Oncogene-induced senescence results in marked metabolic and bioenergetic alterations

Oncogene-induced senescence (OIS) is characterized by permanent growth arrest and the acquisition of a secretory, pro-inflammatory state. Increasingly, OIS is viewed as an important barrier to tumorgenesis. Surprisingly, relatively little is known about the metabolic changes that accompany and therefore may contribute to OIS. Here, we have performed a metabolomic and bioenergetic analysis of Ras-induced senescence. Profiling approximately 300 different intracellular metabolites reveals that cells that have undergone OIS develop a unique metabolic signature that differs markedly from cells undergoing replicative senescence. A number of lipid metabolites appear uniquely increased in OIS cells, including a marked increase in the level of certain intracellular long chain fatty acids. Functional studies reveal that this alteration in the metabolome reflects substantial changes in overall lipid metabolism. In particular, Ras-induced senescent cells manifest a decline in lipid synthesis and a significant increase in fatty acid oxidation. Increased fatty acid oxidation results in an unexpectedly high rate of basal oxygen consumption in cells that have undergone OIS. Pharmacological or genetic inhibition of carnitine palmitoyltransferase 1, the rate-limiting step in mitochondrial fatty acid oxidation, restores a presenescent metabolic rate and, surprisingly, selectively inhibits the secretory, pro-inflammatory state that accompanies OIS. Thus, Ras-induced senescent cells demonstrate profound alterations in their metabolic and bioenergetic profiles, particularly with regards to the levels, synthesis and oxidation of free fatty acids. Furthermore, the inflammatory phenotype that accompanies OIS appears to be related to these underlying changes in cellular metabolism.Key words: oncogene-induced senescence, metabolomics, Ras, fatty acid oxidation     

Oncogene-induced senescence results in marked metabolic and bioenergetic alterations

Oncogene-induced senescence (OIS) is characterized by permanent growth arrest and the acquisition of a secretory, pro-inflammatory state. Increasingly, OIS is viewed as an important barrier to tumorgenesis. Surprisingly, relatively little is known about the metabolic changes that accompany and therefore may contribute to OIS. Here, we have performed a metabolomic and bioenergetic analysis of Ras-induced senescence. Profiling approximately 300 different intracellular metabolites reveals that cells that have undergone OIS develop a unique metabolic signature that differs markedly from cells undergoing replicative senescence. A number of lipid metabolites appear uniquely increased in OIS cells, including a marked increase in the level of certain intracellular long chain fatty acids. Functional studies reveal that this alteration in the metabolome reflects substantial changes in overall lipid metabolism. In particular, Ras-induced senescent cells manifest a decline in lipid synthesis and a significant increase in fatty acid oxidation. Increased fatty acid oxidation results in an unexpectedly high rate of basal oxygen consumption in cells that have undergone OIS. Pharmacological or genetic inhibition of carnitine palmitoyltransferase 1, the rate-limiting step in mitochondrial fatty acid oxidation, restores a pre-senescent metabolic rate and, surprisingly, selectively inhibits the secretory, pro-inflammatory state that accompanies OIS. Thus, Ras-induced senescent cells demonstrate profound alterations in their metabolic and bioenergetic profiles, particularly with regards to the levels, synthesis and oxidation of free fatty acids. Furthermore, the inflammatory phenotype that accompanies OIS appears to be related to these underlying changes in cellular metabolism.     

Controversial aspects of oncogene-induced senescence

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?     

Controversial aspects of oncogene-induced senescence

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?     

3'UTR elements inhibit Ras-induced C/EBPβ post-translational activation and senescence in tumour cells

C/EBPβ is an auto-repressed protein that becomes post-translationally activated by Ras-MEK-ERK signalling. C/EBPβ is required for oncogene-induced senescence (OIS) of primary fibroblasts, but also displays pro-oncogenic functions in many tumour cells. Here, we show that C/EBPβ activation by H-Ras(V12) is suppressed in immortalized/transformed cells, but not in primary cells, by its 3' untranslated region (3'UTR). 3'UTR sequences inhibited Ras-induced cytostatic activity of C/EBPβ, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3'UTR suppressed induction of senescence-associated C/EBPβ target genes, while promoting expression of genes linked to cancers and TGFβ signalling. An AU-rich element (ARE) and its cognate RNA-binding protein, HuR, were required for 3'UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPβ translation controls de-repression by Ras signalling. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPβ activation and OIS to proceed. Our findings reveal a novel mechanism whereby non-coding mRNA sequences selectively regulate C/EBPβ activity and suppress its anti-oncogenic functions.     

