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[Cancer Research 64, 7370-7376, October 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

The Centrosomal Kinase Nek2 Displays Elevated Levels of Protein Expression in Human Breast Cancer

Daniel G. Hayward1, Robert B. Clarke2, Alison J. Faragher1, Meenu R. Pillai1, Iain M. Hagan2 and Andrew M. Fry1

1 Department of Biochemistry, University of Leicester, Leicester; and 2 Paterson Institute for Cancer Research, Manchester, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aneuploidy and chromosome instability are common abnormalities in human cancer. Loss of control over mitotic progression, multipolar spindle formation, and cytokinesis defects are all likely to contribute to these phenotypes. Nek2 is a cell cycle-regulated protein kinase with maximal activity at the onset of mitosis that localizes to the centrosome. Functional studies have implicated Nek2 in regulation of centrosome separation and spindle formation. Here, we present the first study of the protein expression levels of the Nek2 kinase in human cancer cell lines and primary tumors. Nek2 protein is elevated 2- to 5-fold in cell lines derived from a range of human tumors including those of cervical, ovarian, breast, prostate, and leukemic origin. Most importantly, by immunohistochemistry, we find that Nek2 protein is significantly up-regulated in preinvasive in situ ductal carcinomas of the breast as well as in invasive breast carcinomas. Finally, by ectopic expression of Nek2A in immortalized HBL100 breast epithelial cells, we show that increased Nek2 protein leads to accumulation of multinucleated cells with supernumerary centrosomes. These data highlight the Nek2 kinase as novel potential target for chemotherapeutic intervention in breast cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aneuploidy, the possession of more or less than the correct number of chromosomes, is the single most common feature of solid human tumor cells and is generally associated with poor prognostic outcome (1 , 2) . The underlying mechanisms for the generation of aneuploidy, although poorly understood, are likely to include defects in the mitotic machinery used to segregate duplicated chromosomes between daughter cells (3) . Persistent loss of control over chromosome segregation leads to chromosome instability, which is defined as the rate of karyotypic change that occurs within a tumor over time. Ominously, chromosome instability leads to a heterogenous population of cells with respect to genetic content and provides the tumor with a mechanism to select for cells with more malignant or drug resistant properties (4) .

The centrosome plays a critical role in mitotic spindle formation and chromosome segregation, because it is the primary site of microtubule nucleation in cells (5 , 6) . Normal cells enter mitosis with two properly duplicated centrosomes that ensure bipolarity, as well as correct axial positioning, of the spindle. Cancer cells from a wide variety of tumor types exhibit multipolar spindles, and these are often associated with abnormal centrosome number or architecture (7, 8, 9) . In addition, prematurely split centrosomes, unusually positioned centrosomes, and centrosomal proteins with aberrant levels of phosphorylation have all been detected in tumor cells (8 , 10) . Supernumerary centrosomes, and thereby aneuploidy, may be generated either by a direct uncoupling of the centrosome duplication cycle from the cell division cycle or through an indirect failure of cell division that leads to tetraploidization (3 , 11 , 12) . Cells lacking p53 or Rb pocket proteins fail to eliminate tetraploid cells allowing them to progress to the next mitosis where multipolar spindles can form (13) . As a result of centrosome defects, aneuploidy, and chromosome instability being detected in early, even preinvasive, tumors (14 , 15) , there is considerable interest in identifying whether centrosomal proteins are either mutated or abnormally expressed in cancer cells.

