Breast Cancer Research and Treatment

, Volume 132, Issue 2, pp 391–409

KX-01, a novel Src kinase inhibitor directed toward the peptide substrate site, synergizes with tamoxifen in estrogen receptor α positive breast cancer

  • Muralidharan Anbalagan
  • Latonya Carrier
  • Seth Glodowski
  • David Hangauer
  • Bin Shan
  • Brian G. Rowan
Preclinical study


KX-01 is the first clinical Src inhibitor of the novel peptidomimetic class that targets the peptide substrate site of Src providing more specificity toward Src kinase. The present study was designed to evaluate the effects of KX-01 as a single agent and in combination with tamoxifen (TAM) on cell growth and apoptosis of ERα positive breast cancer in vitro and in vivo. Flow cytometry demonstrated that KX-01 induced cell cycle arrest in G2/M phase. Immunofluorescent staining for mitotic phase markers and TUNEL staining indicated that cells had arrested in the mitotic phase and mitotic arrested cells were undergoing apoptosis. KX-01 induced nuclear accumulation of cyclin B1, and activation of CDK1, MPM2, and Cdc25C that is required for progression past the G2/M checkpoint. Apoptosis resulted from activation of caspases 6, 7, 8, and 9. Combinational index analysis revealed that combinations of KX-01 with TAM resulted in synergistic growth inhibition of breast cancer cell lines. KX-01 combined with TAM resulted in decreased ERα phosphorylation at Src-regulated phosphorylation sites serines 118 and 167 that were associated with reduced ERα transcriptional activity. Orally administered KX-01 resulted in a dose dependent growth inhibition of MCF-7 tumor xenografts, and in combination with TAM exhibited synergistic growth inhibition. Immunohistochemical analysis revealed that combinational treatment reduced angiogenesis, and ERα signaling in tumors compared to either drug alone that may underlie the synergistic tumor growth inhibition. Combinations of KX-01 with endocrine therapy present a promising new strategy for clinical management of ERα positive breast cancer.


Breast cancer Src kinase inhibitor KX-01 Tamoxifen Preclinical Synergy 

Supplementary material

10549_2011_1513_MOESM1_ESM.pdf (1.4 mb)
Supplementary material 1 (PDF 1483 kb)


