Clinical & Experimental Metastasis

, Volume 33, Issue 8, pp 829–838 | Cite as

Expression of natural killer cell regulatory microRNA by uveal melanoma cancer stem cells

  • Powrnima Joshi
  • Mitra Kooshki
  • Wayne Aldrich
  • Daniel Varghai
  • Maciej Zborowski
  • Arun D. Singh
  • Pierre L. TriozziEmail author
Research Paper


Natural killer (NK) cells are implicated in the control of metastasis in uveal melanoma, a process that has been ascribed to its cancer stem cell subpopulation. NK cell activation is regulated by specific microRNA (miR). The NK cell sensitivity and regulatory miR production of uveal melanoma cancer stem cells was examined. Cancer stem cells enriched from aggressively metastatic MUM2B uveal melanoma cells by selecting CD271+ cells or propagating as non-adherent spheres in stem-cell supportive were more resistant to NK cell cytolysis than cancer stem cells enriched from less aggressively metastatic OCM1 uveal melanoma cells. Both MUM2B and OCM1 cells expressed and secreted NK cell regulatory miRs, including miR 146a, 181a, 20a, and 223. MUM2B cells expressed and secreted miR-155; OCM1 cells did not. Transfecting MUM2B cells with anti-miR-155 increased NK cell sensitivity. CD271+ cells were identified in the blood of patients with metastatic uveal melanoma and were characterized by low expression of melanocyte differentiation determinants and by the ability to form non-adherent spheres in stem-cell supportive media. These cells also expressed NK cell regulatory miRs, including miR-155. These results indicate that uveal melanoma cancer stem cells can vary in their sensitivity to NK cell lysis and their expression of NK cell regulatory miRs. Circulating CD271+ cells from patients with metastatic uveal melanoma manifest cancer stem cell features and express miRs associated with NK cell suppression, including miR-155, that may contribute to metastatic progression.


CD271 Melanospheres miR-155 Major histocompatibility complex class I molecules MHC class I-related chain A Microphthalmia-associated transcription factor 



This work was supported in part by R21CA175671 from the National Cancer Institute, National Institutes of Health, Bethesda, MD.

Compliance with ethical standards

Conflict of interest

The authors declare that they do not have any competing or financial interests.

Ethical standards

Blood samples were collected from patients with metastatic uveal melanoma and from normal subjects according to protocols approved by the Cleveland Clinic and Wake Forest University Institutional Review Boards. All subjects gave informed consent prior to inclusion in the study. This research was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Supplementary material

10585_2016_9815_MOESM1_ESM.docx (33.9 mb)
Supplementary material 1 (DOCX 34671 kb)


