Small-Molecule Inhibitor BMS-777607 Induces Breast Cancer Cell Polyploidy with Increased Resistance to Cytotoxic Chemotherapy Agents

Published OnlineFirst March 6, 2013; DOI: 10.1158/1535-7163.MCT-12-1079

Molecular Cancer Cancer Therapeutics Insights Therapeutics

Sharad Sharma1,2, Jun-Ying Zeng1,2, Chun-Mei Zhuang1,2, Yong-Qing Zhou4, Hang-Ping Yao5, Xing Hu6, Ruiwen Zhang1,3, and Ming-Hai Wang1,2

Abstract The RON receptor tyrosine kinase is a therapeutic target for cancer treatment. Here, we report therapeutic effect and phenotypic change of breast cancer cells in response to BMS-777607, a RON tyrosine kinase inhibitor. Treatment of breast cancer cells with BMS-777607 at therapeutic doses inhibited cancerous clonogenic growth but had only minimal effect on cell apoptosis. Significantly, BMS-777607 induced extensive polyploidy with multiple sets of chromosomes in cancer cells. This effect is independent of RON expression. Knockdown of RON in T-47D and ZR-75-1 cells by specific siRNA did not prevent polyploid formation. Immunofluorescent analysis of a-tubulin and g-tubulin expression in polyploid cells revealed that BMS-777607 disrupts bipolar spindle formation and causes multipolar-like microtubule assembly. Also, both metaphase equatorial alignment and chromosomal segregation were absent in polyploid cells. These results suggest that cellular mitosis arrests at prophase/pro-metaphase and fails to undergo cytokinesis. By analyzing kinase-inhibitory profiles, aurora kinase B was identified as the target molecule inhibited by BMS-777607. In BMS-777607–treated cells, aurora kinase B was inhibited followed by protein degradation. Moreover, BMS-777607 inhibited Ser10 phosphorylation of histone H3, a substrate of aurora kinase B. Chemosensitivity analysis indicated the resistance of polyploid cells toward chemotherapeutics. Treatment with doxorubicin, bleomycin, methotrex-

ate, and paclitaxel signiﬁcantly increased cellular IC50 values. These ﬁndings highlight the theory that BMS- 777607 acts as a multikinase inhibitor at therapeutic doses and is capable of inducing polyploidy by inhibiting aurora kinase B. Increased resistance of polyploid cells to cytotoxic chemotherapeutics could have a negative impact on targeted cancer therapy using BMS-777607. Mol Cancer Ther; 12(5); 725–36. 2013 AACR.

Introduction tion, survival, and invasion of extracellular matrixes (7). The RON receptor tyrosine kinase belongs to the MET These activities facilitate malignant progression, which is proto-oncogene family (1), which has been implicated in characterized as epithelial-to-mesenchymal transition epithelial tumorigenesis and malignancy (2–4). Overex- (EMT; ref. 8). Accumulated evidence also indicates that pression of RON, accompanied by generation of various RON signaling is integrated into the cellular signaling constitutive RON variants, occurs in different types of machinery that is essential for cancer cell growth and tumors including colon, breast, and pancreatic cancers (5, survival (9–11). In colon and breast cancer cells, knock- 6). Moreover, activated RON transduces signals that reg- down of RON expression by specific siRNA significantly ulate tumorigenic activities including cell growth, migra- attenuates tumor cell growth and increases apoptotic cell death (10–12). These observations provide the rationale for targeting RON as a potential cancer therapy (13, 14). At Authors' Affiliations: 1Cancer Biology Research Center, Departments of present, inhibition of RON by therapeutic monoclonal 2 3 Biomedical Sciences and Pharmaceutical Sciences, School of Pharma- antibodies (mAb) and small-molecule inhibitors (SMI) cy, Texas Tech University Health Sciences Center, Amarillo, Texas; 4Department of Neurosurgery, 5State Key Laboratory for Diagnosis and has been studied in preclinical models (10, 15–17). Partial Treatment of Infectious Diseases at First Affiliated Hospital, Zhejiang inhibition of tumor growth in animal xenograft models University School of Medicine, Hangzhou, Zhejiang; and 6Department of Life Science, Huaihua University, Huaihua, Hunan, PR China has been observed (10, 15–17). Further inhibition is achieved by RON-targeted mAb or SMI in combination Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). with chemotherapeutic agents (15). In light of these findings, we propose that combinational therapy is the opti- Corresponding Author: Ming-Hai Wang, Department of Biomedical Sciences; School of Pharmacy, Texas Tech University Health Sciences mal strategy for RON-targeted cancer treatment to Center, 1406 S. Coulter Street, Suite 1117, Amarillo, TX 79106. Phone: 806- achieve maximal antitumor activity. 356-4750, ext. 231; Fax: 806-356-4034; E-mail: [email protected] SMIs that specifically block RON are under intensive doi: 10.1158/1535-7163.MCT-12-1079 investigation (17, 18). Because of similarities with other 2013 American Association for Cancer Research. tyrosine kinases such as MET, SMIs specific only to RON

