Department of Cell biology, School of Medicine, Jiangsu University, Zhenjiang, China.
Cancer Institute, University of Mississippi Medical Center, Jackson, MS, USA.
Mol Cancer. 2017 Oct 17;16(1):161. doi: 10.1186/s12943-017-0727-3.
The conversion from estrogen-dependent to estrogen-independent state of ER+ breast cancer cells is the key step to promote resistance to endocrine therapies. Although the crucial role of MAPK/ERK signaling pathway in estrogen-independent breast cancer cell growth is well established, the underlying mechanism is not fully understood.
In this study, we profiled lncRNA expression against a focused group of lncRNAs selected from lncRNA database. CRISPR/Cas9 was employed to knockout (KO) linc-RoR in MCF-7 cells, while rescue experiments were carried out to re-express linc-RoR in KO cells. Colony formation and MTT assays were used to examine the role of linc-RoR in estrogen-independent growth and tamoxifen resistance. Western blot and qRT-PCR were used to determine the change of protein and lncRNA levels, respectively. The expression of DUSP7 in clinical specimens was downloaded from Oncomine ( www.oncomine.org ) and the dataset from Kaplan-Meier Plotter ( http://kmplot.com ) was used to analyze the clinical outcomes in relation to DUSP7.
We identified that linc-RoR functions as an onco-lncRNA to promote estrogen-independent growth of ER+ breast cancer. Under estrogen deprivation, linc-RoR causes the upregulation of phosphorylated MAPK/ERK pathway which in turn activates ER signaling. Knockout of linc-RoR abrogates estrogen deprivation-induced ERK activation as well as ER phosphorylation, whereas re-expression of linc-RoR restores all above phenotypes. Moreover, we show that the ERK-specific phosphatase Dual Specificity Phosphatase 7 (DUSP7), also known as MKP-X, is involved in linc-RoR KO-induced repression of MAPK/ERK signaling. Interestingly, linc-RoR KO increases the protein stability of DUSP7, resulting in repression of ERK phosphorylation. Clinical data analysis reveal that DUSP7 expression is lower in ER+ breast cancer samples than that in ER- breast cancer. Moreover, downregulation of DUSP7 expression is associated with poor patient survival.
Taken together, these results suggest that linc-RoR promotes estrogen-independent growth and activation of MAPK/ERK pathway of breast cancer cells by regulating the ERK-specific phosphatase DUSP7. Thus, this study might help not only in establishing a role for linc-RoR in estrogen-independent and tamoxifen resistance of ER+ breast cancer, but also suggesting a link between linc-RoR and MAPK/ERK pathway.
从雌激素依赖性到雌激素非依赖性状态的 ER+乳腺癌细胞的转化是促进内分泌治疗耐药的关键步骤。尽管 MAPK/ERK 信号通路在雌激素非依赖性乳腺癌细胞生长中的关键作用已得到充分证实，但其中的机制尚不完全清楚。
在这项研究中，我们针对一组从 lncRNA 数据库中选择的 lncRNA 进行了 lncRNA 表达谱分析。CRISPR/Cas9 被用于敲除 MCF-7 细胞中的 linc-RoR，而在 KO 细胞中重新表达 linc-RoR 则进行了挽救实验。集落形成和 MTT 测定用于检测 linc-RoR 在雌激素非依赖性生长和他莫昔芬耐药中的作用。Western blot 和 qRT-PCR 分别用于确定蛋白和 lncRNA 水平的变化。临床标本中 DUSP7 的表达从 Oncomine（www.oncomine.org）下载，Kaplan-Meier Plotter（http://kmplot.com）中的数据集用于分析 DUSP7 与临床结局的关系。
我们发现 linc-RoR 作为一种致癌 lncRNA，可促进 ER+乳腺癌的雌激素非依赖性生长。在雌激素剥夺下，linc-RoR 导致磷酸化 MAPK/ERK 通路的上调，进而激活 ER 信号。敲除 linc-RoR 可阻断雌激素剥夺诱导的 ERK 激活和 ER 磷酸化，而重新表达 linc-RoR 则恢复了所有上述表型。此外，我们表明 ERK 特异性磷酸酶 Dual Specificity Phosphatase 7（DUSP7），也称为 MKP-X，参与了 linc-RoR KO 诱导的 MAPK/ERK 信号抑制。有趣的是，linc-RoR KO 增加了 DUSP7 的蛋白稳定性，从而抑制了 ERK 磷酸化。临床数据分析显示，与 ER-乳腺癌相比，DUSP7 在 ER+乳腺癌样本中的表达水平较低。此外，DUSP7 表达下调与患者生存不良相关。
综上所述，这些结果表明，linc-RoR 通过调节 ERK 特异性磷酸酶 DUSP7 促进乳腺癌细胞的雌激素非依赖性生长和 MAPK/ERK 通路的激活。因此，本研究不仅有助于确定 linc-RoR 在 ER+乳腺癌的雌激素非依赖性和他莫昔芬耐药中的作用，还表明 linc-RoR 与 MAPK/ERK 通路之间存在联系。
