ZM447439, a Novel Promising Aurora Kinase Inhibitor

Original Paper Received: October 21, 2008 Accepted after revision: May 7, 2009 Published online: November 14, 2009

Neuroendocrinology DOI: 10.1159/000258705

ZM447439, a Novel Promising Aurora Kinase Inhibitor, Provokes Antiproliferative and Proapoptotic Effects Alone and in Combination with Bio- and Chemotherapeutic Agents in Gastroenteropancreatic Neuroendocrine Tumor Cell Lines I. Georgieva a D. Koychev b Y. Wang a J. Holstein a W. Hopfenmüller c M. Zeitz a P. Grabowski a, d a

Gastroenterologie/Infektiologie/Rheumatologie, Medizinische Klinik I, b Hämatologie/Onkologie und Transfusionmedizin, Medizinische Klinik III, und c Institut für Biometrie und Klinische Epidemiologie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, und d Abteilung für Gastroenterologie/ Onkologie/Endokrinologie, Zentralklinik Bad Berka, Bad Berka, Germany

Key Words Apoptosis ⴢ Aurora kinase inhibitor ⴢ Cell cycle ⴢ Gastroenteropancreatic neuroendocrine tumors ⴢ Prognostic markers ⴢ Proliferation ⴢ ZM447439

Abstract Background: Therapeutic approaches to gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are still not satisfactory. A new direction in treatment options could be the novel aurora kinase inhibitor ZM447439, which was previously reported to interfere with the mitotic spindle integrity checkpoint and chromosome segregation, but does not interfere with other kinases when used up to 5 ␮M. Methods: We evaluated the antineoplastic effects of ZM447439 on growth and apoptosis of the GEP-NET cell lines BON, QGP-1 and MIP-101, representing the different malignant tumor types, using standard cell biological tests as crystal violet assays, caspase activation, DNA fragmentation and cell cycle analysis. Results: ZM447439 dose-dependently inhibited

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proliferation of all three cell lines with IC50 values in the nanomolar to low micromolar range. Moreover, aurora kinase inhibition by ZM447439 potently induced apoptosis, which was accompanied by DNA fragmentation and caspase 3 and 7 activation. Furthermore, we observed cell cycle arrest at G0/G1 phase as well as a block in G2/M transition. In addition, combined treatment with the chemotherapeutic agents streptozocin and cisplatin augmented significantly the antiproliferative effects of those agents. Conclusion: Aurora kinase inhibition by ZM447439 seems to be a promising new therapeutic approach in GEP-NETs, which should be evaluated in further clinical trials. Copyright © 2009 S. Karger AG, Basel

Introduction

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) represent a relatively rare and heterogeneous tumor entity. More than half of GEP-NETs is functionPatricia Grabowski Charité-Universitätsmedizin Berlin Campus Benjamin Franklin, Hindenburgdamm 30 DE–12200 Berlin (Germany) Tel. +49 30 8445 4579, Fax +49 30 8445 4481, E-Mail patricia.grabowski @ charite.de

ally inactive and usually diagnosed once signs and symptoms of tumor metastases occur [1]. The other half of all metastasized GEP-NETs releases large quantities of bioactive amines and/or neurotransmitters and thereby causes hypersecretion syndromes that are characteristic of these tumors. These clinical symptoms can be well controlled generally by somatostatin analogues or interferon. However, therapeutic options to prevent growth and further dissemination of GEP-NETs are limited. Growth behavior of GEP-NETs differs widely, from very slow/moderately growing to very fast growing, aggressive tumors. The distinction between benign and malignant GEP-NETs is very important and has not yet been completely resolved. Klöppel et al. [2] presented an updated World Health Organization system for the classification of GEP-NETs consisting of three main categories: welldifferentiated endocrine tumors (WDETs), well-differentiated endocrine carcinomas (WDECs) and poorly differentiated endocrine carcinomas (PDECs) with the objective to provide a prognostically relevant classification system that assessed tumors according to size, proliferative activity, angioinvasion, organ invasion, metastases, hormone activity and clinical syndromes. This has some impact on therapeutic considerations: Patients with lowdifferentiated neuroendocrine cancers can profit from cytoreductive chemotherapy, whereas patients with slowly growing, well-differentiated endocrine small intestine tumors do not have a survival advantage by chemotherapy [3]. The therapeutic possibilities for this group of patients are completely unsatisfactory and innovative treatment strategies, which are both effective and well tolerated, have to be developed. The aurora family of serine/threonine kinases plays an important role in chromosome alignment, segregation and cytokinesis during mitosis. The family consists of three members: aurora A, B and C. Aurora A is localized in the pericentrosomal region, where it recruits several important components on the forming mitotic spindle during mitosis [4, 5]. So far, aurora A is the only family member that has been reported to act as an oncogene [4, 6, 7]. Aurora B is a chromosomal passenger protein that localizes at centromeres during prometaphase and subsequently relocates to midzone microtubules and midbodies during anaphase and telophase [4, 8]. It forms a complex with the proteins INCENP and survivin, which was shown to be a powerful novel prognostic marker for GEPNET disease by our group [9]. Aurora B plays a role in chromosome alignment, kinetochore microtubule biorientation, activation of the spindle assembly checkpoint and cytokinesis in association with phosphorylation of 2