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have not been reported. SMIs including PHA665752 (19), Zt/g4 and rabbit polyclonal IgG antibody R5029 specific Compound-I (17), and BMS-777607 (18) have been found to RON were used as previously described (5, 15, 23, 24). to inhibit RON-mediated tumorigenic activity using in Mouse or rabbit IgG antibodies specific to MET, a-tubulin, vitro and in vivo models, although the major focus is on g-tubulin, and other signaling proteins were from inhibition of MET signaling. BMS-777607 is a highly Cell Signaling. Mouse or rabbit IgG antibodies specific to selective inhibitor of the MET kinase family (18). The human aurora kinase B (AURK-B), histone H3 phospho- primarily targeted tyrosine kinases as determined by in Ser10 residue, and histone H3 were from BD Transduction vitro enzymatic activities are RON (IC50: 1.8 nmol/L), Laboratories; and PHA665752, compound-I (CP-I), BMS- MET (IC50: 3.9 nmol/L), Tyro-3 (IC50: 4.3 nmol/L), and 777607, and R0-3306 were from Selleck Chemicals. Doxo- AxI (1.1 nmol/L; ref. 18). Compared with other SMIs, rubicin, bleomycin, cisplatin, methotrexate, and paclitaxel BMS-777607 is the only one that inhibits RON kinase were from Fisher Scientific. activity with IC50 at 1.8 nmol/L (17–19). However, BMS-777607 at high concentrations also inhibits Immunoprecipitation and Western blot analysis other protein tyrosine kinases such as Mer (IC50: 14.0 These methods were conducted as previously nmol/L), Flt-3 (IC50: 16 nmol/L), Aurora B (IC50:78 described (23, 24). For immunoprecipitation, cellular pro- nmol/L), Lck (IC50: 120 nmol/L), and VEGFR2 (IC50: teins (250 mg per sample) were mixed with 1.5 mg/mL anti- 180 nmol/L; ref. 18). Aberrant expressions of these pro- RON mAb Zt/g4 coupled to protein G Sepharose beads. teins are known to regulate tumorigenic activities (20, 21). Proteins were separated in an 8% of SDS-PAGE under In this sense, BMS-777607 can be considered as a multi- reduced conditions. RON or other signaling proteins were kinase inhibitor when the therapeutic dose is increased for detected by Western blotting using R5029 or correspond- both in vitro and in vivo experiments. Consistent with this ing antibodies and visualized using enhanced chemilu- notion, studies from a tumor xenograft model of gastric minescent reagents. The membranes also were reprobed C cancer have shown that BMS-777607 at 6.25 mg/kg ( max with antibodies to b-actin to ensure equal sample loading. ¼ 4.5 mmol/L) only partially inhibits the growth of tumor cells expressing MET/RON (18). Therapeutic activity is Assays for chromosome spreading and counting further increased when BMS-777607 is administrated at T-47D or ZT-75-1 cells were treated with 5 mmol/L C ¼ levels up to 50 mg/kg/mouse ( max 43.7 mmol/L; BMS-777607 for 72 hours, suspended in hypotonic solu- ref. 18). We reasoned that the therapeutic effect of BMS- tions, and then preserved with fixative solution (3:1 meth- 777607 is achieved through the coordinated inhibition of anol:acetic acid) according to a previously described multi-protein kinases that function inside tumor cells. In method (25). Cells were then dropped onto glass slides other words, the increased therapeutic efficacy of BMS- and stained with Hema-3 staining solution. Chromo- 777607 is accomplished not only by targeting RON/MET somes were observed under an Olympus BK71 micro- but also by inhibiting additional protein tyrosine kinases. scope and photographed with a charge-coupled device The current study focuses on phenotypic changes of (CCD) camera. The number of chromosomes from indi- breast cancer cells in vitro in response to increased ther- vidual cells was manually counted from individual apeutic doses of BMS-777607. The role of RON in breast samples. cancer pathogenesis and carcinogenesis has been studied extensively through genetic, biochemical, and biologic Immunofluorescent detection of cellular proteins models (2, 3, 14, 22). Overexpression and constitutive This method was conducted as previously described activation of RON are implicated in breast cancer malig- (15). Briefly, cells at 1 104 cells per well in a 24-well plate nancy and drug resistance (2, 3, 14). Thus, the objective of were cultured in RPMI-1640 with 10% FBS and treated this study is to determine whether targeted RON inhibi- with increasing amounts of BMS-777607 for various tion by BMS-777607 achieves a therapeutic effect on breast time periods. To detect AURK-B or histone H3, cells were cancer cells. Specifically, we want to determine tumori- fixed with cold acetone and incubated with individual genic phenotypes of breast cancer cells in response to specific antibodies followed by goat anti-mouse IgG cou- increasing concentrations of BMS-777607, which should pled with fluorescein isothiocyanate (FITC). Normal inhibit not only RON/MET but also other protein tyrosine mouse IgG was used as the control. Cellular immunoflu- kinases. Consequently, knowledge of BMS77607-mediat- orescence was observed under the Olympus BK71 ed inhibition of multi-tyrosine kinases should help us not microscope equipped with DUS/fluorescent apparatus only to improve future therapeutic efficacy of the targeted as previously described (15). RON/MET therapy but also to uncover survival or resistant mechanisms of breast cancer cells in response to Methods for silencing RON mRNA expression multi-kinase inhibition. Synthetic siRNA specific to human RON were acquired from Dharmacon. T-47D or ZR-75-1 cells were cultured Materials and Methods overnight and then transfected with 1 nmol/L of scram- Cell lines and reagents bled or RON-specific siRNA according to the manufac- Breast cancer T-47D, ZR-75-1, and MCF-7 cells were turer’s instructions (Dharmacon). After incubation for 24 from American Type Culture Collection. Mouse mAb or 48 hours, cells were washed and then processed for

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BMS-777607 Induces Breast Cancer Cell Polyploidy

