Most normal mammalian cells have a finite lifespan, thought to constitute a protective mechanism against unlimited proliferation. This phenomenon, called senescence, is driven by telomere attrition, which triggers the induction of tumour suppressors including p16(INK4a) (ref. 5). In cultured cells, senescence can be elicited prematurely by oncogenes; however, whether such oncogene-induced senescence represents a physiological process has long been debated. Human naevi (moles) are benign tumours of melanocytes that frequently harbour oncogenic mutations (predominantly V600E, where valine is substituted for glutamic acid) in BRAF, a protein kinase and downstream effector of Ras. Nonetheless, naevi typically remain in a growth-arrested state for decades and only rarely progress into malignancy (melanoma). This raises the question of whether naevi undergo BRAF(V600E)-induced senescence. Here we show that sustained BRAF(V600E) expression in human melanocytes induces cell cycle arrest, which is accompanied by the induction of both p16(INK4a) and senescence-associated acidic beta-galactosidase (SA-beta-Gal) activity, a commonly used senescence marker. Validating these results in vivo, congenital naevi are invariably positive for SA-beta-Gal, demonstrating the presence of this classical senescence-associated marker in a largely growth-arrested, neoplastic human lesion. In growth-arrested melanocytes, both in vitro and in situ, we observed a marked mosaic induction of p16(INK4a), suggesting that factors other than p16(INK4a) contribute to protection against BRAF(V600E)-driven proliferation. Naevi do not appear to suffer from telomere attrition, arguing in favour of an active oncogene-driven senescence process, rather than a loss of replicative potential. Thus, both in vitro and in vivo, BRAF(V600E)-expressing melanocytes display classical hallmarks of senescence, suggesting that oncogene-induced senescence represents a genuine protective physiological process.

Dysfunction of the endoplasmic reticulum (ER) has been reported in a variety of human pathologies, including cancer. However, the contribution of the ER to the early stages of normal cell transformation is largely unknown. Using primary human melanocytes and biopsies of human naevi (moles), we show that the extent of ER stress induced by cellular oncogenes may define the mechanism of activation of premature senescence. Specifically, we found that oncogenic forms of HRAS (HRAS(G12V)) but not its downstream target BRAF (BRAF(V600E)), engaged a rapid cell-cycle arrest that was associated with massive vacuolization and expansion of the ER. However, neither p53, p16(INK4a) nor classical senescence markers--such as foci of heterochromatin or DNA damage--were able to account for the specific response of melanocytes to HRAS(G12V). Instead, HRAS(G12V)-driven senescence was mediated by the ER-associated unfolded protein response (UPR). The impact of HRAS on the UPR was selective, as it was poorly induced by activated NRAS (more frequently mutated in melanoma than HRAS). These results argue against premature senescence as a converging mechanism of response to activating oncogenes and support a direct role of the ER as a gatekeeper of tumour control.

Cerivastatin is used in the treatment of hypercholesterolemia to inhibit 3-hydroxy 3-methylglutaryl coenzyme A reductase and thus prevent the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible, respectively, for translocation of Ras and Rho to the cell membrane, a step required for their cell signaling, leading to cell proliferation and migration. Recently, it has been suggested that non lipid-related effects of statins could play a beneficial role in cancer therapy. In this study, we have investigated the mechanisms by which statins inhibit cancer and the types of cancers which could benefit from this therapy. In MDA-MB-231 cells, an aggressive breast cancer cell line with spontaneous activation of Ras and NFkappaB and overexpression of RhoA, cerivastatin induced inhibition of both cell proliferation and invasion through Matrigel. This anti-proliferative effect was related to G(1)/S arrest due to an increase in p21(Waf1/Cip1). The anti-invasive effect was observed from 18 h and could be explained by RhoA delocalization from the cell membrane, resulting in disorganization of the actin fibers and disappearance of focal adhesion sites. The importance of RhoA inactivation in both these inhibitory effects was proved by their reversion by GGPP but not by FPP. Moreover, cerivastatin was also shown to induce inactivation of NFkappaB, in a RhoA inhibition-dependent manner, resulting in a decrease in urokinase and metalloproteinase-9 expression, two proteases involved in cell migration. The participation of Ras inactivation is considered a subsidiary mechanism for the effects of cerivastatin, as they were not rescued by FPP. Prolonged treatment of MDA-MB-231 cells with high doses of cerivastatin induced a loss of cell attachment. Interestingly, the effect of cerivastatin was considerably lower on poorly invasive MCF-7 cells. In conclusion, our results suggest that cerivastatin inhibits cell signaling pathways involved in the invasiveness and metastatic properties of highly invasive cancers.