受ERα调控的CLC-3氯通道在乳腺癌Tamoxifen耐药中的作用及机制

Endocrine therapy is one of the most effective targeted treatment strategies fro ER-positive breast caner. Tamoxifen (TAM) is the cornerstone of endocrine therapy. However, TAM resistance is the major obstacle in successful treatment of these breast cancer, and the molecular mechanisms underlying these processes are still fully unknown. We have demonstrated that the regulation of CLC-3 chloride channel by ERα in estrogen promoting proliferation of breast cancer cells. The expression of CLC-3 protein in TAM resistant breast cancer cells was decreased and its distribution was abnormal. Therefore, we assume that reducing the sensitivity of breast cancer to TAM attribute to the ERα expression deletion and/or gene mutations lead to the abnormal regulation of CLC-3 protein expression, subcellular distribution, and channel activity. In this project, we will explore 1) elucidae the inhibition effect of TAM on breast cancer cells through the CLC-3 chloride channel pathway which regulated by ERα? 2) Whether the periodicity of the expression, distribution and channel activity of ERα and CLC-3 protein leads to the cyclical specificity of TAM against breast cancer? 3) Whether ERα dynamically regulates the CLC-3 chloride channel, and then participates in the TAM resistance process? 4) Combine with clinical specimens, we will study the expression and heterogeneity of ERα and ClC-3 chloride channel, analyze the correlation with survival rate of breast cancer patients, so as to evaluate the efficacy and recurrence of TAM. This study will expand the mechanism of TAM against breast cancer and provide a new strategy for the individualized treatment of breast cancer.

他莫昔芬（Tamoxifen，TAM）是ERα阳性乳腺癌内分泌治疗的基石，耐药是其临床应用的最大障碍。我们证实：CLC-3氯通道受ERα的调控参与雌激素促乳腺癌细胞增殖；TAM耐药的乳腺癌细胞CLC-3蛋白表达减少且分布异常。因而我们提出由于ERα表达和/或功能缺失使其调控的CLC-3蛋白表达、分布和通道活性异常，从而降低乳腺癌对TAM的敏感性。在本项目中将探讨：1）TAM抗乳腺癌作用是否经ERα调控CLC-3氯通道实现的？2）ERα和CLC-3蛋白表达、分布和通道活性的周期性是否导致TAM抗乳腺癌的周期特异性？3）ERα是否动态调控CLC-3氯通道而参与TAM耐药进程？4）结合临床病理标本和PDX模型研究ERα与ClC-3蛋白表达、通道活性和关键功能结构域，并与患者生存率的进行相关性分析，评估TAM疗效及复发。本研究将拓展TAM抗乳腺癌作用及耐药机制，为乳腺癌个性化治疗提供新的策略。

原发性或继发性Tamoxifen（TAM）耐药是ER阳性乳腺癌患者治疗失败的最常见的原因。本项目着重探讨ER与ClC-3氯通道的表达状态对乳腺癌内分泌治疗疗效的评估作用及其潜在机制。经过4年的努力，按计划完成课题主要研究内容。1）在细胞水平和乳腺癌临床资料来评估ERα和ClC-3氯通道蛋白的表达状态对乳腺癌内分泌治疗疗效的影响。在细胞水平通过上调和/或下调二者的表达，临床病理标本免疫组化评分，构建4种ERα和ClC-3的表达组合。结果显示，ERαH/ClC-3H组内分泌治疗疗效最佳，而ERαL/ClC-3L则预后最差。ERαH/ClC-3L组的预后及TAM治疗效果也比较差。2）ERα和ClC-3周期性功能和表达影响TAM抑制乳腺癌细胞增殖。研究发现乳腺癌T47D细胞ERα在S期表达最低，ClC-3蛋白在S期表达最多，功能在G1期最强， ERα和ClC-3存在蛋白相互作用。3）ERα和ClC-3联合调控TAM抗乳腺癌作用的机制。首先我们发现TAM阻抑细胞周期于G0/G1期的作用和抑制CyclinD1和CDK4的表达作用在在ERαH/ClC-3H组最强。进一步发现胞内pH值影响细胞周期进程，在pH6.8时G0/G1期比例增加，CyclinD1和CDK4显著减少，而弱碱性pH7.6时S期比例增多，CyclinD1和CDK4表达增加；用Nigericin升高细胞内pH，Cariporide降低胞内pH也呈现类似结果；ClC-3在细胞内主要作为H+-Cl-转运体调节细胞内的pH，在ERαH/ClC-3H组细胞内Cl-浓度相对较低，H+浓度较高，TAM作用后能显著降低H+浓度，增加Cl-浓度。那么，ClC-3是如何调节细胞内pH呢？我们使用String蛋白质相互作用数据库，ClC-3与钠氢转运体调节因子（NHE-1）存在相互作用。在ERαH/ClC-3H组NHE-1表达最少，ERαH/ClC-3H组溶酶体功能则最强促进NHE-1蛋白经溶酶体途径的降解。NHE-1与溶酶体Lamp2蛋白之间存在相互作用的关系。由此我们提出ERα和ClC-3的高表达，通过溶酶体途径降解NHE-1，减少H+外排，增强TAM对乳腺癌细胞周期抑制的作用从而达到较好的抗乳腺癌作用。本项目的完成进一步阐明了乳腺癌内分泌治疗耐药的分子机制，为乳腺癌临床个性化治疗和合理评估内分泌治疗疗效提供了可靠的实验依据。