Western blot analysis or treated with BMS-777607 for express RON or MET. BMS-777607 also caused apoptotic induction of polyploidy. death in T-47D and ZR-75-1 cells, although this effect was relatively weak. Assays for cell growth, viability, apoptotic death, and DNA content Induction of polyploidy by BMS-777607 in breast Cell growth and survival was determined by the clono- cancer cells genic assay. T-47D or ZR-75-1 cells in the presence or An interesting finding after BMS-777607 treatment was absence of BMS777607 were cultured for 10 days to allow the appearance of polyploid cells (Fig. 2A and B). Polyploi- clonogenic growth as previously described (26). The effect dy results from subsequent cycles of DNA replication in the of BMS-777607 or other chemotherapeutic agents on cell absence of cytokinesis and is associated with mitotic check- viability was determined by the MTS assay as previously point defects (29). The minimal amount of BMS-777607 described (27). The effect of BMS-777607 on apoptotic requiredto induce polyploidy is at 1 mmol/Lfor bothT-47D death of T-47D or ZR-75-1 cells was measured using the and ZR-75-1 cells (Fig. 2A). At this concentration, 14% of T- Annexin V/propidium iodide labeling method as previ- 47D and 11% of ZR-75-1 cells underwent polyploidy. The ously described (28). For measuring cellular DNA con- percentages were significantly higher when BMS-777607 tents, cells were treated with BMS-777607 for 3 days, was used at 5.0 mmol/L(78%forT-47Dand82%forZR-75- labeled with propidium iodide, and analyzed by flow 1) to 10.0 mmol/L (86% for T-47D and 88% ZR-75-1). The cytometer as previously described (25). effect of BMS-777607 on polyploidy of MCF-7 cells was relatively weak. Only 36% to 59% of MCF-7 cells were Statistical analysis affected when BMS-777607 was used at 5.0 to 10.0 mmol/L, Graphpad Prism 5 software was used for statistical respectively. Time-dependent polyploidy is shown analysis. Results are shown as mean SD. The data in Fig. 2B. Polyploidy was seen as early as 24 hours after between control and experimental groups were compared 5.0 mmol/L BMS-777607 treatment. At 72 hours, more than using Student t test. Statistical differences at P < 0.05 were 70% of cells underwent polyploidy in T-47D and ZR-75-1 considered significant. cells, respectively.Again,the effectof BMS-777607 onMCF- 7 cells was relatively weak. Polyploid cells were observed Results only 48 hours after BMS-777607 administration and pro- Inhibitory effect of BMS-777607 on breast cancer longed treatment only slightly increased the rate of poly- cells ploid cells. Thus, BMS-777607 can induce polyploidy in T-47D, ZR-751, and MCF-7 were selected as the model breast cancer cells in both dose- and time-dependent man- cell lines due to their variable levels of MET and RON ners and the polyploid cells most likely undergo into a expression as determined by Western blot analysis (Sup- premature senescence phenotype. plementary Fig. S1). We used the clonogenic assay to To determine whether RON expression is required for determine the effect of BMS-777607 on survival and pro- BMS-777607–induced polyploidy, we compared the effect liferation of breast cancer cells. BMS-777607 inhibited of compared BMS-777607 with CP-I and PHA665752 on clonogenic growth of T-47D and ZR-75-1 cells in a polyploidy induction. PHA665752 and CP-I both inhibit dose-dependent manner (Fig. 1A and B). In MCF-7 cells MET/RON with enzymatic IC50 values at 9/68 and 4/9 that barely express RON and MET, BMS-777607 at 5 nmol/L, respectively (17, 19). After treatment, polyploidy mmol/L only slightly affected clonogenic growth was observed only in BMS-777607-treated cells (Supple- (Fig. 1A and B). The apoptotic effect of BMS-777607 on mentary Fig. S3). Both PHA665752 and CP-I had no effect T-47D and ZR-75-1 cells was detected by the Annexin V/ on induction of polyploidy. We further used specific propidium iodide labeling method (28). BMS-777607 at 1.0 siRNA to knockdown RON expression in T-47D and to 2.5 mmol/L had no cytotoxic effect on 3 of the cell lines ZR-75-1 cells (Fig. 2C). Knockdown of RON expression tested (data not shown). However, a slight increase in the had no preventive effect on T-47D cell polyploid forma- rate of apoptotic cell death in T-47D (10.3%) and ZR-75-1 tion after BMS-777607 treatment (Fig. 2D). The rates of cells (9.7%) compared with control MCF-7 cells (2.2%) was polyploidy T-47D cells (79.4%) were comparable with observed when 5 mmol/L BMS-777607 was used (Fig. 1C). those from control (83.5%) or scramble RNA–treated We further conducted time-lapse photography to observe (81.1%) cells. Similar results were observed when ZR- the effect of BMS-777607 on breast cancer cells. Represen- 75-1 cells were used (data not shown). These results tative images from T-47D cells treated with 5 mmol/L indicate that RON expression is not required for BMS- BMS-777607 are shown in Supplementary Fig. S2. 777607–induced breast cancer cell polyploidy. Untreated cells undergoing proliferation served as the To study the fate of polyploid cells, polyploid T-47D control. After BMS-777607 treatment, cell growth inhibi- cells induced by 5 mmol/L BMS-777607 for 72 hours were tion and apoptotic death were observed in a time-depen- collected, cultured in RPMI-1640 with 5%FBS, and dent manner. These results, together with those in Fig. 1, observed for up to 10 days (Fig. 2E). These cells retained show that BMS-777607 treatment ranging from 1 to 5 their polyploid appearance and phenotype without BMS- mmol/L has an inhibitory effect on survival and prolifer- 777607. Moreover, polyploid cells did not undergo obvi- ation of breast cancer T-47D and ZR-75-1 cells, which ous proliferation or death during this period. Similar

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A MCF-7 ZR-75-1 T-47D Control o/ 5.0 μ mol/L 2.5 μ mol/L 1.0 μ mol/L

Figure 1. Effect of BMS-777607 on growth and survival of breast cancer cells. A, the effect of BMS-777607 on survival and proliferation of MCF-7, ZR-75-1, and T-47D cells was determined by clonogenic assay. Brieﬂy, cells (8,000 cells per well) in RPMI-1640 with 5% FBS were cultured in duplicate in a 24-well plate and then treated with different amounts B of BMS-777607 for 10 days. MCF-7 ZR-75-1 T-47D Clonogenic cells were stained with Hema-3 staining solution (Fisher Scientiﬁc) and photographed 100 100 100 using an Olympus BK71 microscope equipped with CCD 80 80 80 camera. B, numbers of clonogenic cells in duplicate from 3 cell lines 60 60 60 were counted. C, apoptotic cell death was measured by the 40 40 40 Annexin V/propidium iodide (% of control)

