Identification of Makorin 1 as a novel SEREX antigen of esophageal squamous cell carcinoma.
BACKGROUND
Esophageal squamous cell carcinoma (SCC) represents one of the most malignant tumors. To improve the poor prognosis, it is necessary to diagnose esophageal SCC at early stages using new tumor markers. SEREX (serological identification of antigens by recombinant cDNA expression cloning) is suitable for large-scale screening of tumor antigens and has been applied for various types of human tumors.


METHODS
Tumor markers of esophageal squamous cell carcinoma (SCC) were screened by SEREX method. The presence of serum anti-makorin 1 (MKRN1) antibodies (s-MKRN1-Abs) was examined by Western blotting using bacterially expressed MKRN1 protein. The expression levels of MKRN1 mRNA in tissues were examined by RT-PCR. The biological activity of MKRN1 was examined by transfection of ras-NIH3T3 mouse fibroblasts with MKRN1 cDNA. Major ubiquitinated proteins in MKRN1-transfected cells were identified by immunoprecipitation with anti-ubiquitin antibody followed by mass spectrometry.


RESULTS
MKRN1 was identified as a novel SEREX antigen of esophageal SCC. Although a total of 18 (25%) of 73 patients with esophageal SCC had s-MKRN1-Abs, none of the 43 healthy donors had a detectable level of s-MKRN1-Abs. There was no correlation between the presence of s-MKRN1-Abs and clinicopathological variables other than histological grading. Well-differentiated tumors were associated significantly with the presence of s-MKRN1-Abs in the patients. The mRNA levels of MKRN1 were frequently higher in esophageal SCC tissues than in the peripheral normal esophageal mucosa. Stable transfection of ras-NIH3T3 cells with MKRN1 cDNA induced prominent morphological changes such as enlargement of the cell body and spreading. Ubiquitination of 80- and 82-kDa proteins were clearly observed in MKRN1-transfected cells but not in the parental cells, which were identified as L-FILIP (filamin A interacting protein 1).


CONCLUSIONS
MKRN1 is a novel SEREX antigen of esophageal SCC, and s-NKRN1-Abs can be a candidate of diagnostic markers of esophageal SCC with high specificity. It is plausible that MKRN1 is involved in carcinogenesis of the well-differentiated type of tumors possibly via ubiquitination of L-FILIP.
Shimada
Hideaki
H
Department of Biochemistry and Genetics, Chiba University, Graduate School of Medicine, Chiba, Japan. hshimada@chiba-cc.jp
Shiratori
Tooru
T
Yasuraoka
Mari
M
Kagaya
Akiko
A
Kuboshima
Mari
M
Nomura
Fumio
F
Takiguchi
Masaki
M
Ochiai
Takenori
T
Matsubara
Hisahiro
H
Hiwasa
Takaki
T
D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004938:Esophageal Neoplasms / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D012261:Ribonucleoproteins / Q000096:biosynthesis*
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D018076:DNA, Complementary; C410808:Makorin ring finger protein 1; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins