theseptin-bindingproteinanillinisoverexpressedin...

The Septin-Binding Protein Anillin Is Overexpressed inDiverse Human TumorsPeter 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 septins 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 higherin tumors than normal tissues (median fold increase 2.58; 95% confidence intervals, 2.19-5.68,P < 0.0001) except in the central nervous systemwhere anillinmRNA levels were lower in tumors.We developed a sensitive reverse transcription-PCR strategy to show that anillin mRNA isexpressed in cell lines and in cDNA panels derived from fetal and adult tissues, thus validating themicroarray data.We compared anillin with Ki67 mRNA expression and found a significant linearrelationship between anillin and Ki67 mRNA expression (Spearmann r f 0.6, P < 0.0001). AnillinmRNA expression was analyzed during tumor progression in breast, ovarian, kidney, colorectal,hepatic, lung, endometrial, andpancreatic tumors and in all tissues therewasprogressive increasein anillin mRNA expression from normal to benign to malignant to metastatic disease. Finally, weused anti-anillin sera and found nuclear anillin immunoreactivity to be widespread in normaltissues, often not correlating with proliferative compartments.These data provide insight into theexistence 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 ofbeing a proliferationmarker. Anillinmay have potential as a novel biomarker.

The process of cytokinesis involves a complex choreography ofregulatory and structural events (1). A compelling body of datasuggests that anillin is a key component that couples filamentsystems during cytokinesis and may influence their spatialorganization (2, 3). Anillin was originally identified as an actin-binding protein by affinity chromatography (4) and its locationis cell cycle regulated in cultured cells, being absent in G0;accumulating in the nucleus during G1, S, and G2; and thenredistributing to the cell cortex at the onset of mitosis (2, 3).With the onset of cytokinesis, anillin localizes to the cleavagefurrow and forms a ring (anillos—Spanish for ring; ref. 2),where it is required for the final phases of cytokinesis (2, 3, 5).

As well as binding actin via its NH2-terminal domain (2),anillin has been shown to bind at least some septins (3, 6) andto associate with myosin II in a phosphorylation-dependentmanner (5).

Recent data indicate that septins have roles in a number ofdisease states, including in neoplasia (7, 8). Mammalian septinsare involved in cytokinesis and in other processes (7) and someseptins can exist in the nucleus (9). As part of our studies ofseptin biology, we investigated the septin-binding protein,anillin. We have used a range of methods to define theexpression of anillin mRNA and protein in human tissues. Wefound that anillin mRNA and protein is expressed in all tissuesand maximally in brain. Whereas anillin message levelscorrelate with Ki67 expression (a well-characterized prolifera-tion marker whose expression is tightly linked to growthfraction; refs. 10, 11), this is not absolute and anillin immuno-reactivity is present in both proliferative and nonproliferativecompartments with highest levels in the brain. Furthermore,anillin mRNA levels are increased in diverse human tumorsand expression increases with tumor progression.

Materials and Methods

Expression microarray experiments. The Affymetrix HG-u133 Gene-Chip was screened for multioligomer probes corresponding to theanillin mRNA sequence (NM_018685). The multioligomer probe set222608_at with the highest proportion of samples called ‘‘present’’ waschosen for downstream analysis on 7,287 fresh frozen human tissuesamples and 292 cell lines with sample preparation and data analysisdone by GeneExpress (GeneLogic, Gaithersburg, MD). Relevant ethical

Human Cancer Biology

Authors’Affiliations: 1Centre for Cancer Research and Cell Biology, QueensUniversity Belfast, Belfast City Hospital, Belfast, United Kingdom; 2Academic Unitof Pathology, University of Leeds, Leeds, United Kingdom; and 3Genentech, Inc.,South San Francisco, CaliforniaReceived 5/9/05; revised 6/27/05; accepted 7/12/05.Grant support: Queen’s University Belfast, Northern Ireland R&D Office, andAction 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 pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: Supplementary data for this article are available at Clinical Cancer ResearchOnline (http://clincancerres.aacrjournals.org/).Requests for reprints: PeterA. Hall, Centre for Cancer Research and Cell Biology,Queens University Belfast, University Floor, Belfast City Hospital, Belfast, BT9 7ABNorthern Ireland, United Kingdom. Phone: 44-28-9063-5397; E-mail: [email protected].

