Mitochondrial Oxidative Stress due to Complex I Dysfunction Promotes Fibroblast Activation and Melanoma Cell Invasiveness

Abstract

Increased ROS (cellular reactive oxygen species) are characteristic of both fibrosis and tumour development. ROS induce the trans-differentiation to myofibroblasts, the activated form of fibroblasts able to promote cancer progression. Here, we report the role of ROS produced in response to dysfunctions of mitochondrial complex I, in fibroblast activation and in tumour progression. We studied human fibroblasts with mitochondrial dysfunctions of complex I, leading to hyperproduction of ROS. We demonstrated that ROS level produced by the mutated fibroblasts correlates with their activation. The increase of ROS in these cells provides a greater ability to remodel the extracellular matrix leading to an increased motility and invasiveness. Furthermore, we evidentiated that in hypoxic conditions these fibroblasts cause HIF-1α stabilization and promote a proinvasive phenotype of human melanoma cells through secretion of cytokines. These data suggest a possible role of deregulated mitochondrial ROS production in fibrosis evolution as well as in cancer progression and invasion.

Figure 1

Oxygen superoxide level and α-SMA in fibroblasts carrying mitochondrial dysfunctions of complex I. (a) Flowcytometer analysis of O₂ ^•− level. Control fibroblasts (HFY) and fibroblasts carrying mutations in NDUFS1 gene (Q522K and R557X/T595A), in NDUFS4 gene (W15X), and in the nuclear PINK gene (W437X) were cultured for 48 hours in low glucose medium and then incubated with 5 mM Mitosox for 10 minutes at 37°C for detection of oxygen superoxide. A flowcytometer analysis is then performed. The results are representative of five experiments with similar results. *P < 0.005 mutated fibroblasts versus control fibroblasts. (b) Fibroblasts seeded on glass coverslips are treated as in (a) and a confocal microscopy analysis is performed. (c) Analysis of α-SMA expression in control fibroblasts (HFY) and fibroblasts carrying mutations. Lysates of cells were subjected to α-SMA immunoblot analysis. An antiactin immunoblot was performed for normalization. (d) Analysis of α-SMA expression in control fibroblasts (HFY) and fibroblasts carrying mutations seeded on glass coverslips by confocal microscopy analysis.

Figure 2

Mutations in NDUFS1 genes (Q522K and R557X/T595A) and in the nuclear PINK gene (W437X) increase fibroblasts migration and invasion.

Figure 3

Mutated fibroblasts induce HIF-1α stabilization in hypoxic conditions.

Figure 4

mRNA levels of VEGF-A, SDF-1, and HGF secreted by fibroblasts cultured in hypoxic condition. (a–c) Quantitative real-time reverse transcription PCR of RNA extracted from fibroblasts cultured for 24 hours in low glucose serum-free medium in hypoxic conditions using primers for human VEGF-A (a), SDF-1 (b), and HGF (c) and GAPDH gene. Results were normalized first to GADPH expression levels and then displayed relative to level in HFY cells. Data are representative of three independent experiments. *P < 0.005 mutated fibroblasts versus control fibroblasts.

Figure 5

Conditioned media from mutated fibroblasts promotes melanoma cells invasiveness; NAC treatment blocks HIF-1α stabilization and reverts the increase of melanoma cells invasiveness.