DTL靶向调控PDCD4促进非小细胞肺癌细胞增殖、侵袭和转移

DTL(Denticleless), which is an ubiquitin ligase substrate receptor, plays a specific recognition role in the degradation of target protein, its role in cancer and molecular mechanism is still unclear. Our preliminary experiments study found that DTL expression is elevated in non-small cell lung cancer (NSCLC) and significantly related to the survival time of NSCLC patients, and can promote proliferation and migration of NSCLC cells. Further, bioinformatics analysis combined with immune mass spectrometry detection found that PDCD4, which regulates the JNK signaling pathway, may be the target protein of DTL. Based on these findings, we propose that "the molecular mechanism by which DTL promotes the development and progression of NSCLC through the ubiquitination of PDCD4, thereby activating the JNK signaling pathway that PDCD4 inhibits." In this project, we will investigate the role of DTL in NSCLC development and metastasis. The molecular mechanism of DTL degrading target protein will be elucidated by affinity purification mass spectrometry, co-immunoprecipitation, target protein half-life period and ubiquitination detection. DTL transgenic mice verified their functional and molecular mechanisms. Combined with the clinical follow-up data of NSCLC patients as well as in vivo and in vitro function detection, we will assess the impact of DTL on NSCLC differentiation, pathological grading, clinical stage, survival time, tumor recurrence and metastasis, and identify its physiological and pathological functions. The results from this proposal will provide theoretical and experimental foundation for the basic research and targeted therapy of NSCLC cancer.

DTL(Denticleless)在靶蛋白泛素化降解过程中发挥着特异性识别作用，其在肿瘤中的作用机制仍不明确。我们的预实验研究发现DTL在非小细胞肺癌(NSCLC)中过表达，与NSCLC病人生存期密切相关，且能促进NSCLC细胞增殖和迁移。生物信息学分析结合免疫共沉淀质谱检测发现PDCD4为DTL的靶蛋白。基于此，我们提出”DTL促进NSCLC发生的分子机制是通过促进抑癌基因PDCD4泛素化降解，进而活化PDCD4抑制的JNK信号通路，最终促进NSCLC的发生发展"这一假说。本课题拟通过：体内外实验确认DTL在NSCLC发生及侵袭中的作用；通过质谱分析、免疫共沉淀、靶蛋白半衰期检测、截短体和功能位点突变等技术揭示DTL促癌的分子机制；DTL转基因小鼠验证其功能及分子机制；分析DTL与NSCLC病理分级、临床分期、预后生存、肿瘤复发和转移之间的关系。为NSCLC的治疗提供新的研究靶点。

CULLIN4 (CUL4)是一种与肿瘤密切相关的E3泛素连接酶，通过与DNA损伤结合蛋白1(DDB1)结合，以DCAF (DDB1 CUL4 associated factors)作为底物受体来识别底物蛋白，促进特异性靶蛋白的泛素化降解。DTL是新近发现的具有重要功能的DCAF，DTL能够促进细胞增殖，与多种肿瘤密切相关。然而，目前对DTL在NSCLC发展中所具有的功能及其分子机制尚未有系统性的研究。本课题在前期实验的基础上，从分子、细胞、模式动物和临床组织标本等层面，系统探索DTL在NSCLC中的作用及其相关分子机制：体内外实验确认DTL在促进NSCLC细胞增殖及侵袭转移中的作用；通过免疫亲和纯化质谱分析、免疫共沉淀、靶蛋白半衰期检测、系列截短体及功能位点突变体构建、泛素化检测等技术手段揭示DTL降解靶蛋白的分子机制，明确DTL促进NSCLC发生发展的分子机制是通过靶向抑癌基因PDCD4，促进PDCD4泛素化降解，进而活化PDCD4抑制的JNK信号通路，最终促进NSCLC的发展；应用可诱导性/组织特异性DTL转基因小鼠研究过表达DTL是否能够诱发小鼠NSCLC，并进一步在体验证DTL对靶蛋白的调控作用及机制；通过NSCLC病理组织采集检测，结合患者临床随访资料，分析DTL与NSCLC病理分级、临床分期、预后生存、肿瘤复发和转移之间的关系，揭示其生理病理学功能及其临床意义。本项目的完成使我们对DTL参与NSCLC调控的作用、分子机理及其临床意义有更加深入的认识，为NSCLC基础研究和靶向治疗提供新的理论基础和实验依据。