Pathology & Oncology Research

, Volume 20, Issue 3, pp 687–695 | Cite as

Possible Prognostic Value of BORIS Transcript Variants Ratio in Laryngeal Squamous Cell Carcinomas – a Pilot Study

  • Renata Novak Kujundžić
  • Ivana Grbeša
  • Mirko Ivkić
  • Božo Krušlin
  • Paško Konjevoda
  • Koraljka Gall Trošelj


BORIS is a paralog of a highly conserved, multi-functional chromatin factor CTCF. Unlike CTCF, which has been shown to possess tumor-suppressive properties, BORIS belongs to the “cancer/testis antigen” family normally expressed only in germ cells and aberrantly activated in a variety of tumors. The consequences of BORIS expression, relative abundance of its isoforms, and its role in carcinogenesis have not been completely elucidated. It activates transcription of hTERT and MYC, genes relevant for laryngeal carcinoma progression. In this study, BORIS expression has been analyzed at the transcriptional level by RT-PCR and protein level by semi-quantitative immunohistochemistry in 32 laryngeal squamous cell carcinomas and adjacent non-tumorous tissue. BORIS was detected in 44 % (14/32) laryngeal squamous cell carcinoma samples, while it was detected only in one normal, tumor-adjacent tissue sample. Tree based survival analysis, using the recursive partitioning algorithm mvpart, extracted the ratio of relative abundance of BORIS transcript variants containing exon 7 (BORIS 7+) and those lacking exon 7 (BORIS 7−) as an independent prognostic factor associated with disease relapse during a 5-year follow-up period. Patients having BORIS 7+/BORIS 7− ratio ≥1 had a higher rate of disease relapse than patients with BORIS 7+/BORIS 7− ratio <1. Hazard ratio for that group, based on Cox Proportional Hazard Regression, was 3.53. This is the first study analyzing expression of BORIS protein and transcript variants in laryngeal squamous cell carcinoma relative to its possible prognostic value for recurrence and overall survival.


Laryngeal squamous cell carcinoma Prognostic value BORIS Transcript variant Immunohistochemistry RT-PCR 



This work was funded by grants 098-0982464-2511 and 108-1081870-1884 from the Ministry of Science, Education and Sport, Republic of Croatia.


