Fig 1: Nuclear FAK prevents p53 activation in response to IZI50 treatment.A Parental (p) and IZI5-conditioned (c) A375 and WM1346 cells were stimulated with IZI50, 5 µM defactinib (iFAK), or both. After 24 h the phosphorylation status of FAK, SRC, PDK1, PI3K, AKT, ERK, cJUN, and p38, as well as processing of caspase-3 and PARP was monitored by Western-blot analysis with β-actin and GAPDH as loading controls. One representative out of three independently performed experiments is shown. B Parental (p) A375 and WM1346 cells were stimulated with IZI50. IZI5-conditioned (c) cells were treated with either 5 µM QVD, 15 µM Nec1s, or both for 1 h prior to combined FAK inhibitor (iFAK) defactinib and IZI50 stimulation. After 24 h apoptosis induction was assessed using a CDDE (n = 3: *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001; n.s. = not significant). C Parental (p) and IZI5-conditioned (c) A375 and WM1346 cells were stimulated with IZI50. After 24 h the status of FAK, p53, p-p53(Ser15), caspase-8, caspase-3, caspase-9 and PARP, respectively, was determined in cytosolic (cyt) and nuclear (nuc) protein fractions by Western-blot analysis, with GAPDH as loading control. One representative out of three independently performed experiments is shown. D Mean expression (rlog counts) of pro-apoptotic BAK, BAX and PUMA genes in parental (p) and IZI5-conditioned (c) A375 and WM1346 cells in response IZI50 treatment for 6 h.
Fig 2: Integrin-mediated FAK activation causes upregulation of MAPK signaling pathways.A Mean expression (rlog counts) of the FAK/PTK2 interaction partners in the GeneWalk regulatory network in the different contexts of A375 and WM1346 cells. p=parental; c=conditioned; IZI = stimulated with 50 ng/ml IZI1551; A = A375; W = WM1346. FAK interaction partners that also represented within the 18 top regulators are marked in yellow. B Mean expression (rlog counts) of PRKDC, IQGAP, ITGAV, MET genes in parental (p) and IZI5-conditioned (c) A375 and WM1346 cells in response to IZI50 treatment for 6 h. C The surface expression of integrin αVβ3 on unstimulated parental (p) and IZI5-conditioned (c) A375 and WM1346 cells was determined by flow cytometry (n = 3: **p ≤ 0.01). D Parental (p) and IZI5-conditioned (c) A375 and WM1346 cells were stimulated with IZI50. After 24 h the phosphorylation status of FAK, SRC, PDK1, PI3K, AKT, ERK, cJUN, and p38 was monitored by Western-blot analysis with α-tubulin as loading control. One representative out of three independently performed experiments is shown. E Parental (p) A375 and WM1346 cells were stimulated with IZI50. IZI-conditioned (c) cells were treated for 1 h with the FAK inhibitor (iFAK; 5 µM) defactinib, the SRC inhibitor (iSrc; 10 µM) dasatinib, the PI3K inhibitor (iPI3K; 20 µM) alpelisib, the PDK1 inhibitor (iPDK1; 10 µM) GSK2334470, and the AKT inhibitor (iAKT; 20 µM) afuresertib, respectively, prior to IZI50 stimulation. After 24 h apoptosis induction was assessed using a CDDE (n = 3: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; n.s. = not significant).
Fig 3: Transcriptomic analysis identifies FAK as key regulator of the metastatic phenotype of IZI5-conditioned MM cells.A Parental (p) and conditioned (c) A375 and WM1346 cells remained untreated or where stimulated with IZI50 for six hrs. Subsequently, RNAseq analysis of three biological replicates was performed and gene expression changes presented by vulcano plots. Genes are represented as dots in −log10 (adjusted p-value) versus log2 (fold-change). Differentially expressed genes with adjusted p-value ≤ 0.1 and abs (fold-change) ≥ 1 are marked in red. B Differentially expressed genes with consistent directionality of change in the parental contexts of both cell lines, but not being differentially expressed in the conditioned contexts (1248 genes), were fed into GeneWalk with default settings. Regulators are represented as dots in ‘Fraction of Relevant GO Terms per Gene’ versus ‘Number of GO Annotations per Gene’. C Mean expression (rlog counts) of the FAK(PKT2) gene in parental (p) and IZI5-conditioned (c) A375 and WM1346 cells in response to IZI50 treatment for 6 h. D Parental (p) and IZI5-conditioned (c) A375 and WM1346 cells were stimulated with IZI50. After 24 h processing of FAK was determined by Western-blot analysis. One representative out of three independently performed experiments is shown. E Parental (p) A375 and WM1346 cells were pretreated or not with 5 µM QVD for 1 h. Subsequently cells were stimulated with IZI50 and cleavage of FAK monitored 24 h later by Western-blot analysis. One representative out of three independently performed experiments is shown. F Wildtype (wt) and caspase-8 knock out (clones #1 and #2) WM115 melanoma cells were stimulated with IZI50 and the protein/cleavage status of FAK and caspase-8 assessed after 24 h by Western-blot analysis. β-actin served as loading controls. One representative out of three independently performed experiments is shown.
