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[Cancer Research 65, 11314-11325, December 15, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

ANLN Plays a Critical Role in Human Lung Carcinogenesis through the Activation of RHOA and by Involvement in the Phosphoinositide 3-Kinase/AKT Pathway

Chie Suzuki1, Yataro Daigo1, Nobuhisa Ishikawa1, Tatsuya Kato1, Satoshi Hayama1, Tomoo Ito2, Eiju Tsuchiya3 and Yusuke Nakamura1

1 Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo; 2 Department of Surgical Pathology, Hokkaido University Graduate School of Medicine, Sapporo; and 3 Kanagawa Cancer Center Research Institute, Kanagawa, Japan

Requests for reprints: Yusuke Nakamura, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372, Fax: 81-3-5449-5433; E-mail: yusuke{at}ims.u-tokyo.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gene expression profile analysis of non–small cell lung cancers (NSCLC) and subsequent functional analyses revealed that human ANLN, a homologue of anillin, an actin-binding protein in Drosophila, was transactivated in lung cancer cells and seemed to play a significant role in pulmonary carcinogenesis. Induction of small interfering RNAs against ANLN in NSCLC cells suppressed its expression and resulted in growth suppression; moreover, treatment with small interfering RNA yielded cells with larger morphology and multiple nuclei, which subsequently died. On the other hand, induction of exogenous expression of ANLN enhanced the migrating ability of mammalian cells by interacting with RHOA, a small guanosine triphosphatase, and inducing actin stress fibers. Interestingly, inhibition of phosphoinositide 3-kinase/AKT activity in NSCLC cells decreased the stability of ANLN and caused a reduction of the nuclear ANLN level. Immunohistochemical staining of nuclear ANLN on lung cancer tissue microarrays was associated with the poor survival of NSCLC patients, indicating that this molecule might serve as a prognostic indicator. Our data imply that up-regulation of ANLN is a common feature of the carcinogenetic process in lung tissue, and suggests that selective suppression of ANLN could be a promising approach for developing a new strategy to treat lung cancers. (Cancer Res 2005; 65(24): 11314-25)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Carcinoma of the lung is one of the most common causes of cancer death worldwide, and non–small cell lung cancer (NSCLC) accounts for nearly 80% of those cases (1). Although many genetic alterations involved in development and/or progression of lung cancer have been reported, the precise molecular mechanism remains unclear (2). Newly developed cytotoxic agents have emerged to offer multiple therapeutic choices for patients with advanced NSCLC, but each of the new regimens can provide only modest survival benefits compared with conventional cisplatin-based therapies (3, 4). Hence, novel therapeutic strategies such as the development of molecular-targeted agents and antibodies, as well as cancer vaccines, are eagerly awaited.

Systematic analysis of expression levels of thousands of genes on a cDNA microarray is an effective approach for identifying molecules involved in pathways of carcinogenesis (5–12); some of these genes or their products will become candidate targets for development of novel anticancer drugs and tumor markers. To isolate such molecules we have been analyzing genome-wide expression profiles of 37 NSCLCs, using pure populations of tumor cells prepared by laser-capture microdissection (5). In the course of those studies, we observed that ANLN, a gene encoding the human homologue of anillin, an actin-binding protein in Drosophila, was overexpressed in the majority of the primary NSCLCs. ANLN is reportedly essential for the formation or organization of actin cables in the cleavage furrow, and it plays an important role in cytokinesis (13).

Small guanosine triphosphatases (GTPase) are important for regulation and coordination of the remodeling of the cytoskeleton (14–16). Three members of the ras-homologue (RHO) family, RHOA, RAC1 (ras-related C3 botulinum toxin substrate 1), and CDC42 (cell division cycle 42) have been extensively studied in terms of their biological functions in mammalian cells. RHOA regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and the formation of actin stress fibers, through recruitment and activation of its effectors that include mDia, ROCK1, and ROCK2 (Rho-associated, coiled coil–containing protein kinases 1 and 2; refs. 17–19). Although RHO activity is important for cellular motility, efficient migration requires a tight balance between activation and deactivation of RAC1, CDC42, and RHOA at appropriate spaces and times in the cellular environment. RHO proteins also participate in the control of gene transcription, cell cycle progression, or antiapoptotic pathways (14). RHOA is activated in some human tumors (15), although the precise mechanism, especially the upstream pathway of RHOA signaling during carcinogenesis, has not been clarified.

On the other hand, phosphoinositide 3-kinases (PI3K) generating specific inositol lipids have been implicated for their important roles in the regulation of cell growth, proliferation, survival, differentiation, and cytoskeletal changes (20, 21). One of the best characterized targets of PI3K lipid products is the protein kinase AKT (v-AKT murine thymoma viral oncogene homologue/protein kinase B). In quiescent cells, AKT resides in the cytosol in a low-activity conformation. Upon cellular stimulation, AKT is translocated to cellular membranes by PI3K lipid products and is activated through phosphorylation by 3' phosphoinositide-dependent kinase-1 (20, 21). The components involved in the PI3K/AKT signaling pathway are often activated in a wide variety of cancers (20, 22). Therefore, a better understanding of the role of the downstream pathway of PI3K/AKT in cancer will lead to the development of more potent and selective inhibitors which should be a useful adjunct to conventional therapies (23).

