SLK knockdown or expression of a dominant negative SLK inhibits cell migration.

<p>Subconfluent MEF 3T3 cells were infected with Adenovirus vectors expressing DN SLK (Ad-HA-KΔC) or LacZ control and subjected to fibronectin (FN) transwell migration assays. (A) Western blot analysis of HA-KΔC expression. Cdc42 was used as a loading control. (B) Polycarbonate membranes were DAPI stained and cells on the underside were enumerated (C) in random fields and expressed as the average/field from triplicate wells. (D) MEF 3T3 cells were transfected with SLK siRNAs and analysed for SLK expression. Western blot analysis of treated lysates indicates that SLK siRNA at 10 pM resulted in a marked knockdown of SLK. Reprobing the membrane with a α-tubulin antibody was used as a control for loading (lower panel). (E–F) Cells were treated with SLK-specific or control siRNAs and assayed for migration through a chamber coated with bovine serum albumin (BSA) (10 µg/ml) (control) or fibronectin (FN) (10 µg/ml). In both cases a 60–70% reduction in migration was observed. (G) Confluent MEF3T3 cells were infected with Adenovirus vectors expressing a scramble or SLK shRNA and manually scratched with a pipet tip. Wound closure was followed for 12 h and the percent closure was evaluated.</p

SLK activation requires FAK/src/MAPK signaling.

<p>(A) Confluent MEF 3T3 monolayers were pre-incubated (60 min) with inhibitors and then scratch wounded in the presence of inhibitors. Cells were collected 60 minutes later and analysed for SLK kinase activity. (A) Treatment with PP2 or PP3 control. (B) FAK-null or wildtype cells were subjected to scratch wound assays as above and assayed for SLK activity. (C) Treatment with U0126 and DMSO control. Phospho-Erk1/2 is shown as a control for U0126 treatment. SLK activation requires FAK/src/MAPK signaling.</p

SLK knockdown results in adhesion stabilization.

<p>Monolayers of MEF3T3 on FN were infected with adenovirus expressing shSLK or an shScramble control and scratch wounded. After 2 hours, the cells were fixed and stained for SLK (A, C, E and G) in combination with vinculin (B and D) or phalloidin (F and H). In addition to reduced SLK staining, shSLK expressing cells showed no SLK immunoreactivity at the leading edge with an increased number of focal adhesions. No overt differences were observed in phalloidin stained samples. Scale bar 10μ.</p

Recruitment of SLK at the leading is c-src-dependent.

<p>Confluent monolayers of FAK wildtype (A–B), FAK-null (C–D), SYF +c-src (E–F) and SYF (G–H) cells were scratch wounded and immunostained for SLK and Rac1. Similarly, MEF3T3 monolayers were pretreated with U0126 (30 min), scratch wounded and stained for SLK and Rac1 (I and J). SLK and Rac1 failed to be recruited to the leading edge in SYF cells. Scale bar 10μ</p

SLK is recruited to the leading edge.

<p>MEF 3T3 monolayers on fibronectin-coated coverslips were scratch wounded and allowed to migrate for 2–3 hours. Monolayers were immunostained for SLK in combination with actin (A–C), paxillin (D–F), α-tubulin (G–I), or Rac1 (J–L). In addition to perinuclear staining, SLK was found to be recruited into membrane ruffles (arrowheads) at the leading edge with the other markers surveyed. SLK was not found in mature adhesion complexes as shown by the lack of co-localization between SLK and paxillin in these structures (arrows). All photomicrographs are shown at 400×. Scale bar 10μ.</p

SLK is activated by scratch wounding and is required for cell migration.

<p>SLK is activated by scratch wounding and is required for cell migration.</p

Patient characteristics.

<p>Patient characteristics.</p

Inhibition of FAK reduces cell viability of EGFR TKI-resistant NSCLC cells.

