Date Published: October 30, 2007
Publisher: Public Library of Science
Author(s): Mark S Cragg, Junya Kuroda, Hamsa Puthalakath, David C. S Huang, Andreas Strasser, Ingo K Mellinghoff
Abstract: BackgroundThe epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.Methods and FindingsWe performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.ConclusionsInhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.
Partial Text: The epidermal growth factor receptor (EGFR) is a type I surface-bound receptor tyrosine kinase of the ErbB receptor family. Its activation by physiological ligands (e.g., EGF) causes EGFR homodimerization or heterodimerization of EGFR with other members of the ErbB family, resulting in activation of diverse signaling molecules such as extracellular signal-regulated protein kinase 1/2 (ERK1/2), protein kinase B (AKT), and signal transducer and activator of transcription proteins (STATs), which regulate cellular proliferation, survival, differentiation, and migration (reviewed in ). EGFR function is commonly dysregulated in a range of solid cancers (e.g., breast, lung, ovarian, bladder, brain, and colon) due to either gene amplification, mutations (resulting in a constitutively active EGFR), or abnormally increased ligand production (reviewed in ). Moreover, enforced expression of mutant EGFR in transgenic mice promoted development of lung carcinomas [2,3]. These observations prompted the development of EGFR inhibitory drugs for cancer therapy. The EGFR tyrosine kinase inhibitors gefitinib (Iressa, AstraZeneca) and erlotinib (Tarceva, Genentech) as well as the monoclonal antibody cetuximab (Erbitux, Merck), which blocks ligand binding, cause substantial regression of a small proportion of non-small cell lung cancers (NSCLCs), particularly those with EGFR mutations that give rise to hyperactive kinases [1,4–6]. Signaling from mutant but not wild-type (WT) EGFR was shown to activate anti-apoptotic pathways, and small interfering RNA-mediated down-regulation of mutant EGFR resulted in the death of these cells , but the mechanisms for tumor cell killing were not examined.
In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib.