Overexpression and nuclear translocation of hypoxia-inducible factor prolyl hydroxylase PHD2 in head and neck squamous cell carcinoma is associated with tumor aggressiveness.
OBJECTIVE
Hypoxia in tumors is associated with poor prognosis and resistance to treatment. The outcome of hypoxia is largely regulated by the hypoxia-inducible factors (HIF-1alpha and HIF-2alpha). HIFs in turn are negatively regulated by a family of prolyl hydroxylases (PHD1-3). The PHD2 isoform is the main down-regulator of HIFs in normoxia and mild hypoxia. This study was designed to analyze the correlation of the expression and subcellular localization of PHD2 with the pathologic features of human carcinomas and HIF-1alpha expression.


METHODS
The expression of PHD2 was studied from paraffin-embedded normal tissue (n = 21) and head and neck squamous cell carcinoma (HNSCC; n = 44) by immunohistochemistry. Further studies included PHD2 mRNA detection and HIF-1alpha immunohistochemistry from HNSCC specimens as well as PHD2 immunocytochemistry from HNSCC-derived cell lines.


RESULTS
In noncancerous tissue, PHD2 is robustly expressed by endothelial cells. In epithelium, the basal proliferating layer also shows strong expression, whereas the more differentiated epithelium shows little or no PHD2 expression. In HNSCC, PHD2 shows strongly elevated expression both at the mRNA and protein level. Moreover, PHD2 expression increases in less differentiated phenotypes and partially relocalizes from the cytoplasm into the nucleus. Endogenously high nuclear PHD2 is seen in a subset of HNSCC-derived cell lines. Finally, although most of the tumor regions with high PHD2 expression show down-regulated HIF-1alpha, regions with simultaneous HIF-1alpha and PHD2 expression could be detected.


CONCLUSIONS
Our results show that increased levels and nuclear translocation of the cellular oxygen sensor, PHD2, are associated with less differentiated and strongly proliferating tumors. Furthermore, they imply that even the elevated PHD2 levels are not sufficient to down-regulate HIF-1alpha in some tumors.
Jokilehto
Terhi
T
Turku Centre for Biotechnology, Turku University, Finland.
Rantanen
Krista
K
Luukkaa
Marjaana
M
Heikkinen
Pekka
P
Grenman
Reidar
R
Minn
Heikki
H
Kronqvist
Pauliina
P
Jaakkola
Panu M
PM
D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D015687:Cell Hypoxia; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D049308:Dioxygenases; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009361:Neoplasm Invasiveness; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D049308:Dioxygenases; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases