IGF-1促进多药耐药蛋白表达介导直肠癌放疗抵抗的分子机制

Preoperative neoadjuvant radiotherapy can significantly improve the prognosis for advanced rectal cancer patients. However, the resistance to irradiation remains to be a very difficult clinical problem. Previously, our research group have successfully established rectal cancer cell and animal models of radioresistance, and applicated molecular biology research to clarify the role of multidrug resistance proteins family members MRP4 and ABCC3 on resistance to neoadjuvant radiotherapy/chemoradiotherapy for rectal cancer. Intriguingly, we found that insulin-like growth factor 1 could obviously up-regulate the expression of ABCC3 in vitro. Therefore,the present project aims to clarify the regulational function of IGF-1 on the expression of MRP family members, and application of RNA interference to specifically inhibit the expression of IGF-1 receptor in rectal cancer cell lines to explore its effect on radiosensitivity both in vivo and in vitro, further research on the functional role and molecular mechanism of IGF-1_ABCC3_caspase-3 apoptosis signaling pathway in resistance to irradiation. Meanwhile, dynamic detection of serum IGF-1 levels pre- and post-irradiation in patients undergoing preoperative neoadjuvant radiotherapy, combined with tissue chips and clinical follow-up data, according to the postoperative pathological tumor regression grades, comprehensive evaluation the clinical significance of IGF-1 detection in predicting the response to neoadjuvant radiotherapy and long-term prognosis of rectal cancer patients. Furthermore, application of the IGF-1 receptor monoclonal antibody to specificly suppress the expression of IGF-1 receptor proteins in nude mice to verify the effect and safety of itself in inducing sensitivity to irradiation. Our goal is to lay the foundation in realization of clinical molecular detection for prospective screening patients, and exogenous application of IGF-1 receptor inhibitors to improve radiosensitivity and survival benefits for advanced rectal cancer patients.

直肠癌术前放疗可显著改善预后，然而放疗抵抗是困扰临床的难题。课题组已建立放疗抵抗的直肠癌细胞和动物模型，并明确了多药耐药蛋白(MRP)家族成员MRP4、ABCC3在直肠癌放化疗耐受中的作用，同时预实验发现胰岛素样生长因子1(IGF-1)能够促进ABCC3表达。本项目拟深入探究IGF-1对MRP家族的调控作用；RNA干扰抑制肠癌细胞中IGF-1受体表达，验证其本身对细胞辐射敏感性的影响；明确IGF-1-ABCC3-caspase-3凋亡信号通路在放疗抵抗中的作用机制；动态检测放疗前后患者血清IGF-1水平，结合组织芯片与临床随访资料，根据病理肿瘤消退分级综合评估IGF-1预测直肠癌放疗反应及预后的临床价值；应用现有IGF-1受体单克隆抗体，裸鼠成瘤体内验证靶向抑制其对放疗的增敏效果及安全性。为实现临床分子检测前瞻性筛选患者，外源性应用IGF-1受体抑制剂提高直肠癌患者放疗反应生存获益奠定基础

项目背景：局部进展期直肠癌手术难度大局部复发率高，术前新辅助放疗可显著改善预后，然而个体放疗敏感性差异较大，因此放疗抵抗是困扰临床的难题，亟需寻找能够精准预估放疗反应的分子标志物，通过多学科协作制订更具针对性和个体化的综合治疗方案，避免无效过度的治疗以减少不良反应，进一步改善预后。.研究内容：回顾性分析已行新辅助放疗的局部进展期直肠癌患者临床病理资料，应用免疫组化检测组织标本中胰岛素样生长因子1受体（IGF-1R）蛋白的表达水平，综合免疫组化染色强度及7项可能影响新辅助治疗疗效的临床病理因素，证明IGF-1R高表达与直肠癌新辅助放疗抵抗呈正相关；细胞株层面依据SF2值区分建立直肠癌新辅助放疗抵抗细胞株SW480、HT-29和放疗敏感细胞株DLD-1、HCT116，运用Western Blot和RT-PCR从蛋白质水平和mRNA层面，进一步验证IGF-1R高表达与放疗抵抗呈正相关；通过流式细胞术染色检测细胞凋亡，克隆形成及CCK8实验检测细胞增殖情况，证实IGF-1R高表达对肿瘤细胞株促进存活，抑制凋亡的作用；与此同时，通过放射模拟照射联合IGF-1R抑制剂NVP-ADW742或体外干扰IGF-1R表达时，两株放疗抵抗细胞的克隆形成数目均明显下降，提示其放射敏感性均得到提高。另在深入机制研究方面，MAPK和PI3K二者均是参与介导细胞增殖分化及生长的核心信号分子，通过Western blot检测发现放疗能激活pErk和pAkt表达，而NVP-ADW742联合放疗，可抑制pErk和pAkt激活。同时放疗联合NVP-ADW742通过增加gammaH2AX酶的含量，以增强DNA双链断裂损伤的程度，从而介导影响DNA损伤修复的途径，最终导致肿瘤细胞不可逆地死亡。最后，我们通过TCGA大肠癌标本数据库，分析270例癌和癌旁标本及正常组织差异基因表达，进一步得到IGF-1R所在信号通路激活及效应分子表达情况，发现PPAR信号通路与直肠癌细胞放疗抵抗相关，为后续工作提供理论基础。.科学意义：IGF-1R在直肠癌细胞株中的高表达水平与放疗抵抗显著相关，对预测直肠癌放疗敏感性具有重要临床意义，IGF-1R抑制剂NVP-ADW742可作为临床潜在的放疗增敏治疗手段，为其转化医学应用提供理论依据。