Controversial aspects of oncogene-induced senescence

Abstract

Oncogene-induced senescence (OIS) is a fail-safe mechanism that is developed to suppress cell proliferation caused by aberrant activation of oncoproteins in normal cells. Most of the available literature considers senescence to be caused by activated RAS or RAF proteins. In the current review, we will discuss some of the controversial aspects of RAS- or RAF-induced senescence in different types of normal cells: are tumor suppressors important for OIS? What is the role of DNA damage in OIS? Are there different types of OIS?

Introduction

Primary cells have a limited lifespan in culture, i.e., replicate only a defined number of times (the so-called Hayflick limit¹) before undergoing a permanent proliferation arrest, termed senescence. This type of senescence was linked to the progressive telomere erosion occurring during replication, and was therefore termed “replicative senescence.”^2-7 The complex role of telomere erosion in cellular senescence and diseases varies depending on the type of cells and species.^8-22 Subsequently, a senescence-like phenotype associated with overexpression of activated RAS proteins were identified as “oncogene-induced senescence” (OIS).^23-25 In addition to proliferation arrest, senescent cells are characterized by increased size, activation of β-galactosidase (SA-β-Gal), emergence of chromatin aggregates enriched with tri-methylated lysine 9 of histone H3, markers for activated DNA damage and enzymes degrading extracellular matrix (ECM). In vitro and in vivo studies have provided strong arguments that OIS serves as the first barrier of defense against cancer development.^2,3,26-30 However, senescence is a double-edged sword that promotes carcinogenesis in some conditions.^3,31,32 Despite a plethora of reports on mechanisms of OIS, or perhaps exactly due to the high number of such reports, the characteristics of senescence are still being debated and so is the function of its key regulators. In this review we will focus on several contentious issues surrounding OIS.

Are Tumor Suppressors Required for OIS?

The tumor suppressors p53 (TP53) and p16^INK4a (INK4A/ARF locus) are essential for the regulation of key cellular processes, including proliferation, differentiation and cell death,^32-37 and oncogene-induced senescence is executed via these tumor suppressors in cultured, normal rodent cells.^24,30,38-45 Accordingly, studies performed in transgenic mice demonstrated an active role of p53 and p16^INK4A in the implementation of OIS in murine models.^34,46-49

In normal human cells, however, the role of these tumor suppressors in senescence induced by activated RAS appears to be less straightforward. For example, several reports demonstrated that depletion of p53 allows the proliferation of HRAS^G12V-expressing normal human fibroblasts.^2,23,50-52 At the same time, HRAS^G12V caused senescence independently of p53 in primary human esophageal keratinocytes.⁵³ Three groups determined that p53 is dispensable for the senescence induced by activated HRAS or NRAS in normal human melanocytes.^54-57 Moreover, one of the groups demonstrated that a direct p53 target, p21^WAF/CIP, is also dispensable for the senescence induced by NRAS^Q61K.⁵⁷ Additionally, normal human mammary epithelial cells were shown to undergo HRAS^G12V-induced senescence via p53-independent mechanism.⁵⁸ Even more paradoxically, it was demonstrated that by inducing quiescence-type cell cycle arrest, activation of p53 can suppress senescence of human cells caused by ectopic expression of p21.^59,60 This can be explained by inhibition of mTOR by p53^43,61-63 (see below).

Like p53, the role of p16^INK4A in OIS of normal rodent cells is well-documented,⁴² and also like p53, the involvement of p16^INK4A in HRAS^G12V-induced senescence of normal human cells is controversial. For instance, p16^INK4A-deficient normal human fibroblasts were refractory to the senescence induced by HRAS^G12V.⁶⁴ Similarly, a separate study showed that oncogenic HRAS^G12V did not cause senescence in freshly isolated human fibroblasts possessing low amounts of p16^INK4A.⁶⁵ In contrast, depletion of p16^INK4A had no effect on senescence induced by oncogenic HRAS or NRAS in normal human melanocytes.^54,55,66 Based on all these data, it appears that the role of p53 and p16^INK4A in RAS-induced senescence is dependent on cell type.

DNA Damage or DNA Damage Response?

Activation of DNA damage response (DDR) is considered one of the major causes of OIS.⁴¹ Until now, two major sources of DNA damage have been described in normal cells undergoing OIS: (1) single- and double-strand DNA breaks (SSB and DSB, respectively) that are caused by prematurely terminated replication forks as a result of DNA hyper-replication induced by aberrant oncogenic signaling,^41,50,52 and (2) oxidative DNA damage, including abasic DNA sites, SSB and DSB, caused by high levels of reactive oxygen species (ROS) induced by activated RAS.^41,67

Recently, another source of DNA damage was described in normal human fibroblasts undergoing HRAS^G12V-induced senescence—depletion of deoxyribonucleoside triphosphate pools.⁷⁹ Specifically, senescent fibroblasts under-expressed several enzymes involved in the de novo deoxyribonucleotide biosynthesis and possessed low levels of deoxyribonucleoside triphosphates (dNTP). Overexpression of thymidylate synthase and ribonucleotide reductase or addition of exogenous deoxyribonucleosides suppressed DNA damage (evidenced by the comet assay), activity of SA-β-Gal and proliferation arrest of normal human fibroblasts expressing HRAS^G12V. Interestingly, a similar role of dNTP pools in control of DNA damage and senescence phenotypes was shown in melanoma cells undergoing senescence due to depletion of C-MYC oncogene.⁸⁰