Number of colony labeling method (28). Cells were 20 20 20 treated with 5 mmol/L BMS- 777607 for 3 days, labeled, and 0 0 0 then analyzed by ﬂow cytometer as 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 previously described (28). Results Concentrations of BMS-777607 (μmol/L) shown here are from 1 of 2 C experiments with similar results. MCF-7 T-47D ZR-75-1 Control BMS Control BMS Control BMS

results were also obtained with polyploid ZR-75-1 cells. Results in Fig. 3A show the DNA contents at different time These results indicate that polyploid cells induced by intervals in MCF-7, T-47D, and ZR-751 cells treated with 5 BMS-777607 are alive and their polyploid phenotypes are mmol/L BMS-777607. The percentages of polyploid cells irreversible during the 10-day period of culture. Thus, with 8N and 16N chromosome contents are shown in BMS-777607–induced polyploidy has a long-lasting effect Supplementary Table S1. Treatment of T-47D and ZR-75-1 on breast cancer cells. cells with BMS-777607 for 24 hours produced a population dominated by cells with 8N chromosome contents (26.0% Chromosome features of BMS-777607–induced for T-47D cells and 14.0% for ZR-75-1 cells). A small polyploidy in breast cancer cells population of cells with 16N chromosome contents also We first used the flow cytometric method to analyze emerged in both cell lines. After treatment for 48 hours, DNA contents in BMS-777607-treated breast cancer cells. the population featured by 16N was significantly

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BMS-777607 Induces Breast Cancer Cell Polyploidy

AC Control 0.5 1.0 2.5 5.0 10.0 μmol/L, BMS T-47D ZR-75-1

MCF-7

kDa Control Scr RNA RON-siRNA Control Scr RNA RON-siRNA T-47D 200 pro-RON β 105 RON -chain ZR-75-1 D Control B BMS Scr RNA 0 816244872h

MCF-7

BMS + Scr RNA siRNA BMS + siRNA T-47D

ZR-75-1

E 0 2 4 6 8 10 Days from T-47D Polyploid cells Polyploid

Figure 2. Induction by BMS-777607 of polyploidy in breast cancer cells. A, BMS-777607 induces polyploidy in breast cancer cells in a dose-dependent manner. Cells (5,000 cells per well) were incubated at 37C in RPMI-1640 with 5% FBS and treated with different amounts of BMS-777607 for 72 hours. Polyploid cells were photographed as described in Fig. 1A. B, time-dependent induction of polyploidy by BMS-777607. Cells were treated with5 mmol/L BMS-777607 for various time intervals. Stained cells were photographed. C, knockdown of RON expression in breast cancer cells by specific siRNA. Cells (2 106 cells in a 60-mm diameter culture dish) were transiently transfected with 1 nmol/L scramble or RON-specific siRNA for 48 hours. Cell lysates (50 mg per sample) were subjected to Western blot analysis using rabbit anti-RON IgG antibody. D, effect of RON expression on BMS-777607– induced polyploidy. T-47D cells were transiently transfected with scramble or RON-specific siRNA for 48 hours as described in C and then treated with 5 mmol/L BMS-777607 for 72 hours. Polyploidy was monitored and photographed. E, fate of polyploid cells in culture. Polyploid T-47D cells were isolated, washed, and then cultured in BMS-777607–free Dulbecco's Modified Eagle Media with 10% FBS for 10 days. Cells were observed and photographed at various times. Results shown here are from 1 of 2 experiments with similar results. increased in T-47D (from 10.0% to 18.5%) and ZR-75-1 results in Fig. 3 show that BMS-777607 at a relatively high cells (from 5.0% to 32.2%). The percentages of cells with concentration induces a dominant population of breast 8N chromosome contents were relatively stable from 48 to cancer polyploid cells with 8N or 16N chromosome 72 hours (T-47D: from 23.0% to 21.0%; ZR-75-1: from contents. 20.0% to 20.0%). The response of MCF-7 cells to BMS- 777607 was different. Treatment of cells for 24 hours only Disruptive effect of BMS-777607 on cellular mitotic caused a small fraction of polyploid cells with 8N chro- spindle assembly and function mosome contents (14.0%). However, the percentage of Results from above studies indicate a delayed progres- cells with 8N chromosome contents increased to 25.5% sion of cellular mitosis in the presence of BMS-777607, after incubation for 48 hours. This population of cells which strongly suggests that microtubule dynamics are increased slightly (29%) 72 hours after stimulation. Inter- affected. We therefore conducted immunofluorescent estingly, BMS-777607 failed to induce a cell population staining of T-47D cells to find any abnormalities of a-tubu- with 16N chromosome contents in MCF-7 cells. Currently, lin and g-tubulin in spindle assembly (Fig. 4A). The we do not know the different responses of MCF-7 and T- untreated control cells exhibited well-formed mitotic 47D/ZR-75-1 cells to BMS-777607. Chromosome count in spindles with chromosomes aligned at the equatorial metaphase spreads confirmed the presence and percen- plate. Both a-tubulin and g-tubulin were shown in a tages of T-47D or ZR-75-1 cells with 8N and 16N chro- bipolar spindle pattern associated with condensed DNA. mosome numbers (Fig. 3B). However, MCF-7 cells with However, in T-47D cells treated with BMS-777607, the 16N chromosome numbers were not observed. Thus, mitoses were severely disorganized, which is marked by

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A Control 24 h 48 h 42 h 5 μmol/L, BMS

