normal control samples of matched tissue type. Of differences in cancer genomics and pathology, but also in how the 115 studies, 102 (89%) showed that EMI1 is significantly EMI1 is misexpressed. overexpressed and 13 (11%) reported that EMI1 is underexpressed Our primary interest in solid cancers led us to test whether EMI1 (Figure 1a and Supplementary Table 1). EMI1 overexpression is overexpression is sustained at the protein level in these cancers. observed in a range of cancers and tumor subtypes (Figure 1b, We confirmed that this is the case using both western blot analysis Supplementary Table 1 and Supplementary Data set 1). Similarly, 22 on primary tumor samples and immunohistochemistry on tissue using the cancer genome atlas (TCGA) breast cancer data set, we microarrays across a panel of five different solid cancer types fi fi nd that EMI1 levels are signi cantly increased in all four major (Figure 1e–g and Supplementary Figure 1). The western blot subtypes (P ⩽ 0.0019, t-test; Figure 1c). analysis indicates that EMI1 is expressed approximately two- to The etiology and genetic makeup of solid and hematological six-fold higher in tumors compared with normal tissue (Figure 1g). cancers differ considerably.14,23 Interestingly, EMI1 mRNA expression is also misregulated differently in these types of cancer. Of the above 100 solid cancer studies, 95 report significant EMI1 EMI1 overexpression is a marker for poor prognosis in human solid overexpression, whereas this fraction is significantly lower for cancers hematological cancers (7 out of 15, 47%; Po0.0001, Fisher’s exact We next compared EMI1 mRNA expression levels in low-grade and test; Figures 1a and d, Supplementary Table 1 and Supplementary high-grade cancers. In high-grade cancers, EMI1 is overexpressed

© 2016 Macmillan Publishers Limited, part of Springer Nature. Oncogene (2016) 5446 – 5455 Emi1 overexpression promotes CIN and tumorigenesis S Vaidyanathan et al 5448 to higher degrees than in low-grade cancers, as observed in 28 Generation of an Emi1 overexpression mouse model data sets for cancers that arise in 11 distinct tissue types (Figure 1c Although the above data demonstrate a strong link between EMI1 and Supplementary Data set 2). overexpression and cancer patient prognosis, they are correlative To more directly assess the prognostic value of EMI1 mRNA and not causative. To study whether Emi1 overexpression overexpression for patient outcome, we performed survival can actively promote solid tumorigenesis in vivo, we generated analysis on 60 data sets. This revealed that EMI1 overexpression a transgenic mouse model in which we could overexpress Emi1 in is a marker for poor prognosis for at least nine forms of solid an inducible manner. First, we cloned the murine Emi1 cDNA into cancers and for several types of patient survival (Figures 1h and i, the pTRE vector, containing a CMV-based tetracycline-regulatory Supplementary Figure 2 and Supplementary Table 2). promoter, an N-terminal HA-tag and a poly-adenylation signal Using our largest data set (n = 3751 patients; Supplementary (Figure 2a). The small HA-tag readily facilitates detection of Table 2), we further assessed the prognostic strength of EMI1 mRNA transgene expression (see below) and the addition of small or overexpression relative to three well-established clinical markers. large tags to the Emi1 N-terminus, which is exposed even when 3 4–6 First, with a P value of 9.32 × 10 − 13 (log-rank test) EMI1 performs bound to the APC/C, does not interfere with Emi1 activity. nearly as well as the well-established prognostic marker KI67 Transfection of this construct into HeLa-Tet-Off cells showed (P =4.15×10− 14). Also, when survival is adjusted for the Notting- that exogenous Emi1 expression was doxycycline-repressible ham Prognostic Index or by Adjuvant! Online, which each predict (Supplementary Figure 4A). We next released a fragment contain- patient outcome using several clinical parameters, as previously ing the inducible Emi1 transgene (Figure 2a) from the pTRE vector, described,24,25 it remains highly significant (P =6.35×10− 5 and injected it into fertilized oocytes and implanted the latter into P =9.29×10− 5; Supplementary Table 3). Importantly, as observed pseudopregnant mice. Of 51 founder animals that were born, by both immunohistochemistry and western blot analysis, these 11 (22%) tested positive for transgene integration, as determined by PCR on their genomic DNA (Supplementary Figure 4B). observations are sustained at the protein level with high-grade/ Southern blot analysis on the genomic DNA of these animals, as metastatic tumors expressing higher EMI1 protein levels than