Identification of the receptor tyrosine kinase AXL in breast cancer as a target for the human miR-34a microRNA

  • Mark Mackiewicz
  • Konrad Huppi
  • Jason J. Pitt
  • Tiffany H. Dorsey
  • Stefan Ambs
  • Natasha J. CaplenEmail author
Brief Report


The identification of molecular features that contribute to the progression of breast cancer can provide valuable insight into the pathogenesis of this disease. Deregulated microRNA expression represents one type of molecular event that has been associated with many different human cancers. In order to identify a miRNA/mRNA regulatory interaction that is biologically relevant to the triple-negative breast cancer genotype/phenotype, we initially conducted a miRNA profiling experiment to detect differentially expressed miRNAs in cell line models representing triple-negative (MDA-MB-231), ER+ (MCF7), and HER-2 overexpressed (SK-BR-3) histotypes. We identified human miR-34a expression as being >3-fold down (from its median expression value across all cell lines) in MDA-MB-231 cells, and identified AXL as a putative mRNA target using multiple miRNA/target prediction algorithms. The miR-34a/AXL interaction was functionally characterized through ectopic overexpression experiments with a miR-34a mimic in two independent triple-negative breast cancer cell lines. In reporter assays, miR-34a binds to its putative target site within the AXL 3′UTR to inhibit luciferase expression. We also observed degradation of AXL mRNA and decreased AXL protein levels, as well as cell signaling effects on AKT phosphorylation and phenotypic effects on cell migration. Finally, we present an inverse correlative trend in miR-34a and AXL expression for both cell line and patient tumor samples.


MicroRNAs AXL miR-34a Triple negative Breast cancer 



This research was supported by the Intramural Research Program (Center for Cancer Research, NCI) of the NIH. The authors thank Drs Stan Lipkowitz, Thomas Reid, Scott Martin, Philip Lorenzi, Amanda Hummon, Michael Difilippantonio, and Paul Meltzer for their useful comments and suggestions. The authors also thank Robert Cornelison, Diane Palmieri, Sarah Anzick, Kristen Gehlhaus, Tamara Jones, Lihui Ou, and Brady Wahlberg for their technical assistance. The authors greatly appreciate the gesture extended by Ashish Lal and Judy Lieberman (Harvard Medical School) in sharing their unpublished data on miR-34a/AXL targeting with us. Finally, the authors gratefully acknowledge the technical assistance that Subu Yerramilli (Qiagen) provided to us in regard to the qRT-PCR analysis of miR-34a primary, precursor, and mature transcripts.

Conflict of interest

No potential conflicts of interest are disclosed.

Supplementary material

10549_2011_1690_MOESM1_ESM.doc (936 kb)
Supplementary material 1 (DOC 936 kb)