2 N 4 N 8 N 4 N 8 N 2 N 4 N 8 N

4 N MCF-7

Figure 3. Effect of BMS-777607 on breast cancer cell cycle and 2 N 8 N 16 N 8 N 16 N 4 N 8 N 16 N chromosome contents. A, analysis T-47D of DNA/chromosome contents 4 N was conducted as previously described (25). Cells (2 106 cells per sample) in RPMI-1640 with 5% FBS were treated with 5 mmol/L 2 N 8 N 16 N 8 N 16 N 4 N 8 N 16 N BMS-777607. After incubation for 24, 48, and 72 hours, cells were 4 N ZR-75-1 collected, ﬁxed, and then stained with propidium iodide. DNA/ chromosome contents in individual samples were analyzed B by FACScan ﬂow cytometer. B, Control, 2N BMS, 2N BMS, 4N BMS, 6N chromosome spreading was conducted by treatment of T-47D cells with 5 mmol/L BMS-777607 for 72 hours. After treatment, cells were collected for chromosome spreading analysis according to a previously described method (25). The number of chromosomes was BMS, 8N BMS, 12N BMS, 16N counted and photographed.

an absence of functional bipolar spindle assembly, highly then allowed to enter cell cycle by removing the inhibitor. condensed/irregular-shaped chromosomes, and a com- The assembly of mitotic spindles was monitored by plete lack of both metaphase equatorial alignment and immunoﬂuorescent staining of a-tubulin and g-tubulin. chromosomal segregation. This effect was observed as As shown in Fig. 4B, T-47D cells entered into cell cycle early as 24 hours after BMS-777607 treatment. Moreover, rapidly after removal of R0-3306. Within 60 minutes after we observed the formation of multipolar spindles, which removal of the inhibitor, the T-47D cells began to form were maintained up to 72 hours after BMS-777607 treat- bipolar mitotic spindles and division was completed after ment. These effects caused cell-cycle arrest in a pro-meta- 120 minutes. However, the removal of BMS-777607 failed phase–like state with dysfunctional mitotic spindle. to relieve and to facilitate T-47D cells entering into telo- Under such conditions, cells underwent polyploidy and phase and cytokinesis (Fig. 4C). These cells were arrested failed to advance into endoreduplication cycle. Similar at metaphase with disorganized multipolar mitotic spin- results also were observed in ZR-75-1 and MCF-7 cells dles. Observation for up to 3 days revealed the persistent (data not shown). Thus, BMS-777607 treatment has a presence of multipolar mitotic spindles in these polyploid disruptive effect on microtubule dynamics and impairs cells (data not shown). These results are consistent with the functional mitotic spindle assembly. those described in Fig. 2E, showing that BMS-777607– Next, we determined whether removal of BMS-777607 treated cells maintained the polyploid phenotype for up to relieves and facilitates cell cycle progress. T-47D cells 10 days. Thus, BMS-777607 has a disruptive effect on the were treated with 5 mmol/L BMS-777607 for 24 hours to assembly of mitotic spindles, displays a long-lasting dis- induce a pro-metaphase arrest. Cells were then extensive- ruptive effect on cell cycles, and causes cells to arrest at the ly washed and allowed to enter mitosis in the absence of metaphase-like state. BMS-777607. Control T-47D cells were synchronized at the G2–M border by pre-incubation for 24 hours with 5 Inhibitory effects of BMS-777607 on aurora kinase B mmol/L R0-3306, a Cdk1 inhibitor (29). Treatment of cells and histone H3 phosphorylation with R0-3306 for up to 24 hours caused a fully reversible Speciﬁc inhibitors of tubulins only cause prolonged G2–M cell-cycle arrest (29). R0-3306–treated cells were metaphase arrest and rarely induce polyploidy (29). The

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A DAPI α-Tubulin DAPI + γ-Tubulin DAPI + DAPI + α-tubulin γ-tubulin α- & γ-tubulins

Control

BMS: 24 h

BMS: 48 h

BMS: 72 h

B C T-47D treated with R0-3306 T-47D treated with BMS DAPI α-Tubulin γ-Tubulin Merged DAPI α-Tubulin γ-Tubulin Merged

Figure 4. Disruptive effect of BMS-777607 on mitotic spindle assembly in breast cancer cells. A, effect of BMS-777607 on a-tubulin and g-tubulin expression and localization. T-47D cells (1 106 cells per dish) in RPMI-1640 with 5% FBS were treated with or without 5 mmol/L BMS-777607 for 24, 48, and 72 hours. Cells were then fixed with cold acetone. Specific mouse IgG antibodies were used to detect a-tubulin or g-tubulin, respectively, followed by FITC-coupled anti-mouse IgG. Cells also were stained for 40,6-diamidino-2-phenylindole (DAPI) to detect DNAs. Analysis of immunofluorescence was conducted using an Olympus BK71 inverted microscope equipped with DSU/fluorescence apparatus and CCD camera. Image was taken at magnification of 200. B and C, T-47D cells were treated 37C with 5 mmol/L of R0-3306 (B) or BMS-777607 (C) for 24 hours, extensively washed, and then cultured in Dulbecco's Modified Eagle Media with 10% FBS for additional 30, 60, and 120 minutes. Expression of a-tubulin and g-tubulin was monitored by immunofluorescent analysis as described above. Image was made at magnification of 400. Results shown here are from 1 of 2 experiments with similar results. induction of prominent polyploidy in breast cancer cells and 72 hours after BMS-777607 treatment. However, the indicates that BMS-777607 inhibits additional signaling amount of AURK-B in BMS-777607–treated cells proteins essential for polyploid formation. To this end, detected by immunofluorescent analysis was progres- we conducted immunofluorescent staining to determine sively reduced in a time-dependent manner. Another AURK-B expression and localization in BMS-777607– interesting finding was that AURK-B was not detected treated T-47D cells according to the kinase profiling of in prominent polyploid cells. Analysis of more than 100 BMS-777607 (ref. 18; Fig. 5A). We also studied the Ser10 polyploid cells without condensed DNA found no pos- phosphorylation of histone H3 (Fig. 5B), which is itive staining of AURK-B. Consistent with these obser- known to be a substrate of AURK-B (30, 31). In control vations, results from Western blot analysis (Fig. 5C) T-47D cells, AURK-B was localized with chromosomes show that BMS-777607 treatment results in reduced in the prophase and metaphase stages and also was AURK-B expression in a time-dependent manner in observed in cytokinesis between the 2 dividing cells T-47D and ZR-75-1 cells. However, this effect was not (Fig.5A,top).Incontrast,AURK-Bexpressionand observed in MCF-7 cells. Ser10 phosphorylation of his- localization in BMS-777607–treated cells were disorga- tone H3 was detected by immunofluorescence staining nized, showing a multipolar-like expression pattern. in mitotic condensed chromosomes in control T-47D AURK-B was associated only with condensed DNA in cells (Fig. 5B). At early stages of BMS-777607 treatment suchexpressionpattern,whichwasdetectedasearlyas (24 hours), we detected Ser10 phosphorylation in his- 24 hours following treatment with BMS-777607 (Fig. tone H3, which was associated with condensed DNAs 5A). The disorganized AURK-B also was observed 48 but not with chromosomes in polyploid cells. However,