low- well as of selected mice that tested negative by PCR and wild-type grade cancers (Figures 1f and g and Supplementary Figure 1). Thus, (WT) control mice, confirmed that at least 9 out of the 11 positive together these data indicate that EMI1 overexpression is a strong animals (18% of 51 founders) carried the transgene (Figure 2b and independent clinical prognostic marker for solid cancers. Supplementary Figure 4C). To be able to induce Emi1 transgene expression in vivo,we Prognostic significance of EMI1 overexpression is only partly cell crossed the TRE-Emi1 transgenic mice with CMV-rtTA (Tet-On cycle dependent system) or CMV-tTA (Tet-Off system) transgenic animals, in which Importantly, as EMI1 is expressed from S phase to early mitosis, it the CMV promoter drives the expression of the reverse tetra- is possible that increased EMI1 expression is simply a reflection of cycline transactivator (rtTA) or the tetracycline transactivator (tTA), 27 its increased abundance in cycling cells. Consistent with this, EMI1 respectively. This generated TRE-Emi1/CMV-rtTA and TRE-Emi1/ expression is associated with KI67 expression, a marker for cycling CMV-tTA bitransgenic mice. In these contexts, Emi1 transgene cells, in at least six different cancer types (P ⩽ 0.0082, Spearman expression could be induced or repressed with doxycycline, correlation; Supplementary Figure 3A). However, for several respectively, as observed in mouse tissues at the mRNA and protein – – reasons, we believe that EMI1 overexpression in tumors is only levels by semi-quantitative reverse transcriptase PCR (RT PCR) and partly cell cycle dependent. First, in tumors, EMI1 levels increase western blot analysis, respectively (Figures 2c and d). more than KI67 levels increase, as observed by a significantly higher EMI1/KI67 expression ratio in tumors than in normal In vivo Emi1 overexpression promotes tumorigenesis tissues (P ⩽ 0.0241, Mann–Whitney test; Supplementary Figure 3B). To assess whether in vivo overexpression of Emi1 is sufficient to Second, in TCGA breast tumors that express normal levels of KI67, initiate tumor formation, we aged groups of TRE-Emi1/CMV-rtTA EMI1 expression is significantly higher than in normal breast tissue and TRE-Emi1/CMV-tTA bitransgenic mice. Each cohort included (P = 9.713 × 10 − 12, t-test; Supplementary Figure 3C). Third, 57 out experimental and control animals in which Emi1 transgene of 107 (53%) TCGA breast tumors with normal KI67 expression expression was induced or not induced by feeding the mice express EMI1 above normal levels (Po0.0001, Fisher’s exact test; a regular or a doxycycline-containing diet after weaning. The Supplementary Figure 3C). Fourth, when we perform survival phenotypes that developed in the Tet-On and Tet-Off back- analysis adjusted for a previously described 42-gene cell cycle grounds were indistinguishable (Supplementary Table 6 and – signature,26 the prognostic strength of EMI1 overexpression Supplementary Figures 5A C). We therefore combined our remains highly significant (P = 0.0175, log-rank test), whereas that analyses on a total of 45 Emi1-overexpressing (Emi1-OE) mice of the KI67 marker for cycling cells does not (Supplementary and 44 genetically identical non-Emi1-overexpressing control (Ctrl) Figure 3D). Fifth, in 14 out of 102 data sets (13.7%) showing EMI1 mice, as well as 16 WT control mice. Emi1-OE mice developed a wide variety of cancerous lesions in a range of organs (Figure 3a overexpression, KI67 expression is not significantly increased and Table 1). Although these developed at long latencies, Emi1-OE (Supplementary Table 4). For instance, one data set shows mice succumbed to cancer significantly earlier than bitransgenic a 2.2-fold increase in EMI1 expression (P = 0.002, t-test), whereas o fi Ctrl (P 0.0001, log-rank test) and WT control mice (P = 0.0004; KI67 levels are not signi cantly altered (P = 0.428; Supplementary Figure 3b and Supplementary Figure 5). Emi1-OE mice also died Table 4). Sixth, in 9 out of 27 data sets comparing low-grade to earlier that Ctrl and WT mice irrespective of the cause of death fi high-grade cancers (33%), EMI1 is signi cantly overexpressed, (Po0.0005; Supplementary Figure 6). In addition, 80% of the whereas KI67 is not (Supplementary Table 5). Finally, Emi1 is Emi1-OE mice developed tumors versus 50% of the bitransgenic fi signi cantly underexpressed in 13 cancer data sets, including Ctrl animals (P = 0.0028, Fisher’s exact test; Figure 3c). Compared highly proliferative leukemias (Supplementary Table 1) and in with Ctrl mice, Emi1-OE mice had a twofold increased tumor three blood cancer data sets reduced, rather than elevated, EMI1 burden and their tumors proliferated more rapidly and were expression significantly correlates with poor patient survival larger at the time of death (P = 0.0006, P = 0.0089, P = 0.0148, (Supplementary Table 2). Thus, collectively these data indicate respectively, t-test; Figures 3d–f). that EMI1 overexpression is a strong prognostic marker for human The cancer incidences by tumor cell of origin were also higher solid cancers and that this is not solely due to its periodic in Emi1-OE mice for all major tumor types: lymphoma, sarcoma, expression in cycling cells. carcinoma and adenoma (Figure 3g). However, the differences