  1. 1.
    Dithmar S, Rusciano D, Lynn MJ, Lawson DH, Armstrong CA, Grossniklaus HE (2000) Neoadjuvant interferon alfa-2b treatment in a murine model for metastatic ocular melanoma: a preliminary study. Arch Ophthalmol 118:1085–1089CrossRefPubMedGoogle Scholar
  2. 2.
    Blom DJ, Luyten GP, Mooy C, Kerkvliet S, Zwinderman AH, Jager MJ (1997) Human leukocyte antigen class I expression: marker of poor prognosis in uveal melanoma. Investig Ophthalmol Vis Sci 38:1865–1872Google Scholar
  3. 3.
    Ericsson C, Seregard S, Bartolazzi A, Levitskaya E, Ferrone S, Kiessling R, Larsson O (2001) Association of HLA class I and class II antigen expression and mortality in uveal melanoma. Investig Ophthalmol Vis Sci 42:2153–2156Google Scholar
  4. 4.
    Vetter CS, Lieb W, Bröcker EB, Becker JC (2004) Loss of nonclassical MHC molecules MIC-A/B expression during progression of uveal melanoma. Br J Cancer 91:1495–1499PubMedPubMedCentralGoogle Scholar
  5. 5.
    Ma D, Luyten GP, Luider TM, Niederkorn JY (1995) Relationship between natural killer cell susceptibility and metastasis of human uveal melanoma cells in a murine model. Investig Ophthalmol Vis Sci 36:435–441Google Scholar
  6. 6.
    Blom DJ, De Waard-Siebinga I, Apte RS, Luyten GP, Niederkorn JY, Jager MJ (1997) Effect of hyperthermia on expression of histocompatibility antigens and heat-shock protein molecules on three human ocular melanoma cell lines. Melanoma Res 7:103–109CrossRefPubMedGoogle Scholar
  7. 7.
    Repp AC, Mayhew ES, Apte S, Niederkorn JY (2000) Human uveal melanoma cells produce macrophage migration-inhibitory factor to prevent lysis by NK cells. J Immunol 165:710–715CrossRefPubMedGoogle Scholar
  8. 8.
    Jager MJ, Hurks HM, Levitskaya J, Kiessling R (2002) HLA expression in uveal melanoma: there is no rule without some exception. Hum Immunol 63:444–451CrossRefPubMedGoogle Scholar
  9. 9.
    Chang SH, Worley LA, Onken MD, Harbour JW (2008) Prognostic biomarkers in uveal melanoma: evidence for a stem cell-like phenotype associated with metastasis. Melanoma Res 18:191–200CrossRefPubMedGoogle Scholar
  10. 10.
    Kalirai H, Damato BE, Coupland SE (2011) Uveal melanoma cell lines contain stem-like cells that self-renew, produce differentiated progeny, and survive chemotherapy. Investig Ophthalmol Vis Sci 52:8458–8466CrossRefGoogle Scholar
  11. 11.
    Thill M, Berna MJ, Grierson R, Reinhart I, Voelkel T, Piechaczek C, Galambos P, Jager MJ, Richard G, Lange C, Gehling UM (2011) Expression of CD133 and other putative stem cell markers in uveal melanoma. Melanoma Res 21:405–416CrossRefPubMedGoogle Scholar
  12. 12.
    Valyi-Nagy K, Kormos B, Ali M, Shukla D, Valyi-Nagy T (2012) Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures. Mol Vis 18:588–592PubMedPubMedCentralGoogle Scholar
  13. 13.
    Matatall KA, Agapova OA, Onken MD, Worley LA, Bowcock AM, Harbour JW (2013) BAP1 deficiency causes loss of melanocytic cell identity in uveal melanoma. BMC Cancer 13:371CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Torres V, Triozzi P, Eng C, Tubbs R, Schoenfiled L, Crabb JW, Saunthararajah Y, Singh AD (2011) Circulating tumor cells in uveal melanoma. Future Oncol 7:101–109CrossRefPubMedGoogle Scholar
  15. 15.
    Murphy GF, Wilson BJ, Girouard SD, Frank NY, Frank MH (2014) Stem cells and targeted approaches to melanoma cure. Mol Asp Med 39:33–49CrossRefGoogle Scholar
  16. 16.
    Civenni G, Walter A, Kobert N, Mihic-Probst D, Zipser M, Belloni B, Seifert B, Moch H, Dummer R, van den Broek M, Sommer L (2011) Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth. Cancer Res 71:3098–3109CrossRefPubMedGoogle Scholar
  17. 17.
    Tseng HC, Arasteh A, Paranjpe A, Teruel A, Yang W, Behel A, Alva JA, Walter G, Head C, Ishikawa TO, Herschman HR, Cacalano N, Pyle AD, Park NH, Jewett A (2010) Increased lysis of stem cells but not their differentiated cells by natural killer cells; de-differentiation or reprogramming activates NK cells. PLoS One 5:e11590CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Tseng HC, Inagaki A, Bui VT, Cacalano N, Kasahara N, Man YG, Jewett A (2015) Differential targeting of stem cells and differentiated glioblastomas by NK cells. J Cancer 6:866–876CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Pietra G, Manzini C, Vitale M, Balsamo M, Ognio E, Boitano M, Queirolo P, Moretta L, Mingari MC (2009) Natural killer cells kill human melanoma cells with characteristics of cancer stem cells. Int Immunol 21:793–801CrossRefPubMedGoogle Scholar
