β2肾上腺素受体介导CREB/RANKL信号通路在骨肉瘤中的作用及机制研究

Osteosarcoma is the most popular primary malignant bone tumors in adolescent, which also take center stage as our research fields. In our previous research work, we found overexpression of beta2 adrenergic receptor (β2-AR) in osteosarcoma cell lines and specimens. Hence, targeting β2-AR possess significant anti-tumor effect on osteosarcoma. Furthermore, Microarray of phosphorylation proteins demonstrate that phosphorylation level of transcription factors CREB declines significantly accompanying with Down-regulation of expression RANKL after targeting inhibition of β2-AR. According to recent researches, CREB/RANKL signaling pathway is more likely to play a critical role in osteosarcoma. However，the mechanism of β2-AR and its regulation effect on CREB/RANKL signaling pathway which may play a important part in downstream in osteosarcoma is remaining to be revealed. We attempt to utilize several osteosarcoma cell lines and animal models to verify the anti-tumor effect on osteosarcoma currently and then make clear the regulation mechanism of the β2-AR regulating CREB/RANKL signaling pathway through transfection、Crispr/Cas9 and CHIP techniques. The third part, clinical significances of the expression of β2-AR and CREB/RANKL signaling pathway in osteosarcoma remain uncovered according to the clinical data accumulated for many years. In general, we are planning to explore deeply and wholly in context of β2-AR and CREB/RANKL signaling pathway in osteosarcoma about their roles and mechanism and finally we will provide a brand new therapy strategy to cure osteosarcoma.

我们前期发现β2肾上腺素受体(β2AR)在骨肉瘤(OS)细胞及临床标本中过表达，靶向β2AR具有显著抗OS效应，同时β2AR阻滞剂普萘洛尔(PPRL)也有明显抗OS作用。进一步CHIP实验发现靶向抑制β2AR后，转录因子CREB磷酸化水平显著降低，同时伴有RANKL表达下调。最新研究发现CREB/RANKL信号通路(CRSP)在OS中具有重要的功能。然而，β2AR对CRSP的调控在OS中的生物学效应及机制尚未阐明。现拟在前期基础上采用细胞株及动物模型验证靶向β2AR抗OS效应；通过CHIP等方法，明确β2AR对CRSP的调控机制；运用体内外实验模型，验证联合靶向β2AR和RANKL的抗OS效应，尤其是PPRL联合RANKL单抗-狄诺塞麦的抗OS效应；结合临床资料，明确β2AR及CRSP在OS中的临床意义。本项目将加深对β2-AR介导的CRSP在OS中作用及机制的认识，为OS提供新治疗策略。

骨肉瘤是青少年最常见的骨骼系统原发性恶性肿瘤，发病的分子机制尚不明确。传统治疗效果遇到瓶颈，仍有部分患者对目前的化疗方案不敏感，早期的肺转移极大地影响了骨肉瘤患者的五年生存率。因此，本研究意在探索骨肉瘤来的分子发病机制以及治疗靶点，为提高骨肉瘤患者的良好预后提供新的策略以及理论支持。我们前期发现β2肾上腺素受体(β2AR)在骨肉瘤细胞及临床标本中过表达，靶向β2AR具有显著抗骨肉瘤效应，同时β2AR阻滞剂普萘洛尔(PPRL)也有明显抗骨肉瘤作用。并且通过CHIP实验发现靶向抑制β2AR后，转录因子CREB磷酸化水平显著降低，同时伴有RANKL表达下调。最新研究发现CREB/RANKL信号通路(CRSP)在骨肉瘤中具有重要的功能。我们采用磷酸化蛋白芯片筛查并验证β2-AR靶向抑制剂ICI118,551处理后U2OS以及143B细胞内磷酸化蛋白水平的变化，发现胞内p-CREB变化最为显著，p-CREB表达水平随着ICI118,551浓度增高而逐渐下降，凋亡标志物cleaved-parp逐渐升高，采用CREB特异性抑制剂KG501抑制该分子活性或者通过siRNA敲低CREB表达均可显著抑制骨肉瘤细胞的增殖能力。为了研究传统化疗药联合靶向β2-肾上腺素受体抑制剂抗骨肉瘤的效果，联用ADM与普萘洛尔的体内、外实验均表明，β2-肾上腺素受体抑制剂也能得到类似ADM抗骨肉瘤效果，并且相对ADM产生更少的副作用，在ADM与β2-肾上腺素受体抑制剂联用组，更能抑制骨肉瘤肿瘤的生长，减少其肺转移的发生。免疫组化结果结合临床资料分析，我们发现β2-肾上腺素受体与骨肉瘤患者的不良预后相关，明确了β2-肾上腺素受体在骨肉瘤发生发展中的重要作用，并且阐明了β2-肾上腺素受体通过影响CREB的磷酸化水平，从而影响下游靶基因参与的对骨肉瘤调控作用。本研究为β2-肾上腺素受体治疗骨肉瘤提供了理论依据，为骨肉瘤提供了新的治疗策略。