towards improved death receptor targeted therapy for ... - TI Pharma
towards improved death receptor targeted therapy for ... - TI Pharma towards improved death receptor targeted therapy for ... - TI Pharma
TRAIL‐induced migration and invasion Non‐canonical TRAIL‐dependent kinase activities are reflected in altered levels of target protein phosphorylation The kinome profiling assay measures kinase activities, but within a cell kinase activities are counterbalanced by the enzymatic activity of phosphatases. Hence it is important to establish if the differences in kinase activities as determined by kinome profiling are reflected in altered levels of phosphorylation of target proteins. In accordance with the PepChip results, Western blot experiments revealed that already within 5 min after TRAIL treatment Src and ERK were phosphorylated, whereas Akt, STAT3 and FAK phoshorylation were detectable at 10 to 15 min post‐treatment in A549 cells (Fig. 3A). However, not all identified possible targets or upstream kinases could be corroborated. For example, TRAIL‐induced VAV2 and JAK2 phosphorylation was not observed by Western Blotting, while we did not find evidence of activation of ROCK (not shown). Basal and TRAIL‐induced phosphorylation of a number of kinases, including Src, Akt, ERK, FAK and STAT3, was variable in the other cell lines (Fig. 3B). In TRAIL resistant SW1573 cells increases in phosphorylation of Src ERK, and FAK by TRAIL were seen. In H460 no detectable effects were observed and in intermediate TRAIL sensitive H322 cells, ERK phosphorylation was slightly elevated after TRAIL exposure (Fig. 3B). Thus the non‐ canonical TRAIL signaling pathway identified by kinome profiling is reflected in meaningful changes in target protein phosphorylation. Figure 3. TRAIL‐induced kinase activation in NSCLC cells; confirming kinases identified by PepChip kinase arrays. (A) the indicated kinases were evaluated for TRAIL‐induced phosphorylation during time by Western blotting in A549 cells using antibodies detecting phosphorylated and total kinase. Numbers represent the quantified bands as ratio of the intensity of bands for the phosphorylated form: band of the total protein. The untreated control was set at 1.0. (B) kinase activation in H460, H322, SW1573, and A549 cells for 15 min after treatment with 50 ng/ml TRAIL determined by Western blotting. ‐ 69 ‐
Chapter 4 Non‐canonical TRAIL responses are mediated through the TRAIL‐R2 Earlier we generated TRAIL variants that selectively engage either TRAIL‐R1 or TRAIL‐R2, named 4C7 and DHER respectively [21;22]. We found these variants to be more potent than wild‐type TRAIL in triggering apoptosis in a tumor cell line‐dependent fashion. We now employed these variants to determine the TRAIL receptor mainly responsible for the induction of non‐canonical pro‐invasive properties of TRAIL. A549 cells express both TRAIL‐R1 and –R2, whereas decoy receptors are hardly detectable on the cell surface (Fig. 4A), in agreement with our previous findings [23]. As shown in Fig. 4B and C, migration and invasion of A549 cells is enhanced when stimulated with the TRAIL‐R2 selective ligand (DHER), and not by TRAIL‐R1 selective 4C7. Thus, the non‐canonical migratory and invasive features of TRAIL in resistant NSCLC cells are predominantly mediated by TRAIL‐ R2. Furthermore, comparing DHER‐ with TRAIL‐treated A549 cells showed a similar kinase phosphorylation profile (Fig. 4D). Figure 4. TRAIL induces migration and invasion in A549 cells mainly via TRAIL‐R2. (A) TRAIL receptor surface expression in A549 cells determined by FACS analysis. (B) effect of TRAIL‐R1 (4C7) and TRAIL‐R2 (DHER) specific TRAIL variants on migration, and (C) invasion are shown. Levels of untreated cells were set at 100%. The averages of experimental triplicates (±s.d.) are shown. *p
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TRAIL‐induced migration and invasion<br />
Non‐canonical TRAIL‐dependent kinase activities are reflected in altered levels of target<br />
protein phosphorylation<br />
The kinome profiling assay measures kinase activities, but within a cell kinase activities<br />
are counterbalanced by the enzymatic activity of phosphatases. Hence it is important to<br />
establish if the differences in kinase activities as determined by kinome profiling are<br />
reflected in altered levels of phosphorylation of target proteins. In accordance with the<br />
PepChip results, Western blot experiments revealed that already within 5 min after TRAIL<br />
treatment Src and ERK were phosphorylated, whereas Akt, STAT3 and FAK<br />
phoshorylation were detectable at 10 to 15 min post‐treatment in A549 cells (Fig. 3A).<br />
However, not all identified possible targets or upstream kinases could be corroborated.<br />
For example, TRAIL‐induced VAV2 and JAK2 phosphorylation was not observed by<br />
Western Blotting, while we did not find evidence of activation of ROCK (not shown). Basal<br />
and TRAIL‐induced phosphorylation of a number of kinases, including Src, Akt, ERK, FAK<br />
and STAT3, was variable in the other cell lines (Fig. 3B). In TRAIL resistant SW1573 cells<br />
increases in phosphorylation of Src ERK, and FAK by TRAIL were seen. In H460 no<br />
detectable effects were observed and in intermediate TRAIL sensitive H322 cells, ERK<br />
phosphorylation was slightly elevated after TRAIL exposure (Fig. 3B). Thus the non‐<br />
canonical TRAIL signaling pathway identified by kinome profiling is reflected in<br />
meaningful changes in target protein phosphorylation.<br />
Figure 3. TRAIL‐induced kinase activation in NSCLC cells; confirming kinases identified by PepChip<br />
kinase arrays. (A) the indicated kinases were evaluated <strong>for</strong> TRAIL‐induced phosphorylation during<br />
time by Western blotting in A549 cells using antibodies detecting phosphorylated and total kinase.<br />
Numbers represent the quantified bands as ratio of the intensity of bands <strong>for</strong> the phosphorylated<br />
<strong>for</strong>m: band of the total protein. The untreated control was set at 1.0. (B) kinase activation in H460,<br />
H322, SW1573, and A549 cells <strong>for</strong> 15 min after treatment with 50 ng/ml TRAIL determined by<br />
Western blotting.<br />
‐ 69 ‐
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