联合阻断核转录因子Gli1活化途径及逆转耐药治疗软骨肉瘤的实验研究

Canonical hedgehog(HH) signaling is characterized by smoothened (SMO)-dependent activation of the transcription factor Gli. Gli1 plays the pivotal role in HH-dependent gene regulation. A growing body of evidence demonstrates aberrant indian hedgehog signaling has been associated with chondrosarcoma development and chemotherapy resistance. We previously showed that targeting Gli1 (using SiGli1) has great impact on cell survival in comparison to the inhibition of SMO (using Cyclopamine); in addition, Gli1 inhibition induced apoptosis as well as autophagy by regulating MAPK and PI3K/mTOR pathways. These indicate that there are crosstalk between canonical Indian hedgehog and non-canonical, oncogene-driven signaling pathways, converging on the activation of Gli1 gene in chondrosarcoma. However, the activation of Gli1 in chondrosarcoma is far from being fully elucidated. It is imperative to acknowledge the role of the complex molecular networks and crosstalk between different components of the tumor microenviroment that can result in the chondrosarcoma development and drug resistance. We plan to apply tissue microarray to identify phospho-proteins correlates with overexpression of Gli1 in chondrosarcoma. SiRNA or small-molecule inhibitors against identified phosphor-proteins of other key pathways combined with HH inhibitors will be investigated in chondrosarcoma cell lines to determine changes in biochemical signaling, cell cycle and cell viability. In vivo antitumor activity was evaluated in chondrosarcoma xenograft model. .Overexpression of Gli1 has been correlated with tumor chemoresistance. Gli1 inhibition may increase the delivery of drugs to chondrosarcoma. The accumulation of intracellular adriamycin will be detected by flow cytometry and the transcripts of MDR and other MDR associated genes will be detected by real time PCR, and the expression of p-gp ,Bcl2, Bcl-xl and Bax will be assayed by using Western blotting. Gli1 knockdown sensitize chondrosarcoma cell to adrimycin revealing a potential strategy to overcome drug resistance in chondrosarcoma.

经典Indian Hedgehog信号通路异常在软骨肉瘤发展和耐药中起到至关重要的作用，Gli是通路的“枢纽”。我们前期实验证实在Gli1水平阻断该通路，对软骨肉瘤细胞增殖的影响显著优于SMO水平阻断；Gli1与MAPK和PI3K/mTOR通路相互交联，促进软骨肉瘤细胞凋亡和自噬。目前软骨肉瘤中哪些重要的非经典通路不依赖于SMO，直接在Gli水平活化Gli1，与经典Hedgehog通路构成相互交联的通路网络发挥生物学效应并不清楚。本课题首先在软骨肉瘤的组织芯片上筛选与Gli1高表达一致的磷酸化蛋白，确定活化Gli1的重要非经典通路。体内和体外实验证实联合阻断通路对软骨肉瘤的治疗价值。软骨肉瘤对化疗不敏感，Gli1高表达与肿瘤细胞耐药相关，本课题研究沉默Gli1是否能逆转阿霉素耐药，并研究其机制。本课题是延伸性研究，为临床联合阻断Gli1活化途径和逆转常规化疗药物耐药提供可靠的实验基础。

软骨肉瘤是继骨肉瘤之后第二位好发的原发恶性骨肿瘤，由于对化疗和放疗不敏感，手术切除目前几乎是治疗软骨肉瘤的唯一有效方法。然而对于手术无法切除或复发、出现远处转移的病人，至今尚无有效控制手段。因此，探索软骨肉瘤发生发展的分子机制具有重要意义，我们前期研究发现 Indian hedgehog（IHH）通路的异常活化与软骨肉瘤的发生显著相关。本课题是在前期研究的基础上，探索影响IHH通路终末转录因子 Gli1 活化的分子机制，并在体外体内验证联合阻断Gli1活化对软骨肉瘤的影响，为临床上软骨肉瘤的靶向治疗提供新靶点及新策略。.本实验通过原代细胞培养探索原代软骨细胞与软骨肉瘤细胞系IHH通路的不同活化状态;免疫共沉淀联合质谱鉴定软骨肉瘤中与 Gli1 相互结合的蛋白，采用蛋白功能区截短实验探索 Gli1的结合肽段;采用 western blot 及免疫荧光探索其影响Gli1 活化的具体机制;转录组测序,CCK8 及流式细胞术研究 Gli1 结合蛋白对肿瘤细胞增殖、凋亡及耐药的影响;免疫组化鉴定软骨肉瘤中 Gli1、Gli1 结合蛋白及相关通路蛋白三者的表达及相关性;通过肿瘤细胞构建异种移植小鼠模型，体内实验验证单独和联合抑制 Gli1 对肿瘤生长的影响。 .结果发现与原代软骨细胞相比，软骨肉瘤细胞对SMO抑制剂不敏感，且 Gli1 的表达不受其调控;软骨肉瘤中 Gli1 可以与 MVP 结合，且与 mTOR,p70 S6K1 形成复 合体，MVP 通过结合 Gli1 的 SUFU binding sites 功能区影响其入核及稳定性;进一步机制验证发现 MVP 可通过激活 mTOR 通路而促进 Gli1 的表达;MVP可参与调控多种肿瘤相关通路，可促进软骨肉瘤细胞增殖及抵抗凋亡，但对肿瘤细胞耐药无明显影响。MVP,Gli1,p-p70s6k1 在软骨肉瘤组织中高表达且三者具有相关性，联合敲除 MVP 及使用 Gli1 抑制剂 GANT61 可显著抑制小鼠肿瘤的生长。 .得出结论：MVP 可通过多种途径影响 Gli1 的表达及稳定性，体内体外实验证实联合抑制MVP及Gli1可显著抑制软骨肉瘤生长，为临床治疗软骨肉瘤提供了新的靶向治疗策略。