Identification of differentially expressed proteins in oral squamous cell carcinoma using a global proteomic approach

A number of protein markers for oral cancer are still not applicable in large populations. Proteomic technologies provide excellent tools for rapid screening of a large number of potential biomarkers in malignant cells. To gain insight into the molecular mechanisms of carcinogenesis and to identify potential biomarkers for oral squamous cell carcinomas (OSCCs), we performed proteomic profiling between human normal oral keratinocytes (HNOKs) and OSCC-derived cell lines (HSC-2 and HSC-3) using fluorescent two-dimensional difference in-gel electrophoresis. Proteins with a > or =2-fold change in expression were considered significant. The spots of interest were digested and identified by matrix-assisted laser desorption/ionization time-of-flight peptide mass finger-printing. Twenty-two proteins were identified as differentially expressed between the HNOKs and OSCC-derived cell lines. Of these, 9 spots were up-regulated and 13 were down-regulated in OSCC-derived cell lines compared to the HNOKs. These spots included the cancer-related proteins; annexin A1, heat shock protein 27, lamin A/C, interleukin 1 receptor antagonist, serine proteinase inhibitor clade B5, stathmin 1, and superoxide dismutase 2. Our results are a first step toward identifying a protein profile of HNOKs and OSCC-derived cell lines. The identified proteins in this experiment may be used in future studies of carcinogenesis or as diagnostic markers and therapeutic targets for OSCC.     

Over-expression of DNA methyltransferases and CDKN2A gene methylation status in squamous cell carcinoma of the oral cavity

The present study was designed to investigate the potential relationship between CDKN2A (p16) gene hypermethylation, which has reported to be frequently observed in oral squamous cell carcinomas (OSCCs), and expression of human DNA methyltransferases (DNMTs: DNMT1, DNMT3A and DNMT3B). Twenty-five pairs of primary OSCCs and matched normal oral mucosa tissues were examined. The p16 gene was hypermethylated (48%) in the tumors showing significant down-regulation of both mRNA and protein expressions. A demethylation assay on 8 OSCC-derived cell lines was also performed by means of treatment with the demethylating agent, 5-aza-2'-deoxycytidine. Four of 5 cell lines showing down-regulation of the p16 gene, revealed re-activation of gene expression after the treatment. In contrast, frequent over-expression of DNMT mRNA expression, also found in the expression of the proteins, was detected: DNMT1 at 72% and DNMT3A at 56%, and DNMT3B at 64%, respectively. However, we could not identify any statistical significance between p16-hypermethylation status in individual tumors and the expression of any of the three DNMTs. These data suggest that hypermethylation of the p16 gene and up-regulation of DNMTs are involved in oral carcinogenesis, but they may be through different mechanisms.     

Sarcoendoplasmic reticulum Ca(2+) ATPase type 2 downregulated in human oral squamous cell carcinoma

Mice with a heterozygous deletion of the Atp2a2 gene (Atp2a2(+/-)) encoding SERCA2 spontaneously develop SCCs of the skin and upper digestive tract, including the oral cavity. To elucidate the contribution of ATP2A2 to human oral carcinogenesis, we analyzed genetic and epigenetic changes as well as mRNA and protein expression in primary OSCCs and OPLs. With the exception of one OSCC-derived cell line showing a 12 bp deletion of ATP2A2, we found no mutations in the coding sequence of the gene in primary OSCCs (n = 52), OPLs (n = 32) and cell lines (n = 8). In immunohistochemistry, however, high frequencies of ATP2A2 downregulation were evident not only in primary OSCCs (42%, 42/100) but also in OPLs (31%, 10/32). Real-time quantitative RT-PCR data were consistent with the protein expression status. Aberrant DNA methylation within ATP2A2 also was detected in 9 of 30 ATP2A2-downregulated OSCCs. Moreover, restoration or elevated expression of the ATP2A2 protein was induced in most of the cell lines showing ATP2A2 methylation after treatment with 5-aza-2'-dC, a DNA demethylating agent. These results suggest that inactivation of the ATP2A2 gene is a frequent and early event during oral carcinogenesis and that loss of expression may be regulated partly by an epigenetic mechanism.     