Figure 2. Generation of an Emi1 overexpression mouse model. (a) DNA fragment used for the generation of Emi1 transgenic mice. The inducible promoter (P) is CMV-based and contains 7 tetracycline-response elements (TRE7). (b) Southern blot showing genomic integration of the transgene in a subset of mice. Equal loading is shown in Supplementary Figure 4C and by the 1210-bp internal control band. The transgene band is 799 bp. (c) Semi-quantitative RT–PCR on RNA from tissues of bitransgenic Tet-On mice on a regular or doxycycline diet for 4 weeks after weaning, as indicated. In, intestine; Li, liver; Lu, lung; Ki, kidney; Th, thymus. (d) Western blots on tissue extracts from bitransgenic TRE-Emi1/rtTA (Tet-On) and TRE-Emi1/tTA (Tet-Off) mice on a regular or doxycycline diet for 4 weeks after weaning, as indicated. were only statistically significant for carcinomas and adenomas to Ctrl tumors, tumors from Emi1-OE mice were highly aneuploid (P =0.0129and P = 0.0133, Fisher’s exact test; Figure 3g). Similarly, (Figures 4a and b). the tumor burden was elevated for all four of these types of cancer, yet the differences were significant only for carcinomas and Tumors in Emi1-overexpressing mice show evidence of CIN adenomas (P =0.0056and P =0.0073, t-tests; Figures 3h and i and To study tumor cell mitoses in vivo, we subjected tissue sections Supplementary Figures 7A and B). In addition, the fraction of animals fi from Emi1-OE and Ctrl tumors to hematoxylin and eosin staining with liver tumors signi cantly increased by more than 2.5-fold, from and immunofluorescence using markers for DNA, microtubules – ’ 11 29% (P = 0.0396, Fisher s exact test; Supplementary Figure 7C) and centrosomes. Aside from normal anaphases, we observed fi and a signi cantly higher number of Emi1-OE female mice anaphases with anaphase bridges, lagging chromosomes ’ developed lesions in the ovaries (P = 0.0276, Fisher s exact test; and chromosomes that did not segregate with the main Supplementary Figure 7D). However, as the liver tumors included chromosome masses—henceforth termed ‘not in mass’ sarcomas and carcinomas and the ovarian lesions comprised cysts, (Figures 5a and b). Anaphase bridges did not occur at a cystadenomas and hemangiosarcomas, they originated from significantly elevated frequency and the total number of different cell types. anaphases per mitosis was also not increased in Emi1-OE tumors Primary cancers in Emi1-OE mice were more aggressive than in (Figure 5b and Supplementary Figure 8A). In contrast, the Ctrl animals, as evidenced by their increased proliferation rate frequencies of lagging and ‘not in mass’ chromosomes were ’ (Figure 3e) and propensity to metastasize (P = 0.0343, Fisher s significantly higher than in Ctrl tumors (t-test; Figure 5b). Overall, exact test; Figure 3j). The latter may in fact be an underestimation, 56% of Emi1-OE tumors exhibited mitotic abnormalities com- because micro-metastases may have been missed. Consistent with pared with 14% of Ctrl tumors (P = 0.0101, t-test; Figure 5b). an Emi1 overexpression-induced increase in malignancy, cancer- Emi1-OE tumors also showed a wider range of mitotic associated immunological abnormalities (other than lymphoma abnormalities with multiple types of abnormalities co- fl fi or leukemia), such as in ammation and lymphocyte in ltration, occurring in 10% of the cells (Figure 5b) and significantly more fi ’ were also signi cantly elevated (P = 0.0003, Fisher s exact test; abnormalities per mitosis (P = 0.0047, t-test; Figure 5c). In Figure 3k). Finally, Emi1-OE mice showed an increased incidence addition, the numbers of lagging chromosomes and chromo- of hyperplasia and neoplasia, such as mammary and prostate somes not segregating with the main chromosome masses per ’ intraepithelial neoplasias (P = 0.0009, Fisher s exact test; Figure 3l; cell were significantly elevated (P = 0.0335 and P = 0.0043, t-test; not included in Table 1). Such abnormalities have the potential to Figures 5d and e). Emi1-OE tumor cells also showed a broader develop into