SSB engage serine/threonine-protein kinase ATR (ataxia telangiectasia and Rad3-related protein), which activates checkpoint kinase 1 (CHK1). CHK1, in turn, phosphorylates CDC25, one of the key regulators of cell cycle progression, causing its degradation.⁶⁸ DSB activate another serine/threonine protein kinase, ATM (ataxia telangiectasia mutated).^69,70 Activated ATM phosphorylates histone H2AX, a variant of H2A histone,^71,72 p53 tumor suppressor^73-75 and CHK2 kinase.⁷⁶

Both ATR and ATM pathways are activated in normal human fibroblasts expressing HRAS^G12V.^50,52,77,78 Moreover, shRNA-mediated depletion of ATM or CHK2 rendered normal human fibroblasts resistant to proliferation arrest and other senescence phenotypes caused by RAS, thus demonstrating the causal role of DDR activation in mediation of RAS-induced senescence.^52,78

Alternatively, Pospelova et al. demonstrated that DDR in senescent cells can also be activated independently of DNA damage. For instance, they found that senescence induced in E1A+Ras-transformed rodent fibroblasts by sodium butyrate and in human fibrosarcoma by p21 was accompanied by increased nuclear staining for phospho-ATM and γ-H2AX in the absence of actual DNA damage based on the comet assay.⁸¹ Furthermore, ATM activation preceded γ-H2AX staining and developed after emergence of senescence phenotypes. The authors termed this phenomenon “pseudo-DNA damage response.” Most importantly, mTOR inhibitor rapamycin decreased both pseudo-DDR and senescent phenotypes.⁸¹ mTOR (mammalian target of rapamycin) belongs to the PIKK family of Ser/Thr kinases. It is activated by the RAS-PI3K-AKT pathway⁸² and functions as a sensor of intracellular nutrients, ATP and redox status. mTOR regulates cellular growth and proliferation by controlling protein synthesis and cellular metabolism,^83-85 growth, aging and diseases.^86-90 Inhibition of mTOR (e.g., by rapamycin) decelerates both replicative and oncogene-induced senescence^60,91 as well as conversion from quiescence to senescence.^92-94 Conversely, activation of mTOR induced senescence independently from DNA damage,⁹³ whereas DNA damage-independent activation of DDR has been reported by other groups,^95,96 albeit not in senescent cells. Therefore, these studies suggest that DNA damage per se may be dispensable for the senescence induced by oncogenic RAS as long as DDR is activated. On the other hand, in normal human mammary epithelial cells, overexpression of HRAS^G12V causes senescence independently of ATM and CHK2,⁵⁸ whereas chemical inhibition of CHK activity did not affect NRAS^Q61K-induced senescence in normal human melanocytes.⁵⁷ Thus, like p53 and p16^INK4A, DDR itself may be dispensable for RAS-induced senescence in certain types of normal cells.

Different Types of OIS

In humans, the best example of OIS is represented by nevi, naturally occurring aggregations of arrested melanocytes. Genetic analysis of nevi revealed that they contain activating mutations in BRAF, NRAS or HRAS genes.^66,97-100 In particular, mutations in HRAS have been frequently detected in Spitz nevi.¹⁰¹ Congenital nevi often harbor mutations in NRAS gene,^102,103 whereas acquired nevi are composed of melanocytes carrying BRAF mutations.⁹⁹ The same mutations in these genes were found in malignant melanomas albeit surprisingly at frequencies lower than that in benign nevi,^99,104,105 suggesting that malignant melanomas, which are frequently derived from nevi, have developed a mechanism(s) for suppressing OIS. Interestingly, the frequency of the above mutations in malignant melanomas varies significantly. Approximately 60% and 20% of human melanomas contain mutations in BRAF and NRAS, respectively, while mutations in HRAS are very rare.⁵⁵ The reasons for such heterogeneity are not completely understood.

Accordingly, overexpression of BRAF^V600E, NRAS^Q61R, HRAS^G12V in normal human melanocytes resulted in classical OIS that, surprisingly, was independent of p53, p21^CIP/WAF, p16^INK4A or p14^{ARF 54–57}. Instead, it has been demonstrated that OIS caused by the above oncogenes in human melanocytes could be at least partially abrogated by ectopic expression of the oncogenic transcription factor C-MYC⁵⁴ or by knockdown of its negative regulator PP2A-B56α, one of the subunits of PP2A tumor suppressor complex.¹⁰⁶ Intriguingly, despite similar degree of upregulation of C-MYC in all studied melanocytic populations, senescence phenotypes were effectively suppressed only in cells expressing BRAF^V600E; NRAS^Q61R-induced senescence was affected only partially, and HRAS^G12V-dependent senescence phenotypes remained unchanged.¹⁰⁶ These data are in agreement with a previous study demonstrating that a negative regulator of cell proliferation and/or viability, the unfolded protein response, can be induced in normal human melanocytes by HRAS^G12V or NRAS^Q61R (albeit less effectively) but not by BRAF^V600E.⁵⁶ UPR is an adaptive signaling pathway that is activated by numerous stimuli, including oxidative and metabolic stresses.¹⁰⁷ Ectopic expression of C-MYC in normal human melanocytes did not affect the UPR pathway, which may account for the failure of C-MYC to effectively suppress NRAS^Q61R-senescence or even partially suppress senescence caused by HRAS^G12V.^54,106

Thus, oncogene-induced senescence in human melanocytes involves activation of different senescence programs that may be suppressed with different efficiency by other dominant oncogenes, such as C-MYC. These observations may offer a possible mechanistic explanation for the reported difference in mutation frequencies of HRAS, NRAS and BRAF genes in human melanomas.