Human Cancer Biology

The -Binding Protein Anillin Is Overexpressed in Diverse Human Tumors Peter A. Hall,1Christopher B. Todd,1Paula L. Hyland,1Simon S. McDade,1Heike Grabsch,2 Mit Dattani,2 Kenneth J. Hillan,3 and S.E. Hilary Russell1

Abstract Anillin is an actin-binding protein that can bind and is a component of the cytokinetic ring. We assessed the anillin expression in 7,579 human tissue samples and cell lines by DNA micro- array analysis. Anillin is expressed ubiquitously but with variable levels of expression, being high- est in the central nervous system. The median level of anillin mRNA expression was higher in tumors than normal tissues (median fold increase 2.58; 95% confidence intervals, 2.19-5.68, P < 0.0001)except in the central nervous system where anillin mRNA levels were lower in tumors. We developed a sensitive reverse transcription-PCR strategy to show that anillin mRNA is expressed in cell lines and in cDNA panels derived from fetal and adult tissues, thus validating the microarray data. We compared anillin with Ki67 mRNA expression and found a significant linear relationship between anillin and Ki67 mRNA expression (Spearmann r f 0.6, P < 0.0001). Anillin mRNA expression was analyzed during tumor progression in breast, ovarian, kidney, colorectal, hepatic, lung, endometrial, and pancreatic tumors and in all tissues there was progressive increase in anillin mRNA expression from normal to benign to malignant to metastatic disease. Finally, we used anti-anillin sera and found nuclear anillin immunoreactivity to be widespread in normal tissues, often not correlating with proliferative compartments. These data provide insight into the existence of nonproliferation-associated activities of anillin and roles in interphase nuclei. Thus, anillin is overexpressed in diverse common human tumors, but not simply as a consequence of being a proliferation marker. Anillin may have potential as a novel biomarker.

The process of involves a complex choreography of As well as binding actin via its NH2-terminal domain (2), regulatory and structural events (1). A compelling body of data anillin has been shown to bind at least some septins (3, 6) and suggests that anillin is a key component that couples filament to associate with myosin II in a phosphorylation-dependent systems during cytokinesis and may influence their spatial manner (5). organization (2, 3). Anillin was originally identified as an actin- Recent data indicate that septins have roles in a number of binding protein by affinity chromatography (4) and its location disease states, including in neoplasia (7, 8). Mammalian septins is cell cycle regulated in cultured cells, being absent in G0; are involved in cytokinesis and in other processes (7) and some accumulating in the nucleus during G1, S, and G2; and then septins can exist in the nucleus (9). As part of our studies of redistributing to the cell cortex at the onset of mitosis (2, 3). septin biology, we investigated the septin-binding protein, With the onset of cytokinesis, anillin localizes to the cleavage anillin. We have used a range of methods to define the furrow and forms a ring (anillos—Spanish for ring; ref. 2), expression of anillin mRNA and protein in human tissues. We where it is required for the final phases of cytokinesis (2, 3, 5). found that anillin mRNA and protein is expressed in all tissues and maximally in brain. Whereas anillin message levels correlate with Ki67 expression (a well-characterized prolifera-

1 tion marker whose expression is tightly linked to growth Authors’ Affiliations: Centre for Cancer Research and Cell Biology, Queens fraction; refs. 10, 11), this is not absolute and anillin immuno- University Belfast, Belfast City Hospital, Belfast, United Kingdom; 2Academic Unit of Pathology, University of Leeds, Leeds, United Kingdom; and 3Genentech, Inc., reactivity is present in both proliferative and nonproliferative South San Francisco, California compartments with highest levels in the brain. Furthermore, Received 5/9/05; revised 6/27/05; accepted 7/12/05. anillin mRNA levels are increased in diverse human tumors Grant support: Queen’s University Belfast, Northern Ireland R&D Office, and and expression increases with tumor progression. Action Cancer; and Pathological Society of Great Britain and Ireland (M. Dattani). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Expression microarray experiments. The Affymetrix HG-u133 - Online (http://clincancerres.aacrjournals.org/). Chip was screened for multioligomer probes corresponding to the Requests for reprints: PeterA. Hall, Centre for Cancer Research and Cell Biology, Queens University Belfast, University Floor, Belfast City Hospital, Belfast, BT9 7AB anillin mRNA sequence (NM_018685). The multioligomer probe set Northern Ireland, United Kingdom. Phone: 44-28-9063-5397; E-mail: peter.hall@ 222608_at with the highest proportion of samples called ‘‘present’’ was qub.ac.uk. chosen for downstream analysis on 7,287 fresh frozen human tissue F 2005 American Association for Cancer Research. samples and 292 cell lines with sample preparation and data analysis doi:10.1158/1078-0432.CCR-05-0997 done by GeneExpress (GeneLogic, Gaithersburg, MD). Relevant ethical