malignant lesions. Taken together, in vivo over- distribution of cells with multiple lagging and 'not in mass' expression of Emi1 promotes the development of a broad range chromosomes (Figures 5f and g). of solid cancers and cancer-associated abnormalities, such as Aside from the above abnormalities, we noticed that mitotic metastasis and inflammation. cells in Emi1-OE tumors were often in early mitosis, as their chromosomes were condensed while their centrosomes had not Tumors in Emi1-overexpressing mice are aneuploid yet separated (Figure 5h and Supplementary Figures 8B and C). Using peri-centromeric probes against chromosomes 12, 16 and Compared with Ctrl tumors, the frequency of cells in early mitosis 17, we next performed interphase-fluorescent in situ hybridization increased 13-fold, from 2.6 to 35% (P = 0.0298, t-test; Figure 5h). (FISH) on the mouse tumor sections. This revealed that, in contrast Thus, in vivo Emi1 overexpression promotes CIN by inducing

Figure 3. In vivo Emi1 overexpression promotes tumorigenesis. (a) Macroscopic and hematoxylin and eosin (H&E)-stained microscopic images of selected lesions from Emi1-overexpressing mice. Scale bars, 1 cm. (b) Tumor-free survival of Emi1-overexpressing (Emi1-OE, red), non-Emi1- overexpressing control (Ctrl, green) and wild-type control (WT, black) mice. P values: log-rank test. See also Supplementary Figure 5. (c) Cancer penetrance. P value: Fisher’s exact test. (d) Tumor burden. Mean values (μ) are indicated ± s.e.m. P value: t-test. (e) Tumor growth rate. Means ± s.e.m. are shown. P value: t-test. (f) Time line of tumor development in Ctrl and Emi-OE mice. P value: t-test. (g) Cancer penetrance per major tumor type. Lymph, lymphoma; Sarc, sarcoma; Carcin, carcinoma; Aden, adenoma. P values: Fisher’s exact test; NS, not signiﬁcant. (h, i) Carcinoma and adenoma burden as in d.(j-l) Incidences of tumor-bearing animals with metastases (j), mice with cancer-associated immunological abnormalities (k) and hyperplastic or neoplastic lesions (l). P values: Fisher’s exact tests.

lagging and missegregating chromosomes and these defects may Table 1. Incidence of tumors in Emi1-overexpressing and Ctrl mice have an early mitotic origin.

a Cancerous lesion Ctrl Emi1-OE Prometaphase and metaphase defects underlie EMI1 overexpression-mediated CIN #%#% To further investigate how EMI1 overexpression promotes CIN, Lymphoma, unspecified type 10 23.8 14 33.3 we overexpressed EMI1 in Hela cells that stably expressed GFP- Histiocytic sarcoma 5 11.9 5 11.9 labeled histone H2B (Supplementary Figure 9A) and monitored Lung adenoma 1 2.4 4 9.5 cell division using live-cell microscopy. This unveiled several Hepatocellular carcinoma 0 0.0 3 7.1 striking differences between EMI1-OE and control cells. First, Lung carcinoma 2 4.8 3 7.1 EMI1-OE cells progressed through mitosis more slowly (P = 0.0004, Carcinoma, unspecified type 0 0.0 2 4.8 t-test) due to delays in both chromosome alignment at the Hemangiosarcoma 0 0.0 2 4.8 fi metaphase plate and subsequent anaphase onset (P = 0.0008 and Sarcoma, unspeci ed type 3 7.1 2 4.8 P = 0.0028, respectively; Figures 6a and b). On average, the time Duodenum adenoma 0 0.0 1 2.4 Harderian gland adenoma 0 0.0 1 2.4 interval between nuclear envelope breakdown and anaphase Hepatocellular adenoma 0 0.0 1 2.4 onset was ~ 6 min longer in EMI1-OE cells, whereas the time from Histiocytic lymphoma 0 0.0 1 2.4 anaphase onset to completion of cytokinesis was not affected Liver carcinoma, unspecified type 0 0.0 1 2.4 (Figure 6b). Multiple myeloma 0 0.0 1 2.4 EMI1-OE cells also showed significant increases in anaphase Myxosarcoma 0 0.0 1 2.4 bridges, lagging or 'not in mass' chromosomes, mitotic arrest and Ovary cystadenoma 0 0.0 1 2.4 cell death (P = 0.0048, P = 0.0297 and P = 0.0027, Fisher’s exact test; Round cell sarcoma 0 0.0 1 2.4 – Seminal vesicle adenocarcinoma 0 0.0 1 2.4 Figures 6c e). We also observed small increases in the formation of micronuclei, binucleate cells and tripolar spindles. However, Abbreviation: Ctrl, control. aThis includes adenomas but not hyperplasias those were not statistically significant (Supplementary Figures 9B- or neoplasias, such as mammary or prostate intraepithelial neoplasias D). Overall, there was a profound increase in the frequency of (MINs and PINs). abnormal mitoses (P = 0.0004; Figure 6f). Taken together, EMI1

Oncogene (2016) 5446 – 5455 © 2016 Macmillan Publishers Limited, part of Springer Nature. Emi1 overexpression promotes CIN and tumorigenesis S Vaidyanathan et al 5451 overexpression delays chromosome alignment at the metaphase plate and anaphase onset and this is followed by chromosome missegregation.

