Supplementary Video S1: PF-228's effect on NCI-446 SCLC cell line migration. NCI-H446 cell lines were grown to confluence in 12-well plates. Cell monolayers were wounded using a micropipette tip and floating cells were washed off with PBS. After overnight incubation, PF-228 at a concentration of 3µM or DMSO was added to culture medium. Cell movements within wounded area were monitored for 12h starting from the time drug was added using a Zeiss Axiovert 200M microscope (Zeiss, Thornwood, NY) at x200 magnification. Images were captured every 15-minutes from five different fields randomly selected in each well. They show that PF-228 decreased migration velocity in NCI-H446 cell lines.
ARTICLE ABSTRACT
Small cell lung cancer (SCLC) has a poor prognosis. Focal adhesion kinase (FAK) is a non–receptor tyrosine kinase regulating cell proliferation, survival, migration, and invasion, which is overexpressed and/or activated in several cancers, including SCLC. We wanted to determine whether FAK contributes to SCLC aggressive behavior. We first evaluated the effect of FAK small-molecule inhibitor PF-573,228 in NCI-H82, NCI-H146, NCI-H196, and NCI-H446 SCLC cell lines. PF-573,228 (0.1–5 μmol/L) inhibited FAK activity by decreasing phospho-FAK (Tyr397), without modifying total FAK expression. PF-573,228 decreased proliferation, decreased DNA synthesis, induced cell-cycle arrest in G2–M phases, and increased apoptosis in all cell lines. PF-573,228 also decreased motility in adherent cell lines. To make sure that these effects were not off-target, we then used a genetic method to inhibit FAK in NCI-H82 and NCI-H446, namely stable transduction with FAK shRNA and/or FAK-related nonkinase (FRNK), a splice variant lacking the N-terminal and kinase domains. Although FAK shRNA transduction decreased total and phospho-FAK (Tyr397) expression, it did not affect proliferation, DNA synthesis, or progression through cell cycle. However, restoration of FAK-targeting (FAT) domain (attached to focal adhesion complex where it inhibits pro-proliferative proteins such as Rac-1) by FRNK transduction inhibited proliferation, DNA synthesis, and induced apoptosis. Moreover, although FAK shRNA transduction increased active Rac1 level, FRNK reexpression in cells previously transduced with FAK shRNA decreased it. Therefore, FAK appears important in SCLC biology and targeting its kinase domain may have a therapeutic potential, while targeting its FAT domain should be avoided to prevent Rac1-mediated protumoral activity.
