Anti-tumor and anti-metastasis efficacy of E6201, a MEK1 inhibitor, in preclinical models of triple-negative breast cancer

  • Jangsoon Lee
  • Bora Lim
  • Troy Pearson
  • Kuicheon Choi
  • Jon A. Fuson
  • Chandra Bartholomeusz
  • Linda J. Paradiso
  • Thomas Myers
  • Debu Tripathy
  • Naoto T. UenoEmail author
Preclinical study



Triple-negative breast cancer (TNBC) lacks the receptor targets estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, and thus, it does not respond to receptor-targeted treatments. TNBC has higher recurrence, metastasis, and mortality rates than other subtypes of breast cancer. Mounting data suggest that the MAPK (also known as RAS-RAF-MEK-ERK) pathway is an important therapeutic target in TNBC.


To evaluate anti-tumor and anti-metastasis efficacy of E6201, we used cell proliferation assay, soft agar assay, cell cycle assay, Annexin V staining assay, immunoblotting analysis, immunohistochemistry, migration assay, invasion assay, mammary fat pad xenograft, and experimental and spontaneous metastasis xenograft models. We also evaluated the anti-tumor efficacy of E6201 plus CDK4/6 inhibitor, mTOR inhibitor, or ATR inhibitor.


E6201 inhibited TNBC cell colony formation, migration, and invasion in a dose-dependent manner. E6201 induced G1 cell cycle arrest and apoptosis. E6201 inhibited TNBC xenograft growth and inhibited TNBC lung metastasis and improved mouse survival in experimental metastasis and spontaneous metastasis assays. Immunohistochemical staining demonstrated that E6201 decreased the metastatic burden in the lung and decreased phosphorylated ERK expression in a dose-dependent manner. Combination of E6201 with CDK4/6 inhibitor or mTOR inhibitor enhanced E6201’s in vitro anti-tumor efficacy.


These results indicate that E6201 exhibits anti-tumor efficacy against TNBC in vitro and anti-metastasis efficacy against TNBC in vivo. These results provide a rationale for further clinical development of E6201 as a MAPK-pathway-targeted therapy for TNBC.


E6201 MEK inhibitor MAPK pathway Triple-negative breast cancer Metastasis 



We thank Sunita Patterson and Stephanie Deming of the Department of Scientific Publications at MD Anderson Cancer Center for editing the manuscript.


This work was supported by the Morgan Welch Inflammatory Breast Cancer Research Program, the State of Texas Rare and Aggressive Breast Cancer Research Program, the National Institutes of Health/National Cancer Institute (Grant CA123318), MD Anderson’s Cancer Center Support Grant (P30CA016672, used the Characterized Cell Line Core Facility and Flow Cytometry and Cellular Imaging Facility), and Spirita Oncology, LLC.

Compliance with ethical standards

Conflict of interest

Naoto T. Ueno has a research agreement with Spirita Oncology, LLC. Linda J. Paradiso and Thomas Myers are employees of Spirita Oncology, LLC. All other authors declare no potential conflicts of interest.

Research involving in animal rights

Animal studies and procedures were approved by The University of Texas MD Anderson Cancer Center Animal Care and Use Committee. Protocol #: 00000968-RN02 (approval date 5/1/2015, expiration date 4/22/2021).

Research involving in human rights

This article does not contain any studies with human participants by any of the authors.

Supplementary material

10549_2019_5166_MOESM1_ESM.pptx (1.8 mb)
Supplemental Figure 1. Anti-proliferation effect of MEK inhibitors in TNBC cell lines. TNBC cells were treated with selumetinib, pimasertib, or trametinib for 5 days, and viability was measured by using CellTiter-Blue and sulforhodamine-B assays. Data shown are representative of three experiments with similar results. Each point represents the mean of three independent experiments; error bars indicate standard deviation. Supplemental Figure 2. FLT3 inhibition does not affect TNBC cell proliferation, migration, or invasion. a Western blotting of cells treated with E6201 for the indicated times. b Proliferation assay. TNBC cell lines were treated with quizartinib for 5 days, and viability was measured by using CellTiter-Blue. c and d Migration and invasion assays. TNBC cells (1×105/well) were added into trans-wells with or without quizartinib for 6 hr (migration, c) or 24 hr (invasion, d). Migration and invasion were evaluated by using ImageJ software. Data shown are representative of three experiments with similar results; error bars indicate standard deviation. Supplementary material 1 (PPTX 1826 KB)


