Enzastaurin inhibits invasion and metastasis in lung cancer by diverse molecules

Abstract

Background: Enzastaurin (Enz) is a serine/threonine kinase inhibitor blocking protein kinase C (PKC)beta/AKT pathway. However, an ability of this compound to inhibit cancer invasion and metastasis is not yet clearly elucidated.

Methods: The ability of Enz to inhibit invasion and metastasis, and to target molecules was investigated in non-small cell lung cancer (NSCLC) by RT-PCR validated microarray, Matrigel, and in vivo chorionallantoic membrane (CAM) assays.

Results: Enzastaurin significantly reduced migration, invasion, and in vivo metastasis to lungs and liver (CAM assay) of diverse NSCLC cell lines. Genes promoting cancer progression (u-PAR, VEGFC, and HIF1alpha) and tumour suppression (VHL, RASSF1, and FHIT) of NSCLC were significantly (P<0.05) down- or upregulated after Enz treatment in H460, A549, and H1299 cells, respectively. Luciferase/chromatin immunoprecipitation analysis showed that Enz transcriptionally controls urokinase-type plasminogen activator receptor (u-PAR) expression by promoter inhibition through Sp1, Sp3, and c-Jun(AP-1). Moreover, siRNA knockdown of u-PAR re-sensitised Enz-resistant cells and induced apoptosis, suggesting u-PAR as a marker of Enz resistance.

Conclusion: This study shows that Enz inhibits migration, invasion, and in vivo metastasis by targeting u-PAR, besides further targeting progression-related and tumour-suppressor genes in NSCLC. Together with u-PAR being a novel putative marker of Enz response, these data encourage molecularly tailored clinical studies on Enz in NSCLC therapy.

Figure 1

Enzastaurin inhibits NSCLC proliferation, migration, and invasion. (A) Cell proliferation of NSCLC after Enz treatment. Data are expressed as the percentage of control (treated with DMSO) cells. Points, mean±s.d. of three experiments. Resulting IC₅₀ values for Enz for all NSCLC cell lines investigated are represented in the box. (B) Apoptosis analysis of H460, A549, H1299 after Enz treatment. After 48 h of Enz treatment, cells were screened by flow cytometry and percent of apoptotic cells was calculated against to DMSO-treated cells. (C) Western blot analysis of cells treated with EGF, Enz, or with the combination of both agents. Specific antibodies against the indicated molecules were used, together with anti-beta-Actin as a loading control. (C and D) Percentage of migrating cells (would-healing assay) and invading cells (Matrigel assay). Cells were treated as described in the Materials and Methods section. DMSO-treated samples served as a control (100%), and other treated samples were calculated and plotted as a percentage of this value. ^*P<0.05.

Figure 2

Enzastaurin induces the expression of tumour-suppressor genes. Real-time PCR quantification of (A) VHL, (B) RASSF1, and (C) FHIT mRNA in H460 and A549 cells after 24–48 h of DMSO or Enz (IC₅₀) treatment. Relative gene expression was normalised against β-Actin mRNA. ^*P<0.05.

Figure 3

Effect of enzastaurin on the expression of pro-invasive genes. Real-time PCR quantification of (A) HIF1α, (B) VEGFC, and (C) u-PAR mRNA in H460, A549, and H1299 cells after 24–48 h of DMSO or Enz (IC₅₀) treatment. Relative gene expression was normalised against β-Actin mRNA. ^*P<0.05.

Figure 4

Enzastaurin inhibits u-PAR promoter activity and u-PAR protein expression. (A) Total amounts of u-PAR protein as evaluated by ELISA as described in the Materials and Methods section. Each value represents the average of triplicate measurements. (B) Luciferase activity assay for H460, A549, and H1299 cells. Data are presented as the mean±s.d. of three independent experiments performed in quadruplicate. ^*P<0.05. (C) Luciferase activity assay of A549 cells transfected with wild-type (wt) u-PAR promoter, and Sp/AP1 binding site mutated reporter constructs. Values represent the mean of three independent experiments performed in triplicate; bars, s.d. (D) Real-time PCR quantification of u-PAR promoter binding after chromatin immunoprecipitation (ChIP) with Sp1, Sp3, p-c-Jun, or IgG antibodies in the presence, or absence, of Enz (24 h). (E and F) Cell viability (MTT) and apoptosis assay. At 24 h after transfection with si-u-PAR/sh-u-PAR, cells were treated with Enz for 48 h and the results compared to DMSO-treated controls for MTT assay, represented as percentage and for apoptosis cell were screened by flow cytometry and percent of apoptotic cells were calculated against to DMSO-treated cells.

Figure 5

Enzastaurin inhibits NSCLC metastasis in vivo. (A–C) Number of metastasising cells in the livers and lungs of chicken embryos treated intravenously with different concentrations of Enz (2 or 4 μM) or DMSO, respectively. Human Alu sequences were amplified and quantified by real-time PCR. ^**P<0.001; ^*P⩽0.05.