LXR激动剂逆转肺癌细胞对EGFR-TKI的继发耐药及机制研究

EGFR-TKI is the most widely used targeted therapy in lung cancer patients and it has significantly improved the overall survival of Chinese lung cancer patients. Unfortunately, almost all lung cancer patients who were initially response to EGFR-TKI therapy will develop acquired resistance to this targeted therapy and the main mechanism is the reactivation of EGFR signaling downstream pathways. Many researchers have focused on reversion of lung cancer cells' acquired resistance to EGFR-TKI therapy. The liver X receptors, LXRα and LXRβ are additional ligand-activated members of the nuclear hormone receptor superfamily. Evidences have emerged that activation of LXR inhibits various types of cancer cells' proliferation. More importantly, recent researches have proved that LXR agonist could significantly inhibit the activation of EGFR downstream pathways, which indicates that LXR agonist is a potential drug that could reverse the acquired resistance to gefitinib. Our preliminary study also demonstrates that addition of LXR agonist could inhibit lung cancer cells (resistance to gefitinib) proliferation, induce cancer cells' apoptosis and significantly increase cancer cells' sensitivity to gefitinib. Based on the close relationship of LXR and downstream of EGFR signaling pathway and our preliminary results, we would explore the effect of LXR on EGFR and the mechanism to provide more options for clinical lung cancer target therapy. We would establish EGFR-TKI-resistant lung cancer cells and investigate the effect of LXR on EGFR-TKI-resistant lung cancer cells and resistant model in nude mice via orthotopic unplantation of EGFR-TKI-resistant fluorescent lung cancer cells.

表皮生长因子受体-酪氨酸激酶抑制剂（EGFR-TKI）是应用最广泛的肺癌靶向药物，但目前存在的问题是几乎所有治疗有效的患者最终都会对EGFR-TKI治疗产生继发耐药，而耐药的机制主要是EGFR下游通路被再度激活。肝X受体（LXR）属于核受体家族， LXR激动剂能显著抑制EGFR下游通路的激活。我们前期实验发现，LXR激动剂与EGFR-TKI（吉非替尼）联合应用能明显抑制肺癌耐药细胞的增殖，增加耐药细胞的凋亡率，并显著增加耐药细胞对吉非替尼的敏感性。本项目拟在前期研究的基础上，拟通过以下方法进一步探讨LXR激动剂逆转肺癌细胞对EGFR-TKI的作用，并阐明其机制，为临床提高肺癌靶向治疗疗效提供可靠的理论依据。①建立肺癌耐药细胞系，研究LXR激动剂对肺癌耐药细胞的影响，并探讨其分子作用机制；②通过耐药细胞系荧光肺癌原位裸鼠模型，进一步探讨LXR激动剂在逆转继发耐药中的作用。

EGFR 下游通路重新激活是表皮生长因子受体-酪氨酸激酶抑制剂（EGFR-TKI）继发耐药的主要原因。肝X 受体（LXR）属于核受体家族,研究表明LXR激动剂能显著抑制EGFR下游通路的激活。为了探讨LXR激动剂是否能逆转耐药细胞对吉非替尼的耐药，本研究的主要研究内容和结果如下：. 1）成功建立了HCC827细胞吉非替尼继发耐药株，采用荧光定量PCR、Western blot、直接测序以及ddPCR等技术对HCC827细胞吉非替尼继发耐药株分析了常见耐药机制，结果发现单克隆细胞株HCC827-8-1中耐药机制未明。进一步用基因芯片筛选在HCC827和HCC827-8-1细胞中差异表达的miRNA和lncRNA,用细胞和分子生物学技术证实了miR-149-5p、LINC00635-001、NONHSAT093968和NONHSAT127642均在HCC827-8-1细胞株耐药形成中起着重要作用。. 2）用细胞和分子生物学技术证实了LXR激动剂可逆转吉非替尼原发耐药肺腺癌细胞株A549和多株HCC827吉非替尼继发耐药细胞对吉非替尼的耐药，其机制为LXR激动剂通过激活LXRβ受体，抑制EGFR下游PI3K/AKT/NF-κB信号通路，促进吉非替尼对EGFR下游信号进一步抑制，诱导细胞周期阻滞和/或细胞凋亡，进而发挥对肿瘤细胞的杀伤作用。. 3）用裸鼠皮下肿瘤模型证实了LXR激动剂T0901317通过抑制AKT活性来抑制A549细胞的增殖增生。用裸鼠荧光肺癌原位移植瘤模型证实T0901317联合吉非替尼用药后不仅能抑制肿瘤生长还能抑制肿瘤转移，用ELISA技术发现这种抑制肿瘤转移的作用可能T0901317抑制血清中MMP9表达水平相关。. 本研究中发现了多个参与EGFR-TKI耐药的非编码RNA，并证实了LXR 激动剂与EGFR-TKI联合用药可以提高肺癌靶向治疗效果，这都将为解决EGFR-TKI继发耐药问题提供了可靠的理论和实验依据。