  1. 1.
    Sakorafas GH, Farley DR, Peros G (2008) Recent advances and current controversies in the management of DCIS of the breast. Cancer Treat Rev 34(6):483–497. doi:10.1016/j.ctrv.2008.03.001 PubMedCrossRefGoogle Scholar
  2. 2.
    Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M, Cronin WM, Vogel V, Robidoux A, Dimitrov N, Atkins J, Daly M, Wieand S, Tan-Chiu E, Ford L, Wolmark N (1998) Tamoxifen for prevention of breast cancer: report of the national surgical adjuvant breast and bowel project p-1 study. J Natl Cancer Inst 90(18):1371–1388PubMedCrossRefGoogle Scholar
  3. 3.
    Swerdlow AJ, Jones ME (2005) Tamoxifen treatment for breast cancer and risk of endometrial cancer: a case-control study. J Natl Cancer Inst 97(5):375–384. doi:10.1093/jnci/dji057 PubMedCrossRefGoogle Scholar
  4. 4.
    Decensi A, Gandini S, Serrano D, Cazzaniga M, Pizzamiglio M, Maffini F, Pelosi G, Daldoss C, Omodei U, Johansson H, Macis D, Lazzeroni M, Penotti M, Sironi L, Moroni S, Bianco V, Rondanina G, Gjerde J, Guerrieri-Gonzaga A, Bonanni B (2007) Randomized dose-ranging trial of tamoxifen at low doses in hormone replacement therapy users. J Clin Oncol 25(27):4201–4209. doi:10.1200/JCO.2006.09.4318 PubMedCrossRefGoogle Scholar
  5. 5.
    Biscardi JS, Ishizawar RC, Silva CM, Parsons SJ (2000) Tyrosine kinase signalling in breast cancer: epidermal growth factor receptor and c-src interactions in breast cancer. Breast Cancer Res 2(3):203–210PubMedCrossRefGoogle Scholar
  6. 6.
    Reissig D, Clement J, Sanger J, Berndt A, Kosmehl H, Bohmer FD (2001) Elevated activity and expression of src-family kinases in human breast carcinoma tissue versus matched non-tumor tissue. J Cancer Res Clin Oncol 127(4):226–230PubMedCrossRefGoogle Scholar
  7. 7.
    Elsberger B, Tan BA, Mitchell TJ, Brown SB, Mallon EA, Tovey SM, Cooke TG, Brunton VG, Edwards J (2009) Is expression or activation of src kinase associated with cancer-specific survival in er-, pr- and her2-negative breast cancer patients? Am J Pathol 175(4):1389–1397. doi:10.2353/ajpath.2009.090273 PubMedCrossRefGoogle Scholar
  8. 8.
    Fox EM, Bernaciak TM, Wen J, Weaver AM, Shupnik MA, Silva CM (2008) Signal transducer and activator of transcription 5b, c-src, and epidermal growth factor receptor signaling play integral roles in estrogen-stimulated proliferation of estrogen receptor-positive breast cancer cells. Mol Endocrinol 22(8):1781–1796. doi:10.1210/me.2007-0419 PubMedCrossRefGoogle Scholar
  9. 9.
    Hiscox S, Morgan L, Green T, Nicholson RI (2006) Src as a therapeutic target in anti-hormone/anti-growth factor-resistant breast cancer. Endocr Relat Cancer 13 Suppl 1:S53–S59. doi:10.1677/erc.1.01297 PubMedCrossRefGoogle Scholar
  10. 10.
    Desouki MM, Rowan BG (2004) Src kinase and mitogen-activated protein kinases in the progression from normal to malignant endometrium. Clin Cancer Res 10(2):546–555PubMedCrossRefGoogle Scholar
  11. 11.
    Shah YM, Rowan BG (2005) The src kinase pathway promotes tamoxifen agonist action in Ishikawa endometrial cells through phosphorylation-dependent stabilization of estrogen receptor (alpha) promoter interaction and elevated steroid receptor coactivator 1 activity. Mol Endocrinol 19(3):732–748. doi:10.1210/me.2004-0298 PubMedCrossRefGoogle Scholar
  12. 12.
