吉非替尼诱导ULBP1高表达提高肺腺癌细胞对CIK细胞敏感性的机制研究

Molecular targeted therapy, represented by EGFR-TKI, is a milestone in the treatment of non-small cell lung cancer(NSCLC). However, drug resistance to EGFR-TKI is inevitable. Cytokine-induced killer cells(CIKs) are induced and amplified from peripheral blood mononuclear cells (PBMCs) with multiple cytokines, which exhibited a powerful killing activity both in vitro and in vivo and transfusion of these cells have become an adjuvant treatment for tumors. NKG2D is an activating receptor that can bind to NKG2DLs(such as MIC A/B,ULBP1-6) that are expressed on the surface of tumor cells and plays a crucial role in antitumor responses.We have found that CIKs overexpress NKG2D and our previous studies have also shown that co-EGFR-TKI and CIK have synergistic effects. Pretreatment of gefitinib can induce lung cancer cells to express NKG2D ligand ULBP1, and thus enhance its sensitivity to CIK cells, but the mechanism remains unclear. SHP2 is a drug resistance-related protein in lung cancer, and our pre-experiment found that SHP2 expression is negatively correlated with ULBP1 expression. Combined with the literatures and our previous studies, we speculated that gefitinib inhibited SHP2, down-regulated the transcriptional inhibitory factor Sp3 via AKT pathway, leading to high expression of ULBP1 and enhanced CIK immune recognition on lung cancer cells. Therefore, we hope to establish lung adenocarcinoma cell lines with stable high/low SHP2 expression through vector construction, RNA interference, detect the changes of AKT/Sp3/ULBP1 pathway and sensitivity to CIK cells before and after gefitinib treatment and verified by animal experiments. The study helped to identify potential targets for inhibiting EGFR-TKI resistance, laying the foundation for exploring new approaches to lung cancer treatments.

EGFR-TKI靶向治疗是非小细胞肺癌治疗的里程碑，但耐药不可避免。CIK细胞高表达NKG2D，与肺癌细胞表面NKG2D配体识别是免疫杀伤的基础。前期研究发现：联合EGFR-TKI和CIK治疗具有协同效应。预实验发现吉非替尼可诱导肺癌细胞高表达NKG2D配体ULBP1，加强其对CIK细胞杀伤敏感性，但机制不清。SHP2是我们研究证实的肺癌顺铂耐药相关蛋白，预实验发现SHP2与ULBP1表达呈负相关。结合文献报道，我们推测吉非替尼抑制SHP2，经AKT通路下调转录抑制因子Sp3活性，导致ULBP1高表达，提高CIK对肺癌细胞的免疫识别。我们拟通过载体构建、RNAi分别建立高/低表达SHP2的肺腺癌细胞系，检测吉非替尼处理前后AKT/Sp3/ULBP1通路变化及对CIK细胞的敏感性变化，并通过动物实验加以验证。研究有助于发现潜在的抑制EGFR-TKI耐药的靶点，为优化肺癌联合治疗模式奠定基础。

以EGFR-TKI为代表的分子靶向治疗，被认为晚期非小细胞肺癌治疗进展中的里程碑事件。然而，EGFR-TKI不可避免地出现耐药。因此，创新探索新的联合治疗模式，挖掘潜在的逆转或延缓耐药的治疗靶点，具有重要的临床意义。我们前期回顾性临床研究发现：EGFR-TKI联合CIK治疗有助于延长患者的无疾病进展生存期，预实验提示EGFR-TKI联合CIK细胞可明显提高对肺腺癌细胞的杀伤活性。因此，在前期研究基础、文献报道和科研假设基础上，我们以对吉非替尼敏感的PC9肺腺癌细胞系和对吉非替尼耐药的PC9/GR肺腺癌细胞系为研究对象，构建了高低表达SHP2的肺腺癌细胞系，通过细胞实验、FACS、Western blot、裸鼠成瘤等方法，证实吉非替尼通过下调SHP2的表达，进而导致下游的Sp1、Sp3蛋白表达下调，降低其对ULBP1表达的抑制作用，诱导ULBP1的高表达，为CIK细胞发挥免疫杀伤作用提供作用靶点。这一研究结果有助于阐明SHP2参与肺腺癌分子靶向药物耐药的作用机制及联合治疗有效的分子基础，从而为肺腺癌的联合治疗提供新的潜在靶点。

内容获取失败，请点击重试