Oncogene activation in melanocytes links reactive oxygen to multinucleated phenotype and senescence

Abstract

Contrary to malignant melanoma, nevi are a benign form of melanocytic hyperproliferation. They are frequently observed as precursor lesions of melanoma, but they also feature biochemical markers of senescence. In particular, evidence for oncogene-induced melanocyte senescence as natural means to prevent tumorigenesis has been obtained in nevi with mutated B-Raf V600E. Here, we demonstrate that strong oncogenic growth factor receptor signalling drives melanocytes into senescence, whereas weaker signals keep them in the proliferative state. Activation of oncogene-induced senescence also produces multinucleated giant cells, a long known histological feature of nevus cells. The protein levels of the senescence mediators, p53 and pRB, and their upstream activators do not correlate with senescence. However, strong oncogene signalling leads to pronounced reactive oxygen stress, and scavenging of reactive oxygen species (ROS) efficiently prevents the formation of multinucleated cells and senescence. Similarly, expression of oncogenic N-RAS results in ROS generation, DNA damage and the same multinuclear senescent phenotype. Hence, we identified oncogenic signalling-dependent ROS production as critical mediator of the melanocytic multinuclear phenotype and senescence, both of them being hallmarks of human nevus cells.

Tumor angiogenesis is caused by single melanoma cells in a reactive oxygen species and NF-κB dependent manner

“…High levels of Xmrk generate a substantial ROS load (Leikam et al, 2008). It is known that EGF receptors, a group of RTKs that includes Xmrk, can mediate ROS induction by activating NADPH oxidases (Browe and Baumgarten, 2006).…”

“…We have previously shown that HERmrk can induce the generation of reactive oxygen species (ROS) (Leikam et al, 2008;Leikam et al, 2013) and that Xmrk-transgenic fish melanomas display a ROS-responsive protein signature (Lokaj et al, 2009). As ROS are potent activators of NF-kB, we investigated their role in NF-kB induction in Hm cells.…”

“…Mouse melanocytes transfected with HERmrk (melan-a Hm) were cultured as described previously (Leikam et al, 2008). For small molecular inhibitor experiments, Hm cells were starved for 3 days in 2,5% starving medium [Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin (400 U/ml), streptomycin (50 mg/ml) and 2.5% dialyzed fetal calf serum (FCS, Invitrogen)].…”

“…Cells were then stimulated with 100 ng/ml EGF for 24 h or remained unstimulated. Immunofluorescence was performed as described before (Leikam et al, 2008). Anti-NF-kB p65 (E498) antibody (1:100; Cell Signaling) and Alexa Fluor 488 goat anti-rabbit IgG (1:1000; Invitrogen) were used.…”

Nano-Scaled Particles of Titanium Dioxide Convert Benign Mouse Fibrosarcoma Cells into Aggressive Tumor Cells

“…A recent study revealed that a multinucleated phenotype is brought about by oncogenic signaling-dependent ROS production in melanocytes. 55 We have to determine whether ROS formation is due to activation of signaling cascade(s) besides ROS formation by the nanoparticles themselves.…”