A number of cell cycle-regulated protein kinases have been localized to the centrosome including Cdk1, Plk1, and Aurora-A. Each of these kinases is active in mitosis and required for mitotic progression and correct bipolar spindle formation (16) . Overexpression of active and catalytically inactive versions of Plk1 and Aurora-A leads to mitotic defects, the generation of aneuploid cells, and supernumerary centrosomes (17 , 18) . Plk1 (e.g., refs. 19, 20, 21 ) and Aurora-A (18 , 22, 23, 24, 25) also exhibit elevated mRNA and protein expression in a wide variety of tumors and cancer cell lines and can induce transformation on overexpression in model systems (18 , 25 , 26) . Another centrosomal kinase that is regulated in a cell cycle-dependent manner is Nek2 (NIMA-related kinase 2; ref. 27 ). Nek2 is expressed in human cells as two splice variants, Nek2A and Nek2B, both of which localize to the centrosome (28) . The combined abundance and activity of the two forms peaks in S and G2 phase of the cell cycle, whereas Nek2A is specifically targeted for proteasomal destruction in mitosis (29) . Nek2A also has a binding site for the catalytic subunit of PP1, and hyperactivation of Nek2A at the onset of mitosis is thought to be dependent on inactivation of PP1 (30) . Overexpression of active Nek2A leads to premature centrosome splitting (31 , 32) , whereas overexpression of kinase-dead Nek2A causes the formation of centrosomal abnormalities, monopolar spindles, and aneuploidy (33) . These data, combined with the characterization of C-Nap1 (34 , 35) , a centrosomal substrate of Nek2, suggest a physiologic role for Nek2A in regulating the separation of centrosomes at the G2-M transition. Studies with the Xenopus laevis homologues of Nek2 indicate that Nek2B may primarily be required for assembly and maintenance of centrosomes in early embryos in a kinase-independent manner (36 , 37) .

Despite its importance in centrosome regulation and spindle formation, detailed analysis of Nek2 expression in tumors has not been performed. The only relevant data comes from two microarray studies that looked at the expression of a range of genes in certain cancer models. These revealed that Nek2 mRNA is significantly elevated in Ewing tumor (pediatric osteosarcoma) cell lines and upon transformation of low-grade follicular lymphoma to the more aggressive diffuse large B-cell lymphoma (38 , 39) . Here, we use specific Nek2 antibodies to demonstrate that Nek2 expression is elevated between 2- and 5-fold in cell lines derived from a variety of different cancer types and is significantly increased in the majority of breast tumors including early preinvasive tumors. We also show that overexpression of Nek2 in nontransformed breast cells can induce aneuploidy. These data lend support to the hypothesis that deregulation of centrosomal proteins in general, and Nek2 in particular, could be contributory factors in cancer progression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection.
HeLa, HBL100, MCF-7, MDA-MB-231, MDA-MB-468, and T47D cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Paisley, United Kingdom) supplemented with 10% fetal calf serum (FCS, Invitrogen) and 2 mmol/L glutamine (Invitrogen); ZR75–1, SKOV3, OVCAR5, DU-145, and PNT2-C2 were cultured in RPMI 1640 (Invitrogen) with 10% FCS and 4 mmol/L glutamine; K562, KCL22, U937, HL60, Ros, Rec, and Riva were cultured in RPMI 1640 (Invitrogen) with 10% FCS and 2 mmol/L glutamine; PC-3 cells were cultured in Ham’s F12 (Invitrogen) with 7% FCS and 4 mmol/L glutamine (PC-3). U2OS osteosarcoma T-REx cells (Invitrogen) and tetracycline-inducible Nek2A cells lines were cultured and induced with tetracycline as described previously (33) . Primary T cells were obtained as described previously (28) . HBL100 cells were transfected using FuGENE 6 transfection reagent according to the manufacturer’s instructions (Roche, Lewes, United Kingdom).

Cell Synchronization and Reverse Transcription-PCR.
HeLa cells were chemically synchronized in different phases of the cell cycle and analyzed by flow cytometry as described previously (28) . Total RNA was isolated from synchronized HeLa cells using TRI reagent (Sigma, St. Louis, MO) according to the manufacturer’s instructions and treated with amplification grade DNaseI (Roche) to avoid DNA contamination. Semiquantitative reverse transcription-PCR (RT-PCR) reactions were then established as described previously (36) using primers specific for human Nek2A and Nek2B (28) . Specifically, 1 µg (for Nek2A) and 2 µg (for Nek2B) of total RNA was used for cDNA synthesis and PCR reactions (94°C, 30 seconds; 50°C, 30 seconds; and 72°C, 45 seconds) carried out for 30 cycles.

