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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-3082 |
| Date | 15 May 2009 |
| Creators | Lungu, Gina Florentina |
| Contributors | Stoica, Gheorghe |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | electronic, application/pdf, born digital |
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