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Fig. 1. Electronic Northern blot for anillin mRNA expression in 7,287 tissue samples and 282 cell lines (green, normal; blue, diseased nontumor; and red, tumor).

permissions were obtained and the samples obtained with due regard RNA extraction, reverse transcription, PCR, and cloning. Total RNA for ethical issues. Diagnoses of the human tissue samples were verified was extracted from cell lines by standard methods (RNA Stat-60; Tel- by internal review by the Department of Pathology at Genentech. A Test) and was subjected to RQ1 DNase digestion (Promega, Madison, Ki67-specific probe set (212020_s_at) was used as a control data set WI). cDNA was generated using random hexamer priming and because Ki67 expression is cell cycle regulated (10, 11). Statistical Moloney murine leukemia virus reverse transcriptase (Invitrogen, analysis was carried out using GraphPad Prism 4 software. Analysis by Paisley, Scotland). Relevant negative controls were always included. the Kruskal-Wallis test was done comparing the three GeneExpress In other experiments, adult or fetal human normal tissue cDNA panels database categories (normal, nonmalignant diseased, and malignant) in (BD Biosciences, San Jose, CA) were used as template for PCR each tissue type. Pairwise comparisons of categories used the Dunn reactions. PCR amplification was carried out using a primer pair posttest. Spearman correlation coefficient of relevant anillin and Ki67 for anillin [F3 5V-ATGCAGGAACTCAATAAC GAAA-3V and R1 5V- data sets was also calculated in each tissue type. AGGCTTTCCAATAGGTTTGTAGCA-3V] and a primer pair for h-actin

Fig. 2. Scatter plots of anillin and Ki67 mRNA expression in colorectum (A), lung (B), breast (C), lymphoid (D), WBC (E), and brain (F). Colorectal samples: n =567; Spearman r = 0.5225; 95% confidence interval,0.4580to0.5815;P < 0.0001; lung samples: n = 371;Spearman r = 0.6587; 95% confidence interval 0.5950 to 0.7142; P < 0.0001; lymphoid samples: n =286; Spearman r = 0 .7111; 9 5 % , c o n f i d e n c e interval, 0.6466 to 0.7655; P < 0.0001; breast samples: n = 428; Spearman r = 0.6616; 95% confidence interval, 0.6029 to 0.7131; P < 0.0001;WBC samples: n =516; Spearman r = 0.7239; 95% confidence interval, 0.6787 to 0.7637; P < 0.0001; central nervous system samples: n = 1,297; Spearman r = À0.1062; 95% confidence interval, À0.1613 to À0.05042; P < 0.0001.