EMI1 overexpression is a strong marker for CIN in human solid cancers Based on the expression levels of 70 genes, the CIN70 gene expression signature is a widely used measure of CIN in human tumor samples.28–30 Consistent with previous studies,29 we find that the CIN70 signature stratifies breast cancer subtypes according to clinical outcome (Supplementary Figure 10). Interestingly, this pattern is similar to the one for EMI1 expression (Figure 1c). We therefore compared CIN70 score and EMI1 expression level directly. This revealed a strong linear correlation between these parameters (r =0.7792, Po0.0001, Pearson’s correlation; Figure 7a). Using a total of 2099 samples, we also analyzed TCGA data sets from seven other forms of cancer. For these cancers, the EMI1 expression levels and CIN70 score relationships were also linear and highly significant (0.6349 ⩽ r ⩽ 0.9166, all P values o0.0001, Pearson’s correlation; Figures 7b-h). Furthermore, 6 CIN70 genes ranked in the top 15 (40%) of most significantly co-expressed genes with EMI1 (Figure 7i and Supplementary Data set 3). EMI1 is not among the 70 genes whose expression levels contribute to the CIN70 score.28 To compare how EMI1 would rank among the CIN70 genes, we plotted the expression level of each of the CIN70 genes individually against the CIN70 score, as in Figures 7a–h for EMI1, and calculated each R2 for best fit for four solid cancer data sets (Supplementary Data set 4). Surprisingly, some CIN70 genes poorly correlated with the CIN70 signature with Figure 4. Tumors from Emi1-overexpressing mice are aneuploid. the average R2 for the CIN70 genes ranging from 0.037 to 0.742. (a) Interphase-FISH on tumor tissue sections from mice, as 2 indicated. 4,6-Diamidino-2-phenylindole (DAPI): white; chromo- With an average R of 0.562, EMI1 ranks in the 74.4th percentile in somes 12, 16 and 17: blue, green and red, respectively. this range of the widely accepted top 70 markers of CIN (Figure 7j (b)Quantification of the fractions of aneuploid cells in tumor and Supplementary Data set 4). This indicates that EMI1 is tissue sections. Each bar reflects the average of at least 100 cells a stronger marker for CIN in human solid cancers than most well- per tumor. P value: t-test. established ones.

Figure 5. Tumors from Emi1-overexpressing mice show evidence of chromosome missegregation. (a) Microscopic images of mitotic cells in hematoxylin and eosin (H&E)-stained mouse tumor sections showing examples of normal and abnormal mitoses. Some cells simultaneously showed more than one abnormality, as exemplified in the image on the right with both an anaphase bridge—chromatin connecting the two main chromosome masses—and a chromosome separate from and not segregating with the two chromosome masses (‘not in mass’). Scale bar, 5 μm. (b–e) Quantification of mitotic abnormalities (Ctrl n = 146, Emi1-OE n = 114 mitoses): frequencies and types of mitotic abnormalities (b; P value summaries based on frequencies irrespective of whether abnormality occurred as single event or in conjunction with other abnormalities), numbers of abnormalities per mitosis (c), lagging chromosomes per cell (d) and chromosomes not segregating with the two chromosomal masses per cell (e). P values: t-test. (f, g) Distributions of the numbers of lagging chromosomes and chromosomes not in mass per cell, respectively. P values: t-test. (h) Fractions of cells in early mitosis. P value: t-test. NS, not significant; *Po0.05; **Po0.01; ****Po0.0001.

Figure 6. In vitro EMI1 overexpression delays mitotic progression and promotes chromosome instability. (a) Time-lapse microscopic images of histone H2B-GFP-expressing Hela cells that do (EMI1-OE) or do not (Ctrl) overexpress EMI1. Time is indicated in hours:minutes with nuclear envelope breakdown (NEBD) set at time point zero. Note the difference in timing between the Ctrl and EMI1-OE cells. (b) Quantiﬁcation of time-lapse microscopy analysis on 100 cells per condition. Time is shown in minutes. Error bars: s.e.m. P values: t-test. (c) Frequencies of cells with lagging chromosomes (top left image), anaphase bridges (bottom left image) or chromosomes not in mass. P-value: Fisher’s exact test. (d–f) Frequencies of cells that arrest in mitosis, die and show abnormal mitoses (not including mitotic delay), respectively. P values: Fisher’s exact test. See also Supplementary Figure 9.

