Skip to main content

Advertisement

SpringerLink
Log in

Co-expression of CD44+/RANKL+ tumor cells in the carcinogenesis of oral squamous cell carcinoma

  • Original Article
  • Published:
Odontology Aims and scope Submit manuscript

Abstract

Receptor activator of nuclear factor-kappa (RANK)/receptor activator of nuclear factor-kappa B ligand (RANKL) signaling helps putative cancer stem cells (CSC) to maintain their stemness. Expression of CD44 and RANKL was analyzed in oral squamous cell carcinoma specimen (n = 191). Moreover, RANKL expression was measured in cancer cell lines (BICR3, BICR56) by immunohistochemistry and western blot analysis. Scanned images were digitally analyzed using ImageJ and the immunomembrane plug-in. CD44 and RANKL expression on protein level was correlated with clinical characteristics and impact on survival. RANKL was co-labeled with CD44 in immunohistochemical and immunofluorescence double labeling experiments. Although high CD44+/RANKL+ co-expression was significantly associated with clinicopathological factors and worse survival, multivariate analysis did not demonstrate high CD44+/RANKL+ co-expression as independent prognostic factor. Immunohistochemical and immunofluorescence double labeling experiments revealed RANKL expression by CD44+ cancer cells. RANKL specificity was confirmed by western blot analysis. For the first time, this study provides evidence that RANKL expression in OSCC might be associated with disease recurrence and a cell compartment measured by CD44+/RANKL+ co-expression within the mucosal epithelial basal layer cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

OSCC:

Oral squamous cell carcinoma

HE:

Hematoxylin and eosin

RANKL:

Receptor activator of NF-κB ligand

ROC:

Receiver operating characteristics analysis

AUC:

Area under the curve analysis

References

  1. Choi S, Myers JN. Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy. J Dent Res. 2008;87(1):14–32.

    Article  PubMed  Google Scholar 

  2. da Silva SD, Ferlito A, Takes RP, Brakenhoff RH, Valentin MD, Woolgar JA, Bradford CR, Rodrigo JP, Rinaldo A, Hier MP, Kowalski LP. Advances and applications of oral cancer basic research. Oral Oncol. 2011;47(9):783–91.

    Article  PubMed  Google Scholar 

  3. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 2008;8(10):755–68.

    Article  PubMed  Google Scholar 

  4. Grimm M, Krimmel M, Polligkeit J, Alexander D, Munz A, Kluba S, Keutel C, Hoffmann J, Reinert S, Hoefert S. ABCB5 expression and cancer stem cell hypothesis in oral squamous cell carcinoma. Eur J Cancer. 2012;48(17):3186–97.

    Article  PubMed  Google Scholar 

  5. Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ. Efficient tumour formation by single human melanoma cells. Nature. 2008;456(7222):593–8.

    Article  PubMed Central  PubMed  Google Scholar 

  6. Dean M. Cancer stem cells: implications for cancer causation and therapy resistance. Discov Med. 2005;5(27):278–82.

    PubMed  Google Scholar 

  7. Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA. 2007;104(3):973–8.

    Article  PubMed Central  PubMed  Google Scholar 

  8. Zhang Z, Filho MS, Nor JE. The biology of head and neck cancer stem cells. Oral Oncol. 2012;48(1):1–9.

    Article  PubMed Central  PubMed  Google Scholar 

  9. Ghatak S, Misra S, Toole BP. Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells. J Biol Chem. 2005;280(10):8875–83.

    Article  PubMed  Google Scholar 

  10. Misra S, Toole BP, Ghatak S. Hyaluronan constitutively regulates activation of multiple receptor tyrosine kinases in epithelial and carcinoma cells. J Biol Chem. 2006;281(46):34936–41.

    Article  PubMed  Google Scholar 

  11. Sheridan C, Kishimoto H, Fuchs RK, Mehrotra S, Bhat-Nakshatri P, Turner CH, Goulet R Jr, Badve S, Nakshatri H. CD44+/CD24− breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res BCR. 2006;8(5):R59.

    Article  Google Scholar 

  12. Omara-Opyene AL, Qiu J, Shah GV, Iczkowski KA. Prostate cancer invasion is influenced more by expression of a CD44 isoform including variant 9 than by Muc18. Lab Invest. 2004;84(7):894–907.

