However, the selectivity was not as marked as that observed for the first-generation EGFR-TKIs

However, the selectivity was not as marked as that observed for the first-generation EGFR-TKIs. effectively downregulated cell surface EGFR and its downstream signals, and finally exerted antitumor effect in NSCLC cells harboring secondary (T790M/del19) and tertiary (C797S/T790M/del19) mutated EGFR, which exhibit acquired resistance to first- and third generation EGFR-TKIs, respectively. M-COPA also downregulated MET expression potentially involved in the acquired BTF2 resistance to EGFR-TKIs bypassing the EGFR pathway blockade. These results provide the first evidence that targeting the Golgi apparatus might be a promising therapeutic strategy to overcome the vicious cycle of TKI resistance in EGFR-mutated NSCLC cells downregulating cell surface RTK expression. and and [33]. These results prompted us to examine whether M-COPA might also be effective against tumor cells harboring an activating somatic mutation in a specific RTK gene. Uridine 5′-monophosphate In the present study, we examined the effect of M-COPA on NSCLC cells harboring an EGFR activating mutation, especially those exhibiting acquired resistance to EGFR-TKIs. We report the first evidence that M-COPA has a preferential antitumor effect on NSCLC cells harboring activating L858R and del19 mutations, but also those with a T790M/del19 double mutation and C797S/T790M/del19 triple mutation, which exhibit resistance to first- and third-generation EGFR-TKIs, respectively. M-COPA markedly downregulated the cell surface expression of EGFR irrespective of its mutation status, and also downregulated MET expression exclusively observed in EGFR-TKI-resistant cells. These results suggest that Golgi-targeted drugs might provide a novel therapeutic option for treating EGFR-activated NSCLC cells, and especially for overcoming TKI resistance by multiple mechanisms, by downregulating the cell surface expression of both mutated EGFR and MET involved in the EGFR-bypassing option pathway. RESULTS M-COPA inhibits the cell surface expression of EGFR and EGFR-downstream signal transduction pathways in NSCLC cell lines harboring an activating EGFR mutation First, we examined the effect of M-COPA treatment around Uridine 5′-monophosphate the cell surface expression of EGFR protein in NSCLC cell lines harboring an activating EGFR mutation by FCM analysis. As shown in Figure ?Determine1A,1A, the cell surface expression of EGFR was detected in the NSCLC cell lines NCI-H3255 (L858R), PC-9 (del19) and NCI-H1975 (T790M/L858R), and the expression levels were decreased in a dose-dependent manner upon treatment with M-COPA. In contrast, the baseline expression of EGFR at the cell surface was relatively low in NCI-H460 (EGFR-wild type and KRAS mutated) and its expression was only slightly affected upon treatment with M-COPA Open in a separate window Physique 1 M-COPA downregulates cell surface EGFR and its downstream signaling in EGFR-addicted cell lines(A) EGFR expression around the cell surface was measured by FCM analysis. Cells were treated with M-COPA at the indicated concentrations for 24 h, and stained with a PE-conjugated anti-EGFR antibody. Lines and areas are used to indicate drug concentrations: black solid lines with dark gray area, no drug; black dotted lines, 30 nM; black dashed lines, 100 nM; black long dashed lines, 300 nM; black chain lines with light gray area, 1000 nM; and gray solid lines, stained with isotype-control IgG. Experiments were performed at least twice and representative results are indicated. (B) Expression levels of total proteins and the phosphorylated forms of EGFR signaling molecules, including Akt, ribosomal S6 protein (S6), MEK, and ERK were examined by immunoblot analysis, upon treatment with M-COPA. Cells were treated with M-COPA at the indicated concentrations for 24 h, and cell extracts were prepared. Proteins in the cell extract were separated by SDS-PAGE and electroblotted onto a membrane. The membrane was then probed with antibodies against the indicated proteins. Experiments were performed at least twice and representative results are indicated. To clarify the effect of M-COPA on EGFR-downstream signal transduction pathways, we examined the expression levels of total proteins and phosphorylated forms of EGFR itself, Akt-mTOR pathway factors (Akt and ribosomal S6 protein) and MEK-ERK pathway factors Uridine 5′-monophosphate (MEK and ERK) by immunoblot analyses. As shown in Figure ?Physique1B,1B, the phosphorylated forms of EGFR, Akt (S473 and T308), S6 (S235/236 and S240/244), MEK and ERK were markedly reduced at an M-COPA concentration of 30 nM or higher in PC-9 cells, and 100 nM or higher in NCI-H3255 and NCI-H1975 cells, respectively..