Biomarker For Oral Squamous Cell Carcinoma Research Articles

Increased expression of Cathepsin B in oral squamous cell carcinoma

Previously, an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelial cells (HIOECs), a line of cancerous HB96 cells and another type of cell (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells. Cathepsin B was one of the significantly up-regulated proteins accompanying cellular transformation. Cathepsin B was further validated for its expression in the three cell lines and in clinical samples of tumour tissues and their adjacent normal epithelia from 30 primary OSCC patients. Western blot analysis and real-time PCR detected increased Cathepsin B protein and mRNA levels in the cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry and real-time PCR showed elevated Cathepsin B protein and mRNA levels in the tumour tissues over the adjacent non-malignant epithelia from OSCC patients. The results presented here suggest that the expression of Cathepsin B increases along with the cancerisation in OSCC both in vitro and in vivo, and it may serve as a candidate biomarker of OSCC.

Identification of increased NBS1 expression as a prognostic marker of squamous cell carcinoma of the oral cavity

Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide; however, accurate molecular markers to predict its prognosis are still limited. We previously demonstrated that overexpression of the DNA double-strand break repair protein NBS1 is a prognostic marker of advanced head and neck squamous cell carcinoma (HNSCC). Therefore, we aimed to investigate the feasibility of using NBS1 as a biomarker in OSCC. In this study, we enrolled 148 OSCC for immunohistochemical (IHC) and clinical analysis. Data from 58 advanced non-oral-cavity HNSCC (NO-HNSCC) cases were also included for comparison due to the biological and clinical discrepancy between OSCC and HNSCC originated from the other sites (e.g. pharynx or larynx). First, we validated the NBS1 IHC results by real-time RT-PCR analysis, and an excellent correlation between the results of these two assays confirmed the reliability and robustness of IHC procedures and interpretation. NBS1 overexpression was an independent prognostic marker in both OSCC and NO-HNSCC cases. In OSCC, the prognostic significance of NBS1 was shown regardless of T stage and lymph node status. Increased NBS1 expression correlated with advanced T stage and recurrence/metastasis. NBS1 overexpression correlated with the phosphorylation levels of Akt and its downstream target mammalian target of rapamycin (mTOR). These results clearly illustrate the expression profile of NBS1 in OSCC and NO-HNSCC, and highlight the role of NBS1 in HNSCC irrespective of the primary sites. It also indicates the practicability of application of NBS1 as a marker in OSCC.

Identification of a truncated cystatin SA-I as a saliva biomarker for oral squamous cell carcinoma using the SELDI ProteinChip platform

New and more consistent biomarkers of oral squamous cell carcinoma (OSCC) are needed to improve early detection of the disease and to monitor patient management. The aim of this study was to detect new OSCC tumor markers in saliva. Unstimulated saliva, collected from patients with primary stage I OSCC as matched pre-and post-treatment samples, was used in the analysis. A surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in the saliva samples. This analysis revealed 26 proteins with significantly different expression levels in the pre-and post-treatment samples (P<0.05). A 14 kDa protein detected in pre-treatment saliva from the OSCC patients was identified as a truncated cystatin SA-I, with deletion of three amino acids from the N-terminus. The authors propose that ProteinChip analysis may provide a reliable screening test for early diagnosis of OSCC and that truncated cystatin SA-I might be a useful tumor biomarker for OSCC.

Proteomic profiling of differential display analysis for human oral squamous cell carcinoma: 14‐3‐3 σ Protein is upregulated in human oral squamous cell carcinoma and dependent on the differentiation level

Oral squamous cell carcinoma (OSCC) has an absolute majority of all oral cancer. We used proteomic technology to analyze the protein expression profile in OSCC tissues and accompanying surrounding normal tissues in four oral locations (buccal mucosa, gingival mucosa, oral floor, and tongue). Ten protein spots were overexpressed more strongly in cancer tissues than normal ones, and were identified as proliferating cell nuclear antigen, 14-3-3 ε, 14-3-3 σ, proteasome subunit α type 5, translationally controlled tumor protein, eukaryotic translation initiation factor 3 subunit, macrophage capping protein, and mitochondrial isocitrate dehydrogenase subunit α. Macrophage capping protein and mitochondrial isocitrate dehydrogenase subunit α had two spots. Especially, we focused on 14-3-3 σ protein, one of the eight identified proteins, and assessed its expression level in four oral locations of OSCC by using differential display methods. The expression level of 14-3-3 σ protein was upregulated in four locations of oral cavity. Eight proteins which we identified in this study may play an important role in OSCC carcinogenesis and progression and could be used as diagnostic biomarkers of OSCC.

