Identification of novel molecular regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in breast cancer cells by RNAi screening.
Identification of novel molecular regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in breast cancer cells by RNAi screening.
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DOI：
10.1186/bcr3645
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发表时间：
2014-04-17
期刊：
影响因子：
--
通讯作者：
Lipkowitz S
中科院分区：
文献类型：
--
作者：
Garimella SV;Gehlhaus K;Dine JL;Pitt JJ;Grandin M;Chakka S;Nau MM;Caplen NJ;Lipkowitz S
肿瘤坏死因子相关凋亡诱导配体（TRAIL）与其受体TRAIL受体1（TRAIL-R1）和TRAIL受体2（TRAIL-R2）结合，通过激活caspase-8和下游执行者caspase-3和caspase-7（caspase-3/7）导致细胞凋亡。具有间充质表型的三阴性乳腺癌（TNBC）细胞系对TRAIL敏感，而其他乳腺癌细胞系是抗性的。控制乳腺癌细胞中TRAIL敏感性的潜在机制尚未完全了解。在这里，我们进行了小干扰RNA（siRNA）筛选，以确定乳腺癌细胞中TRAIL通路的分子调节剂。我们在间充质TNBC细胞系MB 231中对人激酶组（691个基因）、磷酸酶组（320个基因）和约300个另外的基因进行了siRNA筛选。siRNA转染后48小时，相对于阴性对照siRNA（siNeg），在不存在或存在TRAIL的情况下对每种siRNA进行测量半胱天冬酶-8活性、半胱天冬酶-3/7活性或细胞活力的平行筛选。在代表上皮TNBC（MB 468）、HER 2扩增乳腺癌（SKBR 3）和雌激素受体阳性乳腺癌（T47 D）的细胞系中筛选基因子集。通过使用小分子抑制剂研究了TRAIL途径的选定假定负调节剂。在MB 231中的初步筛选确定了150个基因，包括83个激酶、4个磷酸酶和63个非激酶，作为TRAIL的潜在负调节因子。所鉴定的基因参与许多关键的细胞过程，包括细胞凋亡、生长因子受体信号传导、细胞周期调控、转录调控和DNA修复。基因网络分析确定了四个基因（PDPK 1，IKBKB，SRC和BCL 2L 1），形成负调控相互作用网络中的关键节点。在代表不同乳腺癌亚型和对TRAIL敏感性的其他细胞系中鉴定的基因子集的二次筛选验证并扩展了这些发现。此外，我们证实了SRC或BCL 2L 1的小分子抑制与TRAIL组合使乳腺癌细胞对TRAIL诱导的细胞凋亡敏感，包括对TRAIL诱导的细胞毒性具有抗性的细胞系。这些数据确定了新的分子调控TRAIL诱导的乳腺癌细胞凋亡，并提出了战略，增强应用TRAIL作为治疗乳腺癌。
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells. We conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors. The primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity. These data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.
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影响因子：
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作者：
Engels, IH;Stepczynska, A;Wesselborg, S
通讯作者：
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