NUMB通过Hedgehog及Notch信号通路调控多发性骨髓瘤肿瘤干细胞及耐药性的机制研究

Multiple myeloma (MM) is a malignant neoplasm of plasma cells in bone marrow, which leads to bone destruction and marrow failure. Treatment of MM has evolved rapidly with the introduction of new drugs, such as: immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), monoclonal antibodies, and histone deacetylase (HDAC) inhibitors. However, MM is still incurable due to drug resistance and recurrence. The activation of Hedgehog and Notch signaling is related to multiple myeloma stem cell (MMSC) and drug resistance, although the mechanism of their activation is not clear. . NUMB plays a role in the determination of cell fates during development. The encoded protein, whose degradation is induced in a proteasome-dependent manner by MDM2, is a membrane-bound protein that has been shown to associate with NOTCH1 (Notch signaling) and GLI1 (Hedgehog signaling). Several articles show that NUMB downregulation leads to the activation of Hh and Notch signaling in some tumors. We screened the GEO database and found that NUMB is significantly down-regulated in MM. Furthermore, it correlates with the progression of MM (from MGUS, SMM to MM). Our preliminary results suggest that antagonizing NUMB leads to upregulation of Hh signaling and MMSC. Therefore, we proposed that the activation of Hh and Notch signaling triggered by NUMB down-regulation is the key mechanism of MMSC generation, maintenance and MM drug resistance. . Firstly, we will detect the expression level of NUMB in primary plasma cells from MM patients and healthy donors, which is aimed at exploring clinical significance of NUMB, especially for disease staging and drug resistance. Secondly, we will explore the contribution of NUMB in activating Hh and Notch signaling of MM cells. Next, the relationship between NUMB and MMSC / drug resistance will be verified though cell biology experiments and animal experiment. Finally, we will explore the influence of asymmetric distribution of NUMB caused by mitosis on the formation of MMSC. Taken together, our project will provide the theory for the development of anti-MM drugs targeting NUMB or its upstream and downstream molecular.

多发性骨髓瘤（MM）的耐药和复发不可避免。Hedgehog（Hh）和Notch信号通路异常激活与MMSC、耐药相关，但激活机制仍不清楚。衔接蛋白NUMB是一个抑癌因子，其下调可激活某些肿瘤的Hh及Notch信号通路，但其与MM的关联未见报道。我们分析GEO数据库发现NUMB在MM中显著下调，与疾病过程相关；且初步研究发现拮抗NUMB可上调Hh信号通路和MMSC。故我们推测，NUMB下调可能激活Hh及Notch信号通路从而促进MMSC及MM耐药。本课题从NUMB的临床意义入手，研究其与MM疾病分期和耐药的关联；通过细胞生物学、动物模型等方法，解析NUMB对MM细胞Hh和Notch信号通路的负调节作用，阐述其对MMSC和MM耐药的调控作用和机制；并深入探索有丝分裂后NUMB不对称分布对MMSC生成的影响和机制，为以NUMB或其上下游为靶点的抗MM药物研发提供理论依据，以有效防治MM耐药和复发。

多发性骨髓瘤（MM）是一种不可治愈的疾病，其耐药和复发不可避免。Hedgehog（Hh）和Notch信号通路的异常激活与肿瘤干细胞、耐药相关，但激活机制仍不清楚。衔接蛋白NUMB是一个抑癌因子，其下调可通过抑制某些肿瘤的Hh及Notch信号通路成分的泛素化降解从而激活上述信号通路，但其与MM的关联未见报道。我们分析GEO数据库发现NUMB在MM中显著下调，与疾病过程相关。本课题组对有明确骨髓活检确诊的42例MM患者的活检标本进行NUMB、GLI1（Hh通路核心转录因子）和NICD（Notch受体胞内域）的免疫组化染色，证实在MM细胞中NUMB与GLI1、NUMB与NICD的表达水平呈现负相关关系，且GLI1与NICD的表达呈现显著正相关关系。此外我们通过分析NUMB、GLI1与NICD表达水平与MM患者临床病理因素的相关性，证实NUMB与MM患者D-S分期相关，且NUMB的下调表达在MM的ISS III期患者中比例较高。随后，我们通过慢病毒转染分别构建了两种人MM细胞株RPMI8226及U266的NUMB过表达细胞株及其对照，NUMB下调表达细胞株及其对照的稳转细胞株。Western blot 实验证实在MM细胞中NUMB的过表达抑制Hh信号通路及Notch信号通路的表达，同时NUMB的下调表达促进Hh信号通路及Notch信号通路的表达。细胞功能实验则证实NUMB的过表达会抑制MM细胞的增殖，促进其凋亡，并抑制肿瘤干细胞成分（克隆集落形成实验及流式细胞术检测的侧群细胞比例），同时NUMB的下调表达会观察到与其相反的生物学效应。我们也证实NUMB的过表达增加MM细胞对一线化疗药物硼替佐米的药物敏感性，同时NUMB的下调增加了硼替佐米的耐药性。我们的研究首次证实了NUMB蛋白在多发性骨髓瘤中的表达意义，它的下调与MM分期和疾病进展密切相关。由于NUMB的下调激活MM的肿瘤干细胞相关信号通路，所以对MM肿瘤干细胞和化疗药物耐药性的意义重大，为MM的耐药和复发机制形成了一种新的解释。我们的研究为后续过表达NUMB蛋白MM的DNA相关药物、mRNA相关药物的研发提供了研究基础。