Ubiquitous mitochondrial creatine kinase downregulated in oral squamous cell carcinoma

In this study, we performed two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of fly mass spectrometry to identify the protein(s) associated with the development of oral squamous cell carcinomas (OSCCs) by comparing patterns of OSCC-derived cell lines with normal oral keratinocytes (NOKs), and found that downregulation of ubiquitous mitochondrial creatine kinase (CKMT1) could be a good candidate. Decreased levels of CKMT1 mRNA and protein were detected in all OSCC-derived cell lines examined (n=9) when compared to those in primary normal oral keratinocytes. Although no sequence variation in the coding region of the CKMT1 gene with the exception of a nonsense mutation in exon 8 was identified in these cell lines, we found a frequent hypermethylation in the CpG island region. CKMT1 expression was restored by experimental demethylation. In addition, when we transfected CKMT1 into the cell lines, they showed an apoptotic phenotype but no invasiveness. In clinical samples, high frequencies of CKMT1 downregulation were detected by immunohistochemistry (19 of 52 (37%)) and quantitative real-time RT-PCR (21 of 50 (42%)). Furthermore, the CKMT1 expression status was significantly correlated with tumour differentiation (P<0.0001). These results suggest that the CKMT1 gene is frequently inactivated during oral carcinogenesis and that an epigenetic mechanism may regulate loss of expression, which may lead to block apoptosis.     

High expression of EZH2 is associated with tumor proliferation and prognosis in human oral squamous cell carcinomas

Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes and is important in cell cycle regulation. Overexpression of EZH2 protein has been associated with biological malignancy of prostate cancer and several other cancers. The aim of the present study was to evaluate the expression of EZH2 protein in human oral normal mucosa, dysplasia and oral squamous cell carcinoma (OSCC) with clinicopathological profiles. EZH2 expression was assessed by Western blotting and immunohistochemistry in 3 OSCC cell lines, 10 normal mucosae, 50 dysplasias and 102 OSCCs. The labeling indices (LIs) of EZH2, Ki-67, P53, and the apoptotic index (AI) were evaluated by immunohistochemistry and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) method. Western blot analysis detected EZH2 protein as a single band at 91kDa in the 3 OSCC cell lines, but it was almost absent in non-tumoral oral mucosae. The LI of EZH2 was highest in the OSCCs, followed by the dysplasias (p<0.05) and normal mucosae (p<0.05) with significant difference. The LI of EZH2 correlated with the clinical stage, tumor size, lymph node metastasis and LIs of Ki-67 and P53, but not with the AI in OSCCs, and inversely correlated with the histological differentiation of OSCCs. The survival rate calculated by the Kaplan-Meier method revealed that OSCC patients with higher EZH2 expression showed poorer prognosis than those with a lower EZH2 expression (p<0.01). These results suggest that overexpression of EZH2 is correlated with malignant potential and poor prognosis in OSCCs. EZH2 might serve as a novel biomarker for predicting prognosis in patients with OSCCs.     

Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines.

One of the most important components of G1 checkpoint is the retinoblastoma protein (pRB110). The activity of pRB is regulated by its phosphorylation, which is mediated by genes such as cyclin D1 and p16/MTS1. All three genes have been shown to be commonly altered in human malignancies. We have screened a panel of 26 oral squamous cell carcinomas (OSCC), nine premalignant and three normal oral tissue samples as well as eight established OSCC cell lines for mutations in the p16/MTS1 gene. The expression of p16/MTS1, cyclin D1 and pRB110 was also studied in the same panel. We have found p16/MTS1 gene alterations in 5/26 (19%) primary tumours and 6/8 (75%) cell lines. Two primary tumours and five OSCC cell lines had p16/MTS1 point mutations and another three primary and one OSCC cell line contained partial gene deletions. Six of seven p16/MTS1 point mutations resulted in termination codons and the remaining mutation caused a frameshift. Western blot analysis showed absence of p16/MTS1 expression in 18/26 (69%) OSCC, 7/9 (78%) premalignant lesions and 8/8 cell lines. One cell line, H314, contained a frameshift mutation possibly resulting in a truncated p16/MTS1 protein. pRB was detected in 14/25 (56%) of OSCC but only 11/14 (78%) of these contained all or some hypophosphorylated (active) pRB. In premalignant samples, 6/8 (75%) displayed pRB, and all three normal samples and eight cell lines analysed contained RB protein. p16/MTS1 protein was undetectable in 10/11 (91%) OSCCs with positive pRB. Overexpression of cyclin D1 was observed in 9/22 (41%) OSCC, 3/9 (33%) premalignant and 8/8 (100%) of OSCC cell lines. Our data suggest p16/MTS1 mutations and loss of expression to be very common in oral cancer cell lines and less frequent in primary OSCC tumours. A different pattern of p16/MTS1 mutations was observed in OSCC compared to other cancers with all the detected p16/MTS1 mutations resulting in premature termination codons or a frameshift. The RB protein was expressed in about half (44%) of OSCCs and its expression inversely correlated with p16/MTS1 expression. In conclusion, we show that abnormalities of the RB pathway are a common mechanism of oral carcinogenesis.     