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[BA67 5V-GGGAGAGCGGGAAATCGTGCGTGACATT-3V and BA68 5V- all cell lines but with varying intensity indicative of differing GATGGAGTT GAAGGTAGTTTCGTC-3V]. PCR products were analyzed levels of anillin mRNA expression. This was confirmed using on a 1% agarose gel and visualized with ethidium bromide. Real-time a real-time quantitative RT-PCR method (Fig. 3B), and quantitative PCR was done using ABsolute QPCR SYBR Green Mix the results of both assays are positively correlated (r = 0.87; (ABgene, Cambrigde, United Kingdom) on a GRI Lightcycler using a primer pair for anillin [F4 5V-TGGCTAATTGTACCAGTCG TCAGA-3V and R1 5V-AGGCTTTCCAATAGGTT TGTAGCA-3V] and normalized to h-actin [primer pair BA2F-P 5V-GGCTCCG GCATGTGCAAG BA3R-P 5V- CCTCGGTCAGCAG CACGG]. A full-length anillin construct was gener- ated by PCR, cloned into pcDNA3.1/V5-His-Topo, and sequenced on both strands by standard methods (Applied Biosystems, Foster City, CA). production, characterization, and immunohistochemistry. Polyclonal anti-anillin sera (S3 and S4) were raised to keyhole limpet hemocyanin– conjugated peptides [781LKNEGPQRKNKASPQ796 and 1017YWTYPDDEKRKNPIG1031] using standard methods (Eurogentec, Liege, Belgium), purified with Melon gel (Pierce Biotechnology Rock- ford, IL), and affinity purified by standard methods. Protein lysates derived from cell lines were resolved on polyacrylamide gels, transferred to nitrocellulose filters, and probed with relevant immune or preimmune sera. Protein bands were detected by enhanced chemilu- minescence (12). Full-length anillin cDNA were expressed in coupled in vitro transcription/translation reactions was similarly resolved and detected with anti-V5 epitope tag antibody. Cell lines (HeLa, CaOV3, OTN14, A2780, MDA-MB-361, MCF7, RKO, H630, and 293T) were grown under standard conditions. After fixation [methanol (À20jC) or 2% paraformaldehyde in PBS for 10 minutes], cells were rinsed with PBS, permeabilized in PBS with 0.1% Triton X-100, and stained with anti-anillin sera [rabbit sera S3 or S4 (above) or goat serum Ab5910 (AbCam, Cambridge, United Kingdom)]. Bound antibody was detected with FITC-conjugated anti- rabbit or anti-goat sera (DakoCytomation, Ely, United Kingdom) with a 4V,6-diamidino-2-phenylindole nuclear counterstain. Cells were visual- ized on a Leica FW4000 microscope system. In other experiments, pellets of cell lines (HeLa, CaOV3, A2780, MCF7, RKO, and 293T) were fixed in 10% formalin, embedded in wax, and processed for immunohistologic analysis. Immunohistochemistry was done on paraffin sections of cell pellets and normal tissue microarrays using standard methods (13) with ethical approval from the Northern Ireland Local Research Ethics Committee.

Results

Anillin mRNA is transcribed from 24 exons of the anillin gene (ANLN, NM_018685) that span 84 kb on 7p14.2. This encodes a basic (isoelectric point, 8.3) 1,125-residue protein with a predicted molecular weight of 123,477.72 Da. A single Affymetrix probe set (222608_at) maps to 3Vend of the anillin mRNA. Figure 1 shows an electronic Northern blot based on the analysis of 7,287 fresh frozen human tissue samples and 292 cell lines. Anillin mRNA is expressed in all tissues, albeit at varying levels. Given the association of anillin with cytokinesis, we compared expression of anillin with that of Ki67. There is a statistically significant, but modest, correlation between anillin and Ki67 mRNA expression (r is typically f0.6; see Fig. 2A-E). The multiple regression coefficient (r2) is typically f0.36, indicating only a proportion of the variance in anillin is related to Ki67 expression and, by inference, proliferation. In the brain (Fig. 2F), this correlation is lost as there is high-level expression of anillin mRNA but Ki67 expression is negligible as would be expected because the vast majority of cells of the adult central nervous system are postmitotic. We examined the expression of anillin mRNA in human Fig. 3. Analysis of anillin mRNA expression in cell lines by semiquantitative (A)and by real-time quantitative RT-PCR (B).The real-time quantitative RT-PCR method tumor cell lines by reverse transcription-PCR (RT-PCR; was used to study anillin mRNA expression in cDNA libraries from of fetal (C)and Fig. 3A). PCR products of the expected size are detected in adult (D) tissues.

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Fig. 4. Anillin mRNA expression increases from normal to hyperplasia to benign to malignant tumors in different clinical scenarios including ovary (A), breast (B), kidney (C), colorectal (D), pancreas (E), liver (F), lung (G), and endometrium (H).

