Elsevier

Clinica Chimica Acta

Volume 436, 25 September 2014, Pages 121-129
Clinica Chimica Acta

S100A8 as potential salivary biomarker of oral squamous cell carcinoma using nanoLC–MS/MS

https://doi.org/10.1016/j.cca.2014.05.009Get rights and content

Highlights

  • NanoLC–MS/MS identified salivary S100A8 as a specific marker of OSCCs.

  • High level of salivary S100A8 appeared over 95% in late stage OSCC.

  • AUROC curve indicated high accuracy of S100A8-based ELISA in predicting OSCC.

Abstract

Background

Oral squamous cell carcinoma (OSCC) shows low 5-year survival; early treatment greatly reduces mortality and morbidity. Saliva is a non-invasive sample, with good potential to discover biomarkers for early detection.

Methods

NanoLC–MS/MS served to analyze saliva proteome from control subjects (n = 35) and OSCC patients T1 (n = 29), T2 (n = 36), T3 (n = 14) and T4 (n = 21) stages. Identified biomarkers were verified by Western blot and ELISA assays.

Results

NanoLC–MS/MS analysis of salivary proteins between 10 and 15 kDa identified S100A8, hemoglobin delta and gamma-G globin in T3 and T4 stage OSCC as well as S100A7 in T1 and T2 stage OSCC. Western blot and ELISA indicated positive correlation between salivary S100A8 increment and tumor size stage. High level of S100A8 appeared in 3.4, 13.9, 92.9, and 100% of saliva OSCC patients with T1, T2, T3, and T4 stages, respectively. Significant increase of salivary S100A7 was observed in 20.7% and 11.1% of those with T1 and T2, respectively. AUROC curve indicated high sensitivity, specificity and accuracy of S100A8-based ELISA as a detector.

Conclusions

NanoLC–MS/MS, Western blot and ELISA manifested salivary S100A8 as a specific and sensitive marker for detection of OSCC patients. Salivary S100A8 protein could be applicable in developing OSCC diagnostics.

Introduction

Oral squamous cell carcinoma (OSCC) is predominant in oral cancer, with > 300,000 cases annually worldwide that account for 3% of malignancies in men and 2% in women [1], [2], [3]. Tobacco, alcohol, and betel quid are common risk factors for causing large areas of mucosal change, synergistically triggering oral carcinogenesis, even developing a secondary upper aerodigestive tract cancer [4], [5], [6], [7], [8], [9]. Histological and clinical data indicate multi-step changes: leukoplakia, erythroplakia, hyperkeratosis, dysplasia, even carcinoma [10]. Despite advances in surgery, radiotherapy, and chemotherapy, overall average 5-year survival rate for patients has not improved significantly (still approximately 50%) over the past 30 years, far lower than that of laryngeal or nasopharyngeal carcinoma [11], [12]. Appropriate treatment for those with pre-malignant oral lesions proves more effective, significantly raising the survival rate to 80–90% [12]. Consequently, early diagnosis of oral cancer, distinguishing between malignant or premalignant lesions, is crucial to reduce the mortality and morbidity. Biopsy of suspicious lesions offers the gold specimens for the discovery of molecular biomarkers, but non-uniform appearance of cancerous and precancerous lesions allows the difficulty in choosing the location of biopsy, affecting the accuracy of potential OSCC markers [13], [14]. Developing credible, accurate, cost-effective, and noninvasive techniques for early detection is essential.

S100 A1-14 and B, a group of small acidic proteins, contain EF-hand calcium-binding motifs [15], modulating multiple biological properties in distinct cell- and tissue-types via binding with Ca2 +, Zn2 +, and Cu2 +. S100 proteins involve in calcium homeostasis and cytoskeletal dynamics, as well as regulate cell proliferation and transcriptional factor activity [16]. S100A4 protein regulates myosin dynamics by inhibiting protein kinase C (PKC)-mediated phosphorylation on C-terminus of myosin heavy chain [17], [18]. Secreted S100A4 is a candidate maker predicting metastatic and prognostic potential in breast cancer [19]. S100A7 is up-regulated in inflammatory epidermis, correlating with epithelial malignancies: e.g., breast, skin, esophagus, head and neck [20]. S100A8 and S100A9 can be synthesized and secreted by granulocytes, monocytes, and macrophages, identified as cytokine-like and transcriptional factor-like molecules affecting expression of tumor necrosis factor-α, interleukin-1, and matrix metalloproteinases [21], [22], [23], [24]. Up-regulation of S100A8 and S100A9 is found in gastric, colorectal, breast, lung, pancreatic, and prostate cancer, correlating with inflammation cell proliferation and metastatic processes in tumor development [25], [26], [27]. S100B also inhibits PKC-mediated phosphorylation on p53, reducing tumor suppressor activity by suppressing p53-dependent transcription activation [28], [29]. Altered expression of S100 proteins is thereby associated with cancer development; secreted form of S100 proteins could act as potential cancer markers.

Tissue microarray indicated significant up-expression of S100A8 in severe oral dysplasias and OSCC tissue [30], [31]. Proteomic analysis of normal and OSCC tissues suggested S100A7 as a positive marker for OSCC carcinogenesis and early tumor progression that can be confirmed by immunofluorescence and quantitative RT-PCR analyses [32]. Sharp decrease of S100A4 mRNA was evident in OSCC tissues [33]. Earlier we identified S100A8, transferrin, and zinc finger protein 497 as salivary biomarkers, using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) [30]. Salivary samples from such patients showed elevated S100A8, necessary for further probe or correlation with oral cancer tumor grade. This study rated the potential of S100 proteins as salivary markers via proteomic analysis of low molecular weight salivary proteins, using nanoLC–MS/MS. Protein profile indicated change of S100A7 and S100A8 as unique markers in saliva of oral cancer patients. Western blot and direct binding ELISA further examined levels of S100A7 and S100A8 in their saliva while evaluating potency of these markers.

