Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics.
Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics.
复制标题
吉非替尼诱导的表达突变体EGFR的NSCLC细胞系杀死需要BIM，并且可以通过BH3 Mimetics增强。
DOI：
10.1371/journal.pmed.0040316
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发表时间：
2007-10
期刊：
影响因子：
15.8
通讯作者：
Strasser A
中科院分区：
文献类型：
--
作者：
Cragg MS;Kuroda J;Puthalakath H;Huang DC;Strasser A
表皮生长因子受体（EGFR）在细胞增殖、分化和存活的控制中发挥着关键作用。 EGF-EGFR 信号传导异常，例如导致 EGFR 过度活跃或导致野生型受体过度表达的突变，已在多种癌症中发现，包括肺癌、乳腺癌和结肠癌。吉非替尼等 EGFR 抑制剂已被证明可成功治疗某些癌症，特别是 EGFR 基因内含有激活突变的非小细胞肺癌 (NSCLC)，但导致肿瘤消退的分子机制仍不清楚。因此，我们希望描述这些机制。我们进行了生化和遗传学研究，以研究 EGFR 酪氨酸激酶活性抑制剂（例如吉非替尼）抑制人类 NSCLC 生长的机制。我们发现吉非替尼触发了内在的（也称为“线粒体”）细胞凋亡信号，涉及 BAX 的激活和细胞色素 c 的线粒体释放，最终释放 caspase 级联反应。吉非替尼通过转录和翻译后机制导致促凋亡 BH3 蛋白 BIM（也称为 BCL2 样 11）的水平快速增加。药物抑制剂实验表明，阻断 MEK–ERK1/2（丝裂原激活蛋白激酶激酶 - 细胞外信号调节蛋白激酶 1/2）信号传导，但不阻断 PI3K（磷脂酰肌醇 3 激酶）、JNK（c-Jun N 末端激酶或丝裂原激活蛋白激酶 8）或 AKT（蛋白激酶 B），对于 BIM 激活至关重要。通过 RNA 干扰，我们证明 BIM 对于吉非替尼诱导的 NSCLC 细胞杀伤至关重要。此外，我们发现添加 BH3 模拟物 ABT-737 可以增强吉非替尼诱导的细胞凋亡。 EGFR 酪氨酸激酶抑制剂已被证明可用于治疗某些癌症，特别是 EGFR 激酶结构域中具有激活突变的 NSCLC，但杀死肿瘤细胞的机制仍不清楚。在本文中，我们证明促凋亡 BH3 蛋白 BIM 的激活对于杀死肿瘤细胞至关重要，而 EGFR-MEK-ERK 信号级联的关闭对于 BIM 激活至关重要。此外，我们证明添加 BH3 模拟物可显着增强 EGFR 酪氨酸激酶抑制剂吉非替尼对 NSCLC 细胞的杀伤作用。这种方法似乎代表了许多甚至所有致癌酪氨酸激酶所共享的范例，并为癌症治疗提出了一种强大的新策略。 Andreas Strasser 及其同事证明，促凋亡 BH3 蛋白 BIM 的激活对于杀死肿瘤细胞至关重要，而 EGFR−MEK−ERK 信号级联的关闭对于 BIM 激活至关重要。通常，细胞分裂（产生新细胞）和细胞死亡保持良好平衡，以保持人体良好的工作状态。但有时细胞的遗传物质会发生变化（突变），使它们不受控制地分裂形成癌症——危及生命的、杂乱的细胞团。表皮生长因子受体 (EGFR) 是一种在细胞分裂中起关键作用且经常在肿瘤中发生突变的蛋白质。在正常细胞中，蛋白质信使与 EGFR 结合并激活其酪氨酸激酶。然后，这种酶将磷酸基团添加到蛋白质中的酪氨酸（一种氨基酸）上，形成信号级联（例如 MEK-ERK 信号级联）的一部分，告诉细胞分裂。在 EGFR 发生突变的癌症中，信号传导过度活跃，因此癌细胞分裂得比应有的要多得多。例如，一些非小细胞肺癌（NSCLC，最常见的肺癌类型）的 EGFR 酪氨酸激酶内具有激活突变。使用吉非替尼和厄洛替尼等 EGFR 酪氨酸激酶抑制剂 (TKI) 治疗会诱导这些肿瘤中的细胞停止生长并死亡。这种细胞死亡会导致肿瘤缩小（消退）并延长此类非小细胞肺癌患者的预期寿命。不幸的是，TKI 治疗很少能治愈 NSCLC，因此找到一种方法来增强 TKI 对癌细胞的作用将是有用的。为此，需要充分了解这些药物导致癌细胞死亡和肿瘤消退的分子机制。在这项研究中，研究人员结合使用生化和遗传学方法来研究吉非替尼如何杀死带有突变 EGFR 的 NSCLC 细胞。研究人员首先测量了 NSCLC 细胞系（在培养皿中无限生长的肿瘤细胞）对吉非替尼诱导的细胞凋亡的敏感性。吉非替尼在两种表达突变型 EGFR 的细胞系中引起广泛的细胞凋亡，但在一种表达正常 EGFR 的细胞系中则不然。接下来，他们研究了吉非替尼在最敏感的细胞系（H3255）中诱导细胞凋亡的机制。细胞凋亡通过两条主要途径被激活。 “内在”途径的标志包括 BAX 蛋白的激活和细胞色素 c 从称为线粒体的亚细胞区室中释放。吉非替尼治疗在 H3255 细胞中诱导了这两种事件。当促凋亡 BH3-only BCL-2 蛋白（例如 BIM；“BH3-only”描述了这些蛋白的结构）与抗凋亡 BCL2 蛋白结合时，BAX（BCL-2 蛋白家族的促凋亡成员）被激活。吉非替尼治疗通过增加 BIM 蛋白的产生并去除其中的磷酸基团，从而增加 BIM 活性，从而快速增加 H3255 和 HCC827 细胞（但不包括吉非替尼耐药细胞）中的 BIM 活性。 MEK-ERK 信号级联的药物阻断（而非其他 EGFR 信号级联）也会导致 BIM 的积累。相比之下，使用 RNA 干扰技术阻断 BIM 表达可减少吉非替尼诱导的细胞凋亡。最后，吉非替尼和一种名为 ABT-737 的 BH3 模拟化合物（与 BIM 一样，与抗凋亡 BCL-2 蛋白结合）的组合比单独使用吉非替尼引起更多的细胞凋亡。这些发现（以及Gong等人和Costa等人报道的结果）表明促凋亡BH3蛋白BIM的激活对于吉非替尼诱导杀死携带EGFR酪氨酸激酶突变的NSCLC细胞至关重要。他们还表明，吉非替尼对 EGFR-MEK-ERK 信号级联的抑制对于 BIM 激活至关重要。由于这些发现来自对 NSCLC 细胞系的研究，因此需要在新鲜分离的肿瘤细胞和人体生长的肿瘤中进行证实。然而，模拟 BH3 作用的化合物增强吉非替尼诱导的 NSCLC 细胞杀伤作用的证明表明，TKIs 和影响细胞凋亡激活内在途径的药物的组合可能为治疗酪氨酸激酶突变驱动肿瘤生长的癌症提供强大的策略。请通过此摘要的在线版本访问这些网站：http://dx.doi.org/10.1371/journal.pmed.0040316。 Ingo Mellinghoff 的观点讨论了本文和两篇相关研究文章 关于表皮生长因子受体、细胞凋亡和 BCL2 蛋白的维基百科页面（请注意，维基百科是任何人都可以编辑的免费在线百科全书；有多种语言版本） CancerQuest 提供来自埃默里大学的癌症各个方面的信息（多种语言） 美国国家癌症研究所为患者和专业人士提供的肺癌信息（英语和西班牙语） 为英国癌症研究中心的患者提供的肺癌信息，包括关于肺癌的信息TKI 治疗 来自 Cancerbackup 的厄洛替尼和吉非替尼患者信息