DISCUSSION that in many solid cancers EMI1 overexpression occurs through We find that EMI1 overexpression is widespread in human solid defective Rb pathway signaling. P53 pathway defects can also lead cancers and strongly correlates with poor patient prognosis in a to overexpression of E2F target genes through crosstalk to the Rb pathway31 and each of these pathways are impaired in up to 50% manner that is only partly cell cycle dependent. Consistent with 32 this, Emi1-overexpressing mice develop a wide variety of solid of human cancers. Thus, aberrations in the two most frequently cancer types, most significantly adenomas and carcinomas. mutated pathways in human solid cancers can each contribute to Compared with isogenic control mice, Emi1 overexpression EMI1 overexpression and, as we show here, contribute to ‘ 13,33 significantly increases cancer penetrance, tumor size, overall oncogene-induced mitotic stress' leading to CIN and cancer tumor burden, carcinoma and adenoma burden, metastatic progression. Emi1-overexpressing mice develop tumors at long latencies. potential, cancer-associated immunological changes and the This is characteristic for CIN mouse models. In the absence of incidence of pre-malignant lesions. Our data indicate that, rather cooperating mutations, most CIN models develop cancer at than being a cell proliferation- or cancer-associated bystander, – latencies of a year or more.9 13 Consistent with recent work,34 this Emi1 overexpression actively promotes genomic instability and suggests that cells may need time to acquire specific genetic or tumorigenesis in vivo. genomic aberrations before malignant transformation occurs. Our Interestingly, reduced EMI1 mRNA expression often predicts inducible overexpression model will also enable us to test whether poor prognosis for hematological malignancies. Consistent with Emi1 overexpression-induced tumors are ‘oncogene-addicted’, this, Emi1 overexpression in mice does not predispose to blood requiring continued Emi1 overexpression, or whether transient cancer development. These differences could provide an explana- Emi1-induced CIN is sufficient to sustain tumor growth, as tion for the widely different aneuploidy signatures and etiologies 35 14,23 observed in another CIN model. of these cancer types. We note, however, that we have not Emi1 overexpression-induced tumors show a significant demonstrated decreased EMI1 protein levels in human blood increase in cells in early mitosis. Consistent with this, our live- cancers or increased Emi1 levels in Emi1 transgenic mouse cell imaging experiments show that Emi1 overexpression delays hematopoietic lineages. Hence, it remains possible that hemato- the timing between nuclear envelope breakdown and chromo- logical malignancies do not develop in the mice, because Emi1 some alignment. This is probably caused by chromosome levels are not induced in these cells. Nonetheless, it would be misalignment. Previous studies have shown that Emi1 degrada- interesting to see whether reduced Emi1 expression levels tion in early mitosis is required for progression beyond predispose to hematological cancers in vivo. prometaphase.36 Elevated levels of Emi1 may therefore cause Genomic amplifications and mutations involving EMI1 are rare the prometaphase delay in vitro and explain the high number of in human solid cancers. As EMI1 is an E2F target gene,15 it is likely early mitotic cells in transgenic mouse tumors in vivo.Emi1-

Figure 7. EMI1 expression strongly correlates with a CIN signature in human cancers and is a stronger marker of CIN than most well- established ones. (a–h) Scatter plots of EMI1 expression levels and corresponding CIN70 scores for TCGA data sets of: (a) breast carcinoma, (b) ovarian cancer, (c) glioblastoma multiforme, (d) low-grade glioma, (e) colon, (f) kidney, (g) lung and (h) endometrium cancers. Sample size and r for linear regression are indicated. P values: Pearson’s correlation coefficient. (i) Top 15 genes most significantly co-expressed with EMI1 in breast cancer. CIN70 genes are highlighted. See Supplementary Data set 3 for extended list. (j) Performance of EMI1 as a marker for CIN in comparison to the 70 CIN70 genes. overexpressing cells may subsequently arrest at metaphase. Finally, the current study’s identification of EMI1 overexpression Alternatively, if anaphase ensues, their chromosomes are often as both a common phenomenon in human cancers and an in vivo lagging or do not segregate with the main chromosome masses, driver of CIN suggests that it could be used as a means to induce thus facilitating CIN. excessively high levels of CIN in cancer cells, an approach that has Since its definition, the CIN70 signature has often been used both previously been proposed for the eradication of tumor cells.37,38 to assess the level of CIN in human tumors and to predict clinical Thus, our findings not only provide novel mechanic insights into – outcome.28 30 For the TCGA breast cancer cohort,22 