PHD3 regulates differentiation, tumour growth and angiogenesis in pancreatic cancer.
OBJECTIVE
Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown.


METHODS
PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.


RESULTS
PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis.


CONCLUSIONS
Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.
Department of General Surgery, University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg 69120, Germany.
Y
Y
Su
M
M
Loos
N
N
Giese
O J
OJ
Hines
I
I
Diebold
A
A
Görlach
E
E
Metzen
S
S
Pastorekova
H
H
Friess
P
P
Büchler
D000230:Adenocarcinoma / Q000473:pathology / Q000601:surgery; D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D021441:Carcinoma, Pancreatic Ductal / Q000201:enzymology / Q000473:pathology / Q000601:surgery; D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049308:Dioxygenases / Q000235:genetics* / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000502:physiology; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D051379:Mice; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000473:pathology*; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000473:pathology* / Q000601:surgery; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis
D017304:Annexin A5; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D042461:Vascular Endothelial Growth Factor A; D049308:Dioxygenases; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D053148:Caspase 3