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Hatakeyama K.,Shizuoka Cancer Center Research Institute | Ohshima K.,Shizuoka Cancer Center Research Institute | Fukuda Y.,Tokyo University of Agriculture and Technology | Ogura S.-I.,Tokyo Institute of Technology | And 3 more authors.
Proteomics | Year: 2011

Splicing variation enhances proteome diversity and modulates cancer-associated proteins. Thus, the identification of alternative splice forms is significant for discovery of new cancer-related biomarkers. However, relatively few screening approaches of alternative splicing via proteomics have been reported. In the present study, we describe a combined analysis with proteome and transcriptome to simultaneously identify cancer-related splicing variants and splicing variant-derived protein fragments that are differentially expressed in a highly metastatic gastric cancer cell line MKN45P versus its parental cell line MKN45. We found three potential alternative-spliced genes using MS-based shotgun method and two different microarray platforms. Among them, aldolase C, fructose-bisphosphate (ALDOC) was predicted to have novel alternative splice forms. We successfully identified and validated novel splice forms of ALDOC gene by RT-PCR and DNA sequencing analyses, the expression level of which were higher in MKN45P than in MKN45. Furthermore, the protein fragment derived from the validated splicing variant was identified using custom-built data set including sequences of ALDOC variants in MS/MS analysis. Our combined analysis will be a promising technique for screening of cancer-related splicing variants and their protein isoforms. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Yamada H.,Hamamatsu University School of Medicine | Shinmura K.,Hamamatsu University School of Medicine | Ito H.,Showa University | Kasami M.,Shizuoka Cancer Center Hospital and Research Institute | And 8 more authors.
Cancer Science | Year: 2011

Germline point or small frameshift mutations of the CDH1 (E-cadherin) gene are known to cause familial gastric cancer (FGC), but the frequency of CDH1 mutations is low in Japanese patients with FGC. Because recent studies have reported germline large genomic deletions of CDH1 in European and Canadian patients with FGC, in the present study we examined DNA samples from 13 Japanese patients with FGC to determine whether similar germline changes were present in CDH1 in this population. Using a sequencing analysis, a 1-bp deletion (c.1212delC), leading to the production of a truncated protein (p.Asn405IlefsX12), was found in an FGC family; immunohistochemical analysis revealed the loss of CDH1 protein expression in the tumors in this family. Using a combination of multiplex ligation-dependent probe amplification (MLPA) and RT-PCR analyses, we also found a large genomic deletion (c.164-?-387+?del), leading to the loss of exon 3 and the production of a truncated protein (p.Val55GlyfsX38), in another FGC family. The functional effects of the detected mutations were examined using a slow aggregation assay. Significant impairment of cell-cell adhesion was detected in CHO-K1 cells expressing Ile405fsX12- and Gly55fsX38-type CDH1 compared with cells expressing wild-type CDH1. Our results suggest that the p.Asn405IlefsX12 and p.Val55GlyfsX38 mutations of the CDH1 gene contribute to carcinogenesis in patients with FGC. This is the first report of CDH1 germline truncating mutations in Japanese patients with FGC. Screening for large germline rearrangements should be included in CDH1 genetic testing for FGC. © 2011 Japanese Cancer Association. Source


Ohshima K.,Shizuoka Cancer Center Research Institute | Kanto K.,Shizuoka Cancer Center Research Institute | Hatakeyama K.,Shizuoka Cancer Center Research Institute | Ide T.,Shizuoka Cancer Center Research Institute | And 6 more authors.
Proteomics | Year: 2014

Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis- and endocytosis-like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ-521) and metastatic (AZ-P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC-MS/MS and Western blot analyses, polyadenylate-binding protein 1 (PABP1) was found to be predominantly abundant in AZ-P7a exosomes. The amount of exosomal PABP1 in AZ-P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin-proteasome pathway (MG-132 and PYR-41). Treatment of AZ-P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1-immunoreactive products cleaved via a proteolysis-like process. Taken together, these results suggest that AZ-P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome-mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Wakabayashi-Nakao K.,Shizuoka Cancer Center Research Institute | Hatakeyama K.,Shizuoka Cancer Center Research Institute | Ohshima K.,Shizuoka Cancer Center Research Institute | Yamaguchi K.,Shizuoka Cancer Center Hospital and Research Institute | Mochizuki T.,Shizuoka Cancer Center Research Institute
Biomedical Research (Japan) | Year: 2014

Carcinoembryonic antigen (CEA), an oncofetal cell surface glycoprotein, has been widely used as a human tumor marker due to its high expression in tumors and secretion to serum. It belongs to the immunoglobulin superfamily named CEA-related cell adhesion molecule (CEACAM) family. Members of this family are detected in various cancers and have been shown to be involved in cancer growth and invasion. In this study, we examined the mRNA expression profiles of CEACAM family members including CEACAM1, CEACAM3, CEACAM4, CEACAM5 (CEA), CEACAM6, CEACAM7, and CEACAM8 in various tumor cell lines. Our screening data indicated that the mRNA expression patterns of CEACAMs in TT cells, which are derived from medullary thyroid carcinoma (MTC), were distinct from other tumor cell lines. Additionally, CEACAM4 was only expressed in TT cells, in which two novel splice variants of CEACAM4 were expressed. These findings suggested that production of CEA and CEA-related molecules in MTC may be distinct from other tumor-based production of those molecules and that the specific expression of CEACAM4 would make possible to differentiate between MTC and other CEA-producing tumors. © 2014 Biomedical Research Press. Source


Hatakeyama K.,Shizuoka Cancer Center Research Institute | Wakabayashi-Nakao K.,Shizuoka Cancer Center Research Institute | Ohshima K.,Shizuoka Cancer Center Research Institute | Sakura N.,Shizuoka Cancer Center Research Institute | And 2 more authors.
BMC Research Notes | Year: 2013

Background: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is an oncofetal cell surface glycoprotein. Because of its high expression in cancer cells and secretion into serum, CEA has been widely used as a serum tumor marker. Although other members of CEACAM family were investigated for splice variants/variants-derived protein isoforms, few studies about the variants of CEACAM5 have been reported. In this study, we demonstrated the existence of novel CEACAM5 splice variants and splice variant-derived protein isoforms in gastrointestinal cancer cell lines. Results: We identified two novel CEACAM5 splice variants in gastrointestinal (pancreatic, gastric, and colorectal) cancer cell lines. One of the variants possessed an alternative minor splice site that allowed generation of GC-AG intron. Furthermore, CEA protein isoforms derived from the novel splice variants were expressed in cancer cell lines and those protein isoforms were secreted into the culture medium. Although CEA protein isoforms always co-existed with the full-length protein, the secretion patterns of these isoforms did not correlate with the expression patterns. Conclusions: This is the first study to identify the expression of CEA isoforms derived from the novel splice variants processed on the unique splice site. In addition, we also revealed the secretion of those isoforms from gastrointestinal cancer cell lines. Our findings suggested that discrimination between the full-length and identified protein isoforms may improve the clinical utility of CEA as a tumor marker. © 2013 Hatakeyama et al.; licensee BioMed Central Ltd. Source

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