Skip to main content

Proteomic analysis to identify cytokeratin 18 as a novel biomarker of nasopharyngeal carcinoma

Journal of Cancer Research and Clinical Oncology volume 135, pages 1763–1775 (2009)Cite this article

Abstract

Purpose

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers.

Methods

Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cytokeratin 18 in the above two tissues as well as 4 NPC cell lines was determined by Western blotting. Immunohistochemistry was also performed to detect the expression of cytokeratin 18 in 62 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. siRNA and in vitro cell invasion assay were used to check the correlation between the expression of cytokeratin 18 and invasive ability of NPC.

Results

Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cytokeratin 18 in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Significant cytokeratin 18 down-regulation was observed in NPC versus NNET (P = 0.000), whereas significant cytokeratin 18 up-regulation was observed in lymph node metastasis versus primary NPC (P = 0.001). In addition, cytokeratin 18 down-regulation was significantly correlated with poor histological differentiation (P = 0.000), whereas cytokeratin 18 up-regulation was significantly correlated with advanced clinical stage (P = 0.019), recurrence (P = 0.000), and regional lymph node metastasis (P = 0.001), and distant metastasis (P = 0.000). And down-regulated cytokeratin 18 expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Furthermore, survival curves showed that patients with cytokeratin 18 up-regulation had a poor prognosis (P = 0.000). Univariate analysis (Cox’s proportional hazards model) showed that WHO histologic type (P = 0.025), lymph node metastasis (P = 0.007), distant metastasis (P = 0.005), recurrence (P = 0.000), and cytokeratin 18 (P = 0.000) were significantly associated with the prognosis of NPC. Multivariate analysis confirmed that lymph node metastasis (P = 0.012), distant metastasis (P = 0.009), recurrence (P = 0.006), and cytokeratin 18 (P = 0.001) were independent prognostic indicators.

Conclusions

The data suggest that cytokeratin 18 is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

39,95 €

Price includes VAT (Finland)

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

NPC:

Nasopharyngeal carcinoma

NNET:

Normal nasopharyngeal epithelial tissues

LCM:

Laser capture microdissection

2-DE:

Two-dimensional gel electrophoresis

References

  • Ahmad A, Stefani S (1986) Distant metastases of nasopharyngeal carcinoma: a study of 256 male patients. J Surg Oncol 33:194–197. doi:10.1002/jso.2930330310

    Article  PubMed  CAS  Google Scholar 

  • Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333. doi:10.1002/elps.200305844

    Article  PubMed  CAS  Google Scholar 

  • Cheng Y, Zhang J, Li Y, Wang Y, Gong J (2007) Proteome analysis of human gastric cardia adenocarcinoma by laser capture microdissection. BMC Cancer 7:191

    Article  PubMed  Google Scholar 

  • Cheung HW, Jin DY, Ling MT, Wong YC, Wang Q, Tsao SW et al (2005) Mitotic arrest deficient 2 expression induces chemosensitization to a DNA-damaging agent, cisplatin, in nasopharyngeal carcinoma cells. Cancer Res 65:1450–1458. doi:10.1158/0008-5472.CAN-04-0567

    Article  PubMed  CAS  Google Scholar 

  • Chu YW, Seftor EA, Romer LH, Hendrix MJ (1996) Experimental coexpression of vimentin and keratin intermediate filaments in human melanoma cells augments motility. Am J Pathol 148:63–69

    PubMed  CAS  Google Scholar 

  • Cintorino M, Tripod SA, Santopietro R, Antonio P, Lutfi A, Chang F (2001) Cytokeratin expression patterns as an indicator of tumour progression in oesophageal squamous cell carcinoma. Anticancer Res 21:4195–4201

    PubMed  CAS  Google Scholar 

  • Elliott BE, Meens JA, SenGupta SK, Louvard D, Arpin M (2005) The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res 7:R365–R373

