Roles of Hypoxia-Inducible Factor-1α (Hif-1α) Versus Hif-2α ... : Hepatology (original) (raw)

Liver Biology/Pathobiology

Roles of Hypoxia-Inducible Factor-1α (Hif-1α) Versus Hif-2α in the Survival of Hepatocellular Tumor Spheroids

Menrad, Heidi1; Werno, Christian1; Schmid, Tobias1; Copanaki, Ekaterini2; Deller, Thomas2; Dehne, Nathalie1; Brüne, Bernhard1,*

1_Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt, Germany_

2_Institute of Clinical Neuroanatomy, Neuroscience Center, Goethe-University Frankfurt, 60590 Frankfurt, Germany_

*Address reprint requests to: Goethe-University Frankfurt, Faculty of Medicine, Biochemistry I, Theodor-Stern-Kai 7, Bldg. 74, 60590 Frankfurt, Germany. Email:[email protected]; fax +49-69-6301 4203

Received 22 September 2009; Accepted 27 January 2010

Published online 19 February 2010 in Wiley InterScience (www.interscience.wiley.com).

Supported by Deutsche Forschungsgemeinschaft Grant no. SFB815; ECCPS. Sander-Foundation Grant number: 2007.070.2.

Potential conflict of interest: Nothing to report.

Additional Supporting Information may be found in the online version of this article.

Abstract

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-1α and HIF-2α in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF-1α enhanced expression of HIF-2α and vice versa. Unexpectedly, knockdown of HIF-1α or HIF-2α increased cell viability as well as spheroid size and decreased caspase-3 activity. Antiapoptotic Bcl-XL expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF-1α-knockdown cells. Furthermore, an HIF-2α-knockdown significantly increased Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression in an HIF-1α-dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF-2α-knockdown spheroids and mito-/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3-methyladenine restored cell death in HIF-2α-knockdown clones comparable to wildtype cells. Conclusion : An HIF-1α-knockdown increases HIF-2α expression and shifts the balance of Bcl-2 family members toward survival. The knockdown of HIF-2α raises autophagic activity and attenuates apoptosis by enhancing HIF-1α expression. Our data indicate that enhanced expression of one HIF-isoform causes a survival advantage in hepatocellular carcinoma development. Hepatology 2010