Role Pacritinib aml of c Src in the process was first examined since Src is altered in NSCLC. H1650 SPAdh cells were treated with EGFR or Src TKIs and the levels of Oct4 and Sox2 was assessed by western blotting. EGFR inhib ition by 500 nM gefitinib or 200 nM BIBW as well as in hibition of Src activity by 200 nM dasatinib or 1 uM PP2 markedly reduced Sox2 expression. Oct4 level was not affected. These results were verified by immunoflorescence experiments. Similar to Oct4, there was no significant difference in Nanog expression. how ever, the number Sox2 positive cells were significantly decreased in response to the treatment of Inhibitors,Modulators,Libraries EGFR and Src TKIs. Inhibition of EGFR as well as Src signaling resulted in decreased phosphorylation of EGFR, Src, ERK and Akt.

Contribution of Inhibitors,Modulators,Libraries ERK and Akt pathways to EGFR mediated induction of Sox2 was next examined in H1650SPAdh cells. Phosphorylation of ERK was suppressed by MEK inhibitor PD98059 and AKT phosphorylation Inhibitors,Modulators,Libraries was suppressed by the PI3 kinase in hibitor, LY294002. However, PI3 Kinase inhibited H1650SPAdh cells also resulted in slight inhibition in ERK phosphorylation. A similar observation has been reported in earlier studies where PI3 Kinase sig naling was demonstrated to regulate the ERK phosphor ylation in T cell receptor signaling and PDGFR mediated signaling. However, as shown in Figure 5B, inhibition of MEK activity did not affect the levels of Sox2 while Inhibitors,Modulators,Libraries the PI3 kinase inhibition, markedly reduced its levels with corresponding reduction in SP fre quency and ABCG2 expression. These results were confirmed using siRNAs to Src and Akt.

As shown in Figure 5E, SP frequency was signifi cantly downregulated in both Akt and Src siRNA trans fected A549, H1650 and H1975 cells as compared Inhibitors,Modulators,Libraries to the control siRNA transfected cells, with a corresponding re duction in ABCG2 expression. Similar inhibi tory effects were observed upon silencing of two other Src family members, Fyn and Yes. To determine whether Src or Akt signaling facilitates self renewal of SP cells, sphere formation assay was con ducted on SP cells in presence or absence of Src inhibi tors Dasatinib or PP2, MEK inhibitor PD98059 as well as Akt inhibitor LY294002. As shown in Figures 5G and 5H, Src kinase inhibitors KRX-0401 dasatinib or PP2, as well as PI3K Akt inhibitor LY294002 showed a significant decrease in sphere formation. MEK in hibition by PD98059 did not have any significant effect on self renewal. The average size of the spheres formed was found to be 7 10 folds smaller than the untreated cells. Collectively, these data indicated that inhibition of EGFR Src Akt signaling results in depletion of Sox2 ex pression and decreased self renewal of SP cells.