肿瘤微环境中胞外BAG3对胰腺导管腺癌（PDAC）进展的影响及其机制研究

BAG3 is highly expressed in many tumors including PDAC, and its expression has been reported to be closely associated with prognosis of patients with PDAC. Very recently it has been reported that BAG3 and its antibody is detectable in serum of patients with PDAC, indicating that in some circumstance BAG3 can be released from cytoplasm, where it is generally located. The extracellular function of BAG3 is completely unknown till now. For the first time, we found that extracellular BAG3 promoted proliferation, increased phosphorylation of p38 MAPK and nuclear translocation of NF-kB, as well as promoted secretion of some cytokines, including GM-CSF, GM-CSF, IL-4, MCP-1, MCSF, SDF-1, and IGFBP3 in PDAC cells. We also screened the potential interacting partner of extracellular BAG3 and found that recombinant BAG3 coimmunoprecipitated a protein band located between 37kD and 52 kD (simply noted as p37-52) from plasma membrane proteins of PDAC cells. Conditional medium from PDAC incubated with extracellular BAG3 promoted macrophage recruitment, but decreased phagocytosis of macrophages. In addition, we also found that the tumor microenvironment containing extracellular BAG3, macrophage and PDAC induced EMT of PDAC. Therefore, we supposed that extracellular BAG3 might activate intracellular p38 MAPK and NF-kB signal pathways via interaction with membrane surface p37-52, and then promote secretion of some cytokines, by which recruit and remold macrophages. Recruited and remolded macrophages further feedback PDAC to induce its EMT and affect its progression. The current project aims to verify the scientific hypothesis, which might provide potential tumor microenvironmental target for PDAC therapy and/or prevention.

BAG3在胰腺导管腺癌（PDAC）中高表达且与预后密切相关。最新报道PDAC患者血清中检测到BAG3，但其胞外功能不清。我们最近首次发现胞外BAG3与PDAC细胞膜蛋白(暂命名p37-52)结合，激活p38 MAPK和NF-kB信号通路，促进PDAC增殖及某些细胞因子分泌。胞外BAG3孵育PDAC的条件培养基促进巨噬细胞募集，抑制其吞噬功能。胞外BAG3、PDAC和巨噬细胞组成的肿瘤微环境诱导PDAC发生上皮细胞间质转分化（EMT）。因此，我们推测胞外BAG3通过与PDAC表面分子p37-52结合，激活p38和NF-kB信号通路，促进PDAC分泌某些细胞因子，进而招募巨噬细胞并对其进行改造；而被改造的巨噬细胞进一步反馈作用于PDAC，诱导其发生EMT，最终影响PDAC的进展。本项目旨在证实该科学假说，以期为进一步探索如何利用微环境中相关靶点预防及诊治PDAC提供可能的研究方向。

Bcl-2相关永生化（Bcl-2 associated athanogene, BAG）蛋白是一类进化保守的抗凋亡蛋白家族，BAG3是BAG家族的重要成员。BAG3参与许多生命活动的调节如凋亡、氧化应激、自噬等。与健康分化细胞的有氧代谢不同，恶性肿瘤细胞在有氧情况下依旧依赖糖酵解将葡萄糖转化为乳酸进而用来满足旺盛的生物合成需求；而这一现象也被称为有氧糖酵解，即“瓦博格效应”。本项目证实BAG3对胰腺癌增殖和葡萄糖代谢存在明确的调节作用，并且发现己糖激酶HK2的表达随BAG3的下调而下调；在胰腺导管腺癌中BAG3呈高表达，且BAG3的表达与患者的不良预后密切相关。在胰腺癌导管腺癌细胞BxPC3、SW1990两种细胞中，敲低BAG3后胰腺导管腺癌细胞的有氧糖酵解被削弱（线粒体呼吸的增强以及糖酵解的削弱，与此同时两种细胞的葡萄糖消耗及乳酸生成量均有所减少）；而过表达BAG3能够增强胰腺导管腺癌BxPC3、SW1990中的有氧糖酵解，促进肿瘤细胞的快速增殖。我们进一步证实BAG3通过作用于HK2 mRNA 的3/UTR并调节其稳定性，进而影响胰腺导管腺癌细胞的糖酵解水平；而且HK2 mRNA稳定性的调节与miRNA无关。HK2 3/UTR 2077-2083位点存在一段特殊的持续降解元件（CDE）共有序列（UCYRYGA），而此前有报道RNA结合蛋白Roquin可以与含有CDE元件的mRNA相结合并介导mRNA的降解，利用RIP等实验手段证实BAG3可以与Roquin竞争性结合HK2 mRNA的CDE区；敲低BAG3后，Roquine募集至HK2 mRNA加速了HK2 mRNA的降解。我们还证实BAG3与RNA结合蛋白IMP3存在内源性的直接结合，能够协同IMP3稳定HK2 mRNA从而在转录后水平调节HK2的表达进而促进肿瘤细胞的有氧糖酵解和增殖。以上结果提示BAG3作为胰腺导管腺癌诊疗新靶点的可能性。