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.

Oncogene-induced cellular senescence (OIS) is emerging as a potent cancer-protective response to oncogenic events, serving to eliminate early neoplastic cells from the proliferative pool. Using combined genetic and bioinformatic analysis, we find that OIS is linked specifically to the activation of an inflammatory transcriptome. Induced genes included the pleiotropic cytokine interleukin-6 (IL-6), which upon secretion by senescent cells acted mitogenically in a paracrine fashion. Unexpectedly, IL-6 was also required for the execution of OIS, but in a cell-autonomous mode. Its depletion caused the inflammatory network to collapse and abolished senescence entry and maintenance. Furthermore, we demonstrate that the transcription factor C/EBPbeta cooperates with IL-6 to amplify the activation of the inflammatory network, including IL-8. In human colon adenomas, IL-8 specifically colocalized with arrested, p16(INK4A)-positive epithelium. We propose a model in which the context-dependent cytostatic and promitogenic functions of specific interleukins contribute to connect senescence with an inflammatory phenotype and cancer.

VHL, NF-1, c-Ret, and Succinate Dehydrogenase Subunits B and D act on a developmental apoptotic pathway that is activated when nerve growth factor (NGF) becomes limiting for neuronal progenitor cells and requires the EglN3 prolyl hydroxylase as a downstream effector. Germline mutations of these genes cause familial pheochromocytoma and other neural crest-derived tumors. Using an unbiased shRNA screen we found that the kinesin KIF1B␤ acts downstream from EglN3 and is both necessary and sufficient for neuronal apoptosis when NGF becomes limiting. KIF1B␤ maps to chromosome 1p36.2, which is frequently deleted in neural crest-derived tumors including neuroblastomas. We identified inherited loss-of-function KIF1B␤ missense mutations in neuroblastomas and pheochromocytomas and an acquired loss-of-function mutation in a medulloblastoma, arguing that KIF1B␤ is a pathogenic target of these deletions.[Keywords: Apoptosis; kinesin; neuroblastoma; pheochromocytoma; prolyl hydroxylase] Supplemental material is available at http://www.genesdev.org.

Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF V600E -induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF V600E -driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15INK4B . This treatment also eliminated subpopulations resistant to targeted BRAF V600E inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF V600E inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.

Of the RAF family of protein kinases, BRAF is the only member to be frequently activated by mutation in cancer. A single amino acid substitution (V600E) accounts for the vast majority and results in constitutive activation of BRAF kinase function. Its expression is required to maintain the proliferative and oncogenic characteristics of BRAF E600 -expressing human tumour cells. Although BRAF E600 acts as an oncogene in the context of additional genetic lesions, in primary cells it appears to be associated rather with transient stimulation of proliferation. Eventually, BRAF E600 signalling triggers cell cycle arrest with the hallmarks of cellular senescence, as is illustrated by several recent studies in cultured cells, animal models and benign human lesions. In this review, we will discuss recent advances in our understanding of the role of BRAF E600 in benign and malignant human tumours and the implications for therapeutic intervention.

The Wnt-signaling cascade is required for several crucial steps during early embryogenesis, and its activity is modulated by various agonists and antagonists to provide spatiotemporalspecific signaling. Naked cuticle is a Wnt antagonist that itself is induced by Wnt signaling to keep Wnt signaling in check. Little is known about the regulation of this antagonist. We have recently shown that the protein phosphatase 2A regulatory subunit PR72 is required for the inhibitory effect of Naked cuticle on Wnt signaling. In the present study, we show that PR130, which has an N terminus that differs from that of PR72 but shares the same C terminus, also interacts with Naked cuticle but instead functions as an activator of the Wnt-signaling pathway, both in cell culture and during development. We find that PR130 modulates Wnt signal transduction by restricting the ability of Naked cuticle to function as a Wnt inhibitor. Our data establish PR130 as a modulator of the Wntsignaling pathway and suggest a mechanism of Wnt signal regulation in which the inhibitory activity of Naked cuticle is determined by the relative level of expression of two transcripts of the same protein phosphatase 2A regulatory subunit.development ͉ protein phosphatase 2A ͉ Xenopus

Mutant RAS (RAS(V12)) is known to transform most immortal cells but to induce premature senescence in primary cells. RAS(V12)-induced senescence in murine cells depends on the induction of the ARF/p53 and the retinoblastoma (Rb) family tumor suppressor pathways. We and others have shown previously that oncogene-induced senescence in vitro can be used as a tool to identify new cancer-related genes. In addition, we have shown that oncogene-induced senescence corresponds to an in vivo tumor suppressive mechanism. Therefore, we extended our search for novel genes that bypass of RAS(V12)-induced senescence, with the help of a previously designed unbiased functional screen with cDNA expression libraries. In this screen, we expected to find new mediators feeding into the p53 or Rb pathways or novel signaling factors. We report here the identification of leukemia/lymphoma related factor (Lrf) encoding a transcription factor with a BTB/POZ domain and Krüppel-like zinc fingers. This gene was previously identified as a potential oncogene that is overexpressed in human cancer. We find that LRF enhances E2F-dependent transcription and that it synergizes with RAS(V12) in activating E2F. Indeed, LRF-mediated bypass of RAS(V12)-induced senescence is accompanied by the induction of several E2F-target genes, including Cyclin E, Cyclin A and p107. Unexpectedly, LRF exerted this activity independent of several critical senescence inducers, such as p19(ARF), p21(CIP) and p16(INK4A). We show that CYCLIN E is necessary for LRF-mediated bypass, suggesting that it corresponds to a critical mediator of LRF-driven oncogenic transformation. Thus, LRF bypasses RAS(V12)-induced senescence in a CYCLIN E-dependent manner, which conceivably contributes to its role in cancer.