Here, we report evidence to indicate that ANLN plays a significant role in pulmonary carcinogenesis through interaction with RHOA in the cytoplasm and cleavage furrows, as well as through the PI3K/AKT pathway–dependent nuclear function, and suggests that this molecule represents a potential target for the development of novel therapeutic drugs and prognostic markers for lung cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and clinical samples. The 23 human lung cancer cell lines used for this study included seven adenocarcinomas (A549, LC319, PC-3, PC-9, PC-14, A427, and NCI-H1373); two bronchioloalveolar cell carcinomas (NCI-H1666 and NCI-H1781); seven squamous cell carcinomas (RERF-LC-AI, SK-MES-1, EBC-1, LU61, NCI-H520, NCI-H1703, and NCI-H2170); two adenosquamous carcinomas (NCI-H226 and NCI-H647); one large cell carcinoma (LX1); and four small cell lung cancers (DMS114, DMS273, SBC-3, and SBC-5). All cells were grown in monolayers in appropriate media supplemented with 10% FCS and were maintained at 37°C in an atmosphere of humidified air with 5% CO2. Human small airway epithelial cells (SAEC) used as a normal control were grown in optimized medium (SAGM) purchased from Cambrex Bio Science, Inc. (East Rutherford, NJ). Primary NSCLC samples, of which 22 were classified as adenocarcinomas, 14 as squamous cell carcinomas, and 1 as adenosquamous carcinoma, had been obtained earlier with informed consent from 37 patients.

A total of 285 formalin-fixed primary NSCLCs [stage I-IIIA; 190 males and 95 females; age, 63.2 ± 10.1 years (mean ± SD); range, 26-84] and adjacent normal lung tissue samples used for immunostaining on tissue microarrays were obtained with informed consent from patients undergoing surgery, from 1993 to 1999 at Hokkaido University and its affiliated hospitals (Sapporo, Japan). These patients received curative resection of their primary cancers, and among them, only patients with positive lymph node metastasis (stage IIA-IIIA) were treated with cisplatin-based adjuvant chemotherapies after their surgery. This study and the use of all clinical materials mentioned were approved by individual institutional ethical committees.

Selection of a candidate gene and analysis by semiquantitative RT-PCR. On the basis of their expression profiles on a genome-wide cDNA microarray (5), we selected genes that showed expression levels ≥5-fold than in normal lung in >50% of the tumors examined. ANLN was among the genes qualifying for that list (≥5-fold expression in 86.7% of NSCLCs analyzed by cDNA microarray), and we subsequently confirmed its overexpression in lung tumor cells by semiquantitative RT-PCR. We prepared appropriate dilutions of each single-stranded cDNA prepared from mRNAs of clinical lung cancer samples, taking the level of ß-actin (ACTB) expression as a quantitative control. The primer sets for amplification were ACTB-F (5'-GAGGTGATAGCATTGCTTTCG-3') and ACTB-R (5'-CAAGTCAGTGTACAGGTAAGC-3') for ACTB, and ANLN-F1 (5'-GCTGCGTAGCTTACAGACTTAGC-3') and ANLN-R1 (5'-AAGGCGTTTAAAGGTG ATAGGTG-3') for ANLN. All reactions involved initial denaturation at 94°C for 2 minutes followed by 21 cycles (for ACTB) or 30 cycles (for ANLN) at 95°C for 30 seconds, 58°C to 62°C for 30 seconds, and 72°C for 45 seconds on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA).

Northern blot analysis. Human multiple-tissue blots (BD Biosciences Clontech, Palo Alto, CA) were hybridized with a 32P-labeled PCR product of ANLN. The full-length cDNA of ANLN was prepared by RT-PCR using primers ANLN-F2 (5'-CCCAAGCTTGGGGCCACCATGGATCCGTTTACGGAGAAAC-3') and ANLN-R2 (5'-TGCTCTAGAGCAAGGCTTTCCAATAGGTTTGTAG-3'). Prehybridization, hybridization, and washing were done according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at room temperature for 96 hours.

Western blotting. Rabbit antibodies specific for ANLN were raised by immunizing rabbits with glutathione S-transferase (GST)-fused human ANLN protein, and were purified using standard protocols. We used an enhanced chemiluminescence Western blotting analysis system (Amersham Biosciences, Uppsala, Sweden). Cells were maintained in serum-free medium for 24 hours after transfection of plasmids, then lysed in appropriate amounts of lysing buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0), 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate-Na, plus protease inhibitor]. Proteins separated by SDS-PAGE were electroblotted onto nitrocellulose membranes (Amersham Biosciences) and incubated with antibodies. A sheep anti-mouse IgG-horseradish peroxidase (HRP) antibody (Amersham Biosciences) and a goat anti-rabbit IgG-HRP antibody (Amersham Biosciences) served as the secondary antibodies for these experiments.

Immunocytochemical analysis. Cultured cells were washed twice with PBS(–), fixed in 4% paraformaldehyde solution for 60 minutes at room temperature, and then rendered permeable with PBS(–) containing 0.1% Triton X-100 for 1.5 minutes. Prior to the primary antibody reaction, cells were covered with blocking solution [3% bovine serum albumin in PBS(–)] for 60 minutes to block nonspecific antibody binding. After the cells were incubated with antibodies to human ANLN, a goat anti-rabbit secondary antibody conjugated to FITC (Cappel, Durham, NC) or rhodamine (Cappel) was added to reveal endogenous ANLN. Nuclei were stained with 4',6-diamidino-2-phenylindole. The antibody-stained cells were viewed with a laser-confocal microscope (TSC SP2 AOBS: Leica Microsystems, Wetzlar, Germany). To visualize actin filaments, we added Alexa594-conjugated phalloidin (Molecular Probes, Eugene, OR) after the incubation with secondary antibodies. To determine the cell cycle–dependent localization of ANLN, synchronization at the G1-S boundary was achieved with aphidicolin block. Cells were blocked with 1 µg/mL of aphidicolin (Sigma-Aldrich, Co., St. Louis, MO) for 24 hours, released from the block by four washes in PBS. These cells were cultured in medium and harvested for analysis at 1.5, 4.5, and 9 hours after the withdrawal of aphidicolin.

