Identification and distribution of thioredoxin‐like 2 as the antigen for the monoclonal antibody MC3 specific to colorectal cancer

Abstract

MC3 is a colorectal cancer (CRC)-specific mAb previously prepared in our laboratory that can detect CRC with high sensitivity and specificity. However, the target antigen for MC3 had not been identified due to technological limitations. In the present study, immunocytochemistry and immunohistochemistry revealed the expression patterns of MC3 antigen (MC3-Ag) in colon cancer cell lines and CRC tissues. Western blotting analysis showed that the MC3 antibody reproducibly recognized two approximately 30 kDa proteins in the total cell lysates of human colon carcinoma cell lines SW480 and HT-29. Using a proteomic approach, we identified two MC3 immunoreactive spots as two isoforms of thioredoxin-like 2 (Txl-2) protein. Further paired immunostaining showed that Txl-2 had the same expression profile as probed by the MC3 antibody. Western blotting also showed that both antibodies could detect the same two bands, further verifying that Txl-2 is the antigen of MC3 antibody. Additionally, tissue arrays revealed the expression patterns of Txl-2 in various normal and cancer tissues. Further analysis showed that Txl-2 mRNA was elevated in 18 cases of CRC tissues compared to paracancerous tissues and adjacent normal tissues.

Thioredoxin-Like Protein 2 Is Overexpressed in Colon Cancer and Promotes Cancer Cell Metastasis by Interaction with Ran

“…As shown in Table 1, the increased Txl-2 expression also correlated with tumor stages consisting of tumor size (T) ( p < 0.001), lymph node invasion (N) ( p < 0.001), and distant metastasis (M) ( p = 0.022). To determine whether Txl-2 isoforms have differential expression rates, a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis was performed in 50 pairs of colon cancer samples using the method we reported previously (25). We found that Txl-2b was the most frequently presented Txl-2 isoform in tumor tissues (88%, 44/50), as compared to Txl-2a (8%, 4/50, p < 0.001) and Txl-2c (20%, 10/50, p < 0.001) (Fig.…”

“…Specifically, an increased expression of Trx-1 in CRC has 1 been shown to be associated with poor prognosis in patients with liver metastasis (30). We have recently demonstrated an increased expression of Txl-2 in colon cancer (25), but its specific role in colon cancer and the underlying mechanisms are still unknown.…”

“…Of these, Txl-2b was upregulated in colon cancer tissues compared with normal mucosa, while Txl-2c was almost exclusively present in colon cancer tissues, and full length Txl-2 seldom occurred in both normal mucosa and colon cancer tissues (25). Thus, it would be interesting to investigate whether these three transcript variants show differential effects on the functional properties of colon cancer cells.…”

“…Importantly, these biomarkers can serve as targets for developing novel cancer therapeutics (11). In this context, we have previously identified thioredoxin-like protein 2 (Txl-2) as the target of the monoclonal antibody MC3 that we have generated for the detection of colon cancer (25).…”

“…It is well-recognized that mRNA splicing can lead to production of protein isoforms with oncogenic properties (32).Three different isoforms have been identified for Txl-2, including full length Txl-2 (Txl-2a), Txl-2b (splicing variant lacking exons 1 and 5), and Txl-2c (splicing variant lacking exons 1, 4, and 5) (25,37). Of these, Txl-2b was upregulated in colon cancer tissues compared with normal mucosa, while Txl-2c was almost exclusively present in colon cancer tissues, and full length Txl-2 seldom occurred in both normal mucosa and colon cancer tissues (25).…”