Clinical lung cancer

Clinical lung cancer. contrasting previous observations of constitutive binding between inactive Src and TKI-sensitive L858R MT EGFR. Next, we demonstrate that Src inhibition restores TKI sensitivity in CS-exposed NSCLC cells, preventing EGFR auto-phosphorylation in the presence of Erlotinib. Furthermore, we show that over-expression of a dominant-negative Src (Y527F/K295R) restores TKI sensitivity to A549 exposed to CS. Importantly, the TKI resistance that emerges even in CS-exposed L858R EGFR expressing NSCLC cells could be eliminated with Src inhibition. Together, these findings offer new rationale for using Src inhibitors for treating TKI-resistant NSCLC commonly observed in smokers. and 0.05 in respect to control (-). TKI-resistant phenotype emerges when Src is bound to the EGFR in its active, open state We next tested whether the CS-induced interaction between EGFR and Src (21) is dependent on Src activity. Serum-starved A549 cells were incubated with 10 M PP1-PP2 for 30 min. and 5,6-Dihydrouridine then treated with smoke, as before. Cells Rabbit Polyclonal to CSFR (phospho-Tyr699) were lysed, Src was IPed and the immuno-precipitates were IBed for total Src and total EGFR. As shown in Sup. Fig. 3A, EGFR was pull down together with Src in the presence of Src inhibitors, demonstrating that Src kinase activity is not mandatory for physical binding between Src and EGFR that occurs under CS exposure, similar to findings from a previous study with H2O2-induced oxidative stress (29). Subsequently, we investigated whether EGFR kinase activity is required for its binding to Src. To test this and to circumvent the fact that the kinase activity of the EGFR cannot be inhibited by TKIs upon CS exposure (as shown above in Fig. 2B), we transiently over-expressed either wild type (WT) or a kinase-dead (KD) mutant EGFR (K721A-EGFR) in CHO cells which do not endogenously express significant amounts of EGFR or other ErbB family members. 24h after transfection, cells were exposed to either EGF or CS, as before, and EGFR was IPed and IBed for pY416-Src, pY1173-EGFR, and total EGFR. Intriguingly, though the KD EGFR cannot be activated/ phosphorylated by either EGF or CS xposure, active Src still directly binds to 5,6-Dihydrouridine the KD EGFR following CS exposure (Sup. Fig. 3B), thus demonstrating that the kinase activity of EGFR and its subsequent auto-phosphorylation is not essential for its interaction with Src. To further confirm the requirement of Src in the emergence of EGFR resistance to TKIs, GFP-fused dominant-negative Src (DN-Src; K295R/Y527F) or a constitutively active form of Src (CA-Src; Y527F) were transiently expressed in A549 cells. Binding of these two Src constructs to EGFR was tested in 5,6-Dihydrouridine the presence of EGF or CS exposure. Intriguingly, our co-IP studies demonstrated that both GFP-tagged Src constructs (CA- and DN-Src) bind to EGFR in the presence and absence of CS exposure (Fig. 4), reiterating our previous observations that Src remained bound to the aberrantly-activated WT EGFR under CS stimulation despite Src inhibition. Moreover, it reaffirmed that an open conformation of Src is sufficient for its interaction/binding to the WT EGFR. Open in a 5,6-Dihydrouridine separate window Figure 4 CS-dependent TKI resistance of WT EGFR harboring NSCLC cells is caused by Src activationA549 cells were transiently-transfected with WT EGFR and either GFP-tagged Dominant-Negative (DN) Src or GFP-tagged Constitutively-Active (CA) Src; 24h post-transfection the cells were serum starved and incubated for 30 with 1M Erlotinib and then exposed to either EGF or CS, as before. A. EGFR was IPed from the total cell lysates and IBed for total Src (GFP) and total receptor. B. EGFR was IPed from the total cell lysates and IBed for total receptor, total tyrosine (Y) phosphorylation level (p-YEGFR) and specific Y site phosphorylation levels (p-Y1173, p-Y1068, p-Y845). Next, we further tested whether Src activity is required.

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