Role of hypoxia-inducible factor 1alpha in gastric cancer cell growth, angiogenesis, and vessel maturation.
BACKGROUND
Hypoxia-inducible factor 1 (HIF-1), a heterodimer comprising the oxygen-regulated subunit, HIF-1alpha, and HIF-1beta, mediates transcription of the gene for vascular endothelial growth factor (VEGF). Overexpression of HIF-1alpha is associated with tumor angiogenesis and tumor cell proliferation and invasion. We examined the effects of inhibiting HIF-1alpha activity on angiogenesis and human gastric cancer growth in vivo.


METHODS
Human gastric cancer TMK-1 cells were stably transfected with pHIF-1alphaDN, an expression plasmid encoding a dominant-negative form of HIF-1alpha that dimerizes with endogenous HIF-1beta to produce HIF-1 complexes that cannot activate transcription, or with the empty expression vector (pCEP4). Two clones of pHIF-1alphaDN-transfected cells, DN2 and DN3, were tested in all experiments. We used an enzyme-linked immunosorbent assay to measure VEGF secretion by transfected cells cultured in hypoxic (1% O2) or nonhypoxic (20% O2) conditions. We used subcutaneous and orthotopic mouse tumor models to examine the growth of tumors derived from injected pHIF-1alphaDN-or pCEP4-transfected cells. Tumor cell proliferation, vessel area (a measure of functional vascular volume), and tumor endothelial cell association with pericyte-like cells (a measure of vessel maturation) were analyzed by immunohistochemical or immunofluorescent staining. All statistical tests were two-sided.


RESULTS
DN2 cells and DN3 cells secreted less VEGF than pCEP4-transfected TMK-1 cells when cultured in nonhypoxic or hypoxic conditions (e.g., DN2 versus pCEP4 in nonhypoxic conditions: 645 pg of VEGF/10(6) cells versus 1591 pg of VEGF/10(6) cells, difference = 946 pg of VEGF/10(6) cells [95% confidence interval [CI] = 640 to 1251 pg of VEGF/10(6) cells; P =.006]; DN2 versus pCEP4 in hypoxic conditions: 785 pg of VEGF/10(6) cells versus 2807 pg of VEGF/10(6) cells, difference = 2022 pg of VEGF/10(6) cells [95% CI = 1871 to 2152 pg of VEGF/10(6) cells; P<.001]). In the subcutaneous tumor model, tumors derived from DN2 or DN3 cells had lower final volumes, weights, and vessel areas, less tumor endothelial cell association with desmin-positive cells, and fewer proliferating tumor cells than tumors derived from pCEP4-transfected cells. In the orthotopic tumor model, tumors derived from DN2 cells had smaller volumes and less vessel area and maturation than tumors derived from pCEP4-transfected cells.


CONCLUSIONS
Inhibition of HIF-1alpha activity impairs gastric tumor growth, angiogenesis, and vessel maturation.
Stoeltzing
Oliver
O
Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston 77030-4009, USA.
McCarty
Marya F
MF
Wey
Jane S
JS
Fan
Fan
F
Liu
Wenbiao
W
Belcheva
Anna
A
Bucana
Corazon D
CD
Semenza
Gregg L
GL
Ellis
Lee M
LM
D000818:Animals; D017209:Apoptosis; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D015153:Blotting, Western; D002455:Cell Division; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control; D011897:Random Allocation; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D013274:Stomach Neoplasms / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014183:Transplantation, Heterologous; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
C497396:ARNT protein, human; C497397:Arnt protein, mouse; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator