Oncogene-induced senescence was first demonstrated in primary human fibroblasts (13
) and subsequently multiple reports describing the occurrence of this phenomenon both in vitro
and in vivo
) have appeared. Contrasting evidence showing oncogene induced transformation and increased proliferation of primary cells also exists (37
). These variations can be due to differences in the inherent abilities of particular cell types to respond to oncogene activation, however few studies have compared two cell types simultaneously. An alternative is that the extent and duration of oncogene activation dictates a cell’s decision to proliferate or prematurely growth arrest. This has been observed in vitro
in NIH3T3 fibroblasts (39
) and in vivo
in the mammary epithelium where low levels of activated oncogene cause proliferation and high levels induce growth arrest (15
). In this study, we show that stable and sustained Ras activation, independent of expression levels, leads to senescence bypass in primary endothelial cells, while simultaneously inducing growth arrest in primary melanocytes. These data strongly argue that the induction of senescence by Ras, is likely to be dependent upon specific factors relevant to each individual cell type.
Senescence response in melanocytes has been primarily attributed to the accumulation of the tumor suppressor p16INK4a
). We found no induction of p16 in endothelial cells whereas the senescent melanocytes readily accumulated p16. In addition to p16, Ras induced senescence in primary fibroblasts is also accompanied by the accumulation of tumor suppressor p53 (13
). However, we find that p53 is not induced upon Ras activation (data not shown) nor is its target gene p21CIP/WAF
, suggesting p53 regulation may be differentially regulated among cell types. We also have not observed several other causative mechanisms described to induce premature senescence, e.g., the attenuation of Ras signaling events (21
), or activation of p38 (24
). As vascular heterogeneity is well established, we considered that cells from different vascular beds might show heterogeneous responses. However, we found that all of the key findings were also observed in primary cultures of HDMVECs, a cell type relevant to formation of hemangioma and angiosarcoma (Supplementary Fig. S6
). These results highlight a unique and conserved response of endothelial cells to Ras activation. Importantly, while the inherent response of the endothelial cell in isolation may be the same, the varying microenvironment of the tissue may influence the predisposition of the endothelium to manifest an altered phenotype.
Cellular senescence acts as an initial barrier preventing a benign tumor from progressing to a malignant state (40
). Interestingly, senescent cells are only detected in benign but not metastatic tumors (12
) supporting the notion that senescence functions as a mechanism of tumor suppression. Our findings suggest this may not be an effective checkpoint in endothelial cells and could explain the prevalence of Ras mutations in endothelial tumors (42
). Endothelial tumors are characterized by rapidly proliferating endothelial cells (44
) that have an atypical morphology and may appear as cellular sheets (45
). Our findings demonstrate that sustained Ras expression in primary endothelial cells results in enhanced proliferation, autonomous growth, and enhanced endothelial cell survival. Moreover, the ability to organize into properly formed vascular structures is compromised, with cells forming sheet-like structure like those seen in some endothelial tumors (45
). Our data do not support the notion that acquisition of Ras mutations would be sufficient to induce cellular transformation, as several additional growth checkpoints were intact.
Abnormal vessels are prevalent in disorders linked to increased Ras activity. Mutations in RASA1
genes, which down-regulate Ras, have been linked to familial hemangiomas, aneurysms, cerebral vascular malformations and an increased risk of stroke (4
). Our data suggest that pro-proliferative signaling, defective apoptosis, as well abnormal morphogenic programming could contribute to disease conditions. Moreover, Ras mutations, even without gross manifestations, might contribute to an abnormal and unstable capillary vasculature. Recent data has demonstrated that short term adenoviral infection with activated Ras constructs results in enhanced angiogenesis in a mouse ear model. The adenoviral infection in these experiments targets both the endothelial and stromal compartments and the time of expression is limited (28
); this is an important distinction since we find the morphogenic phenotype reversible. Thus microenvironmental cues, expression levels, and duration of signaling may all contribute to a failure to form normal vascular structures.
We find that Ras is sufficient to drive proliferation in the absence of mitogens and this requires ERK signaling. However, our data clearly show that activation of Raf/ERK signaling through either Ras effector mutants or activated Raf is not sufficient to drive proliferation or support survival signaling. However, these sustained signals do not interfere with normal vessel morphogenesis. In contrast, chronic activation of PI-3′-kinase signaling appears to alter morphogenesis. This finding is consistent with results from the transgenic expression of activated Akt in mice (26
). These mice have altered vascular density, morphogenesis, and permeability. Inhibition of PI-3′-kinase activity is associated with a restoration of a normalized vasculature in these mice. Pro-survival signaling alone may not be the cause of the morphogenic defects following chronic Ras signaling; rather it appears that a dynamically regulated change in cellular programming is occurring. This is based on several observations: 1) treatment with U0126 inhibits Ras induced survival and decreases the cell number in the morphogenesis assay with no effect on the defect in morphogenesis induced by Ras; 2) activation of Akt completely protects cells from apoptotic stimuli, however partial morphogenic responses are seen in these cells, albeit severely compromised; and 3) the H-RasV12C40
mutant induces a defect similar to H-RasV12
, though the levels of Akt activation are much lower. Thus, PI-3′-kinase related signals, in addition to Akt, may be involved.
Aberrantly formed blood vessels are known to occur in tumors and have been implicated in metastasis (3
). It seems feasible that sustained and high concentrations of angiogenic growth factors could result in sustained activation of Ras signaling. The pro-mutagenic microenvironment of the tumor bed undergoing therapeutic interventions (rapid proliferation in the presence of mutagens and often radiation) could also result in de novo
acquisition of endothelial Ras mutations. Indeed cytogenetic abnormalities in tumor endothelial have been detected (47
) and suggested as a potential barrier to therapy. Thus, mitigating or minimizing Ras signaling in the tumor vasculature may be an effective and important adjuvant therapeutic approach to vascular normalization.