  1. 1.
    Loukinov DI, Pugacheva E, Vatolin S, Pack SD, Moon H, Chernukin I, Mannan P, Larsson E, Kanduri C, Vostrov AA, Cui H, Niemitz EL, Rasko JE, Docquier FM, Kistler M, Breen JJ, Zhuang Z, Quitschke WW, Renkawitz R, Klenova EM, Feinberg AP, Ohlsson R, Morse HC 3rd, Lobanenkov VV (2002) BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proc Natl Acad Sci U S A 99:6806–6811PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Vatolin S, Abdullaev Z, Pack SD, Flanagan PT, Custer M, Loukinov DI, Pugacheva E, Hong JA, Morse H 3rd, Schrump DS, Risinger JI, Barrett JC, Lobanenkov VV (2005) Conditional expression of the CTCF-paralogous transcriptional factor BORIS in normal cells results in demethylation and derepression of MAGE-A1 and reactivation of other cancer-testis genes. Cancer Res 65:7751–7762PubMedGoogle Scholar
  3. 3.
    Hong JA, Kang Y, Abdullaev Z, Flanagan PT, Pack SD, Fischete MR, Adnani MT, Loukinov DI, Vatolin S, Risinger JI, Custer M, Chen GA, Zhao M, Nguyen DM, Barrett JC, Lobanenkov VV, Schrump DS (2005) Reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter coincides with derepression of this cancer-testis gene in lung cancer cells. Cancer Res 65:7763–7774PubMedGoogle Scholar
  4. 4.
    Hoffmann MJ, Müller M, Engers R, Schulz WA (2006) Epigenetic control of CTCFL/BORIS and OCT4 expression in urogenital malignancies. Biochem Pharmacol 72:1577–1588PubMedCrossRefGoogle Scholar
  5. 5.
    Klenova EM, Morse HC, Ohlsson R, Lobanenkov VV (2002) The novel BORIS + CTCF gene family is uniquely involved in epigenetics of normal biology and cancer. Semin Cancer Biol 12:399–414PubMedCrossRefGoogle Scholar
  6. 6.
    Ohlsson R, Renkawitz R, Lobanenkov V (2001) CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet 17:520–527PubMedCrossRefGoogle Scholar
  7. 7.
    Klenova E, Ohlsson R (2005) Poly(ADP-ribosyl)ation and epigenetics: is CTCF PARt of the plot? Cell Cycle 4:96–101PubMedCrossRefGoogle Scholar
  8. 8.
    Jelinic P, Stehle J-C, Shaw P (2006) The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation. PLoS Biol 4:1910–1922CrossRefGoogle Scholar
  9. 9.
    Fiorentino FP, Macaluso M, Miranda F, Montanari M, Russo A, Bagella L, Giordano A (2011) CTCF and BORIS regulate Rb2/p130 gene transcription: a novel mechanism and a new paradigm for understanding the biology of lung cancer. Mol Cancer Res 9:225–233PubMedCrossRefGoogle Scholar
  10. 10.
    Filipova GN, Fagerlie S, Klenova EM, Myers C, Dehner Y, Goodwin G, Neiman PE, Collins SJ, Lobanenkov VV (1996) An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. Mol Cell Biol 16:2802–2813Google Scholar
  11. 11.
    Pugacheva EM, Suzuki T, Pack SD, Kosaka-Suzuki N, Yoon J, Vostrov AA, Barsov E, Strunnikov AV, Morse HC III, Loukinov D, Lobanenkov V (2010) The structural complexity of the human BORIS gene in gametogenesis and cancer. PLoS One 5:e13872PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Renaud S, Loukinov D, Alberti L, Vostrov A, Kwon Y-W, Bosman FT, Lobanenkov V, Benhattar J (2011) BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells. Nucleic Acids Res 39:862–873PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Ulaner GA, VuTH LT, Hu J-F, Yao X-M, Yang Y, Gorlick R, Meyers P, Healey J, Ladanyi M, Hoffman AR (2003) Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site. Hum Mol Genet 12:535–549PubMedCrossRefGoogle Scholar
  14. 14.
    D’Arcy V, Abdullaev ZK, Pore N, Docquier F, Torrano V, Chernukin I, Smart M, Farrar D, Metodiev M, Fernandez N, Richard C, Delgado DM, Lobanenkov V, Klenova E (2006) The potential of BORIS detected in the leukocytes of breast cancer patients as an early marker of tumorigenesis. Clin Cancer Res 12:5978–5986PubMedCrossRefGoogle Scholar
  15. 15.
    Looijenga LH, Hersmus R, Gillis AJ, Pfundt R, Stoop HJ, van Gurp RJ, Veltman J, Beverloo HB, van Drunen E, van Kessel AG, Pera RR, Schneider DT, Summersgill B, Shipley J, McIntyre A, van der Spek P, Schoenmakers E, Oosterhuis JW (2006) Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene. Cancer Res 66:290–302PubMedCrossRefGoogle Scholar
  16. 16.
    Risinger JI, Chandramouli GVR, Maxwell GL, Custer M, Pack S, Loukinov D, Aprelikova O, Litzi T, Schrump DS, Murphy SK, Berchuck A, Lobanenkov V, Barrett JC (2007) Global expression analysis of cancer/testis genes in uterine cancers reveals a high incidence of BORIS expression. Clin Cancer Res 13:1713–1719PubMedCrossRefGoogle Scholar
  17. 17.
    Woloszynska-Read A, James SR, Link PA, Yu J, Odunsi K, Karpf AR (2007) DNA methylation-dependent regulation of BORIS/CTCFL expression in ovarian cancer. Cancer Immun 7:21PubMedCentralPubMedGoogle Scholar
  18. 18.
    D’Arcy V, Pore N, Docquier F, Abdullaev ZK, Chernukhin I, Kita GX, Rai S, Smart M, Farrar D, Pack S, Lobanenkov V, Klenova E (2008) BORIS, a paralogue of the transcription factor, CTCF, is aberrantly expressed in breast tumors. Br J Cancer 98:571–579PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Kholmanskikh O, Loriot A, Brasseur F, De Plaen E, De Smet C (2008) Expression of BORIS in melanoma: lack of association with MAGE-A1 activation. Int J Cancer 122:777–784PubMedCrossRefGoogle Scholar
  20. 20.