The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms. We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737. Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy. Andreas Strasser and colleagues demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR−MEK−ERK signaling cascade is critical for BIM activation. Normally, cell division (which produces new cells) and cell death are finely balanced to keep the human body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. One protein with a critical role in cell division that is often mutated in tumors is the epidermal growth factor receptor (EGFR). In normal cells, protein messengers bind to EGFR and activate its tyrosine kinase. This enzyme then adds phosphate groups to tyrosine (an amino acid) in proteins that form part of signaling cascades (for example, the MEK–ERK signaling cascade) that tell the cell to divide. In cancers that have mutations in EGFR, signaling is overactive so the cancer cells divide much more than they should. Some non-small cell lung cancers (NSCLC, the commonest type of lung cancer), for example, have activating mutations within the EGFR tyrosine kinase. Treatment with EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib induces the cells in these tumors to stop growing and die. This cell death causes tumor shrinkage (regression) and increases the life expectancy of patients with this type of NSCLC. Unfortunately, treatment with TKIs rarely cures NSCLC, so it would be useful to find a way to augment the effect that TKIs have on cancer cells. To do this, the molecular mechanisms that cause cancer-cell death and tumor regression in response to these drugs need to be fully understood. In this study, the researchers have used a combination of biochemical and genetic approaches to investigate how gefitinib kills NSCLC cells with mutated EGFR. The researchers first measured the sensitivity of NSCLC cell lines (tumor cells that grow indefinitely in dishes) to gefitinib-induced apoptosis. Gefitinib caused extensive apoptosis in two cell lines expressing mutant EGFR but not in one expressing normal EGFR. Next, they investigated the mechanism of gefitinib-induced apoptosis in the most sensitive cell line (H3255). Apoptosis is activated via two major pathways. Hallmarks of the “intrinsic” pathway include activation of a protein called BAX and cytochrome c release from subcellular compartments known as mitochondria. Gefitinib treatment induced