Estrogen receptor-α36 is involved in development of acquired tamoxifen resistance via regulating the growth status switch in breast cancer cells
Estrogen receptor-α36 is involved in development of acquired tamoxifen resistance via regulating the growth status switch in breast cancer cells
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DOI：
10.1016/j.molonc.2013.02.001
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发表时间：
2013-06-01
影响因子：
6.6
通讯作者：
Teng, Lisong
中科院分区：
文献类型：
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作者：
Li, Guangliang;Zhang, Jing;Teng, Lisong
获得性他莫昔芬（TAM）耐药限制了TAM在乳腺癌患者中的治疗获益。从雌激素依赖性生长到生长因子依赖性生长的转变是这一过程中的关键步骤。然而，这种开关背后的分子机制仍然知之甚少。在这项研究中，我们建立了一个TAM耐药细胞亚系（MCF-7/TAM）从雌激素受体-α（ER-α 66）阳性乳腺癌MCF-7细胞，培养ER-α 66阳性MCF-7细胞在培养基加1 μ M TAM超过6个月。MCF-7/TAM细胞的增殖速度加快，体外迁移和侵袭能力增强。与亲本MCF-7细胞相比，雌激素受体α 36（ER-α 36），一种新的ER-α 66的36 kDa变体，在这种体外模型中显着过表达。同时，MCF-7/TAM细胞中表皮生长因子受体（EGFR）的mRNA和蛋白表达均显著上调，而ER-α 66的表达则显著下调。在随后的研究中，我们通过稳定转染在MCF-7细胞中过表达ER-α 36，发现ER-α 36转染的MCF-7细胞（MCF-7/ER-α 36）与空载体转染的MCF-7细胞（MCF-7/V）相比，同样表现出对TAM的敏感性降低、增殖速率加快以及体外迁移和侵袭能力增强。实时定量PCR和Western blotting分析显示，与MCF-7/V细胞相比，MCF-7/ER-α 36细胞在mRNA水平和蛋白水平上EGFR表达增加，而ER-α 66表达降低。MCF-7/ER-α 36细胞中的这种变化可以通过中和抗ER-α 36抗体处理来逆转。此外，在MCF-7/TAM细胞中敲低ER-α 36表达导致增殖率降低以及体外迁移和侵袭能力降低。ER-α 36表达下调的MCF-7/TAM细胞中EGFR mRNA和蛋白表达降低，ER-α 66 mRNA表达增加。此外，阻断MCF-7/ER-α 36细胞中EGFR/ERK信号通路可部分恢复ER-α 66的表达，提示EGFR/ERK信号通路在下调ER-α 66表达中具有调节作用。总之，我们的研究结果首次表明ER-α 36在上调EGFR表达和下调ER-α 66表达中的调节作用，这可能是乳腺肿瘤生长状态转换的潜在机制，有助于产生获得性TAM耐药。ER-α 36可能成为TAM耐药乳腺肿瘤治疗的新靶点。(C)2013年欧洲生物化学学会联合会。Elsevier B. V.出版，保留所有权利。
Acquired tamoxifen (TAM) resistance limits the therapeutic benefit of TAM in patients with hormone-dependent breast cancer. The switch from estrogen-dependent to growth factor-dependent growth is a critical step in this process. However, the molecular mechanisms underlying this switch remain poorly understood. In this study, we established a TAM resistant cell sub line (MCF-7/TAM) from estrogen receptor-alpha (ER-alpha 66) positive breast cancer MCF-7 cells by culturing ER-alpha 66-positive MCF-7 cells in medium plus 1 mu M TAM over 6 months. MCF-7/TAM cells were then found to exhibit accelerated proliferation rate together with enhanced in vitro migratory and invasive ability. And the estrogen receptor-alpha 36 (ER-alpha 36), a novel 36-kDa variant of ER-alpha 66, was dramatically overexpressed in this in vitro model, compared to the parental MCF-7 cells. Meanwhile, the expression of epidermal growth factor receptor (EGFR) in MCF-7/TAM cells was significantly up-regulated both in mRNA level and protein level, and the expression of ER-alpha 66 was greatly down-regulated oppositely. In the subsequent studies, we overexpressed ER-alpha 36 in MCF-7 cells by stable transfection and found that ER-alpha 36 transfected MCF-7 cells (MCF-7/ER-alpha 36) similarly exhibited decreased sensitivity to TAM, accelerated proliferative rate and enhanced in vitro migratory and invasive ability, compared to empty vector transfected MCF-7 cells (MCF-7/V). Real-time qPCR and Western blotting analysis revealed that MCF-7/ER-alpha 36 cells possessed increased EGFR expression but decreased ER-alpha 66 expression both in mRNA level and protein level, compared to MCF-7/V cells. This change in MCF-7/ER-alpha 36 cells could be reversed by neutralizing anti-ER-alpha 36 antibody treatment. Furthermore, knock-down of ER-alpha 36 expression in MCF-7/TAM cells resulted in reduced proliferation rate together with decreased in vitro migratory and invasive ability. Decreased EGFR mRNA and protein expression as well as increased ER-alpha 66 mRNA expression were also observed in MCF-7/TAM cells with down-regulated ER-alpha 36 expression. In addition, blocking EGFR/ERK signaling in MCF-7/ER-alpha 36 cells could restore the expression of ER-alpha 66 partly, suggesting a regulatory function of EGFR/ERK signaling in down-regulation of ER-alpha 66 expression. In conclusion, our results indicated for the first time a regulatory role of ER-alpha 36 in up-regulation of EGFR expression and down-regulation of ER-alpha 66 expression, which could be an underlying mechanism for the growth status switch in breast tumors that contribute to the generation of acquired TAM resistance. And ER-alpha 36 could be considered a potential new therapeutic target in breast tumors which have acquired resistance to TAM. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.