P < 0.005). The quantitative RT-PCR method was then used compared with normal tissues (see Supplementary Table S1). to screen cDNA panels derived from fetal (Fig. 3C) and adult There is higher expression of anillin mRNA in tumors (Fig. 3D) normal tissues. Anillin mRNA is ubiquitously compared with normal tissue controls and the fold inductions expressed in fetal development and in adult tissues, but at are larger than for Ki67 (mean fold increase, 3.54 versus 1.61 varying levels consistent with the data from the microarray for Ki67). The comprehensive nature of the GeneLogic database analysis (Fig. 1). For example, these data show that anillin allowed the identification, in eight tissues, samples that mRNA is highest in brain; and with high levels in testis and reflected tumor progression with data from normal, hyper- placenta; intermediate levels in skeletal muscle, thymus, plastic, benign, malignant, and metastatic samples (Fig. 4). ovary, and intestine (small and large); and lower levels in There is a progressive increase in median anillin mRNA heart, brain, lung, liver, kidney, pancreas, spleen, and expression during tumor progression in breast, colorectal, prostate. Comparison of the real-time RT-PCR expression endometrial, liver, lung, renal, kidney, ovarian, and pancreatic levels with the median signal intensity in microarray studies cancer (in all cases, P < 0.0002 by ANOVA). Of note is that this show a positive linear correlation (r = 0.81, P < 0.0001), analysis with the Ki67 data set gives a similar but less strong validating the microarray data. association with disease progression. From the complete microarray data set, samples from tissues Anillin protein expression was analyzed in cell lines using a in which tumors are not represented, or were represented in polyclonal anti-anillin serum and also a commercial goat small numbers, or where there is lack of homogeneity in the anti-anillin serum (ab5910) raised to the COOH-terminal 13 categories (e.g., blood vessels, heart, head and neck, gall residues of anillin (1112WQPDACYK PIGKP1125). Ab5910 gave bladder) were excluded. This left a data set (n = 5,856) where no signal in Western blots. The rabbit anti-anillin serum anillin expression was defined by tissue, and then into normal, recognizes a band of f180 kDa in lysates of HeLa, MCF7, nontumor diseased, and tumor categories. Samples were and RKO cells (Fig. 5A) and the signal intensity correlates grouped by histologic diagnosis. Ki67 mRNA expression in with mRNA levels (Fig. 3A). The electrophoretic mobility is this series of samples is consistently elevated in tumors less than predicted as reported previously but is the same as

Fig. 5. Characterization of anti-anillin sera.Western blot of anillin in lysates of cell lines (A). A species of f180 kDa is seen and levels correlate with those determined by RT-PCR (lane1, HeLa; lane 2, MCF7; lane 3, RKO).Protein expressed in in vitro transcription translation reactions indicates that anillin has a mobility comparable with that seen by the anti-anillin serum (lane 4, negative control; lane 5, in vitro transcription/translation ^ expressed protein).The slight reduction in mobility is due to theV5-epitope tag. Anillin expression, as detected with serum S4 by immunofluorescence, is present at the midbody in cytokinesis (B) and is found in the nucleus of interphase cells (C)as expected.

www.aacrjournals.org 6783 Clin Cancer Res 2005;11(19) October 1, 2005 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology that of in vitro transcription/translation–expressed anillin Nuclear staining could be seen in some cells derived from all protein. Immunofluorescence studies of cultured cells using germ layers and in cells from continually renewing, condition- a polyclonal rabbit anti-anillin serum and the commercial ally renewing, and quiescent populations. Similarly, cytoplas- antiserum (ab5910) show staining patterns identical to that mic immunoreactivity could be seen in an overlapping reported in other systems (2, 3). Anillin expression was at the distribution. The most striking observation is the lack of clear cytokinetic ring in telophase (Fig. 5B), but is present in the correlation of anillin immunoreactivity with proliferative nucleus in interphase (Fig. 5C). Taken together, the data compartments. Anillin expression is marked in the central indicate that both reagents detect anillin protein in human nervous system and in quiescent populations such as para- cells. thyroid, liver, salivary gland, and prostate. The amount of We undertook an immunohistologic analysis of anillin staining is variable with some cells showing strong nuclear protein expression in normal human tissues (Fig. 6). Both sera expression, whereas others do not. The meiotic cells of the testis gave equivalent results and anillin immunoreactivity was seen and ovary show intense nuclear anillin immunoreactivity. in all adult tissues consistent with the mRNA data. Immuno- With regard to cytoplasmic expression of anillin, three reactivity took three forms—nuclear, cytoplasmic, or mixed. patterns were observed. In general, diffuse cytoplasmic staining