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histone H3 [10]. Recent studies described aberrant expression of aurora A and B in a variety of solid tumors, including prostate [11], colon [12], pancreas [13], breast [14] and thyroid cancers [15]. Aurora kinases A and B were reported as an attractive anti-cancer drug target [16–19], and aurora C was localized to the chromosome passenger complex [20]. Usually, after treatment with DNA-damaging agents, cells pause at the G2/M checkpoint of the cell cycle to repair DNA damage before proceeding through mitosis. On the other hand, aurora B levels are increased in cells in G2/M phase – including those treated with DNA-damaging agents [21, 22]. Therefore, we hypothesized that addition of an aurora kinase inhibitor may potentate the anti-proliferative effect of a cytotoxic drug. Previous studies with the novel, highly selective aurora kinase B inhibitor, ZM447439, have reported an accumulation of cells with 4N DNA content in a variety of human cancer cell lines (A549, MCF-7, DLD-1, HeLa and U2OS) [21]. ZM447439 has been proven to be an effective apoptosisinducing agent in acute myeloid leukemia cell lines and Hep2 cancer cell lines [23, 24]. Little is known about the functional role of aurora kinases in GEP-NETs. The aim of this study was therefore to determine the preclinical profile of the aurora kinase B inhibitor, ZM447439, as a candidate for anti-cancer therapy in this special tumor entity.

Materials and Methods Cell Lines and Drugs Human pancreatic BON cells, which were established from a human pancreatic carcinoid tumor, provide a useful model to study the biology of NETs in vitro [25]. BON cells were grown in a 1:1 mixture of DMEM and Ham’s F-12 medium containing 10% FCS (Biochrom, Berlin, Germany) and 1% L-glutamine. The human well-differentiated pancreatic carcinoma cell line, QGP-1 [26], was cultured in RPMI 1640 (PAA, Cölbe, Germany) containing 10% fetal bovine serum, 100 U/ml penicillin and streptomycin and 1% L-glutamine. MIP-101 [27], an undifferentiated colorectal carcinoma cell line, was kindly provided from I. Tai (Vancouver, B.C., Canada). MIP-101 was grown in DMEM containing 10% fetal bovine serum. The same medium was used for culturing LCC-18, Colo205 and LS-174-T colon carcinoma cell lines. The colon carcinoma cell lines NCI-H716, NCI-H747 and Colo320 were cultured in RPMI 1640 (PAA) containing 10% FCS. The human colon adenocarcinoma cell line, HT 29, was used as negative control and was cultured in RPMI 1640 containing 10% fetal bovine serum. All cell lines were kept at 37 ° C in a humidified atmosphere (5% CO2). ZM447439 was purchased from Tocris, Ellisville, Mo., USA. Cisplatin, 5-fluorouracil (5-FU), doxorubicin, streptozocin and octreotide were purchased from Sigma (Steinheim, Germany).

