新型癌基因 COPS3 通过自噬与 Beclin1 和 Raf-1 相互作用调节骨肉瘤的转移。
Novel oncogene COPS3 interacts with Beclin1 and Raf-1 to regulate metastasis of osteosarcoma through autophagy.
机构信息
Musculoskeletal Tumor Center, Peking University People's Hospital, No. 11 Xizhimen South Street, Beijing, 100044, China.
Beijing Key Laboratory of Musculoskeletal Tumor, Beijing, 100044, China.
出版信息
J Exp Clin Cancer Res. 2018 Jul 3;37(1):135. doi: 10.1186/s13046-018-0791-6.
BACKGROUND
Expression of COP9 signalosome subunit 3 (COPS3), an oncogene overexpressed in osteosarcoma, has been demonstrated to be significantly correlated with tumor metastasis. However, the underlying mechanism by which COPS3 promotes metastasis of osteosarcoma and its role in autophagy remain unknown.
METHODS
The expression of COPS3 was detected in primary osteosarcoma tissues and matching lung metastasis tissues by immunohistochemistry (IHC). The effect of COPS3 on the metastasis of osteosarcoma cells was investigated by transwell, wound healing assays and animal studies. Indicated proteins was analyzed by western blotting when COPS3 was knockdown or overexpressed. The COPS3 Interacting protein was determined by immunoprecipitation assay. The relationship between COPS3 and autophagy was detected by western blotting and immunofluorescence.
RESULTS
We found that knockdown of COPS3 significantly reduced the lung metastasis of osteosarcoma cells in a mouse model, coinciding with downregulation of mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling. The silencing of COPS3 also inhibited the epithelial-mesenchymal transition (EMT) through the 90 kDa ribosomal S6 kinases (RSK), a family of signal transduction proteins downstream of MEK/ERK. Reciprocal immunoprecipitation assays revealed that COPS3 directly interacts with Raf-1, an upstream regulator of MEK/ERK. Surprisingly, Beclin1, an important autophagic protein, appeared in the COPS3-immunoprecipitates, along with the autophagic markers LC3-I and LC3-II. Loss of COPS3 completely inhibited HO-induced autophagic flux and reduced Beclin1 expression. Additionally, autophagy inhibitor or silencing of Beclin1 both decreased cell metastasis.
CONCLUSIONS
Taken together, these data reveal a novel function of COPS3 in the regulation of autophagy and highlight the relationship between autophagy and metastasis in osteosarcoma cells.
背景
在骨肉瘤中过表达的癌基因 COP9 信号osome 亚基 3（COPS3）的表达已被证明与肿瘤转移显著相关。然而，COPS3 促进骨肉瘤转移的潜在机制及其在自噬中的作用尚不清楚。
方法
通过免疫组织化学（IHC）检测原发性骨肉瘤组织和匹配的肺转移组织中 COPS3 的表达。通过 Transwell、划痕愈合实验和动物研究研究 COPS3 对骨肉瘤细胞转移的影响。当 COPS3 被敲低或过表达时，通过 Western blot 分析指示蛋白。通过免疫沉淀实验确定 COPS3 相互作用蛋白。通过 Western blot 和免疫荧光检测 COPS3 与自噬的关系。
结果
我们发现，COPS3 的敲低显著降低了骨肉瘤细胞在小鼠模型中的肺转移，同时下调丝裂原活化蛋白激酶（MEK）和细胞外信号调节激酶（ERK）信号。COPS3 的沉默也通过 MEK/ERK 下游的信号转导蛋白家族 90kDa 核糖体 S6 激酶（RSK）抑制上皮-间充质转化（EMT）。相互免疫沉淀实验显示，COPS3 与 Raf-1 直接相互作用，Raf-1 是 MEK/ERK 的上游调节因子。令人惊讶的是，Beclin1，一种重要的自噬蛋白，与自噬标记物 LC3-I 和 LC3-II 一起出现在 COPS3-免疫沉淀物中。COPS3 的缺失完全抑制了 HO 诱导的自噬流并降低了 Beclin1 的表达。此外，自噬抑制剂或 Beclin1 的沉默均降低了细胞转移。
结论
综上所述，这些数据揭示了 COPS3 在自噬调节中的新功能，并强调了自噬与骨肉瘤细胞转移之间的关系。
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