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The Hallmarks of Aging

Cells continually experience stress and damage from exogenous and endogenous sources, and their responses range from complete recovery to cell death. Proliferating cells can initiate an additional response by adopting a state of permanent cell-cycle arrest that is termed cellular senescence. Understanding the causes and consequences of cellular senescence has provided novel insights into how cells react to stress, especially genotoxic stress, and how this cellular response can affect complex organismal processes such as the development of cancer and ageing.

Cellular senescence: when bad things happen to good cells

Methods We conducted a multicenter, phase 1, dose-escalation trial of PLX4032 (also known as RG7204), an orally available inhibitor of mutated BRAF, followed by an extension phase involving the maximum dose that could be administered without adverse effects (the recommended phase 2 dose). Patients received PLX4032 twice daily until they had disease progression. Pharmacokinetic analysis and tumor-response assessments were conducted in all patients. In selected patients, tumor biopsy was performed before and during treatment to validate BRAF inhibition. Results A total of 55 patients (49 of whom had melanoma) were enrolled in the dose-escalation phase, and 32 additional patients with metastatic melanoma who had BRAF with the V600E mutation were enrolled in the extension phase. The recommended phase 2 dose was 960 mg twice daily, with increases in the dose limited by grade 2 or 3 rash, fatigue, and arthralgia. In the dose-escalation cohort, among the 16 patients with melanoma whose tumors carried the V600E BRAF mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response and 1 had a complete response. Among the 32 patients in the extension cohort, 24 had a partial response and 2 had a complete response. The estimated median progression-free survival among all patients was more than 7 months. Conclusions Treatment of metastatic melanoma with PLX4032 in patients with tumors that carry the V600E BRAF mutation resulted in complete or partial tumor regression in the majority of patients. (Funded by Plexxikon and Roche Pharmaceuticals.)

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Inhibition of mutated, activated BRAF in metastatic melanoma.

Cellular senescence is a tumor-suppressive mechanism that permanently arrests cells at risk for malignant transformation. However, accumulating evidence shows that senescent cells can have deleterious effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescence-associated secretory phenotype (SASP) that turns senescent fibroblasts into proinflammatory cells that have the ability to promote tumor progression.

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The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression

Tumour-associated cell cycle defects are often mediated by alterations in cyclin-dependent kinase (CDK) activity. Misregulated CDKs induce unscheduled proliferation as well as genomic and chromosomal instability. According to current models, mammalian CDKs are essential for driving each cell cycle phase, so therapeutic strategies that block CDK activity are unlikely to selectively target tumour cells. However, recent genetic evidence has revealed that, whereas CDK1 is required for the cell cycle, interphase CDKs are only essential for proliferation of specialized cells. Emerging evidence suggests that tumour cells may also require specific interphase CDKs for proliferation. Thus, selective CDK inhibition may provide therapeutic benefit against certain human neoplasias.

Cell cycle, CDKs and cancer: a changing paradigm

Cellular senescence, a growth-arrest program that limits the lifespan of mammalian cells and prevents unlimited cell proliferation, is attracting considerable interest because of its links to tumour suppression. Using a mouse model in which the oncogene Ras is activated in the haematopoietic compartment of bone marrow, Braig et al. show that cellular senescence can block lymphoma development. Genetic inactivation of the histone methyltransferase Suv39h1 that controls senescence by ‘epigenetic’ modification of DNA-associated proteins, or a pharmacological approach that mimics loss of this enzyme, allow the formation of malignant lymphomas in response to oncogenic Ras. This work has important implications for both tumour development and tumour therapy. Michaloglou et al. report that oncogene-induced senescence may be a physiologically important process in humans, keeping moles in a benign state for many years: unchecked they develop into malignant melanomas. Chen et al. also find that cellular senescence blocks tumorigenesis in vivo: they show that acting together, the p53 tumour suppressor and the cellular senescence system can prevent prostate cancer induction in mice by the PTEN mutation. Collado et al. show that cellular senescence is a defining feature of Ras-initiated premalignant tumours; this could prove valuable in the diagnosis and prognosis of cancer. See the web focus
 . Most normal mammalian cells have a finite lifespan1, thought to constitute a protective mechanism against unlimited proliferation2,3,4. This phenomenon, called senescence, is driven by telomere attrition, which triggers the induction of tumour suppressors including p16INK4a (ref. 5). In cultured cells, senescence can be elicited prematurely by oncogenes6; 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 BRAF7, 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)8,9,10. This raises the question of whether naevi undergo BRAFV600E-induced senescence. Here we show that sustained BRAFV600E expression in human melanocytes induces cell cycle arrest, which is accompanied by the induction of both p16INK4a and senescence-associated acidic β-galactosidase (SA-β-Gal) activity, a commonly used senescence marker. Validating these results in vivo, congenital naevi are invariably positive for SA-β-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 p16INK4a, suggesting that factors other than p16INK4a contribute to protection against BRAFV600E-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, BRAFV600E-expressing melanocytes display classical hallmarks of senescence, suggesting that oncogene-induced senescence represents a genuine protective physiological process.

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BRAFE600-associated senescence-like cell cycle arrest of human naevi

Almost half a century after the first reports describing the limited replicative potential of primary cells in culture, there is now overwhelming evidence for the existence of “cellular senescence” in vivo. It is being recognized as a critical feature of mammalian cells to suppress tumorigenesis, acting alongside cell death programs. Here, we review the various features of cellular senescence and discuss their contribution to tumor suppression. Additionally, we highlight the power and limitations of the biomarkers currently used to identify senescent cells in vitro and in vivo.

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The essence of senescence

Oncogene-Induced Senescence Relayed by an Interleukin-Dependent Inflammatory Network

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.

BRAF E600 in benign and malignant human tumours

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 p15(INK4B). 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.

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Chrysiis Michaloglou

Papers

BRAFE600-associated senescence-like cell cycle arrest of human naevi

The essence of senescence

Oncogene-Induced Senescence Relayed by an Interleukin-Dependent Inflammatory Network

BRAF E600 in benign and malignant human tumours

Abrogation of BRAFV600E-induced senescence by PI3K pathway activation contributes to melanomagenesis