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Enzastaurin inhibits invasion and metastasis in lung cancer by diverse molecules.

Körner A, Mudduluru G, Manegold C, Allgayer H - Br. J. Cancer (2010)

Bottom Line: However, an ability of this compound to inhibit cancer invasion and metastasis is not yet clearly elucidated.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery Mannheim/Molecular Oncology of Solid Tumors (German Cancer Research Center-DKFZ-Heidelberg), Mannheim Medical Faculty, Ruprecht-Karls-University Heidelberg, Mannheim 68167, Germany.

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.

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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.
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fig3: 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.

Mentions: 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.


Enzastaurin inhibits invasion and metastasis in lung cancer by diverse molecules.

Körner A, Mudduluru G, Manegold C, Allgayer H - Br. J. Cancer (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2966618&req=5

fig3: 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.
Mentions: 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.

Bottom Line: However, an ability of this compound to inhibit cancer invasion and metastasis is not yet clearly elucidated.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery Mannheim/Molecular Oncology of Solid Tumors (German Cancer Research Center-DKFZ-Heidelberg), Mannheim Medical Faculty, Ruprecht-Karls-University Heidelberg, Mannheim 68167, Germany.

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.

Show MeSH
Related in: MedlinePlus