    Rouanet P, Linares-Cruz G, Dravet F, Poujol S, Gourgou S, Simony-Lafontaine J, Grenier J, Kramar A, Girault J, Le Nestour E, Maudelonde T (2005) Neoadjuvant percutaneous 4-hydroxytamoxifen decreases breast tumoral cell proliferation: a prospective controlled randomized study comparing three doses of 4-hydroxytamoxifen gel to oral tamoxifen. J Clin Oncol 23(13):2980–2987. doi:10.1200/JCO.2005.06.064 PubMedCrossRefGoogle Scholar
  13. 13.
    Vultur A, Buettner R, Kowolik C, Liang W, Smith D, Boschelli F, Jove R (2008) Ski-606 (bosutinib), a novel src kinase inhibitor, suppresses migration and invasion of human breast cancer cells. Mol Cancer Ther 7(5):1185–1194. doi:10.1158/1535-7163.MCT-08-0126 PubMedCrossRefGoogle Scholar
  14. 14.
    Shor AC, Keschman EA, Lee FY, Muro-Cacho C, Letson GD, Trent JC, Pledger WJ, Jove R (2007) Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on src kinase for survival. Cancer Res 67(6):2800–2808. doi:10.1158/0008-5472.CAN-06-3469 PubMedCrossRefGoogle Scholar
  15. 15.
    Kim H, Laing M, Muller W (2005) c-Src-null mice exhibit defects in normal mammary gland development and ERalpha signaling. Oncogene 24(36):5629–5636. doi:10.1038/sj.onc.1208718 PubMedCrossRefGoogle Scholar
  16. 16.
    Feng W, Webb P, Nguyen P, Liu X, Li J, Karin M, Kushner PJ (2001) Potentiation of estrogen receptor activation function 1 (af-1) by Src/JNK through a serine 118-independent pathway. Mol Endocrinol 15(1):32–45PubMedCrossRefGoogle Scholar
  17. 17.
    Jr DH (2007) Kxo1: compositions for treating cell proliferation disorders. 7300931 B2, 27Google Scholar
  18. 18.
    Hennequin LF, Allen J, Breed J, Curwen J, Fennell M, Green TP, Lambert-van der Brempt C, Morgentin R, Norman RA, Olivier A, Otterbein L, Ple PA, Warin N, Costello G (2006) N-(5-chloro-1, 3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2 h-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor. J Med Chem 49(22):6465–6488. doi:10.1021/jm060434q PubMedCrossRefGoogle Scholar
  19. 19.
    Li Z, Carrier L, Belame A, Thiyagarajah A, Salvo VA, Burow ME, Rowan BG (2009) Combination of methylselenocysteine with tamoxifen inhibits MCF-7 breast cancer xenografts in nude mice through elevated apoptosis and reduced angiogenesis. Breast Cancer Res Treat 118(1):33–43. doi:10.1007/s10549-008-0216-x PubMedCrossRefGoogle Scholar
  20. 20.
    Chou TC (1991) Synergism and antagonism in chemotherapy. The median-effect principle and the combination index for quantitation of synergism and antagonism. Academic Press, San DiegoGoogle Scholar
  21. 21.
    Chen Y, Guggisberg N, Jorda M, Gonzalez-Angulo A, Hennessy B, Mills GB, Tan CK, Slingerland JM (2009) Combined Src and aromatase inhibition impairs human breast cancer growth in vivo and bypass pathways are activated in AZD0530-resistant tumors. Clin Cancer Res 15(10):3396–3405. doi:10.1158/1078-0432.CCR-08-3127 PubMedCrossRefGoogle Scholar
  22. 22.
    Finn RS, Dering J, Ginther C, Wilson CA, Glaspy P, Tchekmedyian N, Slamon DJ (2007) Dasatinib, an orally active small molecule inhibitor of both the src and abl kinases, selectively inhibits growth of basal-type/“triple-negative” breast cancer cell lines growing in vitro. Breast Cancer Res Treat 105(3):319–326. doi:10.1007/s10549-006-9463-x PubMedCrossRefGoogle Scholar
  23. 23.
    Doree M, Hunt T (2002) From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner? J Cell Sci 115(Pt 12):2461–2464PubMedGoogle Scholar
  24. 24.
    Berry LD, Gould KL (1996) Regulation of Cdc2 activity by phosphorylation at T14/Y15. Prog Cell Cycle Res 2:99–105PubMedCrossRefGoogle Scholar
  25. 25.
    Ramalingam S, Natarajan G, Schafer C, Subramaniam D, May R, Ramachandran I, Queimado L, Houchen CW, Anant S (2008) Novel intestinal splice variants of rna-binding protein CUGBP2: isoform-specific effects on mitotic catastrophe. Am J Physiol Gastrointest Liver Physiol 294(4):G971–G981. doi:10.1152/ajpgi.00540.2007 PubMedCrossRefGoogle Scholar
  26. 26.
    Bonnet J, Mayonove P, Morris MC (2008) Differential phosphorylation of Cdc25c phosphatase in mitosis. Biochem Biophys Res Commun 370(3):483–488. doi:10.1016/j.bbrc.2008.03.117 PubMedCrossRefGoogle Scholar
  27. 27.
    Kuang J, Ashorn CL, Gonzalez-Kuyvenhoven M, Penkala JE (1994) Cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor. Mol Biol Cell 5(2):135–145PubMedGoogle Scholar
  28. 28.
    Castedo M, Perfettini JL, Roumier T, Kroemer G (2002) Cyclin-dependent kinase-1: linking apoptosis to cell cycle and mitotic catastrophe. Cell Death Differ 9(12):1287–1293. doi:10.1038/sj.cdd.4401130 PubMedCrossRefGoogle Scholar
  29. 29.
    Pascreau G, Arlot-Bonnemains Y, Prigent C (2003) Phosphorylation of histone and histone-like proteins by aurora kinases during mitosis. Prog Cell Cycle Res 5:369–374PubMedGoogle Scholar
  30. 30.
    Roninson IB, Broude EV, Chang BD (2001) If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Drug Resist Updat 4(5):303–313. doi:10.1054/drup.2001.0213 PubMedCrossRefGoogle Scholar
  31. 31.
    Adjei AA CR, Kurzrock R, Gordon GS, Hangauer D, et al. (2009) Results of a phase i trial of KX2-391, a novel non-atp competitive substrate-pocket directed src inhibitor, in patients with advanced malignancies. J Clin Oncol 27 (suppl; abstr 3511):1Google Scholar
  32. 32.
    Hiscox S, Jordan NJ, Smith C, James M, Morgan L, Taylor KM, Green TP, Nicholson RI (2009) Dual targeting of src and er prevents acquired antihormone resistance in breast cancer cells. Breast Cancer Res Treat 115(1):57–67. doi:10.1007/s10549-008-0058-6 PubMedCrossRefGoogle Scholar
  33. 33.
    Morgan L, Gee J, Pumford S, Farrow L, Finlay P, Robertson J, Ellis I, Kawakatsu H, Nicholson R, Hiscox S (2009) Elevated src kinase activity attenuates tamoxifen response in vitro and is associated with poor prognosis clinically. Cancer Biol Ther 8(16):1550–1558PubMedCrossRefGoogle Scholar
  34. 34.
    Chu I, Sun J, Arnaout A, Kahn H, Hanna W, Narod S, Sun P, Tan CK, Hengst L, Slingerland J (2007) P27 phosphorylation by src regulates inhibition of cyclin e-cdk2. Cell 128(2):281–294. doi:10.1016/j.cell.2006.11.049 PubMedCrossRefGoogle Scholar
  35. 35.
    Herynk MH, Beyer AR, Cui Y, Weiss H, Anderson E, Green TP, Fuqua SA (2006) Cooperative action of tamoxifen and c-Src inhibition in preventing the growth of estrogen receptor-positive human breast cancer cells. Mol Cancer Ther 5(12):3023–3031. doi:10.1158/1535-7163.MCT-06-0394 PubMedCrossRefGoogle Scholar
  36. 36.
    Filardo EJ, Quinn JA, Frackelton AR Jr, Bland KI (2002) Estrogen action via the g protein-coupled receptor, GPR30: Stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis. Mol Endocrinol 16(1):70–84PubMedCrossRefGoogle Scholar
  37. 37.
    Song RX, Zhang Z, Santen RJ (2005) Estrogen rapid action via protein complex formation involving ERalpha and Src. Trends Endocrinol Metab 16(8):347–353. doi:10.1016/j.tem.2005.06.010 PubMedCrossRefGoogle Scholar
  38. 38.
    Weigel NL, Moore NL (2007) Steroid receptor phosphorylation: a key modulator of multiple receptor functions. Mol Endocrinol 21(10):2311–2319. doi:10.1210/me.2007-0101 PubMedCrossRefGoogle Scholar
  39. 39.
