BQ788拮抗EDN3抑制GSLC迁移对GBM放疗的辅助作用研究

Glioblastoma multiforme (GBM, WHO grade IV) is the most common and most aggressive type of primary brain tumor in humans. It remains virtually incurable despite extensive surgical excision and post-operative adjuvant radiotherapy and chemotherapy. Glioblastoma stem-like cells (GSLC), capable of initiating and sustaining tumor growth, are responsible for post-treatment tumor recurrence and therapeutic resistance. Although CD133/prominin, a normal neural stem cell (NSC) marker, is not an obligatory marker for GSLC, tumors initiated by CD133+ GSLC often recapitulate the histopathological features of the patient tumors from which the cells were derived, indicating the ability to self-renew and reproduce the cellular heterogeneity found in human GBM tumors. Functional and molecular studies revealed that the slow-growing CD133+ GSLC, derived from patients' primary tumors that are recurrent and had previous treatment, expressed a series of tumor suppressor/quiescence-associated genes but are capable of clonal self-renewal and spontaneous re-entry to the cell cycle to generate highly proliferative CD133- progeny that can populate tumor spheres in cultures and reconstitute a malignant tumor in mouse brain. Otherwise, the slow-growing CD133+ GSLC express both radial glial cell (RGC) and neural crest cell (NCC)-associated genes. And it was observed that GSLC migration was increased by radiation. Based on these indications, we therefore hypothesize that GSLC use their stem-like pathways to self-renew and migrate for escaping surgery operation or radiation followed by regenerating a new tumor on the peripheral area after treatment removal. Targeting their special signaling pathway may be an effective method to neutralize their functions. Recently, we found that endothelin 3 (EDN3), an essential mitogen for NCC development and migration, is highly produced by GSLC. In normal NCC cultures, EDN3/EDNRB signaling prevents the premature differentiation of crest-derived precursors. In vivo, EDN3/EDNRB signaling is required for NCC migration during ENS development. Mutations in EDN3 or EDNRB can lead to the abnormal development of ENS and melanocytes, implying the loss of function in these two genes may interfere with tumor development by EDN3/EDNRB signaling-dependent GSLC. Indeed, blocking EDN3/endothelin receptor B (EDNRB) signaling by EDNRB antagonist (BQ788) or EDN3 small interfering RNA (siRNA), leads to functional impairment of tumor-sphere formation and cell spreading/migration in culture. Since incorporating GSLC-targeted therapies into conventional cancer treatments may have a clinical implication for the prevention of tumor recurrence. Thus, it is necessary to go on clarifying EDN3/EDNRB signaling mechanism in GSLC and determine whether addition of EDNRB antagonist can sensitize radiation therapy in treatment of established GBM tumors in vivo.

胶质母细胞瘤（GBM）占脑肿瘤的20%，即使实施手术和完整的放化疗方案，患者的平均寿命仅14个月，是人类在神经肿瘤领域面临的最大挑战，胶质母细胞瘤干细胞（GSLC）在GBM细胞中虽占很小比例，却是治疗抵抗和肿瘤复发的根源。GSLC的细胞迁移和自我更新复制是其一种重要的生长方式，也是肿瘤躲避手术和放疗，造成GBM复发的原因之一。因GSLC和一般GBM细胞的基因表达差异，造成其介导细胞增殖、迁移的信号通路的差异，对于一般肿瘤细胞的干预方法并不适用于GSLC。项目申请人最近参与发现EDN 3- EDNRB信号通路作用于GSLC分化的初期，维持其干细胞特性。作为新发现的GSLC特有信号通路，对其干预可抑制GSLC迁移、复制。本项目拟进一步研究其作用机制，并初步验证EDNRB拮抗剂BQ788（临床中黑色素瘤治疗药物）作为潜在的肿瘤干细胞治疗药物在GBM中的药用价值及其对GBM放疗疗效的辅助作用。

胶质瘤干细胞（GSLC）是胶质瘤中最为恶性的一种细胞，被认为是胶质瘤放疗抵抗的主要原因。本项目即针对胶质瘤干细胞放疗抵抗的机制做研究，期望通过杀灭胶质瘤干细胞来实现对胶质瘤的放疗增敏作用。本项目研究了一种新药BQ788对GSLC的促进分化，诱导凋亡作用，从而通过BQ788联合放疗增加了对GSLC的杀灭作用。另外，我们还建立筛选出了三株更具有放疗抵抗性的GSLC，并进行放疗抵抗性的鉴定。并从TNFR1/TRADD/NF-κB通路和TRAIL/DR5通路方面进一步研究了GSLC的放疗抵抗性。通过研究发现，筛选出的更具有放疗抵抗性的细胞株干细胞标记物增加。NF-κB的抑制剂PDTC与放疗联合作用可明显增加GSLC的凋亡率。我们还发现，GSLC对TRAIL诱导的凋亡不敏感，通过放疗可以上调TRAIL受体DR5的表达。通过TRAIL与放疗的联合作用，可明显杀灭GSLC。.因为BQ788在黑色素瘤中已经进入临床应用，通过此发现，我们认为BQ788在胶质瘤的治疗中也具有重要的作用。可以通过BQ788与放疗联合增加GSLC对放疗的敏感性。另外，我们还发现另外两种放疗增敏剂：PDTC和TRAIL，这两种药物都经过我们证实可以明显增强放疗对GSLC的杀灭作用。其中TRAIL具有良好的肿瘤特异性，在临床上有很好的应用前景。