we confirm that tumorigenesis, they also have important implications for cancer the CIN70 signature stratifies breast cancer subtypes according diagnosis and potentially therapy. to clinical outcome. We demonstrate that EMI1 expression has a clinical stratification power similar to the 70-gene CIN70 signature. In addition, EMI1 overexpression is a stronger predictor of CIN in MATERIALS AND METHODS human cancers than most CIN70 genes. This highlights that EMI1 Data set analysis and statistics expression may be used as a strong diagnostic marker. Alternatively, For EMI1 expression analysis, data from previous studies were used and inclusion of EMI1 in the CIN70 signature would increase its strength analyzed as described below and previously.21,22,39–43 All data sets are as a clinical prognostic tool. publicly accessible (GEO: http://www.ncbi.nlm.nih.gov/geo, TCGA: https://

© 2016 Macmillan Publishers Limited, part of Springer Nature. Oncogene (2016) 5446 – 5455 Emi1 overexpression promotes CIN and tumorigenesis S Vaidyanathan et al 5454 tcga-data.nci.nih.gov, DCC: http://caintegrator.nci.nih.gov). Expression dif- western blot analysis as described.31 For semi-quantitative RT–PCR, ferences were determined by Fisher’s exact, one-way analysis of variance Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, or t-tests, as indicated. Under- or overexpression of EMI1 and MKI67 were USA) and the following primers were used: GAPDH: Quantitect primer mix considered different if their levels were statistically significantly reduced or (Qiagen, Venlo, the Netherlands), Emi1 transgene: F: 5′-CTTACGATGTACCG elevated (t-test) by at least 1.5-fold compared with reference tissue. GATTAC-3′,R:5′-CCCACAATTGGTGAGTCGATG-3′. Antibodies used for For TCGA expression and survival analysis, level 3 Agilent data were used. western blot analysis were against HA (1:2000; Covance (Princeton, NJ, Expression outliers were identified and removed based on the standard USA), HA.11, clone 16B12), mouse Emi1 (1:1000; Zymed (Carlsbad, CA, box-whisker’s plot formulae that uses inter quartile range (IQR). Only USA), 38-5000), human Emi1 (1:1,000; IHC antibody listed above) and actin expression data falling into the following range would be taken for further (1:5000; Sigma-Aldrich (St Louis, MO, USA), A2066). analysis to avoid the effect of skewed data: Q1–(1.5*IQR)4expression o data Q3+(1.5*IQR). For survival analysis, expression data were grouped Pathology and fluorescence in situ hybridization into low and high as previously described.44 R and shell scripts were used to segregate the data into two. Survival curves were plotted where the Mouse tumor volumes were measured following necropsy and calculated π 3/2 46 survival was significantly different between the two groups. In R, the library using the formula /6x(LxW) , as described previously. Where tumor surv was used to fit(survfit) the survival curves and compute probability sizes were compared between mice, only tumors with elliptic shape were using the log-rank test (survdiff). All survival curves were re-plotted in included and if the mouse developed multiple tumors, only the largest fi fi GraphPad Prism (La Jolla, CA, USA). Adjustment of prognostic strength for tumor was included. Tumors were formalin- xed and paraf n-embedded. Nottingham prognostic index, Adjuvant! and proliferation was performed Tissue sections were subjected to blinded pathological analysis after H&E – fl as previously described.24 26 staining and to interphase-FISH using uorescently labeled peri- centromeric probes for chromosomes 12, 16 and 17 to count chromosome numbers per nucleus on at least 100 nuclei per tumor. Aneuploidy was Immunohistochemistry defined as: (1) 0 or 1 FISH signal for each of the three chromosomes, as Tissue microarray slides with fixed paraffin-embedded human tumor long as the total number of FISH signals is at least 1 or (2) ⩾ 3 copies of at tissue and matched normal control tissue sections (US Biomax, Rockville, least one chromosome. MD, USA) were subjected to antigen retrieval in sodium citrate buffer, permeabilized in 0.01% Triton-X in phosphate-buffered saline (PBS) for Immunofluorescence 10 min, washed twice in PBS and blocked in 10% BSA in PBS for 1 h. Emi1 fi fi μ antibody (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-365212) Formalin- xed, paraf n-embedded tissues were sectioned at 4 m, incubation was performed overnight at 4 °C. Endogenous horseradish de-waxed 2 × 10 min in 100% xylene and rehydrated 2 × 5 min in 100% ethanol, 1 × 1 min each in: 90, 80, 70, 50, 30% ethanol, 1 × 5 min in distilled peroxidase was blocked in 0.3% H2O2 in PBS for 10 min followed by two PBS washes. Goat anti-Mouse IgG HRP (Life Technologies, Carlsbad, CA, water. Antigen retrieval was carried out in 0.01 M sodium citrate, pH 6.0 USA) was applied at 1:1000 for 1 h followed by two PBS washes. DAB with 0.05% Tween-20 at 105 °C in a decloaking chamber for 15 and 20 min (3,3'-diaminobenzidin; Biocare