Role of hypoxia and hypoxia induced factors in the development of breast cancer brain metastasis

Abstract

Here we studied the role of hypoxia and hypoxia-induced factors in the development of breast cancer brain metastasis by using ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. We detected hypoxia noninvasively by using a novel spectroscopic photoacoustic tomography technology (SPAT). Sprague-Dawley rats inoculated intracranially with ENU1564, a carcinogen-induced rat mammary adenocarcinoma cell line, were imaged with SPAT three weeks post inoculation. Proteins important for tumor angiogenesis and invasion were detected in hypoxic brain foci identified by SPAT and were elevated compared with control brain. We showed that HIF-1α, MMP-9, VEGF-A, and VEGFR2 (Fkl-1) protein and mRNA expression levels were higher (P < 0.05) in brain tumor tissues compared to normal brain. We also found an increased expression of HIF-1α proteins, MMP-9, VEGF-A and VEGFR2 mRNA and proteins in hypoxic ENU1564 cells in vitro. We also demonstrated the involvement of PI3K-Akt pathway in hypoxic regulation of MMP-9 and VEGF but not VEGFR2 by using specific PI3K inhibitor. Using MEK1/2 inhibitor we showed that hypoxic regulation of MMP-9, VEGF-A and VEGFR2 also involve MEK1/2-ERK pathway. We also investigated the effect of fibroblast growth factor-1 (FGF-1), one of the factors known to be upregulated by hypoxia, on the expression of MMP-9 in ENU1564 cell line. We observed that FGF-1 induces an increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. Next, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-κBp65, and enhances degradation of cytoplasmic IκBα. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data showed that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-κB and AP-1 in FGF-1-treated cells and that mutation of either NF-κB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1.