肾母细胞瘤中的自噬调控与新型生物标记筛选

Autophagy is a self-digesting process that is crucial for the maintenance of cellular homeostasis, and is currently being intensively investigated in the field of tumor genesis and treatment. By far there is no report about the deregulation of autophagy in nephroblastoma. However, in Wilm′s tumor, a major subtype of childhood nephroblastoma, there is mal-expression of Bcl-2, p53 and beta-catenin, all of which are key cell death/proliferation related molecules, and have autophagy-regulating effects as well. Hence we hypothesize that autophagy deregulation plays a role in nephroblastoma development and progression. In the pathological types currently with poor prognosis, development of new treatment strategies is of great significance. A prospective research is proposed here, tissues from nephroblastoma patients will be grouped according to the pathological diagnosis and the expression of autophagy-related proteins, and the autophagic level will be systematically examined, its relationship with the type and the prognosis of nephroblastoma will be established. Regulating autophagy in nephroblastoma cells may hamper cell growth and lead to new strategies in chemical therapy. In this project, the in vivo and in vitro effects of autophagy induction/ prohibition in combination with traditional chemical therapeutic agents will be studied, as well as the effectiveness of novel anti-tumor strategies discovered in previous research, i.e., autophagy inhibitors in combination with proteasome inhibitors or ER stress inducers. The specific molecular pathways involved will also be investigated. In another line, tumor-derived exosomes will be isolated from nephroblastoma cell culture medium and from the serum and urine samples from patients. In search of new nephroblastoma biomarkers, exosome proteins and microRNA compositions will be analyzed in tumors, in comparison with normal cell culture medium and samples from healthy individuals. Upon completion of this project, the status of the autophagy regulation and the underlying molecular mechanism in childhood nephroblastoma will be established, and the results obtained will guide the development of new clinical therapeutic strategies as well as new diagnostic/prognostic biomarkers.

自噬是一种维持细胞内稳态的自我消化机制。自噬障碍参与恶性肿瘤发生发展。在肾母细胞瘤中，有自噬调控功能的几种细胞周期相关蛋白Bcl-2、p53、beta-catenin均存在异常表达，故推测该肿瘤中存在自噬异常。本课题拟检测不同类型与化疗前后肾母细胞瘤组织中的自噬水平，从病理学和自噬相关蛋白表达角度对标本进行分类和分析，判断自噬水平与肿瘤病理类型及病程的关系。另外，鉴于通过调控自噬可能抑制肿瘤生长，在高恶性度肾母细胞瘤模型中，使用自噬调控辅助传统化疗药物，以及在前期研究中有效的新型抑瘤方案（自噬抑制剂与蛋白酶体抑制剂或内质网应激诱导剂联用），探索药物的体内外抑瘤效果与相关分子机制。另外，从肿瘤细胞培养液与病人血浆、尿液中分离肿瘤细胞来源的外泌体，比较其蛋白与miRNA成分与非肿瘤的区别。课题的完成将对肾母细胞瘤中自噬水平的动态变化做出系统评价，并为开发新的临床治疗方案和筛选生物标记提供基础。

【背景】肾母细胞瘤（NB）是发病率最高的小儿腹部恶性肿瘤，其发病率占儿科恶性肿瘤的6%。NB整体预后较好，但高恶性度类型死亡率高、常规放化疗影响患儿长期生存质量。本研究根据NB中普遍存在Bcl-2表达升高，p53变异，以及beta-catenin激活，推测这几种分子的异常表达可能通过下游信号分子异常调控改变细胞自噬水平，且自噬可能作为新的治疗靶点。.【研究内容】检测NB组织中自噬水平，并通过调控自噬，在传统的治疗方案基础上提出更有效低毒的抑瘤方案。具体包括以下几方面：①建立NB组织库，检测自噬相关基因蛋白和mRNA表达变化，包括Beclin 1、Atg5、Atg7、LC3、p62等；②在NB细胞系中，通过自噬调控药物联合传统化疗药物，探索是否可以通过调控自噬增强化疗效果和降低毒性；③在裸鼠模型中，观察自噬抑制药物联用化疗药对体内生长NB的抑制效果；④拓展研究：将研究对象拓展至其它几种常见儿童实体肿瘤；并探索了调控自噬是否是中药蛇六谷的抗肿瘤机制。.【主要结果】①通过检测自噬相关基因蛋白与mRNA表达，发现未化疗NB普遍存在自噬抑制，化疗有激活自噬的作用，可能参与化疗耐药；②传统抗有丝分裂化疗药长春新碱联合自噬抑制药物氯喹或自噬诱导药物rapamycin，抑瘤效果均呈现协同效应；通过自噬相关基因ATG7敲减而特异性抑制自噬，证明多靶点药物rapamycin对NB的抑制是通过激活自噬起效；③在裸鼠模型中验证了氯喹与抗有丝分裂药可协同抑制体内生长NB。④发现几个自噬相关基因表达改变是几种儿童实体肿瘤中普遍存在的现象，自噬有可能作为新的化疗靶点。⑤ 发现中抗癌药蛇六谷提取物的机制与诱导自噬有关。.【科学意义】首先，通过本研究，促成本单位建成了标准化管理运行的儿童实体肿瘤组织库，为后续研究和多中心研究提供了资源储备。本研究首次系统检测了NB组织中几种自噬相关基因的表达变化，发现化疗前NB存在自噬抑制，化疗可激活自噬。同时，在体内外实验中，均证明自噬抑制药物（氯喹）可以协同化疗药物进一步抑制NB。本研究结果为NB发生和耐药的机制提供了新线索，并在创新治疗方法方面取得了一定突破，提示自噬可能成为NB新的治疗靶点，可提高化疗效果、降低毒副作用，减少耐药性。研究结果可拓展到其它几种儿科实体肿瘤，包括神经母细胞瘤、髓母细胞瘤，视网膜母细胞瘤。