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Description:
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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 . |