Enzastaurin (in DMSO) was a generous gift from Eli Lilly (Indianapolis, IN, USA). All cell lines (H460, LXF289, A427, H1395, A549, and H1299) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in ATCC-recommended medium (10% FBS, 37°C, 5% CO2). Fertilised special pathogen-free eggs were from Charles River (Hilden, Germany). Antibodies used for supershift/chromatin immunoprecipitation analysis (ChIP) experiments were from Santa Cruz Biotechnology (Santa Cruz, CA, USA): p-c-Jun (sc-822x), JunD (sc-74x), JunB (sc-46x), c-Fos (sc-52x), FosB (sc-7203x), Fra-1 (sc-22794x), Fra-2 (sc-604x), Sp1 (sc-59x), Sp3 (sc-644x), and unspecific IgG (sc-2338). Western blot antibodies against phospho-PKC(PAN) 9371, phospho-p44/42(Thr202/Tyr204) 4370, p44/42 9102, phospho-GSK3β(Ser9) 9336, and phospho-Akt(Ser473) 4058 were from Cell Signaling, Danvers, MA, USA, and Akt1 (sc-1618) and β-Actin (sc-1616R) were from Santa Cruz Biotechnology.

Cells (3 × 10³) per well were seeded in a 96-well plate in a total volume of 100 μl medium with 10% FBS. After 24 h, cells were treated either with DMSO or Enz at concentrations between 0 and 15 μ. After 72 h, the growth inhibitory effect was evaluated using 20 μl per well of CellTiter96 AqueousOneSolution (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Cells (3 × 10⁵) per well were seeded in a 24-well plate in a total volume of 500 μl medium (10% FBS). After 24 h, the medium was changed to serum-free and a wound created in the monolayer by using a pipette tip. Cells were washed twice (PBS), and incubated with fresh medium supplemented with DMSO or Enz. In case of EGF stimulation, EGF (100 n) was added 4 h after initial DMSO or Enz treatment. Digital pictures were taken at 0 and 48 h. To quantify migration, we calculated the diameter of the wound as the average of three different locations.

Cells (1 × 10⁵), starved in serum-free medium (24 h), were plated on transwell chambers precoated with 10 μg Matrigel. DMSO or Enz was applied to bottom and top chambers. In case of EGF, cells were pretreated (4 h) with DMSO or Enz, followed by EGF (100 n). 10% FBS medium in the bottom chamber was used as a chemoattractant. After 24 h, noninvading cells were removed with cotton swabs. Invaded cells were trypsinised and counted using the ATP luminescence-based motility-invasion assay as described (Leupold et al, 2007).

Total RNA was isolated as described (Mudduluru and Allgayer, 2008), and probe labelling and array hybridisation on Illumina Human Sentrix-8 BeadChip arrays (Illumina, San Diego, CA, USA) were conducted according to Illumina's recommended sample labelling procedure based on the modified Eberwine protocol (Eberwine et al, 1992). Scanning was carried out using a Beadstation array scanner, adjusted to a scaling factor of 1 and PMT settings at 430. Data extraction was carried out for all beads individually, and outliers were removed when median absolute deviation was >2.5. All remaining data points were used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated. Data analysis was carried out by normalisation of the signals using the quantile normalisation algorithm without background subtraction, and differentially regulated genes defined by calculating the standard deviation differences of a given probe in a one-by-one comparison of samples.

Cells were transiently transfected with 500 ng of −398/+51 u-PAR WT/Sp1- or AP1-mutant plasmid, or with pGL3 basic (Allgayer et al, 1999), and 50 ng of Renilla-luciferase plasmid as a transfection control using Effectene (Qiagen, Hilden, Germany). After 24 h, cells were treated with DMSO or Enz for another 24 h, and reporter assays performed using the Dual-Luciferase reporter assay system (Promega).

Total RNA isolation and cDNA synthesis was performed as described (Mudduluru and Allgayer, 2008). The u-PAR (Hs00182181; Applied Biosystems, Carlsbad, CA, USA) mRNA was quantified using TaqMan Universal PCR Master Mix and β-Actin (Human ACTB 4333762F; Applied Biosystems) as housekeeping gene. Quantification of HIF-1α, VEGFC, VHL, RASSF1, FHIT, Sp1, Sp3, and c-Jun mRNA was performed using SYBR Green-PCR-Master-Mix (Applied Biosystems). Relative expression of these genes was calculated by the 2^−ΔΔCT method. Primer sequences are provided in Supplementary Table 1.

ELISA and western blot analysis were performed as described (Leupold et al, 2007; Mudduluru and Allgayer, 2008). In brief, for ELISA, cells were either treated with DMSO or with Enz for 24 h. For western blot analysis, cells were serum starved for 24 h and treated with DMSO or Enz for another 24 h. Cells were stimulated with EGF (100 n) for 15 min. Cells were washed twice with ice-cold PBS and lysed with lysis buffer (Biosource, Camarillo, CA, USA). Protein concentration was determined by BCA (Pierce, Rockford, IL, USA). The u-PAR protein was assayed using the Imubind-u-PAR-ELISA kit (American Diagnostica, Hauppauge, NY, USA).

si-u-PAR(ID289377; Ambion, Austin, TX, USA) was used to knockdown the expression of u-PAR. A nonspecific si-RNA (scrambled) served as a negative control. Sh-RNA vectors of sh-u-PAR (RHS4430-99290516), and non-silencing control (RHS4346) were purchased from Open Biosystems (Huntsville, AL, USA). A549 and H1299 cells were transiently transfected using RNAiFect or Effectene, respectively (Qiagen). At 24 h after transfection, cells were used for MTT and Matrigel assays as described.

Cells (1 × 10⁶ per ml) were treated with Enz (as the respective IC₅₀ values) and harvested after 48 h either with or without transfections. Apoptosis induction by Enz was measured by the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) as described (Querfeld et al, 2006). According to the manufacturer's instructions, cells were incubated with 5 μl per test FITC-conjugated annexin V in the presence of 5 μl per test PI, and further screened by flow cytometry (BD Biosciences, San Diego, CA, USA). Annexin V-positive PI-positive cells correspond to apoptotic cells and are represented as a percent of apoptotic cells against DMSO-treated cells.

Gel shifts were performed as described (Mudduluru and Allgayer, 2008), using 5 μg of nuclear extract of DMSO- or Enz-treated (IC₅₀, 24 h) A549 cells. Chromatin immunoprecipitation was performed according to the manufacturer's protocol (Upstate, Lake Placid, NY, USA) as described (Leupold et al, 2007; Mudduluru and Allgayer, 2008), with 2 μg specific (Sp1, Sp3, and p-c-Jun) and nonspecific IgG overnight, using aliquots of precleared lysates. DNA was purified and eluted with 100 μl elution buffer (Qiagen reaction purification kit). PCR was performed according to Leupold et al (2007), for region −190/−171 (u-PAR-AP1). Amplification of region −152/−135 (u-PAR-Sp1/Sp3/AP-2α-like) was performed by SYBR Green Master Mix, with the specific primers: For 5′-AGGCAATCTGGGGACAGAG-3′ and Rev 5′-GGACTCCTCCCAGACGTTTT-3′.

The chorionallantoic membrane (CAM) assay was performed as described (Leupold et al, 2007), with few modifications. Briefly, 2 × 10⁶ cells were inoculated on the CAM of 10-day-old chicken embryos. On days 12 and 14, chicken embryos were treated intravenously with different concentrations of Enz (2 or 4 μ) or DMSO, respectively. Untreated embryos served as a control. On day 16, the embryos were killed. Metastasis was determined by harvesting lungs and liver, and processing the tissue for genomic human DNA by quantitative alu-PCR.

Statistical significance of differences between Enz- vs DMSO-treated samples were calculated with the Student's t-test (P⩽0.05 was considered as statistically significant, P⩽0.1 as a trend).

Six human NSCLC cell lines were tested for their sensitivity to Enz regarding proliferation (Figure 1A). Large cell-type H460 and adenocarcinoma LXF289 cells showed the highest sensitivity. A moderate response was observed in H1395 (adenocarcinoma) and A427 (squamous carcinoma) cells, whereas A549 (squamous carcinoma) and H1299 (adenocarcinoma) cells showed comparatively more resistance to Enz. Mean IC₅₀ values of the cells are presented in the box in Figure 1A. Similarly, viable cells were counted after Enz treatment, confirming the IC₅₀ concentrations determined by MTT assays (data not shown). Next, to determine if the calculated IC₅₀ concentrations of Enz were able to induce apoptosis and inhibit the known target molecules of Enz, apoptotic assays and western blot analysis were performed. H460, A549, and H1299 cells were treated for 48 h with their respective IC₅₀ concentrations. Enzastaurin (IC₅₀ concentrations) induced apoptosis of H460, A549, and H1299 cells compared with the DMSO-treated samples was shown in Figure 1B. Because EGFR-induced signalling represents a major pathway driving NSCLC progression, we in addition sought to investigate the ability of Enz to inhibit essential target molecules following EGF stimulation. H460, A549, and H1299 cells were pretreated either with DMSO, or with Enz, for 24 h, and stimulated, or unstimulated, with EGF (100 nmol l⁻¹) for 15 min before harvesting the cells. As results, we found that Enz treatment can inhibit the phosphorylation of PKC, AKT, p42/44, and GSK3β both in the presence and in the absence of EGF (Figure 1C, Supplementary Figure 1).

To determine whether Enz affects the migration and invasion of NSCLC cells in vitro, we evaluated the migratory (wound-healing assay) and invasive (Transwell in vitro invasion assay) capacity of H460, A549, and H1299 cells, after treatment. Enzastaurin- or Enz/EGF-treated cells displayed a significantly reduced migration and invasion as compared to DMSO- and EGF-stimulated control cells; respectively (P<0.02; Figure 1D), showing that Enz inhibits EGF-induced cell migration and invasion in addition to spontaneous invasion in NSCLC. This prompted further experiments to study changes in gene expression upon Enz treatment, to delineate major target molecules mediating Enz-inhibited invasion.

To analyse changes in gene expression after Enz treatment, we applied Illumina Human Sentrix-8 BeadChip assays. A549 cells were either treated with DMSO, or with Enz, for 24 or 48 h at the same concentration as used for inhibiting invasion. Total RNA was isolated, labelled, and hybridised to the arrays. After 24 h, 1255 genes (616 up, 639 down), and after 48 h, 2711 genes (1477 up, 1234 down) were significantly (⩾1.5-fold) deregulated after Enz treatment (Supplementary Tables 2 and 3), respectively. Comparing our microarray results with data present in the literature, we found that a considerable number of genes that have been described to be either up- or downregulated specifically in NSCLC in previous publications were significantly altered upon Enz treatment (Table 1). These genes include genes regulating cell proliferation, structure, adhesion, migration, invasion, or angiogenesis, or are cell-cycle regulators. We found that all of these genes as described in Table 1 were inversely regulated upon Enz treatment in A549 cells as compared to their gene expression status reported in previous publications on NSCLC for the untreated situation. Interestingly, three tumour suppressors (FHIT, RASSF1, and VHL), which have been reported to be downregulated in NSCLC (Wikman et al, 2002; Hayes et al, 2006), were upregulated upon Enz treatment, which was validated by RT–PCR (Figure 2A–C) at the mRNA level. Also, HIF1α, VEGFC, and u-PAR (Chen et al, 2001; Werle et al, 2004; Hayes et al, 2006), genes that are known to be key regulators of various aspects of carcinogenesis, invasion, and metastasis, were significantly downregulated after Enz treatment. The expression of these genes was validated in NSCLC cell lines through real-time PCR, which confirmed their cell line-independent regulation upon Enz treatment (Figure 3A–C). Taken together, Enz negatively regulates essential genes that are reportedly relevant for NSCLC, induces the expression of three NSCLC-related tumour-suppressors, and inhibits three essential genes of invasion and metastasis at the mRNA level.

