针对TP53杂合性缺失结直肠癌的靶向药物研究

Thetumor suppressor TP53 gene is frequently inactivated by mutation or deletion in a majority of human tumors. Therefore, identification of vulnerabilities conferred by TP53 deletion or mutation is a major challenge to target p53 aberrancy in human cancer. We demonstrate that genomic deletion of TP53 frequently encompasses neighboring essential genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is alwaysco-deleted with TP53 in human cancers. Hemizygous loss of TP53/POLR2A occurs in 53% of colorectal cancers (CRC), 62% of breast cancers, 75% of ovarian cancers, and 41% of pancreatic cancers. POLR2A is specifically inhibited by α-Amanitin—— a peptide toxin found in the death cap mushroom (Amanita phalloides). POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer (CRC). Suppression of POLR2A selectively inhibits proliferation, survival and tumorigenic potential of CRC cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-Amanitin have been limited due to its liver toxicity. However, α-Amanitinis no longer toxic when coupled to antibodies. Therefore, α-Amanitin-based antibody drug conjugates (ADCs) are highly effective therapeutic agents with significantly reduced toxicity. Our study has shown that low doses of α-Amanitin-conjugated anti-EpCAM (Epithelial Cell Adhesion Molecule) antibody lead to complete tumor regression in murine models of human CRC with hemizygous deletion of POLR2A. This topic tries to improve the new therapeutic strategy through the study of drug resistance, chemosensitivity and related cancer treatment, which will provide the foundation for future clinical trials.

在人类的恶性肿瘤中，抑癌基因TP53会出现高频率的突变和缺失而失去活性，因此，在TP53突变或缺失后，寻找肿瘤的治疗方式是一个巨大的挑战。我们前期研究发现在53%的结直肠癌、62%的乳腺癌、75%的卵巢癌和41%的胰腺癌中都会出现TP53和POLR2A共同的杂合性缺失。POLR2A基因能够特定的被一种来自毒蘑菇鬼笔鹅覃的真菌毒素——鹅膏覃碱所抑制。在TP53杂合子缺失的结直肠癌细胞中，抑制POLR2A的表达可以通过TP53非依赖性方式来抑制肠癌细胞的增殖和致瘤潜能。但由于其肝脏毒性，鹅膏蕈碱在以往的临床应用中受到限制。基于鹅膏覃碱的抗体药物偶联物（ADCs）在显著减轻毒性的同时能够成为高效的治疗药物。本课题试图通过对药物耐药性、提高化疗敏感性及相关肿瘤治疗效果的研究，试图完善这一新颖的治疗策略，为以后的临床试验提供基础。

在人类的恶性肿瘤中，抑癌基因TP53会出现高频率的突变和缺失而失去活性，因此，在TP53突变或缺失后，寻找肿瘤的治疗方式是一个巨大的挑战。结直肠癌已成为世界第三大癌症，我们前期研究发现在53%的结直肠癌中都会出现TP53的杂合性缺失。尽管近几年对结直肠癌的研究正在不断深入，在诊断和治疗方面，也取得了重大进展，但五年生存率仍然不高。化疗是结直肠癌术后首选的治疗方式，但其耐药是获益受限的临床瓶颈问题，本研究从结直肠癌易对化疗药物5-Fu产生耐药这个临床问题出发，首先通过构建多株结直肠癌5-Fu耐药的细胞系，来评估其发生耐药的潜在机制，为后续逆转耐药的小分子化合物的筛选奠定基础。通过筛选，发现中药黄芩的主要有效成分黄芩素具有很好的逆转5-Fu耐药的作用，其与5Fu联合用药时具有显著的协同增效作用。随后对其机制和潜在靶点，进行了进一步研究。利用蛋白芯片对黄芩素作用靶点进行高通量筛选，通过分子对接、CETSA等实验证实了脯氨酸脱氢酶PRODH是其作用靶点之一。在制剂方面，由于黄芩素难溶于水的这一生物学特性限制了其在体内的应用，本研究将黄芩素包裹在脂质体材料中，根据动态光散射及电子透射显微镜拍摄的图像看，粒径在150nm左右，EE可达到95%。随后在裸鼠结直肠癌移植瘤模型中证实了黄芩素的纳米制剂能够逆转5-Fu耐药的这一结果。本研究基于前期从小分子化合物靶向药物的研究基础，揭示了黄芩素提高结直肠癌对5-Fu敏感性的作用，并对黄芩素进行纳米制备，通过脂质体的工艺作进一步的延长其稳定性及靶向性，从中西医结合的角度对药物耐药性、提高化疗敏感性进行研究，为结直肠癌的化疗耐药的临床问题的解决方案提供一定的科学依据。