  1. 1.
    Rakha EA, Ellis IO (2009) Triple-negative/basal-like breast cancer: review. Pathology 41:40–47PubMedCrossRefGoogle Scholar
  2. 2.
    Venkitaraman R (2010) Triple-negative/basal-like breast cancer: clinical, pathologic and molecular features. Expert Rev. Anticancer Ther. 10:199–207PubMedCrossRefGoogle Scholar
  3. 3.
    Charafe-Jauffret E, Ginestier C, Monville F, Finetti P, Adélaïde J, Cervera N, Fekairi S, Xerri L, Jacquemier J, Birnbaum D, Bertucci F (2006) Gene expression profiling of breast cancer cell lines identifies potential new basal markers. Oncogene 25:2273–2284PubMedCrossRefGoogle Scholar
  4. 4.
    Kao J, Salari K, Bocanegra M, Choi Y-L, Girard L, Gandhi J, Kwei KA, Hernandez-Boussard T, Wang P, Gazdar AF, Minna JD, Pollack JR (2009) Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery. PLOS One 4:1–16CrossRefGoogle Scholar
  5. 5.
    Kreike B, Van Kouwenhove M, Horlings H, Weigelt B, Peterse H, Bartelink H, Van de Vijver MJ (2007) Gene expression profiling and histopathological characterization of triple-negative/basal-like breast carcinomas. Breast Cancer Res 9:R65PubMedCrossRefGoogle Scholar
  6. 6.
    Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe J-P, Tong F, Speed T, Spellman PT, DeVries S, Lapuk A, Wang NJ, Kuo W-L, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527PubMedCrossRefGoogle Scholar
  7. 7.
    Perou CM, Sørlie T, Eisen MB, Van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Aksien LA, Fluge Ø, Pergamenschikov A, Williams C, Zhu SX, Lønning PE, Børresen-Dale A-L, Brown PO, Botstein D (2000) Molecular portraits of human breast tumours. Nature 406:747–752PubMedCrossRefGoogle Scholar
  8. 8.
    Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, Van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Lønning PE, Børresen-Dale A-L (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98:10869–10874PubMedCrossRefGoogle Scholar
  9. 9.
    Sørlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, Demeter J, Perou CM, Lønning PE, Brown PO, Børresen-Dale A-L, Botstein D (2003) Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 100:8418–8423PubMedCrossRefGoogle Scholar
  10. 10.
    Rakha EA, El-Sayed ME, Green AR, Lee AH, Robertson JF, Ellis IO (2007) Prognostic markers in triple-negative breast cancer. Cancer 109:25–32PubMedCrossRefGoogle Scholar
  11. 11.
    Gluz O, Liedtke C, Gottschalk N, Pusztai L, Nitz U, Harbeck N (2009) Triple-negative breast cancer-current status and future directions. Ann Oncol 20:1913–1927PubMedCrossRefGoogle Scholar
  12. 12.
    Bartel DP (2004) MicroRNAs: genomics biogenesis, mechanism, and function. Cell 116:281–297PubMedCrossRefGoogle Scholar
  13. 13.
    Babar IA, Slack FJ, Weidhaas JB (2008) MiRNA modulation of the cellular stress response. Future Oncol 4:289–298PubMedCrossRefGoogle Scholar
  14. 14.
    Chang TC, Mendell JT (2007) MicroRNAs in vertebrate physiology and human disease. Annu Rev Genomics Hum Genet 8:215–239PubMedCrossRefGoogle Scholar
  15. 15.
    Krutzfeldt J, Stoffel M (2006) MicroRNAs: a new class of regulatory genes affecting metabolism. Cell Metabolism 4:9–12PubMedCrossRefGoogle Scholar
  16. 16.
    Zhao Y, Srivastava D (2007) A developmental view of microRNA function. Trends Biochem Sci 32:189–197PubMedCrossRefGoogle Scholar
  17. 17.
    Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 10:704–714PubMedCrossRefGoogle Scholar
  18. 18.
    He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, Powers S, Cordon-Cardo C, Lowe SW, Hannon GJ, Hammond SM (2005) A microRNA polycistron as a potential human oncogene. Nature 435:828–833PubMedCrossRefGoogle Scholar
  19. 19.
    Garzon R, Calin GA, Croce CM (2009) MicroRNAs in cancer. Annu Rev Med 60:167–179PubMedCrossRefGoogle Scholar
  20. 20.
    Linger RM, Keating AK, Earp HS, Graham DK (2008) TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer. Adv Cancer Res 100:35–83PubMedCrossRefGoogle Scholar
  21. 21.
    Gjerdrum C, Tiron C, Høiby T, Stefansson I, Haugen H, Sandal T, Collett K, Li S, McCormack E, Gjertsen BT, Micklem DR, Aksien LA, Glackin C, Lorens JB (2010) Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. Proc Natl Acad Sci USA 107:1124–1129PubMedCrossRefGoogle Scholar
  22. 22.
