Induction Of Cellular Senescence Research Articles

Friend or foe: emerging role of nuclear factor kappa-light-chain-enhancer of activated B cells in cell senescence.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) proteins are a family of ubiquitously expressed transcription factors that regulate the response to cellular stress. They mediate innate and adaptive immunity through the initiation of an inflammatory response to pro-inflammatory signals. The role of persistent inflammation in aiding tumor development has led to the NF-κB family of transcription factors being strongly implicated in promoting cancer. However, recent studies have now revealed that NF-κB can also function as a tumor suppressor through the induction of cellular senescence. Cellular senescence is a stable cell cycle arrest that normal cells undergo in response to a variety of intrinsic and extrinsic stimuli including: progressive telomere shortening, changes in telomeric structure, or other forms of genotoxic stress. Senescence can compromise tissue repair and regeneration, contributing to tissue and organismal aging via the accumulation of senescent cells, depletion of stem/progenitor cells and secretion of an array of inflammatory cytokines, chemokines, and matrix metalloproteinases. Senescence can also lead to the removal of potentially cancerous cells, thereby acting as a potent tumor suppressor mechanism. Herein, we review the evidence indicating a role for NF-κB in tumor suppression via cellular senescence and suggest that depending upon the subunit expressed, the biological context, and the type and intensity of the signal, NF-κB can indeed promote senescence growth arrest.

Loss of the Osteogenic Differentiation Potential during Senescence Is Limited to Bone Progenitor Cells and Is Dependent on p53

DNA damage can lead to the induction of cellular senescence. In particular, we showed that exposure to ionizing radiation (IR) leads to the senescence of bone marrow-derived multipotent stromal cells (MSC) and osteoblast-like stromal cells (OB–SC), a phenotype associated with bone loss. The mechanism by which IR leads to bone dysfunction is not fully understood. One possibility involves that DNA damage-induced senescence limits the regeneration of bone progenitor cells. Another possibility entails that bone dysfunction arises from the inability of accumulating senescent cells to fulfill their physiological function. Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB–SC could still form mineralize nodules. This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes. Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors. These results are the first to demonstrate that senescence impacts osteogenesis in a cell type dependent manner and suggest that the accumulation of senescent osteoblasts is unlikely to significantly contribute to bone dysfunction in a cell autonomous manner.

76: ADAM17 is activated upon cellular senescence and contributes to the secretory profile of senescent cells by ectodomain shedding

Senescence is the irreversible growth arrest following cellular stressors such as DNA damage. It has become increasingly apparent that senescence represents a crucial tumor suppressor mechanism restricting the proliferation of cells at risk for neoplastic transformation. By secreting numerous cytokines, growth factors as well as proteases – a phenomenon that has been termed Senescence – Associated Secretory Phenotype (SASP) – senescent cells generate a strong proinflammatory environment likely promoting the development of chronic inflammatory diseases and – paradoxically – cancer. The supernatant of senescent cells contains various SASP factors, that are primarily synthesized by the cell as membrane-bound proteins and only afterwards are converted into their soluble counterpart by – as yet unknown – posttranslational means. Here, we show that upon induction of cellular senescence the metalloprotease ADAM17 is activated and releases Amphiregulin and TNFRI from the surface of human cancer cells as well as primary cells by ectodomain shedding. ADAM17 activation involves phosphorylation of its cytoplasmic tail, which is mediated by MAP kinase p38. In addition, these findings have been confirmed in primary embryonic fibroblasts derived from ADAM17 deficient mice suggesting that ADAM17-mediated shedding of SASP components is relevant across species. However, mechanisms governing the SASP are complex, since elevated levels of ICAM-1 are due to increased microparticle release rather than proteolytic ectodomain shedding.

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

HMGA2 expression in white adipose tissue linking cellular senescence with diabetes

There is a clear link between overweight, gain of white adipose tissue, and diabetes type 2 (T2D). The molecular mechanism of the gain of adipose tissue is linked with the expression of high mobility group protein AT-hook 2 (HMGA2), and recent studies revealed an association with a SNP near HMGA2. In this study, we investigated the gene expression of HMGA2, p14 (Arf) , CDKN1A, and BAX in human abdominal subcutaneous white adipose tissue from 157 patients. We found a significant higher HMGA2 expression in obese individuals than in non-obese patients. Furthermore, the HMGA2 expression in white adipose tissue in patient with type 2 diabetes was significantly higher than in nondiabetic patients. There is an association between the DNA-binding nonhistone protein HMGA2 and the risk of developing T2D that remains mechanistically unexplained so far. Likewise, p14(Arf), an inducer of cellular senescence, has been associated with the occurrence of T2D. The data of the present study provide evidence that both proteins act within the same network to drive proliferation of adipose tissue stem and precursor cells, senescence, and increased risk of T2D, respectively.