  1. 1.
    Ismail-Khan R, Bui MM (2010) A review of triple-negative breast cancer. Cancer Control 17(3):173–176Google Scholar
  2. 2.
    Brouckaert O, Wildiers H, Floris G, Neven P (2012) Update on triple-negative breast cancer: prognosis and management strategies. Int J Womens Health 4:511–520Google Scholar
  3. 3.
    Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA et al (2007) Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13(15 Pt 1):4429–4434Google Scholar
  4. 4.
    Santarpia L, Lippman SM, El-Naggar AK (2012) Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin Ther Targets 16(1):103–119Google Scholar
  5. 5.
    Eroglu Z, Ribas A (2016) Combination therapy with BRAF and MEK inhibitors for melanoma: latest evidence and place in therapy. Ther Adv Med Oncol 8(1):48–56Google Scholar
  6. 6.
    Tran KA, Cheng MY, Mitra A, Ogawa H, Shi VY, Olney LP et al (2016) MEK inhibitors and their potential in the treatment of advanced melanoma: the advantages of combination therapy. Drug Des Dev Ther 10:43–52Google Scholar
  7. 7.
    Yang Y, Liu YH, Sun X, Yu MW, Yang L, Cheng PY et al (2017) Risk of peripheral edema in cancer patients treated with MEK inhibitors: a systematic review and meta-analysis of clinical trials. Curr Med Res Opin 33(9):1663–1675Google Scholar
  8. 8.
    Bartholomeusz C, Xie X, Pitner MK, Kondo K, Dadbin A, Lee J et al (2015) MEK inhibitor selumetinib (AZD6244; ARRY-142886) prevents lung metastasis in a triple-negative breast cancer xenograft model. Mol Cancer Ther 14(12):2773–2781Google Scholar
  9. 9.
    Lee J, Galloway R, Grandjean G, Jacob J, Humphries J, Bartholomeusz C et al (2015) Comprehensive two- and three-dimensional RNAi screening identifies PI3K inhibition as a complement to MEK inhibitor AS703026 for combination treatment of triple-negative breast cancer. J Cancer 6(12):1306–1319Google Scholar
  10. 10.
    Muramoto K, Goto M, Inoue Y, Ishii N, Chiba K, Kuboi Y et al (2010) E6201, a novel kinase inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1: in vivo effects on cutaneous inflammatory responses by topical administration. J Pharmacol Exp Ther 335(1):23–31Google Scholar
  11. 11.
    Ikemori-Kawada M, Inoue A, Goto M, Wang YJ, Kawakami Y (2012) Docking simulation study and kinase selectivity of f152A1 and its analogs. J Chem Inf Model 52(8):2059–2068Google Scholar
  12. 12.
    Byron SA, Loch DC, Wellens CL, Wortmann A, Wu J, Wang J et al (2012) Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status. Mol Cancer 11:75Google Scholar
  13. 13.
    Gampa G, Kim M, Cook-Rostie N, Laramy JK, Sarkaria JN, Paradiso L et al (2018) Brain distribution of a novel MEK inhibitor E6201: implications in the treatment of melanoma brain metastases. Drug Metab Dispos 46(5):658–666Google Scholar
  14. 14.
    Tibes R, Borad MJ, Dutcus CE, Reyderman L, Feit K, Eisen A et al (2018) Safety, pharmacokinetics, and preliminary efficacy of E6201 in patients with advanced solid tumours, including melanoma: results of a phase 1 study. Br J Cancer 118(12):1580–1585Google Scholar
  15. 15.
    Zhang W, Borthakur G, Gao C, Chen Y, Mu H, Ruvolo VR et al (2016) The dual MEK/FLT3 inhibitor E6201 exerts cytotoxic activity against acute myeloid leukemia cells harboring resistance-conferring FLT3 mutations. Cancer Res 76(6):1528–1537Google Scholar
