No abstract

Cervical lymph node metastasis represents the major prognosticator for oral cavity squamous cell carcinoma (OSCC). Here, we used an iTRAQ-based quantitative proteomic approach to identify proteins that are differentially expressed between microdissected primary and metastatic OSCC tumors. The selected candidates were examined in tissue sections via immunohistochemistry, and their roles in OSCC cell function investigated using RNA interference. Seventy-four differentially expressed proteins in nodal metastases, including PRDX4 and P4HA2, were identified. Immunohistochemical analysis revealed significantly higher levels of PRDX4 and P4HA2 in tumor cells than adjacent non-tumor epithelia (P < 0.0001 and P < 0.0001, respectively), and even higher expression in the 31 metastatic tumors of lymph nodes, compared to the corresponding primary tumors (P = 0.060 and P = 0.002, respectively). Overexpression of PRDX4 and P4HA2 was significantly associated with positive pN status (P = 0.048 and P = 0.021, respectively). PRDX4 overexpression was a significant prognostic factor for disease-specific survival in both univariate and multivariate analyses (P = 0.034 and P = 0.032, respectively). Additionally, cell migration and invasiveness were attenuated in OEC-M1 cells upon in vitro knockdown of PRDX4 and P4HA2 with specific interfering RNA. Novel metastasis-related prognostic markers for OSCC could be identified by our approach.

Oral cavity squamous cell carcinoma (OSCC) is a devastating disease that accounts for 3% of all cancer cases diagnosed annually. OSCC is usually diagnosed at advanced clinical stages, resulting in poor outcomes. To identify effective biomarkers for improved OSCC diagnosis and/or management, we simultaneously analyzed the OSCC cell secretome and tissue transcriptome. Among the 19 candidates isolated, guanylate-binding protein 1 (GBP1) was selected for further validation using serum samples from OSCC patients and healthy controls. Notably, the serum level of GBP1 was higher in OSCC patients, compared to that in healthy controls. Immunohistochemical analysis further revealed GBP1 overexpression in OSCC tissues, compared with adjacent noncancerous epithelia. Importantly, the higher GBP1 level in OSCC tissue was associated with higher overall pathological stage, positive perineural invasion, and poorer prognosis. Moreover, GBP1 modulated the migration and invasion of OSCC cells in vitro. Our results collectively indicate that integrated analysis of the cancer secretome and transcriptome is a feasible strategy for the efficient identification of novel OSCC markers.

Oral squamous cell carcinoma (OSCC) is the 11th most common cancer worldwide, and is associated with a high death rate. At present, there are no suitable markers for detecting and/or monitoring OSCC in body fluids/tissues. Here, we used 1D SDS-PAGE and MALDI-TOF MS to systematically analyze the secretomes of two OSCC cell lines (OEC-M1 and SCC4). The putative OSCC-related proteins identified in this analysis included the Mac-2 binding protein (Mac-2 BP), which was further found to be overexpressed in OSCC specimens and significantly elevated in the sera of OSCC patients compared to healthy controls. Finally, RNA interference-based knock-down of Mac-2 BP expression in OSCC cells revealed for the first time that Mac-2 BP is involved in regulating growth and motility of OSCC cells.

The profiling of cancer cell secretomes is considered to be a good strategy for identifying cancer-related biomarkers, but few studies have focused on identifying low-molecular-mass (LMr) proteins (<15 kDa) in cancer cell secretomes. Here, we used tricine–SDS-gel-assisted fractionation and LC–MS/MS to systemically identify LMr proteins in the secretomes of five oral cavity squamous cell carcinoma (OSCC) cell lines. Cross-matching of these results with nine OSCC tissue transcriptome datasets allowed us to identify 33 LMr genes/proteins that were highly upregulated in OSCC tissues and secreted/released from OSCC cells. Immunohistochemistry and quantitative real-time PCR were used to verify the overexpression of two candidates, HMGA2 and MIF, in OSCC tissues. The overexpressions of both proteins were associated with cervical metastasis, perineural invasion, deeper tumor invasion, higher overall stage, and a poorer prognosis for post-treatment survival. Functional assays further revealed that both proteins promoted the migration and invasion of OSCC cell lines in vitro. Collectively, our data indicate that the tricine–SDS-gel/LC–MS/MS approach can be used to efficiently identify LMr proteins from OSCC cell secretomes, and suggest that HMGA2 and MIF could be potential tissue biomarkers for OSCC.

The findings in this study indicate that BST2 expression in OSCC tumors is an independent prognostic factor of patient survival and associated with tumor metastasis.

Thymidine kinase (TK) 1 is an enzyme that catalyzes the transfer of the terminal phosphate of ATP to the 5Ј-hydroxyl group of thymidine to form dTMP, which is the salvage pathway for dTTP synthesis. In eukaryotic cells, there are two TK isoenzymes: TK1 and TK2, also called cytosolic and mitochondrial TK, respectively. The amount of cytosolic TK1 is increased significantly in cells during transition from G 1 to S phase (1, 2), whereas the expression level of TK2 is low and is controlled in a cell cycle-independent manner (3, 4). TK1 is highly expressed in dividing or malignant cells, but is absent in quiescent cells (5-8), and its expression is stringently regulated in normal cells, but not in malignant cells (9, 10).Studies on TK1 regulation have provided a good model for understanding the molecular events that coordinate progression through the cell cycle. It is well documented that the transcriptional and translational activation of TK1 gene expression leads to elevation of its activity in the G 1 /S phase (11-18). In addition, other lines of evidence have shown that the C-terminal region of the TK1 polypeptide may determine its stability in cells in different growth states, indicating that cell cycle-dependent degradation is also involved in the regulation of TK1 expression (19 -21). Thus, it is clear that multiple levels of control regulate TK1 expression in eukaryotic cells.Our laboratory has previously reported that TK1 can be phosphorylated in human promyeloleukemia cells in response to growth stimulation (22). The phosphorylated form of TK1 was also detetected by isoelectric focusing gel analysis of TK1 purified from mouse Ehrlich ascites tumor cells (23). The regulation of TK1 phosphorylation was investigated further during the cell cycle. When cells were M phase-arrested by treatment with nocodazole, a microtubule-depolymerizing drug, TK1 became hyperphosphorylated in K562, and HeLa cells (24). Phosphoaminoacid analysis of immunoprecipitated human TK1 polypeptide indicated that serine is the residue involved in mitotic phosphorylation (22,24). Amino acid sequence analysis reveals that several potential phosphorylation sites exist for a variety of serine/threonine protein kinases, including cAMP-dependent protein kinase at serine 194, cyclindependent kinase(s) at serine 13 and serine 231, and protein kinase C at serine 30. In this study, we examined the specific phosphorylated site for human TK1 in mitotically arrested TK-deficient mouse Ltk Ϫ fibroblasts and human carcinoma HeLa cells, and we identified the kinase responsible for the mitotic phosphorylation of hTK1. The physiologic role of the mitotic phosphorylation of hTK1 was also investigated.

The thyroid hormone, 3, 3,5-triiodo-L-thyronine (T 3 ), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T 3 are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T 3 -regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TR␣1 (HepG2-TR␣1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using