Georgieva /Koychev /Wang /Holstein / Hopfenmüller /Zeitz /Grabowski

SOM230 was kindly provided by Novartis Pharma (Nuremberg, Germany). Stock solutions were prepared in DMSO and stored at –20 ° C. Immediately before use, the drugs were diluted to the final concentration in fresh media. In all experiments, the final DMSO concentration was !0.5%, not affecting cell growth. To evaluate the effects of ZM447439, cells were incubated with either control medium or medium containing rising concentrations of ZM447439. Media were changed every other day to ensure constant drug concentrations in the incubation medium. Flow Cytometry The antibodies were purchased from Sigma if not mentioned otherwise. Cells were trypsinized, washed twice with phosphatebuffered NaCl solution (PBS; 140 mM NaCl, 10 mM Na 2HPO4, 2.6 mM KCl and 1.4 mM KH2PO4, pH 7.4). Cells were then fixed in 4% paraformaldehyde for 30 min at room temperature and washed once with PBS; the supernatant was removed, and 100 ␮l Fix and Perm Medium A (Caltag Laboratories, Vienna, Austria) were added. After a 15-min incubation, the cells were washed twice with 1 ml PBS. The supernatant was removed and the respective first antibody was added at the appropriate concentration: antisynaptophysin (1:1,000), anti-chromogranin A (CgA; 1:100), antisyntaxin (1: 1,000), anti-synaptobrevin (VAMP; 1: 100), SNAP25 (1: 1,000), mouse IgG-negative isotype control (Dako, Glostrup, Denmark, 1: 10) and incubated ⬃60 min at room temperature. Afterwards, the cells were washed twice in 1 ml PBS and 4 ␮g/ml secondary AlexaTM488-labeled goat-anti mouse IgG antibody (Molecular Probes, Eugene, Oreg., USA) for 1 h in a dark place on ice. After the incubation, cells were washed twice with 1 ml of PBS. Finally, 300 ␮l of 1! PBS were added to the cells, and cells were transferred into FACS tubes and fluorescence was detected by flow cytometry on a FACSCalibur (BD, Heidelberg, Germany) and analyzed using CellQuest software (BD). The cell samples already prepared for flow cytometry were also used for immunofluorescence. The stained cells were transferred and pelleted onto coverslips using spin-down tubes after centrifugation in a swinging bucket rotor at 10,000 rpm for 10 min and then examined in a Leica fluorescent microscope. Western Blot Analysis Whole cell extracts were prepared by harvesting and lysing the cells with lysis buffer (0.1 SDS, 0.5% sodium deoxycholic acid, 1% Nonidet P-40, 0.1 mM PMSF, 1 ␮g/ml aprotinin and 1 ␮g/ml pepstatin A). The protein content of the lysate was determined using the BCA protein assay kit (Pierce, Rockford, Ill., USA). The cell lysate was mixed with gel loading buffer (62.5 m M Tris-HCl, 10% glycerol, 1% SDS and 2.5% ␤-mercaptoethanol). After boiling for 10 min, the lysates were subjected to SDS/polyacrylamide gel electrophoresis (40 ␮g protein/lane; gel: 12% polyacrylamide, 0.1% SDS and 25 mM Tris-HCl; running buffer: 25 mM Tris base, 50 mM glycine and 0.1% SDS). Proteins were transferred to PVDF membranes by electroblotting using transfer buffer (25 mM Tris base, 50 mM glycine and 20% methanol). Blots were blocked in 5% BSA and then incubated at 4 ° C overnight with the following antibodies: survivin (1: 500, Biozol, Eching, Germany), AIM-1 (1: 250, BD Biosciences, Franklin Lakes, N.J., USA) and ␤-actin (1: 5,000, Sigma-Aldrich). After washing with PBS containing 0.1% Tween-20 and incubating with horseradish peroxidase-coupled anti-IgG antibody (1:10,000) at room temperature for 1 h, the blot was washed extensively and developed using enhanced che-

