We report for the first time about a lipoxygenase enzyme and its arachidonic metabolite positively influencing the level and activity of the transcription factor HIF-1α under hypoxic conditions. 12-LOX also enhances the nuclear localization, DNA binding activity, and transcriptional activation of HIF-1α under hypoxia. The signaling mechanism involved in the activation of HIF-1 by 12-LOX was found to be mediated by the PI3K-Akt pathway, inhibition of which diminishes the ability of 12-LOX to upregulate HIF-1α. Finally, inhibition of HIF-1α by siRNA in 12-LOX overexpressing cells affects the secretion of VEGF in these cells, clearly demonstrating the influence of 12-LOX in eliciting VEGF expression via activation of the HIF-1 pathway.
Immunoblotting performed from cell lysates of 12-LOX overexpressing PC-3 and vector control cells incubated under hypoxic conditions, showed an increase in the level of HIF-1α protein, thereby indicating the presence of a regulatory pathway connecting these molecules. Similar results were also obtained from incubation of wild-type PC-3 cells with exogenously added 12(S)-HETE. Inhibition of 12-LOX activity using a LOX inhibitor ETYA, or two chemically unrelated specific 12-LOX inhibitors baicalein or BMD-122 (data not shown), decreased the level of HIF-1α protein emphasizing the necessity for 12-LOX enzymatic activity for the HIF-1 response. Other eicosanoids have been shown to regulate the expression and function of HIF-1α in a number of cancer cells. For example, COX-2 overexpression resulted in the upregulation of HIF-1 in gastric carcinoma cells. The same study also showed that treatment of gastric carcinoma cells with exogenously added PGE2 upregulated the HIF-1α protein level [15
]. However, it was also observed in prostate cancer PC-3ML cells that PGE2 treatment did not increase the amount of HIF-1α protein, but only led to a nuclear translocation of HIF-1α, enhancing its availability within the nuclear pool [14
]. Studies have also revealed that indomethacin treatment inhibits HIF-1α levels in both PC-3 and Du145 prostate cancer cell lines, but on the contrary were found to be COX-2 independent [16
Real time PCR and northern blotting demonstrated an increase in HIF-1α mRNA in 12-LOX overexpressing cells. Several mechanisms have been proposed that can enhance HIF-1α levels and most of these revolve around the posttranslational stabilization and inhibition of protein degradation [1
]. The presence of Sp1 binding sites on the promoter region of the HIF-1 gene has been identified by systematic sequence analysis [17
]. Promoter mutagenesis experiments performed for studying VEGF expression have revealed the significance of Sp1 on VEGF transcription triggered by 12-LOX [8
] and it is possible that 12-LOX could activate HIF-1α transcription via Sp1. In addition to Sp1, NF-κB binding sites also have been identified in the HIF-1 promoter region, and an NF-κB dependent increase in expression of HIF-1α has been documented in human monocytes in response to bacterial LPS under aerobic conditions [19
]. We have previously reported the stimulation of NF-κB by 12-LOX and 12(S)-HETE in prostate cancer cells and hence it is possible that 12-LOX may enhance HIF-1α transcription via NF-κB [20
Earlier studies showed that the PI3K-Akt pathway is pivotal in mediating 12-LOX induced VEGF expression and secretion [8
]. The present work has revealed that 12-LOX utilizes the same pathway in upregulating HIF-1α levels under hypoxic conditions. Additionally we have demonstrated that mTOR plays a role in 12-LOX mediated upregulation of HIF-1 under hypoxia. An active PI3K-Akt pathway has been linked to the stimulation of HIF-1 under hypoxic conditions, for example in Hep G2 cells, inhibition of the pathway led to a decrease in HIF-1α protein level [21
HIF-1α activated by 12-LOX was found to be transcriptionally active as determined by nuclear localization, DNA binding ability, and functional assays such as promoter assays using the 5xHRE vector and siRNA studies. The ability of 12-LOX to influence VEGF expression via multiple transcription factors is evident in the siRNA studies. The incomplete abolition of VEGF transcription and secretion () upon introduction of HIF-1α siRNA suggests that other transcription factors may also play a role to compensate the loss of HIF-1α. These may include Sp1 and AP2, which were shown to be involved in 12-LOX dependent upregulation of VEGF, as reported previously [8
]. Collectively these findings demonstrate a link between 12-LOX and HIF-1 that may trigger angiogenesis in tumors. By upregulating HIF-1, 12-LOX cannot only turn on the angiogenic switch, but can also regulate other downstream targets of HIF-1α such as GLUT-1. This in turn improves the availability of glucose inside anaerobic cells thereby enhancing metabolism and survival under conditions of high energy demand amidst an environment of low oxygen supply. The summary of these interactions is depicted in .
Summary of regulation of HIF-1α by 12-LOX in hypoxic prostate cancer cells
HIF-1α expression and function was reported to be a poor prognostic indicator in tumors, because this promotes improved cell survival, angiogenesis, and resistance to therapy [5
]. By triggering the HIF-1 pathway, 12-LOX overexpressing cells within a heterogeneous population of cells in a solid tumor may stimulate the angiogenic response. In the absence of tumor suppressors such as p53 and PTEN, activation of this pathway can lead to the formation of highly aggressive tumors that may have a higher potential for metastasis and resistance to therapy. Use of 12-LOX inhibitors, in combination with other therapeutic regimens may prove successful in managing solid tumors under such conditions.