Cell Extraction, Protein Electrophoresis, and Western Blotting.
Whole cell lysates of asynchronous cell populations were prepared in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS]. Briefly, cells were washed in ice-cold 1 x PBS (150 mmol/L NaCl, 2.7 mmol/L KCl, 8 mmol/L disodium hydrogen Pi, and 1.5 mmol/L potassium dihydrogen phosphate) and lysed directly in radioimmunoprecipitation assay buffer on ice for 30 minutes, centrifuged at 4°C, 14,000 rpm, to remove insoluble material and total protein concentration of the resulting supernatant determined by bicinchoninic acid assay (Pierce, Rockford, United Kingdom). Lysate, 50 µg, for each cell line was resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with affinity-purified antibodies raised to Nek2 [antipeptide antibody (40) , 1 µg/ml], C-Nap1 [R63 (34) , 1 µg/ml], and {alpha}-tubulin (Sigma, 1 µg/ml). Blots were developed by enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, United Kingdom).

Immunohistochemistry.
Four-micrometer thick sections of formalin-fixed, paraffin wax-embedded samples of resected tumor or normal breast tissue were mounted on (3-aminopropyl)triethoxysilane coated slides. Sections were dewaxed in three washes of xylene and rehydrated in graded alcohols: 100% EtOH for 10 minutes followed by 95%, 90%, and 70% for 5 minutes each, before incubation in 1 x PBS. Slides were then incubated in a pressure cooker in 10 mmol/L citrate buffer (10 mmol/L citric acid monohydrate; pH 6.0), for 2 or 3 minutes at full pressure to recover antigenic epitopes. Slides were probed with affinity-purified polyclonal rabbit antibodies raised to Nek2 [either antipeptide (40) or R40 (31) , 2 µg/ml] or control rabbit IgGs as an appropriate control (DAKO, Ely, United Kingdom; 2 µg/mL). For other measurements, slides were probed with antibodies raised against the Ki67 proliferation-associated nuclear antigen (clone MIB1, Dako), estrogen receptor {alpha} (clone 1D5, Dako), progesterone receptor (clone PgR636, Dako), and epidermal growth factor receptor (EGFR, NCL-EGFR, Novocastra) at concentrations recommended by the manufacturers. Bound antibodies were detected with a horseradish peroxidase-conjugated secondary antibody system (Envision, DAKO) and 3,3'-diaminobenzidine development according to the manufacturer’s protocol (brown stain). Developed slides were counterstained in hematoxylin (purple stain), dehydrated through graded alcohols, incubated in Histoclear (National Diagnostics, Hessle, United Kingdom), and mounted in Pertex (Cellpath, Newtown, United Kingdom). Slide images were captured with a Zeiss Axioskop microscope using a Zeiss Axiocam and processed using Axiovision and Adobe photoshop software. EGFR expression was graded from – (zero) to ++++ (very high) intensity of membrane staining on most tumor cells. Nek2, estrogen receptor {alpha}, progesterone receptor, and Ki67 were assessed as the percentage of positively stained cells after examination of ≥1,000 tumor cells in randomly selected fields. For example, in tumor sample 2196, 1,077 of 1,147 cells were positively stained for Nek2 (93.9%), 805 of 1,145 cells for estrogen receptor {alpha} (70%), 238 of 1,004 cells for progesterone receptor (24%), and 69 of 1,007 cells for Ki67 (6.9%). Intensity of staining was not assessed for these four antibodies.

