EGFR mutations in lung cancer reside within the intracellular kinase domain . EGFR mutations in GBM cluster in the extracellular domain and comprise of in frame deletions and missense mutations . Each EGFR ectodomain and kinase domain mutations encode oncoproteins using the ability to transform NIH 3T3 cells during the absence of ligand . In this study, we examined the purpose of EGFR for the survival of GBM cells harboring EGFR ectodomain mutations. We demonstrate that EGFR signals are essential for the survival of those cells and that EGFR EC mutants vary markedly from EGFR KD mutants in their sensitivity to ATP internet site aggressive EGFR kinase inhibitors. Missense mutations within the EGFR extracellular domain are observed in 10 15 of GBMs . To find out whether EGFR signals are important for the survival of GBM cells endogenously expressing this kind of mutations, we to begin with sequenced the coding region of EGFR in a panel of GBM cell lines.
We observed two lines with EGFR EC mutations. Both mutations resulted in amino acid substitutions at alanine 289, the most common web-site of extracellular EGFR missense mutations in human GBMs . Alanine was substituted by valine in SF268 cells and by aspartic acid in SKMG3 cells . We examined irrespective of whether depletion of the EGFR protein selleck chemicals HIF inhibitor was sufficient to induce cell death in these lines. Acute infection of SKMG3 and SF268 cells with retroviral shRNA constructs focusing on two distinct places with the EGFR mRNA resulted in loss of EGFR protein expression inside of 72 hours of infection and robust cell death induction immediately after 5 days. EGFR knockdown in human astrocytes and two GBM cell lines with no EGFR mutation didn’t induce cell death .
Of note, SKMG3 cells really don’t express the tumor suppressor protein Phosphatase and Tensin homolog , confirming our earlier findings that PTEN inactivation is not really adequate penlac to relieve EGFR mutant cancer cells from their dependence on EGFR for survival . We conducted comparable experiments with shRNA constructs targeting the EGF receptor family member HER2 considering that HER2 can heterodimerize with EGFR and transmit oncogenic signals in certain cellular contexts . HER2 knockdown didn’t induce a significant volume of cell death as measured by the trypan blue dye exclusion assay and immunoblotting for the cleaved Caspase3 substrate Poly polymerase . HER2 depletion also did not affect EGFR phosphorylation at tyrosine 1068, suggesting that basal EGFR phosphorylation in SF268 and SKMG3 cells will not be the result of trans phosphorylation through the HER2 kinase.
Many prosurvival functions of EGFR have already been attributed to kinase independent properties from the receptor protein . To assess no matter if EGFR kinase action is required for your survival of SKMG3 and SF268 cells, we handled them using the second generation EGFR kinase inhibitor HKI 272 .