  16. 16.
    Carragher NO, Frame MC (2004) Focal adhesion and actin dynamics: a place where kinases and proteases meet to promote invasion. Trends Cell Biol 14(5):241–249Google Scholar
  17. 17.
    Wallace EM, Lyssikatos JP, Yeh T, Winkler JD, Koch K (2005) Progress towards therapeutic small molecule MEK inhibitors for use in cancer therapy. Curr Top Med Chem 5(2):215–229Google Scholar
  18. 18.
    Mitchell C, Yacoub A, Hossein H, Martin AP, Bareford MD, Eulitt P et al (2010) Inhibition of MCL-1 in breast cancer cells promotes cell death in vitro and in vivo. Cancer Biol Ther 10(9):903–917. Google Scholar
  19. 19.
    Chetoui N, Sylla K, Gagnon-Houde JV, Alcaide-Loridan C, Charron D, Al-Daccak R et al (2008) Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis. Mol Cancer Res 6(1):42–52. Google Scholar
  20. 20.
    Konopleva M, Milella M, Ruvolo P, Watts JC, Ricciardi MR, Korchin B et al (2012) MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex. Leukemia 26(4):778–787. Google Scholar
  21. 21.
    Hermanson DL, Das SG, Li Y, Xing C (2013) Overexpression of Mcl-1 confers multidrug resistance, whereas topoisomerase IIbeta downregulation introduces mitoxantrone-specific drug resistance in acute myeloid leukemia. Mol Pharmacol 84(2):236–243. Google Scholar
  22. 22.
    Wu H, Schiff DS, Lin Y, Neboori HJ, Goyal S, Feng Z et al (2014) Ionizing radiation sensitizes breast cancer cells to Bcl-2 inhibitor, ABT-737, through regulating Mcl-1. Radiat Res 182(6):618–625. Google Scholar
  23. 23.
    Kawakami H, Huang S, Pal K, Dutta SK, Mukhopadhyay D, Sinicrope FA (2016) Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer. Mol Cancer Ther 15(12):3015–3027. Google Scholar
  24. 24.
    Bartholomeusz C, Xie X, Pitner MK, Kondo K, Dadbin A, Lee J et al (2015) MEK inhibitor selumetinib (AZD6244; ARRY-142886) prevents lung metastasis in a triple-negative breast cancer xenograft model. Mol Cancer Ther. Google Scholar
  25. 25.
    Arthur JS, Ley SC (2013) Mitogen-activated protein kinases in innate immunity. Nat Rev Immunol 13(9):679–692. Google Scholar
  26. 26.
  27. 27.
    Cheng Y, Tian H (2017) Current Development status of MEK inhibitors. Molecules 22(10):1551Google Scholar
  28. 28.
    Albanell J, Elvin JA, Ali SM, Schrock AB, Chung J, Vergilio J-A et al (2017) BRAF: an emerging target for triple-negative breast cancer. J Clin Oncol 35(15_suppl):1099–1099Google Scholar
  29. 29.
    Rimawi MF, Shetty PB, Weiss HL, Schiff R, Osborne CK, Chamness GC et al (2010) Epidermal growth factor receptor expression in breast cancer association with biologic phenotype and clinical outcomes. Cancer 116(5):1234–1242Google Scholar
  30. 30.
    Law JH, Habibi G, Hu K, Masoudi H, Wang MY, Stratford AL et al (2008) Phosphorylated insulin-like growth factor-i/insulin receptor is present in all breast cancer subtypes and is related to poor survival. Cancer Res 68(24):10238–10246Google Scholar
  31. 31.
    Verbeek BS, Vroom TM, Adriaansen-Slot SS, Ottenhoff-Kalff AE, Geertzema JG, Hennipman A et al (1996) c-Src protein expression is increased in human breast cancer. An immunohistochemical and biochemical analysis. J Pathol 180(4):383–388Google Scholar
  32. 32.
