三阴性乳腺癌雌激素受体α信号通路活化及其生物学特性研究

ER-α re-expression in triple negative breast cancer seems a promising method for endocrine therapy. To our knowledge, the clinical trials concerning ER-α re-expression in triple negative breast cancer patients didn’t achieve expected results, and mostly ended in failure. Triple negative breast cancer is a group of cancers with significantly heterogeneity, and can be divided into six subtypes according to the transcriptomic profiles. ER-α re-expression in triple negative breast cancer whether to change the biological behavior and enhance sensitivity to endocrine therapy of these six subtypes are important outstanding issues. In our previous study, we have confirmed that ER-α re-expression can enhance the tamoxifen sensitivity of triple negative breast cancer MSL subtype cell line MDA-MB-231. In this project, we will firstly employ RNA-seq assay to detect the transcriptomic profiles of lesions from primary triple negative breast cancer and its paired ER-α re-expression metastasis. The primary triple negative breast cancers will be subtyped according their transcriptomic profiles by TNBCtype. The candidate genes regulating the ER-α expression will be picked up by comparing differences between the transcriptomic profiles of primary triple negative breast cancer and its paired ER-α re-expression metastasis. Secondly, we will transfect plasmids to express ER-α in the 6 triple negative breast cancer subtypes cell lines. The biologic characteristics and sensitivity to endocrine drugs of the ER-α re-expressed cell lines will be assessed. Finally, the regulating function of candidate genes on ER-α expression will be validated by shRNA silencing and gene over-expression in breast cancer cell lines. qRT-PCR and western blot assays will be used to detect the expression of ESR1 mRNA and ER-α, respectively. The project will reveal the triple negative breast cancer subtypes which prone to metastatic ER-α re-expression and can benefit from to treat with ER-α re-expression. Meanwhile, the new regulation mechanism of ER-α expression might be found. As a result, these will provide experimental basis for precise treatment strategy in ER-α re-expression treatment in triple negative breast cancer patients.

三阴性乳腺癌（TNBC）ER-α再表达为内分泌治疗带来希望，但临床试验未取得预期结果且大多失败。TNBC异质性明显，以全基因表达谱可分为6亚型，ER-α再表达可否改变6亚型生物学特性并增强内分泌治疗敏感性尚不明确。本课题组已证实ER-α再表达可增强TNBC MSL亚型细胞对他莫昔芬敏感性。本项目拟采用RNA-seq技术检测并分析TNBC原发灶及ER-α再表达转移灶的全基因表达谱，将TNBC分型并筛选调控ER-α表达的候选基因；研究ER-α再表达对6个亚型细胞生物学特性的影响及对内分泌治疗的敏感性；通过shRNA沉默及高表达调控ER-α表达的候选基因，研究候选基因对ER-α表达的调控作用。本项目将揭示容易发生转移灶ER-α再表达及适合ER-α再表达治疗的TNBC亚型，发现调控ER-α表达的新基因，为制定 TNBC的ER-α再表达精准治疗策略提供实验基础。

三阴性乳腺癌不仅预后不良，缺乏针对性的治疗外，原发灶与转移灶之间的不一致也给临床治疗带来了困难。据此本研究回顾分析了270例转移性乳腺癌原发灶与转移灶雌激素受体（ER）与孕激素受体（PR）的表达情况，发现晚期乳腺癌患者原发灶与转移灶的ER与PR有较高的转变率，并发现激素受体表达转变与患者预后有关。课题组预实验研究发现ER-α再表达可增强三阴性乳腺癌 MSL亚型MDA-MB-231细胞对他莫昔芬敏感性。但是三阴性乳腺癌异质性明显，ER-α再表达可否改变其他5个亚型的三阴性乳腺癌细胞的生物学特性并增强内分泌治疗敏感性尚不明确。本课题组进一步探讨了ER-α再表达对其他5个亚型三阴性乳腺癌细胞的生物学行为及对内分泌药物敏感性的影响。研究发现LAR型MDA-MB-453细胞及M型CAL-51细胞能成功转染ESR1质粒并建系，但ER-α再表达对MDA-MB-453细胞及CAL-51细胞形态、细胞增殖能力及对他莫昔芬的敏感性无影响。有研究表明去泛素化酶OTUB1能结合ER-α并去泛素化ER-α蛋白。但去泛素化酶OTUB1是否能调控三阴性乳腺癌细胞ER-α蛋白尚未研究。本研究通过si-OTUB1手段下调6个亚型的三阴性乳腺癌细胞的OTUB1蛋白，发现MSL型MDA-MB-231和M型CAL-51三阴性乳腺癌细胞系下调OTUB1蛋白后能够表达ER-α蛋白，同时能够抑制这两株细胞系的增殖能力。进一步研究发现下调OTUB1后，MDA-MB-231及CAL-51细胞系对他莫昔芬的敏感性增加。综上，课题组再次论证了临床上乳腺癌肿瘤的异质性的存在不仅影响患者的预后，还为临床治疗带来困难。并发现MSL型三阴性乳腺癌可能对ER-α 再表达治疗敏感。同时阐述了OTUB1能调控MSL型及M型三阴性乳腺癌中的ER-α蛋白。这为深入研究三阴性乳腺癌ER-α蛋白再表达治疗提供实验基础，并为其治疗提供的新的潜在治疗靶点。