F2005 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-05-0997

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permissions were obtained and the samples obtained with due regardfor ethical issues. Diagnoses of the human tissue samples were verifiedby internal review by the Department of Pathology at Genentech. AKi67-specific probe set (212020_s_at) was used as a control data setbecause Ki67 expression is cell cycle regulated (10, 11). Statisticalanalysis was carried out using GraphPad Prism 4 software. Analysis bythe Kruskal-Wallis test was done comparing the three GeneExpressdatabase categories (normal, nonmalignant diseased, and malignant) ineach tissue type. Pairwise comparisons of categories used the Dunnposttest. Spearman correlation coefficient of relevant anillin and Ki67data sets was also calculated in each tissue type.

RNA extraction, reverse transcription, PCR, and cloning. Total RNAwas extracted from cell lines by standard methods (RNA Stat-60; Tel-Test) and was subjected to RQ1 DNase digestion (Promega, Madison,WI). cDNA was generated using random hexamer priming andMoloney murine leukemia virus reverse transcriptase (Invitrogen,Paisley, Scotland). Relevant negative controls were always included.In other experiments, adult or fetal human normal tissue cDNA panels(BD Biosciences, San Jose, CA) were used as template for PCRreactions. PCR amplification was carried out using a primer pairfor anillin [F3 5V-ATGCAGGAACTCAATAAC GAAA-3V and R1 5V-AGGCTTTCCAATAGGTTTGTAGCA-3V] and a primer pair for h-actin

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

Fig. 2. Scatter plots of anillin and Ki67mRNA expression in colorectum (A), lung(B), breast (C), lymphoid (D),WBC (E), andbrain (F). Colorectal samples: n = 567;Spearman r = 0.5225; 95% confidenceinterval, 0.4580 to 0.5815; P < 0.0001; lungsamples: 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; 95%, confidenceinterval, 0.6466 to 0.7655; P < 0.0001;breast samples: n = 428; Spearman r =0.6616; 95% confidence interval, 0.6029 to0.7131; P < 0.0001;WBC samples: n = 516;Spearman r = 0.7239; 95% confidenceinterval, 0.6787 to 0.7637; P < 0.0001;central nervous system samples: n = 1,297;Spearman r =�0.1062; 95% confidenceinterval,�0.1613 to�0.05042; P < 0.0001.

Anillin Is Overexpressed in Tumors

www.aacrjournals.org Clin Cancer Res 2005;11(19) October1, 20056781



[BA67 5V-GGGAGAGCGGGAAATCGTGCGTGACATT-3V and BA68 5V-GATGGAGTT GAAGGTAGTTTCGTC-3V]. PCR products were analyzedon a 1% agarose gel and visualized with ethidium bromide. Real-timequantitative PCR was done using ABsolute QPCR SYBR Green Mix(ABgene, Cambrigde, United Kingdom) on a GRI Lightcycler using aprimer pair for anillin [F4 5V-TGGCTAATTGTACCAGTCG TCAGA-3Vand R1 5V-AGGCTTTCCAATAGGTT TGTAGCA-3V] and normalized toh-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 onboth strands by standard methods (Applied Biosystems, Foster City, CA).

Antibody production, characterization, and immunohistochemistry.

Polyclonal anti-anillin sera (S3 and S4) were raised to keyhole limpethemocyanin– conjugated peptides [781LKNEGPQRKNKASPQ796 and1017YWTYPDDEKRKNPIG1031] using standard methods (Eurogentec,Liege, Belgium), purified with Melon gel (Pierce Biotechnology Rock-ford, IL), and affinity purified by standard methods. Protein lysatesderived from cell lines were resolved on polyacrylamide gels, transferredto nitrocellulose filters, and probed with relevant immune orpreimmune sera. Protein bands were detected by enhanced chemilu-minescence (12). Full-length anillin cDNA were expressed in coupled invitro transcription/translation reactions was similarly resolved anddetected 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. Afterfixation [methanol (�20jC) or 2% paraformaldehyde in PBS for10 minutes], cells were rinsed with PBS, permeabilized in PBS with0.1% Triton X-100, and stained with anti-anillin sera [rabbit sera S3 orS4 (above) or goat serum Ab5910 (AbCam, Cambridge, UnitedKingdom)]. Bound antibody was detected with FITC-conjugated anti-rabbit or anti-goat sera (DakoCytomation, Ely, United Kingdom) with a4V,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) werefixed in 10% formalin, embedded in wax, and processed forimmunohistologic analysis. Immunohistochemistry was done onparaffin sections of cell pellets and normal tissue microarrays usingstandard methods (13) with ethical approval from the Northern IrelandLocal Research Ethics Committee.

