Mechanisms of resistance to erbitux (anti-epidermal growth factor receptor) combination therapy in pancreatic adenocarcinoma cells
Mechanisms of resistance to erbitux (anti-epidermal growth factor receptor) combination therapy in pancreatic adenocarcinoma cells
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DOI：
10.1016/j.gassur.2004.09.021
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发表时间：
2004-12-01
影响因子：
3.2
通讯作者：
Vickers, SM
中科院分区：
文献类型：
--
作者：
Arnoletti, JP;Buchsbaum, DJ;Vickers, SM
我们先前证明胰腺癌BxPC-3异种移植物对爱必妥、吉西他滨和放射治疗具有抗性，而MIA PaCa-2异种移植物对相同的治疗高度敏感。在这里，我们阐明了体外机制，可以解释观察到的差异反应表皮生长因子受体（EGFR）表达胰腺癌异种移植爱必妥为基础的联合治疗在体内。用EGFR、ErbB 2、ErbB 3和ErbB 4的抗体探测MIA、PaCa-2和BxPC-3蛋白裂解物。使用抗磷酸化酪氨酸抗体和受体特异性免疫沉淀物通过免疫印迹分析观察组成型ErbB 3活性。用实时聚合酶链反应定量两种细胞系中的erbB 2和erbB 3基因表达。在0 ℃和37 ℃下用爱必妥处理不同孵育时间后，通过流式细胞术测定爱必妥诱导的EGFR内化。MIA PaCa-2和BxPC-3蛋白提取物也用EGF刺激后和爱必妥存在下的抗磷酸丝裂原活化蛋白激酶抗体探测。尽管两种细胞系均表达EGFR和ErbB 2蛋白，但ErbB 3蛋白选择性地由BxPC-3细胞表达，在BxPC-3细胞中也显示出组成性磷酸化的证据。与MIA PaCa-2相比，BxPC-3细胞中erbB 3转录水平增加了10倍。ErbB 4蛋白在两种细胞系中均未检测到。Erbitux在孵育2小时后介导MIA PaCa-2细胞中的EGFR内化，而在BxPC-3细胞中不促进EGFR内化。同样，Erbitux处理MIA PaCa-2细胞后，MAPK p44/42的EGF依赖性磷酸化被阻断，而BxPC-3细胞则没有。爱必妥选择性地干扰EGF诱导的MIA PaCa-2细胞MAPK激活，但不干扰BxPC-3细胞。BxPC-3胰腺癌细胞的持续MAPK激活和EGFR的体外内化受损可能是由于与EGFR的异源二聚化促进的组成性ErbB 3信号传导，这可能解释了体内对基于爱必妥的联合治疗的抗性。(C)2004年，消化道外科学会。
We previously demonstrated that pancreatic adenocarcinoma BxPC-3 xenografts display resistance to treatment with Erbitux, gemcitabine, and radiation, whereas MIA PaCa-2 xenografts are highly sensitive to the same therapy. Here, we elucidate in vitro mechanisms that may explain the observed differential response of epidermal growth factor receptor (EGFR) expressing pancreatic adenocarcinoma xenografts to Erbitux-based combination therapy in vivo. MIA, PaCa-2 and BxPC-3 protein lysates were probed with antibodies to EGFR, ErbB2, ErbB3, and ErbB4. Constitutive ErbB3 activity was visualized by immunoblot analysis using anti-phosphotyrosine antibodies and receptor-specific immunoprecipitates. erbB2 and erbB3 gene expression in both cell lines was quantified with real-time polymerase chain reaction. Erbitux-induced internalization of EGFR was determined by flow cytometry following Erbitux treatment for different incubation times at 0degreesC and 37degreesC. MIA PaCa-2 and BxPC-3 protein extracts were also probed with anti-phospho-mitogen-activated protein kinase antibody after stimulation with EGF and in the presence of Erbitux. Although both cell lines expressed EGFR and ErbB2 protein, ErbB3 protein was selectively expressed by BxPC-3 cells, where it also showed evidence of constitutive phosphorylation. There was a 10-fold increase of erbB3 transcript levels in BxPC-3 cells compared with MIA PaCa-2. ErbB4 protein was not detectable in either cell line. Erbitux mediated EGFR internalization in MIA PaCa-2 cells after 2 hours of incubation, whereas it did not promote EGFR internalization in BxPC-3 cells. Likewise, EGF-dependent phosphorylation of MAPK p44/42 was blocked by Erbitux treatment in MIA PaCa-2 but not BxPC-3 cells. Erbitux selectively interfered with EGF-induced MAPK activation in MIA PaCa-2 but not BxPC-3 cells. Persistent MAPK activation and impaired in vitro internalization of EGFR by BxPC-3 pancreatic cancer cells may be due to constitutive ErbB3 signaling, facilitated by heterodimerization with EGFR, which may explain resistance to Erbitux-based combination therapy in vivo. (C) 2004 The Society for Surgery of the Alimentary Tract.