Costa Daniel B, Halmos Balázs, Kumar Amit, Schumer Susan T, Huberman Mark S, Boggon Titus J, Tenen Daniel G, Kobayashi Susumu
Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
PLoS Med. 2007 Oct;4(10):1669-79; discussion 1680. doi: 10.1371/journal.pmed.0040315.
Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.
To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.
Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.
在大多数对表皮生长因子受体（EGFR）酪氨酸激酶抑制剂（TKI）吉非替尼或厄洛替尼敏感的非小细胞肺癌（NSCLC）患者中存在EGFR突变。这些依赖EGFR的肿瘤最终会对TKI产生耐药性，常见的继发性T790M突变占对吉非替尼获得性耐药肿瘤的一半。然而，参与TKI诱导细胞死亡的关键促凋亡蛋白以及参与耐药的其他继发性突变仍不清楚。本研究的目的是确定EGFR TKI诱导凋亡的机制以及影响这一过程的继发性耐药突变。
为了研究TKI诱导的细胞死亡和耐药机制，我们使用了肺癌细胞系（有或无EGFR突变）、稳定转染EGFR突变构建体的Ba/F3细胞以及来自一名吉非替尼耐药患者的肿瘤样本。在此我们表明，仅含BH3结构域的多肽BIM（也称为BCL2样11）的上调与吉非替尼敏感的EGFR突变肺癌细胞中吉非替尼诱导的凋亡相关。T790M突变阻断了吉非替尼诱导的BIM上调和凋亡。不可逆TKI CL-387,785克服了这种阻断。通过小干扰RNA敲低BIM能够减弱EGFR TKIs诱导的凋亡。此外，从一名携带激活型L858R突变的吉非替尼耐药患者中，我们鉴定出一种新的继发性耐药突变，即与激活突变顺式排列的L747S，它减弱了BIM的上调并减少了凋亡。
我们的结果提供了证据，表明BIM参与了敏感的EGFR突变细胞中TKI诱导的凋亡，并且在含有常见EGFR T790M和新的L747S继发性耐药突变的模型中均观察到BIM上调的减弱和对吉非替尼诱导凋亡的耐药。这些发现还表明，诱导BIM可能在TKI耐药肿瘤的治疗中发挥作用。
