Polo-like kinase1, a New Target for Antisense Tumor Therapy

Biochemical and Biophysical Research Communications 269, 352–356 (2000) doi:10.1006/bbrc.2000.2291, available online at http://www.idealibrary.com on

Polo-like kinase1, a New Target for Antisense Tumor Therapy Robert Elez,* Albrecht Piiper,* Claudio D. Giannini,* Martin Brendel,† and Stefan Zeuzem* *Department of Medicine II and †Institut fu¨r Mikrobiologie, J. W. Goethe-Universita¨t, Theodor-Stern-Kai 7, Haus 75, 60590 Frankfurt, Germany

Received January 31, 2000

The Polo-like kinase 1 (Plk1) is a highly conserved mitotic serine/threonine kinase which is commonly overexpressed in cancer cell lines. Plk1 positively regulates mitotic progression by activating the CDC25CCDK1 amplification loop and by regulating late mitotic events, primarily the ubiquitin-dependent proteolysis. In the present study, an antisense strategy against Plk1 mRNA was developed to specifically inhibit cell proliferation of cancer cells in cell culture and in the nude-mouse tumor model. Among 41 phosphorothioate antisense oligodeoxynucleotides tested, the 20-mer JWG2000 strongly inhibited expression of Plk1 in cultured A549 cells, leading to loss of cell viability, and exhibited anti-tumor activity in nude mice A549 xenograft. JWG2000 did not inhibit growth and viability of primary human mesangial cells and human amnion fibroblasts. © 2000 Academic Press

The Polo-like kinases (Plk) have been implicated in various aspects of cell-division cycle and M-phase progression in various eukaryotic organisms, especially in organization and function of the mitotic apparatus. Plk1 is a mammalian serine/threonine protein kinase whose expression peaks at the onset of mitosis and that is particularly active during the anaphase and telophase (1–3). It is closely related to other mammalian kinases, e.g., Snk/Plk2 (4), Fnk/Prk/Plk3 (5, 6), as well as to Xenopus laevis Plx1 (7), Drosophila melanogaster polo (8, 9), Schizosaccharomyces pombe Plo1 (10), Saccharomyces cerevisiae Cdc5 (11), and Trypanosoma brucei TbPlk (12). In vertebrates, Plks participate in controlling important biochemical steps, i.e., the activation of Cdc25C phosphatase at G2/M phase in meiotic maturation in Xenopus oocytes (13), in the centrosome maturation and spindle assembly, as well as the activation of the Cdk1/cyclin B complex CDC25-Cdk1 amplification loop (14), in bipolar spindle formation (15), DNA checkpoint adaptation (16), induction of cytokinesis-associated 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

septal structures (17) and regulation of the anaphasepromoting complex and cyclin B degradation (13, 18). Because of the essential role of Plk1 in the Cdc25CCdk1 amplification loop, in organizing bipolar spindle assembly, and in activating the anaphase promotion complex (19), Plk1 could serve as a suitable diagnostic and prognostic marker for tumor progression and as target for anti-cancer therapy. Antisense oligodeoxynucleotides (ODNs) offer potential not only for investigation of gene expression, but also as therapeutic agents by altering the intermediary metabolism of RNA, thus modulating transfer of information from gene to protein. Owing to their increased biological stability and ability to trigger RNase H activity phosphorothioate ODNs represent the bestcharacterized antisense therapeuticals and many of them were shown to be potent and sequence-specific inhibitors of gene expression in vitro and in vivo (20). The present study indicates that JWG2000 is a potent and specific 20-mer phosphorothioate ODN antisense inhibitor of Plk1 expression as its anti-proliferative and antitumor activity in cell culture (A549 and Detroit562) and in mouse models xenografted with A549 cells could be clearly established. Thus, JWG2000 might be a suitable candidate for anti-cancer therapy. MATERIALS AND METHODS Oligonucleotide synthesis. We used 19-mer antisense ODNs with fully modified phosphorothioate backbones, synthesized on an automated synthesizer (Perseptive 8909; BioSpring GmbH, Frankfurt, Germany). For deprotection oligonucleotides were treated with concentrated ammonia for 16 h at room temperature. ODNs were purified by reverse phase chromatography. The lyophilisate was precipitated twice with 1 M NaCl/ethanol and then lyophilized again. Treatment of human cancer cell lines with antisense ODNs. The human carcinoma cell lines A549 and Detroit562 used in cell culture and A549 in nude mouse xenografts were obtained from the American Type Culture Collection (ATCC) and from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, respectively. 2.5 ⫻ 10 5 cells suspended in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS), antibiotics, and 2 mM L-glutamine were seeded in T25 cell culture flasks and grown at 37°C in a 5% CO 2–95% air atmosphere in a humidified incubator. On

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the following day, cells (50 – 60% confluent) were treated with the appropriate ODNs dissolved in sterile and de-ionized water for cell culture in the presence of the monocationic lipid 1,2-dioeoyloxy-3(trimethylammonio) propane, Dotap (Roche Diagnostics) in 20 mM Hepes buffer (pH 7.4 with NaOH) and OptiMem medium (Gibco BRL). After 4 h incubation at 37°C, the ODN-containing medium was replaced with normal cell culture medium containing 10% FCS, and the cells were incubated for further 24 or 48 h. After appropriate times following transfection cell viability was determined by trypan blue staining and cell number was determined using a hemacytometer. Transfected and Dotap-only treated cells showed 95–100% viability 24 h after transfection. RNA preparation, Northern blot analysis, and autoradiography. From excised xenograft tumors, portions of 0.5–1.5 g were homogenized in a guanidinium isothiocyanate solution (21). RNA was prepared by ultracentrifugation at 125,000g through a 5.7 M CsCl cushion. For determination of mRNA levels in transfected cells, total RNA was prepared from cells using the RNeasy mini kit (Qiagen) 24 and 48 h after transfection or at the indicated times for in vivo antisense mRNA analysis. 20 ␮g of total RNA was separated by electrophoresis in a denaturing 1% agarose/formaldehyde gel, transferred to nylon membranes and probed with a radioactive PCRgenerated fragment of Plk1. Primers were generated on an Applied Biosystems 380A DNA synthesizer. Radiolabeling of the probe for Northern blotting was carried out by a standard PCR procedure using 150 ␮Ci of [␣- 32P]dCTP (6000 Ci/mmol, Amersham-Pharmacia Biotech). Radiolabeling of the antisense strands was performed using the following primers: Plk1: 5⬘-GAT GTT GGC ACC CTT TCA GC- 3⬘, G3PDH: 5⬘-ATG GTT CAC ACC ATG A CG-3⬘. Hybridization was carried out in QuickHyb (Stratagene) solution for 1 h at 68°C. The membranes were washed two or three times under high stringency conditions in 0.1⫻ SSC, 1% SDS at 65°C for 60 min and subjected to autoradiography overnight (22). Subsequently, the membranes were stripped and rehybridized with another radioactive probe to control for quality and uniformity of the mRNA loading under identical conditions (for instance G3PDH). RNA was normalized using G3PDH mRNA level. Western blot analysis. For Western blot analysis cellular proteins were extracted using 250 ␮l of RIPA buffer (1⫻ PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) per T25 cell culture flask. 25 ␮g of protein was separated by electrophoresis in 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Plk1 and actin were detected using anti-Plk1 (Transduction Laboratories) and anti-actin antibody (Sigma). Immune complexes were visualized using appropriate horseradish peroxidase-conjugated secondary IgG (Jackson Immuno Research) and the chemiluminescence system (Amersham-Pharmacia Biotech). Measurement of cell proliferation and cytotoxicity. Antiproliferative effects were evaluated daily based on the cell number and viability as assessed by direct counting using the trypan-blue exclusion method. Cell viability was measured daily for 4 days following treatment with JWG2000, sense and random control ODNs. Cells were suspended in 0.1% trypan blue and counted. The mean of three counts was taken at each time point and the percentage of viable cells calculated. ODN treatment in the animal tumor model. Animal studies were conducted with groups of 5 athymic nude mice (nu/nu) NMRI (Harlan Winkelmann), 8 –10 weeks old. 2 ⫻ 10 6 A549 or Detroit562 cells were injected subcutaneously and serially passed by at least 3 consecutive transplantations before the start of the antisense therapy. Tumor fragments of about 20 –25 mg were implanted subcutaneously into the left and right flank of the animals under urethane anesthesia. Antisense ODN treatments were initiated when the tumors had reached an average volume of 60 – 80 mm 3 (7–14 days after the last transplantation). ODNs dissolved in saline solution were administrated intravenously daily as a bolus (200 ␮l) into the tail vein at a dose of 15 mg/kg. Tumor size was measured with a caliper and tumor

volume was calculated by the formula V ⫽ ␲ /6 ⫻ larger diameter ⫻ smaller diameter 2 (23). For determining Plk1 mRNA in tumor xenografts, total RNA was prepared from excised tumors and analyzed by Northern blotting as described above.

RESULTS Antisense phosphorothioate ODNs inhibit Plk1 expression. Since the proliferation-associated gene Plk1 is highly expressed in neoplastic cells (27), we used an antisense strategy to down-modulate Plk1 expression in cultured cells. The uptake of ODNs by cells was controlled by imaging fluorescein-labeled JWG2000. This ODN demonstrated high transfection efficiency (⬎90%) in cultured A549 and Detroit 562 cells. The uptake of ODNs by A549 tumor cell xenografts was investigated by injecting fluorescein-labeled JWG2000 into athymic mice. Because of the secondary and tertiary structure not all sequences of a mRNA are equally accessible. Thus, careful selection and extensive testing of ODNs was necessary. 41 antisense phosphorothioate ODNs (each at a concentration of 1–2 ␮M) were screened for their antiproliferative activity in A549 cells (24). In addition to the Plk1 antisense phosphorothioate ODN JWG2000, the following controls were tested: (a) a random sequence from herpes simplex virus (24) and (b) a sense JWG2000 sequence. JWG2000 was the most potent antisense compound since it reduced Plk1 mRNA level by at least 85% (24), and was therefore studied in more detail. Figure 1A shows the doseresponse relation for the effect of JWG2000 on the Plk1 mRNA in A549 cells. A single application of JWG2000 (2 ␮M) reduced the mRNA of Plk1 in A549 cells to less than 10% of the control level. The IC 50 value for JWG2000 to suppress Plk1 expression was 1 ␮M. A similar response to JWG2000 was found in Detroit562 cells (data not shown). Treatment of A549 cells with 2 ␮M JWG2000 resulted in a significant reduction of Plk1 at the protein level, whereas Plk1 was unchanged in cells subjected to lipofection only or when transfected with 2 ␮M sense JWG2000 (Fig. 2). The same blot was also probed with anti-actin antibody to control for the amount of protein in each lane. Reduction of Plk1 expression has antiproliferative effects in cell culture. To determine antiproliferative effects of JWG2000, A549 and Detroit562 cells were transfected either with JWG2000 (2 ␮M 5⬘-CCT GAC CAG CCC ACG CTC C-3⬘), JWG2000 sense or random control or treated with Dotap only. Treatment with Dotap only or sense and random ODN had no adverse effects on the cells. In comparison with the Dotap-only treated cells, JWG2000 treatment reduced the number of viable cells with a percentage of dead cells of up to 80% (Fig. 3A). Treatment with an equal concentration of a random sequence ODN (from herpes

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FIG. 2. Effect of JWG2000 on Plk1 protein in A549 cells. A549 cells were transfected with the indicated amount of ODN (2 ␮M) as described under Materials and Methods. The proteins were extracted 48 h after transfection and subjected to Western blotting analysis. Blots were probed with anti-Plk1 and anti-actin antibodies.

cant inhibition of both tumor size and mass as compared with the control animals (Fig. 4). DISCUSSION

FIG. 1. Effect of the 20-mer antisense (A) and sense control (B) ODN, JWG2000 (5⬘-CCT GAC CAG CCC ACG CTC C-3⬘) on Plk1 mRNA expression in A549 cells. A549 cells were transfected with the indicated amount of ODN as described under Materials and Methods. The mRNA was extracted 24 h after transfection and subjected to Northern blotting analysis. Blots were hybridized with radioactive Plk1probe and (after stripping) with G3PDH probe.

simplex, data not shown) or with sense JWG2000 ODN were without effect (Fig. 3A). Thus, downmodulation of Plk1 expression with JWG2000 was accompanied with decreased proliferation and viability of cancer cells. In contrast, the growth of primary human mesangial cells and human amnion fibroblasts was not significantly affected by JWG2000 treatment (data not shown), indicating that the growth inhibitory effect of JWG2000 is selective for Plk1-overexpressing cancer cells. JWG2000 is antineoplastic in the nude mouse tumor model. To investigate a possible anti-tumor activity of JWG2000 in vivo A549 cells were injected subcutaneously in nude mice, i.e., were used as a human tumor xenograft mouse model. ODN treatment was initiated after at least three passages of established tumors with a minimal tumor volume of 60 – 80 mm 3. Animals received one daily dose of ODNs (15 mg/kg body weight) over 28 days. Tumor volume was measured weekly and tumor weight after sacrificing the animals. Tumor growth was not significantly influenced during the first two weeks of treatment with JWG2000 in two independent in vivo experiments. Upon continuation of the application of JWG2000, however, there was a signifi-

Our results show that the administration of antisense phosphorothioate ODN JWG2000 against Plk1 mRNA can downregulate expression of Plk1 in cell culture. Targeted to the coding region of human Plk1 mRNA JWG2000 was the most efficient antisense inhibitor amongst 41 different human Plk1 antisense phosphorothioate ODNs tested. JWG2000 exhibited an IC 50 value of 1 ␮M and reduced Plk1 mRNA expression in vitro for up to 38 – 48 h. When applied intravenously in mice at a dose of 15 mg/kg of body weight, JWG2000 reduced Plk1 mRNA level in xenografted tumors (data not shown). Known side effects of phosphorothioate ODN administration are hypotension, thrombocytopenia and increased clotting times. None of these effects have been problematic in animal trials, but they would necessitate prolonged infusion times in patients. A further

FIG. 3. Growth inhibition of A549 cells with JWG2000. The cells were treated once (at time 0) with 2 ␮M JWG2000 or with a sense control ODN of JWG2000 (control ODN). Two further negative controls (no treatment and treatment with lipofection (DOTAP) without the antisense compound were also used. The antiproliferative effect, e.g., the cell number, was determined after 24, 48, and 72 h after transfection.

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FIG. 4. Antineoplastic effect of JWG2000 in A549 human tumor xenograft mouse model. Following establishing of tumors in nude mice, JWG2000 and corresponding control ODN were administered once daily by bolus i.v. injection at a dose of 15 mg/kg. Tumor size was determined over a 4-week period following initiation of ODN treatment as described under Materials and Methods (A). (B) Tumor weight at day 28.

disadvantage is that phosphorothioate ODNs can cause numerous non-antisense effects in cell culture studies because of their potential to bind to proteins such as growth factors and their receptors, or to extracellular matrix proteins (25, 26). They may also have non-specific effects on cellular adhesion. For example, phosphorothioate ODNs tend to bind with relatively high affinity to various non-nucleic acid targets, acting as aptamers or decoys and induce unexpected inhibition of a biological process (25), reducing their therapeutic index. Therefore, there is a pressing need for development of novel analogues with reduced nonspecific and thus non-antisense side effects. The development of this approach still requires optimization of the biophysical and biochemical properties of antisense ODNs such as: (a) prolonged in vivo stability and resistance of antisense ODNs and analogues to both proteases and nucleases (28), (b) alteration of the charge, improved solubility and cellular membrane passage, (c) enhanced affinity of the modified, conjugated or radiolabeled ODNs to hybridize to target mRNA, (d) RNA-binding affinity and stability of the mRNA and DNA hybrid and high T m (melting temperature) values for duplexes formed with singlestranded RNA or DNA (29), and (e) and potential of

triggering intracellular nuclease activity, especially that of RNase H. RNase H is an ubiquitous enzyme that degrades the RNA strand of an RNA-DNA duplex. The results of the present study show that interference of JWG2000 with Plk1 expression at the mRNA level led to loss of cell viability, blockage of tumor cell proliferation, and induction of mitotic death in Plk1overexpressing A549 and Detroit562 cell lines. Thus, antisense ODNs against Plk1 mRNA may provide a novel therapeutical concept to inhibit the production of Plk1 protein via antisense-induced degradation of its mRNA. Plk1 seems an excellent target for antisense anti-tumor therapy since it plays a major role in G2/M transition and, via Cdc25C activation and cyclin B degradation in telophase, also controls the cells’ exit from mitosis. Cancer cells with high Plk1 expression and rapid cell cycling will not immediately show the effects of antisense blocking of Plk1 mRNA, but once Plk1 levels diminish, cells will be unable to orderly arrest at G2/M checkpoint due to lack of checkpoint control and, therefore, will eventually die by aberrant mitotic processes. The efficiency of the anti-proliferative action of antisense ODN JWG2000 may be further strengthened by combining it with other antisense ODNs, each specifically aimed at an important cell cycle progression and proliferation target, one of them being Plk1. ACKNOWLEDGMENTS This work was supported by Grant Ze 237/4-2 of the Deutsche Forschungsgemeinschaft. We are grateful to Drs. Chr. Winter and W. Luderschmidt for support and encouragement.

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