Roles of hypoxia-inducible factor-1alpha (HIF-1alpha) versus HIF-2alpha in the survival of hepatocellular tumor spheroids.
Hypoxia-inducible factors (HIFs) provoke adaptation to hypoxic stress occurring in rapidly growing tumor tissues. Therefore, overexpression of HIF-1 or HIF-2 is a common feature in hepatocellular carcinoma but their specific function is still controversially discussed. To analyze HIF function in hypoxia-induced cell death we created a stable knockdown of HIF-1alpha and HIF-2alpha in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF-1alpha enhanced expression of HIF-2alpha and vice versa. Unexpectedly, knockdown of HIF-1alpha or HIF-2alpha increased cell viability as well as spheroid size and decreased caspase-3 activity. Antiapoptotic Bcl-X(L) expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF-1alpha-knockdown cells. Furthermore, an HIF-2alpha-knockdown significantly increased Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression in an HIF-1alpha-dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF-2alpha-knockdown spheroids and mito-/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3-methyladenine restored cell death in HIF-2alpha-knockdown clones comparable to wildtype cells.


CONCLUSIONS
An HIF-1alpha-knockdown increases HIF-2alpha expression and shifts the balance of Bcl-2 family members toward survival. The knockdown of HIF-2alpha raises autophagic activity and attenuates apoptosis by enhancing HIF-1alpha expression. Our data indicate that enhanced expression of one HIF-isoform causes a survival advantage in hepatocellular carcinoma development.
Menrad
Heidi
H
Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt, Germany.
Werno
Christian
C
Schmid
Tobias
T
Copanaki
Ekaterini
E
Deller
Thomas
T
Dehne
Nathalie
N
Brüne
Bernhard
B
D017209:Apoptosis; D001343:Autophagy; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D049108:Cell Enlargement; D002470:Cell Survival; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D018874:Spheroids, Cellular / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D053148:Caspase 3; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000099162:Endothelial PAS Domain-Containing Protein 1; C494811:BCL2L1 protein, human; C090121:BNIP3 protein, human