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

MSI 阳性结肠癌移码突变蛋白的系统鉴定

• 批准号: 14571226
• 负责人: FUJITA Tomonobu
• 金额: $ 2.11万
• 依托单位: KEIO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571226/
• 关键词: Colorectal cancers with microsatellite instability; frameshift-mutated peptides; immunotherapy; tumor antigen; SEREX; フレームシフトタンパク; TGFBRII

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.

由DNA错配修复功能障碍引起的微卫星不稳定性结直肠癌（MSI+CRC）具有独特的临床病理特征，包括肿瘤中T细胞浸润的良好预后。之前我们通过 SEREX（重组 cDNA 表达克隆的血清学分析）证明了移码突变的 CDX2 作为肿瘤抗原可诱导针对 MSI+ CRC 的免疫反应。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。我们利用两个MSI阳性结肠癌组织和自体血清文库从SEREX中分离出26个基因，但通过RT-PCR未发现报告为MSI靶基因和肿瘤特异性基因的基因。我们从8个靶基因（TGFBRII、BAX、TCF-4、CASP-5、SEC63、AIM2、OGT、RAD50）制备重组蛋白，产生由错配修复基因引起的移码突变肽。通过蛋白质印迹和ELISA检测13名MSI+CRC患者血清中存在针对重组蛋白的IgG抗体，但均未显示阳性。然而，患者的组织中靶基因存在一些突变。我们重建了 TGFBRII 的重组蛋白，并使用另外 4 份 MSI+ CRC 血清进行了蛋白质印迹分析。 TGFBRII 突变患者的一种血清与正常和突变的重组蛋白发生反应。因此，MSI产生的肿瘤特异性肽可能参与抗肿瘤免疫反应，但对MSI移码突变蛋白的免疫反应能力可能相对较弱。需要进一步研究来鉴定 MSI+ CRC 中发生移码突变的蛋白质。

项目成果

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Ito, T: "Identification of bladder cancer antigens recognized by IgG antibodies of a patient with metastatic bladder cancer"International Journal of cancer. 108. 712-724 (2004)

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