    Article  PubMed  CAS  Google Scholar 

  • Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR (1996) Laser capture microdissection. Science 274:998–1001. doi:10.1126/science.274.5289.998

    Article  PubMed  CAS  Google Scholar 

  • Franzé B, Linder S, Alaiya AA, Eriksson E, Fujioka K, Bergman AC (1997) Analysis of polypeptide expression in benign and malignant human breast lesions. Electrophoresis 18:582–587

    Article  Google Scholar 

  • Hara A, Okayasu I (2004) Cyclooxygenase-2 and inducible nitric oxide synthase expression in human astrocytic gliomas: correlation with angiogenesis and prognostic significance. Acta Neuropathol 108:43–48. doi:10.1007/s00401-004-0860-0

    Article  PubMed  CAS  Google Scholar 

  • Hesse M, Magin TM, Weber K (2001) Genes for intermediate filament proteins and the draft sequence of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. J Cell Sci 114:2569–2575

    PubMed  CAS  Google Scholar 

  • Hui AB, Or YY, Takano H, Tsang RK, To KF, Guan XY et al (2005) Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma. Cancer Res 65:8125–8133. doi:10.1158/0008-5472.CAN-05-0648

    Article  PubMed  CAS  Google Scholar 

  • Jang JS, Cho HY, Lee YJ, Ha WS, Kim HW (2004) The differential proteome profile of stomach cancer: identification of the biomarker candidates. Oncol Res 14:491–499

    PubMed  CAS  Google Scholar 

  • Kwong J, Lo KW, To KF, Teo PM, Johnson PJ, Huang DP (2002) Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res 8:131–137

    PubMed  CAS  Google Scholar 

  • Li C, Hong Y, Tan YX, Zhou H, Ai JH, Li SJ et al (2004) Accurate qualitative and quantitative proteomic analysis of clinical hepatocellular carcinoma using laser capture microdissection coupled with isotope-coded affinity tag and two-dimensional liquid chromatography mass spectrometry. Mol Cell Proteomics 3:399–409. doi:10.1074/mcp.M300133-MCP200

    Article  PubMed  CAS  Google Scholar 

  • Morifuji M, Taniguchi S, Sakai H, Nakabeppu Y, Ohishi M (2000) Differential expression of cytokeratin after orthotopic implantation of newly established human tongue cancer cell lines of defined metastatic ability. Am J Pathol 156:1317–1326

    PubMed  CAS  Google Scholar 

  • Nagle RB (1996) Intermediate filament expression in prostate cancer. Cancer Metastasis Rev 15:473–482

    Article  PubMed  CAS  Google Scholar 

  • Neubauer H, Clare SE, Kurek R, Fehm T, Wallwiener D, Sotlar K et al (2006) Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioisotope detection. Electrophoresis 27:1840–1852. doi:10.1002/elps.200500739

    Article  PubMed  CAS  Google Scholar 

  • Qian CN, Guo X, Cao B, Kort EJ, Lee CC, Chen J et al (2002) Met protein expression level correlates with survival in patients with late-stage nasopharyngeal carcinoma. Cancer Res 62:589–596

    PubMed  CAS  Google Scholar 

  • Reymond MA, Schlegel W (2007) Proteomics in cancer. Adv Clin Chem 44:103–142. doi:10.1016/S0065-2423(07)44004-5

    Article  PubMed  CAS  Google Scholar 

  • Shanmugaratnam K, Sobin LH (2005) The World Health Organization histological classification of tumours of the upper respiratory tract and ear. Cancer 71:2689–2697. doi:10.1002/1097-0142(19930415)71:8<2689::AID-CNCR2820710843>3.0.CO;2-H (a commentary on the second edition)

    Article  Google Scholar 

  • Shekouh AR, Thompson CC, Prime W, Campbell F, Hamlett J, Herrington CS et al (2005) Application of laser capture microdissection combined with two-dimensional electrophoresis for the discovery of differentially regulated proteins in pancreatic ductal adenocarcinoma. Proteomics 3:1988–2001. doi:10.1002/pmic.200300466

    Article  Google Scholar 

  • Shi W, Pataki I, MacMillan C, Pintilie M, Payne D, O’Sullivan B et al (2002) Molecular pathology parameters in human nasopharyngeal carcinoma. Cancer 94:1997–2006. doi:10.1002/cncr.0679

    Article  PubMed  CAS  Google Scholar 

  • Song LB, Yan J, Jian SW, Zhang L, Li MZ, Li D et al (2002) Molecular mechanisms of tumorigenesis and metastasis in nasopharyngeal carcinoma cell sublines. Ai Zheng 21:158–162

    PubMed  Google Scholar 

  • Su CK, Wang CC (2002) Prognostic value of Chinese race in nasopharyngeal cancer. Int J Radiat Oncol Biol Phys 54:752–758. doi:10.1016/S0360-3016(02)02969-3

    PubMed  Google Scholar 

  • Volk T, Geiger B, Raz A (2005) Motility and adhesive properties of high- and low-metastatic murine neoplastic cells. Cancer Res 44:811–824

    Google Scholar 

  • Walker LC, Harris GC, Holloway AJ, McKenzie GW, Wells JE, Robinson BA (2007) Cytokeratin KRT8/18 expression differentiates distinct subtypes of grade 3 invasive ductal carcinoma of the breast. Cancer Genet Cytogenet 2007(178):94–103

    Article  Google Scholar 

  • Woelfle U, Sauter G, Santjer S, Brakenhoff R, Pantel K (2004) Down-regulated expression of cytokeratin 18 promotes progression of human breast cancer. Clin Cancer Res 10:2670–2674

    Article  PubMed  CAS  Google Scholar 

  • Xu W, Zhang MW, Huang J, Wang X, Xu SF (2005) Correlation between CK18 gene and gastric carcinoma micrometastasis. World J Gastroenterol 11:6530–6534

    PubMed  CAS  Google Scholar 

  • Yau WL, Lung HL, Zabarovsky ER, Lerman MI, Sham JS, Chua DT et al (2006) Functional studies of the chromosome 3p21.3 candidate tumor suppressor geneBLU/ZMYND10 in nasopharyngeal carcinoma. Int J Cancer 119:2821–2826. doi:10.1002/ijc.22232

    Article  PubMed  CAS  Google Scholar 

  • Yu MC, Yuan JM (2002) Epidemiology of nasopharyngeal carcinoma. Semin Cancer Biol 12:421–429. doi:10.1016/S1044579X02000858

    Article  PubMed  Google Scholar 

  • Zhang D, Koay ES (2008) Analysis of laser capture microdissected cells by 2-dimensional gel electrophoresis. Methods Mol Biol 428:77–91. doi:10.1007/978-1-59745-117-8_5

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Emergency Department, Xiangya Hospital, Central South University, 410008, Changsha, China

    Xiang-Min Li

  2. Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 410008, Changsha, China

    Hong Yi & Zhi-Qiang Xiao

  3. Cancer Research Institute, University of South China, 421001, Hengyang, China

    Wei-Guo Huang & Ai-Lan Cheng

Authors
  1. Xiang-Min Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei-Guo Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hong Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ai-Lan Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhi-Qiang Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei-Guo Huang.

Additional information

X.-M. Li, W.-G. Huang and H. Yi contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, XM., Huang, WG., Yi, H. et al. Proteomic analysis to identify cytokeratin 18 as a novel biomarker of nasopharyngeal carcinoma. J Cancer Res Clin Oncol 135, 1763–1775 (2009). https://doi.org/10.1007/s00432-009-0623-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00432-009-0623-3

Keywords

  • Nasopharyngeal carcinoma
  • Biomarker
  • Cytokeratin 18
  • Laser capture microdissection
  • Proteomics
  • Prognosis