Breast cancer expressing the activated HER2/neu is sensitive to gefitinib In vitro and In vivo and acquires resistance through a novel point mutation in the HER2/neu
Breast cancer expressing the activated HER2/neu is sensitive to gefitinib In vitro and In vivo and acquires resistance through a novel point mutation in the HER2/neu
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DOI：
10.1158/0008-5472.can-07-0765
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发表时间：
2007-07-15
期刊：
影响因子：
11.2
通讯作者：
Lonardo, Fulvio
中科院分区：
文献类型：
--
作者：
Piechocki, Marie P.;Yoo, George H.;Lonardo, Fulvio
HER2/neu癌基因是乳腺癌和其他癌症的重要诊断和预后因素以及治疗靶点。我们开发并鉴定了一种乳腺癌细胞系 (Bam1a)，该细胞系过表达激活的 HER2/neu 和 ErbB-3，并且具有与 ErbB-2 遗传特征一致的基因表达谱。我们在体外和体内评估了表皮生长因子受体 (EGFR)/HER2 抑制剂吉非替尼对该乳腺肿瘤系的影响。我们通过蛋白质印迹表征了吉非替尼对 EGFR、HER2 和 ErbB-3 磷酸化的影响，并通过生长、存活和应激途径确定了对下游信号传导的影响以及对增殖、细胞周期和凋亡的影响。吉非替尼治疗以剂量依赖性方式减少 ErbB-3 > EGFR > HER2/neu 的磷酸化以及信号转导器和转录激活剂。通过丝裂原激活蛋白 (MAP)/细胞外信号调节激酶激酶、p44/42 MAP 激酶 ((mapk)) 的下游有丝分裂信号传导以及通过 c-jun-NH2-激酶 (jNK) 1 和 c-jun 的应激信号传导受损（1 μmol/L，4-24 小时），导致细胞周期蛋白 D1、细胞周期蛋白 B1 和细胞周期蛋白 B1 减少，导致 24 小时内细胞停滞和细胞周期停滞。 p(Ser795)Rb 并增加 p27。增殖和集落形成分别在 0.5 和 1 μmol/L 时受到抑制，并且与基因表达谱的改变相关。在 caspase-3 和聚 (ADP) 核糖聚合酶 (PARP) 裂解和细胞凋亡之前，通过 Akt 的生存信号减弱、诱导 he、连接蛋白 43 丢失以及血管内皮生长因子-D 产生减少（> 50% 2 mu mol/L，48 小时）。口服吉非替尼能够防止 Bam1a 肿瘤细胞从可触知的病变中生长，缩小已形成的肿瘤，消除 HER2 和 HER3 磷酸化，并减少体内 MAPK 和 Akt 信号传导。 Bamla 细胞系 IR-5 的变体具有在 5 μmol/L 浓度下生长的能力，吉非替尼已被开发和表征。 IR-5 在 HER2/neu 中具有一个新的点突变，对应于 ATP 结合袋中的 L7261，并与吉非替尼敏感性的对数下降、与 EGFR 和 HER3 的异二聚化增加以及下调受损相关。 IR-5 的基因表达谱显示 EMP-1、NOTCH4、FLT-1、PDGFB 和其他几个基因的表达增加，这些基因可能有助于耐药表型并通过 MAPK 和 Akt 维持信号传导。该模型将有助于理解在针对癌基因成瘾疾病的治疗策略背景下内在药物敏感性和获得性耐药之间的差异。
The HER2/neu oncogene is an important diagnostic and prognostic factor and therapeutic target in breast and other cancers. We developed and characterized a breast cancer cell line (Bam1a) that overexpresses the activated HER2/neu and ErbB-3 and has a gene expression profile consistent with the ErbB-2 genetic signature. We evaluated the effects of the epidermal growth factor receptor (EGFR)/HER2 inhibitor, gefitinib, on this breast tumor line in vitro and in vivo. We characterized the effects of gefitinib on EGFR, HER2, and ErbB-3 phosphorylation by Western blot and determined the effects on downstream signaling through growth, survival, and stress pathways and the effect on proliferation, cell cycle, and apoptosis. Gefitinib treatment diminished phosphorylation of the ErbB-3 > EGFR > HER2/neu and signal transducers and activators of transcriptions in a dose-dependent fashion. Downstream mitogenic signaling through mitogen-activated protein (MAP)/extracellular signal regulated kinase kinase, p44/42 MAP kinase ((mapk)) and stress signaling through c-jun-NH2-kinase (jNK) 1 and c-jun was impaired (1 mu mol/L, 4-24 h), leading to cytostasis and cell cycle arrest within 24 h by decreased cyclin D1, cyclin B1, and p(Ser795)Rb and increased p27. Proliferation and colony formation were inhibited at 0.5 and 1 mu mol/L, respectively, and correlated with altered gene expression profiles. Diminished survival signaling through Akt, induction of him, loss of connexin43, and decreased production of vascular endothelial growth factor-D preceded caspase-3 and poly(ADP)ribose polymerase (PARP) cleavage and apoptosis (> 50% 2 mu mol/L, 48 h). Oral administration of gefitinib was able to prevent the outgrowth of Bam1a tumor cells from palpable lesions, shrink established tumors, eliminate HER2 and HER3 phosphorylation, and decrease MAPK and Akt signaling in vivo. A variant of the Bamla cell line, IR-5, with acquired ability to grow in 5 mu mol/L, gefitinib was developed and characterized. IR-5 bears a novel point mutation in the HER2/neu that corresponds to a L7261 in the ATP-binding pocket and correlates with a log decrease in sensitivity to gefitinib, increased heterodimerization with EGFR and HER3, and impaired down-regulation. Gene expression profiling of IR-5 showed increased expression of EMP-1, NOTCH4, FLT-1, PDGFB, and several other genes that may contribute to the resistant phenotype and sustain signaling through MAPK and Akt. This model will be useful in understanding the differences between intrinsic drug sensitivity and acquired resistance in the context of therapeutic strategies that target oncogene addicted diseases.