Mari Kuboshima

Serological identification of TROP2 by recombinant cDNA expression cloning using sera of patients with esophageal squamous cell carcinoma

We applied serological analysis of recombinant cDNA expression libraries (SEREX) to cases of esophageal squamous cell carcinoma (SCC) to identify tumor antigens. One of the clones identified was TROP2, which is known as calcium signal transducer. To evaluate the clinical significance of serum anti‐TROP2 antibodies (s‐TROP2‐Abs) in patients with esophageal SCC, the presence of s‐TROP2‐Abs was analyzed by Western blotting using bacterially expressed TROP2 protein. We found that 23 of 75 (31%) patients were positive for s‐TROP2‐Abs. Positivity in terms of s‐TROP2‐Abs showed a significant association with tumor size but not with other clinicopathological features. The protein expression levels of TROP2 were much higher in esophageal SCC cell lines as compared to those in normal esophageal mucosa and its immortalized cells although the mRNA expression levels were not necessarily elevated in malignant cell lines and tissues. Immunohistochemical studies showed that the expression of TROP2 protein in esophageal SCC specimens was noticeably higher than that found in mild hyperplasia of esophageal mucosae. Thus, s‐TROP2‐Abs seemed useful in the diagnosis of SCC and may be a candidate for serum tumor markers.

Presence of serum tripartite motif-containing 21 antibodies in patients with esophageal squamous cell carcinoma

SEREX has been applied to esophageal SCC, and the TRIM21 gene was identified as a novel SEREX antigen of esophageal SCC. The presence of s‐TRIM21‐Abs was confirmed by Western blotting using bacterially expressed TRIM21 gene product and was evaluated for clinicopathological significance in patients with esophageal SCC. s‐TRIM21‐Abs were detected in 18 (20%) of 91 patients with esophageal SCC but not in 42 healthy donors. The presence of s‐TRIM21‐Abs was partly associated with tumor size (P = 0.063) and poor survival (P = 0.067). To measure serum antibody levels, ELISA using purified recombinant TRIM21 protein was developed. The levels of s‐TRIM21‐Abs were significantly higher in patients with esophageal SCC than in healthy donors (P = 0.013). s‐TRIM21‐Abs may be a useful tumor marker to diagnose and predict disease progression in patients with esophageal SCC. (Cancer Sci 2006; 97: 380–386)

Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma

BackgroundDiagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available.ResultsFifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (e sophageal c arcinoma S EREX a ntigen)-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a). Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA). The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues.ConclusionsWe have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.

Identification of Makorin 1 as a novel SEREX antigen of esophageal squamous cell carcinoma

BackgroundEsophageal 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.MethodsTumor 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.ResultsMKRN1 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).ConclusionMKRN1 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.

Elevated Serum Antibody Levels against Cyclin L2 in Patients with Esophageal Squamous Cell Carcinoma

Cyclin L2 (CCNL2) (Acc. No.: NM_030937) was detected as a tumor antigen of esophageal squamous cell carcinoma (SCC) by serological identification of antigens by recombinant cDNA expression cloning (SEREX). Serum anti-CCNL2 antibodies were detected by enzyme-linked immunosorbent assay more frequent in patients with esophageal SCC than in healthy donors (32% and 15%, P<0.01). An AlphaLISA further confirmed the significant difference in serum antibody levels between the patients and healthy donors using a different set of serum specimens. The expression levels of CCNL2 mRNA detected by reverse transcription polymerase chain reaction were higher in esophageal SCC tissues than those detected in adjacent normal esophageal tissues. We then analyzed for biological function by the transient transfection of CCNL2 expression plasmids into ras-NIH3T3 mouse fibroblasts followed by analysis via luciferase assay using p53-responsive reporter plasmids. CCNL2 increased the transactivation ability of p53, which was attenuated by protein kinase C (PKC) inhibitors or a dominant negative PKCa. Thus, it is possible that CCNL2 activates p53 via PKCa activation.