AURKA/SMAD4信号通路对结直肠癌西妥昔单抗耐药的影响及分子靶向治疗研究

Cetuximab is the molecular targeted drug and treatment of colorectal cancer. However, the clinical application is limited because of primary and secondary resistance. Therefore, it is clinical significant to study the mechanism of cetuximab resistance in CRC and its reverse strategy. AURKA is recognized as oncogene and SMAD4 is recognized as tumor suppressor, which have been widely reported to be involved in the occurrence and development of colorectal cancer. But the correlation between AURKA/SMAD4 and cetuximab resistance is not clear now. We previously reported that AURKA interacts with SMAD4 and down-regulates its expression in the cancer cells. Recently, we found that AURKA protein levels in CRC cells are related to cetuximab resistance. Moreover, targeting AURKA/SMAD4 pathway could enhance the effect of cetuximab, suggesting that AURKA/SMAD4 pathway is related to cetuximab resistance in CRC. However, the mechanism of regulation between AURKA and SMAD4, and the mechanism of AURKA/SMAD4 pathway mediating cetuximab resistance in CRC are not clear. Therefore, based on the techniques of gene manipulation and phosphorylated proteomics, we plan to elucidate the role and molecular mechanisms of AURKA/SMAD4 pathway on the cetuximab resistance and the reversing effect of AURKA inhibitors in three levels: systemic level, cellular level and molecular level, which will be helpful to offer promising efficient approach for patients with cetuximab resistance and provide a novel path for reversing cetuximab resistance in CRC.

西妥昔单抗是结直肠癌靶向治疗药物，其耐药现象频现限制了它的应用，因此探索西妥昔单抗耐药机制及其逆转策略对结直肠癌治疗具有重要意义。癌基因AURKA和肿瘤抑制基因SMAD4被报道参与结直肠癌的发生发展，但两者与西妥昔单抗耐药的关系仍不清晰。课题组前期发现AURKA可与结合SMAD4并负向调控其表达水平。此外，我们发现AURKA在结直肠癌细胞的表达水平与西妥昔单抗耐药相关，靶向干预AURKA/SMAD4通路可以增强西妥昔单抗的疗效，这初步证明了AURKA/SMAD4信号通路与西妥昔单抗耐药相关，但两者之间调控的确切方式，及介导耐药的分子机制尚不明确。基于此，本项目拟采用基因操控和磷酸化蛋白组学等技术，从分子、细胞和整体水平研究AURKA/SMAD4信号对西妥昔单抗耐药的确切影响，并探索小分子AURKA抑制剂的逆转耐药作用，旨在深入探究西妥昔单抗耐药的分子机制，并为逆转耐药提供新的治疗策略。

西妥昔单抗是结直肠癌靶向治疗药物，其耐药现象频现限制了它的应用，因此探索西妥昔单抗耐药机制及其逆转策略对结直肠癌治疗具有重要意义。本研究从分子、细胞和整体水平，研究AURKA在西妥昔单抗耐药中作用、调控机制，并探索小分子AURKA抑制剂的逆转耐药作用，旨在深入探究西妥昔单抗耐药的分子机制，并为逆转耐药提供新的治疗策略。.主要研究结果如下：.1. AURKA在结直肠癌西妥昔单抗耐药过程中的作用及调控机制.癌基因AURKA在结直肠癌中表达显著增高，且其蛋白水平与西妥昔单抗的敏感性呈负相关。在敏感细胞中过表达AURKA，西妥昔单抗敏感性减弱；在耐药细胞中敲低AURKA，西妥昔单抗敏感性增强。进一步研究发现基因干预AURKA表达在影响西妥昔单抗的疗效，同时，还能够影响SMAD4蛋白表达的水平。而且，在耐药细胞中，重塑SMAD4表达能够提高西妥昔单抗的敏感性。提示，AURKA介导的西妥昔单抗耐药与SMAD4密切相关。蛋白互作研究发现在结直肠癌中AURKA与SMAD4相结合后，其赖氨酸磷酸化水平升高。在AURKA过表达的耐药细胞中，胞浆中的SMAD4发生泛素化修饰，蛋白降解。沉默E3泛素酶SKP2时，SMAD4的表达得以恢复，表明AURKA通过E3泛素酶SKP2调节SMAD4水平。综上所述，AURKA通过磷酸化修饰和泛素化降解SMAD4介导结直肠癌西妥昔单抗耐药。.2. 靶向干预SRC/AURKA信号轴对结直肠癌西妥昔单抗耐药的逆转作用.AURKA抑制剂Alisertib联合西妥昔单抗显著增加耐药细胞对西妥昔单抗的敏感性，抑制HCT-8/AURKA-OE异位荷瘤小鼠肿瘤生长，逆转西妥昔单抗耐药。SRC通路抑制剂SU6656下调AURKA，与Alisertib、西妥昔单抗联用增加耐药细胞对西妥昔单抗的敏感性，抑制HCT-8/AURKA-OE异位荷瘤裸鼠肿瘤生长，逆转西妥昔单抗耐药。.综上所述，研究发现AURKA在西妥昔单抗耐药细胞中表达失调并参与西妥昔单抗耐药进程，并揭示AURKA过表达通过磷酸化修饰和泛素化降解SMAD4而影响西妥昔单抗的敏感性，此外，我们还发现小分子化合物可通过调控AURKA表达逆转西妥昔单抗耐药。本研究为阐明西妥昔单抗耐药的新机制，并为探索新的逆转策略提供了药理依据。