The u-PAR has been reported to be one of the major metastasis-related genes being overexpressed in many cancer types, and also in NSCLC (Beer et al, 2002; Werle et al, 2004). Previous reports including our own have shown that an overexpression of this receptor is associated with a high invasive and metastatic capacity of NSCLC, and also EGF-induced, NSCLC cells, among other cancer types (Nikolova et al, 2009). Therefore, we decided to specifically analyse a putative function of the u-PAR in Enz-regulated invasion, and molecular mediators addressed by Enz to regulate u-PAR in NSCLC. ELISA showed a significant reduction of u-PAR protein after Enz treatment in H460 (P=0.0021), A549 (P=0.02), and H1299 (P=0.0022) cells as compared to the DMSO-treated samples (Figure 4A), in addition to the downregulation of u-PAR mRNA observed in the same cell lines in the previous section. It has been well reported that u-PAR gene expression in malignant cells is largely because of the transcriptional regulation of the gene (Allgayer et al, 1999; Laufs et al, 2006; Leupold et al, 2007). Specifically, it has been reported that the u-PAR promoter, to a major part, is being regulated by transcription factors of the AP-1 and Sp family (Allgayer et al, 1999; Laufs et al, 2006; Leupold et al, 2007). Correspondingly, we found that Enz treatment in A549 and H1299 cells significantly downregulated the expression of Sp1-, Sp3-, and c-Jun-transcription factors (P<0.05; Supplementary Figure 2), which have been reported previously to be major regulators of u-PAR promoter activity. In luciferase assays of H460, A549, and H1299 cells transfected with a reporter plasmid driven by the −398/+51 upstream region of the u-PAR gene, we found a significant reduction in u-PAR wild-type promoter activity after Enz treatment (P<0.008) (Figure 4B). Moreover, site-directed mutagenesis abolishing the binding of either Sp- or AP-1-family members to their respective binding sites within the u-PAR promoter abolished the ability of Enz to induce a further reduction of promoter activity of the respective construct (Figure 4C). This was paralleled by a specific reduction of the binding of either Sp1, Sp3, or AP-1-family members to their specific cis elements within the u-PAR promoter in EMSA analysis (Supplementary Figure 3b and c), which could also be observed for the situation of EGF-induced A549 cells (Supplementary Figure 3a). Finally, to show that Enz is able to regulate transcription factor binding to the natural u-PAR promoter, we performed ChIP analysis for the endogenous u-PAR upstream region. Chromatin immunoprecipitation revealed significantly less binding of especially Sp1, and phospho-c-Jun, to the endogenous u-PAR promoter after Enz treatment (Figure 4D, Supplementary Figure 3d). Taken together, data suggest that Enz inhibits the expression of the u-PAR, at least to a relevant part by suppressing u-PAR promoter activity, by downregulation of the binding of especially Sp1, and c-Jun out of the AP-1 family.

A427, A459, and H1299 are relatively resistant to Enz treatment as compared to other NSCLC cell lines. This observation supported the notion that endogenous u-PAR gene expression might be associated with resistance to Enz. Therefore, we conducted specific knockdown experiments with an si-RNA/sh-RNA against endogenous u-PAR, the efficient knockdown of endogenous u-PAR being confirmed with u-PAR-ELISA (Supplementary Figure 4a and b). We observed that A549 and H1299 cells treated with si-u-PAR/sh-u-PAR were significantly re-sensitised to Enz (A549/IC₅₀⩽7.0 μ; H1299/IC₅₀⩽9.0 μ) when compared to scrambled control-transfected cells (A549/IC₅₀=10 μ; H1299/IC₅₀=12 μ) (Figure 4E). Similarly, viable cells were counted after Enz treatment either with scrambled/sh-RNA or with si-u-PAR/sh-u-PAR knockdown, which confirmed the IC₅₀ concentrations determined by MTT assay (data not shown). Furthermore, u-PAR knockdown cells significantly induced apoptosis when compared with control cells after 48 h of Enz treatment (Figure 4F), and also showed a further decrease in u-PAR protein levels after Enz treatment (Supplementary Figure 4a and b). These data suggest that u-PAR might be one molecular parameter indicating Enz resistance of NSCLC cells.

Our previous results suggested that Enz reduces migration and invasion in vitro. To strengthen these observations, we used the chicken embryo metastasis assay (CAM) (Leupold et al, 2007) to check the ability of Enz to inhibit the formation of distant metastasis in vivo. Towards this end, H460, A549, and H1299 cells were inoculated on the upper CAM of 10-day-old chicken embryos. On day 12 and 14, the embryos were treated intravenously with 2 or 4 μ of Enz, or equal concentrations of DMSO, respectively. On day 16, the embryos were killed, lungs and livers harvested for DNA, and the number of cells metastasised into livers and lungs were measured with a TaqMan-based PCR amplifying human alu-sequences on the chicken background (Leupold et al, 2007). As a result, we found that Enz was able to reduce the formation of distant metastasis significantly in H460 (liver P=0.001, lungs P=0.003), A549 (liver P=0.037, lungs P⩽0.01), and H1299 (liver P=0.02, lungs P=0.05) cells when compared with their respective DMSO controls (Figure 5A–C). These data suggest that Enz, besides migration and invasion, inhibits in vivo metastasis of three NSCLC cell lines.