Fig. 6. Immunohistochemical analysis of anillin expression in normal human tissues. Nuclear anillin immunoreactivity in seminal vesicle epithelium detected with Ab5910 (A)orS4(B). All other preparations show S4 antianillin immunoreactivity in oocytes and ovarian stroma (C), seminiferous tubules (D and E), fallopian tube (F), urothelium of bladder (G), renal tubules (H), parathyroid (I), stomach (J), small intestine and Brunners gland (top left, K), colon (L), placenta (M), cerebral cortex (N), liver (O), myocardium with Z band staining (P), salivary gland (Q), and prostate (R).

Clin Cancer Res 2005;11(19) October 1, 2005 6784 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Anillin Is Overexpressed in Tumors was seen (e.g., fallopian tube, small intestine, liver, prostate, high levels in germ cells, consistent with observations of anillin transitional epithelium, renal tubules, and salivary gland being required for meiotic cytokinesis in Drosophila (17, 19). ducts). However, in the colon and pancreatic ducts and gall We have found that anillin mRNA is consistently over- bladder epithelium, supranuclear cytoplasmic immunore- expressed in tumors derived from diverse sites and have activity was observed, which may represent staining of the shown an increase in anillin mRNA expression during tumor Golgi apparatus. Finally, striking staining of the intercalated progression. These data are supported by other recent reports. discs (Z dics) of the myocardium was observed, a region For example, anillin is overexpressed both breast tumors and enriched for vinculin, neural cell adhesion molecule, and at the transition from in situ to invasive disease (20). h-catenin (14). Relatively weak staining of skeletal muscle is Similarly, anillin is overexpressed in endometrial carcinomas seen and this is also membrane associated, whereas smooth (21), gastric cancer (22), and uveal melanoma (23). However, muscle showed more diffuse cytoplasmic immunoreactivity. the mechanism of anillin overexpression in tumors is not clear. The correlation of anillin and Ki67 mRNA expression Discussion suggests that anillin expression is regulated by cell cycle– dependent factors, but this is unlikely to be the entire We present a detailed characterization by multiple modalities explanation because anillin is expressed in nondividing cells of the expression of anillin mRNA and protein in human cells in many tissues. Another possibility is that the anillin locus and tissues. The data sets generated provide cross-validation might be amplified in neoplasia. Chromosome 7p harbors and paint a clear picture of the widespread distribution of loci amplified in cancer such as the epidermal growth factor anillin mRNA and protein. Our quantitative RT-PCR analysis receptor locus at 7p12.3 but this amplicon is typically <2 Mb shows that anillin mRNA is expressed in diverse tissues and the (24). The anillin locus at chromosome 7p14-15 is 18.5 Mb levels correlate well with those observed by microarray analysis. telomeric to this and is, thus, unlikely to be on the same There is a general, but not absolute, correlation between anillin amplicon. Furthermore, epidermal growth factor receptor and Ki67 mRNA expression. Our immunohistologic data overexpression is much more restricted (25) than seen with indicate that anillin protein is widely expressed in diverse anillin. Finally, flanking the ANLN locus (SEPT7 tissues, including in nonproliferative cells (e.g., neurones, 484,461 bp telomeric and AMPH 1,929,545 bp centromeric) terminally differentiated epithelial, and quiescent stromal are not overexpressed in cancer,4 suggesting that either this cells), as well as in proliferative compartments. Anillin mRNA chromosomal fragment is not amplified or, if it is, the and protein expression cannot, therefore, be simply viewed as amplicon is small. That anillin overexpression is so common yet another marker of proliferation. perhaps suggests a more global role in cellular physiology In cultured cells, anillin protein expression is predominantly, with deregulation in neoplasia. but not exclusively, nuclear, and profound spatial reorganiza- Other proteins associated with cytokinesis are altered in tion of anillin occurs through the cell cycle (2, 3). In G0, levels neoplasia. Kinetochore proteins, such