Matricellular Protein CCN1 Promotes Regression of Liver Fibrosis through Induction of Cellular Senescence in Hepatic Myofibroblasts

Liver fibrosis occurs as a wound-healing response to chronic hepatic injuries irrespective of the underlying etiology and may progress to life-threatening cirrhosis. Here we show that CCN1, a matricellular protein of the CCN (CYR61/CTGF/NOV) family, is accumulated in hepatocytes of human cirrhotic livers. CCN1 is not required for liver development or regeneration, since these processes are normal in mice with hepatocyte-specific Ccn1 deletion. However, Ccn1 expression is upregulated upon liver injuries and functions to inhibit liver fibrogenesis induced by either carbon tetrachloride intoxication or bile duct ligation and promote fibrosis regression. CCN1 acts by triggering cellular senescence in activated hepatic stellate cells and portal fibroblasts by engaging integrin α6β1 to induce reactive oxygen species accumulation through the RAC1-NADPH oxidase 1 enzyme complex, whereupon the senescent cells express an antifibrosis genetic program. Mice with hepatocyte-specific Ccn1 deletion suffer exacerbated fibrosis with a concomitant deficit in cellular senescence, whereas overexpression of hepatic Ccn1 reduces liver fibrosis with enhanced senescence. Furthermore, tail vein delivery of purified CCN1 protein accelerates fibrosis regression in mice with established fibrosis. These findings reveal a novel integrin-dependent mechanism of fibrosis resolution in chronic liver injury and identify the CCN1 signaling pathway as a potential target for therapeutic intervention.

197 ENDOTHELIAL CELL SENESCENCE: THE ROLE OF SIRT6

Introduction The loss of telomere integrity and the consequent induction of cellular senescence have been increasingly implicated in the development of vascular pathologies. Hence unravelling the mechanisms that may attenuate these processes is important to develop strategies against diseases of the cardiovascular system. Sirtuin 6 (SIRT6) belongs to a family of NAD + -dependent deacylases that influence metabolism, stress responses and ageing, and are generally considered to fulfil beneficial functions. Whether SIRT6 has a cytoprotective role in vascular cells is unknown. Here we have investigated the expression of SIRT6 in endothelial cells from different vascular beds and explored its function as an anti-senescence gene. Methods SIRT6 mRNA expression was measured by quantitative polymerase chain reaction. SIRT6 protein localization and levels were determined by immunohistochemistry and immunoblotting, respectively. Silencing of SIRT6 for functional studies was performed by RNA interference. The formation of DNA damage foci and telomere dysfunction-induced foci was detected by immunofluorescence microscopy using anti-gamma H2.AX antibodies. Senescence was evaluated by detection of senescence-associated β-galactosidase. Results Generation of endothelial cells from haematopoietic progenitor cells (HPC) resulted in an increase in SIRT6 mRNA levels. Similarly, compared to HPC, mature endothelial cells derived from different vascular beds also displayed higher levels of this transcript. SIRT6 protein was readily detected in early passage cultured human umbilical vein endothelial cells (HUVEC), in aortic endothelial cells (HAEC), and in normal humna coronary artery endothelium where it displayed a nuclear localization. In cultured cells SIRT6 protein levels increased significantly upon nutrient deprivation and decreased upon senescence. Silencing of SIRT6 in both HUVEC and HAEC diminished cell proliferation, increased the fraction of senescent cells, and impaired the formation of capillary tube networks. Consistent with the induction of a senescent phenotype, SIRT6-silenced cells displayed lower expression levels of eNOS, and higher levels of ICAM-1 and PAI-1. At the molecular level, depletion of SIRT6 led to an increase in nuclear DNA damage and to the formation of telomere-dysfunction induced foci, with no evidence of an increase in intracellular oxidative stress. SIRT6-depleted HUVEC also showed evidence of activation of the p21 signalling pathway. Conclusions This work demonstrates that in endothelial cells SIRT6 confers protection from genomic DNA damage and telomere dysfunction, preventing a decrease in replicative capacity and the onset of premature senescence. Thus, SIRT6 may be important to mantain endothelial homeostatic functions and delay vascular ageing. In this context, our findings that in endothelial cells SIRT6 levels are amenable to up-regulation, may have implications for the development of strategies to combat vascular pathologies.

Environmental stress, ageing and glial cell senescence: a novel mechanistic link to Parkinson's disease?

Exposure to environmental toxins is associated with a variety of age-related diseases including cancer and neurodegeneration. For example, in Parkinson's disease (PD), chronic environmental exposure to certain toxins has been linked to the age-related development of neuropathology. Neuronal damage is believed to involve the induction of neuroinflammatory events as a consequence of glial cell activation. Cellular senescence is a potent anti-cancer mechanism that occurs in a number of proliferative cell types and causes the arrest of proliferation of cells at risk of malignant transformation following exposure to potentially oncogenic stimuli. With age, senescent cells accumulate and express a senescence-associated secretory phenotype (SASP; that is the robust secretion of many inflammatory cytokines, growth factors and proteases). Whereas cell senescence in peripheral tissues has been causally linked to a number of age-related pathologies, little is known about the induction of cellular senescence and the SASP in the brain. On the basis of recently reported findings, we propose that environmental stressors associated with PD may act in part by eliciting senescence and the SASP within non neuronal glial cells in the ageing brain, thus contributing to the characteristic decline in neuronal integrity that occurs in this disorder.

