接头蛋白P62通过RANKL-RANK-NF-κb信号调控骨巨细胞瘤增殖及侵袭的机制研究

Giant cell tumor of bone (GCTB) is a locally invasive bone neoplasm which characterized by osteolytic destruction. The main treatment is surgery aiming for as near complete removal of the tumor as possible without loss of major morbidity. But local recurrence is reported to occur in between 16.7%-39% of cases within 2 years. 1%-3.2% of cases even develop to sarcoma or distant metastasis. In last 3 years, a fully human, monoclonal antibody against RANKL (Receptor activator of nuclear factor B) —Denosumab was approved for the treatment of unresectable GCTB. The results were fantastic, but new trouble also arose, such as rapid recurrence after drug withdrawal, sarcoma transformation and pathological fracture, of which mechanism was not clear. P62 (SQSTM1，A170) protein is an adaptor protein related to bone metabolism. Previous research revealed over-expression of P62 in GCTB tissues, which correlated to RANKL-RANK expression of upstream signaling, and prompted NF-κb activation of downstream signaling. P62 knockdown could decrease the invasion and migration ability of tumor. So we speculate P62 protein may play an adaptor role between RANKL-RANK and NF-κb signaling, which regulates proliferation and invasion of GCTB. Next, we filter GCTB spindle-like stromal cells to detect proliferation level by Cell Calculation, CCK8 and Cell Cycle assay, and invasion level by Transwell migration, Matrigel invasive, Osteoclast induction and Sclerite invasion assay. Secondly, we upregulate or downregulate RANKL and P62 expression by plasmid transfection of GCTB spindle-like stromal cells, and check the change of tumor proliferation or invasion. Meanwhile we analyze activation of NF-κb by Electrophoretic mobility shift assay (EMSA) and immune-fluorescence microscopy. Totally, we judge whether P62 protein regulate invasion of GCTB by RANKL-RANK-NF-κb signaling. Finally, we implant P62-transfected or control GCTB cell in chick embryo chorioallantoic membrane tissue, and inject to femoral marrow cavity of nude mice. After different concentration of Denosumab treated, we calculate tumor size and bone mineral density by Micro-CT imaging, and detect the P62 and other related protein expression in tumor tissue of nude mice model, evaluating whether P62 protein serves as a prognosis factor of Denosumab treatment, which guides tailored medication for GCTB. We also debate whether inhibition of RANKL and P62 could be a new strategy for GCTB target treatment.

骨巨细胞瘤是一种以溶骨破坏为特征的侵袭性骨肿瘤，术后易复发。近年来，RANKL单抗狄诺塞麦疗效好，但出现停药后迅速复发、恶变等新问题，机制不明。P62蛋白是一种骨代谢相关蛋白，我们发现P62蛋白在骨巨细胞瘤组织中过表达，与上游RANKL及RANK信号相关，下游激活NF-κb，据此推测P62蛋白在RANKL-RANK-NF-κb信号中发挥接头作用，调控骨巨细胞瘤增殖及侵袭。下一步，质粒转染上调及下调RANKL、P62表达，观察细胞增殖及侵袭水平变化，并检测下游NF-κb激活情况，判定P62蛋白是否通过RANKL-RANK-NF-κb信号调控骨巨细胞瘤增殖及侵袭。最后，在裸鼠股骨髓腔种植骨巨细胞瘤，Micro-CT检查不同浓度狄诺塞麦处理后肿瘤变化，分析P62等相关蛋白改变，评估P62蛋白能否作为预测狄诺塞麦疗效的指标，指导临床个体化用药，探讨同时抑制RANKL及P62能否成为新的靶向治疗策略

骨巨细胞瘤（GCTB）是一种好发于四肢长骨，并以溶骨破坏为特征的中间型骨肿瘤。单纯手术后复发率高达16.7%-39%，并且2%-5%的患者会发生远处转移。作为治疗骨巨细胞瘤唯一的靶向药物，地舒单抗（Denosumab，狄诺赛麦）通过阻断RANK配体（RANKL）与RANK的结合，抑制破骨细胞分化及激活，同时抑制单核基质细胞增殖，但是下游的机制并不明确。前期研究发现，p62蛋白作为RANKL-RANK信号下游的接头蛋白，其表达水平与GCTB肿瘤临床及病理特征关系密切，本研究主要验证p62蛋白是否通过RANKL-RANK-NF-κb信号通路进行调控。结果显示，p62在GCTB组织中呈高表达，并且与肿瘤的临床预后相关；地舒单抗治疗后，p62蛋白表达下降，下降水平与GCTB复发相关；分离GCTB组织中的单核基质细胞，下调p62蛋白表达能够抑制GCTB肿瘤细胞增殖、侵袭及迁移，并且可以通过NF-κb信号通路进行调控；在细胞及组织水平均证实，下调p62蛋白能够加强地舒单抗的抑瘤作用，同时抑制RANKL及p62表达是一种更加有效的靶向治疗策略。本研究证明p62蛋白可作为预测地舒单抗疗效的指标，并阐明了相关调控机制，提出针对抑制RANKL及p62双靶点治疗GCTB的新策略，为开发针对p62靶点的药物或肿瘤疫苗提供新的理论依据。