as CENP-F, CENP-A, and of anillin protein are negligible, consistent with the report of Nek2 kinase, are overexpressed in tumors (25–28) with the Whitfield et al. (11) who showed that anillin mRNA is cell cycle CENP-F locus being amplified in head and neck tumors (29). regulated in vitro. However, our in vivo data indicate that a more The aurora kinases have been implicated in neoplasia and have complex situation exists with a partial correlation of anillin altered expression in tumor progression (30) as do key mRNA expression with Ki67 mRNA, but that anillin protein is elements of the cytokinesis apparatus, such as localized in both proliferative and quiescent populations. The (31, 32) and polo-like kinases (33). The key cytokinesis- reasons for this are unclear but several possibilities exist. The associated myosin, myosin II, can be altered in neoplasia (34). most likely is the clear distinction of cell cycles in immortalized As well as binding anillin (5), myosin II interacts with the cultured cells in an artificial environment and the highly metastasis-associated protein S100A4 (35) and the nuclear regulated conditions in real tissues (15). In addition, the tumor suppressor menin (36). Clearly, then, the molecular microarray data is based on extraction of mRNA and, hence, machinery associated with cytokinesis can be deranged in represents average levels across a sample rather than allowing neoplasia, often with altered expression. It is also of note that the contribution of distinct cellular populations within the patients with neoplasia may develop autoantibodies to sample to be defined. Furthermore, the issue of protein half-life kinetochore and other cytokinesis-associated proteins (37). It cannot be addressed by static assays and might provide some will be interesting to search for anillin autoantibodies in partial explanation for differences. Finally, translational control patients with cancer. is increasingly recognized as crucial throughout biology and It has been proposed that the nuclear location of anillin in may be perturbed in cancer (16). interphase is simply to keep it spatially separate from cytoplas- It is striking that anillin is expressed widely and often in mic components (2). It would seem more likely that anillin has postmitotic cells. The cell cycle distribution of anillin in active nuclear roles because pools of cytoplasmic anillin exist. Drosophila suggested that in fly development, G0 cells have no Actin is present in the nucleus (38, 39) where it has diverse roles detectable anillin (2). Furthermore, detailed examination of interacting with nuclear proteins including polymerases (40), fly development suggests that anillin only localizes to the nuclei helicases (41), transcriptional regulators (42), chromatin of dividing cells and may mark cells with the potential to remodeling proteins (43), ribonuclear proteins (44), structural divide. Indeed, anillin has been posited to be a marker of proteins (45), and tumor suppressor genes such as p53 (46). somatic stem cells (17, 18). Whereas this may be true of the Actin may also have a role in energy-dependent, vectorial fly, our immunolocalization studies in human tissues are not consistent with this conjecture. However, there are some parallels between flies and man because anillin is expressed at 4 P.A. Hall, unpublished data.

www.aacrjournals.org 6785 Clin Cancer Res 2005;11(19) October 1, 2005 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology movement of multimolecular complexes and organelles around recognized (9), including SEPT6,5 which is an anillin-binding the nucleus (47), and at least one myosin isoform exists in the protein (6). Given the expression of anillin in the nucleus, nucleus (48). Might anillin have roles in such actin-associated anillin functions may be important in nuclear physiology, and nuclear processes? Interestingly, nuclear septins are now overexpression of anillin may perturb the nucleus in cancer. Anillin may also prove to be a new biomarker of cancer and disease progression. Studies to test this hypothesis are in 5 C.Todd et al., in preparation. progress.

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Peter A. Hall, Christopher B. Todd, Paula L. Hyland, et al.

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