    Maiello MR, D’Alessio A, Bevilacqua S, Gallo M, Normanno N, De Luca A (2015) EGFR and MEK blockade in triple negative breast cancer cells. J Cell Biochem 116(12):2778–2785Google Scholar
  33. 33.
    Nakai K, Hung MC, Yamaguchi H (2016) A perspective on anti-EGFR therapies targeting triple-negative breast cancer. Am J Cancer Res 6(8):1609–1623Google Scholar
  34. 34.
    Bartholomeusz C, Oishi T, Saso H, Akar U, Liu P, Kondo K et al (2012) MEK1/2 inhibitor selumetinib (AZD6244) inhibits growth of ovarian clear cell carcinoma in a PEA-15-dependent manner in a mouse xenograft model. Mol Cancer Ther 11(2):360–369Google Scholar
  35. 35.
    Chen X, Zheng Z, Chen L, Zheng H (2017) MAPK, NFkappaB, and VEGF signaling pathways regulate breast cancer liver metastasis. Oncotarget 8(60):101452–101460Google Scholar
  36. 36.
    Shao GL, Wang MC, Fan XL, Zhong L, Ji SF, Sang G et al (2018) Correlation between Raf/MEK/ERK signaling pathway and clinicopathological features and prognosis for patients with breast cancer having axillary Lymph node metastasis. Technol Cancer Res Treat. Google Scholar
  37. 37.
    Torres-Adorno AM, Lee J, Kogawa T, Ordentlich P, Tripathy D, Lim B et al (2017) Histone deacetylase inhibitor enhances the efficacy of MEK inhibitor through NOXA-mediated MCL1 degradation in triple-negative and inflammatory breast cancer. Clin Cancer Res 23(16):4780–4792Google Scholar
  38. 38.
    Jing J, Greshock J, Holbrook JD, Gilmartin A, Zhang X, McNeil E et al (2012) Comprehensive predictive biomarker analysis for MEK inhibitor GSK1120212. Mol Cancer Ther 11(3):720–729Google Scholar
  39. 39.
    Nagaria TS, Shi C, Leduc C, Hoskin V, Sikdar S, Sangrar W et al (2017) Combined targeting of Raf and Mek synergistically inhibits tumorigenesis in triple negative breast cancer model systems. Oncotarget 8(46):80804–80819Google Scholar
  40. 40.
    Sato N, Wakabayashi M, Nakatsuji M, Kashiwagura H, Shimoji N, Sakamoto S et al (2017) MEK and PI3K catalytic activity as predictor of the response to molecularly targeted agents in triple-negative breast cancer. Biochem Biophys Res Commun 489(4):484–489Google Scholar
  41. 41.
    Duncan JS, Whittle MC, Nakamura K, Abell AN, Midland AA, Zawistowski JS et al (2012) Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer. Cell 149(2):307–321Google Scholar
  42. 42.
    Mirzoeva OK, Das D, Heiser LM, Bhattacharya S, Siwak D, Gendelman R et al (2009) Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition. Cancer Res 69(2):565–572Google Scholar
  43. 43.
    Adjei AA, Cohen RB, Franklin W, Morris C, Wilson D, Molina JR et al (2008) Phase I pharmacokinetic and pharmacodynamic study of the oral, small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers. J Clin Oncol 26(13):2139–2146Google Scholar
  44. 44.
    Rinehart J, Adjei AA, Lorusso PM, Waterhouse D, Hecht JR, Natale RB et al (2004) Multicenter phase II study of the oral MEK inhibitor, CI-1040, in patients with advanced non-small-cell lung, breast, colon, and pancreatic cancer. J Clin Oncol 22(22):4456–4462Google Scholar
  45. 45.
    Hoeflich KP, O’Brien C, Boyd Z, Cavet G, Guerrero S, Jung K et al (2009) In vivo antitumor activity of MEK and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models. Clin Cancer Res 15(14):4649–4664Google Scholar
