Identification of PRDX4 and P4HA2 as Metastasis-Associated Proteins in Oral Cavity Squamous Cell Carcinoma by Comparative Tissue Proteomics of Microdissected Specimens Using iTRAQ Technology

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.

MicroRNA-205 Targets Tight Junction-related Proteins during Urothelial Cellular Differentiation

“…iTRAQ Experiments-Equal amount of proteins from each treatment were digested with trypsin and labeled with iTRAQ reagents (Applied Biosystems) as described previously (43). Two four-plex iTRAQ comparisons were set up as follows.…”

“…Comparison B: Ant-709 was labeled with iTRAQ reagent 114, Ant-205 with 115, control-1 with 116, and control-2 with 117. The labeled peptides from each iTRAQ comparison were then pooled and fractionized on a reverse phase capillary column with an acetonitrile gradient in the presence of about 0.1% NH 4 OH (Gemini C18 column, 3 ϫ 100 mm, 3 m; Phenomenex, Torrence, CA) carried on a Breeze HPLC instrument (Waters, Milford, MA) (43). The effluents were collected and then pooled into ϳ60 fractions dependent on the signal intensity of OD 280.…”

“…MS-based Protein Identification and Quantification-Peptide samples were analyzed on a nano-LC-LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher) as described previously (43). The MS and MS/MS raw data were converted to MGF peak list files with DTASuperCharge 1.37 using SmartPicking with 200 segment size (Th) and a max search level of 8.…”

“…Consequently, the proteins were identified by more than two distinct peptides. Quantitative information of proteins was simultaneously obtained using the default settings of the Libra quantitation tool built in the TPP platform as described previously (43). In this study, only the distinct peptides and their reporter ions with an intensity of higher than 20 were included for protein quantification.…”

Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer

“…Immunohistochemical Staining-Immunohistochemical (IHC) staining of 5-m-thick consecutive sections of formalin fixed, paraffinembedded tissue specimens was performed as previously described (20). Antigens for carbonic anhydrase 2 (CA2), phosphoglycerate kinase 1 (PGK1), 14 -3-3 (SFN), 4F2 cell-surface antigen heavy chain (SLC3A2/CD98), transgelin-2 (TAGLN2), thioredoxin (TXN), and stathmin (STMN1) were retrieved by incubating with Bond Epitope Retrieval Solution 1 of the Bond-Max Automated Immunostainer (Vision BioSystems, Melbourne, Australia) at 100°C for 20 min.…”