ER-α66及其变异体ER-α36在乳腺癌中共表达增强Tamoxifen抵抗的分子机制研究

ER-α66 and its variant ER-α36 are recently found to be co-expressed in breast cancer tissues and increase tamoxifen resistance which mechanism is not clear.It has been demonstrated that ER-α66 or ER-α36 alone is invovled in estogen non genomic activity and tamoxifen resistance through cross-talk with HER2 signalling in ER-α66 postive or ER-α36 positive breast cancer cells.Our preliminary data showed that ER-α66 and ER-α36 could be co-located on cytoplasm and membrane of ER-α66 and ER-α36 co-expressing breast cancer cells and enhanced estogen or tamoxifen-mediated phosphorylation of MAPK and AKt and tamoxifen-stimulated cell proliferation. It is deduced that tamoxifen resistance in ER-α66 and ER-α36 co-expressing breast cancer cells can be enhanced through ER-α66 or ER-α36-mediated non genomic activity respectively. The aim of our project is to study the mechanism of enhanced tamoxifen ressitance in ER-α66 and ER-α36 co-expressing breast cancer cells. The study results will provide a new mechanism for study on tamoxifen resistance in clinic, determine the role of ER-α36 as an important marker for tamoxifen therapy in ER-α66 postive breast cancer and provide a new theoretical basis on the strategy of combined therapy based on tamoxifen for breast cancer patients.

最近研究发现ER-α66及其新的异构体ER-α36可在乳腺癌组织中共表达增强Tamoxifen(Tam)抵抗，但其机制不清。由于在单独的ER-α66+或ER-α36+乳腺癌细胞中二者均可通过与HER2的"串话"介导E2非基因组活性并参与Tam抵抗，我们的预实验也显示ER-α66和ER-α36可共定位于乳腺癌细胞浆/膜，并增强E2/Tam介导的MAPK的磷酸化和Tam刺激的细胞增殖，故推测两种受体在乳腺癌细胞中共表达可通过各自介导的非基因组活性增强Tam抵抗。本项目拟采用Western blot、Confocal、CoIP等技术研究二者在乳腺癌中共表达增强Tam抵抗的分子机制，项目的实施可为临床Tam抵抗机制的研究提供一个新的重要机制，确定ER-α36作为ER-α66+乳腺癌Tam治疗重要标志物的作用，并为临床乳腺癌病人基于Tam的联合应用治疗策略的实施提供新的理论依据。

Tam是最常见的内分泌治疗一线药物，但临床Tam抵抗十分常见且机制不清。新近研究发现雌激素受体ER-66及其变异体ER-36可在乳腺癌中共表达并增强Tam抵抗，但其分子机制尚不完全清楚。由于ER-66和ER-36均可通过非基因组活性影响对Tam的敏感性，我们推测ER-66和ER-36在乳腺癌中共表达可能通过各自介导的非基因组活性增强对Tam的抵抗。本项目采用筛选和鉴定的稳定转染ER-36的BT474/ER-36、MCF-7/ER-36以及稳定转染ER-66的SKBR3/ER-66乳腺癌细胞及其野生型对照细胞为研究对象，研究了ER-66及其变异体ER-36在乳腺癌中共表达增强Tam抵抗的分子机制。结果发现：ER-66和ER-36在二者共表达的乳腺癌细胞中能与经典的浆膜蛋白caveolin-1 共定位于细胞浆/膜，并且ER-66和ER-36能在细胞浆/膜共定位。在二者共表达的乳腺癌细胞中，E2和Tam诱导的MAPK磷酸化比野生型细胞更强， MAPK/ ERK拮抗剂U0126在两种细胞中均可明显减弱E2和Tam诱导的MAPK磷酸化；ER-66拮抗剂ICI在野生型细胞中具有明显的减弱效应，但在共表达细胞中ICI仅发挥部分减弱效应。用不同剂量的E2处理ER-66和ER-36共表达乳腺癌细胞，采用CoIP检测ER-66和ER-36抗体免疫沉淀物中HER2、Src、Shc的共存情况。结果发现HER2、Src、Shc共存于ER-36，ER-66抗体免疫沉淀物中，E2/Tam处理可增加ER-36/ER-66与HER2、Src、Shc的相互作用。E2/Tam刺激的HER2、Src、ERK磷酸化水平可分别被Src拮抗剂PP2，EGFR拮抗剂AG1478和MAPK/ERK拮抗剂U0126所抑制。Tam刺激的细胞增殖分析发现ER-66和ER-36共表达的乳腺癌细胞中中Tam刺激的细胞增殖比野生型细胞更强。研究结果表明：ER-66和ER-36在乳腺癌中共表达可通过各自介导的非基因组活性增强对Tam的抵抗。该研究结果可为临床Tam抵抗的研究提供一个新的重要机制，并可确定ER-36作为ER-66+乳腺癌Tam治疗的重要标志物的作用，为临床乳腺癌病人基于Tam的联合应用治疗策略的实施提供新的理论依据。