The Function Of FBW7 In The Development Of Acquired Resistance To Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors In Non-small Cell Lung Cancer

Objective:FBW7(F-box and WD repeat domain-containing7, FBW7) is a member of F-box protein family, which serves as a substrate recognition unit of the SCF ubiquitin protein enzyme complex. FBW7 mediates the ubiquitylation of several oncoproteins, and promotes degradation through the ubiquitin-proteasome pathway. FBW7 plays crucial roles in regulating a broad spectrum of biological processes, including cell cycle regulation, cell proliferation, differentiation, apoptosis and tumor metastasis. As a putative tumor suppressor, gene deletions or inactivating mutations of FBW7 have been identified in various types of cancers. In comparison with the epidermal growth factor receptor(EGFR) mutated, Gefitinib sensitive lung cancer cells, our preliminary studies indicate that FBW7 expression is significantly decreased in the Gefitinib resistant lung cancer cells. In order to investigate the function of FBW7 and shed light on the underlying mechanisms, we have performed the following experiments. Method:1. The Gefitinib resistant PC-9GR cells were generated by exposing PC-9 parental cells to stepwise increasing concentrations of Gefitinib. After 6 months of treatment, the PC-9GR cells were tested for T790 M and Met amplification by DNA sequencing and FISH, respectively. 2. Expression of FBW7 in PC-9 and HCC827 parental cells and PC-9GR, HCC827GR5 and HCC827GR6 cells were examined by RT-PCR and Western blot. 3. By using the shRNA assay, FBW7 was knockeddown in PC-9 and HCC827 cells, the PC-9 shFBW7 and HCC827 shFBW7 cells were tested for Gefitinib sensitivity. 4. Identifying the molecular mechanism and investigating roles of Mcl-1 in FBW7-mediated Gefitinib resistance. Results:1. The PC-9GR cells were resistant to Gefitinib and did not harbored T790 M or Met amplification. 2. Expression of FBW7 at mRNA and protein levels was significantly higer in the Gefitinib sensitive PC-9 and HCC827 cells than the Gefitinib resistant PC-9GR, HCC827GR5 and HCC827GR6 cells. 3. Downregulation of FBW7 by shRNA assay reduced Gefitinib-induced apoptosis and led to a relatively resistant phenotype in PC-9 and HCC827 cells. 4. Mcl-1 protein was significantly higher in Gefitinib resistant PC-9GR, HCC827GR5 and HCC827GR6 cells. FBW7 affected Gefitinib sensitivity by regulating Mcl-1 degradation. Conclusion:1. The expression of FBW7 is higher in Gefitinib sensitive PC-9 and HCC827 cells. The PC-9GR cells did not harbored T790 M or Met amplification, suggesting that downregulation of FBW7 might be a new mechanism of Gefitinib resistance. 2. Downregulating FBW7 expression in PC-9 and HCC827 parental cells induced a resistant phenotype and reduced apoptosis, which might be attributed to the stabilization of anti-apoptotic proteins. 3. Downregulation of FBW7 stablized Mcl-1 expression and reduced its degradation. Mcl-1 might participate in the development of Gefitinib resistance.