Hypoxia-inducible factor-1 alpha and vascular endothelial growth factor in BC CTC's
 

http://breast-cancer-research.com/co...df/bcr2452.pdf

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Re: Hypoxia-inducible factor-1 alpha and vascular endothelial growth factor in BC CTC
 

Abstract
Introduction
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-1α (HIF-1α) 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 (VEGFR2), HIF-1α 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-1α or pFAK antibodies, respectively.

Results
pFAK expression in CTCs was detected in 92% of patients whereas expression of VEGF, VEGFR2 and HIF-1α was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGFR2, HIF-1α and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. VEGF mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and 3 triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1α and VEGFR2.

Conclusions
The expression of pFAK, HIF-1α, VEGF and VEGFR2 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.





Conclusions
The data reported in the present study demonstrate that CTCs from patients with metastatic breast cancer express VEGF both at mRNA and protein level. The production of VEGF is probably under the regulation of HIF-1α and/or pFAK as suggested by the observed significant correlation between the expression of these molecules. Moreover, we have also demonstrated that VEGFR2 is also expressed on CTCs’ membrane suggesting an autocrine loop involving VEGF and VEGFR2. The biologic significance of the presence of angiogenic molecules on CTCs is currently unknown. However, based on previous evidence, one might hypothesize that angiogenic pathways present on CTCs could result in evasion of apoptosis, enhancement of metastatic potential [10] and resistance to endocrine therapy [38] In addition, the expression of angiogenic molecules on CTCs could be indicative of the tumor dependence on angiogenesis. Accordingly, anti-VEGF and/or anti-VEGFR2 targeting agents might have therapeutic implications in patients with VEGF- and VEGFR2- expressing CTCs.