    Smith IM, Glazer CA, Mithani SK, Ochs MF, Sun W, Bhan S, Vostrov A, Abdullaev Z, Lobanenkov V, Gray A, Liu C, Chang SS, Ostrow KL, Westra WH, Begum S, Dhara M, Califano J (2009) Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer. PLoS One 4:e4961PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Smiraglia DJ, Smith LT, Lang JC, Rush LJ, Dai Z, Schuller DE, Plass C (2003) Differential targets of CpG island hypermethylation in primary and metastatic head and neck squamous cell carcinoma (HNSCC). J Med Genet 40:25–33PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Schmezer P, Plass C (2008) Epigenetic aspects in carcinomas of the head and neck. HNO 56:594–602PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Larynx D et al (2010) In: Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A (eds) AJCC cancer staging manual, 7th edn. Springer, New York, pp 57–62Google Scholar
  24. 24.
    Grbeša I, Ivkić M, Pegan B, Gall-Trošelj K (2006) Loss of imprinting and promoter usage of the IGF2 in laryngeal squamous cell carcinoma. Cancer Lett 238:224–229PubMedCrossRefGoogle Scholar
  25. 25.
    rpart by Terry M Therneau, Beth Atkinson. R port of rpart by Brian Ripley>. Some routines from vegan – Jari Oksanen < > Extensions and adaptations of rpart to mvpart by Glenn De’ath. (2013) mvpart: Multivariate partitioning. R package version 1.6-1. Accessed 15 Jan 2014
  26. 26.
    R Core Team (2013) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL Accessed 15 Jan 2014
  27. 27.
    Schumacher M, Holländer N, Schwarzer G, Willi Sauerbrei W (2006) Prognostic factor studies. In: Crowley J, Ankerst DP (eds) Handbook of statistics in clinical oncology, 2nd edn. Chapman & Hall/CRC, Boca Raton, pp 289–333Google Scholar
  28. 28.
    Liu X (2013) Survival analysis. Wiley, ChichesterGoogle Scholar
  29. 29.
    Ragin CC, Modugno F, Gollin SM (2007) The epidemiology and risk factors of head and neck cancer: a focus on human papillomavirus. J Dent Res 86:104–114PubMedCrossRefGoogle Scholar
  30. 30.
    Pöschl G, Stickel F, Wang XD, Seitz HK (2004) Alcohol and cancer: genetic and nutritional aspects. Proc Nutr Soc 63:65–71PubMedCrossRefGoogle Scholar
  31. 31.
    Flatley JE, McNeir K, Balasubramani L, Tidy J, Stuart EL, Young TA, Powers HJ (2009) Folate status and aberrant DNA methylation are associated with HPV infection and cervical pathogenesis. Cancer Epidemiol Biomarkers Prev 18:2782–2789PubMedCrossRefGoogle Scholar
  32. 32.
    McCarty KS Jr, Szabo E, Flowers JL, Cox EB, Leight GS, Miller L, Konrath J, Soper JT, Budwit DA, Creasman WT, Seigler HF, McCarty KS Sr (1986) Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors. Cancer Res 46:4244s–4248sPubMedGoogle Scholar
  33. 33.
    Rosa-Garrido M, Ceballos L, Alonso-Lecue P, Abraira C, Delgado MD, Gandarillas A (2012) A cell cycle role for the epigenetic factor CTCF-L/BORIS. PLoS One 7:e39371PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Grbesa I, Marinkovic M, Ivkic M, Kruslin B, Novak-Kujundzic R, Pegan B, Bogdanovic O, Bedekovic V, Gall-Troselj K (2008) Loss of imprinting of IGF2 and H19, loss of heterozygosity of IGF2R and CTCF, and helicobacter pylori infection in laryngeal squamous cell carcinoma. J Mol Med (Berl) 86:1057–1066CrossRefGoogle Scholar
  35. 35.
    Hogarth C, Itman C, Jans DA, Loveland KL (2005) Regulated nucleocytoplasmic transport in spermatogenesis: a driver of cellular differentiation? BioEssays 27:1011–1025PubMedCrossRefGoogle Scholar
  36. 36.
    Nguyen P, Bar-Sela G, Sun L, Bisht KS, Cui H, Kohn E, Feinberg AP, Gius D (2008) BAT3 and SET1A form a complex with CTCFL/BORIS to modulate H3K4 histone dimethylation and gene expression. Mol Cell Biol 28:6720–6729PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Ozaki T, Hanaoka E, Naka M, Nakagawara A, Sakiyama S (1999) Cloning and characterization of rat BAT3 cDNA. DNA Cell Biol 18:503–512PubMedCrossRefGoogle Scholar
  38. 38.
    Manchen ST, Hubberstey AV (2001) Human Scythe contains a functional nuclear localization sequence and remains in the nucleus during staurosporine-induced apoptosis. Biochem Biophys Res Commun 287:1075–1082PubMedCrossRefGoogle Scholar
  39. 39.
    Tsukahara T, Kimura S, Ichimiya S, Torigoe T, Kawaguchi S, Wada T, Yamashita T, Sato N (2009) Scythe/BAT3 regulates apoptotic cell death induced by papillomavirus binding factor in human osteosarcoma. Cancer Sci 100:47–53PubMedCrossRefGoogle Scholar
  40. 40.
    Ehrlich M (2002) DNA methylation in cancer: too much, but also too little. Oncogene 21:5400–5413PubMedCrossRefGoogle Scholar
  41. 41.
    Renaud S, Pugacheva EM, Delgado MD, Braunschweig R, Abdullaev Z, Loukinov D, Benhattar J, Lobanenkov V (2007) Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors. Nucleic Acids Res 35:7372–7388PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Hines WC, Bazarov AV, Mukhopadhyay R, Yaswen P (2010) BORIS (CTCFL) is not expressed in most human breast cell lines and high grade breast carcinomas. PLoS One 5:e9738PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2014

Authors and Affiliations

  • Renata Novak Kujundžić
    • 1
    • 5
  • Ivana Grbeša
    • 1
  • Mirko Ivkić
    • 2
  • Božo Krušlin
    • 3
  • Paško Konjevoda
    • 4
  • Koraljka Gall Trošelj
    • 1
  1. 1.Laboratory for EpigenomicsRuđer Bošković InstituteZagrebCroatia
  2. 2.Department of Otorhinolaryngology and Head and Neck SurgerySestre Milosrdnice University HospitalZagrebCroatia
  3. 3.Department of Pathology «Ljudevit Jurak»Sestre Milosrdnice University HospitalZagrebCroatia
  4. 4.Center for NMRRuđer Bošković InstituteZagrebCroatia
  5. 5.ZagrebCroatia

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