<p>(<b>A</b>) Cell viability was assessed following treatment with PF-228 at varying doses for 48 hours in complete media with 5% FBS. Cell viability was assessed by MTT assay with the viability of DMSO-treated cells set as 100%. Data indicates the mean ± SEM for log[inhibitor] vs. normalized response from two independently performed experiments. (<b>B</b>) HCC4006 cells serially grown in increasing concentrations of erlotinib up to 3 μM were confirmed resistant compared to parental cells following MTT assay of cells treated with increasing concentrations of erlotinib. Data is graphed as the mean of log[inhibitor] vs normalized response ± SEM (N = 2). (<b>C</b>) Erlotinib resistant HCC4006 cells are more sensitive to FAK inhibitor PF-228 than parental HCC4006 cells. Cells were treated with increasing concentrations of PF-228 and cell viability was assessed by MTT assay. Data is presented as the mean of log[inhibitor] vs normalized response ± SEM (N = 2). (<b>D</b>) Relative protein expression between erlotinib resistant (A549, H1299, H1975) and sensitive (HCC827, HCC4006) parental cell lines. (<b>E</b>) A549 cell lysates were immunoprecipitated for endogenous FAK and subjected to <i>in vitro</i> kinase assays in the presence of either DMSO (vehicle control) or PF-228 (1 μM or 5 μM). Autoradiography revealed FAK phosphorylation (FAK AUTORAD) and western blotting indicated total levels of FAK (IB: FAK).</p

Increased apoptosis in NSCLC cells treated with erlotinib and FAK inhibitor PF-228 in combination.

<p>(<b>A</b>) Apoptotic cell fraction as determined by the percentage of cells in SubG1 cell cycle phase using propidium iodide staining and flow cytometric analysis following treatment for 48 hours with either vehicle control (DMSO), erlotinib (10 μM), PF-228 (1 μM or 5 μM), or both erlotinib and PF-228. Data presented is the mean ± SEM from two independently performed experiments. Statistically significant differences were determined by ANOVA. Asterisks denote statistically significant differences compared to control treated cells (** <i>P</i> < 0.01, *** <i>P</i> < 0.001), while significant differences between other treatment groups are displayed as <i>P</i> values above the comparison. (<b>B</b>) Western blot analysis indicating increased levels of cleaved PARP following drug treatment for 48 hours. Cells were treated with either vehicle control (DMSO), erlotinib (10 μM), PF-228 (1 μM or 5 μM), or both erlotinib and PF-228. β-actin was used as loading control. Data presented is representative of results obtained from two independently performed experiments.</p

LKB1 does not appear to affect sensitivity to combination treatment with erlotinib and PF-228.

<p>(<b>A</b>) Confirmation of LKB1 expression in FLAG-LKB1 transfected A549 cells. Stably transfected cells were assessed for LKB1 expression by western blot. β-actin was used as loading control. H1299 cells normally express wild-type LKB1 and were used as positive control. (<b>B</b>) Expression of LKB1 in A549 cells does not alter sensitivity to erlotinib and PF-228. Cell viability was assessed by MTT assay following incubation with erlotinib (10 μM), PF-228 (5 μM) or both drugs for 48 hours. Data is presented as the mean ± SEM of two independently performed experiments. Statistically significant differences were determined by ANOVA (** <i>P</i> < 0.01, *** <i>P</i> < 0.001). (<b>C</b>) Expression of LKB1 in A549 cells does not alter the phosphorylation pattern of Akt following treatment with erlotinib and PF-228. Cells were serum-starved overnight and treated with erlotinib (10 μM) and/or PF-228 at the indicated concentrations for 30 minutes prior to stimulation with EGF (100 ng/ml) for 30 minutes. Levels of phospho- and total Akt were assessed by western blot. β-actin was used as loading control. (<b>D</b>) LKB1 is effectively depleted in H1299 cells following siRNA transfection. Expression of LKB1 was determined by western blot 48 hours post-siRNA transfection. β-actin levels were used as a loading control. Image is representative of two independently performed experiments. (<b>E</b>) Sensitivity of H1299 cells to erlotinib and PF-228 is unaltered following depletion of LKB1. H1299 cells were transfected with 50 nM of either control siRNA or LKB1 siRNA. Cell viability was assessed by MTT assay following a 48 hour treatment with erlotinib (10 μM), PF-228 (5 μM) or both drugs in combination. Data is presented as the mean ± SEM of two independently performed experiments. Statistically significant differences were determined by ANOVA (* <i>P</i> < 0.05, ** <i>P</i> < 0.01). (<b>F</b>) The phosphorylation pattern of Akt is unaltered in response to erlotinib and PF-228 in H1299 cells depleted of LKB1 as compared to control transfected cells. Cells were stimulated with EGF (100 ng/ml) following treatment with erlotinib (10 μM) and/or PF-228 (5 μM) for 30 minutes. Levels of phospho- and total Akt were assessed by western blot. β-actin was used as loading control. Image is representative of two independently performed experiments.</p