Serum proteome analysis for profiling protein markers associated with carcinogenesis and lymph node metastasis in nasopharyngeal carcinoma

Serum is a complex body fluid, containing a large diversity of proteins. More than 10,000 different proteins are present in the human serum and many of them are secreted or shed by cells during different physiology or pathology processes [18]. Consequently, proteomics has raised great expectations for the discovery of biomarkers to improve diagnosis or classification of a wide range of diseases, including cancers [19]. Serum is expected to be an excellent source of protein biomarkers because it circulates through, or comes in contact with, all tissues. During this contact it is likely to pick up proteins secreted or shed by tissues, which has recently been tested and confirmed [20]. However, serum has been termed as the most complex human proteome [21] with considerable differences in the concentrations of individual proteins, ranging from several milligrams to less than one pictogram per milliliter [22]. Another analytical challenge for biomarker discovery arises from the high variability in the concentration and state of modification of some human plasma proteins between different individuals [23]. Despite these limitations, human serum holds immense diagnostic potential. In the last decade, several large-scale projects have been initiated, aimed at characterizing the human plasma/serum proteome.

In the mean time, several serum proteomic studies on NPC have been reported. Cho et al. performed protein chip profiling analysis with surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology on sera from NPC patients and demonstrated that SAA may be a potentially usefully biomarker for NPC [24]. However, the technology for discovery of new cancer biomarkers has recently been questioned for its flaws, such as its qualitative nature, high identification error rate, poor reproducibility and nonspecific absorption matrices [25‐27]. 2D-based comparative proteome analysis, though a new technology for the separation and identification of disease-specific proteins, has been applied successfully to screen potential biomarkers for NPC in cell lines and tumor tissues [3‐8].

Recently, Saeid et al. reported their pioneering work on NPC serum analysis by 2D, revealing the enhanced expression of such relatively abundant proteins as ceruloplasmin (CPL) [9]. However, in 2D study, abundant proteins, such as albumin and IgG, that account for approximately 60–97% of the total serum proteins [28], mask other proteins that migrate to the surrounding areas and limit the loading amount of serum. As albumin and IgG are known to function as carriers and transporters of important proteins such as hormones, cytokines, and lipoproteins within the blood [29‐31], the depletion of these two highly abundant proteins may result in the loss of potentially important proteins bound to them at the same time. In order to release those adsorbed or bound proteins, we sonicated the diluted sera before the depletion and desalting steps as suggested by Quero et al. [32, 33]. The improvements we made in sample preparation enabled us to find some valuable low abundant proteins.

Of the 13 successfully identified protein spots, we focused on 10 up-regulated proteins in NPC for further validation. We also accumulated certain knowledge about three proteins by literature profiling. Finally, we decided to further investigate sICAM-1, HSP70 and SAA, which seemed more associated with our research interest, using both ELISA and IHC to validate their differential expressions. Intriguingly, most of these identified proteins have been reported to be associated with carcinogenesis and tumor metastasis. Our research is herein chiefly concerned with the functional implications of the three proteins to NPC at the serum level.

Although the source of sICAM-1 has not been fully elucidated, researches show it can be released by cancer cells [13, 14] as well as by peripheral blood mononuclear, endothelial, and fibroblastic cells [34]. Proteolytic cleavage of membrane-bound ICAM-1 may be the most likely mechanism for the generation of sICAM-1 [35]. In patients with certain malignancies, the serum sICAM-1 titers have been found elevated in association with tumor growth and distant metastasis of malignant melanoma [36], lung [37], breast [38], gastric [39], hepatocellular [40], and colorectal cancers [41]. Poor survival of cancer patients correlated with a high level of serum sICAM-1 has also been demonstrated [36, 38, 39]. In our study, the positive expression of tissue ICAM-1 and levels of serum sICAM-1 were significantly correlated to the presence, progression, metastasis and mortality of NPC. sICAM-1 possesses most of the necessary extracellular structures to retain the functional activities of ICAM-1 [13, 35]. It has been reported that ICAM-1 on the surface of cancer cells or antigen presenting cells (i.e., macrophages) is a costimulatory factor that stabilizes T-cell receptor-mediated binding between these cells and T lymphocytes [42]. sICAM-1 would work as an immunosuppressive agent by blocking LFA-1 on T lymphocytes, thus rendering it less available for binding with ICAM-1 on the surface of cancer cells [43]. In this manner, the shedding of sICAM-1 may speed up the metastatic process by escaping host immune surveillance. This, probably, presents an additional potential mechanism accounting for high serum levels of sICAM-1 in NPC patients who have metastasized via hematogenous and lymphatic routes. As serum sICAM-1 may be useful for monitoring hematogenous metastasis, measuring the serum sICAM-1 level might be potentially significant in clinic.