ZM447439 in Gastroenteropancreatic Neuroendocrine Tumors

miluminescent detection (Amersham, Uppsala, Sweden). Blots were exposed to Hyperfilm ECL film (Amersham, Uppsala, Sweden) for 1–30 min and analyzed densitometrically using the Image J software (National Institutes of Health, Bethesda, Md., USA). As a loading control, ␤-actin (mouse-anti-human ␤-actin antibody from Sigma, Deisenhofen, Germany) was detected on each blot. Measurement of Growth Inhibition Cell number was evaluated by crystal violet staining, as described [28]. In brief, cells in 96-well plates were fixed with 1% glutaraldehyde. Then cells were stained with 0.1% crystal violet. The unbound dye was removed by washing with water. Bound crystal violet was solubilized with 0.2% Triton X-100. Light extinction which increases linearly with the cell number was analyzed at 570 nm using an ELISA reader. Drug Combination Studies To check for possible additive or synergistic effects of ZM447439 with conventional biotherapeutic and cytotoxic drugs, combination treatment was performed. ZM447439 plus octreotide and SOM 230 in the BON cell line, ZM447439 plus streptozocin or doxorubicin in the QGP-1 cell line and ZM447439 plus cisplatin and 5-FU in the MIP-101 cell line were evaluated. The cytotoxic activities of the combinations were compared to those of each drug alone. For all experiments, cell number was evaluated by crystal violet staining, as described above. Functional Detection of Apoptosis The activity of caspases 3 and 7 was calculated from the cleavage of fluorogenic substrates as described by the manufacturer (Apo-ONE Homogeneous Caspase-3/7 Assay; Promega, Mannheim, Germany). Substrate cleavage was measured fluorometrically with a VersaFluor fluorometer (Bio-Rad, Munich, Germany) with optimal excitation wavelength detection at 499 nm. DNA fragmentation was determined using a DNA laddering kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, after 24 h of incubation, cells were lysed in incubation buffer. The lysed samples were poured into centrifugation tubes containing glass fiber fleece which binds the apoptotic DNA in the presence of guanidine hydrochloride. After centrifugation and additional washing steps, apoptotic DNA was eluted by addition of warm elution buffer and collected by centrifugation. Purified DNA (2 ␮g/sample) was mixed with gel loading buffer and analyzed on agarose gel. Cell Cycle Analysis After harvesting 1 ! 106 single cells in cold PBS, cells were centrifuged twice for 5 min at 1,500 rpm. The pellet was resuspended in 1 ml of ice-cold 70% ethanol in PBS (–20 ° C) and incubated for at least 2 h at –20 ° C. Then the cells were centrifuged and the pellet was resuspended in 1 ml PBS (room temperature) and again pelleted. The pellet was resuspended in 1 ml propidium iodide/Triton X-100 staining solution with RNAse A and incubated 30 min at room temperature in the dark, and then the samples were analyzed flow cytometrically using ModFit software (BD).

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Table 1. Expression of neuroendocrine markers in GEP-NET cell lines

Cell lines

Origin

Synaptophysin

CgA

SNAP25

Synaptobrevin

Syntaxin

HT 29 [35] BON [36] Caco2 [37] Colo205 [38] Colo320 [39] LCC-18 [40] LS-174-T [41] MIP-101 [27] NCI-H716 [42] NCI-H747 [42] QGP-1 [26]

colon carcinoma pancreas NET colon carcinoma colon carcinoma colon carcinoma colon carcinoma colon carcinoma colon carcinoma colon carcinoma colon carcinoma pancreas NEC

– + – – – + – + + – +

– + – – + + – – –/+ – +

– –/+ – – – – – – – – –/+

– –/+ –/+ – – – – –/+ –/+ – –

– + – – – – – + + – +

Cells from 11 GEP-NET cell lines (1 ! 105) were washed, fixed, incubated with 1st and 2nd antibody and mean fluorescence was calculated at FACS against the isotype control. The HT 29 colon cancer cell line was used as a negative control. Here we show representative results of at least three independent experiments made in duplicate.

Results

Fig. 1. Expression of synaptophysin in the cytoplasm of MIP-101 cells (marked with FITC-labeled antibody). The cells labeled with fluorescent antibodies were spun down onto coverslips using spin-down tubes and examined by a Leica fluorescent microscope. Here we show a representative picture of at least three independent stainings.

Statistical Analysis If not stated otherwise, data were expressed as means of at least three independent experiments (8SEM). Significance between controls and treated samples was calculated by Student’s two-sided t test. Caspase 3 measurements were evaluated using the twosided Welch t test. p ! 0.05 was considered statistically significant.

