NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications.
NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications.
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DOI：
10.1186/bcr2900
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发表时间：
2011-06-14
期刊：
影响因子：
--
通讯作者：
Osipo C
中科院分区：
文献类型：
--
作者：
Clementz AG;Rogowski A;Pandya K;Miele L;Osipo C
患有三阴性乳腺癌的女性预后最差，经常出现转移性肿瘤，并且几乎没有靶向治疗选择。Notch-1和Notch-4是在三阴性和其他亚型乳腺癌中过表达的强效乳腺癌基因。PEA 3是一种ETS转录因子，在三阴性和其他乳腺癌亚型中也过表达。我们研究了PEA 3是否可能是MDA-MB-231和其他乳腺癌细胞中Notch受体的关键转录激活因子。采用Real-time PCR和Western blot方法检测siRNA敲除PEA 3后乳腺癌细胞中Notch-1、Notch-2、Notch-3和Notch-4受体的表达。进行染色质免疫沉淀以鉴定募集PEA 3的Notch基因的启动子区域。使用TAM-67和c-Jun siRNA来鉴定c-Jun是Notch-4启动子上PEA 3富集所必需的。使用Notch-4荧光素酶报告基因来确认内源PEA 3或AP-1激活Notch-4启动子区。进行细胞周期分析、台盼蓝排除、膜联蛋白V流式细胞术、集落形成测定和体内异种移植研究以确定经由siRNA靶向PEA 3、经由γ-分泌酶抑制剂的Notch信号传导或两者的生物学意义。在此，我们提供了新的证据，Notch的转录调控PEA 3在乳腺癌。PEA 3激活MCF-7、MDA-MB-231和SKBr 3乳腺癌细胞中的Notch-1转录。PEA 3在PEA 3水平内源性高的MDA-MB-231细胞中激活Notch-4转录。在PEA 3水平低的SKBr 3和BT474乳腺癌细胞中，PEA 3的过表达增加Notch-4转录物。染色质免疫沉淀证实PEA 3在MDA-MB-231细胞中富集在Notch-1和Notch-4启动子上。PEA 3募集至Notch-1是AP-1非依赖性的，而PEA 3募集至Notch-4是c-JUN依赖性的。重要的是，通过γ-分泌酶抑制剂（MRK-003 GSI）联合抑制Notch信号传导和敲低PEA 3使生长停滞在G1期，降低了锚定依赖性和锚定非依赖性生长，并显著增加了体外凋亡细胞。此外，PEA 3敲低或MRK-003 GSI处理显著降低了体内MDA-MB-231异种移植物的肿瘤生长。总之，这项研究的结果首次证明Notch-1和Notch-4是乳腺癌细胞中PEA 3的新转录靶点。靶向PEA 3和/或Notch通路可能为三阴性和可能的其他乳腺癌亚型提供新的治疗策略。
Women with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells. Real-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both. Herein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G1 phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo. Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes.
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影响因子：
4
作者：
Davidson, B;Goldberg, I;Kopolovic, J
通讯作者：
Kopolovic, J
DOI：
10.1073/pnas.96.1.23
发表时间：
1999-01-05
影响因子：
11.1
作者：
Hsieh, JJD;Zhou, SF;Hayward, SD
通讯作者：
Hayward, SD
影响因子：
2.5
作者：
Callahan, R;Raafat, A
通讯作者：
Raafat, A
影响因子：
11.2
作者：
Harrison, Hannah;Farnie, Gillian;Clarke, Robert B.
通讯作者：
Clarke, Robert B.
影响因子：
3.1
作者：
Bojovic, B. Bonnie;Hassell, John A.
通讯作者：
Hassell, John A.