RNA interference assay. We had established a vector-based RNA interference (RNAi) system, psiH1BX3.0, to direct the synthesis of small interfering RNAs (siRNA) in mammalian cells, as reported elsewhere (8, 10, 11). Ten-microgram aliquots of siRNA expression vector were transfected into NSCLC cell lines LC319 and A549, both of which overexpressed ANLN using 30 µL of LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). The transfected cells were cultured for 5 days in the presence of appropriate concentrations of geneticin (G418). Cell numbers and viability were measured by Giemsa staining and triplicate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The target sequences of the synthetic oligonucleotides for RNAi were as follows: control-1 (LUC, luciferase gene from Photinus pyralis), 5'-CGTACGCGGAATACTTCGA-3'; control-2 (Scramble, gene coding for 5S and 16S rRNAs in chloroplasts of Euglena gracilis), 5'-GCGCGCTTTGTAGGATTCG-3'; siRNA-ANLN-1 (si-1), 5'-CCAGTTGAGTCGACATCTG-3'; siRNA-ANLN-2 (si-2), 5'-GCAGCAGATACCATCAGTG-3'; siRNA-RHOA-1, 5'-GCAGGTAGAGTTGGCTTTG-3'; siRNA-RHOA-2, 5'-GAAGGATCTTCGGAATGAT-3'. To validate our RNAi system, individual control siRNAs were tested by semiquantitative RT-PCR to confirm the decrease in expression of the corresponding target genes that had been transiently transfected to COS-7 cells. Down-regulation of ANLN expression by functional siRNA, but not by controls, was also confirmed in the cell lines used for this assay.

Flow cytometry. Cells were plated at densities of 5 x 105cells/100 mm dish, transfected with siRNA expression vectors, and cultured in the presence of appropriate concentrations of geneticin. Cells were trypsinized 5 days after transfection, collected in PBS, and fixed in 70% cold ethanol for 30 minutes. After treatment with 100 µg/mL RNase (Sigma-Aldrich), the cells were stained with 50 µg/mL propidium iodide (Sigma-Aldrich) in PBS. Flow cytometry was done on a Becton Dickinson FACScan and analyzed by ModFit software (Verity Software House, Inc., Topsham, ME). The cells selected from at least 20,000 ungated cells were analyzed for DNA content.

Bromodeoxyuridine incorporation assay. Lung cancer cells (LC319 and A549 cells) transfected with plasmids designed to express ANLN or mock plasmids, were cultured in serum-free medium for 4 hours. The medium was then replaced by RPMI 1640 containing 10% FCS with 10 µmol/L bromodeoxyuridine (BrdUrd). These cells were incubated for 20 hours and fixed; incorporated BrdUrd was measured using a commercially available kit (Cell Proliferation ELISA, BrdUrd; Roche Diagnostics, Basel, Switzerland).

Matrigel invasion assay. NIH3T3 and COS-7 cells, which scarcely express endogenous ANLN, but were transfected with plasmids designed to express ANLN or mock plasmids were grown to the confluent stage in DMEM containing 10% FCS. The cells were harvested by trypsinization, washed in DMEM without the addition of serum or proteinase inhibitor, and suspended in DMEM at concentrations of 1 x 105 cells/mL. Before preparing the cell suspensions, the dried layer of Matrigel matrix (Becton Dickinson Labware, Bedford, MA) was rehydrated with DMEM for 2 hours at room temperature. DMEM (0.75 mL) containing 10% FCS was added to each lower chamber of 24-well Matrigel invasion chambers, and 0.5 mL (5 x 104 cells) of cell suspension was added to each insert of the upper chamber. The plates were incubated for 22 hours at 37°C. After incubation, the chambers were processed and the cells invading through the Matrigel-coated inserts were fixed and stained by Giemsa as directed by the supplier (Becton Dickinson Labware).

Wound migration assay. NIH3T3 cells transfected with plasmids designed to express ANLN, or mock plasmids, were suspended in serum-free DMEM and plated in individual wells of two-well chambers (Becton Dickinson Labware) that had been coated with 10 µg/mL of fibronectin. After 4 hours of incubation, a line of adherent cells were scraped from the bottom of each chamber with a P200 pipette tip to generate wounds, and the medium was replaced with DMEM containing 10% FCS. After the cells were allowed to proliferate and migrate into the wound area for 48 hours, cells in the wound areas were counted under a microscope (DP50, Olympus, Tokyo, Japan).

Activation of RHO. Activation of this enzyme by ANLN was detected using EZ-Detect Rho Activation Kits (Pierce, Rockford, IL). Briefly, LC319 cells transfected with ANLN-expressing plasmids or mock plasmids were cultured for 24 hours, washed, and lysed with lysis buffer. After centrifugation at 16,000 x g, each lysate was mixed with GST-rhotekin-RBD and a "SwellGel Immobilized Glutathione Disc" for affinity-precipitation of activated RHO. The GST pulled-down precipitant that contained activated RHO was washed and boiled with sample buffer to serve for Western blot analysis using anti-RHO (-A, -B, and -C) and anti-ANLN antibodies. We did phosphorimager quantification analysis (Molecular Imager FX: Bio-Rad Laboratories, Hercules, CA), and relative Western blotting band intensities (active RHO levels in ANLN-transfected cells/those in mock-transfected cells) were quantified.

AKT expression plasmids. Constructs of a wild-type and dominant-negative (point mutant) form of AKT with Thr308 and Ser473 to alanines were generated as reported elsewhere (24).