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Characteristics of GEP-NET Cell Lines and Aurora B Expression First, we searched for cancer cell lines possessing neuroendocrine characteristics using flow cytometry. In order to prove neuroendocrine differentiation of the cell lines, we looked for expression of at least two of the following markers: CgA, synaptophysin, syntaxin, SNAP25 and synaptobrevin (table 1; fig. 1). From cell lines meeting these criteria, we chose for all further experiments cell lines which represent the different growth patterns of neuroendocrine tumors. The BON cell line was used as a model for WDETs, whereas QGP-1 cells served as a model for WDECs. So far, there is no established cancer cell line representing PDEC. In a number of experiments, we tried to establish cell lines as models for this special entity (table 1). LCC-18 showed some neuroendocrine characteristics, although it was repetitively negative for syntaxin, SNAP25 and synaptobrevin. Caco2 and NCI-H716 also showed some neuroendocrine characteristics, but they were not sufficient to be included in this study. We report here that the poorly differentiated highly malignant colorectal cancer cell line MIP-101 also expresses neuroendocrine markers. Since the cell lines BON, QGP-1 and MIP-101 represent the different growth patterns of neuroendocrine tumors, they were used for further studies. As shown in figure 2, we detected in all three cell lines selected aurora kinase B expression by Western blot analGeorgieva /Koychev /Wang /Holstein / Hopfenmüller /Zeitz /Grabowski

Fig. 2. Expression of aurora kinase B in NET cell lines. Cell lysates

Aurora B

Proliferation (% of control)

were obtained from BON, QGP-1 and MIP-101 cells and subjected to Western blot analysis. The membranes were sequentially incubated with AIM-1 and actin antibodies. Western blot analysis detected high levels of aurora kinase B expression in GEP-NET cell lines. The WDET BON cell line showed a lower expression of aurora B kinase than QGP-1 (WDEC) and MIP-101 (PDEC).

100

BON

100

80

*

60 40 20

*

80

60

40

40

* MIP-101

Incubation (days)

b

QGP-1

*

0 0

4

* *

20

0 2

MIP-101

80

60

0

QGP-1

100

20

BON 0

a

Actin

2

0

4

Incubation (days)

c

2

0 μM 0.1 μM 0.5 μM 1.0 μM 2.5 μM 5.0 μM

4

Incubation (days)

Fig. 3. Growth inhibition of three different GEP-NET tumor cell lines with ZM447439. Cells from BON (a), MIP-101 (b) and QGP-1 (c) GEP-NETs were incubated on a 96-well plate (1 ! 104) with the novel aurora kinase

inhibitor ZM447439 (0–5 ␮ M). Representative results of at least three independent experiments assessed by crystal violet staining are shown (means 8 SEM of 3 independent experiments). * p ! 0.05 vs. control.

yses, although to a different extent. BON cells expressed significantly less aurora B than the more malignant cell types QGP-1 and MIP-101. Growth-Inhibitory Effects of ZM447439 on Neuroendocrine Gastrointestinal Tumor Cells The effects of ZM447439 inhibition on the growth of GEP-NETs were studied by crystal violet staining. Aurora kinase inhibition with ZM447439 time- and dosedependently inhibited the growth of all three cell lines (fig. 3). After 96 h of incubation with rising concentrations of ZM447439 (0–5 ␮M), a decrease in cell number by 60–99% was observed (MIP-101 –80%; QGP-1 –98%, and BON –50%). The IC50 values of ZM447439 determined after 72 h of continuous exposure to ZM447439 were 3 ␮M (BON), 0.9 ␮M (QGP-1) and 3 ␮M (MIP-101). Combination Treatment of ZM447439 with Conventional Biotherapeutic and Cytotoxic Drugs Furthermore, we investigated the effects of combination treatment of GEP-NET cells with ZM447439 and biotherapeutic/cytotoxic drugs commonly used in clinical ZM447439 in Gastroenteropancreatic Neuroendocrine Tumors

practice: octreotide and SOM230 (in BON cells), streptozocin and doxorubicin (in QGP-1 cells) and cisplatin and 5-FU (in MIP-101 cells). Interestingly, additive, statistically highly significant antiproliferative effects were observed in MIP-101 cells, when sub-IC50 concentrations of ZM447439 were combined with the clinically relevant chemotherapeutic agent cisplatin (83.5 vs. 68.1% for 5 ␮M cisplatin alone; fig. 4b). However, ZM447439 and 5-FU acted in a mutually antagonizing manner, and 5-FU alone had a stronger effect on MIP-101 cell growth than the combination with ZM447439 (fig. 4a). When treating QGP-1 cells with sub-IC50 concentrations of ZM447439 plus doxorubicin, statistically significant additive growth-inhibitory effects were also obtained (fig. 4c). Streptozocin in combination with subIC50 ZM447439 statistically significantly reduced cell growth of QGP-1 by 43.4 versus 12.4% of streptozocin alone (fig. 4d). Applying octreotide alone for 3 days decreased the growth of BON cells by up to 19.1% (2,000 nM). When treating BON cells with a sub-IC50 concentration of ZM447439 plus octreotide (20–2,000 nM), this resulted in additive growth-inhibitory effects of up to 47.7% Neuroendocrinology

