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.
Conclusion: 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.