Original Contribution
HIF-1α stabilization by mitochondrial ROS promotes Met-dependent invasive growth and vasculogenic mimicry in melanoma cells

https://doi.org/10.1016/j.freeradbiomed.2011.05.042Get rights and content

Abstract

The “angiogenic switch” during tumor progression is increasingly recognized as a milestone event in tumorigenesis, although the surprising prometastatic effect of antiangiogenic therapies has recently shaken the scientific community. Tumor hypoxia has been singled out as a possible responsible factor in this prometastatic effect, although the molecular pathways are completely unknown. We report herein that human melanoma cells respond to hypoxia through a deregulation of the mitochondrial release of reactive oxygen species (ROS) by the electron transfer chain complex III. These ROS are mandatory to stabilize hypoxia-inducible factor-1α (HIF-1α), the master transcriptional regulator of the hypoxic response. We found that melanoma cells sense hypoxia-enhancing expression/activation of the Met proto-oncogene, which drives a motogenic escape program. Silencing analyses revealed a definite hierarchy of this process, in which mitochondrial ROS drive HIF-1α stabilization, which in turn activates the Met proto-oncogene. This pathway elicits a clear metastatic program of melanoma cells, enhancing spreading on extracellular matrix, motility, and invasion of 3D matrices, as well as growth of metastatic colonies and the ability to form capillary-like structures by vasculogenic mimicry. Both pharmacological and genetic interference with mitochondrial ROS delivery or Met expression block the hypoxia-driven metastatic program. Hence, we propose that hypoxia-driven ROS act as a primary driving force to elicit an invasive program exploited by aggressive melanoma cells to escape from a hypoxic hostile environment.

Section snippets

Materials

Unless specified all reagents were obtained from Sigma. Hs29-4T cells were from the ATCC, all antibodies were from Santa Cruz Biotechnology, except for the anti-HIF-1α, which was from BD Biosciences. 2′,7′-Dichlorofluorescein diacetate (DCF-DA) was from Molecular Probes and the ATP-competitive inhibitor of Met kinase PHA-665752 (PHA) was from Tocris. Polyvinylidene difluoride was from Millipore and Matrigel was from BD Biosciences. siRNA oligonucleotides targeting HIF-1α (target sequence

Hypoxia induces a more aggressive phenotype in Hs29-4T melanoma cells

A preliminary screening of growth factor receptors (GF-Rs) regulated by hypoxia on Hs29-4T melanoma cells, including HGF-R/Met, VEGF-R1, VEGF-R2, EphA2, EphA3, EphB2, platelet derived growth factor β receptor (PDGF-R), and fibroblast growth factor-2 receptor, indicated that 1% O2 mainly leads to increased expression of Met, showing only a marginal effect on VEGF-R2 and being ineffective at the regulation of the other GF-Rs (Supplementary Fig. 1). Hs29-4T melanoma cells were exposed for 24 h to

Discussion

The results presented in this report lead to these major conclusions: (1) in Hs29-4T human melanoma cells hypoxia leads to a redox-dependent stabilization of HIF-1α, mainly mediated by mitochondrial ROS, and (2) HIF-1α accumulation, in turn, leads to enhanced expression and activation of the proto-oncogene Met and to activation of: (i) the motogenic program, (ii) invasiveness and ability to disseminate metastatic colonies of melanoma cells, and (iii) the skill to mimic the vasculature, forming

Acknowledgments

This work was supported by the Associazione Italiana Ricerca sul Cancro, Istituto Toscano Tumori, Region Toscana, and Fondazione Cassa di Risparmio di Lucca.

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