Takao Kawakami

Synuclein-γ Is Closely Involved in Perineural Invasion and Distant Metastasis in Mouse Models and Is a Novel Prognostic Factor in Pancreatic Cancer

Purpose: Perineural invasion is associated with the high incidence of local recurrence and a dismal prognosis in pancreatic cancer. We previously reported a novel perineural invasion model and distinguished high– and low–perineural invasion groups in pancreatic cancer cell lines. This study aimed to elucidate the molecular mechanism of perineural invasion. Experimental Design: To identify key biological markers involved in perineural invasion, differentially expressed molecules were investigated by proteomics and transcriptomics. Synuclein-γ emerged as the only up-regulated molecule in high–perineural invasion group by both analyses. The clinical significance and the biological property of synuclein-γ were examined in 62 resected cases of pancreatic cancer and mouse models. Results: Synuclein-γ overexpression was observed in 38 (61%) cases and correlated with major invasive parameters, including perineural invasion and lymph node metastasis (P < 0.05). Multivariate analyses revealed synuclein-γ overexpression as the only independent predictor of diminished overall survival [hazard ratio, 3.4 (95% confidence interval, 1.51-7.51)] and the strongest negative indicator of disease-free survival [2.8 (1.26-6.02)]. In mouse perineural invasion and orthotopic transplantation models, stable synuclein-γ suppression by short hairpin RNA significantly reduced the incidence of perineural invasion (P = 0.009) and liver/lymph node metastasis (P = 0.019 and P = 0.020, respectively) compared with the control. Conclusions: This is the first study to provide in vivo evidence that synuclein-γ is closely involved in perineural invasion/distant metastasis and is a significant prognostic factor in pancreatic cancer. Synuclein-γ may serve as a promising molecular target of early diagnosis and anticancer therapy.

Identification by differential tissue proteome analysis of talin-1 as a novel molecular marker of progression of hepatocellular carcinoma.

Objective: Hepatocellular carcinoma (HCC) is characterized by a multistage process of tumor progression. This study addressed its molecular features to identify novel protein candidates involved in HCC progression. Methods: Using liquid chromatography-tandem mass spectrometry, proteomes of 4 early HCCs and 4 non-HCC tissues derived from 2 cases of liver transplant surgery were compared with respect to the separation profiles of their tryptic peptides. Immunohistochemistry was performed on 106 HCC nodules to confirm the results of the proteomic analysis. Results: Statistical analysis of the profiles selected the peptide peaks differentiating HCC from non-HCC. A database search of the tandem mass spectrometry data from those peptide peaks identified 61 proteins, including a cytoskeletal protein, talin-1, as upregulated in HCC. Talin-1 expression levels in HCC nodules were significantly associated with the dedifferentiation of HCC (p = 0.001). A follow-up survey of the examined clinical cases revealed a correlation between talin-1 upregulation and a shorter time to recurrence after resection (p = 0.039), which may be related to the higher rate of portal vein invasion in HCCs with talin-1 up-regulation (p = 0.029). Conclusions: Proteomic analysis led to identification of talin-1 as a promising HCC marker. Talin-1 upregulation is associated with HCC progression and may serve as a prognostic marker.

Proteomic Biomarkers for Acute Interstitial Lung Disease in Gefitinib-Treated Japanese Lung Cancer Patients

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10−25), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control.

A neuronal transmembrane protein LRFN4 induces monocyte/macrophage migration via actin cytoskeleton reorganization.

Leucine‐rich repeat and fibronectin type III domain‐containing (LRFN) family proteins are thought to be neuronal‐specific proteins that play essential roles in neurite outgrowth and synapse formation. Here, we focused on expression and function of LRFN4, the fourth member of the LRFN family, in non‐neural tissues. We found that LRFN4 was expressed in a wide variety of cancer and leukemia cell lines. We also found that expression of LRFN4 in the monocytic cell line THP‐1 and in primary monocytes was upregulated following macrophage differentiation. Furthermore, we demonstrated that LRFN4 signaling regulated both the transendothelial migration of THP‐1 cells and the elongation of THP‐1 cells via actin cytoskeleton reorganization. Our data indicate that LRFN4 signaling plays an important role in the migration of monocytes/macrophages.