  20. 20.
    Liu C, Tang DG (2011) MicroRNA regulation of cancer stem cells. Cancer Res 71:5950–5954CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Cichocki F, Felices M, McCullar V, Presnell SR, Al-Attar A, Lutz CT, Miller JS (2011) Cutting edge: microRNA-181 promotes human NK cell development by regulating Notch signaling. J Immunol 187:6171–6175CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Trotta R, Chen L, Ciarlariello D, Josyula S, Mao C, Costinean S, Yu L, Butchar JP, Tridandapani S, Croce CM, Caligiuri MA (2012) miR-155 regulates IFN-γ production in natural killer cells. Blood 119:3478–3485CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Fehniger TA, Wylie T, Germino E, Leong JW, Magrini VJ, Koul S, Keppel CR, Schneider SE, Koboldt DC, Sullivan RP, Heinz ME, Crosby SD, Nagarajan R, Ramsingh G, Link DC, Ley TJ, Mardis ER (2010) Next-generation sequencing identifies the natural killer cell microRNA transcriptome. Genome Res 20:1590–1604CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Sun X, Zhang J, Hou Z, Han Q, Zhang C, Tian Z (2015) miR-146a is directly regulated by STAT3 in human hepatocellular carcinoma cells and involved in anti-tumor immune suppression. Cell Cycle 14:243–252CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Stern-Ginossar N, Gur C, Biton M, Horwitz E, Elboim M, Stanietsky N, Mandelboim M, Mandelboim O (2008) Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D. Nat Immunol 9:1065–1073CrossRefPubMedGoogle Scholar
  26. 26.
    Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659CrossRefPubMedGoogle Scholar
  27. 27.
    He S, Chu J, Wu LC, Mao H, Peng Y, Alvarez-Breckenridge CA, Hughes T, Wei M, Zhang J, Yuan S, Sandhu S, Vasu S, Benson DM Jr, Hofmeister CC, He X, Ghoshal K, Devine SM, Caligiuri MA, Yu J (2013) MicroRNAs activate natural killer cells through Toll-like receptor signaling. Blood 121:4663–4671CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Achberger S, Aldrich W, Tubbs R, Crabb JW, Singh AD, Triozzi PL (2014) Circulating immune cell and microRNA in patients with uveal melanoma developing metastatic disease. Mol Immunol 58:182–186CrossRefPubMedGoogle Scholar
  29. 29.
    Folberg R, Kadkol SS, Frenkel S, Valyi-Nagy K, Jager MJ, Pe’er J, Maniotis AJ (2008) Authenticating cell lines in ophthalmic research laboratories. Investig Ophthalmol Vis Sci 49:4697–4701CrossRefGoogle Scholar
  30. 30.
    Folberg R, Leach L, Valyi-Nagy K, Lin AY, Apushkin MA, Ai Z, Barak V, Majumdar D, Pe’er J, Maniotis AJ (2007) Modeling the behavior of uveal melanoma in the liver. Investig Ophthalmol Vis Sci 48:2967–2974CrossRefGoogle Scholar
  31. 31.
    Duan F, Lin M, Li C, Ding X, Qian G, Zhang H, Ge S, Fan X, Li J (2014) Effects of inhibition of hedgehog signaling on cell growth and migration of uveal melanoma cells. Cancer Biol Ther 15:544–559CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Truzzi F, Marconi A, Lotti R, Dallaglio K, French LE, Hempstead BL, Pincelli C (2008) Neurotrophins and their receptors stimulate melanoma cell proliferation and migration. J Investig Dermatol 128:2031–2040CrossRefPubMedGoogle Scholar
  33. 33.
    Sullivan RP, Fogel LA, Leong JW, Schneider SE, Wong R, Romee R, Thai TH, Sexl V, Matkovich SJ, Dorn GW 2nd, French AR, Fehniger TA (2013) MicroRNA-155 tunes both the threshold and extent of NK cell activation via targeting of multiple signaling pathways. J Immunol 191:5904–5913CrossRefPubMedGoogle Scholar
  34. 34.
    Elemam NM, Mekky RY, El-Ekiaby NM, El Sobky SA, El Din MA, Esmat G, Abdelaziz AI (2015) Repressing PU.1 by miR-29a∗ in NK cells of HCV patients, diminishes its cytolytic effect on HCV infected cell models. Hum Immunol 76:687–694CrossRefPubMedGoogle Scholar
  35. 35.
    Hallermalm K, Seki K, De Geer A, Motyka B, Bleackley RC, Jager MJ, Froelich CJ, Kiessling R, Levitsky V, Levitskaya J (2008) Modulation of the tumor cell phenotype by IFN-gamma results in resistance of uveal melanoma cells to granule-mediated lysis by cytotoxic lymphocytes. J Immunol 180:3766–3774CrossRefPubMedGoogle Scholar
  36. 36.
    Levati L, Pagani E, Romani S, Castiglia D, Piccinni E, Covaciu C, Caporaso P, Bondanza S, Antonetti FR, Bonmassar E, Martelli F, Alvino E, D’Atri S (2011) MicroRNA-155 targets the SKI gene in human melanoma cell lines. Pigment Cell Melanoma Res 24:538–550CrossRefPubMedGoogle Scholar