    Corney DC, Hwang CL, Matoso A, Vogt M, Flesken-Nikitin A, Godwin AK, Kamat AA, Sood AK, Ellenson LH, Hermeking H, Nikitin AY (2010) Frequent downregulation of miR-34 family in human ovarian cancers. Clin Cancer Res 16:1119–1128PubMedCrossRefGoogle Scholar
  23. 23.
    Guessous F, Zhang Y, Kofman A, Catania A, Li Y, Schiff D, Purow B, Abounader R (2010) MicroRNA-34a is tumor suppressive in brain tumors and glioma stem cells. Cell Cycle 9:1031–1036PubMedCrossRefGoogle Scholar
  24. 24.
    Hu Y, Correa AM, Hoque A, Guan B, Ye F, Huang J, Swisher SG, Wu TT, Ajani JA, Xu XC (2010) Prognostic significance of differentially expressed miRNAs in esophageal cancer. Int J Cancer. doi: 10.1002/ijc.25330.
  25. 25.
    Li N, Fu H, Tie Y, Hu Z, Kong W, Wu Y, Zheng X (2009) miR-34a inhibits migration and invasion by down-regulation of c-Met expression in human hepatocellular carcinoma cells. Cancer Lett 275:44–53PubMedCrossRefGoogle Scholar
  26. 26.
    Li Y, Guessous F, Zhang Y, Dipierro C, Kefas B, Johnson E, Marcinkiewicz L, Jiang J, Yang Y, Schmittgen TD, Lopes B, Schiff D, Purow B, Abounader R (2009) MicroRNA-34a inhibits glioblastoma growth by targeting multiple oncogenes. Cancer Res 69:7569–7576PubMedCrossRefGoogle Scholar
  27. 27.
    Yan D, Zhou X, Chen X, Hu DN, Dong XD, Wang J, Lu F, Tu L, Qu J (2009) MicroRNA-34a inhibits uveal melanoma cell proliferation and migration through downregulation of c-MET. Invest Ophthalmol Vis Sci 50:1559–1565PubMedCrossRefGoogle Scholar
  28. 28.
    Prueitt RL, Boersma BJ, Howe TM, Goodman JE, Thomas DD, Ying L, Pfiester CM, Yfantis HG, Cottrell JR, Lee DH, Remaley AT, Hofseth LJ, Wink DA, Ambs S (2007) Inflammation and IGF-I activate the Akt pathway in breast cancer. Int J Cancer 120:796–805PubMedCrossRefGoogle Scholar
  29. 29.
    Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez-Boussard T, Livasy C, Cowan D, Dressler L, Akslen LA, Ragaz J, Gown AM, Gilks CB, Van de Rijn M, Perou CM (2004) Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10:5367–5374PubMedCrossRefGoogle Scholar
  30. 30.
    Boersma BJ, Howe TM, Goodman JE, Yfantis HG, Lee DH, Chanock SJ, Ambs S (2006) Association of breast cancer outcome with status of p53 and MDM2 SNP309. J Natl Cancer Inst 98:911–919PubMedCrossRefGoogle Scholar
  31. 31.
    Saldanha AJ (2004) Java Treeview—extensible visualization of microarray data. Bioinformatics 20:3246–3248PubMedCrossRefGoogle Scholar
  32. 32.
    Martin SE, Jones TL, Thomas CL, Lorenzi PL, Nguyen DA, Runfola T, Gunsior M, Weinstein JN, Goldsmith PK, Lader E, Huppi K, Caplen NJ (2007) Multiplexing siRNAs to compress RNAi-based screen size in human cells. Nucleic Acids Res 35:e57PubMedCrossRefGoogle Scholar
  33. 33.
    Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11:36–42Google Scholar
  34. 34.
    Sempere LF, Christensen M, Silahtaroglu A, Bak M, Heath CV, Schwartz G, Wells W, Kauppinen S, Cole CN (2007) Altered microRNA expression confined to specific epithelial cell subpopulations in breast cancer. Cancer Res 67:11612–11620PubMedCrossRefGoogle Scholar
  35. 35.
    Radojicic J, Zaravinos A, Vrekoussis T, Kafousi M, Spandidos DA, Stathopoulos EN (2011) MicroRNA expression analysis in triple-negative (ER, PR and Her2/neu) breast cancer. Cell Cycle 10:507–517PubMedCrossRefGoogle Scholar
  36. 36.
    Holland SJ, Powell MJ, Franci C, Chan EW, Friera AM, Atchison RE, McLaughlin J, Swift SE, Pali ES, Yam G, Wong S, Lasaga J, Shen MR, Yu S, Xu W, Hitoshi Y, Bogenberger J, Nör JE, Payan DG, Lorens JB (2005) Multiple roles for the receptor tyrosine kinase axl in tumor formation. Cancer Res 65:9294–9303PubMedCrossRefGoogle Scholar
  37. 37.
    Li Y, Ye X, Tan C, Hongo JA, Zha J, Liu J, Kallop D, Lulam MJ, Pei L (2009) Axl as a potential therapeutic target in cancer: role of Axl in tumor growth, metastasis, and angiogenesis. Oncogene 28:3442–3455PubMedCrossRefGoogle Scholar
  38. 38.
    Zhang YX, Knyazev PG, Cheburkin YV, Sharma K, Knyazev YP, Orfi L, Szabadkai I, Daub H, Kéri G, Ullrich A (2008) AXL is a potential target for therapeutic intervention in breast cancer progressio. Cancer Res 68:1905–1915PubMedCrossRefGoogle Scholar
  39. 39.
    Meric F, Lee W-P, Sahin A, Zhang H, Kung H-J, Hung M-C (2002) Expression profile of tyrosine kinases in breast cancer. Clin Cancer Res 8:361–367PubMedGoogle Scholar
  40. 40.
