Hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients.
BACKGROUND
The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.


METHODS
The expression of cytokeratins (CK), VEGF, vascular endothelial growth factor receptor-2 (VEGF2), HIF-1alpha and phosphorylated-focal adhesion kinase (pFAK) in CTCs from 34 patients with metastatic breast cancer who had detectable CK-19 mRNA-positive CTCs was assessed using double staining experiments and confocal laser scanning microscopy. Peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with either VEGF or VEGFR2 or HIF-1alpha or pFAK antibodies, respectively.


RESULTS
pFAK expression in circulating tumor cells was detected in 92% of patients whereas expression of VEGF, VEGF2 and HIF-1alpha was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGF2, HIF-1alpha and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. Vascular endothelial growth mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1alpha and VEGF2.


CONCLUSIONS
The expression of pFAK, HIF-1alpha, VEGF and VEGF2 in CTCs of patients with metastatic breast cancer could explain the metastatic potential of these cells and may provide a therapeutic target for their elimination.
Kallergi
Galatea
G
Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes, Heraklion, 71110, Greece. kalergi@med.uoc.gr
Markomanolaki
Harris
H
Giannoukaraki
Vicky
V
Papadaki
Maria A
MA
Strati
Areti
A
Lianidou
Evi S
ES
Georgoulias
Vassilis
V
Mavroudis
Dimitris
D
Agelaki
Sofia
S
D000328:Adult; D000368:Aged; D001943:Breast Neoplasms / Q000097:blood* / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D051417:Focal Adhesion Kinase 1 / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D008875:Middle Aged; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000097:blood / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis / Q000235:genetics
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human