Medical, Concord, CA, USA) was applied for cooling. Sections were washed 3 × 5 min in dH2O and blocked in 10% 2 min and tissues were counterstained with hematoxylin. Semiquantitative normal goat serum (Sigma-Aldrich) in PBS (blocking buffer) for 60 min at room temperature in a humidified chamber. Sections were incubated with analysis was performed in a blinded manner using the IHC multiplicative fi quickscore method, as described.45 primary antibodies in blocking buffer in a humidi ed chamber at 4 °C overnight. Primary antibodies were β-tubulin (Abcam (Cambridge, UK), 1:100), γ-tubulin (Sigma-Aldrich, 1:50), 4,6-diamidino-2-phenylindole Human primary tumor protein analysis (Sigma-Aldrich, 0.2 μg/ml). Sections were washed 3 × 20 min in PBS-0.5% With patient informed consent and Institutional Human Research Ethics Tween-20, incubated in secondary antibodies in blocking buffer for 1 h at Approval from the University of Queensland, de-identified frozen primary room temperature in a dark humidified chamber. Secondary antibodies human tumor tissues were obtained from the Wesley Research Institute were Alexa Fluor-488 and Alexa Fluor-594-conjugated goat anti-rabbit Tissue Bank (Toowong, QLD, Australia). Tissue samples were suspended in and goat anti-mouse IgG (Invitrogen, 1:500). Sections were mounted RIPA buffer (1:5 (w/v) ratio) and protein extracted using Precellys24 in in Vectashield (Vector Labs, Burlingame, CA, USA) and imaged using an tubes containing 1mm Zirconia beads (Daintree Scientific, St Helens, TAS, Olympus Fluoview v4.2 confocal microscope and Olympus Fluoview Australia) at 6500 r.p.m. for 3 × 15 s with 15 s gaps. Western blot analysis FV1200 software (Olympus, Tokyo, Japan). was performed as described below. Time-lapse microscopy Generation of transgenic mice Hela cells (American Type Culture Collection, Manassas, VA, USA) stably Mouse experiments and procedures were approved by the institutional expressing histone H2B-GFP (B. Gabrielli, mycoplasma-negative) were animal ethics committee. Inducible Emi1-overexpressing mice (in a mixed transduced with a lentiviral vector containing the human EMI1 ORF. C57BL/6 x 129/Sv background) were generated by injecting a BsrBI- Following puromycin selection (3 days at 2 μg/ml), resistant cell digested fragment (containing an inducible, CMV-based promoter with populations and control cells were subjected to time-lapse microscopy. 5 seven tetracycline-response elements; Figure 2a) into fertilized oocytes, For that, 10 cells were seeded in a six-well plate in DMEM/F12 media followed by implantation into pseudopregnant mice. Founder mice were supplemented with 10% FBS, 2 mML-glutamine, 1 mM sodium pyruvate, tested for transgene genomic integration by PCR (F: 5′-AGCAGAGCTCGTTT 20 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were AGGAACC-3′;R:5′-ACTTCAAGCTCGGAAAGATCAG-3'; 29 cycles of (30 s at maintained at 5% CO2 and 37 °C and monitored with an Olympus time- 94 °C, 30 s at 57 °C, 30 s at 72 °C)) and Southern blot analysis (see below). Two lapse microscope and 100 cells in each condition were analyzed. positive founder mice were crossed with CMV-rtTA (Tet-On) and CMV-tTA 27 (Tet-Off) transgenic mice to create bitransgenic mice. Transgene expression CIN70 analysis was regulated by feeding the mice a regular or doxycycline-containing diet Agilent probe-level expression data (log signal intensity normalized (625 mg/kg; Specialty Feeds, Glen Forrest, WA, USA) after weaning. 2 to reference) were obtained from TCGA (BRCA, CRC, LUAD+LUSC, LGG, KIRP+KISC,UCEC,OV,GBM).22,39–43,47,48 Expression data were obtained after Southern blot analysis background correction, normalization and loess transformation. Agilent Mouse genomic DNA extraction and Southern blotting were performed platform data (G4502A_07_2, G4502A_07_3) were analyzed. Probes corre- 28 according to standard methods. Briefly, for each sample, 8 μg of DNA was sponding to the 70 CIN70 genes and EMI1 were taken from the Agilent ADF digested with SacI and loaded on a 0.8% agarose gel. Following transfer to Data sets. Probe sequences were megablasted against the human NR/NT the blot, a probe of about 750 bp was released from pTRE-HA-Emi1 by SacI database. Probes mapping to multiple or incorrect genes were excluded. digestion, purified, radioactively labeled and hybridized to the membrane. These were: KIF4A: A_23_P148474, A_23_P148475; MT1JP: A_23_P414341, A_24_P74828, A_24_P74830; PTTG1: A_23_P7636, A_24_P72112; UBE2C: A_23_P304770. Of 261 blasted CIN70 probes, 253 (69 genes) mapped to RNA and protein analysis their corresponding genes with significant values to pass quality control. RNA and protein were extracted from mouse tissues and analyzed for Probe level expression data were averaged for each gene and the average transgene and endogenous expression by semi-quantitative RT–PCR and (CIN70 score) was calculated. EMI1 probes were similarly quality checked

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)