    Nagaraja GM, Othman M, Fox BP, Alsaber R, Pellegrino CM, Zeng Y, Khanna R, Tamburini P, Swaroop A, Kandpal RP (2006) Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics. Oncogene 25:2328–2338PubMedCrossRefGoogle Scholar
  41. 41.
    Bommer GT, Gerin I, Feng Y, Kaczorowski AJ, Kuick R, Love RE, Zhai Y, Giordano TJ, Qin ZS, Moore BB (2007) p53-mediated activiation of miRNA34 candidate tumor-suppressor genes. Curr Biol 17:1298–1307PubMedCrossRefGoogle Scholar
  42. 42.
    Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH, Feldmann G, Yamakuchi M, Ferlito M, Lowenstein CJ, Arking DE, Beer MA, Maitra A, Mendell JT (2007) Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Molecular Cell 26(5):745–752.PubMedCrossRefGoogle Scholar
  43. 43.
    Corney DC, Flesken-Nikitin A, Godwin AK, Wang W, Nikitin AY (2007) MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. Cancer Res 67:8433–8438PubMedCrossRefGoogle Scholar
  44. 44.
    He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y, Xue W, Zender L, Magnus J, Ridzon D, Jackson AL, Linsley PS, Chen C, Lowe SW, Cleary MA, Hannon GJ (2007) A microRNA component of the p53 tumour suppressor network. Nature 447:1130–1134PubMedCrossRefGoogle Scholar
  45. 45.
    He X, He L, Hannon GJ (2007) The guardian’s little helper: microRNAs in the p53 tumor suppressor network. Cancer Res 67:11099–11101PubMedCrossRefGoogle Scholar
  46. 46.
    Hermeking H (2007) p53 enters the microRNA world. Cancer Cell 12:414–418PubMedCrossRefGoogle Scholar
  47. 47.
    Raver-Shapira N, Marciano E, Meiri E, Spector Y, Rosenfeld N, Moskovits N, Bentwich Z, Oren M (2007) Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell 26:731–743PubMedCrossRefGoogle Scholar
  48. 48.
    Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen A, Meister G, Hermeking H (2007) Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 6:1586–1593PubMedCrossRefGoogle Scholar
  49. 49.
    Mudduluru G, Ceppi P, Kumarswamy R, Scagliotti GV, Papotti M, Allgayer H (2011) Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer. Oncogene 30:2888–2899Google Scholar
  50. 50.
    Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP (2008) The impact of microRNAs on protein output. Nature 455:64–71PubMedCrossRefGoogle Scholar
  51. 51.
    Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455:58–63PubMedCrossRefGoogle Scholar
  52. 52.
    Fridell YW, Villa J Jr, Attar EC, Liu ET (1998) GAS6 induces Axl-mediated chemotaxis of vascular smooth muscle cells. J Biol Chem 273:7123–7126PubMedCrossRefGoogle Scholar
  53. 53.
    Hafizi S, Dahlbäck B (2006) Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases. Cytokine Growth Factor Rev 17:295–304PubMedCrossRefGoogle Scholar
  54. 54.
    Koorstra J-B, Karikari CA, Feldmann G, Bisht S, Rojas PL, Offerhaus GJ, Alvarez H, Maitra A (2009) The Axl recptor tyrosine kinase confers an adverse prognostic influence in pancreatic cancer and represents a new therapeutic target. Cancer Biol Ther 8:618–626PubMedCrossRefGoogle Scholar
  55. 55.
    Pierce A, Bliesner B, Xu M, Nielsen-Preiss S, Lemke G, Tobet S, Wierman ME (2008) Axl and Tyro3 modulate female reproduction by influencing gonadotropin-releasing hormone neuron survival and migration. Mol Endocrinol 22:2481–2495PubMedCrossRefGoogle Scholar
  56. 56.
    Forbes SA, Bhamra G, Bamford S, Dawson E, Kok C, Clements J, Menzies A, Teague JW, Futreal PA, Stratton MR (2008) The catalogue of somatic mutations in cancer (COSMIC). Curr Protocols Hum Genet 10.11:1–26Google Scholar
  57. 57.
    Welch C, Chen Y, Stallings RL (2007) MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells. Oncogene 26:5017–5022PubMedCrossRefGoogle Scholar
  58. 58.
    Hermeking H (2010) The miR-34 family in cancer and apoptosis. Cell Death Differ 17:193–199PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. (outside the USA)  2011

Authors and Affiliations

  • Mark Mackiewicz
    • 1
  • Konrad Huppi
    • 1
  • Jason J. Pitt
    • 1
  • Tiffany H. Dorsey
    • 2
  • Stefan Ambs
    • 2
  • Natasha J. Caplen
    • 1
    Email author
  1. 1.Genetics Branch, Center for Cancer ResearchNational Cancer Institute, National Institutes of HealthBethesdaUSA
  2. 2.Laboratory of Human Carcinogenesis, Center for Cancer ResearchNational Cancer Institute, National Institutes of HealthBethesdaUSA

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