Activation from the epidermal growth factor receptor (EGFR) in glioblastoma (GBM)

Activation from the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. EGFR conformation on Catharanthine sulfate the other hand potently inhibit EGFR EC mutants and induce cell death in EGFR mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these two cancer types. INTRODUCTION Glioblastoma (GBM) is the most common malignant brain tumor in adults. Most GBM patients succumb to their disease within two years and there is a dire need for the development of novel therapeutics (1). Inhibitors of deregulated signaling pathways are active agents in a variety of human cancers (2 3 Rabbit polyclonal to Rex1 and represent a compelling area of drug development for GBM because many of these tumors harbor genetic alterations in growth factor signaling pathways (4 5 The epidermal growth factor receptor (EGFR) is a member of the EGFR family of receptor tyrosine kinases which also includes HER2 (ErbB2) HER3 (ErbB3) and HER4 (ErbB4) (6). EGFR has generated particular interest as a drug target in GBM because of the high frequency of EGFR alterations in this disease (7) and because ATP-site competitive EGFR kinase inhibitors are active agents in patients with EGFR-mutant lung cancer (8). EGFR kinase inhibitors which received regulatory approval for the treating lung tumor (erlotinib gefitinib) nevertheless have shown unsatisfactory results in individuals with GBM (9). Known reasons for this insufficient response in GBM stay poorly understood you need to include redundancy in signaling pathways (10) and intratumoral heterogeneity (11). One essential difference between EGFR in GBM and lung tumor may be the distribution of mutations inside the EGFR coding series. EGFR mutations in lung tumor have a home in the intracellular kinase domain (KD) (12). EGFR mutations in GBM cluster in the extracellular (EC) domain and include in-frame deletions (such as the common “variant III”) (7) Catharanthine sulfate and missense mutations (13)(Fig. 1A). Both EGFR ectodomain and kinase domain mutations encode oncoproteins Catharanthine sulfate with the ability to transform NIH-3T3 cells in the absence of ligand (13-15). In this study we examined the role of EGFR for the survival of GBM cells harboring EGFR ectodomain mutations. We demonstrate that EGFR signals are essential for the survival of these cells and that EGFR EC mutants differ markedly from EGFR KD mutants in their sensitivity to ATP-site competitive EGFR kinase inhibitors. FIGURE 1 EGFR-knockdown induces cell death in GBM cells with EGFR EC mutations RESULTS 1 mutant GBM cells are EGFR addicted Missense mutations in the extracellular (EC) domain are found in 10-15 % of GBMs (4 5 13 To determine whether EGFR signals are essential for the survival of GBM cells endogenously expressing such mutations we first sequenced the coding region of in a panel of GBM cell lines. We found two lines with EC mutations. Both mutations resulted in amino acid substitutions at alanine 289 the most common site of extracellular EGFR missense mutations in human GBMs (Fig. 1A). Alanine Catharanthine sulfate was substituted by valine (A289V) in SF268 cells and by aspartic acid (A289D) in SKMG3 cells (Suppl. Figure 1). We tested whether depletion of the EGFR protein was sufficient to induce cell death in these lines. Acute infection of SKMG3 and SF268 cells with retroviral shRNA constructs targeting two distinct areas of the EGFR mRNA resulted in loss of EGFR protein expression within 72 hours of infection and robust cell death induction after 5 days. EGFR knockdown in human astrocytes (NHAs)(16) and two GBM cell lines without mutation (SF295 8 did not induce cell death (Fig. 1B). Of note SKMG3 cells do not express the tumor suppressor protein Phosphatase and Tensin homolog (PTEN) confirming our earlier findings that PTEN inactivation is not sufficient to relieve mutant cancer cells from their dependence on EGFR for survival (17). We conducted similar experiments with shRNA constructs targeting the EGF receptor family.