Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy
Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy
复制标题
DOI：
10.1158/1078-0432.ccr-1100-03
复制
发表时间：
2004-01-15
影响因子：
11.5
通讯作者：
Tortora, G
中科院分区：
文献类型：
--
作者：
Ciardiello, F;Bianco, R;Tortora, G
目的：表皮生长因子受体（EGFR）自分泌信号通路参与肿瘤的发生发展。EGFR抑制剂，如C225（西妥昔单抗），一种嵌合人-小鼠抗EGFR单克隆抗体，以及ZD1839（吉非替尼），一种小分子EGFR选择性酪氨酸激酶抑制剂，正处于临床开发的后期。在使用EGFR抑制剂治疗的表达EGFR的癌症中，癌细胞耐药性的潜在出现可能决定了这些药物在一些癌症患者中缺乏活性。血管内皮生长因子（Vascular endothelial growth factor， VEGF）由癌细胞分泌，在肿瘤诱导的内皮细胞增殖和通透性调控中起关键作用。ZD6474是一种小分子VEGF flk-1/KDR （VEGFR-2）酪氨酸激酶抑制剂，对EGFR酪氨酸激酶也有抑制活性。实验设计：实验检测了ZD1839、C225和ZD6474在人GEO结肠癌异种移植胸腺小鼠体内的抗肿瘤活性。通过在长期用ZD1839或C225处理的小鼠体内生长的GEO异种移植物，建立了对EGFR抑制剂耐药的GEO细胞系。流式细胞术检测EGFR的表达。Western blotting检测参与细胞内信号传导的各种蛋白的表达。肿瘤生长数据采用Student’st检验进行统计学显著性评价。所有p都是双面的。结果：尽管在大多数小鼠中，慢性给予最佳剂量的C225或ZD1839有效地阻断了GEO肿瘤的生长，但肿瘤在80-90天内开始缓慢生长，尽管持续治疗。相比之下，用ZD6474持续治疗已建立GEO异种移植物的小鼠，在整个给药期间（长达150天）都能有效抑制肿瘤生长。用ZD1839或C225预处理小鼠也测定了ZD6474的活性。当EGFR抑制剂治疗4周后GEO生长明显时，小鼠再用EGFR抑制剂治疗或用ZD6474治疗。仅在用ZD6474治疗的小鼠中，GEO肿瘤生长被阻断，而在用C225或ZD1839再次治疗的小鼠中，观察到肿瘤进展。在C225或ZD1839治疗期间生长的GEO肿瘤被建立为细胞系（分别为GEO-C225- res和GEO-ZD1839- res）。与亲代GEO细胞相比，这些细胞系的细胞膜相关EGFR表达仅略有降低。Western blot结果显示，bcl-2、bcl-xL、p53、p27、MDM-2、akt、活化磷酸化akt或丝裂原活化蛋白激酶的EGFR配体转化生长因子α的表达无明显变化。然而，GEO- c225 - res和GEO- zd1839 - res细胞的活化磷酸化丝裂原活化蛋白激酶、环氧化酶-2和VEGF的表达均比GEO细胞高5-10倍。C225和ZD1839均未显著影响GEO-C225-RES和GEO-ZD1839-RES在裸鼠体内的生长，但ZD6474有效抑制了GEO-C225-RES和GEO-ZD1839-RES的生长。结论：长期使用选择性EGFR抑制剂治疗GEO异种移植物可导致EGFR抑制剂耐药癌细胞的发展。ZD6474可以抑制EGFR抑制剂耐药肿瘤的生长。这些数据表明，抑制VEGF信号传导具有潜在的抗癌策略，即使在对EGF抑制剂有抗性的肿瘤中也是如此。
Purpose: The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small molecule EGFR-selective tyrosine kinase inhibitor, are in advanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase.Experimental Design: The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical significance using the Student's t test. All Ps were two-sided.Results: Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80-90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pretreated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was observed in mice re-treated with C225 or ZD1839. GEO tumors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO-ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting revealed no major change in the expression of the EGFR ligand transforming growth factor alpha of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-activated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5-10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474.Conclusions: Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474. These data indicate that inhibition of VEGF signaling has potential as an anticancer strategy, even in tumors that are resistant to EGF inhibitors.