Mutations in epidermal growth factor receptor (EGFR) rendering it constitutively active

Mutations in epidermal growth factor receptor (EGFR) rendering it constitutively active is one of the major causes for metastatic non-small-cell lung cancer (NSCLC) and EGFR-targeted therapies utilizing tyrosine kinase inhibitors (TKIs) are often used clinically as the first-line treatment. MET amplification requires EPAS1 since EPAS1 knock-down reduced MET levels. When NSCLC cells expressing T790M EGFR were treated with TKIs reduced EPAS1 levels BMPS significantly enhanced the drug effect whereas over-expression of EPAS1 increased the drug resistant effect. This EPAS1-dependent TKI-resistance was abolished by knocking-down MET suggesting that EPAS1 does not cause TKI-resistance itself but functions to bridge EGFR and MET interactions. Our findings suggest that EPAS1 is a key factor in the EGFR-MET crosstalk in conferring TKI-resistance in NSCLC cases and could be used as a potential therapeutic target in TKI-resistant NSCLC patients. (Fig. 1C middle and bottom panel lane 4). BMPS To rule out the possibility whether this selective interaction between EPAS1 and T790M EGFR was a cell line specific effect we did the same expression and co-immunoprecipitation assay in another NSCLC cell line A549 (Fig. S1). As expected EPAS1 only interacted with T790M but not wild-type EGFR in A549 cells meaning the binding between these 2 proteins is a interaction across different cell lines. Next we investigated whether the interaction between EPAS1 and T790M EGFR was a direct binding or not through protein crosslinking assay using dithio-bismaleimidoethane (DTME) as the crosslinker. HCC827 cells expressing HA-EPAS1 were also transfected with either wild-type or T790M EGFR and protein lysates were subjected to immunoprecipitation with HA antibody after the crosslinking. Same as the previous experiment T790M but not wild-type EGFR was pull-down together with HA-EPAS1 (Fig. 2 middle panel + DTT). Because DTME is a thiol-cleavable crosslinker removing DTT from the sample loading buffer could preserve the BMPS covalent bond between crosslinked protein pairs causing them to migrate slower in SDS-PAGE. Indeed at nonreducing conditions (- DTT) a band could be seen migrating around 250?kDa in the protein precipitate of T790M EGFR and HA-EPAS1 (Fig. BMPS 2 BMPS right panel open arrow heads) but was absent from the equivalent lane at reducing condition (Fig. 2 middle panel + DTT). Judged by its mobility this single band came from the direct crosslinking of HA-EPAS1 and T790M EGFR. Figure 2. EPAS1 directly binds T790M EGFR in protein crosslinking assay. HCC827 cells co-expressing HA-EPAS1 with either wild-type (Myc-EGFR) or T790M (Myc-T790M) Myc-tagged EGFR were incubated Rabbit Polyclonal to ADORA1. with crosslinker DTME (see Materials and Methods) and subjected to … EPAS1 and T790M EGFR interaction up-regulates MET pathway independent of EGF ligand binding In NSCLC cases aberrant activation of MET is the major cause for resistance to EGFR TKIs 27 because MET shares the same downstream pathway as EGFR.15 16 To test whether MET reacts to the interaction of EPAS1 and T790M EGFR we expressed T790M EGFR and EPAS1 in HCC827 cells simultaneously and examined MET protein levels using anti-c-Met antibody. As previously reported expression of wild-type and T790M EGFR alone was sufficient to trigger MET amplification 25 even in the absence of EPAS1 (Fig. 3A lanes 1 and 2 from left). EPAS1 expression combined with wild-type EGFR had no further effects on the level of MET (Fig. 3A lane 3) however when EPAS1 was co-expressed with T790M BMPS EGFR MET amplification was greatly enhanced (Fig. 3A lane 4 comparing with lane 2 and 3). In cells expressing just EPAS1 MET was only mildly activated (Fig. 3A lane 5 comparing with lane 2 and 4). These results have shown that EPAS1 and T790M EGFR interaction synergizes to up-regulate MET signaling pathway. The same results were also acquired from A549 cells (Fig. S2) again indicating this EPAS1 and T790M EGFR synergistic up-regulation of MET indeed reflected a general mechanism in NSCLC cells. Figure 3. EPAS1 interaction with T790M EGFR up-regulates MET independent of ligand binding. (A) Co-expression of T790M but not wild-type EGFR with EPAS1 increased MET levels. Whole cell lysates from HCC827 cells expressing HA-EPAS1 Myc-tagged wild-type EGFR (Myc-EGFR) … Although MET and EGFR both regulate MAPK and PI3K/AKT downstream pathways the 2 2 tyrosine.