Immunohistochemistry and tissue microarrays. Tumor-tissue microarrays prepared from formalin-fixed lung cancers were constructed as published previously (25–28). The staining pattern of ANLN was assessed semiquantitatively as absent (scored as 0) or positive (1+), as well as qualitatively according to nuclear ANLN (n-ANLN) or cytoplasmic ANLN (c-ANLN), by three independent investigators without prior knowledge of the clinical follow-up data. Cases with <10% of n- or c-ANLN-stained tumor cells were judged as ANLN-negative. Cases were accepted as positive only if reviewers independently defined them as such.

To investigate the presence of ANLN protein in tissue microarrays of clinical samples, we stained the sections using Envision+ kit/HRP (DakoCytomation, Glostrup, Denmark). Rabbit antibody to human ANLN was added after blocking endogenous peroxidase and proteins, and the sections were incubated with HRP-labeled anti-rabbit IgG as the secondary antibody. Substrate chromogen was added and the specimens were counterstained with hematoxylin.

Statistical analysis. We attempted to correlate clinicopathologic variables such as age, gender, and pathologic tumor-node-metastasis stage with the expression levels of n- or c-ANLN protein in 285 NSCLCs determined by tissue microarray analysis. Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC, or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for n- or c-ANLN expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were done with the Cox proportional-hazard regression model to determine associations between clinicopathologic variables and cancer-related mortality. First, we analyzed associations between death and possible prognostic factors including age, gender, pT classification, and pN classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced n-ANLN expression into the model, along with any and all variables that satisfied an entry level of P < 0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05. The relationship between n-ANLN expression and survival was also validated by subgroup analysis. The 285 NSCLC cases were divided into two subgroups with or without the adjuvant therapy: group-1 for node-negative cases (stage IA-IB, 195 patients) without any adjuvant treatment and group-2 for node-positive cases (stage IIA-IIIA, 90 patients) who were treated with cisplatin-based adjuvant chemotherapy after surgery. Tumor-specific survival was evaluated among each group of patients with the Kaplan-Meier method, and differences in survival times between the n-ANLN-positive and -negative patients were evaluated with the log-rank test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of ANLN in non–small cell lung cancer tissues and cell lines. We previously screened 23,040 genes on a cDNA microarray to detect transcripts indicating ≥5-fold expression in cancer cells than in normal control cells in >50% of 37 NSCLCs analyzed. Among the up-regulated genes, we identified the ANLN transcript (≥5-fold expression in cancer cells than in normal lung cells in 86.7% of these NSCLC cases) and confirmed its overexpression in 12 representative NSCLC cases by semiquantitative RT-PCR experiments (Fig. 1A, top). We also observed high levels of ANLN expression in all of the 23 lung cancer cell lines we examined, whereas no PCR product was detected in cells derived from normal small airway epithelial cells (Fig. 1A, bottom). Northern blotting using ANLN cDNA as a probe identified a weak band of about 4.0 kb, present only in testis and spinal cord among 24 normal human tissues examined (data not shown).




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  		Figure 1. ANLN expression in primary NSCLCs and lung cancer cell lines (ADC, adenocarcinoma; BAC, bronchioloalveolar cell carcinoma; SCC, squamous cell carcinoma; ASC, adenosquamous carcinoma; LCC, large cell carcinoma; and SCLC, small cell lung cancer), and its subcellular localization and formation of actin stress fibers in NSCLC cells. A, expression of ANLN in clinical samples of NSCLC, examined by semi-quantitative RT-PCR (top); expression of ANLN in lung cancer cell lines (bottom). B, endogenous expression of ANLN in the nucleus (n-ANLN), cytoplasm (c-ANLN), and cleavage furrow, detected by immunocytochemical staining using rhodamine-conjugated secondary antibody. C, cell cycle–dependent localization of endogenous ANLN. Antibodies to ANLN were stained with an anti-rabbit secondary antibody conjugated to FITC (green); nuclei were stained blue with 4',6-diamidino-2-phenylindole. LC319 cells were growth-arrested at G1 phase by incubation with 1 µg/mL of aphidicolin for 24 hours and released from G1 arrest by the removal of aphidicolin. FACS analysis, immunocytochemical staining (top), and Western blotting (bottom) were done at 1.5, 4.5, and 9 hours after the withdrawal of aphidicolin. D, colocalization of endogenous c-ANLN and F-actin in LC319 cells, detected with FITC-conjugated secondary antibody and Alexa594-conjugated phalloidin, respectively (left); LC319 cells 24 hours after transient transfection with ANLN-expressing plasmids (right); ANLN and F-actin distribution were assessed by FITC-immunostaining for ANLN and Alexa594-phalloidin staining, respectively. Additional stress fibers induced by exogenous expression of ANLN are colocalized with ANLN.

 
Subcellular localization of ANLN and formation of actin stress fibers. We did immunocytochemical analysis using anti-ANLN antibody to examine the subcellular localization of ANLN in lung cancer cells. Endogenous ANLN was detected in LC319 and A549 cells in various patterns of localization (Fig. 1B); i.e., ANLN protein was observed in the nuclei and/or cytoplasm (n-ANLN and c-ANLN), in the cortex following nuclear envelope breakdown, in the cleavage furrow during cytokinesis, and in the midbody at late telophase. Because the result of immunocytochemical staining suggested a possible cell cycle–dependent localization of ANLN, we synchronized LC319 cells using aphidicolin and examined ANLN localization during cell cycle progression. The protein was located predominantly in the nucleus at G1 and S phases, and mainly in the cytoplasm at G2 and M phases (Fig. 1C, top). ANLN levels increased at a transition period from G1 to S phases, reaching the maximum level at S phase and then decreasing in G2 and M phases, as shown by Western blot analysis (Fig. 1C, bottom). On the other hand, fiber-like staining in the cytoplasm was observed in a considerable proportion of the cells. We confirmed the colocalization of endogenous ANLN and filamentous actin (F-actin) on stress fibers in these cells by immunostaining, using anti-ANLN antibody and phalloidin, respectively (Fig. 1D, left). Because the actin cytoskeleton plays an important role in cytokinesis and morphology of mammalian cells, we examined the effect of ANLN on the formation of actin stress fibers by transfecting ANLN-expressing plasmids into LC319 and NIH3T3 cells. Fiber-like staining detected with phalloidin was significantly increased after transfection of ANLN, suggesting that overexpression of exogenous ANLN induced many new stress fibers in these cells (Fig. 1D, right).