    Article  PubMed  Google Scholar 

  13. Li Y, Heldin P. Hyaluronan production increases the malignant properties of mesothelioma cells. Br J Cancer. 2001;85(4):600–7.

    Article  PubMed Central  PubMed  Google Scholar 

  14. Sharma B, Singh RK. Emerging candidates in breast cancer stem cell maintenance, therapy resistance and relapse. J Carcinog. 2011;10:36.

    Article  PubMed Central  PubMed  Google Scholar 

  15. Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature. 2001;414(6859):98–104.

    Article  PubMed  Google Scholar 

  16. Dougall WC. Molecular pathways: osteoclast-dependent and osteoclast-independent roles of the RANKL/RANK/OPG pathway in tumorigenesis and metastasis. Clin Cancer Res Off J Am Assoc Cancer Res. 2012;18(2):326–35.

    Article  Google Scholar 

  17. Cross SS, Harrison RF, Balasubramanian SP, Lippitt JM, Evans CA, Reed MW, Holen I. Expression of receptor activator of nuclear factor kappabeta ligand (RANKL) and tumour necrosis factor related, apoptosis inducing ligand (TRAIL) in breast cancer, and their relations with osteoprotegerin, oestrogen receptor, and clinicopathological variables. J Clin Pathol. 2006;59(7):716–20.

    Article  PubMed Central  PubMed  Google Scholar 

  18. Chen G, Sircar K, Aprikian A, Potti A, Goltzman D, Rabbani SA. Expression of RANKL/RANK/OPG in primary and metastatic human prostate cancer as markers of disease stage and functional regulation. Cancer. 2006;107(2):289–98.

    Article  PubMed  Google Scholar 

  19. Lee JA, Jung JS, Kim DH, Lim JS, Kim MS, Kong CB, Song WS, Cho WH, Jeon DG, Lee SY, Koh JS. RANKL expression is related to treatment outcome of patients with localized, high-grade osteosarcoma. Pediatr Blood Cancer. 2011;56(5):738–43.

    Article  PubMed  Google Scholar 

  20. Sasaki A, Ishikawa K, Haraguchi N, Inoue H, Ishio T, Shibata K, Ohta M, Kitano S, Mori M. Receptor activator of nuclear factor-kappaB ligand (RANKL) expression in hepatocellular carcinoma with bone metastasis. Ann Surg Oncol. 2007;14(3):1191–9.

    Article  PubMed  Google Scholar 

  21. Mikami S, Katsube K, Oya M, Ishida M, Kosaka T, Mizuno R, Mochizuki S, Ikeda T, Mukai M, Okada Y. Increased RANKL expression is related to tumour migration and metastasis of renal cell carcinomas. J Pathol. 2009;218(4):530–9.

    Article  PubMed  Google Scholar 

  22. Gonzalez-Suarez E, Jacob AP, Jones J, Miller R, Roudier-Meyer MP, Erwert R, Pinkas J, Branstetter D, Dougall WC. RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature. 2010;468(7320):103–7.

    Article  PubMed  Google Scholar 

  23. Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, Hanada R, Joshi PA, Aliprantis A, Glimcher L, Pasparakis M, Khokha R, Ormandy CJ, Widschwendter M, Schett G, Penninger JM. Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature. 2010;468(7320):98–102.

    Article  PubMed Central  PubMed  Google Scholar 

  24. Kupas V, Weishaupt C, Siepmann D, Kaserer ML, Eickelmann M, Metze D, Luger TA, Beissert S, Loser K. RANK is expressed in metastatic melanoma and highly upregulated on melanoma-initiating cells. J Invest Dermatol. 2011;131(4):944–55.

    Article  PubMed  Google Scholar 

  25. Chuang FH, Hsue SS, Wu CW, Chen YK. Immunohistochemical expression of RANKL, RANK, and OPG in human oral squamous cell carcinoma. J Oral Pathol Med Off Publ Int Assoc Oral Pathol Am Acad Oral Pathol. 2009;38(10):753–8.

    Google Scholar 

  26. Grimm M, Krimmel M, Polligkeit J, Alexander D, Munz A, Kluba S, Keutel C, Hoffmann J, Reinert S, Hoefert S. ABCB5 expression and cancer stem cell hypothesis in oral squamous cell carcinoma. Eur J Cancer. 2012;48(17):3186–97.

    Article  PubMed  Google Scholar 

  27. Sobin LH, Wittekind Ch. UICC TNM classification of malignant tumors. 7th ed. Berlin: Springer; 2010.

    Google Scholar 

  28. Hamilton SR, Aaltonen LA. Pathology and genetics. Tumours of the digestive system. 3rd ed. Lyon: IARC Press Third Edition; 2000.

    Google Scholar 

  29. von Rahden BH, Kircher S, Kafka M, Stuermer L, Reiber C, Gattenlohner S, Germer CT, Grimm M. Glucocorticoid-induced TNFR family-related receptor (GITR)-expression in tumor infiltrating leucocytes (TILs) is associated with the pathogenesis of esophageal adenocarcinomas with and without Barrett’s mucosa. Cancer Biomark. 2010;7(6):285–94.

    Google Scholar 

  30. Edington KG, Loughran OP, Berry IJ, Parkinson EK. Cellular immortality: a late event in the progression of human squamous cell carcinoma of the head and neck associated with p53 alteration and a high frequency of allele loss. Mol Carcinog. 1995;13(4):254–65.

    Article  PubMed  Google Scholar 

  31. Alexander D, Schafer F, Olbrich M, Friedrich B, Buhring HJ, Hoffmann J, Reinert S. MSCA-1/TNAP selection of human jaw periosteal cells improves their mineralization capacity. Cell Physiol Biochem. 2010;26(6):1073–80.

    Article  PubMed  Google Scholar 

  32. Zweig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem. 1993;39(4):561–77.

    PubMed  Google Scholar 

  33. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc. 1958;75:457–87.

    Article  Google Scholar 

  34. Mantel N. Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother Rep. 1966;50(3):163–70.

    PubMed  Google Scholar 

  35. Cox DR. Regression models and life tables. J R Stat Soc. 1972;34:1987–2001.

    Google Scholar 

  36. Shin M, Matsuo K, Tada T, Fukushima H, Furuta H, Ozeki S, Kadowaki T, Yamamoto K, Okamoto M, Jimi E. The inhibition of RANKL/RANK signaling by osteoprotegerin suppresses bone invasion by oral squamous cell carcinoma cells. Carcinogenesis. 2011;32(11):1634–40.

    Article  PubMed  Google Scholar 

  37. Jimi E, Furuta H, Matsuo K, Tominaga K, Takahashi T, Nakanishi O. The cellular and molecular mechanisms of bone invasion by oral squamous cell carcinoma. Oral Dis. 2011;17(5):462–8.

    Article  PubMed  Google Scholar 

  38. Gonzalez-Moles MA, Bravo M, Ruiz-Avila I, Esteban F, Rodriguez-Archilla A, Gonzalez-Moles S, Arias B. Adhesion molecule CD44 as a prognostic factor in tongue cancer. Anticancer Res. 2003;23:5197–202.

    PubMed  Google Scholar 

  39. Stoll C, Baretton G, Soost F, Terpe HJ, Domide P, Lohrs U. Prognostic importance of the expression of CD44 splice variants in oral squamous cell carcinomas. Oral Oncol. 1999;35(5):484–9.

    Article  PubMed  Google Scholar 

  40. Mack B, Gires O. CD44 s and CD44v6 expression in head and neck epithelia. PLoS ONE. 2008;3(10):e3360.

    Article  PubMed Central  PubMed  Google Scholar 

  41. Hirvikoski P, Tammi R, Kumpulainen E, Virtaniemi J, Parkkinen JJ, Tammi M, Johansson R, Agren U, Karhunen J, Kosma VM. Irregular expression of hyaluronan and its CD44 receptor is associated with metastatic phenotype in laryngeal squamous cell carcinoma. Virchows Arch Int J Path. 1999;434(1):37–44.

    Article  Google Scholar 

  42. Ioachim E, Assimakopoulos D, Goussia AC, Peschos D, Skevas A, Agnantis NJ. Glycoprotein CD44 expression in benign, premalignant and malignant epithelial lesions of the larynx: an immunohistochemical study including correlation with Rb, p53, Ki-67 and PCNA. Histol Histopathol. 1999;14(4):1113–8.

    PubMed  Google Scholar 

  43. Harper LJ, Costea DE, Gammon L, Fazil B, Biddle A, Mackenzie IC. Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance. BMC Cancer. 2010;10:166.