Section snippets

Human subjects and saliva collection

In all, 35 subjects without and 100 with OSCC were enrolled for study from February 2007 to March 2014; OSCC patients were diagnosed via biopsy at China Medical University Hospital in Taichung. Subjects gave informed consent prior to saliva collection approved by the Institutional Review Board of China Medical University Hospital (permission number DMR96-IRB-80). Exclusion criteria for OSCC patients and control individuals were followed, as in our prior studies [30], [34]. Table 1 lists

Clinical parameters of OSCC patients and control subjects

A total of 35 controls and 100 OSCC cases were recruited, the latter grouped as T1, T2, T3 and T4, based on tumor size stage of UICC TNM staging system (Table 1). Males formed a majority in all groups; mean age of OSCC cases was over 50 years, with controls (50.2 years) slightly younger. Among OSCC cases, tongue and buccal sites showed most frequent oral cancer lesions; nearly half were N0 stage without tumor cells from regional lymph nodes, 13% N1 and 29% N2 stage. Histologic examination of

Discussion

This study was the first report that nanoLC–MS/MS utilized to identify salivary protein filing of OSCC patients and controls. NanoLC–MS/MS served as powerful tools to discover potential serum, urine, and tissue biomarkers for colon, lung, breast, colorectal, and gastric cancers [36], [37], [38], [39]. In the present study, nanoLC–MS/MS analysis of salivary proteins between 10 and 15 kDa identified S100A8 as a potential salivary biomarker for oral cancer (Fig. 1 and Table 2), in accordance with

Acknowledgments

This work was supported by the National Science Council of Taiwan (NSC101-2320-B-039-036-MY3) and China Medical University (CMU101-ASIA-05 and CMU101-S-24).

References (48)

  • R.T. Greenlee et al.

    Cancer statistics, 2000

    CA Cancer J Clin

    (2000)
  • D.M. Parkin et al.

    Global cancer statistics, 2002

    CA Cancer J Clin

    (2005)
  • Cancer facts and figures 2006

    (2006)
  • P.S. Ho et al.

    Consumption of areca quid, cigarettes, and alcohol related to the comorbidity of oral submucous fibrosis and oral cancer

    Oral Surg Oral Med Oral Pathol Oral Radiol Endod

    (2007)
  • X. Zhang et al.

    A review of betel quid chewing, oral cancer and precancer in Mainland China

    Oral Oncol

    (2007)
  • B.H. Haughey et al.

    Meta-analysis of second malignant tumors in head and neck cancer: the case for an endoscopic screening protocol

    Ann Otol Rhinol Laryngol

    (1992)
  • A.S. Jones et al.

    Second primary tumors in patients with head and neck squamous cell carcinoma

    Cancer

    (1995)
  • A. Jovanovic et al.

    Second respiratory and upper digestive tract cancer following oral squamous cell carcinoma

    Eur J Cancer B Oral Oncol

    (1994)
  • B.S. Tepperman et al.

    Second respiratory and upper digestive tract cancers after oral cancer

    Lancet

    (1981)
  • P.K. Tsantoulis et al.

    Advances in the biology of oral cancer

    Oral Oncol

    (2007)
  • M.T. Canto et al.

    Oral cavity and pharynx cancer incidence rates in the United States, 1975–1998

    Oral Oncol

    (2002)
  • S.J. Silverman

    Demographics and occurrence of oral and pharyngeal cancers. The outcomes, the trends, the challenge

    J Am Dent Assoc

    (2001)
  • C.Y. Yen et al.

    Evaluating the performance of fibronectin 1 (FN1), integrin α4β1 (ITGA4), syndecan-2 (SDC2), and glycoprotein CD44 as the potential biomarkers of oral squamous cell carcinoma (OSCC)

    Biomarkers

    (2013)
  • C.Y. Yen et al.

    Matrix metalloproteinases (MMP) 1 and MMP10 but not MMP12 are potential oral cancer markers

    Biomarkers

    (2009)
  • H.L. Hsieh et al.

    Expression analysis of S100 proteins and RAGE in human tumors using tissue microarrays

    Biochem Biophys Res Commun

    (2003)
  • I. Salama et al.

    A review of the S100 proteins in cancer

    EJSO

    (2008)
  • M. Kriajevska et al.

    Metastasis-associated Mts1 (S100A4) protein modulates protein kinase C phosphorylation of the heavy chain of nonmuscle myosin

    J Biol Chem

    (1998)
  • N. Murakami et al.

    Two distinct mechanisms for regulation of nonmuscle myosin assembly via the heavy chain: phosphorylation for MIIB and mts 1 binding for MIIA

    Biochemistry

    (2000)
  • P.S. Rudland et al.

    Prognostic significance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer

    Cancer Res

    (2000)
  • A. Sneh et al.

    Differential role of psoriasin (S100A7) in estrogen receptor α positive and negative breast cancer cells occur through actin remodeling

    Breast Cancer Res Treat

    (2013)
  • K. Odink et al.

    Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis

    Nature

    (1987)
  • J. Roth et al.

    Complex pattern of the myelo-monocytic differentiation antigens MRP8 and MRP14 during chronic airway inflammation

    Immunobiology

    (1992)
  • P.L. van Lent et al.

    Stimulation of chondrocyte-mediated cartilage destruction by S100A8 in experimental murine arthritis

    Arthritis Rheum

    (2008)
  • P. Youssef et al.

    Expression of myeloid related proteins (MRP) 8 and 14 and the MRP8/14 heterodimer in rheumatoid arthritis synovial membrane

    J Rheumatol

    (1999)
  • Cited by (0)

    View full text