    Castano E, Vorojeikina DP, Notides AC (1997) Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation. Biochem J 326(Pt 1):149–157PubMedGoogle Scholar
  40. 40.
    Arnold SF, Obourn JD, Jaffe H, Notides AC (1995) Phosphorylation of the human estrogen receptor by mitogen-activated protein kinase and casein kinase ii: consequence on DNA binding. J Steroid Biochem Mol Biol 55(2):163–172PubMedCrossRefGoogle Scholar
  41. 41.
    Roche S, Fumagalli S, Courtneidge SA (1995) Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division. Science 269(5230):1567–1569PubMedCrossRefGoogle Scholar
  42. 42.
    Moasser MM, Srethapakdi M, Sachar KS, Kraker AJ, Rosen N (1999) Inhibition of Src kinases by a selective tyrosine kinase inhibitor causes mitotic arrest. Cancer Res 59(24):6145–6152PubMedGoogle Scholar
  43. 43.
    Gonzalez L, Agullo-Ortuno MT, Garcia-Martinez JM, Calcabrini A, Gamallo C, Palacios J, Aranda A, Martin-Perez J (2006) Role of c-Src in human MCF7 breast cancer cell tumorigenesis. J Biol Chem 281(30):20851–20864. doi:10.1074/jbc.M601570200 PubMedCrossRefGoogle Scholar
  44. 44.
    Kasahara K, Nakayama Y, Nakazato Y, Ikeda K, Kuga T, Yamaguchi N (2007) Src signaling regulates completion of abscission in cytokinesis through ERK/MAPK activation at the midbody. J Biol Chem 282(8):5327–5339. doi:10.1074/jbc.M608396200 PubMedCrossRefGoogle Scholar
  45. 45.
    Garcia-Martinez JM, Calcabrini A, Gonzalez L, Martin-Forero E, Agullo-Ortuno MT, Simon V, Watkin H, Anderson SM, Roche S, Martin-Perez J (2010) A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling. Cell Signal 22(3):415–426. doi:10.1016/j.cellsig.2009.10.013 PubMedCrossRefGoogle Scholar
  46. 46.
    Finlay D, Vuori K (2007) Novel noncatalytic role for caspase-8 in promoting Src-mediated adhesion and erk signaling in neuroblastoma cells. Cancer Res 67(24):11704–11711. doi:10.1158/0008-5472.CAN-07-1906 PubMedCrossRefGoogle Scholar
  47. 47.
    Lagadec C, Adriaenssens E, Toillon RA, Chopin V, Romon R, Van Coppenolle F, Hondermarck H, Le Bourhis X (2008) Tamoxifen and trail synergistically induce apoptosis in breast cancer cells. Oncogene 27(10):1472–1477. doi:10.1038/sj.onc.1210749 PubMedCrossRefGoogle Scholar
  48. 48.
    Decensi A, Gandini S, Guerrieri-Gonzaga A, Johansson H, Manetti L, Bonanni B, Sandri MT, Barreca A, Costa A, Robertson C, Lien EA (1999) Effect of blood tamoxifen concentrations on surrogate biomarkers in a trial of dose reduction in healthy women. J Clin Oncol 17(9):2633–2638PubMedGoogle Scholar
  49. 49.
    Park SI, Shah AN, Zhang J, Gallick GE (2007) Regulation of angiogenesis and vascular permeability by src family kinases: opportunities for therapeutic treatment of solid tumors. Expert Opin Ther Targets 11(9):1207–1217. doi:10.1517/14728222.11.9.1207 PubMedCrossRefGoogle Scholar
  50. 50.
    Garvin S, Nilsson UW, Dabrosin C (2005) Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells. Br J Cancer 93(9):1005–1010. doi:10.1038/sj.bjc.6602824 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Muralidharan Anbalagan
    • 1
  • Latonya Carrier
    • 1
  • Seth Glodowski
    • 1
  • David Hangauer
    • 2
  • Bin Shan
    • 3
  • Brian G. Rowan
    • 1
  1. 1.Department of Structural and Cellular BiologyTulane University School of MedicineNew OrleansUSA
  2. 2.Kinex Pharmaceuticals LLCState University of New York at BuffaloBuffaloUSA
  3. 3.Department of MedicineTulane University School of MedicineNew OrleansUSA

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