Advertisement

Clinical & Experimental Metastasis

, Volume 36, Issue 3, pp 271–290 | Cite as

Identification of canonical NFκB (C-NFκB) pathway in uveal melanoma and their relation with patient outcome

  • Mithalesh Kumar Singh
  • Lata Singh
  • Neelam Pushker
  • Neeru Saini
  • Rachna Meel
  • Kunzang Chosdol
  • Sameer Bakhshi
  • Seema Sen
  • Pradeep Venkatesh
  • Bhavna Chawla
  • Jasbir Kaur
  • Seema KashyapEmail author
Research Paper

Abstract

Inflammation in uveal melanoma (UM) is linked to a bad prognosis. It is rare type of cancer, of which the metastases are usually fatal within a year. Infiltration with an inflammatory infiltrate increases with disease progression but does not seem to inhibit metastasis. The Canonical NFκB (C-NFκB) pathway is known to play a crucial role in tumor inflammation. We therefore, studied the expression of canonical NFκB proteins and their prognostic relevance in UM. Our study evaluated the expression of C-NFκB proteins (p65, p50, and c-Rel) by using immunohistochemistry on sections from 75 formalin-fixed UM. Activation of the NFκB subunit was determined on fresh tumor specimens by measuring the DNA-binding activity in nuclei using an NFκB ELISA assay. Real-time PCR was performed on frozen material on 58 tumors. The presence of native C-NFκB heterodimers (p65/p50 and c-Rel/p50) was confirmed by co-immunoprecipitation followed by Western blotting. We observed a high nuclear immunoreactivity of p65, p50, and c-Rel proteins in 54, 60 and 41% UM cases, respectively. Expression of C-NFκB proteins significantly correlated with parameters which are related to the inflammatory environment of UM. Nuclear immunoreactivity of p65 and p50 was associated with lower patient survival (p = 0.041; p = 0.048) while c-Rel was not. Our finding reveals that C-NFκB proteins expressed are more often in UM with inflammation than those without inflammation. Activation of the canonical NFκB pathway is more frequent in high risk UM patients. These observations might help to understand the behaviour of high risk tumors, with upregulation of C-NFκB proteins contributing to tumor aggressiveness.

Keywords

Uveal melanoma NFκB Inflammation Immunohistochemistry p65/p50 c-Rel/p50 heterodimer 

Notes

Acknowledgments

We are very grateful to Mr. Pankaj Kumar for his excellent technical assistance.

Author contributions

MKS and SK are responsible for the conception or design of the work; MKS, LS, KC, NS, and JK contributes the acquisition, analysis, or interpretation of data for the work; NP, BC, RM and PV provides the tissue samples; SB, RM Helps in the follow up of the patients; SS helps in reviewing the histopathology slides; All authors finally approved the manuscript version to be published. SK is the guarantor of the article.

Funding

Mithalesh Kumar Singh supported by Indian Council of Medical Research, which provided a Senior Research Fellowship (SRF) and conducted this research with Grant No. 3/2/2/327/2015-NCD-III.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Ethical approval

Ethical approval obtained from the Institute’s Ethical Committee, All India Institute of Medical Sciences (Ref. No. IESC/T-417/2015) and carried out by the declaration of Helsinki principles.

Research involving human participants and informed consent

Written consent obtained from all the patients.

Supplementary material

10585_2019_9969_MOESM1_ESM.tif (133 kb)
Supplementary material 1—Supplementary Figure 1 a Nuclear Immunoreactivity patterns of C-NFκB proteins uveal melanoma; b Relative immunoreactivity patterns of C-NFκB proteins in group I and group II uveal melanoma (TIFF 132 kb)

