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Role of Telomeric TRF2 in Orosphere Formation and CSC Phenotype Maintenance Through Efficient DNA Repair Pathway and its Correlation with Recurrence in OSCC

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Abstract

The major problem to effective treatment of oral cancer is the presence of therapy resistance. Presence of cancer stem cell in the bulk of tumor have been implicated in therapeutic resistance. In this study, we report a non-telomeric role of TRF2 in formation of oral cancer spheroids and CSC phenotype maintenance via an efficient DNA damage repair mechanism in the presence of chemotherapeutic insult. We report reduced sphere formation efficiency and reduced spheroid size in TRF2 silenced oral cancer cell lines. TRF2 silenced orospheres further reported reduced proliferative capacity as compared to non-silenced orospheres. Furthermore, TRF2 silencing hampered the migratory potential of oral cancer cell line and also reduced the expression of several CSC markers like CD44, Oct4, Sox2, KLF4 and c-Myc along with β-catenin and hTERT molecules both in Cal27 cell line and generated orospheres. TRF2 silencing impaired efficient DNA damage repair capacity of non-orospheric and orospheric cells and repressed ERCC1 expression levels when treated with Cisplatin. TRF2 overexpression was also observed to correlate with poor overall survival and disease relapse of OSCC patients. In silico studies further identified several amino acid residues that show high binding affinity and strong protein-protein interactions among TRF2 and CSC marker KLF4. Hence, our report confirms a non-telomeric role of TRF2 in spheroid generation, maintenance of CSC phenotype and efficient DNA damage repair capacity contributing to chemotherapy resistance in oral cancer cell line. We further iterate the use of TRF2 as a prognostic marker in OSCC for faster detection and improved survival.

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Fig. 1: TRF2 overexpression linked to poor survival and disease relapse.
Fig. 2: High expression of shelterin component and telomerase in orospheres.
Fig. 3: Reduced spheroid formation efficiency, size and proliferative capacity in absence of TRF2.
Fig. 4: Impaired migratory potential of Cal27 oral cancer cells.
Fig. 5: Transiently silencing TRF2 hampers efficient DNA damage repair capacity in the presence of Cisplatin.
Fig. 6: Loss of TRF2 function affects Cancer Stem Cell expression status.
Fig. 7: TRF2 silencing represses CSC marker expression in orospheres.
Fig. 8: Interaction between TRF2 and KLF4 proteins.

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Acknowledgements

This work was supported by extramural funding from Department of Biotechnology (DBT), Government of India (Grant No.:BT/PR/14659/MED/31/108/2010) and Board of Research in Nuclear Science, Department of Atomic Energy, Government of India (Grant No.:2013/35/45/BRNS). We also acknowledge the technical support of the MSSB (Molecular Stress and Stem Cell Biology) group, School of Biotechnology.

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  1. Arka Saha and Souvick Roy contributed equally to this work.

Authors and Affiliations

  1. Molecular Stress and Stem Cell Biology Group, KIIT School of Biotechnology, Disease Biology Lab, KIIT University, Bhubaneswar, Odisha, 751024, India

    Arka Saha, Souvick Roy, Shomereeta Roy, SwatiShree Padhi & Birendranath Banerjee

  2. Department of Surgical Oncology, All India Institute of Medical Sciences, (AIIMS), Bhubaneswar, Odisha, 751019, India

    Madhabananda Kar

  3. Centre for Computational Biology and Bioinformatics, School of Life Sciences, Central University of Himachal Pradesh, Shahpur, Himachal Pradesh, 176206, India

    Shweta Thakur

  4. Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow, Uttar Pradesh, 226025, India

    Yusuf Akhter

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Supplementary Figure 1

Interaction between TRF2 and CD44 proteins. A) Schematic representation of interacting amino acid residues of TRF2 and CD44 protein as analysed by Cluspro 2.0 molecular docking software. B-E) Molecular docking studies showing interaction of amino acid residues TRF2 and CD44 proteins. TRF2 amino acid residues are coded in green color, KLF4 amino acid residues are coded in pink color. F) Figure showing protein backbone of TRF2 in blue color. Direct inter residue interactions shown with solid lines and the water-mediated interactions in dash lines. Green denotes minimally frustrated regions, highly frustrated regions are in red and neutral regions are not indicated. G) Figure showing projection of local frustration distribution in the amino acid sequences of TRF2 protein. The number of contacts within 5 Å of the C-α of each residue is plotted, as classified according to their frustration index. H) Figure showing protein backbone of CD44 in blue color. Direct inter residue interactions are shown with solid lines and the water-mediated interactions in dash lines. Green denotes minimally frustrated regions, highly frustrated regions are in red and neutral regions are not shown. I) Figure showing projection of local frustration distribution in the amino acid sequences of KLF4 protein. The number of contacts within 5 Å of the C-α of each residue is plotted, as classified according to their frustration index. No interacting residues of high frustrations between TRF2 and CD44 were observed. (PNG 469 kb)

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Saha, A., Roy, S., Kar, M. et al. Role of Telomeric TRF2 in Orosphere Formation and CSC Phenotype Maintenance Through Efficient DNA Repair Pathway and its Correlation with Recurrence in OSCC. Stem Cell Rev and Rep 14, 871–887 (2018). https://doi.org/10.1007/s12015-018-9823-z

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