Oncogene‐induced senescence and melanoma: where do we stand?

In recent years, many groups have detected biomarkers of cellular senescence in a plethora of neoplastic lesions, in model systems, and humans. Indeed, we have come to realize that oncogene-induced senescence (OIS) acts as a potent barrier to oncogenic transformation, operating alongside cell death programs. We have begun to uncover some of its underlying principles, but many fundamental questions remain. In this perspective, some of the 'knowns' and 'unknowns' of OIS are discussed, with a focus on melanomagenesis.     

NORE1A is a double barreled Ras senescence effector that activates p53 and Rb

Although Ras is a potent oncogene in human tumors it has the paradoxical ability to promote Oncogene Induced Senescence (OIS). This appears to serve as a major barrier to Ras driven transformation in vivo. The signaling pathways used by Ras to promote senescence remain relatively poorly understood, but appear to invoke both the p53 and the Rb master tumor suppressors. Exactly how Ras communicates with p53 and Rb has remained something of a puzzle. NORE1A is a direct Ras effector that is frequently downregulated in human tumors. We have now found that it serves as a powerful Ras senescence effector. Moreover, we have defined signaling mechanisms that allows Ras to control both p53 and Rb post-translational modifications via the NORE1A scaffolding molecule. Indeed, NORE1A can be detected in complex with both p53 and Rb. Thus, by coupling Ras to both tumor suppressors, NORE1A forms a major component of the Ras senescence machinery and serves as the missing link between Ras and p53/Rb.     

Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability.     

SUMO-modified nuclear cyclin D1 bypasses Ras-induced senescence

Oncogene-induced senescence represents a key tumor suppressive mechanism. Here, we show that Ras oncogene-induced senescence can be mediated by the recently identified haploinsufficient tumor suppressor apoptosis-stimulating protein of p53 (ASPP) 2 through a novel and p53/p19^Arf/p21^waf1/cip1-independent pathway. ASPP2 suppresses Ras-induced small ubiquitin-like modifier (SUMO)-modified nuclear cyclin D1 and inhibits retinoblastoma protein (Rb) phosphorylation. The lysine residue, K33, of cyclin D1 is a key site for this newly identified regulation. In agreement with the fact that its nuclear localization is required for its oncogenic activity, we show that nuclear cyclin D1 is far more potent than wild-type (WT) cyclin D1 in bypassing Ras-induced senescence. Thus, this study identifies SUMO modification as a positive regulator of nuclear cyclin D1, and reveals a new way by which cell cycle entry and senescence are regulated.     

NQO1 Stabilizes p53 in Response to Oncogene-Induced Senescence

Cellular senescence is a state of permanent cellular arrest that provides an initial barrier to cell transformation and tumorigenesis. In this study, we report that expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a cytoplasmic 2-electron reductase, is induced during oncogene-induced senescence (OIS). Depletion of NQO1 resulted in the delayed onset of senescence. In contrast, ectopic expression of NQO1 enhanced the senescence phenotype. Analysis of the mechanism underlying the up-regulation of NQO1 expression during senescence identified that NQO1 promotes p53 accumulation in an MDM2 and ubiquitin independent manner, which reinforces the cellular senescence phenotype. Specifically, we demonstrated that NRF2/KEAP1 signaling regulates NQO1 expression during OIS. More importantly, we confirmed that depletion of NQO1 facilitates cell transformation and tumorigenesis, which indicates that NQO1 takes part in the senescence barrier and has anti-oncogenic properties in cell transformation.     