Results

Anillin mRNA is transcribed from 24 exons of the anillin gene(ANLN, NM_018685) that span 84 kb on chromosome 7p14.2.This encodes a basic (isoelectric point, 8.3) 1,125-residueprotein with a predicted molecular weight of 123,477.72 Da.A single Affymetrix probe set (222608_at) maps to 3Vend of theanillin mRNA. Figure 1 shows an electronic Northern blot basedon the analysis of 7,287 fresh frozen human tissue samples and292 cell lines. Anillin mRNA is expressed in all tissues, albeit atvarying levels. Given the association of anillin with cytokinesis,we compared expression of anillin with that of Ki67. There is astatistically significant, but modest, correlation between anillinand 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 relatedto Ki67 expression and, by inference, proliferation. In the brain(Fig. 2F), this correlation is lost as there is high-level expressionof anillin mRNA but Ki67 expression is negligible as would beexpected because the vast majority of cells of the adult centralnervous system are postmitotic.

We examined the expression of anillin mRNA in humantumor cell lines by reverse transcription-PCR (RT-PCR;Fig. 3A). PCR products of the expected size are detected in

all cell lines but with varying intensity indicative of differinglevels of anillin mRNA expression. This was confirmed usinga real-time quantitative RT-PCR method (Fig. 3B), andthe results of both assays are positively correlated (r = 0.87;

Fig. 3. Analysis of anillinmRNA expression in cell lines by semiquantitative (A) andby real-time quantitative RT-PCR (B).The real-time quantitative RT-PCRmethodwas used to study anillin mRNA expression in cDNA libraries from of fetal (C) andadult (D) tissues.





P < 0.005). The quantitative RT-PCR method was then usedto screen cDNA panels derived from fetal (Fig. 3C) and adult(Fig. 3D) normal tissues. Anillin mRNA is ubiquitouslyexpressed in fetal development and in adult tissues, but atvarying levels consistent with the data from the microarrayanalysis (Fig. 1). For example, these data show that anillinmRNA is highest in brain; and with high levels in testis andplacenta; intermediate levels in skeletal muscle, thymus,ovary, and intestine (small and large); and lower levels inheart, brain, lung, liver, kidney, pancreas, spleen, andprostate. Comparison of the real-time RT-PCR expressionlevels with the median signal intensity in microarray studiesshow a positive linear correlation (r = 0.81, P < 0.0001),validating the microarray data.

From the complete microarray data set, samples from tissuesin which tumors are not represented, or were represented insmall numbers, or where there is lack of homogeneity in thecategories (e.g., blood vessels, heart, head and neck, gallbladder) were excluded. This left a data set (n = 5,856) whereanillin expression was defined by tissue, and then into normal,nontumor diseased, and tumor categories. Samples weregrouped by histologic diagnosis. Ki67 mRNA expression inthis series of samples is consistently elevated in tumors

compared with normal tissues (see Supplementary Table S1).There is higher expression of anillin mRNA in tumorscompared with normal tissue controls and the fold inductionsare larger than for Ki67 (mean fold increase, 3.54 versus 1.61for Ki67). The comprehensive nature of the GeneLogic databaseallowed the identification, in eight tissues, samples thatreflected tumor progression with data from normal, hyper-plastic, benign, malignant, and metastatic samples (Fig. 4).There is a progressive increase in median anillin mRNAexpression during tumor progression in breast, colorectal,endometrial, liver, lung, renal, kidney, ovarian, and pancreaticcancer (in all cases, P < 0.0002 by ANOVA). Of note is that thisanalysis with the Ki67 data set gives a similar but less strongassociation with disease progression.

Anillin protein expression was analyzed in cell lines using apolyclonal anti-anillin serum and also a commercial goatanti-anillin serum (ab5910) raised to the COOH-terminal 13residues of anillin (1112WQPDACYK PIGKP1125). Ab5910 gaveno signal in Western blots. The rabbit anti-anillin serumrecognizes a band of f180 kDa in lysates of HeLa, MCF7,and RKO cells (Fig. 5A) and the signal intensity correlateswith mRNA levels (Fig. 3A). The electrophoretic mobility isless than predicted as reported previously but is the same as

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

Fig. 5. Characterization of anti-anillin sera.Western blot of anillin in lysates of cell lines (A). A species off180 kDa is seen and levels correlate with those determined byRT-PCR (lane1, HeLa; lane2,MCF7; lane 3, RKO). Protein expressed in in vitro transcription translation reactions indicates that anillinhas amobility comparablewith that seenby the anti-anillin serum (lane 4, negative control; lane 5, in vitro transcription/translation ^ expressed protein).The slight reduction inmobility is due to theV5-epitope tag.Anillin expression, as detected with serum S4 by immunofluorescence, is present at themidbody in cytokinesis (B) and is found in the nucleus of interphase cells (C) asexpected.





that of in vitro transcription/translation–expressed anillinprotein. Immunofluorescence studies of cultured cells usinga polyclonal rabbit anti-anillin serum and the commercialantiserum (ab5910) show staining patterns identical to thatreported in other systems (2, 3). Anillin expression was at thecytokinetic ring in telophase (Fig. 5B), but is present in thenucleus in interphase (Fig. 5C). Taken together, the dataindicate that both reagents detect anillin protein in humancells.