Fig 4: FAK inhibition sensitizes resistant mutBRAF and mutNRAS melanoma to mutation-specific targeted kinase inhibition.A Parental mutBRAF Malme3M and WM3248 MM cells were conditioned to 1 µM BRAF inhibitor dabrafenib (Dabra); mutNRAS SkMel2 and SkMel147 cells to 1 nM MEK inhibitor trametinib (Trame). mutBRAF parental (p) and conditioned (c) cells were treated with 10 µM dabrafenib, dabrafenib + 1 µM trametinib, 2.5, 5, or 10 µM of the iFAK defactinib alone and in combination with 10 µM dabrafenib. Parental (p) and conditioned (c) mutNRAS cells were treated with 1 µM trametinib, trametinib + 10 µM dabrafenib, 2.5, 5 or 10 µM of the iFAK defactinib alone and in combination with trametinib. Cell death was monitored by PI (1 µg/ml) uptake using IncuCyte® live-cell analysis for 48 h. B Three mutBRAF (M45, M53, M59) and three mutNRAS (M10, M20, M32) cell samples freshly isolated from patients with relapsed melanoma metastases were treated as in A according to their mutation status. Cell death was monitored by PI (1 µg/ml) uptake using IncuCyte® technology for 48 h. C The same patient-derived metastatic cell samples as in (B) were treated with IZI50, 2.5, 5, or 10 µM of the iFAK defactinib alone and in combination with IZI50. Cell death was monitored by PI (1 µg/ml) uptake using IncuCyte® technology for 48 h. For each experiment n = 3 is shown (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; n.s. = not significant).
Fig 5: TRAIL-resistance and tumor outgrowth of IZI5-conditioned MM cells depends on FAK.A FAK was depleted in parental (p) and IZI5-conditioned (c) A375 and WM1346 cells using RNA interference (siFAK), with siLacZ as scrambled control. Transient knockdown of FAK was monitored 24 h later by Western-blot analysis, with β-actin as loading control. One representative out of three independently performed experiments is shown. B Cells as in A were stimulated with IZI50 and apoptosis induction determined 24 h later using a CDDE (n = 3: ***p ≤ 0.001; ****p ≤ 0.0001; n.s. = not significant). C Unstimulated cells as in A were subjected to a proliferation assay and cell numbers quantified at the indicated time points (n = 3: **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001). D GFP-expressing IZI5-conditioned (c) A375 and WM1346 cells were left untreated or treated with the FAK inhibitor defactinib (iFAK, 5 µM). Migration of cells was monitored by scratch assay (scale bar = 100 µm) and gap closure quantified after 24 h (n = 3: **p ≤ 0.01; ***p ≤ 0.001). E GFP-expressing spheroids consisting of IZI5-conditioned (c) mutBRAF (A375, Malme3M) and mutNRAS (WM1346, WM1366) melanoma cells, respectively, were individually embedded into 3D dextran-based gel-matrices and stimulated with IZI50 and 5 µM defactinib (iFAK) every other day over 8 days (scale bar = 200 µm). At day eight cell death was visualized by addition of 6.7 µg/ml PI. Confocal images of individual spheroids were taken at day one and eight and the tumor volume (green) versus tumor death (red) quantified at day eight (n = 3: *p ≤ 0.05; ***p ≤ 0.001).
Supplier Page from Cell Signaling Technology for SignalSilence ® FAK siRNA I