Wnt/β-catenin与EGFR信号通路交互作用在NSCLC吉非替尼获得性耐药中的作用和机制研究

Our previous researches showed that the protein expression of β-catenin and transcriptional activity of Wnt/β-catenin signal pathway were significantly higher in gefitinib acquired resistant non-small cell lung cancer (NSCLC) cell line PC9/AB2 than that in sensitive cell line PC9. Furthermore, knocking down the expression of β-catenin would elevate the sensitivity of resistant cells to gefitinib and influence the expression of EGFR and its downstream pathways. Thus, we assume that cross-talking between Wnt/β-catenin and EGFR signal pathways may be involved in EGFR-TKIs resistance in NSCLC. As far as we know, there is no other reports in this field in NSCLC. In this program, we intend to use the method of microarray to compare mRNA expression of genes in Wnt/β-catenin and EGFR signal pathways between gefitinib acquired resistant NSCLC cell lines including PC9/AB2, PC9/AB11 and PC9/BB4 and gefitinib sensitive cell line PC9. Then by silencing or upregulating the expression of abnormal expression genes in Wnt/β-catenin signal pathway selected from microarray, we will investigate the inflence of Wnt signal pathway on the sensitivity to gefitinib and study the expression change of EGFR signal pathway. Furthermore, the role and mechanisms of Wnt/β-catenin and EGFR signal pathway cross-talking in gefitinib acquired resistance will be further verifed by animal and clinical specimen studies. We expect this study will be helpful to provide the potential strategy to reverse EGFR-TKIs resistance in NSCLC.

我们前期研究发现NSCLC吉非替尼获得性耐药株PC9/AB2中β-catenin蛋白表达和Wnt/β-catenin信号通路转录活性都明显高于敏感株PC9，干扰β-catenin表达可提高耐药细胞对吉非替尼的敏感性并影响EGFR及下游蛋白表达。我们推测Wnt/β-catenin与EGFR信号通路间的交互作用可能参与NSCLC EGFR-TKIs获得性耐药。该方向尚无其它报道。本课题拟采用基因芯片比较敏感株PC9和三株耐药株Wnt/β-catenin和EGFR信号通路基因mRNA表达，沉默或上调筛选的Wnt/β-catenin通路表达异常基因，研究Wnt通路对药物敏感性的影响及对EGFR通路的调控作用，探讨两条通路间的交互作用在EGFR-TKIs获得性耐药中的作用及机制；结果经动物实验和临床标本加以验证。本研究有望为逆转NSCLC EGFR-TKIs获得性耐药提供潜在的候选靶点。

本研究采用二代测序RNA-seq检测NSCLC吉非替尼敏感株PC-9和PC-9加吉非替尼培养诱导而成的耐药株PC-9-R转录组的表达差异，研究吉非替尼耐药机制。我们发现耐药株PC-9-R中Wnt/β-catenin通路和EGFR及下游通路多个基因表达异常，β-catenin上游调控基因Wnt3a、Wnt5a表达上调，p-GSK3β表达增高，细胞核内β-catenin及下游c-myc表达增高，p-EGFR表达增高。我们还发现耐药株PC-9-R中乙醛脱氢酶ALDH3A1表达明显上调，干扰沉默其表达后可下调Wnt3a、β-catenin、p-GSK3β、p-EGFR及下游p-AKT、p-MAPK、p-MEK1/2、p-stat3表达，而干扰Wnt3a和β-catenin表达不影响ALDH3A1表达。敏感株PC-9吉非替尼用药后会诱导ALDH3A1表达，但没有耐药株中表达显著；随着吉非替尼药物浓度增高和培养时间延长，耐药株PC-9-R中ALDH3A1有更高蛋白表达，撤药后ALDH3A1表达降低。上述研究结果提示吉非替尼可诱导ALDH3A1表达，吉非替尼在细胞中蓄积通过活化Wnt/β-catenin和EGFR及下游通路诱导药物耐受。在肺腺癌人体组织样本中未用药肺腺癌患者ALDH3A1阴性或弱阳性、少量阳性，少部分呈中或强阳性表达。本研究揭示了肺腺癌ALDH3A1与Wnt/β-catenin和EGFR的相互调控关系，促进对靶向药物表皮生长因子受体-酪氨酸激酶抑制剂耐药机制的认识。ALDH3A1和β-catenin基因有可能成为肺腺癌预测耐药的新指标和药物干预靶点。