We undertook an immunohistologic analysis of anillinprotein expression in normal human tissues (Fig. 6). Both seragave equivalent results and anillin immunoreactivity was seenin all adult tissues consistent with the mRNA data. Immuno-reactivity took three forms—nuclear, cytoplasmic, or mixed.

Nuclear staining could be seen in some cells derived from allgerm layers and in cells from continually renewing, condition-ally renewing, and quiescent populations. Similarly, cytoplas-mic immunoreactivity could be seen in an overlappingdistribution. The most striking observation is the lack of clearcorrelation of anillin immunoreactivity with proliferativecompartments. Anillin expression is marked in the centralnervous system and in quiescent populations such as para-thyroid, liver, salivary gland, and prostate. The amount ofstaining is variable with some cells showing strong nuclearexpression, whereas others do not. The meiotic cells of the testisand ovary show intense nuclear anillin immunoreactivity.

With regard to cytoplasmic expression of anillin, threepatterns were observed. In general, diffuse cytoplasmic staining

Fig. 6. Immunohistochemical analysis of anillinexpression in normal human tissues. Nuclear anillinimmunoreactivity in seminal vesicle epitheliumdetected with Ab5910 (A) or S4 (B). All otherpreparations show S4 antianillin immunoreactivity inoocytes and ovarian stroma (C), seminiferoustubules (D and E), fallopian tube (F), urothelium ofbladder (G), renal tubules (H), parathyroid (I),stomach (J), small intestine andBrunners gland (topleft, K), colon (L), placenta (M), cerebral cortex (N),liver (O), myocardiumwith Z band staining (P),salivary gland (Q), and prostate (R).





was seen (e.g., fallopian tube, small intestine, liver, prostate,transitional epithelium, renal tubules, and salivary glandducts). However, in the colon and pancreatic ducts and gallbladder epithelium, supranuclear cytoplasmic immunore-activity was observed, which may represent staining of theGolgi apparatus. Finally, striking staining of the intercalateddiscs (Z dics) of the myocardium was observed, a regionenriched for vinculin, neural cell adhesion molecule, andh-catenin (14). Relatively weak staining of skeletal muscle isseen and this is also membrane associated, whereas smoothmuscle showed more diffuse cytoplasmic immunoreactivity.

Discussion

We present a detailed characterization by multiple modalitiesof the expression of anillin mRNA and protein in human cellsand tissues. The data sets generated provide cross-validationand paint a clear picture of the widespread distribution ofanillin mRNA and protein. Our quantitative RT-PCR analysisshows that anillin mRNA is expressed in diverse tissues and thelevels correlate well with those observed by microarray analysis.There is a general, but not absolute, correlation between anillinand Ki67 mRNA expression. Our immunohistologic dataindicate that anillin protein is widely expressed in diversetissues, including in nonproliferative cells (e.g., neurones,terminally differentiated epithelial, and quiescent stromalcells), as well as in proliferative compartments. Anillin mRNAand protein expression cannot, therefore, be simply viewed asyet another marker of proliferation.

In cultured cells, anillin protein expression is predominantly,but not exclusively, nuclear, and profound spatial reorganiza-tion of anillin occurs through the cell cycle (2, 3). In G0, levelsof anillin protein are negligible, consistent with the report ofWhitfield et al. (11) who showed that anillin mRNA is cell cycleregulated in vitro . However, our in vivo data indicate that a morecomplex situation exists with a partial correlation of anillinmRNA expression with Ki67 mRNA, but that anillin protein islocalized in both proliferative and quiescent populations. Thereasons for this are unclear but several possibilities exist. Themost likely is the clear distinction of cell cycles in immortalizedcultured cells in an artificial environment and the highlyregulated conditions in real tissues (15). In addition, themicroarray data is based on extraction of mRNA and, hence,represents average levels across a sample rather than allowingthe contribution of distinct cellular populations within thesample to be defined. Furthermore, the issue of protein half-lifecannot be addressed by static assays and might provide somepartial explanation for differences. Finally, translational controlis increasingly recognized as crucial throughout biology andmay be perturbed in cancer (16).