Telomere dysfunction puts the brakes on oncogene-induced cancers

EMBO J 31 13, 2839–2851 (2012); published online May082012Senescence represents a major tumour suppressor checkpoint activated by telomere dysfunction or cellular stress factors such as oncogene activation. In this issue of The EMBO Journal, Suram et al (2012) reveal a surprising interconnection between oncogene activation and telomere dysfunction induced senescence. The study supports an alternative model of tumour suppression, indicating that oncogene-induced accumulation of telomeric DNA damage contributes to the induction of senescence in telomerase-negative tumours.Telomere shortening limits the proliferative capacity of primary human cells after 50–70 cell divisions by induction of replicative senescence activated by critically short, dysfunctional telomeres. Different mechanisms were thought to initiate senescence in response to oncogene activation, which occurs abruptly within a few cell doublings (Serrano et al, 1997). Oncogene-induced senescence (OIS) involves an activation of DNA damage signals at stalled replication forks induced by DNA replication stress (Bartkova et al, 2006; Di Micco et al, 2006). Replication fork stalling in response to oncogene activation preferentially affects common fragile sites of the DNA (Tsantoulis et al, 2008). The ends of eukaryotic chromosomes—the telomeres–represent common fragile sites that are sensitive to replication fork stalling (Sfeir et al, 2009). These data made it tempting to speculate whether replication fork stalling at telomeres was causatively involved in OIS. Studies on replicative senescence in human fibroblast also supported this possibility showing that mitogenic signals amplify DNA damage responses in senescent cells (Satyanarayana et al, 2004).Multiple studies revealed experimental evidences that senescence suppresses tumour progression in mouse models and early human tumours (for review see Collado and Serrano, 2010). The relative contribution of OIS and telomere dysfunction induced senescence (TDIS) to tumour suppression and possible interconnections between the two pathways at the level of checkpoint induction were not investigated in previous studies. In this issue of The EMBO Journal, Suram et al (2012) describe the presence of TDIS in human precursor lesions but not in the corresponding malignant tumours. Mechanistically, the study shows that oncogenic signals cause replication fork stalling, resulting in telomeric DNA damage accumulation and activation of DNA damage checkpoints reminiscent to TDIS. Telomerase expression does not rescue replication fork stalling but prevents the accumulation of DNA damage at telomeres allowing a bypass of OIS.The study has several important implications for molecular pathways and therapeutic approaches in cancer that need to be further explored (Figure 1):Open in a separate windowFigure 1Traditional and new models of senescence in tumour suppression. (A) Traditional model of replicative senescence: Telomerase-negative tumour cell clones experience telomere shortening as a consequence of cell division. After a lack period depending on the initial telomere length, tumour cells accumulate telomere dysfunction and activation of senescence impairs tumour growth. Telomerase activation represents a late event allowing tumour progression. (B) New model of oncogene induced, telomere-dependent senescence: Oncogene activation leads to abrupt accumulation of DNA damage at telomeres resulting in senescence and tumour suppression. Telomerase-positive stem cells could be resistant to OIS and may be selected as the cell type of origin of tumour development.(i) Telomere length independent roles of telomeres in tumour suppressionThe classical model of telomere-dependent tumour suppression indicates that proliferation-dependent telomere shortening leads to telomere dysfunction, activation of DNA damage checkpoints, and induction of senescence suppressing the growth of telomerase-negative tumour clones. Studies on mouse models supported this concept showing that telomere shortening impairs the progression of initiated tumours in a telomere length-dependent manner (Feldser and Greider, 2007). The new data from Suram et al (2012) indicate that oncogene-induced replication fork stalling activates a telomere-dependent senescence checkpoint, which is independent of telomere length. The study shows that replication forks stall in response to oncogene activation throughout the genome. However, stalled replication forks are resolved in non-telomeric regions, whereas fork stalling inside telomeres leads to un-repairable DNA damage in telomerase-negative cells. These findings are in line with recent publication showing accumulation of un-repairable DNA damage in telomeric DNA in response to aging and stress-induced DNA damage (Fumagalli et al, 2012).(ii) Telomere length independent roles of telomerase in tumour progressionFollowing the classical model telomeres in tumour suppression (Figure 1A), telomerase re-activation is required for tumour progression by limiting telomere dysfunction and the induction of DNA damage checkpoints in response to telomere shortening. The new data from Suram et al (2012) indicate that telomerase has an additional telomere length independent role in tumour progression. The study shows that catalytically active telomerase prevents the activation of DNA damage signals originating from stalled replication forks inside telomeres in response to oncogene activation (Figure 1B). The exact mechanisms of telomerase-dependent healing of stalled replication forks at telomeres remain to be elucidated. It is also unclear whether telomerase activity can prevent any type of DNA damage at telomeres as an over-expression of TERT could not suppress irradiation-induced cellular senescence or the persistence of telomeric DDR following irradiation, H₂O₂, or chemotherapy induced DNA damage (Hewitt et al, 2012).The data could provide a plausible explanation for the increased tumorigenesis in telomerase transgenic mice—a finding which is difficult to explain by telomere length dependent effects of telomerase given the long telomere reserves in mouse tissues (Gonzalez-Suarez et al, 2001). According to the findings of Suram et al (2012), anti-telomerase therapies could have immediate anti-cancer effects in tumours depending on telomerase-mediated healing of stalled replication forks at telomeres. Specific markers for this dependency could be of clinical value. In addition, the data support the concept that somatic stem cells could represent the cell type of origin of cancers. In contrast to differentiated somatic cells, tissues stem cells are often telomerase-positive, indicating that stem cells might be less sensitive to OIS.     