References

  1. 1.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674PubMedCrossRefGoogle Scholar
  2. 2.
    Maat W, Ly LV, Jordanova ES, De Wolff-Rouendaal D, Schalij-Delfos NE, Jager MJ (2008) Monosomy of chromosome 3 and an inflammatory phenotype occur together in uveal melanoma. Invest Ophthalmol Vis Sci 49(2):505–510PubMedCrossRefGoogle Scholar
  3. 3.
    Niederkorn JY, Wang S (2005) Immunology of intraocular tumors. Ocul Immunol Inflamm 13(1):105–110PubMedCrossRefGoogle Scholar
  4. 4.
    Hawkins BS (2004) The collaborative ocular melanoma study (COMS) randomized trial of pre-enucleation radiation of large choroidal melanoma: IV. Ten-year mortality findings and prognostic factors. COMS report number 24. Am J Ophthalmol 138(6):936–951PubMedCrossRefGoogle Scholar
  5. 5.
    Collaborative Ocular Melanoma Study Group (2006) The COMS randomized trial of iodine 125 brachytherapy for choroidal melanoma: V Twelve-year mortality rates and prognostic factors: COMS report no. 28. Arch Ophthalmol 124(12):1684–1693CrossRefGoogle Scholar
  6. 6.
    van Essen TH, van Pelt SI, Bronkhorst IH, Versluis M, Némati F, Laurent C, Luyten GP, van Hall T, van den Elsen PJ, van der Velden PA, Decaudin D, Jager MJ (2016) Upregulation of HLA expression in primary uveal melanoma by infiltrating leukocytes. PLoS ONE 11(10):e0164292PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    de Lange MJ, Nell RJ, Lalai RN, Versluis M, Jordanova ES, Luyten GPM, Jager MJ, van der Burg SH, Zoutman WH, van Hall T, Velden PA (2018) Digital PCR-based T-cell quantification-assisted deconvolution of the microenvironment reveals that activated macrophages drive tumor inflammation in uveal melanoma. Mol Cancer Res 16(12):1902–1911PubMedCrossRefGoogle Scholar
  8. 8.
    Mlecnik B, Bindea G, Pages F, Galon J (2011) Tumor immunosurveillance in human cancers. Cancer Metast Rev 30(1):5–12CrossRefGoogle Scholar
  9. 9.
    Bronkhorst IH, Jager MJ (2013) Inflammation in uveal melanoma. Eye (Lond). 27(2):217–223PubMedCrossRefGoogle Scholar
  10. 10.
    Maat W, Ly LV, Jordanova ES, de Wolff-Rouendaal D, Schalij-Delfos NE, Jager MJ (2008) Monosomy of chromosome 3 and an inflammatory phenotype occur together in uveal melanoma. Invest Ophthalmol Vis Sci 49(2):505–510PubMedCrossRefGoogle Scholar
  11. 11.
    Bronkhorst IH, Ly LV, Jordanova ES, Vrolijk J, Versluis M, Luyten GP, Jager MJ (2011) Detection of M2-macrophages in uveal melanoma and relation with survival. Invest Ophthalmol Vis Sci 52(2):643–650PubMedCrossRefGoogle Scholar
  12. 12.
    Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454(7203):436–444PubMedCrossRefGoogle Scholar
  13. 13.
    Karin M (2006) Nuclear factor-κB in cancer development and progression. Nature 441(7092):431–436PubMedCrossRefGoogle Scholar
  14. 14.
    Yu H, Kortylewski M, Pardoll D (2007) Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment. Nat Rev. Immunol 7(1):41–51PubMedCrossRefGoogle Scholar
  15. 15.
    Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E, Ben-Neriah Y (2004) NF-kB functions as a tumour promoter in inflammation-associated cancer. Nature 431(7007):461–466PubMedCrossRefGoogle Scholar
  16. 16.
    Karin M, Cao Y, Greten FR, Li ZW (2002) NF-κB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2(4):301–310PubMedCrossRefGoogle Scholar
  17. 17.
    Karin M, Delhase M (2000) The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatory signalling. Semin Immunol 12(1):85–98PubMedCrossRefGoogle Scholar
  18. 18.
