氨肽酶P2作为恶性肿瘤转移分子标志及其促进宫颈癌转移作用机制研究

We have screened a novel tumor-homing peptide named TMTP1, which specifically targets metastasis, using FliTrx bacterial display system in our previous study. Furthermore, XPNPEP2 also has been screened and identified as the receptor of TMTP1 in our team (data to be published). Our primary results demonstrated that XPNPEP2 expressed at higher level in metastatic cancer cells of para-cancer nest or lymph nodes compared with that in primary cancer cells. In addition, 99mTC-HYNIC/EDDA-TMTP1 successfully targeted highly metastasis as a radiopharmaceutic imaging agent of highly metastatic cervical cancer xenograft in nude mice. The expression profile of XPNPEP2 in malignant primary and metastatic sites needs to be further investigated as a potent receptor of TMTP1 and aminopeptidase. Moreover, whether it participate in promoting tumor metastasis or its mechanisms, also needs further investigation. In this project, we planed to collect plenty of clinical primary and metastatic cervical caner samples and evaluate the expression levels of XPNPEP2, to exemplify whether it could serve as a novel tumor metastasis marker. Next, we would investigate whether XPNPEP2 promotes tumor metastasis through EMT or it degrades ECM of tumor cells. Finally, a novel drug, doxorubicin -TMTP1 would be synthesized and evaluated its targeting cytoxity and biodistribution in a cervical cancer bearing SCID mice model.

我们用细菌鞭毛肽库成功筛选到高转移肿瘤靶向肽TMTP1，并进一步筛选和鉴定出其潜在受体氨肽酶p2（XPNPEP2），初步的研究显示XPNPEP2在乳腺癌、宫颈癌等癌旁和淋巴结转移灶中高表达，放射性核素鍀99标记TMTP1可对高转移宫颈癌裸鼠皮下瘤进行分子显像，XPNPEP2作为一种针对脯氨酸残基的水解酶和肿瘤转移靶向肽TMTP1的潜在受体,是否参与促进肿瘤转移及其分子机制值得深入研究。本项目拟从妇科最常见的宫颈癌入手，大量收集样本研究宫颈癌等原发灶和转移灶中XPNPEP2表达情况，明确XPNPEP2能否作为宫颈癌乃至多种恶性肿瘤转移的一种新的分子标志，研究其是否通过促进肿瘤细胞EMT和水解肿瘤细胞外基质中粘附蛋白参与肿瘤转移，并在此基础上，对高表达XPNPEP2的宫颈癌小鼠脚垫淋巴结转移模型中，用TMTP1偶联的阿霉素进行靶向治疗，观察其疗效和毒性。

我们在前面的研究中利用噬菌体鞭毛肽库成功筛选对高转移肿瘤具有特异性识别和显著的靶向性的TMTP1，并进一步利用pulldown技术鉴定出其潜在受体XPNPEP2。初步研究显示XPNPEP2在乳腺癌、宫颈癌等癌旁和肿瘤转移淋巴结中高表达。在此基础上提出假设一，作为TMTP1与高转移肿瘤细胞结合的潜在受体，XPNPEP2能否成为肿瘤转移的一种分子标志；假设二，XPNPEP2是否通过肿瘤细胞上皮间质转化促进肿瘤转移；假设三，作为一种氨肽酶，XPNPEP2是否通过分解基质中的黏附蛋白进一步促进肿瘤细胞侵袭和转移。本研究中，通过收集大量宫颈癌、乳腺癌等原位灶和转移灶，免疫组化分析XPNPEP2的表达水平及与肿瘤转移的相关性；利用western blot、RT-PCR和免疫荧光检测宫颈癌细胞系中XPNPEP2的表达，流式分析与TMTP1的亲和力；通过lentivirus系统过表达或敲除宫颈癌细胞株的XPNPEP2，检测其与TMTP1的亲和力，迁移和侵袭能力，EMT相关分子和表型、细胞基质骨架等微环境的改变；利用裸鼠模型检测XPNPEP2在体内对肿瘤转移的影响；合成（99m）TC-HYNIC/EDDA-TMTP1进行SPECT显像；合成Doxorubicin-TMTP1在宫颈癌小鼠模型中探究其药物分布和靶向抗肿瘤效应。我们发现XPNPEP2在宫颈癌的原位灶和转移灶的表达显著高于正常的宫颈和CIN，并且其表达与宫颈癌的FIGO分期和淋巴转移密切相关；高转移的肿瘤细胞XPNPEP2表达增加，过表达XPNPEP2可增强癌细胞的迁移和侵袭能力，敲除XPNPEP2可以减弱其引起的肿瘤细胞迁移和侵袭能力；在小鼠模型中，XPNPEP2过表达促进肿瘤转移，（99m）TC-HYNIC/EDDA-TMTP1可以清楚地显示出肿瘤原位灶和转移灶，Doxorubicin-TMTP1也展示出肿瘤区的累积和靶向肿瘤杀伤效应。在本研究中，我们明确了XPNPEP2可以通过EMT促进肿瘤转移，为其作为宫颈癌转移的分子标志监测肿瘤转移和复发提供了事实依据，也为将来开发针对XPNPEP2的小分子药物提供了试验证据；同时，TMTP1作为一种靶向肽，其偶联的显像剂和治疗剂均展示出良好的诊疗效果，为将来TMTP1偶联其他诊疗剂实现临床转化建立了基础。