Laser-capture Microdissection and Protein Extraction for Protein Fingerprint of OSCC and OLK

To find new biomarkers and establish histopathology protein fingerprint models for early detection of oral squamous cell carcinoma (OSCC), laser capture microdissection (LCM) technology was utilized in 21 OSCC tissues and 7 oral leukoplaque (OLK) tissues as well as their adjacent normal tissues. Each sample was then detected by SELDI-TOF-MS technology and CM10 protein chip as well as bioinformatics tools. Three proteomic biomarker patterns were identified. Pattern 1 distinguishes patients with OLK from healthy individuals. Pattern 2 distinguishes patients with OSCC from healthy individuals. Pattern 3 distinguishes patients with OSCC from patients with OLK. The analysis yielded both a specificity and a sensitivity of 90.48% for pattern 1, a specificity of 100.00% and a sensitivity of 85.71% for pattern 2, and a specificity of 100.00% and a sensitivity of 85.71% for pattern 3. Proteome mass/charge 3714, 3515, and 4944 built the distinguished protein peaks between the OSCC tumor and adjacent normal tissues. The accuracy of the blind prediction was 90.48% (38/42). M/Z 15122 and 7569 built the distinguished protein peaks between the OLK and adjacent normal tissues. M/Z 3738 and 11366 built the distinguished protein peaks between the OSCC and the OLK. By employing LCM technology combined with SELDI-TOF-MS technology and bioinformatics approaches, histopathology would not only facilitate the discovery of better biomarkers for OSCC and OLK, but also provide a useful tool for molecular diagnosis by potential biomarker.

Salivary Proteomics for Oral Cancer Biomarker Discovery

This study aims to explore the presence of informative protein biomarkers in the human saliva proteome and to evaluate their potential for detection of oral squamous cell carcinoma (OSCC). Whole saliva samples were collected from patients (n = 64) with OSCC and matched healthy subjects (n = 64). The proteins in pooled whole saliva samples of patients with OSCC (n = 16) and matched healthy subjects (n = 16) were profiled using shotgun proteomics based on C4 reversed-phase liquid chromatography for prefractionation, capillary reversed-phase liquid chromatography with quadruple time-of-flight mass spectrometry, and Mascot sequence database searching. Immunoassays were used for validation of the candidate biomarkers on a new group of OSCC (n = 48) and matched healthy subjects (n = 48). Receiver operating characteristic analysis was exploited to evaluate the diagnostic value of discovered candidate biomarkers for OSCC. Subtractive proteomics revealed several salivary proteins at differential levels between the OSCC patients and matched control subjects. Five candidate biomarkers were successfully validated using immunoassays on an independent set of OSCC patients and matched healthy subjects. The combination of these candidate biomarkers yielded a receiver operating characteristic value of 93%, sensitivity of 90%, and specificity of 83% in detecting OSCC. Patient-based saliva proteomics is a promising approach to searching for OSCC biomarkers. The discovery of these new targets may lead to a simple clinical tool for the noninvasive diagnosis of oral cancer. Long-term longitudinal studies with large populations of individuals with oral cancer and those who are at high risk of developing oral cancer are needed to validate these potential biomarkers.

Quantification of Serum Proteins of Metastatic Oral Cancer Patients Using LC-MS/MS and iTRAQ Labeling

Metastasis is a critical event in oral squamous cell carcinoma (OSCC) progression. In this study, we have performed quantitative analysis of serum proteins from non-metastatic (lymph-node metastasis free) and metastatic OSCC patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with iTRAQ labeling (isobaric tagging for relative and absolute quantitation). To eliminate highly abundant proteins, the serum samples were initially separated by SDS-PAGE and only low abundant protein bands were excised for subsequent in-gel tryptic digestion. The resulting peptides were then extracted from each sample gels and labeled with iTRAQ reagent 114 (control), 116 (non-metastatic) and 117 (metastatic), respectively. Afterwards, the labeled samples were combined and subjected to LC-MS/MS analysis using linear ion trap (LIT) MS with pulsed Q collision induced dissociation (PQD). A total of 64 proteins were identified and quantified by this approach. Our study showed that iTRAQ labeling and LIT-MS with PQD is a valuable approach to quantification of serum proteins. We also demonstrated the presence of differentially expressed serum proteins between non-metastatic and metastatic OSCCs that may be further validated as biomarkers for metastatic OSCC. However, in order to comprehensively quantify low abundant serum proteins, a more efficient approach is needed to deplete highly abundant proteins prior to quantitative serum proteome analysis of OSCC.