5

120 80



100

60

*

40

*

20 0

a

+ – ZM447439 5 (5 μM)

– 10

– 25

+ 5

+ 10

100 80

*

40 20

b

5-FU

*

60

0

+ 25 μM

*

*

+ – ZM447439 5 (0.5 μM)

– 10

– 50

+ 5

+ 10

+ 50 nM

*

*

+ 10

+ 25 μM

Doxorubicin

120


100 Proliferation (% of control)

120 100 80 60 40

*

*

20 0

c

* * + – ZM447439 5 (0.5 μM)

– 10

– 25

+ 5

80 60 40

*

20

*

*

+ 10

+ 25 μM

Cisplatin

0

d

+ – ZM447439 2.5 (0.5 μM)

– 10

– 25

+ 2.5

Streptozocin

Fig. 4. Antiproliferative effects of ZM447439-based combination treatment with established chemo- and biotherapy assessed by crystal violet staining. MIP-101 cells were incubated with increasing concentrations of 5-FU (a) and cisplatin (b); QGP-1 cells were incubated with doxorubicin (c) and streptozocin (d), and BON cells with octreotide (e). Percent inhibition was plotted and the concentration of each compound that induced 25, 50 and 75% growth inhibition (IC25, IC50 and IC75) was calculated (data not shown). Combination treatments of either octreotide, cisplatin or streptozocin with a sub-IC50 concentration of ZM447439 were performed, and cell numbers were determined after 3 days of incubation. Data are given as percentage of untreated controls, which were set at 100% (means 8 SEM of 5 independent experiments). * p ! 0.05 vs. control.

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120 100

* 80

* 60 40 20 0

e

+ – ZM447439 20 (2 μM)

– 200

– 2,000

+ 20

+ 200

+ 2,000 nM

Octreotide


100 Cell proportion (%)

Fig. 5. ZM447439 induced cell cycle arrest in GEP-NET cell lines. BON, QGP-1 and MIP-101 cells (1 ! 106/ml) were plated in 24-well plates and cultured with ZM447439. After 48 h, cells were harvested and cell cycle distribution was measured by flow cytometry. Results represent one of the experiments performed three times. ZM447439 dose-dependently arrested GEP-NET cells in the G0/G1 and G2/M phase of the cell cycle, thereby decreasing the proportion of cells in the S phase. Moreover, especially in BON cells an apoptosis-specific increase in the sub-G1-peak was observed at higher concentrations. * p ! 0.05 vs. control.

*

*

*

60 40

* *

20 0

0

2

5

*

* *

0

QGP-1

Caspase 3/7 activity (% of control)

pendent effects of ZM447439. Caspase 3 activity was evaluated in BON and MIP101 cells after 48 h of continuous exposure to ZM447439. Caspase 3 and 7 activation is shown as percentage of untreated controls (means 8 SEM of 5 independent experiments for each cell line). * p ! 0.05 vs. controls, which were set at 100%.

2

5

0

2

MIP-101

5

**

BON

*

24 h 48 h

*

120

*

80

* 40

0

*

*

*

2.5 μM

compared with the treatment with ZM447439 alone (fig. 4e). Interestingly, ZM447439 and SOM230 had an antagonizing effect on each other, and SOM230 alone had a stronger effect on BON cell growth than the combination with ZM447439 (data not shown). ZM447439 and Cell Cycle Regulation To test whether induction of cell cycle arrest contributed to the antiproliferative potency of ZM447439 in GEP-NET cancer cells, we performed flow-cytometric cell cycle analysis. After 48 h of incubation, ZM447439 dose-dependently arrested GEP-NET cells in the G 0/G1 and G2/M phase of the cell cycle, thereby decreasing the proportion of cells in the S phase (fig. 5). Moreover, especially in BON cells, an apoptosis-specific increase in the sub-G1 peak was observed at higher concentrations. ZM447439 in Gastroenteropancreatic Neuroendocrine Tumors

