Time-course effect of estradiol and ERa17p on Early Gene expression in SKBR3 cells. Homo sapiens

ERα17p is a synthetic peptide corresponding to the sequence P295LMIKRSKKNSLALSLT311 of the estrogen receptor alpha (ERα) and initially synthesized to mimic its calmodulin binding site. ERα17p was subsequently found to elicit estrogenic responses in E2-deprived ERα-positive breast cancer cells, increasing proliferation and E2-dependent gene transcription. Surprisingly, in E2-supplemented media, ERα17p induced apoptosis and modified the actin network, influencing thereby cell motility. Here, we report that ERα17p induces a massive early (3h) transcriptional activity in breast cancer cell lines SKBR3). Remarkably, about 75% of the significantly modified transcripts were also modified by E2, confirming the pro-estrogenic profile of ERα17p. The different ER spectra of the used cell lines allowed us to extract a specific ERα17p signature related to ERα and its variant ERα36. With respect to ERα, the peptide activates nuclear (cell cycle, cell proliferation, nucleic acid and protein synthesis) and extranuclear signaling pathways. In contrast, through ERα36 it exerts inhibitory events on inflammation and cell cycle and inhibition of EGFR signaling. This is the first work reporting ERα36 specific transcriptional effects. The fact that a number ERα17p-induced transcripts is different from those activated by E2 revealed that the apoptosis and actin modifying effects of ERα17p are independent from the ER-related actions of the peptide. Overall design: Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2 (10-6M) or ERa17p in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturer’s instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.

ERα17p是一种合成肽，对应雌激素受体α（estrogen receptor alpha, ERα）的P295LMIKRSKKNSLALSLT311序列，最初合成的目的是模拟其钙调蛋白结合位点。后续研究发现，ERα17p可在剥夺了雌二醇（E2）的ERα阳性乳腺癌细胞中引发雌激素样反应，促进细胞增殖并增强E2依赖的基因转录。令人意外的是，在添加了E2的培养基中，ERα17p反而可诱导细胞凋亡并重塑肌动蛋白网络，进而影响细胞运动能力。
本研究报道，ERα17p可在乳腺癌细胞系SKBR3中诱导大规模的早期（3小时）转录组激活。值得注意的是，约75%的显著差异表达转录本同时可被E2调控，这证实了ERα17p的类雌激素活性。本次实验使用的细胞系具有不同的ER表达谱，借此我们得以提取出与ERα及其变体ERα36相关的特异性ERα17p转录特征。相较于ERα，该肽可激活核内（细胞周期、细胞增殖、核酸与蛋白质合成）及核外信号通路；与之相反，通过ERα36，该肽则可对炎症反应、细胞周期产生抑制作用，并阻断表皮生长因子受体（EGFR）信号通路。本研究首次报道了ERα36特异性的转录调控效应。部分由ERα17p诱导的转录本与E2激活的转录本存在差异，这表明ERα17p所引发的细胞凋亡与肌动蛋白重塑效应，独立于该肽的ER相关调控作用。
整体实验设计：将细胞置于含10%活性炭吸附胎牛血清的培养基中孵育4小时后，再转移至添加了10%活性炭吸附胎牛血清的RPMI 1640培养基中，分别在有/无10^-6 M E2或ERα17p的条件下孵育3小时。按照制造商说明书，使用Nucleospin II柱（德国达姆施塔特马切雷-纳格尔公司）提取总RNA。参考Affymetrix基因芯片表达分析技术手册中的实验流程，使用HGU133A Plus 2芯片对RNA进行标记与杂交，共分析54675条转录本。信号通过Affymetrix微阵列芯片扫描仪进行检测。