Yamasaki Fumiyuki, Johansen Mary J, Zhang Dongwei, Krishnamurthy Savitri, Felix Edward, Bartholomeusz Chandra, Aguilar Richard J, Kurisu Kaoru, Mills Gordon B, Hortobagyi Gabriel N, Ueno Naoto T
Breast Cancer Translational Research Laboratory, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030-4009, USA.
Cancer Res. 2007 Jun 15;67(12):5779-88. doi: 10.1158/0008-5472.CAN-06-3020.
Erlotinib (Tarceva), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has clinical activity in advanced lung cancer, but disease that initially responds to erlotinib eventually progresses. The mechanism of this acquired resistance is unclear. We established two erlotinib-resistant pools of A-431 cells, a well-characterized epidermoid cancer cell line that constitutively overexpresses EGFR and is sensitive to erlotinib, by continuous exposure to erlotinib over a 6-month period. The extent of EGFR gene amplification or mutation of the EGFR tyrosine kinase domain was not altered in the resistant cells. Intracellular erlotinib concentrations, determined by liquid chromatography-tandem mass spectrometry, were almost the same in all three cell lines. Immunoprecipitation with EGFR antibody followed by detection with phosphotyrosine antibody revealed that erlotinib effectively reduced EGFR phosphorylation in both parental cells and resistant cells. Erlotinib induced mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) and suppressed phosphorylated Akt (Ser(473)) but not in the erlotinib-resistant cells. Overexpression of MMAC1/PTEN by transfection with Ad.MMAC1/PTEN or by pharmacologic suppression of Akt activity restored erlotinib sensitivity in both resistant pools. Further, transfection of parental A-431 cells with constitutively active Akt was sufficient to cause resistance to erlotinib. We propose that acquired erlotinib resistance associated with MMAC1/PTEN down-regulation and Akt activation could be overcome by inhibitors of signaling through the phosphatidylinositol 3-kinase pathway.
厄洛替尼（特罗凯）是一种表皮生长因子受体（EGFR）酪氨酸激酶抑制剂，对晚期肺癌具有临床活性，但最初对厄洛替尼有反应的疾病最终会进展。这种获得性耐药的机制尚不清楚。我们通过在6个月的时间里持续暴露于厄洛替尼，建立了两个A-431细胞的厄洛替尼耐药细胞群，A-431是一种特征明确的表皮样癌细胞系，其EGFR持续过表达且对厄洛替尼敏感。耐药细胞中EGFR基因扩增程度或EGFR酪氨酸激酶结构域的突变未发生改变。通过液相色谱-串联质谱法测定，所有三种细胞系中的细胞内厄洛替尼浓度几乎相同。用EGFR抗体进行免疫沉淀，然后用磷酸酪氨酸抗体进行检测，结果显示厄洛替尼可有效降低亲本细胞和耐药细胞中的EGFR磷酸化水平。厄洛替尼可诱导多种晚期癌症中1/磷酸酶和张力蛋白同源物（MMAC1/PTEN）发生突变，并抑制磷酸化的Akt（Ser(473)），但在厄洛替尼耐药细胞中则不然。通过用Ad.MMAC1/PTEN转染或通过药物抑制Akt活性来过表达MMAC1/PTEN，可恢复两个耐药细胞群对厄洛替尼的敏感性。此外，用组成型活性Akt转染亲本A-431细胞足以使其对厄洛替尼产生耐药性。我们提出，与MMAC1/PTEN下调和Akt激活相关的获得性厄洛替尼耐药可通过磷脂酰肌醇3-激酶途径信号传导抑制剂来克服。
