Platelet-type 12-lipoxygenase induces MMP9 expression and cellular invasion via activation of PI3K/Akt/NF-κB

Abstract

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of death in males in the United States. Using human prostate cancer specimens, the authors have previously shown that elevated expression levels of 12-lipoxygenase (12-LOX) occurred more frequently in advanced stage, high-grade prostate cancer, suggesting that 12-LOX expression is associated with carcinoma progression and invasion. Previous reports from their group and others have shown that 12-LOX is a positive modulator of invasion and metastasis; however, the mechanism remains unclear. In this work, a new link between 12-LOX and the matrix metalloproteinase 9 (MMP9) in prostate cancer angiogenesis is reported. This study demonstrated that overexpression of 12-LOX in prostate cancer PC-3 cells resulted in elevated expression of MMP9 mRNA, protein and secretion. Exogenous addition of 12(S)-hydroxy eicosatetraenoic acid, the sole and stable end product of arachidonic acid metabolism by 12-LOX, is able to increase MMP9 expression in wild-type PC-3 cells. Furthermore, using pharmacological and genetic inhibition approaches, it was found that 12-LOX activates phosphoinositol 3 kinase (PI3K)/Akt, which results in nuclear factor-kappa B (NF-κB)-driven MMP9 expression, ensuing in enhanced chemoattraction of endothelial cells. Specific inhibitors of 12-LOX, PI3K or NF-κB inhibited MMP9 expression in 12-LOX-expressing PC-3 cells and resulted in the blockade of the migratory ability of endothelial cells. In summary, the authors have identified a new pathway by which overexpression of 12-LOX in prostate cancer cells leads to augmented production of MMP9 via activation of PI3K/Akt/NF-κB signaling. The role of 12-LOX-mediated MMP9 secretion in endothelial cell migration may account for the proangiogenic function of 12-LOX in prostate cancer.

Keywords: 12-lipoxygenase; MMP9; NF-κB; angiogenesis; matrix metalloproteinase; prostate cancer.

Figure 1

Western blot analysis of nL8 cells shows higher 12-LOX (a) and MMP9 (b) protein levels compared to neo-α control cells. nL8 cells show an elevated mRNA level of MMP9 by using real-time PCR (d and f). Gelatin zymogram analysis of culture supernatant of nL8 cells demonstrated an elevated gelatinolytic activity compared to neo-α control cells (g). Tumor sections from nL8 cells or neo-α cells subcutaneously injected into mice were immunostained using antibodies specific for 12-LOX and MMP9, which show brown, positive staining compared to neo-α cell-injected tumor (h). Arrows (→) indicate positive staining of 12-LOX and MMP-9. Slides stained with secondary antibodies (IgG control) are shown as an indication of the staining specificity (scale bar: 50 µm). ^#p < 0.05 according to Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Exogenous 12(S)-HETE induces the MMP-9 expression in PC3 cells. PC-3 wild-type cells were exposed to the indicated concentrations of 12(S)-HETE for 18 hr, after which conditioned medium was collected and measured by ELISA using MMP9 R&D assay kit (a). MMP9 protein levels increase in 12(S)-HETE-treated wild-type PC-3 cells in a dose-dependent manner (b). Gelatin zymo-gram analysis of conditioned medium of 12-HETE-treated wild-type PC-3 cells shows elevated gelatinase activity in a dose-dependent manner (d). The results are representative of at least three independent experiments. *p <0.01 according to Student’s t-test.

Figure 3

12-LOX-induced MMP9 expression is mediated through the PI3K/Akt/NF-κB pathway. nL8 cells were exposed to PI3K inhibitor (20 µM), NF-κB inhibitor (20 µM), 12-LOX inhibitor (20 µM) and solvent control (S.C.) for 18 hr. After treatment, cells were lysed and subjected to Western blot analysis with an anti-MMP9 antibody (a). The blot was reprobed with actin as a protein loading control (b). MMP9 was detected in cell-conditioned medium by zymography (c). nL8 cells were exposed to 20 µM LY294002 or MG-132, and Baicalein shows a decrease in MMP9 levels (d) compared to vehicle control. ^#p < 0.05 according to Student’s t-test.

Figure 4

12-LOX induces MMP9 expression in NF-κB p50- and p65-dependent manner. nL8 cells were transfected with NF-κB p65 or p50 siRNAs or scrambled siRNA. After 72 hr, nuclear protein extracts were subject to Western blot analysis with anti-p65 and anti-p50 antibodies (a and c). Actin was used as an internal control (b and d). The effect of NF-κB p65 or p50 siRNAs on mRNA levels of MMP9 in nL8 cells was determined by RT-PCR (e). GAPDH was used as an internal control (f). Protein expression levels of MMP9 in conditioned media were determined by immunoblot analysis (g). Gelatin zymogram assay was performed on the cells transfected with siRNAs for p65 and p50 to analyze gelatinolytic activities of secreted MMP9 (i). The effect of NF-κB p65 or p50 siRNA on MMP9 levels in nL8 cells was determined by ELISA. nL8 cells were transfected with NF-κB p65 or p50 siRNAs or scrambled siRNA. After 72 hr, MMP9 levels were determined by ELISA (j). ^#p <0.05 according to Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

Effect of PI3K/Akt/NF-κB/12-LOX inhibitors on nL8 cells and HUVEC migration. nL8 cells were exposed to PI3K/Akt/NF-κB and 12-LOX inhibitors. After 24 hr, conditioned medium (CM) from 12-LOX-transfected PC-3 cells and vector controls were harvested and concentrated (6×) using Centricon-10. HUVEC migration assay was carried out through exposure to CM from neo-α and nL8 cells in the presence or absence of PI3K/Akt/NF-κB and 12-LOX inhibitors. CM from nL8 cells demonstrated significant increase in HUVEC migration compared to empty vector (ev). CM from nL8 cells after exposure to PI3K/Akt/NF-κB and 12-LOX inhibitors demonstrates significant decrease in HUVEC migration compared to nontreated nL8 cell CM (a). Representative phase contrast microscopy images are shown in correlation with the invasion assay (b). Scale bar: 50 µm.

Figure 6

Stimulation of endothelial cell migration by nL8 is blocked by an MMP9-neutralizing antibody. Conditioned media (CM) from nL8 and neo-α cells were harvested and concentrated. The migrated endothelial cells in response to the CM are shown. This increased stimulation of endothelial cell migration was blocked by the pretreatment with a MMP9-neutralizing antibody. The migration of endothelial cells was expressed as the percentage of the control in which no CM was added (a). Column, the average percentage of migration compared to the medium control; bar, SD from six membranes. *p < 0.01 and ^#p < 0.05 according to Student’s t-test. Representative phase contrast microscopy images are shown in correlation with the invasion assay (b). Scale bar: 50 µm.