Kong Anthony, Calleja Véronique, Leboucher Pierre, Harris Adrian, Parker Peter J, Larijani Banafshé
Cell Biophysics Laboratory, Lincoln's Inn Fields laboratories, London Research Institute, Cancer Research UK, London, United Kingdom.
PLoS One. 2008 Aug 6;3(8):e2881. doi: 10.1371/journal.pone.0002881.
The response rate to EGFR tyrosine kinase inhibitors (TKIs) may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses.
Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET) which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs) of EGFR, AG1478 and Iressa (Gefitinib) decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment-induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa.
These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs) in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients.
在表皮生长因子受体（EGFR）表达本身作为反应指标并不充分的癌症患者中，EGFR酪氨酸激酶抑制剂（TKIs）的反应率可能较低且不可预测。对这种耐药性的潜在机制了解有限。此外，虽然TKIs可抑制HER2过表达的乳腺肿瘤细胞生长，但在生理剂量下它们并不能完全抑制HER2的致癌功能。
在此，我们提供了一种分子机制，即由于自分泌配体释放，HER2致癌功能如何通过替代HER受体激活而逃避TKIs的抑制。使用监测原位HER受体磷酸化的荧光共振能量转移（FRET）以及经典生化分析，我们发现EGFR的特异性酪氨酸激酶抑制剂（TKIs）AG1478和易瑞沙（吉非替尼）通过抑制EGFR/HER3二聚化来降低EGFR和HER3的磷酸化。随之而来的是，我们证明HER4的裂解以及HER4/HER2的二聚化与通过HER2/HER3对HER3的重新激活同时发生，导致在现在耐药的存活细胞中HER2持续磷酸化。发现这些药物治疗诱导的过程是由包括神经调节蛋白和β细胞素在内的配体释放介导的，这些配体通过HER2激活HER3和HER4。虽然抗β细胞素抗体与易瑞沙联合使用可增强对耐药细胞的抗增殖作用，但神经调节蛋白和β细胞素等配体使敏感的SKBR3细胞对易瑞沙产生耐药性。
这些结果证明了药物诱导的自分泌事件在维持HER2磷酸化以及介导乳腺癌细胞对EGFR酪氨酸激酶抑制剂（TKIs）耐药性方面导致替代HER受体激活的作用，从而明确了克服患者耐药性的治疗机会。