  37. 37.
    Liu F, Kong X, Lv L, Gao J (2015) TGF-β1 acts through miR-155 to down-regulate TP53INP1 in promoting epithelial-mesenchymal transition and cancer stem cell phenotypes. Cancer Lett 359:288–298CrossRefPubMedGoogle Scholar
  38. 38.
    Ji J, Zheng X, Forgues M, Yamashita T, Wauthier EL, Reid LM, Wen X, Song Y, Wei JS, Khan J, Thorgeirsson SS, Wang XW (2015) Identification of microRNAs specific for epithelial cell adhesion molecule-positive tumor cells in hepatocellular carcinoma. Hepatology 62:829–840CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Zhang GJ, Xiao HX, Tian HP, Liu ZL, Xia SS, Zhou T (2013) Upregulation of microRNA-155 promotes the migration and invasion of colorectal cancer cells through the regulation of claudin-1 expression. Int J Mol Med 31:1375–1380PubMedGoogle Scholar
  40. 40.
    Kong W, He L, Richards EJ, Challa S, Xu CX, Permuth-Wey J, Lancaster JM, Coppola D, Sellers TA, Djeu JY, Cheng JQ (2014) Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer. Oncogene 33:679–689CrossRefPubMedGoogle Scholar
  41. 41.
    Tomellini E, Lagadec C, Polakowska R, Le Bourhis X (2014) Role of p75 neurotrophin receptor in stem cell biology: more than just a marker. Cell Mol Life Sci 71:2467–2481CrossRefPubMedGoogle Scholar
  42. 42.
    Cheli Y, Bonnazi VF, Jacquel A, Allegra M, De Donatis GM, Bahadoran P, Bertolotto C, Ballotti R (2014) CD271 is an imperfect marker for melanoma initiating cells. Oncotarget 5(5272–83):39Google Scholar
  43. 43.
    Bergeron MA, Champagne S, Gaudreault M, Deschambeault A, Landreville S (2012) Repression of genes involved in melanocyte differentiation in uveal melanoma. Mol Vis 18:1813–1822PubMedPubMedCentralGoogle Scholar
  44. 44.
    Landreville S, Lupien CB, Vigneault F, Gaudreault M, Mathieu M, Rousseau AP, Guerin SL, Salesse C (2011) Identification of differentially expressed genes in uveal melanoma using suppressive subtractive hybridization. Mol Vis 17:1324–1333PubMedPubMedCentralGoogle Scholar
  45. 45.
    Melamed I, Kelleher CA, Franklin RA, Brodie C, Hempstead B, Kaplan D, Gelfand EW (1996) Nerve growth factor signal transduction in human B lymphocytes is mediated by gp140trk. Eur J Immunol 26:1985–1992CrossRefPubMedGoogle Scholar
  46. 46.
    Dome B, Timar J, Dobos J, Meszaros L, Raso E, Paku S, Kenessey I, Ostoros G, Magyar M, Ladanyi A, Bogos K, Tovari J (2006) Identification and clinical significance of circulating endothelial progenitor cells in human non-small cell lung cancer. Cancer Res 66:7341–7347CrossRefPubMedGoogle Scholar
  47. 47.
    Vroling L, Lind JS, de Haas RR, Verheul HM, van Hinsbergh VW, Broxterman HJ, Smit EF (2010) CD133+ circulating haematopoietic progenitor cells predict for response to sorafenib plus erlotinib in non-small cell lung cancer patients. Br J Cancer 102:268–275CrossRefPubMedGoogle Scholar
  48. 48.
    Damato B, Coupland SE (2009) Translating uveal melanoma cytogenetics into clinical care. Arch Ophthalmol 127:423–429CrossRefPubMedGoogle Scholar
  49. 49.
    Onken MD, Worley LA, Tuscan MD, Harbour JW (2010) An accurate, clinically feasible multi-gene expression assay for predicting metastasis in uveal melanoma. J Mol Diagn 12:461–468CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Ferracin M, Veronese A, Negrini M (2010) Micromarkers: miRNAs in cancer diagnosis and prognosis. Expert Rev Mol Diagn 10:297–308CrossRefPubMedGoogle Scholar
  51. 51.
    Cubillos-Ruiz JR, Baird JR, Tesone AJ, Rutkowski MR, Scarlett UK, Camposeco-Jacobs AL, Anadon-Arnillas J, Harwood NM, Korc M, Fiering SN, Sempere LF, Conejo-Garcia JR (2012) Reprogramming tumor-associated dendritic cells in vivo using miRNA mimetics triggers protective immunity against ovarian cancer. Cancer Res 72:1683–1693CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Jackson A, Linsley PS (2010) The therapeutic potential of microRNA modulation. Discov Med 9:311–318PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Lerner Research InstituteCleveland Clinic FoundationClevelandUSA
  2. 2.Comprehensive Cancer CenterWake Forest UniversityWinston-SalemUSA
  3. 3.Taussig Cancer InstituteCleveland Clinic FoundationClevelandUSA
  4. 4.Cole Eye InstituteCleveland Clinic FoundationClevelandUSA
  5. 5.Wake Forest School of MedicineWinston-SalemUSA

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