疟原虫ΔCSP蛋白抑制肺癌细胞自噬在其增敏EGFR靶向治疗中的作用和机制研究

The EGFR-TKI (Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor) was demonstrated to be effective anticancer agents in the clinical treatment of lung cancer, however, tumor resistance arises rapidly. Therefore, current research is focus on searching for new ways to sensitization or delay the drug resistance. Our research group has been committed to the research in lung cancer treatment by using malaria parasite. Funded under the previous national natural science foundation item, we found that the inhibiting effect of gefitinib on A549 could be improved by ΔCSP (crcumsporozoite protein) of Plasmodium yoelii. More interestingly, we found the inhibiting effect of gefitinib was also impoved by ΔCSP on the H1975 cell (T790M mutation). Results of transcriptome sequencing indicated that the mRNA level of autophagy related proteins was significantly decreased in the H1975 expressing ΔCSP and the sensitization effect of ΔCSP could be significantly inhibited by autophagy inducing caused by rapamycin. All these results indicated that ΔCSP-sensitization effect on the gefitinib was mediated by the down-regulated the mRNA level of autophagy related proteins, but the molecular mechanism is still unclear. In this study, we would like to confirm the abovementioned hypothesis by using technology of ATG5-knockout cell and imaging in vivo, and test whether ΔCSP downregulates the autophagy-related proteins through binding to the HSPA8. This study could not only reveal new mechanism of EGFR-TKI sensitization, but also provide novel clues to design prophylactic stragies against durg-resistant.

EGFR-TKI在肺癌临床治疗中面临的最大难题是迅速耐药，寻找新的方法来增敏或延缓耐药是当前研究的焦点。课题组一直致力于疟原虫抗肺癌研究，在前一个面上项目的资助下，我们发现疟原虫ΔCSP蛋白能够增敏吉非替尼对A549的抑制作用。进而发现ΔCSP同样能够显著增敏吉非替尼对H1975细胞(T790M突变)的抑制作用；转录组测序分析发现，ΔCSP能够下调H1975细胞自噬相关蛋白的表达水平；雷帕霉素诱导自噬能够抑制ΔCSP增敏吉非替尼。提示我们ΔCSP蛋白极有可能是通过抑制自噬增敏EGFR-TKI，但其具体机制并不清楚。本课题拟利用ATG5敲除及体内活体成像技术，进一步证实ΔCSP通过抑制自噬增敏吉非替尼；采用Co-IP技术探讨HSPA8在ΔCSP抑制自噬中的分子机制。深入探讨这一机制不但有助于揭示新的EGFR-TKI增敏机制，还能为设计新的EGFR-TKI增敏措施提供重要的理论依据和研究策略。

耐药是导致不能手术的非小细胞肺癌患者治疗无效和预后不良的主要原因。EGFR-TKI治疗虽然延长了EGFR阳性突变患者的生存时间，但也存在耐药性。课题组前期研究发现，CSP能显著增敏TNF-ɑ对肺癌细胞A549的杀伤。在本研究中，我们发现过表达CSP的NSCLC细胞对吉非替尼更敏感，伴有明显的细胞凋亡和细胞活力下降。吉非替尼治疗后，OE-CSP组细胞自噬受到显著抑制，自噬相关蛋白ATG5和LC3表达量降低。自噬诱导剂雷帕霉素作用于OE-CSP细胞后，细胞活力和干性恢复。在肺癌皮下瘤模型中，CSP抑制自噬导致细胞生长受到抑制，肿瘤体积明显小于对照组。进一步探查机制，我们发现OE-CSP组细胞自噬相关蛋白mRNA水平并未明显变化，但蛋白水平则明显下降，这暗示我们CSP下调ATG5,LC3蛋白的表达是通过翻译后修饰途径。最后，我们发现CSP是通过泛素蛋白酶体途径降解LC3和ATG5从而下调自噬。用泛素特异性抑制剂TAK243处理细胞后，OE-CSP组的细胞活力、凋亡和细胞生长情况与对照组相当。综上所述，我们发现CSP通过泛素-蛋白酶体途径下调细胞自噬，从而提高吉非替尼的疗效。

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