Anti-FIRs (PUF60) auto-antibodies are detected in the sera of early-stage colon cancer patients

Abstract

Anti-PUF60, poly(U)-binding-splicing factor, autoantibodies are reported to be detected in the sera of dermatomyositis and Sjogren's syndrome that occasionally associated with malignancies. PUF60 is identical with far-upstream element-binding protein-interacting repressor (FIR) that is a transcriptional repressor of c-myc gene. In colorectal cancers, a splicing variant of FIR that lacks exon2 (FIRΔexon2) is overexpressed as a dominant negative form of FIR. In this study, to reveal the presence and the significance of anti-FIRs (FIR/FIRΔexon2) antibodies in cancers were explored in the sera of colorectal and other cancer patients. Anti-FIRs antibodies were surely detected in the preoperative sera of 28 colorectal cancer patients (32.2% of positive rates), and the detection rate was significantly higher than that in healthy control sera (Mann-Whitney U test, p < 0.01). The level of anti-FIRs antibodies significantly decreased after the operation (p < 0.01). Anti-FIRs antibodies were detected in the sera of early-stage and/or recurrent colon cancer patients in which anti-p53 antibodies, CEA, and CA19-9 were not detected as well as in the sera of other cancer patients. Furthermore, the area under the curve of receiver operating characteristic for anti-FIRs antibodies was significantly larger (0.85) than that for anti-p53 antibodies or CA19-9. In conclusions, the combination of anti-FIRs antibodies with other clinically available tumor markers further improved the specificity and accuracy of cancer diagnosis.

Keywords: auto-antibodies; cancer biomarker; colorectal cancer; far-upstream element-binding protein-interacting repressor (FIR) = poly(U)-binding-splicing factor (PUF60).

Figure 1. Auto-antibodies against FIR/FIRΔexon2 purified proteins were detected in the sera of colorectal cancer patients

(A) Expression of FIRs proteins were examined by western blotting in tumor (T) and adjacent non-tumor (N) tissue samples from colon cancers and colon polyps' tissues. Representative cases were indicated. (B) Bands' intensities were quantified using Scion Image imaging analysis software (National Institutes of Health. USA) and the average band intensity of proteins normalized to the corresponding β-actin were shown. (C) FIR and FIRΔexon2 purified proteins were prepared as antigens to detect the auto-antibodies against FIR/FIRΔexon2 in colorectal cancer patients' sera by western blotting (upper panel). The anti-FIRs (6B4) antibody was used as a positive control (middle panel). Molecular weight of bands detected by patients' sera were exactly same size with those detected by anti-FIRs (6B4) antibodiy in western blot (middle panel) shown in CBB (Coomassie Brilliant Blue) staining (lower panel). (D) Auto-antibodies against FIR or FIRΔexon2 in colorectal cancer patients' sera were further confirmed in dose dependent manner by western blotting. FIRwt: FIR purified protein. BSA: Bovine serum albumin.

Figure 2. Detection of the anti-FIRs antibodies in the serum of colon cancer patients

(A) The relative expression of anti-FIRs antibodies in the serum was detected against 2, 10, 50 ng of FIRΔexon2 purified proteins by dot blot analysis. The intensities of the dot signals were quantified by Lumi Vision Imager analysis software (Aisin Seiki Co., Ltd. Aichi, Japan). The relative intensity of the signals was compared between colorectal cancer patients and healthy subjects. (B) The dot blot membranes were stripped and reprobed with anti-FIRs (6B4) antibody for the confirmation of the dose of antigens. Dot blot intensities were standardized by positive control anti-FIRs (6B4) antibody. (C) Dose dependent curves of each patient were shown. (D) The levels of anti-FIRs antibodies in esophageal cancer (EC), colorectal cancer (CC) and were examined by AlphaLISA. Healthy subjects (HS) were as control. P values were calculated by Mann-Whitney U test.

Figure 3. Diagnostic value of anti-FIRs antibodies in the sera of colorectal and esophageal cancer patients

(A) The detection frequencies of anti-FIRs antibodies, His-tagged-FIRΔexon2 purified proteins as antigens, in the sera of early/advanced stages of colorectal cancer patients and healthy subjects were shown. (B) ROC (receiver operating characteristic) curve of colorectal cancer is indicated. The area under the curve (AUC) for anti-FIRs antibodies is 0.85, for anti-p53 antibody is 0.61, for CEA is 0.87 and for CA19-9 is 0.67. The best cutoff values for anti-FIRs antibodies, anti-p53 antibody, CEA and CA19-9 are 0.926%, 0.719 U/ml, 1.953 ng/ml and 12.8 U/ml, respectively. (C) The detection frequencies of anti-FIRs antibodies in the different stages of esophageal cancer patients and healthy subjects were also shown in graphical view. (D) The detection frequencies of anti-FIRs antibodies and three tumor markers in the different stages of colorectal cancer patients were shown in graphical view. The combination of three markers with anti-FIRs antibodies further increased the detection rate up to 74.7%.

Figure 4. Evaluation of postoperative monitoring in colorectal cancer

(A) Detection frequency of anti-FIRs antibodies in CRC sera before and after surgical operation. After surgical operation, anti-FIRs antibodies were detected in 19 cases out of 28 cases that were anti-FIR antibody positive before operation. (B) The level of anti-FIRs antibodies was significantly decreased after operation. (C) Comparison of the levels of anti-FIRs antibodies and clinically adopted tumor markers before and after surgical operation. The level of anti-FIRs antibodies was significantly decreased after operation.

Figure 5. Anti-FIRΔexon2 or anti-FIR antibodies were detected in the sera of various cancer patients

Purified proteins were prepared by Nus-tag FIR or -FIRΔexon2 as antigens. Alpha counts against FIR or FIRΔexon2 proteins were indicated in healthy subjects (n = 92), breast cancer (n = 82), pancreatic cancer (n = 80). bile duct cancer (n = 77), gall bladder stones (n = 48), pancreatic neuroendocrine tumor (n = 24), esophageal cancer (n = 91), gastric cancer (n = 90), and colorectal cancer (n = 89) patients.