Immunofluorescence microscopy.
HBL100 or MDA-MB-468 cells were fixed in cold methanol and processed for indirect immunofluorescence microscopy as described previously (33) . Primary antibodies used were anti-myc monoclonal (1:2,000; Cell Signaling Technologies, Beverly, MA), anti-{gamma}-tubulin (1:2,000; Sigma), or anti-C-Nap1 (R63 1 µg/ml; ref. 34 ). Secondary antibodies were Alexa Fluor 594 goat antimouse or Alexa Fluor 488 goat antirabbit (1 µg/mL; Invitrogen). DNA was stained with Hoechst 33258 (0.2 µg/mL; Calbiochem, San Diego, CA). Images were captured on a Nikon TE300 microscope with an ORCA-ER (Hamamatsu, Hamamatsu, Japan) CCD camera using Openlab 3.1.4 software (Improvision, Coventry, United Kingdom).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nek2 Protein Expression Is Elevated in a Variety of Human Cancer Cell Lines.
To determine whether the protein expression level of the Nek2 kinase is altered in human cancer, we first performed Western blots on extracts prepared from a panel of human cancer cell lines with a polyclonal anti-Nek2 antibody. This previously characterized antibody was raised against a peptide representing amino acids 278–299 of Nek2 and, therefore, detects both Nek2A and Nek2B (40) . For controls, Western blots were also performed on extracts of peripheral T lymphocytes, primary human umbilical vein endothelial cells, and immortalized breast and prostate epithelial cells (HBL100 and PNT2-C2, respectively). These four nontransformed cell types showed very similar levels of expression of Nek2 proteins with Nek2A consistently expressed at higher levels than Nek2B. In contrast, elevated expression of Nek2 proteins was found in 10 of 17 (59%) cancer cell lines including those of ovarian (SKOV3 and OVCAR5), leukemic (K562, KCL22, RIVA, and Rec), breast (MCF-7 and MDA-MB-468), prostate (PC3), and cervical (HeLa) origin (Fig. 1A)Citation . Those cell lines having increased expression of Nek2A generally showed similar increases in Nek2B suggesting either amplification of the gene or up-regulated transcription (Fig. 1, B and C)Citation . None of the cell lines tested had altered expression of the centrosomal Nek2 substrate, C-Nap1 (Fig. 1A)Citation .



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  		Fig. 1. Expression of centrosomal proteins Nek2 and C-Nap1 in cancer cell lines. A. The expression levels of Nek2 and C-Nap1 were determined by Western blotting total lysates of asynchronous cell populations equalized for protein content and are presented with respect to that seen in primary cells (HUVEC). Relative expression levels were determined by densitometric scanning over the linear range of enhanced chemiluminescence autoradiographs using NIH Image 1.62 and denoted as follows: +, expressed at equal abundance to that seen in HUVECs; ++, overexpressed up to 2-fold; +++, >2-fold overexpressed; –, expressed at lower abundance than in HUVECs; ND, not determined. Each cell line was analyzed in at least three independent experiments. B and C, representative Western blots of cancer cell lysates. Total cell lysate, 50 µg, was separated on SDS-polyacrylamide gels, Western blotted, and probed with Nek2 (antipeptide antibody; 1 µg/mL) or {alpha}-tubulin (1 µg/mL) antibodies. D. Semiquantitative RT-PCR was performed using specific primers against Nek2A (top) or Nek2B (bottom) on equalized amounts of total RNA collected from HeLa cells synchronized in the indicated phases of the cell cycle (M, G1, S, and G2) or on pGEM-Nek2A plasmid (P). Samples were separated on agarose gels and visualized with ethidium bromide. HUVEC, human umbilical vascular endothelial cell

 
In a recent microarray study, Nek2 was identified as a potential E2F4 transcription factor target gene (41) . E2F4 acts as a transcriptional repressor during G1 and G0 through recruitment of the Rb pocket proteins p107 and p130. These tumor suppressors, in turn, recruit histone deacetylases, which promote a closed state of chromatin preventing access to transcriptional activators. Importantly, Nek2 mRNA is elevated in mouse embryo fibroblasts lacking both p107 and p130 supporting the hypothesis that Nek2 transcription is tightly regulated under normal cell cycle conditions (41) . To directly test whether Nek2 transcription is cell cycle regulated, we performed semiquantitative RT-PCR on RNA isolated from extracts of synchronized HeLa cells (Fig. 1D)Citation . Conditions for semiquantitative RT-PCR were carefully established through serial dilution of total RNA added and optimization of PCR cycle number (36) and synchronization was confirmed by flow cytometry (data not shown). As predicted from the binding of E2F4, the abundance of both Nek2A and Nek2B mRNAs was significantly lower in G1 than in S and G2. The mRNAs were undetectable in mitotically arrested cells.

Nek2 Antigen Retrieval in Formaldehyde-Fixed Cells.
Whereas studies on cell lines are informative, it was important to determine whether Nek2 protein levels were altered in primary human tumors. For this purpose, we decided to use an immunohistochemical staining approach with the antipeptide Nek2 polyclonal antibody (40) . However, the archival tumor samples we wished to use were stored as formaldehyde-fixed, paraffin-embedded tissue, and we had determined previously that this antibody did not detect Nek2 after formaldehyde fixation (data not shown). To investigate a viable antigen retrieval protocol, we made use of a tetracycline-inducible Nek2A cell line generated previously in our laboratory (33) . After 24-hour induction with tetracycline, parental U2OS and U2OS:Nek2A cells were harvested, fixed with formaldehyde, embedded in agarose, and sectioned. Sections were then either untreated or subjected to an antigen retrieval procedure using citrate buffer before staining with either control or anti-Nek2 antibodies (Fig. 2)Citation . After antigen retrieval, Nek2 staining was strongly detected in the U2OS:Nek2A cell line and weakly in the parental U2OS cells, whereas the control antibodies gave no stain. A similar antigen retrieval approach was also shown to be successful on formaldehyde-fixed HBL100 breast cells (data not shown).



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  		Fig. 2. Antigen retrieval enables Nek2 staining after formaldehyde fixation. U2OS osteosarcoma cells stably transformed with a construct expressing kinase-dead Nek2A-K37R-myc-His (U2OS:Nek2A; B, D, F, and H) or the parental U2OS cell line (U2OS; A, C, E, and G) were fixed with formaldehyde before embedding in paraffin wax. Sections were then cut and stained with antibodies raised to Nek2 (A–D; 3.75 µg/mL) or control IgGs (E–H) before (A, B, E, and F) or after (C, D, G, and H) antigen retrieval. 3,3'-diaminobenzidine staining (brown) indicates that Nek2 is recognized in the stable cell line only after processing for antigen retrieval (D). The cell-to-cell variability of staining reflects the cell cycle dependent expression of Nek2A. Scale bar, 10 µm.

 
Nek2 Is Overexpressed in Primary Human Breast Tumors.
Major karyotypic changes, including gains or losses of whole chromosomes, are by far the most common lesion in breast cancer and are strongly associated with a bleak clinical outcome (42) . Using the antigen retrieval methods determined above and the polyclonal antipeptide Nek2 antibody, we therefore analyzed Nek2 protein expression in archival samples of 20 human breast cancers. Strikingly, Nek2 protein expression was elevated significantly in the majority of these breast tumor samples. Elevated Nek2 expression was clearly detected in ductal carcinoma in situ (DCIS) tumor cells as compared with normal breast ductal cells or stromal cells within the same sections (Fig. 3, A and C)Citation . These latter cells showed weak levels of staining as would be expected for a protein known to be ubiquitously expressed. DCIS is an early step in the pathology of breast cancer in which a luminal epithelial cell lining the duct has undergone an early transformation event and proliferated, although the carcinoma is still bounded by the basement membrane surrounding the duct (Fig. 3, B and D)Citation . Strong Nek2 staining was both restricted to the carcinoma and dependent on the presence of primary antibodies to Nek2 as sequential DCIS tumor sections incubated with control rabbit IgG fraction exhibited no specific staining (Fig. 3, E and F)Citation .



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  		Fig. 3. Nek2 expression is elevated in DCIS breast tumors. Breast tumor sections were stained with polyclonal antipeptide Nek2 (A–E; 2 µg/mL) or mock antibodies (F; control IgGs, 2 µg/mL) after antigen retrieval. Images taken at low (A) and high (B–F) magnification are shown. DCIS tumor cells (arrowed, T, A, and C) show highly elevated Nek2 expression as compared with normal breast duct tissue (arrowed, N, A, and C). The carcinoma cells in DCIS are bounded by an intact basement membrane (arrowed, B and D). To demonstrate antibody specificity, sequential tumor sections were cut and stained with Nek2 (E) or mock (F) antibodies after antigen retrieval. Scale bar in A, 100 µm, and in F (for B–F), 30 µm.

 
Analysis of invasive ductal carcinoma and invasive lobular carcinoma tissue also revealed a significant increase in the expression of Nek2 protein in tumor cells compared with controls (Fig. 4, A–DCitation , and data not shown). In invasive carcinomas, the tumor cells are present throughout the stroma and are no longer confined within ducts or lobules. This breakdown of normal histology and invasion of surrounding tissue is an important stage in the establishment of metastatic tumors. To verify the staining pattern that we had observed with the antipeptide Nek2 antibody, a second independent polyclonal antibody was used. This antibody was raised to a bacterially expressed fragment representing approximately the COOH-terminal two-thirds of the Nek2A protein (31) . Again, after antigen retrieval, carcinoma cells in DCIS tumors stained strongly for Nek2, whereas normal ductal tissue and stromal cells did not (Fig. 4, E and F)Citation .



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  		Fig. 4. Nek2 expression is elevated in invasive carcinomas of the breast. Invasive lobular carcinoma breast tumor sections were cut and stained with antibodies raised to Nek2 (A and B; 2 µg/mL) after antigen retrieval. Images taken at low (A) and high (B) magnification are shown. Sequential tumor sections stained with Nek2 (C) or mock (D) antibodies demonstrate the specificity of the response in the invasive tumor cells. Sequential tumor sections were also stained with a second independent polyclonal antibody raised to Nek2 (E; R40; 2 µg/mL) or mock antibodies (F; control IgGs; 2 µg/mL) after antigen retrieval. A DCIS carcinoma engorged with tumor cells is stained strongly for Nek2 (arrowed T in E), whereas normal ducts (arrowed N in E) and surrounding stroma are not. Scale bar in D, 20 µm, and in F, 100 µm.

 
Thus far, we have analyzed 20 breast tumor samples, including DCIS, invasive ductal carcinoma, and invasive lobular carcinoma tumors, in which areas of both tumor and normal tissue could be detected for comparison of expression levels. Sixteen (80%) exhibited significantly elevated Nek2 protein expression, whereas 4 (20%) gave no more staining than the normal tissue (Table 1)Citation . Within those tumors that showed elevated Nek2 expression, all had increased expression in >70% of tumor cells (n > 1000). We compared the extent of cells overexpressing Nek2 in these breast cancers with data that had been obtained previously on estrogen, progesterone, and EGFR expression. However, no obvious correlation was apparent between the expression of Nek2 and any of these markers. For example, of the 16 tumors that were strongly Nek2 positive (>70%), 9 were estrogen receptor {alpha} positive (>70%), whereas 7 were estrogen receptor {alpha} negative. Likewise, some tumors that were Nek2 positive were progesterone receptor and EGFR positive, whereas others were progesterone receptor and EGFR negative. Clearly, however, our current sample size is too small to draw any strong conclusion about the correlative expression of these markers. Finally, no correlation was detected between expression of Nek2 and Ki67, a marker for cell proliferation. Whereas one might predict that cells expressing Nek2 would be proliferating, we note that there was no obvious correlation either between expression of proliferation markers (Ki67 or PCNA) and the overexpression of the other mitotic kinases, Plk1 and Aurora-A (19 , 21 , 22 , 43) .


