Lang-Ming Chi

Enhanced Interferon Signaling Pathway in Oral Cancer Revealed by Quantitative Proteome Analysis of Microdissected Specimens Using 16O/18O Labeling and Integrated Two-dimensional LC-ESI-MALDI Tandem MS

Oral squamous cell carcinoma (OSCC) remains one of the most common cancers worldwide, and the mortality rate of this disease has increased in recent years. No molecular markers are available to assist with the early detection and therapeutic evaluation of OSCC; thus, identification of differentially expressed proteins may assist with the detection of potential disease markers and shed light on the molecular mechanisms of OSCC pathogenesis. We performed a multidimensional 16O/18O proteomics analysis using an integrated ESI-ion trap and MALDI-TOF/TOF MS system and a computational data analysis pipeline to identify proteins that are differentially expressed in microdissected OSCC tumor cells relative to adjacent non-tumor epithelia. We identified 1233 unique proteins in microdissected oral squamous epithelia obtained from three pairs of OSCC specimens with a false discovery rate of <3%. Among these, 977 proteins were quantified between tumor and non-tumor cells. Our data revealed 80 dysregulated proteins (53 up-regulated and 27 down-regulated) when a 2.5-fold change was used as the threshold. Immunohistochemical staining and Western blot analyses were performed to confirm the overexpression of 12 up-regulated proteins in OSCC tissues. When the biological roles of 80 differentially expressed proteins were assessed via MetaCore™ analysis, the interferon (IFN) signaling pathway emerged as one of the most significantly altered pathways in OSCC. As many as 20% (10 of 53) of the up-regulated proteins belonged to the IFN-stimulated gene (ISG) family, including ubiquitin cross-reactive protein (UCRP)/ISG15. Using head-and-neck cancer tissue microarrays, we determined that UCRP is overexpressed in the majority of cheek and tongue cancers and in several cases of larynx cancer. In addition, we found that IFN-β stimulates UCRP expression in oral cancer cells and enhances their motility in vitro. Our findings shed new light on OSCC pathogenesis and provide a basis for the future development of novel biomarkers.

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.

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.

Secretome-based identification of Mac-2 binding protein as a potential oral cancer marker involved in cell growth and motility.

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.

Identification of Guanylate-Binding Protein 1 as a Potential Oral Cancer Marker Involved in Cell Invasion Using Omics-Based Analysis

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.

Overexpression of BST2 is associated with nodal metastasis and poorer prognosis in oral cavity cancer

Bone marrow stromal cell antigen 2 (BST2) was one of the proteins that were found to be related to tumor metastasis in our previous proteomic study. Now we examine its clinical role on the oral cavity squamous cell carcinoma (OSCC).

Low-molecular-mass secretome profiling identifies C-C motif chemokine 5 as a potential plasma biomarker and therapeutic target for nasopharyngeal carcinoma.

UNLABELLED Cancer cell secretome profiling has been shown to be a promising strategy for identifying potential body fluid-accessible cancer biomarkers and therapeutic targets. However, very few reports have investigated low-molecular-mass (LMr) proteins (<15kDa) in the cancer cell secretome. In the present study, we applied tricine-SDS-gel-assisted fractionation in conjunction with LC-MS/MS to systemically identify LMr proteins in the secretomes of three nasopharyngeal carcinoma (NPC) cell lines. We examined two NPC tissue transcriptome datasets to identify LMr genes/proteins that are highly upregulated in NPC tissues and also secreted/released from NPC cells, obtaining 35 candidates. We verified the overexpression of four targets (LSM2, SUMO1, RPL22, and CCL5) in NPC tissues by immunohistochemistry and demonstrated elevated plasma levels of two targets (S100A2 and CCL5) in NPC patients by ELISA. Notably, plasma CCL5 showed good power (AUC 0.801) for discriminating NPC patients from healthy controls. Additionally, functional assays revealed that CCL5 promoted migration of NPC cells, an effect that was effectively blocked by CCL5-neutralizing antibodies and maraviroc, a CCL5 receptor antagonist. Collectively, our data indicate the feasibility of the tricine-SDS-gel/LC-MS/MS approach for efficient identification of LMr proteins from cancer cell secretomes, and suggest that CCL5 is a potential plasma biomarker and therapeutic target for NPC. BIOLOGICAL SIGNIFICANCE Both LMr proteome and cancer cell secretome represent attractive reservoirs for discovery of cancer biomarkers and therapeutic targets. Our present study provides evidence for the practicality of using the tricine-SDS-PAGE/LC-MS/MS approach for in-depth identification of LMr proteins from the NPC cell secretomes, leading to the discovery of CCL5 as a potential plasma biomarker and therapeutic target for NPC. We believe that the modified GeLC-MS/MS approach used here can be further applied to explore extremely low-abundance, extracellular LMr proteins with important biological functions in other cell lines and biospecimens.

Identification of protein kinase CK2 as a potent kinase of Epstein–Barr virus latent membrane protein 1

The C-terminus of latent membrane protein 1 (LMP1) can be phosphorylated in vivo. However, the protein kinase responsible for LMP1 phosphorylation has not yet been identified. In this study, GST fusion proteins containing the C-terminus of LMP1 were generated and used as substrates to survey the kinases that phosphorylate LMP1. Among several purified protein kinases tested, only protein kinase CK2 (CK2) could specifically phosphorylate LMP1. Using the in-gel kinase assay in the absence and presence of a selective CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole, CK2 was determined to be the major kinase to phosphorylate LMP1 in lymphoma and epithelial cell lines. This is the first study to show that CK2 is a potent kinase to phosphorylate LMP1 in vitro.

Overexpression of caldesmon is associated with lymph node metastasis and poorer prognosis in patients with oral cavity squamous cell carcinoma

A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC.

Identification of the lamin A/C phosphoepitope recognized by the antibody P-STM in mitotic HeLa S3 cells

BackgroundLamins A and C, two major structural components of the nuclear lamina that determine nuclear shape and size, are phosphoproteins. Phosphorylation of lamin A/C is cell cycle-dependent and is involved in regulating the assembly–disassembly of lamin filaments during mitosis. We previously reported that P-STM, a phosphoepitope-specific antibody raised against the autophosphorylation site of p21-activated kinase 2, recognizes a number of phosphoproteins, including lamins A and C, in mitotic HeLa cells.ResultsHere, using recombinant proteins and synthetic phosphopeptides containing potential lamin A/C phosphorylation sites in conjunction with in vitro phosphorylation assays, we determined the lamin A/C phosphoepitope(s) recognized by P-STM. We found that phosphorylation of Thr-19 is required for generating the P-STM phosphoepitope in lamin A/C and showed that it could be created in vitro by p34cdc2/cyclin B kinase (CDK1)-catalyzed phosphorylation of lamin A/C immunoprecipitated from unsynchronized HeLa S3 cells. To further explore changes in lamin A/C phosphorylation in living cells, we precisely quantified the phosphorylation levels of Thr-19 and other sites in lamin A/C isolated from HeLa S3 cells at interphase and mitosis using the SILAC method and liquid chromatography-tandem mass spectrometry. The results showed that the levels of phosphorylated Thr-19, Ser-22 and Ser-392 in both lamins A and C, and Ser-636 in lamin A only, increased ~2- to 6-fold in mitotic HeLa S3 cells.ConclusionsCollectively, our results demonstrate that P-STM is a useful tool for detecting Thr-19-phosphorylated lamin A/C in cells and reveal quantitative changes in the phosphorylation status of major lamin A/C phosphorylation sites during mitosis.

Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3

Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D3A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D3A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D3A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both 16O/18O- and 14N/15N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D3A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT.