DT-13基于脂肪微环境胶原HLCCs/LCCs交联构型的抗乳腺癌转移机制研究

Collagen is the key component of extracellular matrix(ECM), recently, researches showed that collagen fibers formed covalent intermolecular cross-links and aligned like 'bundle' after post-translational modifications, further to pave a 'Highway' for cancer cell metastasis. PLOD2 catalysis the hydroxylation of collagen lysine residues as a post-translational event in collagen biosynthesis, and the latter forms hydroxylation aldehyde-derived collagen cross-links (HLCCs) which is much more stable than lysine aldehyde-derived cross-links (LCCs). The HLCCs/LCCs collagen configuration is the potential factor for cancer metastasis. Saponin monomer 13 of dwarf lilyturf tuber (DT-13), one of the saponin monomers of dwarf lilyturf tuber is isolated from Ophiopogon japonicus (Thunb.). Previous work confirmed that DT-13 is an active monomer that has anti-metastatic role (articles have been published, the patent has been authorized). Recently, we found DT-13 could also inhibit the hydroxylation modifications of collagen in adipocyte-breast cancer microenvironment. In this project, we are going to investigate the key component of ECM, collagen and explore the effect of collagen HLCCs/LCCs configuration on breast cancer metastasis based on adipocyte-breast cancer microenvironment at molecular, cellular, animal and clinical levels. Meanwhile, we intended to comprehensively evaluate DT-13 anti-metastasis effect on the theoretical basis of adipocyte-breast cancer microenvironment. Overall, clarifying the mechanism of DT-13 anti-metastatic effect of breast cancer metastasis has extremely significant for the research and development of DT-13.

胶原是细胞外基质最主要成分，近年研究发现胶原蛋白修饰后共价交联，呈束状排列，为肿瘤转移铺建了一条“高速公路”。PLOD2羟基化修饰胶原赖氨酸残基，后者形成HLCCs交联构型比未羟基化修饰的LCCs构型更稳定，HLCCs/LCCs构型的比例是影响肿瘤转移的潜在因素。DT-13是从短葶山麦冬中分离得到的单体皂苷，申请者前期大量工作证实DT-13是一个具有抗乳腺癌转移作用的中药活性单体。近期研究发现，DT-13对脂肪微环境中胶原羟基化修饰相关基因有一定抑制作用。本课题拟在分子、细胞、整体动物及临床样本水平，从脂肪-乳腺癌微环境入手，以细胞外基质主要成分胶原为研究对象，探讨微环境中胶原HLCCs/LCCs构型对乳腺癌转移的影响；并在该理论基础上从脂肪-乳腺癌微环境角度全面评价DT-13抗乳腺癌转移作用，阐明DT-13抗乳腺癌转移机制，对DT-13的研究、开发都具有十分重要意义。

胶原是细胞外基质最主要成分，近年研究发现赖氨酸羟化酶（PLOD2）羟基化修饰胶原形成共价交联，使胶原呈束状排列，为肿瘤转移铺建一条“高速公路”。DT-13是从短葶山麦冬中分离得到的皂苷单体。申请者前期研究发现，DT-13对脂肪微环境胶原羟基化修饰相关基因有一定调控作用。本课题在细胞、分子、动物和临床标本四个层次，从乳腺癌最主要的间质细胞—脂肪细胞入手，探讨微环境中胶原排列构型对乳腺癌转移的影响；并在该理论基础上从脂肪-乳腺癌微环境角度全面探讨DT-13抗乳腺癌转移机制。本项目研究发现：一方面，脂肪-乳腺癌细胞共培养促进脂肪细胞去分化，使脂肪细胞体积变小、细胞内脂滴数量减少，部分细胞形态发生“纤维样”改变；而脂肪-乳腺癌微环境通过促进CAAs细胞高表达PLOD2，介导CAAs来源的胶原排列发生线型重构；体内脂肪-乳腺癌细胞共注射模型可以促进乳腺癌转移，而PLOD2抑制剂能够抑制脂肪-乳腺癌微环境介导的脂肪细胞胶原线型排列，并抑制乳腺癌发生肺转移；机制研究发现：乳腺癌细胞来源的PAI-1通过激活脂肪细胞中PI3K/AKT信号通路，并促进转录因子FOXP1与PLOD2启动子区结合，介导脂肪细胞PLOD2高表达和脂肪来源胶原线型重构；另一方面，从脂肪微环境对乳腺癌PLOD2作用的角度研究，发现脂肪细胞分泌的leptin和IL-6促进乳腺癌细胞高表达PLOD2，从而介导乳腺癌细胞发生EMT，直接增强乳腺癌细胞的转移能力；机制研究发现：leptin和IL-6通过激活乳腺癌细胞中PI3K/AKT和JAK/STAT3通路，促进PLOD2表达升高，进一步在临床乳腺癌标本中证实了PLOD2与乳腺癌的不良预后和恶性进展相关； DT-13可以直接抑制乳腺癌细胞PLOD2表达，也可以通过抑制脂肪-乳腺癌微环境中IL-6和leptin介导PLOD2表达而发挥抗乳腺癌转移作用。本项目研究阐述了脂肪-乳腺癌微环境相互作用，既可以通过激活脂肪细胞PLOD2表达介导了脂肪微环境的胶原构型重排，从而发挥间接促乳腺癌转移作用；也可以激活乳腺癌细胞自身PLOD2水平发挥直接促乳腺癌转移作用；而DT-13则通过抑制乳腺癌细胞PLOD2表达，发挥抑制脂肪促乳腺癌转移作用。