背景:化疗耐药是治疗结直肠癌(CRC)的一个重要障碍,然而,CRC化学耐药的分子基础仍然知之甚少,阻碍新的治疗干预措施的发展。含有TuGTP结合域的延伸因子2(EFTUD2)已成为涉及各种癌症类型的潜在致癌因子,促进肿瘤生长和存活。然而,其在调节CRC细胞对化疗敏感性中的具体作用尚不清楚。
方法:进行公共数据集分析和内部样本验证,以评估EFTUD2在5-氟尿嘧啶(5-FU)化疗耐药的CRC细胞中的表达以及EFTUD2作为CRC预后指标的潜力。实验都是在体外,包括MTT测定,EdU细胞增殖试验,TUNEL检测,和克隆形成测定和体内,使用细胞来源的异种移植模型,进行以阐明EFTUD2在CRC细胞对5-FU治疗的敏感性中的功能。通过分子对接研究了EFTUD2与致癌转录因子c-MYC相互调控的分子机制,泛素化测定,染色质免疫沉淀(ChIP),双荧光素酶报告分析,和共免疫沉淀(Co-IP)。
结果:我们发现EFTUD2表达与5-FU耐药呈正相关,病理分级较高,CRC患者预后不良。我们还在体外和体内证明了EFTUD2敲低使CRC细胞对5-FU治疗敏感,而EFTUD2的过度表达削弱了这种敏感性。机械上,我们发现EFTUD2通过阻止其泛素介导的蛋白酶体降解与c-MYC蛋白物理相互作用并稳定c-MYC蛋白。有趣的是,我们发现c-MYC与EFTUD2基因的启动子区直接结合,激活它的转录。利用救援实验,我们进一步证实,EFTUD2对5-FU抗性的影响取决于c-MYC的稳定性。
结论:我们的发现揭示了一个涉及EFTUD2/c-MYC轴的正反馈环,通过增加EFTUD2转录和稳定c-MYC癌蛋白,阻碍了5-FU化疗在CRC细胞中的疗效。这项研究强调了EFTUD2作为克服CRC患者化疗耐药的有希望的治疗靶点的潜力。
BACKGROUND: Chemoresistance presents a significant obstacle in the treatment of colorectal cancer (CRC), yet the molecular basis underlying CRC chemoresistance remains poorly understood, impeding the development of new therapeutic interventions. Elongation factor Tu GTP binding domain containing 2 (EFTUD2) has emerged as a potential oncogenic factor implicated in various cancer types, where it fosters tumor growth and survival. However, its specific role in modulating the sensitivity of CRC cells to chemotherapy is still unclear.
METHODS: Public dataset analysis and in-house sample validation were conducted to assess the expression of EFTUD2 in 5-fluorouracil (5-FU) chemotherapy-resistant CRC cells and the potential of EFTUD2 as a prognostic indicator for CRC. Experiments both in vitro, including MTT assay, EdU cell proliferation assay, TUNEL assay, and clone formation assay and in vivo, using cell-derived xenograft models, were performed to elucidate the function of EFTUD2 in sensitivity of CRC cells to 5-FU treatment. The molecular mechanism on the reciprocal regulation between EFTUD2 and the oncogenic transcription factor c-MYC was investigated through molecular docking, ubiquitination assay, chromatin immunoprecipitation (ChIP), dual luciferase reporter assay, and co-immunoprecipitation (Co-IP).
RESULTS: We found that EFTUD2 expression was positively correlated with 5-FU resistance, higher pathological grade, and poor prognosis in CRC patients. We also demonstrated both in vitro and in vivo that knockdown of EFTUD2 sensitized CRC cells to 5-FU treatment, whereas overexpression of EFTUD2 impaired such sensitivity. Mechanistically, we uncovered that EFTUD2 physically interacted with and stabilized c-MYC protein by preventing its ubiquitin-mediated proteasomal degradation. Intriguingly, we found that c-MYC directly bound to the promoter region of EFTUD2 gene, activating its transcription. Leveraging rescue experiments, we further confirmed that the effect of EFTUD2 on 5-FU resistance was dependent on c-MYC stabilization.
CONCLUSIONS: Our findings revealed a positive feedback loop involving an EFTUD2/c-MYC axis that hampers the efficacy of 5-FU chemotherapy in CRC cells by increasing EFTUD2 transcription and stabilizing c-MYC oncoprotein. This study highlights the potential of EFTUD2 as a promising therapeutic target to surmount chemotherapy resistance in CRC patients.