Only a few studies were done on proteomic analysis of the malignant transformation mechanism from precancerous lesion with different stage of dysplasia into invasive cancer. We have performed a comparative proteomic analysis to profile differentially expressed proteins in the transformation process. Using GO analysis, we further analyzed the biological process and pathway network of these proteins, which can generate a new insight into systemic biology in carcinogenesis.
Oral squamous carcinoma, like esophageal adenocarcinoma, has been associated with the presence of precancerous lesion with different stage of dysplasia, thus providing a good model to elucidate every stage of carcinogenesis in more detail. In our study, each pair of precancerous and cancer tissues was from the same patient, which provides an opportunity to eliminate or at least reduce heterogeneity. Using the proteomic approach, we have identified 85 differently expressed gene products and found some proteins are related to apoptosis, response to stimulus, metabolic regulation and etc. We can thus conclude that these proteins may play an important role in malignant transformation process. Our most significant finding was that several proteins in the same protein families and homologs were identified in this transformation process, such as peroxiredoxins (Peroxiredoxin-3 and 4), Annexin family (A1, A 3, A 4, A 5, A 8), Rho GDP-dissociation inhibitor 1 and 2, Heat shock protein family (70 kDa protein 1, 71 kDa protein, and Heat shock protein beta-1), PA28 homolog (PA28 a and b), Protein S100 family (A7, A8, A9, A10, A11, and A16). Among which, the annexins and S100 are two super-families of closely related calcium and membrane-binding proteins and their relationship with carcinogenesis has been widely studied. They have a diverse range of cellular functions including vesicle trafficking, cell division, apoptosis, calcium signaling and growth regulation.
Many studies have revealed the annexins to be among the genes whose expression is differentially altered in neoplasia. Some annexins showed increased expression in specific types of tumors, while others displayed loss of expression. In our report, the expression level of annexin A1, 3, 4, 5, 8 were all decreased while annexin A8 showed increased expression. Annexin A1 has been extensively studied in vitro
and in vivo
. The loss of expression of annexin A1 in our study confirmed previous findings in head and neck squamous carcinomas[12
]. Expression of annexin A3 has only been studied in a limited number of tumor types with only one report regarding its expression in head and neck cancer [13
]. For annexin A4, a few studies reported its increased expression in clear cell renal cancer and colorectal cancer by using a combination of proteomics tools [14
]. The change of Annexin A5 was also observed in our study, which has been considered as one of the signals on the surface of the apoptotic cells and has been used as a probe for apoptosis [16
]. Annexin A8 has been shown to be consistently over-expressed in acute promyelocytic leukaemia, breast cancers, pancreatic cancer by a combination of gene expression microarrays and immunohistochemistry. The expression of annexin A4, 5, and 8 in head and neck cancer has been reported in this study for the first time, which was consistent with the results obtained in other studies concerning their expression in other cancers [17
The S100 proteins are a multi-gene calcium-binding family of proteins comprising 20 known human members. There has been growing interest in the S100 protein family and their relationship with different cancers. While the precise role of S100 proteins in the development and promotion of cancer remains unclear, it is evident that the S100 proteins have a variety of intracellular and extracellular roles, and that disruption of any one of these functions may contribute to carcinogenesis. There is evidence that these proteins play a major role in tumor metastasis by interacting with a number of different proteins, including matrix metalloproteinase, cytoskeletal proteins, p53, Jab1, Cox-2 and BRCA1. In this study, we have identified a series of members including S100A7, 8, 9, 10, 11, and 16 with differential expression in OSCC tissues. S100A7 (psoriasin) was a member first characterized as being highly expressed in psoriatic keratinocytes [19
]. There is accumulating evidence that S100A7 is up-regulated in bladder cancer skin tumors and some invasive carcinomas. Its expression is associated with a poorer prognosis and reduced survival [20
]. On the contrary, other reports in OSCC showed its expression was associated with a better prognosis based on the finding that S100A7 is highly expressed in pre-invasive, well-differentiated and early staged OSCC, but little or no expression was found in poorly differentiated, later-staged invasive tumors [23
]. Other reports showed that S100A7 inhibits both OSCC cell proliferation in vitro and tumor growth/invasion in vivo [24
]. These results were echoed by our study, in which S100A7 was identified to be down-regulated in the transformation process form precancerous dysplasia to invasive cancer. Therefore, unlike in other tumors, our data suggests S100A7 to be a tumor suppressor in OSCC. The detailed function should be further elucidated. S100A8 and S100A9 which form a heterodimer complex 90 are up-regulated in many cancers and have been implicated in the metastatic process including gastric cancer, prostate cancer, colorectal cancer, and breast cancer [25
]. In OSCC, one study reported there was more than a 10-fold over-expression of S100 A8 in HPV18+ OSCC [28
]. For S100A11, its function has been somewhat controversial. In bladder carcinoma and renal carcinoma, its expression is related to tumor suppression, and decreased expression of S100A11 has been associated with an increase in histopathological grade, poorer prognosis and decreased survival [29
]. However, in prostate cancer and breast cancer it is thought to be a tumor promoter. Its increased expression in prostate cancers has been shown to be associated with advanced pathological stage [30
]. There is only one report about the gene expression of S100A11 related to its diverse functions [31
]. S100A16 protein, a new and unique member of the EF-hand Ca (2+)-binding proteinswas found to accumulate within nucleoli and be translocated to the cytoplasm in response to Ca (2+) stimulation [32
]. Here we report for the first time the expression of S100A16 protein in carcinogenesis from precancerous dysplasia to OSCC. It is possible that each S100 protein may play multiple roles in tumourigenesis and metastasis. This highlights the need for an improved understanding of the S100 family, before the design of S100 protein-targeted therapies can be achieved.
Proteasomes are large complexes that carry out crucial roles in many cellular pathways by degrading proteins in the cytosol and nucleus of eukaryotic cells [33
]. Proteasomes are activated by protein complexes that bind to the end rings of subunits. PA28 (also known as 11S or REG) has been shown to bind specifically to and activate 20S proteasomes against model peptide substrates [34
]. The biological roles of PA28 are less well understood. There are three PA28 homologs, called a, b and g. Although PA28a and b subunits are expressed in many organs, they are particularly abundant in immune tissues and are virtually absent from the brain. By the late 1990s, PA28ab was found to contribute to Class I presentation, based on the high levels of PA28ab in immune tissues, the IFNg induction of PA28ab and many components of the class I pathway, and the direct production of some Class I epitopes by PA28ab-proteasome complexes[35
]. Our results also reinforce the connection between PA28ab and cellular immunity by showing that the key proteins in the MHC I antigen presenting pathway like PA28, proteosome, HSP70 and HSP90 have been detected up-regulated in OSCC tissues. An earlier study showed that PA28g expression correlated with cell proliferation. Recently, some researchers have gained more insight into the role of PA28g in apoptosis [37
]. These findings were paralleled by studies suggesting that PA28g functions in cell cycle progression and has an immune role [38
]. Two-hybrid screens have identified several proteins that interact with PA28g as well. Interestingly, all these findings suggest that PA28g is an anti-apoptotic factor. Less is known about how PA28g may suppress apoptosis in oral carcinesogenesis. In our validation study, Three homologs were all included. The results have confirmed the up-regulation of PA28 in carcinogenesis by comparison between several OSCC cell lines and oral keratinocytes. In our further studies, we would valuate the PA28 ab complex and PA28 g immunostaining pattern in different stage of tissue samples from normal, precancerous to infiltrative OSCC. Moreover, the relationships of immunostaning with survival rate and recurrence will be analyzed.