    Beinke S, Ley SC (2004) Functions of NF-kappaB1 and NF-kappaB2 in immune cell biology. Biochem J 382(Pt 2):393–409PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Hayden MS, Ghosh S (2008) Shared principles in NF-kappaB signaling. Cell 132(3):344–362PubMedCrossRefGoogle Scholar
  20. 20.
    Ben-Neriah Y, Karin M (2011) Inflammation meets cancer, with NF-κB as the matchmaker. Nat Immunol 12(8):715–723PubMedCrossRefGoogle Scholar
  21. 21.
    Hoesel B, Schmid JA (2013) The complexity of NF-κB signaling in inflammation and cancer. Mol Cancer 12(1):86PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Howe LR (2007) Inflammation and breast cancer. Cyclooxygenase/prostaglandin signaling and breast cancer. Breast Cancer Res 9(4):210PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Shattuck-Brandt RL, Richmond A (1997) Enhanced degradation of I-kappaB alpha contributes to endogenous activation of NF-kappaB in Hs294T melanoma cells. Cancer Res 57(14):3032–3039PubMedGoogle Scholar
  24. 24.
    Tai DI, Tsai SL, Chang YH, Huang SN, Chen TC, Chang KS, Liaw YF (2000) Constitutive activation of nuclear factor kappaB in hepatocellular carcinoma. Cancer 89(11):2274–2281PubMedCrossRefGoogle Scholar
  25. 25.
    Wang W, Abbruzzese JL, Evans DB, Larry L, Cleary KR, Chiao PJ (1999) The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Clin Cancer Res 5(1):119–127PubMedGoogle Scholar
  26. 26.
    Smyth MJ, Dunn GP, Schreiber RD (2006) Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol 90:1–50PubMedCrossRefGoogle Scholar
  27. 27.
    Dror R, Lederman M, Umezawa K, Barak V, Pe’er J, Chowers I (2010) Characterizing the involvement of the nuclear factor kappa B (NF kappa B) transcription factor in uveal melanoma. Invest Ophthalmol Vis Sci 51(4):1811–1816PubMedCrossRefGoogle Scholar
  28. 28.
    Kaliki S, Shields CL, Shields JA (2015) Uveal melanoma: estimating prognosis. Indian J Ophthalmol 63(2):93–102PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Jager MJ, Ly LV, El Filali M, Madigan MC (2011) Macrophages in uveal melanoma and in experimental ocular tumor models: friends or foes? Prog Retin Eye Res 30(2):129–146PubMedCrossRefGoogle Scholar
  30. 30.
    Mäkitie T, Summanen P, Tarkkanen A, Kivelä T (2001) Tumor-infiltrating macrophages (CD68(+) cells) and prognosis in malignant uveal melanoma. Invest Ophthalmol Vis Sci 42(7):1414–1421PubMedGoogle Scholar
  31. 31.
    Kivela T, Simpson RE, Grossniklaus HE (2016) Uveal melanoma. In: Amin MB (ed) AJCC cancer staging 429 manual. Springer, New York, pp 805–817Google Scholar
  32. 32.
    Rosen T, Schulkin J, Power M, Tadesse S, Norwitz ER, Wen Z, Wang B (2015) Comparative immunohistochemistry of placental corticotropin-releasing hormone and the transcription factor RelB-NFκB2 between humans and nonhuman primates. Comp Med 65(2):140–143PubMedPubMedCentralGoogle Scholar
  33. 33.
    Li Zhiqin, Han Chunfang, Feng Jing (2017) Relationship of the expression levels of XIAP and p53 genes in hepatocellular carcinoma and the prognosis of patients. Oncol Lett 14(4):4037–4042PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Singh MK, Pushker N, Meel R, Chodsol K, Sen S, Bakhshi S, Singh L, Kashyap S (2018) Does NEMO/IKKγ protein have a role in determining prognostic significance in uveal melanoma? Clin Transl Oncol 20(12):1592–1603PubMedCrossRefGoogle Scholar
  35. 35.
    Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A (2009) Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis 30(7):1073–1081PubMedCrossRefGoogle Scholar
  36. 36.
    Huang DB, Vu D, Ghosh G (2005) NF-B RelB forms an intertwined homodimer. Structure 13(9):1365–1373PubMedCrossRefGoogle Scholar