    Article  PubMed Central  PubMed  Google Scholar 

  44. Mackenzie IC. Growth of malignant oral epithelial stem cells after seeding into organotypical cultures of normal mucosa. J Oral Pathol Med. 2004;33(2):71–8.

    Article  PubMed  Google Scholar 

  45. Richard V, Pillai MR. The stem cell code in oral epithelial tumorigenesis: ‘the cancer stem cell shift hypothesis’. Biochim Biophys Acta. 2010;1806(2):146–62.

    PubMed  Google Scholar 

  46. Ishizawa K, Rasheed ZA, Karisch R, Wang Q, Kowalski J, Susky E, Pereira K, Karamboulas C, Moghal N, Rajeshkumar NV, Hidalgo M, Tsao M, Ailles L, Waddell TK, Maitra A, Neel BG, Matsui W. Tumor-initiating cells are rare in many human tumors. Cell Stem Cell. 2010;7(3):279–82.

    Article  PubMed Central  PubMed  Google Scholar 

  47. Clay MR, Tabor M, Owen JH, Carey TE, Bradford CR, Wolf GT, Wicha MS, Prince ME. Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase. Head Neck. 2010;32(9):1195–201.

    Article  PubMed Central  PubMed  Google Scholar 

  48. Krishnamurthy S, Dong Z, Vodopyanov D, Imai A, Helman JI, Prince ME, Wicha MS, Nor JE. Endothelial cell-initiated signaling promotes the survival and self-renewal of cancer stem cells. Cancer Res. 2010;70(23):9969–78.

    Article  PubMed Central  PubMed  Google Scholar 

  49. Chen YC, Chen YW, Hsu HS, Tseng LM, Huang PI, Lu KH, Chen DT, Tai LK, Yung MC, Chang SC, Ku HH, Chiou SH, Lo WL. Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer. Biochem Biophys Res Commun. 2009;385(3):307–13.

    Article  PubMed  Google Scholar 

  50. Kelly PN, Dakic A, Adams JM, Nutt SL, Strasser A. Tumor growth need not be driven by rare cancer stem cells. Science. 2007;317(5836):337.

    Article  PubMed  Google Scholar 

  51. Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol. 2004;5(7):738–43.

    Article  PubMed  Google Scholar 

  52. O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445(7123):106–10. doi:10.1038/nature05372.

    Article  PubMed  Google Scholar 

  53. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB. Identification of human brain tumour initiating cells. Nature. 2004;432(7015):396–401.

    Article  PubMed  Google Scholar 

  54. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003;100(7):3983–8.

    Article  PubMed Central  PubMed  Google Scholar 

  55. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74.

    Article  PubMed  Google Scholar 

  56. Body JJ, Facon T, Coleman RE, Lipton A, Geurs F, Fan M, Holloway D, Peterson MC, Bekker PJ. A study of the biological receptor activator of nuclear factor-kappaB ligand inhibitor, denosumab, in patients with multiple myeloma or bone metastases from breast cancer. Clinical Cancer Res Off J Am Assoc Cancer Res. 2006;12(4):1221–8.

    Article  Google Scholar 

  57. Jimi E, Shin M, Furuta H, Tada Y, Kusukawa J. The RANKL/RANK system as a therapeutic target for bone invasion by oral squamous cell carcinoma (review). Int J Oncol. 2013;42(3):803–9.

    PubMed  Google Scholar 

Download references

Acknowledgments

We thank Julia Grimm, Mohammed Saleh, and Beshr Shokri for their technical assistance. The authors have no financial disclosures to report.

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Department of Oral and Maxillofacial Surgery, University Hospital Tübingen, Osianderstrasse 2-8, 72076, Tübingen, Germany

    Martin Grimm, Cornelius Renz, Adelheid Munz, Sebastian Hoefert, Michael Krimmel & Siegmar Reinert

Authors
  1. Martin Grimm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cornelius Renz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adelheid Munz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sebastian Hoefert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Krimmel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Siegmar Reinert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martin Grimm.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Grimm, M., Renz, C., Munz, A. et al. Co-expression of CD44+/RANKL+ tumor cells in the carcinogenesis of oral squamous cell carcinoma. Odontology 103, 36–49 (2015). https://doi.org/10.1007/s10266-013-0133-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10266-013-0133-2

Keywords

Search

Navigation