肿瘤相关成纤维细胞经自噬促进乳腺癌侵袭转移的作用机制研究

Invasion and metastasis of breast cancer has always been one of the main obstacles which troubled therapies for the breast cancer to be successfully used in clinic. It is proved that the epithelial-interstitial transformation induced by tumor stromal cells provides necessary conditions for invasion and metastasis of breast cancer. How to block the EMT process mediated by tumor stroma cells should be the major challenge of current cancer therapy and prevention. Relevant research indicated that carcinoma-associated fibroblasts (CAFs) were closely related to the EMT process of breast cancer cells in the tumor microenvironment, which would be one of the key factors for invasion and metastasis of breast cancer. Accordingly, the project is focused on the effects of the formation and autophagy of CAFs in the EMT process of breast cancer cells to explore the mechanisms of tumor invasion and metastasis induced by CAFs autophagy. Furthermore, based on the superiority of CAFs as a target for tumor therapy and its key role to promote the EMT process of breast cancer cells in the tumor microenvironment, the measures to selectively inhibit the formation of CAFs and block the autophagy of CAFs will be evaluated referring to previous studies on TGF-β1-induced autophagy promoted the formation of CAFs phenotype in tumor microenvironment of breast cancer. The prospective result is to provide a new strategy to promote anti-tumor response.

乳腺癌侵袭转移始终是困扰乳腺癌治疗成功用于临床的主要障碍。肿瘤间质细胞介导的上皮-间质转化（EMT）为乳腺癌的侵袭和转移提供了重要支撑。如何阻断肿瘤间质细胞介导的EMT过程是现今肿瘤治疗领域的难题与挑战。研究表明，存在于肿瘤微环境的肿瘤相关成纤维细胞（CAFs）与乳腺癌细胞发生EMT的过程密切相关，是导致乳腺癌侵袭转移的关键因素之一。据此，本课题拟通过重点考察肿瘤微环境中CAFs形成和CAFs自噬对肿瘤细胞发生EMT过程的影响，深入解析其介导乳腺癌侵袭转移的内在机制；并基于CAFs在乳腺癌细胞发生EMT过程中的重要作用以及CAFs突出的靶标优势；借鉴课题组前期有关TGF-β1诱导自噬促进肿瘤微环境CAFs形成的研究基础，进一步探讨特异性抑制CAFs形成和CAFs自噬的措施，并确证其抑制乳腺癌细胞发生EMT导致阻断肿瘤侵袭转移的有效性与可行性，以期为提高乳腺癌治疗效果提出新的策略。

肿瘤相关成纤维细胞(CAFs)在乳腺癌的发生、转移和侵袭中发挥着重要作用。然而，自噬是否通过诱导上皮间质转化(epithelial-mesenchymal transition, EMT)作为肿瘤的促进机制仍存在争议，其机制尚不明确。在本研究中，我们研究了自噬或FAP-α是否在乳腺癌细胞侵袭、肺转移和EMT中是必需的，以及其潜在的机制。我们采用H2O2处理NIH3T3成纤维细胞的体外模型，证实在肿瘤微环境氧化应激下，TGF-β1可通过自噬将成纤维细胞转化为CAFs。通过雷帕霉素、3-甲基腺嘌呤或Atg5下调自噬调节CAFs标记物的表达，提示自噬参与TGF-β1诱导的CAFs激活的保护机制。在此基础上，我们建立了间接共培养模型和相应的体内培养模型。结果发现，TGF-β1激活的CAFs可促进肿瘤侵袭、肺转移和EMT，这两种模型均通过自噬和FAP-α的过表达发挥作用，而自噬抑制剂3-甲基腺嘌呤可阻断TGF-β1激活的CAFs诱导的这些作用。此外，LC3β和EMT标记物vimentin在混合异种移植中共同定位也表明TGF-β1激活的CAFs部分通过自噬促进肿瘤生长、肺转移和EMT程序。此外，敲低FAP-α可逆转EMT，抑制TGF-β1激活的CAFs诱导的肿瘤侵袭和肺转移。以上结果说明自噬和FAP-α都是乳腺癌细胞侵袭和转移所必需的。靶向自噬或FAP-α都可以作为改善人类乳腺癌预后的潜在途径。总之，本课题研究从分子生物学水平探讨自噬和CAFs标志蛋白FAP-α对乳腺癌侵袭转移通路的干预机制，为临床安全用药提供实验依据。