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AB DAPI Aurora B Merge DAPI p-H-H3 Merge

Control

BMS: 24 h

BMS: 48 h

BMS: 72 h

CD MCF-7 ZR-75-1 T-47D BMS-777607 Lactacystin 0 24 48 72 0 24 48 72 0 24 48 72 (h): 5 μmol/L BMS Aurora-B Aurora B kinase

T-47D Actin p-Histone H3 Aurora B Histone H3 Actin

Actin ZR-75-1

Figure 5. Regulatory effect of BMS-777607 on aurora kinase B expression and histone H3 phosphorylation in breast cancer T-47D cells. A and B, AURK-B expression and histone H3 Ser10 phosphorylation in polyploid cells were analyzed by immunofluorescence using specific antibodies. T-47D cells (5,000 cells per culture chamber) were treated at 37C with 5 mmol/L BMS-777607. After treatment for 24, 48, and 72 hours, cells were fixed with cold acetone and used to detect AURK-B expression and histone H3 Ser10 phosphorylation using specific antibodies to AURK-B and phospho-Ser10 of histone H3, respectively. Cells stained with 40,6-diamidino-2-phenylindole (DAPI) for DNAs were used as the control. Immunofluorescence was observed using the Olympus BK71 microscope equipped with DSU/fluorescence apparatus. C, Western blot analysis of AURK-B and histone H3 was carried out by treatment of cells with 5 mmol/L BMS-777607 followed by use of specific antibodies to detect AURK-B, histone H3 phospho-Ser10, and histone H3, respectively. The membrane was also reprobed with anti-b-actin antibody as the loading control. D, preventive effect of lactacystin on BMS-777607–induced reduction of AURK-B. T-47D and ZR-75-1 cells were treated for 72 hours with 5 mmol/L BMS-777607 in the presence or absence of 5 mmol/L of lactacystin. Expression of AURK-B was measured by Western blot analysis using AURK-B- specific mouse IgG antibody. Actin was used as the loading control. Results shown here are from 1 of 2 experiments with similar results.

after incubation of cells for 48 to 72 hours, Ser10 phos- Increased resistance of BMS-777607–induced phorylation was progressively reduced in BMS-777607– polyploid cells in response to cytotoxic treated cells (Fig. 5B). This observation was further chemotherapeutics confirmed by Western blot analysis showing that Ser10 To determine sensitivity of polyploid cells to cytotoxic phosphorylation in histone H3 was progressively chemotherapeutics, polyploid cells were collected from diminished in a time-dependent manner (Fig. 5C). Nev- BMS-777607–treated T-47D and ZR-75-1 cells and then ertheless, the reduction was not associated with histone treated for 72 hours with various amounts of chemoagents H3 protein expression. We used proteoasome inhibitor including doxorubicin, bleomycin, cisplatin, paclitaxel, lactacystin to determine whether BMS-777607–induced and methotrexate. Analysis of cell viability indicated that reduction of AURK-B is mediated by intracellular pro- polyploid T-47D and ZR-75-1 cells developed a reduced tein degradation. As shown in Fig. 5D, lactacystin effec- sensitivity in responsetocytotoxicchemotherapeuticscom- tively blocked BMS-777607–induced reduction of pared with those of control cells (Fig. 6). The IC50 values of AURK-B in both T-47D and ZR-75-1 cells, which indi- polyploid T-47D cells and ZR-75-1 cells significantly cates that the decrease of AURK-B after BMS-777607 increased in response to doxorubicin, bleomycin, paclitax- treatment is caused by proteoasome-mediated protein el, and methotrexate when compared with those of control degradation. Taken together, results in Fig. 5 show that T-47D or Z-75-1 cells (Supplementary Table S2). The only BMS-777607 inhibits AURK-B function and induces its chemotherapy agent to which polyploid cells did not show protein degradation. BMS-777607 also suppressed a significant increase in IC50 values is cisplatin. Thus, BMS- AURK-B–mediated Ser10 phosphorylation of histone 777607–induced polyploid cells developed cellular resis- H3. tance to chemoagent-induced cellular cytotoxicity.

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125 T-47D N 125 125 125 125 T-47D P 100 100 100 100 100

75 75 75 75 75

50 50 50 50 50 % Cell survival

Cell viability (% control) 25 Cell viability (% control) 25 Cell viability (% control) 25 Cell viability (% control) 25 25

0 0 0 0 0

0 0 0 1 2 4 8 0 50 10 50 16 10 50 0 1 5 100 200 500 100 200 500 100 200 500 0.5 2.5 10 25 50 1,000 2,000 1,000 2,000 1,000 Doxorubicin conc (nmol/L) Bleomycin conc (nmol/L) Cisplatin conc (nmol/L) MTX conc (nmol/L) Paclitaxel (nmol/L)

125 125 125 125 125 ZR-75-1 N ZR-75-1 P 100 100 100 100 100

75 75 75 75 75

50 50 50 50 50 % Cell survival

Cell viability (% control) 25 Cell viability (% control) 25 Cell viability (% control) 25 Cell viability (% control) 25 25

0 0 0 0 0 0 0 0 0 1 5 0 1 2 4 8 10 50 50 10 50 16 10 25 50 100 200 500 100 200 500 100 200 500 0.5 2.5 1,000 2,000 1,000 2,000 1,000 Doxorubicin conc (nmol/L) Bleomycin conc (nmol/L) Cisplatin conc (nmol/L) MTX conc (nmol/L) Paclitaxel (nmol/L)

Figure 6. Cytotoxic effects of different chemotherapy agents on BMS-777607–induced breast cancer polyploid cells. T-47D and ZR-75-1 cells (3 106 cells in 100-mm diameter culture dishes) were treated with 5 mmol/L BMS-777607 for 72 hours. Polyploid cells were collected, washed, and cultured at 5,000 cells per well in a 96-well culture plate. Regular T-47D and ZR-75-1 cells (5,000 cells/well) without BMS-777607 treatment were used as the control. Cells were treated for 72 hours with different amounts of doxorubicin, bleomycin, cisplatin, paclitaxel, or methotrexate in triplicate followed by the MTS assay to determine cell viability (27). The IC50 values from individual drugs were calculated as previously described (27). The calculated IC50 values of bleomycin for control T-47D and ZR-75-1 cells were 5,783 and 6,134 nmol/L, respectively. Results shown here are from 1 of 3 experiments with similar results.

Discussion tics. Thus, BMS-777607–directed growth inhibition of The major finding in this study is the induction of breast cancer cells is associated with generation of poly- polyploidy by BMS-777607 in breast cancer cells, which ploid cells that are resistant to certain chemoagents. Such results in increased resistance to cytotoxic chemothera- activities could have an impact on therapeutic efficacy of peutics. Preclinical studies have shown that RON is over- BMS-777607 against tumors in vivo. expressed and activated in breast cancer cells, which Targeted inhibition of RON for potential cancer treat- facilitates malignant tumor progression (13). Here, we ment has achieved significant success in preclinical stud- determined the therapeutic effect of BMS-777607 on ies (15–18). We and others have shown that therapeutic RON-mediated signaling in breast cancer cells. BMS- antibodies specific to RON, administered repeatedly as a 777607 inhibited growth and survival of T-47D and ZT- single agent, inhibited tumor growth mediated by colon, 75-1 cells in a dose-dependent manner. It also caused breast, and pancreatic cancer cells in mouse tumor xeno- apoptosis, although the effect was relatively weak. These graft models (15, 16). Similar results were also observed in activities contributes directly to BMS-777607–mediated studies using specific SMIs (10, 17). BMS-777607 is a inhibition of RON/MET signaling, as BMS-777607 had tyrosine kinase inhibitor targeting RON with the enzy- marginal effect on growth, survival, and apoptosis of matic IC50 of 1.8 nmol/L (18). Studies using BMS-777607 MCF-7 cells that barely express RON or MET. To our to target MET has shown the effectiveness in MET-medi- surprise, BMS-777607 at therapeutic doses caused exten- ated tumor cell migration, matrix invasion, and distance sive polyploidy in breast cancer cells. This effect was not metastasis (32, 33). However, the effect of BMS-777607 on related to RON expression because knockdown of RON RON-mediated tumorigenesis is unknown. We found expression by specific siRNA did not prevent polyploidy that BMS-777607 has a growth-inhibitory effect on breast formation. In contrast, this effect was directly related to cancer cells expressing RON. However, the ability of BMS-777607–mediated inhibition of AURK-B, a vital reg- BMS-777607 to induce apoptosis is relatively weak. Thus, ulator for mitosis (29). Furthermore, we showed that BMS-777607 has the ability to target breast cells expressing polyploid cells induced by BMS-777607 display a che- RON for potential therapeutic application. moresistant phenotype, which renders tumor cells less BMS-777607induced polyploidy is featured by accumu- sensitive to cytotoxic activities of various chemotherapeu- lation of 4N, 8N, and 16N DNA contents with

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Sharma et al.