Examination of Oral Cancer Biomarkers by Tissue Microarray Analysis

To validate the DNA microarray results on a subset of genes that could potentially serve as biomarkers of oral squamous cell carcinoma (OSCC) by examining their expression with an alternate quantitative method and by assessing their protein levels. Based on DNA microarray data from our laboratory and data reported in the literature, we identified 6 potential biomarkers of OSCC to investigate further. We used quantitative real-time polymerase chain reaction to examine expression changes of CDH11, MMP3, SPARC, POSTN, TNC, and TGM3 in OSCC and histologically normal control tissues. We further examined validated markers at the protein level by immunohistochemical analysis of OSCC tissue microarray sections. Quantitative real-time polymerase chain reaction analysis revealed upregulation of CDH11, SPARC, POSTN, and TNC gene expression and decreased TGM3 expression in OSCC tissue compared with control tissue; MMP3 was not found to be differentially expressed. In tissue microarray immunohistochemical analyses, SPARC (secreted protein, acidic, rich in cysteine), periostin, and tenascin C exhibited increased protein expression in tumor tissue compared with control tissue, and their expression was primarily localized within tumor-associated stroma rather than tumor epithelium. Conversely, transglutaminase 3 protein expression was found only within keratinocytes in control tissue and was significantly downregulated in cancer cells. Of 6 potential gene markers of OSCC, initially identified by DNA microarray analyses, differential expression of CDH11, SPARC, POSTN, TNC, and TGM3 were validated by quantitative real-time polymerase chain reaction. Differential expression and localization of proteins encoded by SPARC, POSTN, TNC, and TGM3 were clearly shown by tissue microarray immunohistochemical analysis.

Overexpression of PAX5 in oral carcinogenesis

The oncogenic transcription factor PAX5 is an important developmental regulator and is implicated in the pathogenesis of several malignancies. The PAX5 gene is involved in medulloblastoma, non-Hodgkin's lymphoma, transitional cell carcinoma of the bladder, neuroblastoma, breast cancer and SCC. In the current study, to determine the potential involvement of PAX5 in oral squamous-cell carcinoma (OSCC) and leukoplakias, we evaluated the status of PAX5 mRNA and protein expression in OSCC cell lines, human primary OSCCs, and leukoplakias by real-time quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. A significant increase in PAX5 expression was observed in all OSCC-derived cell lines examined compared to human normal oral keratinocytes (HNOKs). In immunohistochemistry, 78% of tumors and 42% of leukoplakias examined were positive for PAX5, while no immunoreaction was observed in corresponding normal tissues. The results suggest that PAX5 plays an important role during oral carcinogenesis, especially in the early stage, and that the gene may have potential as a biomarker and therapeutic target for OSCC.

A concerted cell and serum proteomic approach for the identification of oral squamous cell carcinoma biomarkers

5512 Background: Head and neck squamous cell carcinoma is the sixth most common cause of cancer deaths in the world. Despite extensive research, the 5-year survival rates have not changed significantly over the last decade. Presently, the lack of serum biomarkers for head and neck carcinoma limits early diagnosis and treatment monitoring of advanced disease. In this study, we used a proteomic approach on serum from mice bearing human oral squamous cell carcinoma xenografts and from conditioned cell culture medium from matched cell lines. Methods: Oral squamous cell carcinomas, with distinct invasive phenotypes, and adjacent normal tissue were obtained from human patients, and were transplanted orthotopicaly into tongues of RAG-2/γ(c) mice. After a 20% weight loss, the mice were sacrificed by exsanguinations. Two distinct proteomic protocols were used to analyze the mouse serum. The first involved albumin depletion followed by two-dimensional gel electrophoresis and MALDI. The second arm utilized the same albumin depletion followed by multidimensional-protein-information-technology (ESI-LC and MS/MS). The top candidate proteins, which were differentially expressed in the cancer bearing mice compared to matched controls were then validated by Western blot, immunoprecipitation, immunofluorescence, and/or immunohistochemistry analyses using mouse serum and conditioned medium from matched cell lines. Results: Orthotopic implantation of human tumor tissues in mouse tongue was successful in 100% of the mice tested. The human origin of these tumors was confirmed pathologically. A correlation between disease stages and invasiveness was observed. We identified over one hundred proteins as being differentially expressed between control and cancer-bearing mice (p &lt; 0.05), including proteins involved in cell cytoskeleton signaling. The expression of these proteins was then validated in mouse serum, tissue xenografts, and conditioned medium from oral cancer matched established cell lines. Conclusion: We report the first proteomic in-vivo model of oral cancer for the identification of low molecular weight serum biomarkers. We identified candidates that can be exploited as potential markers for diagnosis of human oral squamous cell carcinoma. No significant financial relationships to disclose.

Immunohistochemical analysis of P57(kip2), p53 and hsp60 expressions in premalignant and malignant oral tissues

To investigate the expressions and significances of P57(kip2), p53 and hsp60 proteins in the dysplasia-carcinoma sequence of oral mucosa. A retrospective study was performed in 10 cases of normal oral mucosa, 79 cases of leukoplakia and 67 cases of squamous cell carcinoma (SCC). The expressions of P57(kip2), p53 and hsp60 proteins were detected in the tissue samples of these populations using immunohistochemical method. P57(kip2) expression was decreased in oral leukoplakia with moderate or severe dysplasia, and further decreased in oral SCC. Negative expression of P57(kip2) was significantly associated with advanced tumor size, the occurrence of lymph node metastasis and the advanced clinical stage in oral SCC. The overall 5-year survival rate in the P57(kip2) positive group was significantly higher than that in the P57(kip2) negative group. P57(kip2) expression was decreased in oral leukoplakia with moderate or severe dysplasia, and further decreased in oral SCC. It was a remarkable progressive and prognostic biomarker in oral SCC.