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  		Table 1 Nek2, Ki67, and hormone receptor expression levels in breast tumors

 
Elevated Expression of Nek2A Induces Aneuploidy in Immortalized Breast Cells.
To determine whether altered Nek2 expression might contribute to aneuploidy and chromosome instability in breast cancer, we analyzed the consequences of ectopic expression of Nek2A in nontransformed breast cells. HBL100 cells are derived from normal breast epithelia but are immortal due to the presence of SV40 large T antigen (44) . Immunofluorescence microscopy revealed that <1% of untransfected HBL100 cells had supernumerary centrosomes or abnormal nuclei, whereas a breast cancer cell line (MDA-MB-468) had ~5% cells with supernumerary centrosomes (data not shown). After 72 hours of transfection with myc-tagged Nek2A, ~9% HBL100 cells (n = 900) exhibited gross nuclear abnormalities being either multinucleated or with extra chromosomal material (Fig. 5A)Citation . Staining with antibodies against {gamma}-tubulin (Fig. 5, B–E)Citation and C-Nap1 (data not shown) revealed that the multinucleated cells invariably contained extra centrosomes suggesting that they arose through an aborted mitosis or failed cytokinesis. Cells transfected with myc-tagged lamin A showed only ~1% nuclear defects (n = 1200; Fig. 5ACitation ). Ectopically expressed Nek2A colocalized with {gamma}-tubulin at the centrosome in HBL100 cells as expected (Fig. 5, B–E)Citation . Therefore, these results support the hypothesis that elevated Nek2 levels can contribute to errors in mitotic progression and/or chromosome segregation that generate aneuploid cells.



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  		Fig. 5. Elevated expression of Nek2A induces multinucleation in HBL100 cells. A, histogram showing percentage of multinucleated cells after 72-hour transfection of either myc-LaminA or myc-Nek2A; 100–300 transfected cells were counted in at least four independent experiments. Bars, ±SD and the P = 0.01 (Student’s paired t test). B–E, merged images of multinucleated HBL100 cells expressing myc-Nek2A stained with antibodies against the myc tag (red) and {gamma}-tubulin (green). DNA was stained with Hoechst 33258 (blue). An enlargement of the {gamma}-tubulin stain is shown in the inset in each panel demonstrating the presence of supernumerary centrosomes. Scale bar, 5 µm.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The majority of tumor cells in human cancers exhibit centrosome abnormalities. These typically include increased centrosome size, number, and microtubule nucleation capacity (7, 8, 9) . Importantly, centrosome aberrations correlate with or even precede the generation of aneuploidy and the acquisition of a chromosome instability phenotype in breast and prostate tumors (14 , 15) . This has led to the hypothesis that deregulation of centrosome function could be a major contributory factor to the genetic instability and loss of tissue differentiation that drive most cancer progression.

Here, we show for the first time elevated protein expression of the centrosomal kinase Nek2 in cancer cell lines and primary breast tumors. This adds to previous mRNA studies demonstrating elevated Nek2 expression in Ewing tumor cell lines and diffuse large B-cell lymphomas (38 , 39) . In our analysis of 17 cancer cell lines, we found up-regulated expression in breast, ovarian, leukemic, prostate, and cervical cancer cells. The level of up-regulation was 2- to 5-fold with respect to its abundance in primary or immortalized untransformed cell lines. Similar levels of protein overexpression were reported for Aurora-A in pancreatic, breast, and colorectal tumor cell lines (18 , 24 , 25) . The HBL100 and PNT2-C2 immortalized cell lines do contain SV40 antigens that could alter expression from E2F responsive genes (44 , 45) . However, these cell lines had equivalent expression of Nek2 to the two primary cell types and significantly less than related cancer cell lines. In primary breast tumors, elevated Nek2 protein was detected with two independent antibodies in both in situ and invasive carcinomas. Its up-regulation in DCIS tumors indicates that alteration of Nek2 protein levels occurs in breast tumors before invasion and metastasis. We note that centrosomal defects, aneuploidy, and chromosome instability have all been observed with high frequency in these early stage tumors (15) . From our limited data, however, we saw no correlation between Nek2 and estrogen or progesterone receptor expression.