  37. 37.
    Pallares J, Martinez-Guitarte JL, Dolcet X, Llobet D, Rue M, Palacios J, Prat J, Matias-Guiu X (2004) Abnormalities in the NF-kB family and related proteins in endometrial carcinoma. J Pathol 204(5):569–577PubMedCrossRefGoogle Scholar
  38. 38.
    Ross JS, Kallakury BV, Sheehan CE, Fisher HA, Kaufman RP Jr, Kaur P, Gray K, Stringer B (2004) Expression of nuclear factor-skappa B and I kappa B alpha proteins in prostatic adenocarcinomas: correlation of nuclear factor-kappa B immunoreactivity with disease recurrence. Clin Cancer Res 10(7):2466–2472PubMedCrossRefGoogle Scholar
  39. 39.
    Li L, Xu-Monette ZY, Ok CY, Tzankov A, Manyam GC, Sun R, Visco C, Zhang M et al (2015) Prognostic impact of c-Rel nuclear expression and REL amplification and crosstalk between c-Rel and the p53 pathway in diffuse large B-cell lymphoma. Oncotarget 15(6):23517–23580Google Scholar
  40. 40.
    Moorchung N, Kunwar S, Ahmed KW (2014) An evaluation of nuclear factor kappa B expression in colorectal carcinoma: an analysis of 50 cases. J Cancer Res Ther 10(3):631–635PubMedGoogle Scholar
  41. 41.
    Durairajan S, Walter CEJ, Samuel MD, Palani D, Pasupati S, Johnson T (2018) Differential expression of NF-κB heterodimer RelA/p50 in human urothelial carcinoma. PeerJ.  https://doi.org/10.7717/peerj.5563 PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Collaborative ocular melanoma study group (1998) Histopathologic characteristics of uveal melanomas in eyes enucleated from the Collaborative Ocular Melanoma Study. COMS report no 6. Am J Ophthalmol 125(6):745–766CrossRefGoogle Scholar
  43. 43.
    AJCC Ophthalmic Oncology Task Force (2015) International validation of the American Joint Committee on Cancer’s 7th edition classification of uveal melanoma. JAMA Ophthalmol 133(4):376–383CrossRefGoogle Scholar
  44. 44.
    Zhou Y, Eppenberger-Castori S, Eppenberger U, Benz CC (2005) The NFkappaB pathway and endocrine-resistant breast cancer. Endocr Relat Cancer 12(Suppl 1):37–46CrossRefGoogle Scholar
  45. 45.
    Zhou XL, Fan W, Yang G, Yu MX (2014) The clinical significance of PR, ER, NF- kappa B, and TNF- alpha in breast cancer. Dis Mark.  https://doi.org/10.1155/2014/494581 CrossRefGoogle Scholar
  46. 46.
    Qu Y, Zhou F, Dai X, Wang H, Shi J, Zhang X, Wang Y, Wei W (2011) Clinicopathologic significances of nuclear expression of nuclear factor-κB transcription factors in retinoblastoma. J Clin Pathol 64(8):695–700PubMedCrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Mithalesh Kumar Singh
    • 1
  • Lata Singh
    • 2
  • Neelam Pushker
    • 3
  • Neeru Saini
    • 4
  • Rachna Meel
    • 3
  • Kunzang Chosdol
    • 5
  • Sameer Bakhshi
    • 6
  • Seema Sen
    • 1
  • Pradeep Venkatesh
    • 3
  • Bhavna Chawla
    • 3
  • Jasbir Kaur
    • 7
  • Seema Kashyap
    • 1
    Email author
  1. 1.Department of Ocular Pathology, Dr. R. P. Centre for Ophthalmic SciencesAll India Institute of Medical SciencesNew DelhiIndia
  2. 2.Departrment of BiosciencesJamia Millia IslamiaNew DelhiIndia
  3. 3.Department of Ophthalmology, Dr. R. P. Centre for Ophthalmic SciencesAll India Institute of Medical SciencesNew DelhiIndia
  4. 4.Functional Genomics UnitInstitute of Genomics and Integrative BiologyNew DelhiIndia
  5. 5.Department of BiochemistryAIIMSNew DelhiIndia
  6. 6.Department of Medical Oncology, IRCHAIIMSNew DelhiIndia
  7. 7.Department of Ocular Biochemistry, Dr. R. P. Centre for Ophthalmic SciencesAll India Institute of Medical SciencesNew DelhiIndia

Personalised recommendations