corresponding numbers of chromosome in T-47D, ZR-75- for chromosome condensation and cell-cycle progression 1, and MCF-7 cells. The increase of 4N DNA content cells during mitosis (30). We discovered that after BMS-777607 is likely caused by defect in cytokinesis. However, the treatment, the dynamic association of AURK-B with cen- appearance of 8N and 16N DNA is most likely manifested tromeres had disappeared in polyploid cells. Instead, by other mechanisms. Increased endoreplication without AURK-B is associated with condensed and disorganized proper mitosis and/or cytokinesis is a potential cause, chromosomes in prophase/pro-metaphase leading to the which leads to formation of 8N and 16N DNA contents. multipolarized expression pattern. Such irregular appear- Endoreplication, also known as endoreduplication, is a ance highly resembled to that of mitotic spindle micro- form of cell cycle in which mitosis is skipped and cells tubules observed in analysis of a-tubulin and g-tubulin. repeatedly replicate their DNA, resulting in cell polyploi- Again, the disorganized multipolar patterns of AURK-B dy with 8N and 16N DNA contents (29). It appears that were detected as early as 24 hours and then progressively BMS-777607 has the ability to increase endoreplication in diminished after prolonged BMS-777607 treatment. Con- breast cancer cells. sistent with these findings, Western blot analysis showed Induction of breast cancer cell polyploidy by BMS- that BMS-777607 treatment results in decreased AURK-B 777607 is manifested by BMS-777607–targeted inhibition expression in T-47D and ZR-75-1 cells, which is mediated of AURK-B. We have shown that PHA665752 and com- through an intracellular proteomic degradation mecha- pound-I, both MET/RON-specific inhibitors (17, 19), have nism. This conclusion is based on our studies showing that no effect on induction of polyploidy. Moreover, knock- inhibition of proteomic degradation activity by lactacystin down of RON expression by specific siRNA did not effectively prevents BMS-777607–mediated AURK-B prevent BMS-777607–induced polyploidy, which sug- reduction. We reason that the interaction of BMS- gests that RON is not involved in BMS-777607–induced 777607 with AURK-B triggers the intracellular proteomic polyploidy. We also found that BMS-777607 induces degradation mechanism, leading to degradation of polyploidy in MCF-7 cells, although this activity is rela- AURK-B. tively weak compared with its effect on T-47D and ZR-75- Resistance of BMS-777607–induced polyploid cells to 1 cells. Analysis of BMS-7776-7–targeted kinase profiles various chemotherapeutics indicates the existence of a has directly confirmed that BMS-777607 impairs AURK-B protective survival mechanism in breast cancer cells. activity with IC50 value at 78 nmol/L (18). The fact that Previous studies have shown that chemotherapy-induced BMS-777607 is highly effective in induction of polyploidy resistance, characterized by senescence and prolonged in breast cancer T-47D and ZR-75-1 cells suggests that cell-cycle arrest, is responsible for the survival of RON/MET-expressing breast cancer cells may be highly cancer cells (34, 35). Results from this study indicate that sensitive to BMS-777607–targeted inhibition of AURK-B. BMS-777607–induced polyploid cells could use a similar In this sense, BMS-777607 can be considered as a multiple survival mechanism against the cytotoxic effect of che- kinase inhibitor affecting not only RON and MET but also motherapy agents. As shown in Fig. 6, polyploid T-47D AURK-B activity. Such inhibitory effects constitute a and ZR-75-1 cells both displayed a significant increase in mechanism of action by which BMS-777607 exerts its IC50 values against 4 chemoagents including doxorubicin, effect on phenotypes of breast cancer cells. bleomycin, paclitaxel, and methotrexate, when compared The evidence that BMS-777607 affects cell cycle and with control cells. Because the mechanisms of action mitosis comes from our analysis of mitotic spindle assem- differ among these 3 chemoagents, BMS-77767–induced bly and function. Using immunofluorescence to analyze polyploid cells seem to develop a common resistance a-tubulin and g-tubulin expression and localization, we mechanism, which reduces cellular sensitivity in response discovered that BMS-777607 treatment results in disorga- to cytotoxic activities of different chemotherapy agents. nized mitotic spindle assembly in polyploid cells. The Such mechanism could protect cancer cells from bipolar spindle assembly was completely disrupted and cytotoxic death and facilitate their survival in a hostile replaced with multipolarized mitotic spindles with con- environment. densed and disorganized multiple chromosomes. This effect was observed as early as 24 hours and lasted up to Disclosure of Potential Conflicts of Interest 72 hours after BMS-777607 treatment. Another interesting No potential conflicts of interest were disclosed. observation is that after removal of BMS-777607, the multipolarized spindles continued to appear in polyploid cells 3 days after BMS-777607 treatment. These results Authors' Contributions suggest that BMS-777607 affects microtubule dynamics. Conception and design: S. Sharma, Y.-Q. Zhou, R. Zhang, M.-H. Wang Development of methodology: S. Sharma, J.-Y. Zeng, C.-M. Zhuang, Y.-Q. The effect on polyploidy is long-lasting and difficult to Zhou, H.-P. Yao, X. Hu, M.-H. Wang reverse. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Sharma, J.-Y. Zeng, C.-M. Zhuang, Y.-Q. Zhou, We also analyzed the inhibitory effect of BMS-777607 on X. Hu, M.-H. Wang AURK-B and its substrate histone H3. AURK-B associates Analysis and interpretation of data (e.g., statistical analysis, biostatis- with spindle microtubules and is essential during chro- tics, computational analysis): S. Sharma, J.-Y. Zeng, C.-M. Zhuang, Y.-Q. Zhou, X. Hu, M.-H. Wang mosome segregation (31). In addition, AURK-B–mediated Writing, review, and/or revision of the manuscript: S. Sharma, Y.-Q. phosphorylation of histone H3 at Ser10 residue is crucial Zhou, R. Zhang, M.-H. Wang

734 Mol Cancer Ther; 12(5) May 2013 Molecular Cancer Therapeutics

BMS-777607 Induces Breast Cancer Cell Polyploidy

Administrative, technical, or material support (i.e., reporting or orga- Key Laboratory for Diagnosis & Treatment of Infectious Diseases in First nizing data, constructing databases): S. Sharma, J.-Y. Zeng, C.-M. Zhuang, Afﬁliated Hospital of Zhejiang University School of Medicine, Hangzhou, X. Hu, M.-H. Wang P. R. China (M.H. Wang). R. Zhang was supported by NIH grants R01 Study supervision: M.-H. Wang CA112029 and CA121211. Support also was provided by the Zhejiang Provincial Science & Technology Foundation grant #2009C33150 (H.P. Acknowledgments Yao). The costs of publication of this article were defrayed in part by the The assistance of Ms. Susan Denney (Texas Tech University Health payment of page charges. This article must therefore be hereby marked Sciences Center School of Pharmacy, Amarillo, TX) in editing the manu- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate script is greatly appreciated. this fact. Grant Support This work was supported in part by NIH grant R01 CA91980, funds Received November 6, 2012; revised February 28, 2013; accepted from the Amarillo Area Foundation, and subproject #2011ZZ01 from State February 28, 2013; published OnlineFirst March 6, 2013.

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736 Mol Cancer Ther; 12(5) May 2013 Molecular Cancer Therapeutics

Small-Molecule Inhibitor BMS-777607 Induces Breast Cancer Cell Polyploidy with Increased Resistance to Cytotoxic Chemotherapy Agents

Sharad Sharma, Jun-Ying Zeng, Chun-Mei Zhuang, et al.

Mol Cancer Ther 2013;12:725-736. Published OnlineFirst March 6, 2013.

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