抑制肺腺癌新靶标ROR1逆转EGFR-TKI获得性耐药的作用及机制

Recent study has shown that EGFR tyrosine kinase inhibitors (EGFR-TKIs) are particularly effective in lung adenocarcinoma patients harboring sensitizing EGFR mutations, but after treatment most patients develop a secondary resistance. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is aberrantly expressed in primary lung adenocarcinoma tissues and cell lines, and is involved in anti-apoptosis process irrespective of their EGFR status. Based on our previous study which targeting ROR1 with monoclonal antibodies in B-cell malignancies, ROR1 has shown potent anti-apoptosis functions. In this project we will comprehensively detect the expression level of ROR1 in primary lung adenocarcinoma tissues and clarify the association of ROR1 expression and activity levels with tumor TNM stages and EGFR mutation status. We will determine ROR1 expression and activity levels among lung adenocarcinoma cell lines with varing degree of erlotinib resistance. After inhibiting ROR1, we will detect erlotinib IC50 value and proliferation rate in lung adenocarcinoma cell lines with erlotinib acquired resistance which could determine whether ROR1 mediate an EGFR-independent growth pathway in tumor cells. In addition we will clarify the inhibiting roles and molecular mechanisms of ROR1 synergistically with erlotinib in the treatment of lung adenocarcinoma cells with erlotinib required resistance in vitro and in vivo. In this way our study could help to provide with a new target and feasible management for the treatment of lung adenocarcinoma, especially those with EGFR-TKI required resistance and the same time pave the way for further developing anti-ROR1 monoclonal antibodies or small molecular ROR1 inhibitors in the personal treatment of lung adenocarcinoma.

利用EGFR酪氨酸激酶抑制剂治疗肺腺癌的疗效受EGFR突变类型影响，并面临获得性耐药问题。近年发现受体酪氨酸激酶样孤儿受体1（ROR1）具备肿瘤特异性，高表达于肺腺癌，可促细胞增殖和抗凋亡，且不受EGFR突变类型限制。申请人前期对慢性B淋巴细胞性白血病进行了ROR1抗体靶向治疗研究，已获得较好结果。本课题拟分析肺腺癌患者ROR1表达和活性与肿瘤分期、EGFR突变类型等的关联；检测厄洛替尼不同耐药程度肺腺癌细胞ROR1表达和活性的差异；通过干扰ROR1表达，分析肺癌细胞对厄洛替尼耐药的变化及干扰对细胞增殖和凋亡的影响，明确 ROR1可否为获得性耐药细胞提供旁路生长刺激信号；通过体内外实验相映证，明确靶向ROR1可否协同或增强厄洛替尼对获得性耐药肿瘤细胞的抑制作用及机制。该研究将为肺腺癌的靶向治疗提供新的潜在靶点，并为ROR1特异性抗体或小分子抑制剂对肺腺癌的个体化治疗提供理论和实验基础。

一、项目的背景.近年来表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）的出现给肺腺癌的治疗带来了新的转机，但存在原发性和获得性耐药问题。受体酪氨酸激酶样孤儿受体1（ROR1）是近年新发现的与多种肿瘤生长转移密切相关的原癌基因。研究认为ROR1是肺腺癌致病的要害因素，并且极有可能成为新的治疗靶点，用于肺腺癌尤其是对EGFR-TKI耐药患者的治疗。..二、主要研究内容.1..明确肺腺癌患者肿瘤组织ROR1表达水平与肿瘤的病理分期的关系。.2..明确ROR1可否为erlotinib获得性耐药的肿瘤细胞提供旁路生长刺激信号。.3..明确抑制ROR1可否协同或增强erlotinib对肺腺癌细胞株的抑制增殖/促进凋亡作用及分子机制。.4..明确ROR1胞外Kringle功能区的结构，有利于ROR1的结构和功能研究。..三、重要结果及其科学意义.（一）重要结果.1..通过37例不同病理分期的肺腺癌肿瘤组织、癌旁组织，及10例正常肺组织，发现高达65%的肺腺癌患者表达ROR1，而肺鳞癌患者几乎不表达或仅低水平表达ROR1，表明ROR1主要表达于肺腺癌细胞。.2..来自erlotinib耐药、敏感以及获得性耐药的肺腺癌细胞株的结果表明，ROR1的表达水平与肺腺癌细胞株是否存在EGFR-TKI耐药性并无直接联系，说明ROR1的靶向治疗适用于所有ROR1+肺腺癌患者。.3..体外细胞株实验发现阻断ROR1能显著诱导肿瘤细胞的凋亡和生长抑制，证实ROR1可为erlotinib获得性耐药的肿瘤细胞提供旁路生长刺激信号，并且PI3K/AKT/mTOR信号通路参与了ROR1介导的肺腺癌细胞增殖。.4..体外试验进一步证实采用siRNA干扰ROR1的表达后，可以增强erlotinib通过PI3K/AKT/mTOR信号通路促进肺癌细胞株的细胞毒性和凋亡作用。.5..通过液相核磁技术明确ROR1胞外Kringle功能区的骨架和侧链三维结构，为下一步设计针对ROR1的治疗性抗体或小分子化合物奠定结构基础。..（二）科学意义.对ROR1阳性的肺腺癌，采用单独靶向ROR1或靶向ROR1且联合EGFR-TKI的治疗方法，一方面可以为肺腺癌提供新的潜在治疗靶点并逆转肺腺癌对EGFR-TKI的获得性耐药；另一方面可以为下一步利用ROR1特异性抗体或小分子抑制剂进行肺腺癌个体化治疗奠定理论和实验基础。