SAA is an acute-phase protein with various isoforms in a molecular mass range of 11–14. In normal individuals, SAA is produced by hepatocytes in the liver [16]. After secreted into serum, it rapidly binds to high-density lipoprotein, with 90% of the protein particles bound [15]. A review of the literature shows that only a low level of SAA can be found in the sera of healthy individuals, despite the ubiquitous nature of SAA [44]. This is in sharp contrast to the patients with neoplastic diseases, such as those with renal [45] and colorectal [46] cancers, who showed dramatic elevation of serum SAA. The prognostic significance of SAA for other cancers has also been found by conventional radioimmunoassay, in line with the findings of this study [47]. Cho et al. demonstrated that SAA may be a potentially usefully biomarker for NPC [24]. The report fully confirmed relatively huge concentrations of SAA in serum (0.2–2 g/l) that are thousands of times higher than classical cancer biomarkers (such as CEA, PSA, CA125, etc.) originating from tumor cells. In a similar way, we further confirmed that serum SAA was much elevated in NPC patients, particularly at the process of lymph node metastases when compared with non-LNM patients. However, such biomarkers (acute-phase reactants) are not commonly considered as cancer-specific ones and expected to be elevated in other malignant diseases or inflammatory diseases as well [26, 27]. In previous reports, SAA was found elevated in different malignancies, such as cancers of kidney, colon and prostate, as well as in leukemias and lymphomas [24, 48]. Therefore, SAA may represent a cancer epiphenomenon, unlikely to be of much clinical use in diagnosing and monitoring cancer [49], but it will be interesting to explore whether the rapid production of SAA in the liver or the epithelia of different organs [50] may be related to the stimulation by cytokines abundantly present in the NPC cells. It is also meaningful to investigate how SAA is produced at different stages of clinical manifestation in NPC patients.

HSP70 is the main protein produced during cellular response to varied stresses, such as heat shock, ischemia/reperfusion, and oxidative changes [51, 52]. Because HSP70 confers cell protection against different stresses, it has been hypothesized that it plays a protective role in tumor growth in vivo [53, 54]. Indeed, HSP70 is overexpressed in human tumors of varied origins [55], such as colorectal [56], breast [57, 58], prostate [59], liver [60] cancers and melanoma [61]. LNM and poor survival of cancer patients correlated with HSP70 overexpression has also been documented [57, 58, 61‐63]. It is suggested that HSP70 is needed for in vivo tumor progression. In addition, HSP70 may be released from tumor cells involving involves both active secretion and passive release from necrotic cells [64]. Physiological mechanisms include co-secretion in exosomes of HSC70 with TRF and the active HSP70 secretion by the nonclassical pathway employed by cytokines [65]. Therefore, HSP70 is detectable in serum so that it could potentially be used as a biomarker for diagnosis or disease classification. Abe et al. reported that HSP70 is a marker of prostate cancer, and may be used in conjunction with PSA to identify patients with early-stage prostate cancer [59]. However, to the best of our knowledge, the association between serum level of HSP70 and NPC status has not been reported. Since our findings demonstrated that there was a modest association between serum HSP70 level and NPC staging, the serum HSP70 level should not be considered as an independent prognostic factor in NPC patients, although it might be a prognostic predictor by univariate analysis. In short, serum HSP70 may have an adjunctive clinical value in monitoring tumor progression and evaluating prognosis in NPC patients, though its clinical application as a major tumor marker is limited.

In conclusion, our serum proteomic analysis, using a comprehensive pretreatment strategy, provides a practical and exemplary tool of screening progression-associated serum proteins in NPC research. After comparing 2D image analyses for healthy volunteers, non-LNM NPC and LNM NPC patients, we successfully identified 13 differentially expressed protein spots. We further explored the differential expressions of three of the proteins, namely, sICAM-1, HSP70 and SAA, by ELISA at serum and IHC at tissue, though more work should be completed to pinpoint the direct correlation between serum level of sICAM and HSP70 and their expression level in the tissues. We suggest that the three proteins may be potential serum biomarkers which can serve as effective target points for early diagnosis and therapy of NPC patients, though further clinical research should be done before the potential come true.