Effects of ANLN on the growth of non–small cell lung cancer cells. To assess whether ANLN is essential for the growth or survival of lung cancer cells, we designed and constructed plasmids to express siRNA against ANLN (siRNA-ANLN-1 and -2) and two control plasmids [siRNAs for Luciferase (LUC), or Scramble (SCR)], and transfected each of them into LC319 and A549 cells. The amount of ANLN transcript in the lung cancer cells transfected with siRNA-ANLN-1 or -2 was significantly decreased in comparison with cells transfected with either of the two control siRNAs (representative data of LC319 was shown in Fig. 2A); transfection of siRNA-ANLN-1 or -2 also resulted in significant decreases in colony numbers and cell viability measured by colony formation and MTT assays (P = 5.9 x 10–7 and P = 1.8 x 10–5, respectively; unpaired t test; Fig. 2B and C). Moreover, the cells treated with siRNA-ANLN-1 showed larger morphology and multiple nuclei (Fig. 2D, top). To further clarify the molecular mechanism(s) of this phenotype, we did flow cytometry using LC319 cells that had been transfected with siRNA-ANLN-1 and found that the proportion of cells with a DNA content of 4 to 16 N was significantly higher in cultures transfected with siRNA-ANLN-1 than in cultures transfected with control siRNA (LUC; Fig. 2D, bottom).



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  		Figure 2. Inhibition of growth of NSCLC cells by siRNA against ANLN. A, expression of ANLN in response to siRNA-ANLN-1 (si-1), -2 (si-2), or control siRNAs against luciferase (LUC) or scramble (SCR) in LC319 cells, analyzed by semiquantitative RT-PCR. B, colony-formation assays of LC319 cells transfected with specific siRNAs for ANLN (si-1 and si-2) or control plasmids (si-LUC and si-SCR). C, viability of LC319 cells evaluated by MTT assay in response to si-1 or -2, in comparison with that to controls (P = 5.9 x 10–7 and P = 1.8 x 10–5, respectively; unpaired t test). D, microscopic observation of LC319 cells transfected with si-1 or -LUC (top). Arrows, cells treated with siRNA-ANLN-1 (si-1), where multinucleation and larger cell morphology are notable; flow cytometric analysis of LC319 cells transfected with si-1 or -LUC (bottom). The proportion of cells with a DNA content of 4 to 16 N in cultures transfected with si-1 was significantly higher than in cultures transfected with control siRNA (si-LUC). Assays were done thrice in triplicate wells.

 
To further investigate the effects of ANLN on the regulation of cell cycle progression, we did BrdUrd-incorporation assays using LC319 and A549 cells transiently transfected with ANLN-expressing plasmids. DNA synthesis seemed to be enhanced by the induction of ANLN expression in a dose-dependent manner, in both cell lines (P = 0.05 and P = 0.04, respectively; unpaired t test; Fig. 3A).



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  		Figure 3. Promotion of DNA synthesis and activation of cellular motility by ANLN. A, incorporation of BrdUrd in LC319 or A549 cells transiently transfected with ANLN. After 20 hours of incubation with BrdUrd, DNA synthesis seemed to be promoted in a dose-dependent manner in both lines of transfected cells (P = 0.05 and P = 0.04, respectively; unpaired t test). B, Matrigel invasion assay demonstrating enhancement of invasiveness of NIH3T3 and COS-7 cells after transfection with human ANLN expression plasmids (P = 0.02 and P = 0.01, respectively; unpaired t test). Left, Giemsa staining (x100); right, the relative number of cells migrating through the Matrigel-coated filters (cells with ANLN expressing plasmids/cells with mock plasmids). Assays were done thrice in triplicate wells.

 
Effect of ANLN on cellular motility. Because the immunocytochemical analysis had indicated that ANLN protein and F-actin were located together on stress fibers, and induction of exogenous ANLN expression had promoted the formation of actin stress fibers in mammalian cells, we did Matrigel invasion assays to determine whether ANLN might have some role in cellular motility. Invasion through Matrigel of NIH3T3 and COS-7 cells transfected with ANLN expression vectors was significantly promoted (P = 0.02 and P = 0.01, respectively; unpaired t test; Fig. 3B). Wound migration assays using NIH3T3 cells transfected with plasmids designed to express either ANLN or mock plasmid also showed significant activation of migration among ANLN-expressing cells (data not shown).

To clarify the mechanism responsible for ANLN-activated cellular motility, we carried out the following assays. Because a small GTPase, RHOA, is known to control the formation of actin structures, we first examined a possible interaction between RHOA and ANLN. As shown in Fig. 4A, we determined by immunocytochemical analysis that endogenous RHOA and ANLN were located together in the cytoplasm and in cleavage furrows in LC319 lung cancer cells. We confirmed the association of endogenous RHOA with exogenously expressed ANLN by immunoprecipitation assays (Fig. 4B).



