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

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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.

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Acknowledgments

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

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.

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Authors and Affiliations

  1. Department of Ocular Pathology, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India

    Mithalesh Kumar Singh, Seema Sen & Seema Kashyap

  2. Departrment of Biosciences, Jamia Millia Islamia, New Delhi, India

    Lata Singh

  3. Department of Ophthalmology, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India

    Neelam Pushker, Rachna Meel, Pradeep Venkatesh & Bhavna Chawla

  4. Functional Genomics Unit, Institute of Genomics and Integrative Biology, New Delhi, India

    Neeru Saini

  5. Department of Biochemistry, AIIMS, New Delhi, India

    Kunzang Chosdol

  6. Department of Medical Oncology, IRCH, AIIMS, New Delhi, India

    Sameer Bakhshi

  7. Department of Ocular Biochemistry, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India

    Jasbir Kaur

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  1. Mithalesh Kumar Singh
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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.

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Correspondence to Seema Kashyap.

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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.

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10585_2019_9969_MOESM1_ESM.tif

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)

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Singh, M.K., Singh, L., Pushker, N. et al. Identification of canonical NFκB (C-NFκB) pathway in uveal melanoma and their relation with patient outcome. Clin Exp Metastasis 36, 271–290 (2019). https://doi.org/10.1007/s10585-019-09969-y

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