  46. 46.
    Leung EY, Kim JE, Askarian-Amiri M, Rewcastle GW, Finlay GJ, Baguley BC (2014) Relationships between signaling pathway usage and sensitivity to a pathway inhibitor: examination of trametinib responses in cultured breast cancer lines. PLoS ONE 9(8):e105792Google Scholar
  47. 47.
    Van Swearingen AED, Sambade MJ, Siegel MB, Sud S, McNeill RS, Bevill SM et al (2017) Combined kinase inhibitors of MEK1/2 and either PI3K or PDGFR are efficacious in intracranial triple-negative breast cancer. Neuro Oncol 19(11):1481–1493Google Scholar
  48. 48.
    Corcoran RB, Andre T, Atreya CE, Schellens JHM, Yoshino T, Bendell JC et al (2018) Combined BRAF, EGFR, and MEK Inhibition in patients with BRAF(V600E)-mutant colorectal cancer. Cancer Discov 8(4):428–443Google Scholar
  49. 49.
    Gaudio E, Tarantelli C, Kwee I, Barassi C, Bernasconi E, Rinaldi A et al (2016) Combination of the MEK inhibitor pimasertib with BTK or PI3K-delta inhibitors is active in preclinical models of aggressive lymphomas. Ann Oncol 27(6):1123–1128Google Scholar
  50. 50.
    Lee MS, Helms TL, Feng N, Gay J, Chang QE, Tian F et al (2016) Efficacy of the combination of MEK and CDK4/6 inhibitors in vitro and in vivo in KRAS mutant colorectal cancer models. Oncotarget 7(26):39595–39608Google Scholar
  51. 51.
    Zhao H, Cui K, Nie F, Wang L, Brandl MB, Jin G et al (2012) The effect of mTOR inhibition alone or combined with MEK inhibitors on brain metastasis: an in vivo analysis in triple-negative breast cancer models. Breast Cancer Res Treat 131(2):425–436Google Scholar
  52. 52.
    Balko JM, Giltnane JM, Wang K, Schwarz LJ, Young CD, Cook RS et al (2014) Molecular profiling of the residual disease of triple-negative breast cancers after neoadjuvant chemotherapy identifies actionable therapeutic targets. Cancer Discov 4(2):232–245Google Scholar
  53. 53.
    Brufsky A, Miles D, Zvirbule Z, Eniu A, Lopez-Miranda E, Seo JH et al (2018) Abstract P5-21-01: Cobimetinib combined with paclitaxel as first-line treatment for patients with advanced triple-negative breast cancer (COLET study): Primary analysis of cohort I. Cancer Research. SABCS17-P5-21-01Google Scholar
  54. 54.
    Chumsri S, Polley M-Y, Anderson SL, O’Sullivan CCM, Colon-Otero G, Knutson KL et al (2018) Phase I/II trial of pembrolizumab in combination with binimetinib in unresectable locally advanced or metastatic triple negative breast cancer. J Clin Oncol 36(5_suppl):TPS17–TPS17Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jangsoon Lee
    • 1
    • 3
  • Bora Lim
    • 1
    • 3
  • Troy Pearson
    • 1
  • Kuicheon Choi
    • 1
  • Jon A. Fuson
    • 1
  • Chandra Bartholomeusz
    • 1
    • 3
  • Linda J. Paradiso
    • 2
  • Thomas Myers
    • 2
  • Debu Tripathy
    • 1
  • Naoto T. Ueno
    • 1
    • 3
    Email author
  1. 1.Section of Translational Breast Cancer Research, Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, Department of Breast Medical OncologyThe University of Texas MD Anderson Cancer CenterHoustonUSA
  2. 2.Spirita Oncology, LLCNatickUSA
  3. 3.Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, Department of Breast Medical OncologyThe University of Texas MD Anderson Cancer CenterHoustonUSA

Personalised recommendations