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  		Figure 4. Interaction of ANLN with RHOA and evidence for RHO activation. A, colocalization of endogenous ANLN and RHOA, detected by immunocytochemical staining using anti-ANLN antibody (FITC) and anti-RHOA antibody (rhodamine). B, immunoprecipitation (IP) of exogenous ANLN and endogenous RHOA from cell extracts of lung cancer line LC319. LC319 cells were transfected with mock or ANLN and subjected to ANLN-IP with anti-ANLN antibody, followed by immunoblotting (IB) with anti-RHOA antibody (top). Aliquots of cell lysates were subjected directly to immunoblotting to confirm the expression of each protein (bottom three panels). C, RHO activation induced by binding with ANLN. LC319 cells were transfected with mock or ANLN and immunoblotted directly to confirm expression of each protein (top three panels). Aliquots of cell lysates were incubated with GST-rhotekin-RBD and subjected to a GST pull-down assay, then immunoblotted with anti-RHO or anti-ANLN antibodies (bottom two panels).

 
Like other GTPases, RHO is active when bound to GTP and inactive when bound to GDP. Upon binding to GTP, RHO interacts with downstream effectors such as rhotekin. On that basis, we attempted to investigate a possible interaction of ANLN with the active form of RHO by performing a GST pull-down assay, using lysate from LC319 cells transfected with ANLN-expressing plasmids, and GST-fusion rhotekin to affinity-precipitate the complex containing GTP-RHO (active form). An immune-complex containing ANLN and RHO was detected by Western analysis using antibodies raised against either of the proteins. When LC319 cells were transfected with plasmids designed to express ANLN, we observed 2-fold activation of RHO (active RHO levels in ANLN-transfected cells/those in mock-transfected cells) as well as interaction between ANLN and the active form of RHO (Fig. 4C). To investigate the effect of RHOA on ANLN-driven cell proliferation and/or motility, we designed and constructed plasmids to express siRNA against RHOA (siRNA-RHOA-1 and -2), and transfected each of them or two control plasmids (siRNAs for LUC or SCR) into LC319 cells. The amount of RHOA transcript in the lung cancer cells transfected with siRNA-RHOA-1 or -2 was significantly decreased in comparison with cells transfected with either of the two control siRNAs. Transfection of siRNA-RHOA resulted in only a slight decrease in colony number, however, it significantly reduced stress fiber–formation and induced larger cell morphology with double-sized nuclei. The results implied that ANLN-RHOA complex were mainly involved in cellular motility and cytokinesis of lung cancers.

Phosphoinositide 3-kinase/AKT-dependent nuclear localization of nuclear ANLN. Our data (shown above) suggests a cell cycle–dependent nuclear localization of ANLN, but its function in the nucleus is unclear. Activation of PI3K and AKT, an essential downstream component of PI3K-mediated oncogenic signaling, is known to promote entry into the cell cycle and is considered to provide a tumor-progressive signal through regulation of various proteins involved in cell cycle regulation as well as proapoptotic factors (20, 22). In addition, ANLN contains two regions which are similar to the consensus phosphorylation site for AKT kinase (RXRXXXS45 and RXXRXXS659; ref. 29). ANLN protein was detected as double bands by Western blotting when they were separated for longer times by SDS-PAGE. Therefore, we first incubated extracts from LC319 cells in the presence or absence of protein phosphatase (New England Biolabs, Beverly, MA) and analyzed the molecular weight of ANLN protein by Western blot analysis. Expectedly, the measured weight of the majority of ANLN protein in the extracts treated with phosphatase was smaller than that in the untreated cells. The data indicated that ANLN was possibly phosphorylated in lung cancer cells (Fig. 5A, right). We then examined a possible involvement of nuclear ANLN in the PI3K/AKT pathway. LY294002 (Sigma-Aldrich; 20 µmol/L for 16 hours), a specific inhibitor of the catalytic subunit of PI3K, which is directed at the ATP-binding site of the kinase (30), decreased AKT phosphorylation and induced the G1 arrest of lung cancer cells as expected (Fig. 5A, left and Fig. 5B). The level of total ANLN as well as phosphorylated ANLN was decreased according to the AKT inhibition (Fig. 5A). As shown in Fig. 1C, ANLN was predominantly located in the nucleus at G1 phase, but inhibition of the AKT phosphorylation markedly reduced the amount of ANLN in the nucleus (Fig. 5C). Similar results were also obtained by another selective AKT-inhibitor, NL71-101 (Calbiochem/Merck KGaA, Darmstadt, Germany; 12 µmol/L for 12 hours). Moreover, we observed the decrease of total ANLN as well as phosphorylated ANLN in lung cancer cells transfected with dominant-negative AKT construct (24) in comparison with the cells with wild-type AKT (data not shown). We then did nuclear/cytosolic ANLN extraction experiments with Western blot analysis using cell lysates prepared from LC319 cells treated with the PI3K/AKT inhibitors using different doses of the agents (LY294002: 10, 20, and 40 µmol/L for 16 hours; NL71-101: 6, 12, and 24 µmol/L for 12 hours) or at several time points (LY294002: 4, 8, 12, and 16 hours after 20 µmol/L treatment; NL71-101: 1, 2, 4, 6, and 12 hours after 12 µmol/L treatment). We confirmed that the level of total ANLN as well as phosphorylated ANLN in the nuclear fraction was mainly decreased in a dose- and time-dependent manner, although total ANLN levels in the cytosolic fraction were only slightly decreased (data not shown). These results implied the possibility that nuclear localization of ANLN, and probably its stability, are regulated by the PI3K/AKT signaling.



