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