Profiling of differentially expressed cancer-related genes in esophageal squamous cell carcinoma (ESCC) using human cancer cDNA arrays: overexpression of oncogene MET correlates with tumor differentiation in ESCC.

PURPOSE: To examine the global gene expression of cancer-related genes in esophageal squamous cell carcinoma (ESCC) through the use of Atlas Human Cancer Array membranes printed with 588 well-characterized human genes involved in cancer and tumor biology. EXPERIMENTAL DESIGN: Two human ESCC cell lines (HKESC-1 and HKESC-2) and one morphologically normal esophageal epithelium tissue specimen from the patient of which the HKESC-2 was derived were screened in parallel using cDNA expression arrays. The array results were additionally validated using semiquantitative PCR. The overexpression of oncogene MET was studied more extensively for its protein expression by immunohistochemistry in the two ESCC cell lines and their corresponding primary tissues and 61 primary ESCC resected specimens. Sixteen of these 61 ESCC cases also had available the corresponding morphologically normal esophageal epithelium tissues and were also analyzed for MET expression. The clinicopathological features associated with overexpression of the MET gene were also correlated. RESULTS: The results of cDNA arrays showed that 13 cancer-related genes were up-regulated > or =2-fold (CDC25B, cyclin D1, PCNA, MET, Jagged 2, Integrin alpha3, Integrin alpha6, Integrin beta4, Caveolin-2, Caveolin-1, MMP13, MMP14, and BIGH3) and 5 genes were down-regulated > or =2-fold (CK4, Bad, IGFBP2, CSPCP, and IL-1RA) in both ESCC cell lines at the mRNA level. Semiquantitative RT-PCR analysis of 9 of these differentially expressed genes, including the MET gene, gave results consistent with cDNA array findings. The immunostaining results of the expression of MET gene showed that MET was overexpressed in both ESCC cell lines and their corresponding primary tumors at the protein level, validating the cDNA arrays findings. The results of the clinical specimens showed that the MET gene was overexpressed in ESCC compared with normal esophageal epithelium in 56 of 61 cases (92%). Moreover, the overexpression of MET protein was more often seen in well/moderately differentiated than in poorly differentiated ESCC. CONCLUSIONS: Multiple cancer-related genes are differentially expressed in ESCC, the oncogene MET is overexpressed in ESCC compared with normal esophageal epithelium, and its protein overexpression correlates with tumor differentiation in ESCC.     

Mutual paracrine effects of oral squamous cell carcinoma cells and normal oral fibroblasts: induction of fibroblast to myofibroblast transdifferentiation and modulation of tumor cell proliferation

Several lines of evidence demonstrated that the stroma surrounding the tumors plays an important role in the growth and progression of several neoplasms, including oral squamous cell carcinomas (OSCC). We evaluated the presence of myofibroblasts in OSCC and determined whether their presence is associated with clinicopathological features of the tumors. We also investigated the mutual paracrine effects of tumor cells and myofibroblasts on fibroblast-myofibroblast transdifferentiation and tumor cell proliferation. Immunohistochemical analysis showed the approximately 60% of the OSCCs contained myofibroblasts in the stroma of the tumor. Abundant presence of myofibroblasts significantly correlated with N stage, disease stage, regional recurrence, and proliferative potential of the tumor cells. Using OSCC cell lines and primary oral normal fibroblasts (ONF), we demonstrated that tumor cells induced transdifferentiation of ONFs to myofibroblasts via secretion of transforming growth factor-beta 1 (TGF-beta 1). In turn, myofibroblasts secreted factors that stimulated OSCC cell proliferation, as revealed by measuring BrdU incorporation and Ki67 expression. The results of the study suggest that during tumor invasion OSCC-derived TGF-beta 1 promote fibroblast-myofibroblast transdifferentiation, and that tumor cellular proliferation can be induced by factors released from myofibroblasts, which may favor tumor growth.