MiR-34a/AXL介导非小细胞肺癌EGFR-TKI获得性耐药的分子机制

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the standard first-line treatment for EGFR mutated non-small cell lung cancer (NSCLC) patients. However, most patients who benefit clinically will eventually relapse after a short period time use of EGFR-TKIs. This is generally termed "acquired resistance". Except the secondary EGFR gene mutation-T790M and MET amplification, other mechanisms of acquired resistance remain largely unknown. Recently, AXL has been proved to be associated with acquired resistance to TKIs. AXL, together with Tyro3 and Mer, composes the TAM family of receptor tyrosine kinases (RTKs). AXL was initially cloned from leukemia cells and it was demonstrated to be over-expressed in a variety of human cancers, including in NSCLC. AXL over-expression is generally associated with proliferation, anti-apoptosis, metastasis and poor clinical outcome of many cancers. It has also been found to regulate drug resistance in a number of cancers, including breast cancer, myeloid leukemia and NSCLC. In our previous work, we observed that AXL was over-expressed in gefitinib resistant cell lines-HCC827/GR and PC9/GR, both of which were generated based on gefitinib sensitive cell lines-HCC827 and PC9 (both harboring Exon 19 deletion in EGFR gene). Based on these observations, we postulate that AXL may regulate acquired resistance via activating downstream signaling pathways such as PI3K/AKT、MAK/ERK and JAT/STAT, resulting in bypassing the inhibitory effect of EGFR downstream signaling pathways. Moreover, we also found that miR-34a was significantly down-regulated in both of gefitinib resistant cell lines. Computational microRNA(miR) target prediction software indicates that AXL is one of miR-34a potential targets. Therefore, miR-34a may regulate the expression of AXL and AXL over-expression may activate the downstream signaling pathways to confer resistance to gefitinib in lung cancer cells. In this project, we plan to up-regulate or down-regulate AXL in lung cancer cells, as to observe the activation of AXL downstream signaling pathways-PI3K/AKT, MEK/ERK and JAT/STAT. We also construct the AXL plasmids and use luciferase reporter system to prove that AXL is the downstream target of miR-34a in lung cancer cells. All the results will be further verified on animal models and clinical samples. In summary, this project will try to clarify the molecular mechanism of acquired resistance to EGFR-TKIs and may provide potential targets for reversing the resistance to EGFR-TKIs.

表皮生长因子受体抑制剂（EGFR-TKI）已成为EGFR突变型晚期非小细胞肺癌的一线标准治疗，但获得性耐药却成为临床治疗上的难题。AXL是编码受体酪氨酸激酶的基因，其蛋白被证实在多种肿瘤组织中异常表达，并与肿瘤细胞的增殖、凋亡、转移及耐药相关。我们前期研究发现在吉非替尼耐药细胞株中AXL高表达，而miR-34a明显低表达，生物信息学软件提示AXL是miR-34a的潜在靶点，因此推测miR-34a可能调控AXL的表达，AXL高表达后通过活化下游信号通路而导致耐药。本课题拟进一步上调或者下调AXL，观察肺癌细胞内AXL下游信号通路PI3K/AKT、MEK/ERK及JAT/STAT通路的变化，探讨AXL导致获得性耐药的机制；并构建载体验证AXL是miR-34a的下游靶点，明确AXL的调控机制；同时建立动物模型和收集临床标本进行验证，为AXL或miR-34a作为逆转TKI耐药的治疗靶点提供思路。

背景：AXL是编码受体酪氨酸激酶的基因，并与肿瘤细胞的增殖、凋亡、转移及耐药相关。本课题拟上调或者下调AXL，观察肺癌细胞内AXL下游信号通路的变化，探讨AXL导致获得性耐药的机制；并构建载体验证AXL是miR-34a的下游靶点，明确AXL的调控机制；同时建立动物模型和收集临床标本进行验证，为AXL或miR-34a作为逆转TKI耐药的治疗靶点提供思路。.研究内容： （1）细胞及临床标本中检测AXL及其配体Gas6的表达. （2）探讨AXL导致EGFR突变型NSCLC细胞耐药的分子机制. （3）探讨EGFR突变性肺癌细胞株中AXL的调控. （4）动物体内验证AXL导致耐药及其调控.重要结果：（1）在吉非替尼耐药的细胞株，AXL表达明显升高（是吉非替尼敏感细胞的3倍以上）；如果在耐药细胞株中AXL的表达被抑制（siRNA），其下游通路的p-AKT及p-ERK1/2表达明显减少，且耐药细胞株部分恢复对吉非替尼敏感；如果在敏感细胞株中过表达AXL，则细胞会产生耐药；我们还发现AXL和NSCLC的分化和肿瘤大小及预后相关；. （2）在吉非替尼耐药的细胞株中，AXL的表达被抑制后，miRNA表达会发生变化；其中变化最明显的为miR-374a 和 miR-548b；在NSCLC中miR-374a调控Wnt5a，而miR-548b调控CCNB1；在耐药细胞株中，miR-374a被下调后会抑制细胞的转移，而miR-548b则会诱导细胞的周期停止，而miR-374a 和 miR-548b和NSCLC细胞.的EMT及凋亡相关；. （3）长链RNA FOXF1-AS1在NSCLC组织中低表达，在NSCLC细胞中高表达FOXF1-AS1可以抑制细胞转移；FOXF1-AS1可以调控NSCLC细胞的EMT和干细胞特性；.关键数据及意义：（1）AXL和NSCLC的预后相关；将来可作为预后因子；. （2）AXL可以调控miR-374a 和 miR-548b，进而调控NSCLC的EMT，干细胞特性，凋亡和TKI耐药，从而可以作为将来逆转耐药及转移相关的治疗靶点