Systemic identification of mutated proteins with frame-shifted from MSI positive colon carcinoma

DNA错配修复功能障碍所致的微卫星不稳定性(MSI+CRC)结直肠癌具有独特的临床病理特征，包括肿瘤内T细胞浸润，预后良好。此前，我们通过SEREX(重组基因表达克隆的血清学分析)证明了移码突变的CDX2是诱导抗MSI+CRC的免疫反应的肿瘤抗原。本研究筛选了编码区微卫星发生移码突变的免疫原性蛋白。我们从SEREX和两个MSI阳性结肠癌组织和自体血清文库中分离到26个基因，但RT-PCR没有发现MSI靶基因和肿瘤特异性基因。我们从8个靶基因(TGFBRII、Bax、TCF-4、CASP-5、SEC63、AIM2、OGT、Rad50)中制备了重组蛋白，使错配修复基因引起的突变多肽发生了移码。用MSI+CRCS检测13例患者血清中抗重组蛋白的抗体，经免疫印迹和酶联免疫吸附试验检测均为阳性。然而，患者组织中的靶基因发生了一些突变。我们构建了TGFBRII的重组蛋白，并用另外4份MSI+CRCS血清进行了Western印迹分析。1例TGFBRII突变患者血清与正常和突变重组蛋白反应。因此，MSI产生的肿瘤特异性多肽可能参与了抗肿瘤免疫反应，但对MSI中移码突变蛋白的免疫应答能力可能相对较弱。需要进一步的研究来鉴定在MSI+CRCs中发生移码突变的蛋白质。

Colorectal cancers with microsatellite instability (MSI+ CRCs) caused by dysfunction of DNA mismatch repair have unique clinicopathological characteristics including good prognosis with T-cell infiltration in tumor. Previously we demonstrated frame-shifted mutated CDX2, as tumor antigens that induce immune response against MSI+ CRC, by SEREX (serological analysis of recombinant cDNA expression cloning). In this study, the immunogenic proteins with frameshift mutated in the microsatellite of the coding region were screened. We isolated 26 genes from SEREX with two libraries of MSI positive colon cancer tissues and autologus sera, but found no genes reported as MSI target genes and tumor specific genes by RT-PCR. We made recombinant proteins from 8 target genes (TGFBRII, BAX, TCF-4, CASP-5, SEC63, AIM2, OGT, RAD50), which make frame-shifted mutated peptide caused by mismatch repair genes. The presence of IgG antibody against recombinant proteins examined by MSI+ CRCs sera of 13 patients by western blotting and ELISA, but none of them showed positive. However, the tissues with patients had some mutation in the target genes. We reconstructed recombinant protein of TGFBRII, and we performed Western blot analysis using another 4 MSI+ CRCs sera. One serum from patient with TGFBRII mutation reacted with normal and mutated recombinant protein. Therefore, tumor specific peptides generated by MSI may be involved in anti-tumor immune responses, but the abilities of immuno response to proteins with frameshift mutated in MSI may be relatively weak. Further investigation is required to identify the proteins with frameshift mutated in MSI+ CRCs.