G2/M S G0/G1

*

160

Fig. 6. Proapoptotic, dose- and time-de-

*

80

5 μM MIP-101

2.5 μM

5 μM BON

Proapoptotic Effects of ZM447439 To determine how ZM447439 affects caspase-dependent apoptosis levels in GEP-NET cells, we evaluated the activation of caspases 3 and 7, key enzymes in the apoptotic signaling cascade. Again, cells were treated with increasing concentrations of ZM447439, and caspase 3 and 7 activities were determined after 24 and 48 h, respectively. In BON cells, ZM447439 (0–5 ␮M) induced a 50% increase in caspase 3 and 7 activity after 24 h of incubation. However, in MIP101 and QGP-1 cells, ZM447439-induced caspase 3 and 7 activity showed a comparable dose dependency but a less marked increase in enzyme activity (fig. 6). To further substantiate the antiapoptotic effect of ZM447439 in GEP-NETs, we determined DNA fragmentation as a biochemical hallmark of apoptosis. The effect was evaluated after 48 h of incubation with ZM447439 Neuroendocrinology

7

41 kDa aurora kinase B

16 kDa survivin 42 kDa ␤-actin –

2 BON

M

A

B

C

– 2 MIP-101

–

0.5 QGP-1

ZM447439 (μM)

D

Fig. 7. ZM447439-induced DNA ragmentation in GEP-NET cell lines. Representative tracings of three independent experiments are shown. A = Negative control; B–D = MIP-101 (B), BON (C) and QGP-1 (D) treated with 5 ␮ M of ZM447439; M = molecular weight marker (100 bp).

Fig. 8. Effects of ZM447439 on the aurora kinase B and survivin expression. BON, MIP-101 and QGP-1 cells were treated with rising concentrations of ZM447439 (0–2 ␮ M) for 48 h. Changes in the expression of aurora kinase B and survivin were analyzed by Western blotting. Representative results of three independent experiments are shown.

and was more pronounced in MIP-101 and QGP-1 than in BON cells (fig. 7).

eloid leukemia, acute lymphoblastic leukemia and chronic myeloid leukemia, aberrantly expressed aurora kinases A and B [30]. Since aurora kinases form a complex with the antiapoptotic protein survivin, which we have shown previously to be of prognostic significance in GEP-NET disease, we hypothesized that an aurora kinase inhibitor would be a feasible approach in these tumors. Here, we provide evidence that ZM447439 may be a promising anti-cancer agent for GEP-NET monotherapy as well as in combination with conventional cytotoxic agents. ZM447439 inhibited GEP-NET cell growth in a timeand dose-dependent manner in all three cell lines, QGP1, MIP-101 and BON, but induced low levels of caspase 3 and 7 activation and DNA fragmentation, the last being consistent with the results from previous studies [31]. Surprisingly, apoptotic effects were also observed in the well-differentiated neuroendocrine cell lines (BON and QGP-1), probably due to pathway signals leading to apoptosis after induction of cell cycle arrest. The antiproliferative effects observed were due to cell cycle arrest in S phase and accumulation of cells with 4N DNA content. Sub-IC50 concentrations of ZM447439 enhanced the antiproliferative effect of cisplatin, doxorubicin and streptozocin. The most pronounced effect was observed in the QGP-1 cell line where the addition of ZM447439 to 2.5 ␮M of streptozocin reduced cell growth almost 90%. With the addition of ZM447439 to doxorubicin, the lack of response to doxorubicin was offset. This is very interesting because QGP-1, a proposed cell model of advanced WDEC, is often resistant to aggressive protocols, including streptozocin and doxorubicin. The somatostatin ana-

Effects of ZM447439 on Aurora Kinase B and Survivin Expression As previously mentioned, the experimental aurora kinase inhibitor ZM447439 produced a dose-dependent inhibition of aurora kinase B function when used at subIC50 concentrations. However, blocking the function of aurora kinase B led to its compensatory overexpression in MIP-101 cells, which was followed by survivin overexpression in this cell line. In BON and QGP-1 cell lines, the aurora kinase B inhibition led to a dose-dependent aurora kinase B protein downregulation followed by survivin downregulation in both cases (fig. 8).

Discussion

Treatment possibilities of GEP-NETs are still not satisfactory and the prognosis of these patients is also not evident with the current diagnostic tools. Thus, novel therapeutic options are needed. Previous studies found that aurora kinases A and B were aberrantly expressed in a variety of solid tumors, including prostate [11], colon [12], pancreas [13], breast [14] and thyroid cancers [15]. In addition, increased levels of aurora kinases correlated with advanced clinical stage in patients with prostate cancer, as well as in patients with head and neck squamous cell carcinoma [11, 29]. Recently, it has been shown that hematological malignant cells, including those from acute my8

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logue octreotide, which has only poor antiproliferative effects as monotherapy, inhibited cell growth in BON cells when combined with ZM447439. Interestingly, SOM230, a new somatostatin analogue which engages five somatostatin receptors, did not exert such effects in the BON cell line (data not shown). As expected, cisplatin had a strong growth-inhibiting effect on the MIP-101 cell line, serving as a model of PDECs in this study, but addition of sub-IC50 concentrations of ZM447439 inhibited cell growth even more effectively. This might be helpful in clinical practice, facilitating the reduction of cisplatin doses and consequently side effects, too. The main side effect of other aurora kinase inhibitors used in clinical trials is a transient myelodepression. This may be avoided by administering lower doses of both drugs. Intriguing and surprising was the antagonizing effect on cell growth in MIP-101 cells of combination treatment with ZM447439 and 5-FU, a drug commonly used in the treatment of colorectal adenocarcinomas and proposed for the treatment of PDECs. A possible explanation may be the fact that 5-FU is a pyrimidine analogue which inhibits thymidylate synthase and thus RNA synthesis, but does not induce effects on DNA molecules, whereas ZM447439 induces a G2/M cell cycle block inhibiting the function of aurora kinases A and B, which participate in DNA repair. The DNA-intercalating agents streptozocin, cisplatin and especially doxorubicin [32, 33] induce G2/ M-phase arrest, a phase during which cells are overexpressing aurora kinases A and B in order to overcome the G2/M block; thus the addition of ZM447439 might account for the profound effect on tumor cell growth. Blocking the function of aurora kinase B using ZM447439 led to its overexpression in MIP-101. In BON and QGP-1 cells, aurora kinase B inhibition led to dosedependent aurora kinase B and survivin downregulation in both cases. Intriguingly, the last two cell lines possess no p53 mutations (p53 mutation database by Thierry Soussi, version 2007). In MIP-101, however, p53 mutations were established in exon 7, codon 241 [34]. Apparently, the mechanism for dynamic regulation of survivin may be the modulation of aurora kinase B, which might be additionally modulated by the p53 status [4], being one possible explanation for the upregulation of aurora kinase B in MIP-101 cells in the absence of survivin upregulation. Another explanation may be a possible ‘on/off’ mechanism of the interaction between survivin and aurora kinase B. However, further studies are needed to confirm these findings. Existing cell models for WDEC include the rat RIM-1 and the mouse STC-1 cell lines, but the results from these

cell lines are not transferable to human GEP-NETs and, moreover, there is no cell model for PDEC. We propose that both MIP-101 and LCC-18 could be used as a model for PDEC, but we preferred MIP-101 for its faster growth and simple growth conditions. QGP-1 can be used as a WDEC model. In conclusion, our study provides the first evidence that the aurora kinase B inhibitor ZM447439 potently inhibits the growth of human gastrointestinal NET cells. Combined with the overexpression of aurora B in solid tumors shown in previous studies and the fact that the compound specifically blocks aurora kinases at nanoand micromolar concentrations, this in vitro model is well suited for combination treatment approaches and our results warrant further studies on aurora kinase inhibitors in the treatment of GEP-NET. The combination of sub-IC50 concentrations of ZM447439 and DNA-interacting cytotoxic agents could be extremely valuable in clinical practice, thus increasing the benefits of therapy while decreasing adverse reactions to either agent.

ZM447439 in Gastroenteropancreatic Neuroendocrine Tumors

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Acknowledgments We thank J. Stehr for excellent technical assistance and Dr. K. Maaser for the critical revision of the paper. Furthermore, we thank Novartis Pharma for providing us SOM 230. This study was supported by grants from the Cancer Society of Berlin (Ernst von Leyden Fellowship to I.G.), the DAAD (German Academic Exchange Service) and the Sonnenfeld foundation (grant to I.G.).

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