线粒体自噬缺陷诱导乳腺癌骨转移前微环境的形成及其分子机制

Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. The formation of pre- metastatic bone niche provide a "nutrition centers" for subsequent breast cancer cell colonization. However, the molecular interactions governing the early events of osteolytic lesion formation are currently unclear. ULK1 is critical for the induction of mitophagy under hypoxia, In our preliminary study, we found that ULK1 protein levels decreased in breast cancer tissues, and the patients with low expression ULK1 are more susceptible to bone metastasis. ERK kinase may be involved in the ULK1 ubiquitin proteasome degradation. And in experimental bone metastasis animal models, ULK1-knockdown tumor-bearing mice had significantly increased numbers of pre-metastatic focal osteolytic lesions compared to WT tumor-bearing mice. Human inflammation antibody array showed increased secretion of specific cytokines in breast cancer cells with ULK1 knockdown. In this proposal, we will further explore mechanisms that phosphorylation of ULK1 by ERK kinase triggers its interaction with SCFβ-TRCP, and subsequent ubiquitination and destruction. We will establish the mitophagy-deficient models, and investigate the loss of mitophagy induces breast cancer pre-metastatic bone lesions by disrupting the balance between osteoclasts and osteoblasts，and confirm the pre-metastatic preparation of secondary sites facilitate subsequent breast cancer cells colonization. We will identify the key specific inflammatory cytokines secreted by mitophagy-deficient cells, and extend the molecular mechanisms that regulate myeloid-derived suppressor cells / macrophage cells differentiation into osteoclasts by these cytokines. We will propose potential therapeutic strategies by using antibodies targeting the key specific inflammatory cytokines alone and /or with inhibitors targeting ERK kinase, and validate their anti-tumor efficacy in mice bone metastasis models. Overall, this study will improve our understanding of mitophagy and provide new strategies for breast cancer bone metastasis treatment.

乳腺癌骨转移是患者治疗失败和死亡的主要原因。骨转移前微环境的形成为转移干预带来新思路，但形成机制有待阐明。ULK1介导了缺氧条件下线粒体自噬的发生。我们发现ULK1在乳腺癌低表达，且低表达的患者易发生骨转移，ERK激酶可能介导了其泛素化蛋白酶体降解；在实验性骨转移模型上，ULK1敲低组的小鼠易形成转移前溶骨性病灶；炎症因子芯片显示ULK1缺失时多种因子分泌增加。本课题拟阐明ERK磷酸化ULK1导致其发生β-TRCP介导的泛素化降解的机制；构建线粒体自噬缺陷的肿瘤模型，探讨其通过破坏破骨细胞与成骨细胞间的平衡形成转移前溶骨性病灶，并明确该病灶促进癌细胞定植于骨；鉴定在其中起关键作用的特异性炎症因子，及其促使骨髓源性抑制细胞/巨噬细胞分化为破骨细胞的分子机制；明确阻断特异性炎症因子的抗体和ERK抑制剂单药或联用对骨转移的影响，从中筛选出最佳用药方案，为乳腺癌骨转移的防治提供新策略。

乳腺癌骨转移是患者治疗失败和死亡的主要原因。本课题重点探讨了ULK1介导的线粒体自噬缺陷在乳腺癌骨转移中的作用与分子机制。本项目取得成果如下：① 阐明线粒体自噬相关基因ULK1在乳腺癌中低表达的分子机制：发现ERK1/2通过磷酸化ULK1，促进ULK1与E3泛素连接酶β-TRCP结合，介导ULK1的K48型泛素化修饰，并促进ULK1发生蛋白酶体途径降解；乳腺癌患者石蜡标本免疫组化结果显示，ULK1与p-ERK存在显著负相关。② 明确线粒体自噬缺陷促进乳腺癌溶骨性病灶形成及骨转移的发生：发现在缺氧条件下，ULK1缺失介导的线粒体自噬缺陷促进乳腺癌细胞的迁移和侵袭能力，并促进小鼠骨转移发生, 且线粒体自噬缺陷组小鼠出现大量溶骨性病灶及TRAP染色阳性的成熟破骨细胞。③ 阐明线粒体自噬缺陷通过激活炎症体促进破骨细胞分化和成熟的分子机制：发现ULK1缺失介导的线粒体自噬缺陷肿瘤细胞的培养基上清显著促进巨噬细胞分化为成熟的破骨细胞；线粒体自噬缺陷通过激活NLRP3炎症小体，促进IL-1及IL-6的释放，促进破骨细胞分化及骨转移的发生。④ 阐明线粒体自噬缺陷诱导NLRP3炎症体异常激活的分子机制：发现线粒体自噬缺陷介导的ROS异常累积显著促进NLRP3炎症体激活；发现ROS通过激活PKC/IκB/NF-κB通路，促进NLRP3等的转录，继而激活NLRP3炎症体；动物实验发现ROS清除剂或NLRP3抑制剂显著抑制线粒体自噬缺陷介导的骨转移。⑤ 证实了干预线粒体自噬缺陷对乳腺癌骨转移的防治作用：发现MEK抑制剂曲美替尼（trametinib）通过抑制ERK1/2激酶活性回复肿瘤细胞的线粒体自噬能力；发现trametinib抑制NLRP3及Cleaved-Caspase1的蛋白水平，降低IL-1β及IL6的分泌，抑制巨噬细胞分化为成熟的破骨细胞；动物实验发现trametinib可上调ULK1表达及线粒体自噬，显著抑制破骨细胞形成及乳腺癌骨转移，并有效延长小鼠生存期。本研究阐明乳腺癌中ULK1低表达的机制，发现ULK1介导的线粒体自噬缺陷是乳腺癌骨转移的重要驱动因素，为线粒体自噬缺陷的干预治疗提供潜在靶点, 为乳腺癌骨转移的防治提供新策略。