The underlying cause for an increase in Nek2 expression is unclear. However, it is intriguing to speculate that loss of transcriptional control through inactivation of the tumor suppressors p107 and/or p130 may be one reason. By analyzing the abundance of Nek2 mRNA in synchronized cells, we found that there was a reduced amount in G1 and M phase compared with S and G2 phase. This result is in agreement with the observation that the promoter region of the human Nek2 gene, located at chromosome 1q32.2–1q41 (28) , binds the E2F4 transcriptional repressor (41) . This would be expected to lead to a decrease in transcription in G1 and G0 through recruitment of the p107/p130 pocket proteins. Indeed, cells lacking p107 and p130 exhibit elevated levels of Nek2 mRNA (41) . Due to the significant number of mitotic and G2-M checkpoint proteins shown to have E2F4 binding sites, loss of p107 and p130 may well cause up-regulation of many cell cycle regulators (41) . Because loss of Rb pocket proteins also promotes survival of tetraploid cells (13) , we propose that these tumor suppressors may be critical in preventing the accumulation of centrosome abnormalities and aneuploidy.

Previous studies have shown that Nek2 contributes to assembly and maintenance of centrosomes and to bipolar spindle formation (33 , 36 , 37) . Therefore, inappropriately high expression of Nek2 might interfere with either centrosome integrity or chromosome segregation. In nondividing cells, this could contribute to the loss of differentiated cell morphology and breakdown in tissue architecture typical of invasive breast carcinomas (8 , 15) . In dividing cells, this could lead to aneuploidy and chromosome instability. Indeed, overexpression of Nek2A in the nontransformed HBL100 cell line did induce the generation of multinucleated cells at a level that was 9-fold higher than control transfections. Because these multinucleated cells also had supernumerary centrosomes, it seems reasonable to predict that they have failed cytokinesis, perhaps as a result of some earlier defect in mitosis. Overexpression of Aurora A and Plk1 also leads to multinucleation, and it has been proposed that this may provide a major route to both tetraploidization and centrosome amplification (17) . As well as perturbing spindle formation, altered expression of Nek2 might interfere with other mitotic processes. Nek2 has been reported to interact with the kinetochore proteins Hec1 and Mad1 suggesting a role in the spindle checkpoint (46 , 47) . Meanwhile, studies performed on homologues of Nek2 in lower eukaryotes suggest that these kinases may promote mitotic entry through recruitment of cell cycle regulators to the centrosome and, possibly, have a direct role in triggering cytokinesis (48, 49, 50) . Future studies will now be needed to determine whether inappropriate expression of Nek2A can also cause cellular transformation or tumor formation in animal models.


   ACKNOWLEDGMENTS
 
We thank A. Cramer (Paterson Institute for Cancer Research, Manchester) for technical assistance with immunohistochemistry and Dr. S. Shackleton (University of Leicester) for the lamin A plasmid. We are most grateful to Prof. R. Walker (Glenfield, Hospital, Leicester), Drs. N. Smith, R. Chopra, and N. Clarke (Paterson Institute for Cancer Research, Manchester) and the DSMZ Cell Bank (Braunschweig, Germany) for providing cell lines.


   FOOTNOTES
 
Grant support: Cancer Research United Kingdom (C1420/A4582). A. M. Fry is a Lister Institute Research Fellow.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A. Faragher is currently at the MRC Toxicology Unit, Hodgkin Building, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom; M. Pillai is currently at the Department of Immunology, Institute of Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, United Kingdom.

Requests for reprints: Andrew M. Fry, Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom. Phone: 44-116-252-5024; Fax: 44-116-252-3369; E-mail: amf5{at}le.ac.uk

Received 3/18/04. Revised 7/19/04. Accepted 8/17/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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