Abstract
Oncogene-induced senescence acts as a barrier against tumour formation and has been implicated as the mechanism preventing the transformation of benign melanocytic lesions that frequently harbour oncogenic B-RAF or N-RAS mutations. In the present study we systematically assessed the relative importance of the tumour suppressor proteins p53, p21(Waf1), pRb and p16(INK4a) in mediating oncogene-induced senescence in human melanocytes. We now show that oncogenic N-RAS induced senescence in melanocytes is associated with DNA damage, a potent DNA damage response and the activation of both the p16(INK4a)/pRb and p53/p21(Waf1) tumour suppressor pathways. Surprisingly neither the pharmacological inhibition of the DNA damage response pathway nor silencing of p53 expression had any detectable impact on oncogene-induced senescence in human melanocytes. Our data indicate that the pRb pathway is the dominant effector of senescence in these cells, as its specific inactivation delays the onset of senescence and weakens oncogene-induced proliferative arrest. Furthermore, we show that although both p16(INK4a) and p21(Waf1) are upregulated in response to N-RAS(Q61K), the activities of these CDK inhibitors are clearly distinct and only the loss of p16(INK4a) weakens senescence. We propose that the ability of p16(INK4a) to inhibit the cyclin D-dependent kinases and DNA replication, functions not shared by p21(Waf1), contribute to its role in senescence. Thus, in melanocytes with oncogenic signalling only p16(INK4a) can fully engage the pRb pathway to alter chromatin structure and silence the genes that are required for proliferation.
Highlights
The response of primary human melanocytes to the oncogenic, melanoma-associated N-RASQ61K mutant was evaluated by stably transducing N-RASQ61K into human epidermal melanocytes
LM, light microscopy. (B) Human epidermal melanocytes infected with lentiviruses expressing N‐RASQ61K or Copepod GFP (copGFP) were stained with DAPI and antibodies to H3K9Me, 15 days post transduction. (C) Expression of the indicated proteins was determined by western blot analysis 15 days after infection of human epidermal melanocytes with lentiviruses expressing N‐RASQ61K or copGFP control
We show that N-RASQ61K induces senescence in human melanocytes that was associated with markers of DNA damage response, and involved the activation of both the p53 and pRb pathways
Summary
Fewer than 5% of patients with distant visceral metastases from cutaneous melanoma survive 12 months and there are no effective drug treatments [1]. Cutaneous melanoma arises from benign melanocytic lesions (benign naevi) or de novo from melanocytes of the skin [2]. Mutations activating the N-RAS or B-RAF kinase components of the mitogen-activated protein kinase (MAPK) pathway are found in approximately 15% and 60% of human melanomas, respectively [3,4,5]. Greater than 89% of B-RAF mutations in melanoma alter a single amino acid (V600E and V600K), whereas highly recurrent mutations affecting Gly-12, Ala-18 and Gln account for approximately 12%, 5% and 70% of melanoma-associated N-RAS mutations, respectively [6]. The B-RAFV600E and N-RASQ61K mutations are found in up to 80% and 55% of benign naevi, respectively [7, 8] and benign naevi display several markers of senescence, including positive senescenceassociated ß-galactosidase (SA-ß-Gal) activity and p16INK4a expression [9, 10]. The presence of senescent cells in human benign naevi remains controversial [11], accumulating evidence suggests that www.impactaging.com
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