Acute infection of SKMG3 and SF268 cells with retroviral shRNA constructs targeting two distinct areas on the EGFR mRNA resulted in loss of EGFR protein expression within 72 hours of infection and robust cell death induction soon after 5 days. EGFR knockdown in human astrocytes and two GBM cell lines with out EGFR mutation didn’t induce cell death. Of note, SKMG3 cells never express the tumor suppressor protein Phosphatase and Tensin homolog, confirming our earlier findings that PTEN inactivation isn’t enough to alleviate EGFR mutant cancer cells from their dependence on EGFR for survival. We conducted very similar experiments with shRNA constructs targeting the EGF receptor member of the family HER2 given that HER2 can heterodimerize with EGFR and transmit oncogenic signals in specific cellular contexts.
HER2 knockdown did not induce a significant amount of cell death as measured from the trypan blue dye exclusion assay and immunoblotting for your cleaved Caspase3 substrate Poly polymerase. HER2 depletion also didn’t impact EGFR selleck chemicals phosphorylation at tyrosine 1068, suggesting that basal EGFR phosphorylation in SF268 and SKMG3 cells will not be the consequence of trans phosphorylation through the HER2 kinase. A few prosurvival functions of EGFR have been attributed to kinase independent properties in the receptor protein. To assess irrespective of whether EGFR kinase exercise is needed for the survival of SKMG3 and SF268 cells, we treated them with all the 2nd generation EGFR kinase inhibitor HKI 272. This drug irreversibly inhibits EGFR because it kinds covalent interactions with cysteines in the ATP cleft within the kinase domain. HKI 272 induced cell death in SF268 and SKMG3 cells, but not in EGFR wildtype GBM, lung cancer cells, or human astrocytes.
To lengthen our observations with HKI 272 to a second EGFR kinase inhibitor, we repeated our experiments with CI 1033. Like HKI 272, CI 1033 is definitely an irreversible, Amonafide ATP internet site competitive inhibitor of ErbB receptors and inhibits phosphorylation of wildtype EGFR in intact cells with equivalent potency as HKI 272. To our surprise, CI 1033 failed to induce cell death in either SF268 or SKMG3 cells. Immunoblots of entire cell lysates from SKMG3 cells taken care of with either inhibitor showed that CI 1033 inhibited EGFR phosphorylation less efficiently than HKI 272. We wondered no matter whether the differential impact of HKI 272 and CI 1033 on EGFR was special to GBM cells with EGFR EC mutations. We hence also compared the exercise of the two compounds in HCC827 lung cancer cells which harbor a deletion inside the EGFR kinase domain. In contrast to our findings in GBM cells, CI 1033 a lot more potently inhibited EGFR phosphorylation and even more potently induced cell death than HKI 272. Each inhibitors induced cell death at submicromolar concentrations in HCC827 cells, constant with all the reported hypersensitivity within the EGFR746 750 mutant to ATP web site aggressive EGFR kinase inhibitors in vitro and in lung cancer patients.