TAp73/DNp73表达失衡在多发性骨髓瘤DNA损伤诱导细胞凋亡中的作用及机制研究

Aberrant DNA damage response pathways are implicated in the development and drug-resistance induction of multiple myeloma. Our previous study showed that even in the presence of great DNA insult, myeloma cells consistently evade apoptosis even under basal conditions. The ABL1/p73 is an alternative pathway which triggers apoptosis following DNA damage in myeloma cells. Strikingly, even in the presence of elevated ABL1 expression apoptosis was undetectable in the myeloma cells tests. As the down-stream of ABL1, the expression balance of p73 isoform TAp73 and DNp73 was broken. TAp73 was down-regulated and DNp73 was up-regulated significantly in myeloma cells. Kaplan-Meier analyses showed that myeloma patients with higher expression of DNp73 had a significantly inferior survival. shRNA knock-down DNp73 expression suppressed myeloma cells proliferation and promoted the sensitivity of myeloma cells to DNA damage reagent doxorubicin induced apoptosis. ChIP-seq and gene expression profiles (GEP) of tumor cells in myeloma patients suggested that transcription factor NF-κB and alternative splicing factor SLU7 contributed to DNp73 over-expression in myeloma cells. It is our hypothesis that down-regulation of TAp73 and up-regulation of DNp73 promotes myeloma cells proliferation and against DNA damage response induced apoptosis. Activation of NF-κB transcription factor and higher level of splicing factor SLU7 increased DNp73 up-regulation in myeloma cells. DNp73 competitive combined with ABL1 kinase and suppressed myeloma cells apoptosis induced by DNA damage response. This mechanism also contributed to drug-resistance of myeloma cells. Therefore, this project will investigate TAp73 and DNp73 functions of cell proliferation and drug resistance in multiple myeloma. Combined with RIP, ChIP-seq and TAP/MS high-throughput assay, Co-IP experiments, as well as 5TGM1 mouse myeloma model, the DNp73 key signaling pathways and the role of regulatory molecules will be clarified. The results will provide theoretical and experimental foundation for the search for new therapeutic targets in multiple myeloma.

ABL1/p73信号通路是MM细胞DNA损伤诱导细胞凋亡的重要途径。前期研究发现p73蛋白异构体在MM细胞中表达失衡，TAp73表达降低，DNp73表达升高并具有临床预后意义。并且证实敲减DNp73表达可抑制MM细胞增殖，增加其对DNA损伤药物的敏感性。ChIP-seq及表达谱芯片结果显示转录因子NF-κB和选择性剪切因子SLU7参与DNp73基因表达调控。据此我们提出TAp73/DNp73表达失衡是MM细胞抵抗DNA损伤诱导细胞凋亡的重要原因，高表达DNp73竞争性结合ABL1激酶，抑制细胞凋亡，促进MM细胞增殖与耐药，导致患者生存期缩短的科学假设。本研究拟从基因、蛋白水平，体外及体内实验等多层次探讨TAp73/DNp73表达失衡在MM细胞DNA损伤细胞凋亡中的分子机制。并结合RNA-seq等高通量检测方法，探明DNp73作用关键信号通路及调控分子，为寻找新的治疗靶点提供理论及实验基础。

基因组的稳定性对于多细胞生物的发育、组织和器官的动态平衡至关重要。基因组失稳与肿瘤等多种疾病的发生紧密相关。多发性骨髓瘤（multiple myeloma, MM）是中老年人群常见的血液系统肿瘤，大约占血液系统恶性肿瘤的10%，基因组不稳定和染色体异常是MM等许多血液系统肿瘤重要的生物学特征。DNA损伤是诱发基因组失稳的主要因素，生理状态下DNA损伤发生后细胞通过一系列的应答反应，包括细胞周期停滞、DNA修复以及细胞凋亡等维持基因组的稳定性；然而肿瘤细胞可通过复杂的调控机制逃逸DNA损伤引起的细胞凋亡（DNA damage-induced apoptosis）而继续存活。多柔比星（阿霉素）、依托泊甙等DNA损伤药物仍是我国MM治疗一线用药，MM细胞耐药是导致治疗失败的重要原因，但是其耐药分子机制目前尚不明确。.本项目探讨了DNp73表达异常参与调控MM肿瘤细胞DNA损伤诱导细胞凋亡及其在MM细胞耐药中的分子机制。通过体外、体内及MM临床患者标本检测等实验研究，结果发现⑴ DNp73高表达MM细胞增殖明显加快，对多种药物治疗不敏感。DNp73高表达促进逆转药物诱导的MM细胞细胞周期阻滞，促进细胞存活。机制研究发现⑵ DNp73的高表达促进下游癌基因c-Myc表达和活性上升，抑制细胞周期蛋白p21CIP和p27KIP以及p53信号通路活性，从而促进MM细胞增殖加快、抵抗DNA药物诱导的细胞周期停滞和细胞凋亡。⑶ DNp73在MM细胞中的高表达与耐药发生有关，研究发现DNp73高表达可抑制MM细胞miRNA-15a分子表达，miR-15a低表达患者生存时间明显缩短，并介导对药物治疗敏感性降低。⑷ ChIP-qPCR检测分析发现MM肿瘤细胞持续激活的NF-kB信号通路，是导致DNp73基因及蛋白表达上调的重要原因。.本研究明确MM细胞高表达DNp73促进细胞增殖存活，降低对治疗药物敏感性；机制研究阐明DNp73激活下游Myc信号通路，参与MM细胞增殖凋亡调控，并且介导MM细胞对DNA损伤药物耐药性的产生。同时本研究也明确转录因子NF-kB活性升高，是导致MM细胞DNp73高表达的重要分子机制。通过本研究，为明确MM细胞耐药机制，研发靶向治疗药物提供实验及理论基础。