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  		Figure 5. PI3K/AKT-dependent stabilization and nuclear localization of n-ANLN. A-C, reduction of n-ANLN level by inhibition of the PI3K/AKT pathway. Phosphorylated and dephosphorylated levels of ANLN detected in LC319 cells by phosphatase assay coupled with Western blot analysis (A, right). Western blotting (A, left), FACS analysis (B), and immunocytochemical staining (C) of LC319 cells treated with PI3K inhibitor, LY294002 (20 mmol/L, 16 hours).

 
Association of nuclear ANLN expression with poor prognosis. We did immunohistochemical analysis with anti-ANLN polyclonal antibody using tissue microarrays prepared from 285 NSCLCs (stage IA-IIIA), all of which had been surgically resected primary tumors. Of the 285 cases, 267 (93.6%) revealed positive ANLN staining only in the cytoplasm (c-ANLN-positive), 128 (44.9%) revealed staining in both the cytoplasm (c-ANLN) and the nucleus (n-ANLN). Nucleus-only staining was not observed in any of the cases (Fig. 6A). We then examined the association of ANLN expression with clinical outcomes. Statistical analysis revealed no significant correlation of c- or n-ANLN expression with pT or pN factors among the lung cancer patients examined. However, we found a striking association between n-ANLN expression and tumor-specific 5-year survival using the Kaplan-Meier method (P < 0.0001 by the log-rank test; Fig. 6B). Univariate analysis showed that pT, pN, gender, and n-ANLN expression were each significantly related to the decreased tumor-specific survival of the 285 NSCLC patients. Furthermore, n-ANLN staining was determined to be an independent prognostic factor by multivariate analysis using the Cox proportional hazard model (P = 0.001). To further analyze the prognostic value of n-ANLN in more homogeneous populations of patients, we validated the relationship between n-ANLN expression and survival by subgroup analysis based on tumor stage and the status of adjuvant treatment. We divided the 285 NSCLC cases into two subgroups with or without the adjuvant therapy (see Materials and Methods): group-1 for node-negative cases (stage IA-IB, 195 patients) who had no adjuvant treatment and group-2 for node-positive cases (stage IIA-IIIA; 90 patients) who were treated with cisplatin-based adjuvant chemotherapy after surgery. Of the 195 cases in group 1, 81 (41.5%) revealed positive staining in the nucleus (n-ANLN), and 47 (52.2%) of the 90 cases in group 2 were judged to be n-ANLN-positive. We confirmed that n-ANLN expression is significantly associated with poor prognosis in the group-1 patients (Fig. 6 C; P = 0.005 by the log-rank test), as well as with that in the group-2 patients (Fig. 6 D; P = 0.018 by the log-rank test).



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  		Figure 6. Association of n-ANLN overexpression with poor outcomes in NSCLC. A, immunohistochemical evaluation of representative samples from surgically resected NSCLC tissues [adenocarcinomas (ADC) and squamous cell carcinomas (SCC)], using anti-ANLN polyclonal antibody on tissue microarrays. c-ANLN, localized in cytoplasm; n-ANLN, present in nuclei. Arrows, examples of cells expressing n-ANLN. B, tissue microarray and Kaplan-Meier analysis of tumor-specific survival times among 285 NSCLC patients (stage IA-IIIA) according to the presence or absence of n-ANLN (P < 0.0001, log-rank test). C and D, validation of the relationship between n-ANLN expression and survival by subgroup analysis of the 285 patients based on tumor stage and the status of adjuvant treatment. C, Kaplan-Meier analysis of tumor-specific survival period among group-1 patients for node-negative NSCLC cases (stage IA-IB, 195 patients) according to presence or absence of n-ANLN (P = 0.005; log-rank test). D, Kaplan-Meier analysis of tumor-specific survival period among group-2 for node-positive cases (stage IIA-IIIA, 90 patients) according to presence or absence of n-ANLN (P = 0.018; log-rank test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Molecular-targeted drugs are expected to be highly specific to malignant cells, and have minimal adverse effects due to their well-defined mechanisms of action. Following a promising strategy to identify appropriate molecular targets for the development of such drugs, we combined genome-wide expression analysis for selecting genes that were overexpressed in lung cancer cells with high-throughput screening of loss-of-function effects by means of the RNAi technique (5–8, 10, 11). In addition, we used tissue microarrays to analyze hundreds of archived clinical samples for validation of the potential target proteins (9–12). Using this systematic approach, we found ANLN to be frequently overexpressed in clinical NSCLC samples as well as cell lines, and showed that overexpression of this gene product plays an indispensable role in the growth and progression of lung cancer cells.

ANLN was initially characterized as a human homologue of anillin, a Drosophila actin-binding protein (13). The human ANLN cDNA encodes a 1,125–amino acid protein that includes an actin-binding domain and a COOH-terminal domain with pleckstrin homology. It also contains several consensus nuclear localization sequences and one consensus SH3-binding motif. ANLN localizes to the cleavage furrow and is supposed to play an important role there during cytokinesis. In our experiments, ANLN localized not only to the cytoplasm but also to nuclei in some proportion of cancer cells; it was present at the cortex following breakdown of the nuclear envelope, as well as in the cleavage furrow during cytokinesis. The localization seems to be drastically altered in a cell cycle–dependent manner. As reported previously, ANLN is likely to play an important role in cell cycle progression; in late phases, it may assemble the actin and myosin contractile ring that separates daughter cells, through interaction with at least two other furrow proteins, actin and septins (13). We observed that NSCLC cells treated with ANLN siRNA showed furrow regression and larger cell morphology, and became multinucleated, probably due to dysfunction of the cytokinetic process as a consequence of prevention of the assembly of the contractile ring. We also documented the interaction of endogenous RHOA with ANLN, not only in the cytoplasm of NSCLC cells, but also in the cleavage furrow and the midbody, indicating that cell growth was promoted through ANLN-RHOA interaction and acceleration of cytokinesis.

