ABCC3表达与进展期直肠癌新辅助放疗敏感性的关系

Preoperative neoadjuvant radiotherapy can reduce the TNM staging, increase anal sphincter preservation rate and reduce local recurrence rate of locally advanced rectal cancer, while individual radiosensitivity is significantly different. Therefore, it urgently needs for effective molecular markers to screen proper patients with avoiding invalid or excessive treatment. Based on our previous National Natural Science Foundation (30973460) that multidrug resistance-associated protein 4 (MRP4) is significantly correlated with radioresistance of rectal cancer, and further screening with high-throughput gene microarray, this study intends to detect ATP binding cassette protein 3 (ABCC3) mRNA and protein expression in specimens of locally advanced rectal cancer patients who received preoperative neoadjuvant radiotherapy retrospectively, combined with the tumor regression grade TRG and disease free survival rates DFS for assessment of ABCC3 expression on radiosensitivity and long-term prognosis; meanwhile detect changes of ABCC3 expression in paired samples of biopsy and surgery before and after neoadjuvant radiotherapy prospectively to analyze the impact of radiotherapy on its expression, thus make it clear that ABCC3 expression is significantly associated with judgment for clinical radiosensitivity. For the experiments in vivo and in vitro, application of shRNA silences ABCC3 expression in colorectal cancer cells, after the intervention with gradient dose irradiations, we detect functional changes of cell proliferation, cell apoptosis, cell cycle arrest, and nude mice tumor model corresponding phenotypic changes to explain the role of ABCC3 in the resistance to radiotherapy, then further analyze the role of ABCC3 expression in regulating caspase apoptotic pathway, and in the transportation of glutathione (GSH) and reactive oxidative stress (ROS),to reveal the molecular mechanisms of ABCC3 on radiosensitivity in locally advanced rectal cancer. We expect to lay a theoretical foundation for the application of ABCC3 screening radiation-sensitive patients to guide clinical develop individualized treatment plan.

术前新辅助放疗可降低进展期直肠癌TNM分期，增加保肛率并减少局部复发，但个体敏感性差异显著，因此亟需分子标志物筛选患者，避免无效、过度的治疗。本研究在前期工作基础上，拟回顾性检测新辅助放疗直肠癌标本中ABCC3的mRNA及蛋白表达，结合肿瘤消退分级TRG和无病生存率DFS评估ABCC3对放疗反应的影响；前瞻性检测放疗前后活检与手术配对标本中ABCC3表达变化，分析放疗对其表达的影响，从而明确ABCC3表达对于临床放疗敏感性判断的意义；同时，在体内和体外实验中，从射线处理后的肠癌细胞细胞增殖、凋亡、周期阻滞等功能变化和裸鼠成瘤模型的相应表型改变阐释ABCC3在肠癌放疗抵抗中的作用；进一步分析ABCC3在caspase凋亡通路的调节作用，及其在谷胱甘肽GSH转运中的调节方式，揭示ABCC3在直肠癌放疗抵抗中的分子机制，为应用ABCC3筛选放疗敏感患者，指导临床制定个体化治疗奠定理论基础。

研究背景：进展期直肠癌手术难度大、术后控便功能差、局部复发率高，是结直肠外科研究的重点问题。新辅助治疗即术前放化疗可以显著改善患者预后，降低肿瘤分期，增加保肛率并减少局部复发。然而放化疗敏感性差异较大，放化疗抵抗是困扰临床的难题，因此亟需寻找能够准确评估放化疗反应的分子标志物，多学科协作制定更具针对性个体化的综合治疗方案，避免无效过度的治疗并减少副反应，进一步改善预后。研究内容：回顾性分析行新辅助放化疗进展期直肠癌患者临床病理资料，并应用免疫组化检测标本中ABCC3蛋白表达水平，按照5分法肿瘤消退分级标准评价其新辅助治疗后肿瘤病理退缩程度，并分析11项可能影响新辅助治疗疗效的临床病理因素，随访长期生存情况评综合评估ABCC3表达水平对进展期直肠癌放化疗反应及长期预后的影响。细胞水平应用小干扰RNA抑制结直肠癌细胞株中ABCC3表达，梯度剂量射线照射与化疗药物干预后，克隆形成实验检测细胞增殖，单击多靶模型SF=1-(1-e–D/D0)N拟合剂量生存曲线，计算一系列放疗敏感参数，流式细胞术与Hoechst 33342染色检测细胞凋亡、细胞周期阻滞等功能变化；动物水平应用裸鼠成瘤验证体外研究结果；机制研究方面进一步分析ABCC3所在凋亡信号通路激活及效应分子表达情况，阐述ABCC3在进展期直肠癌放化疗抵抗中的分子作用途径。进一步的评估细胞内活性氧的含量和照射后caspase-3依赖的凋亡通路活性改变。关键数据：单因素分析表明高表达ABCC3（免疫活性分数大于6）显著相关于抵抗放化疗不良病理反应（肿瘤消退级≤2与≥3，P = 0.002），并在多变量Cox分析模型中显示其预示着较差的长期预后（5年总生存率：HR = 1.612，95% CI，1.094对2.375，P = 0.016；5年无病生存率：HR = 1.513，95% CI，1.041对2.200，P = 0.030）。抑制ABCC3表达能够明显减少照射后细胞内活性氧的从细胞中释放，并显著增加活化的聚ADP核糖聚合酶和caspase-3的表达，揭示了潜在的分子作用机制。科学意义：ABCC3在结直肠癌细胞中的表达水平与放化疗抵抗显著相关，对预测直肠癌放化疗敏感性及预后具有重要临床意义，可作为前瞻性筛选患者的有效分子标志物，ABCC3可能是治疗晚期直肠癌的一个有吸引力的研究目标，为下一步其转化医学应用提供了理论依据。