both these events in H3255 cells. BAX (a proapoptotic member of the BCL-2 family of proteins) is activated when proapoptotic BH3-only BCL-2 proteins (for example, BIM; “BH3-only” describes the structure of these proteins) bind to antiapoptotic BCL2 proteins. Gefitinib treatment rapidly increased BIM activity in H3255 and HCC827 cells (but not in gefitinib-resistant cells) by increasing the production of BIM protein and the removal of phosphate groups from it, which increases BIM activity. Pharmacological blockade of the MEK–ERK signaling cascade, but not of other EGFR signaling cascades, also caused the accumulation of BIM. By contrast, blocking BIM expression using a technique called RNA interference reduced gefitinib-induced apoptosis. Finally, a combination of gefitinib and a BH3-mimicking compound called ABT-737 (which, like BIM, binds to antiapoptotic BCL-2 proteins) caused more apoptosis than gefitinib alone. These findings (and those reported by Gong et al. and Costa et al.) indicate that activation of the proapoptotic BH3-only protein BIM is essential for gefitinib-induced killing of NSCLC cells that carry EGFR tyrosine kinase mutations. They also show that inhibition of the EGFR–MEK–ERK signaling cascade by gefitinib is essential for BIM activation. Because these findings come from studies on NSCLC cell lines, they need confirming in freshly isolated tumor cells and in tumors growing in people. However, the demonstration that a compound that mimics BH3 action enhances gefitinib-induced killing of NSCLC cells suggests that combinations of TKIs and drugs that affect the intrinsic pathway of apoptosis activation might provide a powerful strategy for treating cancers in which tyrosine kinase mutations drive tumor growth. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040316. A perspective by Ingo Mellinghoff discusses this article and two related research articles Wikipedia pages on epidermal growth factor receptor, apoptosis, and BCL2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages) CancerQuest provides information on all aspects of cancer</ext-link> from Emory University (in several languages) US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish) Information for patients from Cancer Research UK on lung cancer including information on treatment with TKIs Information for patients from Cancerbackup on erlotinib and gefitinib
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影响因子：
158.5
作者：
Lynch, TJ;Bell, DW;Haber, DA
通讯作者：
Haber, DA
影响因子：
4.8
作者：
Komatsu, N;Watanabe, T;Ozawa, K
通讯作者：
Ozawa, K
影响因子：
11.4
作者：
Akiyama, T;Bouillet, P;Tanaka, S
通讯作者：
Tanaka, S
影响因子：
8
作者：
Essafi, A;de Mattos, SF;Lam, EWF
通讯作者：
Lam, EWF
影响因子：
4.8
作者：
Favata, MF;Horiuchi, KY;Trzaskos, JM
通讯作者：
Trzaskos, JM