Caveolin-1介导乳腺癌干细胞化疗抵抗及干性维持的作用和分子机制研究

Multidrug resistance-induced chemotherapy failure and metastasis are the main reasons leading to breast cancer death. Recent numerous studies demonstrated that cancer stem cells (CSCs) had strong drug resistance ability and enriched expression of ABC transporters. Identification and characterization of critical signalings involved in regulating CSCs’ drug resistance has become a hot direction in improving chemotherapeutic efficacy. Caveolin-1 is the essential constituent protein of specialized membrane invaginations called caveolae. Numerous findings indicated that caveolin-1 acted as a molecular hub integrating multiple molecules implicated in cancer development. However, the significance of caveolin-1 in regulating CSCs and drug resistance is largely remained unknown. Our preliminary study found that caveolin-1 had enriched expression on breast CSCs and was significantly elevated after chemotherapy both in vitro and in vivo. Caveolin-1 silencing could greatly promote drug influx in breast CSCs and enhance their differentiation. Meanwhile, clinical study also demonstrated that caveolin-1 was positively correlated to β-catenin/ABCG2 pathway. Therefore we hypothesized that caveolin-1 could promote breast cancer drug resistance and regulate CSCs fate decision through activating β-catenin/ABCG2 signaling. To confirm the hypothesis, this proposal will include three special objectives: (1) To validate whether caveolin-1 can regulate breast CSCs drug resistance and self-renewal in vitro and in vivo; (2) To test the influences of caveolin-1 on breast CSCs’ differentiation and its role in regulating drug resistance; (3) To explore the molecular interactions and pathways between caveolin-1 and β-catenin/ABCG2 signaling. If the hypothesis can be validated, we will not only provide novel strategies for preventing drug resistance or chemosensitization, but also benefit for improving breast cancer chemotherapeutic efficacy and clinical outcomes.

肿瘤干细胞具有高度耐药性，并富集表达ABC转运蛋白，解析肿瘤干细胞耐药的关键分子及调控通路成为逆转耐药的新思路。Caveolin-1是细胞小窝的主要构成及功能蛋白，其在细胞信号转导及肿瘤发展过程中发挥枢纽作用，但目前其与肿瘤干细胞及耐药调控的关系尚不清楚。我们前期研究发现caveolin-1在乳腺癌干细胞上富集表达，其表达抑制可显著增加干细胞内化疗药物含量并促进其分化，同时与β-catenin/ABCG2信号通路正性相关，由此我们设想caveolin-1可通过β-catenin/ABCG2通路介导乳腺癌干细胞耐药并调控其更新分化能力。本项目拟通过体内外基因重组、流式分选及三维培养等技术探讨caveolin-1调控乳腺癌干细胞耐药的关键作用及其对更新分化能力的影响，并解析caveolin-1调控β-catenin/ABCG2信号通路的分子机制。研究的开展将为耐药预防或化疗增敏提供新策略。

耐药是乳腺癌治疗失败和患者死亡的主要原因，鉴定肿瘤耐药靶标是提高疗效及改善预后的关键环节。Caveolin-1是构成细胞小窝的组分蛋白，近年来有研究报道其在肿瘤耐药细胞中表达上调，而在致癌过程中发挥抑癌基因功能。肿瘤干细胞在肿瘤发生和耐药介导中都发挥关键作用，然而caveolin-1在乳腺癌干细胞中的表达水平及功能调控尚不清楚。本项目首先确定了Caveolin-1对乳腺癌干细胞耐药特性和自我更新的正向调节能力，其上调能增加乳腺癌干细胞群落数量、化疗抵抗能力和体内成瘤能力；同时乳腺癌干细胞分化过程中Caveolin-1表达下降，Caveolin-1下调促进其分化，而其上调抑制其分化。分子机制研究发现caveolin-1通过抑制β-catenin蛋白酶体降解途径激活乳腺癌干细胞的自我更新和化疗抵抗能力。体内研究证明caveolin-1下调能显著增加乳腺癌干细胞化疗敏感性。此部分研究证明caveolin-1在肿瘤干细胞中呈现富集表达状态，但其在正常干细胞中的表达状态及功能调控尚未阐释。通过构建CAV1 KO的乳腺癌自发转基因小鼠我们证实CAV1敲除能显著加速乳腺癌发生发展及导管形成，此与乳腺癌干细胞群落增加密切相关。Caveolin-1在乳腺癌干细胞中的表达水平显著低于正常乳腺干细胞，分子机制研究表明caveolin-1下调可通过阻断VHL介导的c-Myc泛素化降解激活糖酵解，从而加速乳腺癌的发生发展。临床研究亦显示caveolin-1低表达乳腺癌患者总生存期显著缩短，由此我们推论caveolin-1在乳腺癌中的表达遵循正常干细胞>肿瘤干细胞>普通肿瘤细胞，并提出Caveolin-1的双向功能调节模型，Caveolin-1是一种细胞应激保护蛋白，在乳腺癌发生早期，其水平下降可降低细胞的应激保护能力，促使恶性转化发生；而在乳腺癌晚期， Caveolin-1上调可增强细胞的应激保护能力，促使细胞耐药和转移的发生。在此基础上，我们进一步发现黄芪提取物黄芪甲苷可通过靶向抑制caveolin-1激活eNOS/NO/3-NT通路诱导细胞凋亡，增加紫杉醇化疗敏感性，再次验证了caveolin-1的耐药调节作用，并提示其抑癌基因功能可能与细胞内氧化压力密切相关。此项目的开展丰富了caveolin-1肿瘤分子生物学行为及临床意义，对于化疗增敏药物的开发具有一定的积极意义。