Inverse relationship between Sec14l3 mRNA/protein expression and allergic airway inflammation

Bronchial asthma is an inflammatory disease of the airways. The Sec14l3 gene, encoding a 45-kDa secretory protein, is specifically expressed in airway epithelium. Here, we report on the kinetics of Sec14l3 expression following allergic inflammation of the lung. Brown Norway rats were sensitized by intraperitoneal injection of ovalbumin, followed by challenge with aerosolized ovalbumin after a 3-week interval. This animal model showed many features similar to human allergic asthma: an increase in inflammatory cells such as eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid and histopathological alteration of lung tissue, exhibiting infiltration of these inflammatory cells and degeneration and necrosis of alveolar epithelium. These parameters reached their maximal level 24h after allergen challenge. In contrast, quantitative polymerase chain reaction analyses demonstrated a rapid and significant reduction of Sec14l3 mRNA in lung tissue and maximum reduction (to 1.4% of the control) was observed at 24h. Pretreatment with dexamethasone significantly suppressed both the Sec14I3 mRNA reduction and all of the inflammatory changes. The 45-kDa secretory protein was identified in the supernatant of BAL fluids. Two-dimensional gel images of the supernatant proteome also revealed down-regulation of the protein following inflammation (to approximately 30% of the control at 24h). Thus, Sec14l3 expression is highly and inversely associated with the progression of airway inflammation. Sec14l3 mRNA and protein may function in the homeostasis of airway epithelial cells under normal conditions.

Disease proteomics toward bedside reality

The human genome has been sequenced, and investigation of its products has become possible in a sequencebased framework. More than 200000 protein species are expressed in the body from ≈30000 human genes. The term proteome, coined as a linguistic equivalent to the concept of genome, is used to describe the complete set of proteins that is expressed, and modified following expression, by the entire genome in a cell at any one time. Protein types and amounts expressed in a body vary greatly depending upon whether it is healthy or ill. Therefore, proteomics is attracting an increasing interest in its application to better understanding of disease processes, to development of new biomarkers for diagnosis and early detection of disease, and to accelerate drug development. There are numerous opportunities for medicine, although it is quite challenging to meet the needs for high sensitivity and high throughput required for disease-related investigations.

Alteration of protein composition in mouse thymocytes by signals through T‐cell receptor

To avoid destructive autoimmunity, T‐cell precursors (thymocytes) expressing autoreactive T‐cell receptor are deleted in the thymus via an apoptotic process by the signals from the T‐cell receptor‐CD3 complexes. In order to analyze the apoptotic mechansism, we established a cell‐free system using the lysates from mouse thymocytes treated in vivo with anti‐CD3 monoclonal antibody (mAb). The soluble cytosolic high molecular mass protein fraction from the anti‐CD3‐treated thymocytes revealed an activity that directly induces nuclear apoptotic morphological changes and DNA fragmentation. This fragmentation activity was not observed in the fraction from the thymocytes without anti‐CD3 treatment. Proteins in both fractions were separated by two‐dimensional electrophoresis. The silver‐stained gels revealed differences in protein spots. These protein spots were identified by database searching of mass spectrometric (MS) and tandem mass spectrometric (MS/MS) data obtained from in‐gel tryptic digests of the spots, using an integrated system of liquid chromatography/electrospray ionization/ion‐trap mass spectrometry. In this study, the high mobility group protein HMG2 was identified as one of the cytosolic proteins that is increased by the signals from the T‐cell receptor, and heterogeneous nuclear ribonucleoprotein A2/B1 and glyceraldehyde 3‐phosphate dehydrogenase were found to be decreased by the signals.

Proteomic approach to apoptotic thymus maturation

Apoptosis is an essential process for selection of T lymphocytes specific for foreign antigen in the process of mammalian thymus maturation. Proteomics, a comprehensive study of proteins expressed in a cell, will facilitate the systematic analysis of protein molecules related to such a complicated biological system. Protein expression profiles including information about protein signatures, localization and their quantitative changes with extracellular stimulations are extremely useful to construct intracellular pathway models resulting in the apoptotic cell death.

Application of chemical selective cleavage methods to analyze post-translational modification in proteins

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post‐translationally modified groups in peptides or proteins. The modified groups cover the external modifications N‐formyl, N‐acetyl, N‐pyroglutamyi residues and C‐terminal‐α amide, as well as the internal modifications such as O‐acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post‐translational modified groups in the protein.

Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a.

An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26-41) alone is not enough, and that amino acids 12-41 from the N-terminus of ASB2a is essential for binding. ASB2a(12-41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12-41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated β-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins.