Results and Discussion Resistance to RON-Targeted Tyrosine Kinase Inhibitor BMS-777607-Induced Polyploidy by Colon Cancer Cells with Retained Chemosensitivity A.G.M. Mostofa, Hang-Ping Yao, and Ming-Hai Wang   Departments of Biomedical Sciences, Texas Tech University HSC School of Pharmacy, Amarillo, TX 79106; Viral Oncogenesis Section at SKLDTID, First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, P. R. China Abstract Results and Discussion Fig 1. Effect of BMS-777607 on RON phosphorylation (A) and survival and proliferation of CRC cells (B). Fig 3: Resistance of HT29 and SW620 but not HCT116 cells to BMS-777607-induced disruptive effect on mitotic spindle assembly and signaling protein expression. p-AuKB AuKB p-histone H3 histone H3 β-Actin HCT116 HT29 SW620 Control 1 µM 2.5 µM 5 µM 0.5 µM BMS-777607 BMS-777607 BMS-777607 B 100 79 47 52 21 100 97 93 83 72 100 123 131 91 107 Intensity (%) 100 104 127 124 117 100 103 114 102 109 100 123 131 91 107 Intensity (%) 100 109 104 <5 <3 100 108 109 117 103 100 114 98 97 83 Intensity (%) 100 101 84 89 77 100 111 119 116 104 100 87 83 94 76 Intensity (%) Specific tyrosine kinase inhibitors targeting MET and RON signaling are potential therapeutics for treatment of cancer cells harboring aberrant signaling of these receptors. Here we studied colorectal cancer (CRC) cells with abnormal MET/RON signaling in response to BMS-777607, a unique tyrosine kinase inhibitor targeting the MET/RON superfamily with a polyploidy-inducing activity. CRC cell lines HT29, HCT116, and SW620 were used as the model. We observed in vitro studies that HT29 and SW620 were naturally resistant to BMS-777607-induced polyploidy but retained their sensitivity to different chemotherapeutics. In contrast, HCT116 cells developed extensive polyploidy after BMS777607 treatment, which results in cells insensitive to chemocytotoxicity. To understand the underlying mechanisms, we performed various studies which revealed that BMS-777607-induced disorganization of microtubule assembly was present in HCT116 cells but not in HT29 and SW620 cells. Resistance to BMS-777607-induced inhibition of aurora kinase B (AurKB), altered localization of polo-like kinase-1, and abnormal accumulation of survivin was also detected in HT29 and SW620 cells. Moreover, AurKB from HT29 and SW620 cells but not HCT116 cells was insensitive to proteolytic degradation after BMS-777607 treatment and inhibition of AuKB phosphorylation. These observations indicate the impairment in binding of BMS77607 to AuKB in resistant but not in sensitive cells. We further performed tumor xenografts studies using HCT116 and HT29 cells as the models. Interestingly, an additive effect was observed in mice treated with BMS-777607 in combinaiton with 5-fluorouracil on tumor growth mediated by HT29 cells but not by HCT116 cells. We conclude from these studies that primary resistance to BMS-induced polyploidy exists in colorectal cancer cells and BMS-777607 in combination with chemoagents is able to achieve the maximal effect on CRC cells, which are naturally resistant to polyploidy. 0.5 µM 0 5 20 100 200 nM BMS-777607 RON β-chain RON160 β-chain β-actin HT29 HCT116 SW620 A DAPI α-tubulin DAPI+ Aurora B α-tubulin+ HT29 +BMS HT29 HCT116 +BMS HCT116 A HCT116 HT29 SW620 Control 2.5 µM 5.0 µM 10.0 µM B Fig 6:Chemosensitivity of CRC cells in response to cytotoxic therapeutics after BMS-777607 treatment. 5-fluorouracil (μM) Oxaliplatin (μM) Methotrexate (nM) Doxorubicin (μM) HCT116 HCT116 + BMS HT29 HT29 + BMS SW620 SW620 + BMS % cell viability HT29 +BMS HHT29 HCT116 +BMS HCT116 DAPI α-tubulin DAPI+ PLK1 α-tubulin+ B Materials and Methods Cell Lines and Reagents: Human CRC cell lines HCT116, HT29, and SW620 were from American Type Cell Culture. Antibodies specific to AuKB, polo-like kinase (PLK)-1, surviving and other signaling proteins were from Cell Signaling. BMS-777607 was from Selleck Chemicals. Anti-RON antibodies were previously produced by us. Western blotting & immunofluorescence analyses: Cellular proteins from cell