It is striking that anillin is expressed widely and often inpostmitotic cells. The cell cycle distribution of anillin inDrosophila suggested that in fly development, G0 cells have nodetectable anillin (2). Furthermore, detailed examination offly development suggests that anillin only localizes to the nucleiof dividing cells and may mark cells with the potential todivide. Indeed, anillin has been posited to be a marker ofsomatic stem cells (17, 18). Whereas this may be true of thefly, our immunolocalization studies in human tissues are notconsistent with this conjecture. However, there are someparallels between flies and man because anillin is expressed at

high levels in germ cells, consistent with observations of anillinbeing required for meiotic cytokinesis in Drosophila (17, 19).

We have found that anillin mRNA is consistently over-expressed in tumors derived from diverse sites and haveshown an increase in anillin mRNA expression during tumorprogression. These data are supported by other recent reports.For example, anillin is overexpressed both breast tumors andat the transition from in situ to invasive disease (20).Similarly, anillin is overexpressed in endometrial carcinomas(21), gastric cancer (22), and uveal melanoma (23). However,the mechanism of anillin overexpression in tumors is notclear. The correlation of anillin and Ki67 mRNA expressionsuggests that anillin expression is regulated by cell cycle–dependent factors, but this is unlikely to be the entireexplanation because anillin is expressed in nondividing cellsin many tissues. Another possibility is that the anillin locusmight be amplified in neoplasia. Chromosome 7p harborsloci amplified in cancer such as the epidermal growth factorreceptor locus at 7p12.3 but this amplicon is typically <2 Mb(24). The anillin locus at chromosome 7p14-15 is 18.5 Mbtelomeric to this and is, thus, unlikely to be on the sameamplicon. Furthermore, epidermal growth factor receptoroverexpression is much more restricted (25) than seen withanillin. Finally, genes flanking the ANLN locus (SEPT7484,461 bp telomeric and AMPH 1,929,545 bp centromeric)are not overexpressed in cancer,4 suggesting that either thischromosomal fragment is not amplified or, if it is, theamplicon is small. That anillin overexpression is so commonperhaps suggests a more global role in cellular physiologywith deregulation in neoplasia.

Other proteins associated with cytokinesis are altered inneoplasia. Kinetochore proteins, such as CENP-F, CENP-A, andNek2 kinase, are overexpressed in tumors (25–28) with theCENP-F locus being amplified in head and neck tumors (29).The aurora kinases have been implicated in neoplasia and havealtered expression in tumor progression (30) as do keyelements of the cytokinesis apparatus, such as citron kinase(31, 32) and polo-like kinases (33). The key cytokinesis-associated myosin, myosin II, can be altered in neoplasia (34).As well as binding anillin (5), myosin II interacts with themetastasis-associated protein S100A4 (35) and the nucleartumor suppressor menin (36). Clearly, then, the molecularmachinery associated with cytokinesis can be deranged inneoplasia, often with altered expression. It is also of note thatpatients with neoplasia may develop autoantibodies tokinetochore and other cytokinesis-associated proteins (37). Itwill be interesting to search for anillin autoantibodies inpatients with cancer.

It has been proposed that the nuclear location of anillin ininterphase is simply to keep it spatially separate from cytoplas-mic components (2). It would seem more likely that anillin hasactive nuclear roles because pools of cytoplasmic anillin exist.Actin is present in the nucleus (38, 39) where it has diverse rolesinteracting with nuclear proteins including polymerases (40),helicases (41), transcriptional regulators (42), chromatinremodeling proteins (43), ribonuclear proteins (44), structuralproteins (45), and tumor suppressor genes such as p53 (46).Actin may also have a role in energy-dependent, vectorial

4 P.A. Hall, unpublished data.





movement of multimolecular complexes and organelles aroundthe nucleus (47), and at least one myosin isoform exists in thenucleus (48). Might anillin have roles in such actin-associatednuclear processes? Interestingly, nuclear septins are now

recognized (9), including SEPT6,5 which is an anillin-bindingprotein (6). Given the expression of anillin in the nucleus,anillin functions may be important in nuclear physiology, andoverexpression of anillin may perturb the nucleus in cancer.Anillin may also prove to be a new biomarker of cancer anddisease progression. Studies to test this hypothesis are inprogress.5 C.Todd et al., in preparation.

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2005;11:6780-6786. Clin Cancer Res Peter A. Hall, Christopher B. Todd, Paula L. Hyland, et al. Diverse Human TumorsThe Septin-Binding Protein Anillin Is Overexpressed in

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