We then focused on the effect of ANLN on the activation of RHO, which is known to control the formation of actin structures (17), and found that overexpression of exogenous ANLN promoted the formation of actin stress fibers in mammalian cells. Those data suggested that ANLN might activate RHO signaling through interaction with RHOA, significantly promoting reorganization of the actin cytoskeleton; that process could be responsible for the activation of cellular migration which we observed in Matrigel invasion and wound migration assays.

RAS and RHO GTPases are well studied signaling molecules; members of the RHO family regulate a diverse set of biological activities including actin organization, focal complex/adhesion assembly, cell motility, cell polarity, gene transcription, and cell cycle progression (31, 32). RHO proteins are overexpressed in several types of human tumor, and some growth factors including epidermal growth factor, hepatocyte growth factor, lysophosphatidic acid, platelet-derived growth factor, and transforming growth factor-ß can activate RHO proteins (33–35). Several classes of cell adhesion molecules including integrins, cadherins, and members of the immunoglobulin superfamily also affect RHO activities (36–38). Moreover, guanine nucleotide-exchange factors might abnormally activate RHO proteins and their downstream effectors, resulting in neoplastic transformation (39, 40). A functional association between ANLN and activation of RHOA remains to be clarified, but our data suggests that aberrant activation of RHOA by overexpressed ANLN promotes the migratory activity of mammalian cells through reorganization of the actin cytoskeleton; this process could contribute to the invasiveness and metastatic potential of cancer cells. It should also be noted that we observed activation of ANLN in nearly half of the series of pancreatic tumors and invasive gastric cancers we examined (data not shown).

We found that nuclear localization and stability of n-ANLN are regulated by PI3K/AKT signaling pathway. The PI3K/AKT signal transduction cascade has been investigated extensively for its roles in oncogenic transformation. Initial studies suggested the involvement of both PI3K and AKT in the suppression of apoptosis. However, more recent evidences have implicated their roles in regulation of cell cycle progression (20, 22). AKT triggers a network that positively regulates G1-S cell cycle progression through inactivation of GSK3-ß, leading to increased cyclin D1, and inhibition of Forkhead family transcription factors and the tumor suppressor tuberin, leading to a reduction of p27Kip1 (21, 23). The identification of ANLN as a component of the PI3K/AKT signaling pathway provides new insights into mechanisms whereby hyperactivation of this pathway might contribute to lung carcinogenesis. It remains to be elucidated whether ANLN could be directly phosphorylated by AKT or by some kinase(s) in the AKT pathway. According to two previous reports, both possibilities are reserved; for example, some growth factor signaling activates nuclear factor {kappa}ß (NF-{kappa}B) through PI3K to the AKT kinase and the I{kappa}B kinase (IKK; PI3K/AKT/IKK-NF-{kappa}B pathway; refs. 41, 42). We also observed through our tissue microarray experiments that lung cancer patients with n-ANLN-positive tumors showed shorter cancer-specific survival times than patients whose tumors were negative for n-ANLN, thus, independently confirming the effect of n-ANLN on the promotion of the malignant nature of lung cancer cells. Survival times in patients with NSCLC could be affected by several factors including tumor stage or adjuvant therapy received. One of the determinants of chemosensitivity is known to be the activation of drug resistance genes in cancer cells. In fact, overexpression of the nucleotide excision repair genes, which are crucial in the repair of cisplatin-DNA adducts, is reported to negatively influence the effectiveness of cisplatin-based therapy (43–45). Importantly, the subgroup analysis of our tissue microarray data based on the tumor stage and the status of adjuvant treatment further confirmed that n-ANLN expression is significantly associated with poor prognosis of patients with early-stage NSCLCs (stage I) who received no adjuvant chemotherapy, as well as that of patients with lymph-node metastasis who were treated with adjuvant chemotherapy after surgery. Although additional studies involving more subcategorized groups based on type of adjuvant treatment might help in assessing the relevance of n-ANLN to drug resistance and its availability to more personalized chemotherapy, the result of this validation clearly indicates that n-ANLN is a significant prognostic factor even in patients with NSCLC at stage I. Prospective studies on assessing molecular markers (7, 9–12), identified by our group in patients treated with several standardized protocols, are in progress in our institute. The precise molecular mechanism of ANLN transport to the nucleus and its direct association with the PI3K/AKT signaling, and whether n-ANLN has an additional, nucleus-specific function are not clear, but our data raise a possibility that n-ANLN could contribute to the highly malignant phenotype of lung cancer cells by activating the novel pathways including a n-ANLN regulated by PI3K/AKT signaling or by stimulating some unidentified signaling pathway(s).

In summary, we have shown that ANLN interacts with and activates RHOA, and that this complex is likely to be essential for the growth-promoting pathway and aggressive features of lung cancers as well as for cell division. Moreover n-ANLN, whose nuclear localization and stability are regulated by PI3K/AKT signaling, seems to regulate the malignant potential of cancer cells. Our data uncovering the signaling network of ANLN spanning from extracellular environment to the nucleus should illuminate biochemical events contributing to malignant transformation of lung cancer cells. The evidence reported here strongly implies that the ANLN-RHOA and/or PI3K/AKT-ANLN pathway(s) could be a good molecular target for designing a novel biomarker and for developing therapeutic drugs for lung cancer. Specific siRNAs should be one of the options to investigate for interfering with this pathway.


   Acknowledgments
 
Grant support: "Research for the Future" Program Grant of the Japan Society for the Promotion of Science (no. 00L01402) to Y. Nakamura.

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.

Received 5/ 2/05. Revised 9/26/05. Accepted 9/29/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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