lysate were separated in an 8% or 12% SDS-PAGE under reduced conditions. Signaling proteins were detected using specific antibodies corresponding to individual proteins. Immunofluorescence detection was performed on treated or untreated cells with various amounts of BMS-777607 at different time point. Cells were fixed with 4% paraformaldehyde solution and permeabilized with 0.1% triton X, followed by incubation with individual primary and secondary antibodies. Assays for cell growth, viability, and DNA content: The effect of BMS-777607 on CRC cell growth and survival was determined by the clonogenic assay. The activity of BMS-777607 and individual chemotherapeutics on cell viability was determined by the MTT assay. For measuring cellular DNA contents, cells were treated with drug, labeled with propidium iodide, and analyzed by an Accuri Flow Cytometer. Methods for determining AuKB stability: Cells were pretreated with 50 μg/ml cycloheximide for 30 min, followed by addition of 5 μM BMS-777607. Cell lysates prepared at different time intervals from individual samples were subjected to Western blot analysis. The half-life of AuKB was calculated on the basis of protein intensity measured quantitatively by using analytical software (ImageJ 1.48). Mouse xenograft CRC model and drug treatment: Female athymic nude mice (6 to 8 weeks old) were used to develop xenograft tumors of CRC cells. Treatment began when all tumors have reached an average tumor volume of 50 to 100 mm3. Mice in the BMS-777607 group were administrated once daily by oral dosing of 6.25 mg/kg for fourteen consecutive days. Mice in the 5-FU group were i.p. administered at a dose of 125 mg/kg on days 5, 14, and 23. Mice in the combination treatment group were treated with BMS-777607 plus 5-FU at the same doses and schedules as described above. Tumor volume was measured every five days, calculated as the product of length x width x height, and expressed as mean tumor volume (mm3). Statistical analysis: GraphPad Prism 6 software was used for statistical analysis. Results are shown as mean ± SD. The data between control and experimental groups were compared using Student t test. Statistical differences at p < 0.05 were considered significant. Fig 2: Differential responsiveness of CRC cells to BMS-777607-induced polyploidy (A) and cell cycle changes (B). Fig 7:Therapeutic activity of BMS-777607, 5-FU, and their combinations on tumor growth induced by HCT116 and HT29 cells in mouse xenograft models Control 2.5 µM 5.0 µM HT29 HCT116 SW620 %poly: 2.7 ± 0.6 6.3 ± 1.2 30.4 ± 2.3 %poly: 0 ± 0 1.5 ± 0.5 2.7 ± 1.2 %poly: ND ND 10 µM 84.6 ± 7.8 14.9 ± 3.6 A Fig 4: Effect of BMS-777607 on expression of AuKB, PLK1, and survivin by CRC cells. Mean tumor volume (mm3) Days of treatment Control Treated with BMS-777607 Treated with 5-Fu Treated with BMS + 5-Fu Injection with 5-Fu Oral dosing of BMS a: HT29 model b: HCT116 model HCT116 HT29 SW620 β-Actin Survivin PLK1 AuKB 0 24 48 72 0 24 48 72 0 24 48 72 hours Fig 5: Increased stability and resistance of aurora kinase B to proteolytic degradation (A) and inhibition of phosphorylation (B) in BMS-777607-treated HT29 and SW620 cells. Conclusion Some CRC cells are naturally resistant to BMS-777607 induced polyploidy. After BMS-777607 treatment, aberrant expression and localization of AuKB, PKL-1, and survivin were observed in sensitive cells but not in resistant cells. In the resistant cells, BMS-777607 was unable to induce proteolytic degradation and dephosphorylation of AuKB. Impaired binding of BMS-777607 to AuKB could be the underlying mechanism of resistance. Cells resistant to polyploidy remained sensitive to different chemotherapeutics and showed additive response to combination therapy. HCT116 SW620 Relative cell counts Relative fluorescence intensity BMS-777607: 0 μM 2.5 μM 5 μM 10 μM 8N 16N HT29 B 0 0.5 1 2 4 8 (h) Aurora-B β-Actin HCT116 HT29 SW620 CHX alone 0 0.5 1 2 4 8 (h) CHX+ BMS-777607 A Acknowledgement Supported in part by NIH R01 CA91980 and Chinese Natural Sciences Foundation