Subversion of autophagy by Kaposi's sarcoma-associated herpesvirus impairs oncogene-induced senescence

Acute oncogenic stress can activate autophagy and facilitate permanent arrest of the cell cycle through a failsafe mechanism known as oncogene-induced senescence (OIS). Kaposi's sarcoma-associated herpesvirus (KSHV) proteins are known to subvert autophagic pathways, but the link to Kaposi's sarcoma pathogenesis is unclear. We find that oncogenic assault caused by latent KSHV infection elicits DNA damage responses (DDRs) characteristic of OIS, yet infected cells display only modest levels of autophagy and fail to senesce. These aberrant responses result from the combined activities of tandemly expressed KSHV v-cyclin and v-FLIP proteins. v-Cyclin deregulates the cell cycle, triggers DDRs, and if left unchecked can promote autophagy and senescence. However, during latency v-FLIP blocks v-cyclin-induced autophagy and senescence in a manner that requires intact v-FLIP ATG3-binding domains. Together, these data reveal a coordinated viral gene expression program that usurps autophagy, blocks senescence, and facilitates the proliferation of KSHV-infected cells.     

DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

Oncogene-induced senescence (OIS) is a stable cell cycle arrest that occurs in normal cells upon oncogene activation. Cells undergoing OIS express a wide variety of secreted factors that affect the senescent microenvironment termed the senescence-associated secretory phenotype (SASP), which is beneficial or detrimental in a context-dependent manner. OIS cells are also characterized by marked epigenetic changes. We globally assessed histone modifications of OIS cells and discovered an increase in the active histone marks H3K79me2/3. The H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) was necessary and sufficient for increased H3K79me2/3 occupancy at the IL1A gene locus, but not other SASP genes, and was downstream of STING. Modulating DOT1L expression did not affect the cell cycle arrest. Together, our studies establish DOT1L as an epigenetic regulator of the SASP, whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.     

Oxidative stress induces an ATM-independent senescence pathway through p38 MAPK-mediated lamin B1 accumulation

We report crosstalk between three senescence-inducing conditions, DNA damage response (DDR) defects, oxidative stress (OS) and nuclear shape alterations. The recessive autosomal genetic disorder Ataxia telangiectasia (A-T) is associated with DDR defects, endogenous OS and premature ageing. Here, we find frequent nuclear shape alterations in A-T cells, as well as accumulation of the key nuclear architecture component lamin B1. Lamin B1 overexpression is sufficient to induce nuclear shape alterations and senescence in wild-type cells, and normalizing lamin B1 levels in A-T cells reciprocally reduces both nuclear shape alterations and senescence. We further show that OS increases lamin B1 levels through p38 Mitogen Activated Protein kinase activation. Lamin B1 accumulation and nuclear shape alterations also occur during stress-induced senescence and oncogene-induced senescence (OIS), two canonical senescence situations. These data reveal lamin B1 as a general molecular mediator that controls OS-induced senescence, independent of established Ataxia Telangiectasia Mutated (ATM) roles in OIS.