Abstract 2607: BMI1 and dominant negative p53 cooperate to suppress AKT-mediated oncogene-induced senescence and promote transformation in human neural stem cells.

Abstract Purpose: BMI-1 is required for the maintenance of normal brain stem cells and is associated with aggressive brain tumors such as glioblastoma multiforme (GBM). BMI1 is a polycomb group protein that can modify chromatin to silence the pro-senescence tumor suppressor p16INK4a. The PI3K/AKT pathway is also associated with clinically aggressive brain tumors. Interestingly, persistent activation of AKT can cause oncogene-induced senescence. Dominant negative R248W p53 mutation blocks induction of the tumor suppressor p21CIP1 and is found in aggressive brain tumors such as GBM. Telomere maintenance through telomerase enzyme activity is also found in aggressive brain tumors. We investigated if constitutively active AKT, dominant negative p53, BMI1, and hTERT can cooperate to bring about the transformation of human neural stem and progenitor cells, and in particular if BMI1 can suppress AKT induced senescence. Experimental Design: We used lentivirus to introduce singly and in combination BMI1, R248W p53, hTERT and constitutively active AKT into human cortex derived stem and progenitor cells. We assayed the effects of one, two, three or four oncogenes on the proliferation and induction of cellular senescence. We measured proliferation using bromodeoxyurine (BrdU) and senescence by western blotting for p16INK4a and p21 CIP1. Results: Increased expression of BMI1 alone did not have a significant effect on human neural stem cell proliferation, but did lead to the suppression of p16INK4a. AKT alone transduced cells showed no increased proliferation compared to control cells, but did show upregulation of p16INK4a. AKT alone expressing cells stopped growing after several passages in culture, in association with increased p16 INK4a expression. Cells transduced with BMI1 and AKT initially grew at a similar rate to control cells, but after several passages, similar to AKT alone transduced cells, BMI1/AKT cells stopped proliferating and expressed p21CIP1and p16INK4a. Control cells continued to proliferate for multiple additional passages. Cells transduced with R248W p53/hTERT/BMI1/AKT proliferated at an increased rate compared to control cells (17% BrdU positive for pBABE control cells compared to 37% for R248W p53/hTERT/BMI1/AKT p=0.0003, t-test). These cells did not undergo senescence, did not induce p16INK4a or p21CIP1 and continued proliferating more than control or single oncogene transduced cells long after other cells had senesced. Human neural stem cells transduced with R248W p53/hTERT/BMI1/AKT formed aggressive orthotopic xenograft glial tumors, while control cells did not form tumors. Conclusions: BMI1 alone cannot suppress AKT induced senescence in human cortex derived neural stem cells. But BMI1 combined with R248W p53 and hTERT caused suppression of AKT induced senescence, and resulting cells showed signs of oncogenic transformation Citation Format: Dacheng Ding, Ulf Kahlert, Jarek Maciaczyk, Guido Nikkhah, Charles Eberhart, Eric Raabe. BMI1 and dominant negative p53 cooperate to suppress AKT-mediated oncogene-induced senescence and promote transformation in human neural stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2607. doi:10.1158/1538-7445.AM2013-2607

Abstract 4044: MUC4 knockdown induces cellular senescence in head and neck cancer cells.

Abstract The limited effectiveness of therapy for patients with advanced stage Head and Neck Squamous Cell Carcinoma (HNSCC) or recurrent disease is a reflection of an incomplete understanding of the molecular basis of HNSCC pathogenesis. Cellular senescence is an extremely stable form of cell cycle arrest that limits the proliferation of damaged cells and may act as a natural barrier to cancer progression. MUC4, a high molecular weight glycoprotein, is overexpressed in many cancers and is implicated in cell proliferation, adhesion, anti-apoptosis, cell cycle regulation, migration and invasion of various carcinomas, but nothing is known about its clinical relevance and the mechanism through which it regulates cancer progression in HNSCC. Therefore, the present study was aimed to investigate clinical relevance and a potential role of MUC4 in cellular senescence in HNSCCs. Using immunohistochemical analysis, we observed a significant up regulation of MUC4 in 79% (68/86) of HNSCC tissue samples compared to only 10% (1/10) of benign tissues. Further, we observed high expression of MUC4 in a majority of HNSCC cell lines tested. Knockdown (KD) of MUC4 with specific shRNA in HNSCC cells markedly decreased cell proliferation. The doubling time increased from 26 h to 36 h and 34 h to 42 h in MUC4 silenced UMSCC-1 and SCC-10B cells, respectively as compared to their control counterparts. MUC4 KD in UMSCC-1 and UMSCC-10B cells resulted in accumulation of cells at the G0/G1 phase with concomitant decrease in the expression of cell cycle regulating proteins like Cyclin-E and Cyclin-D1, and decrease in BrDU incorporation. More importantly, MUC4 KD resulted in the induction of cellular senescence in both cell lines as indicated by an increase in the number of flat, enlarged and senescence-associated β-galactosidase (SA-β-Gal) positive cells. Further, MUC4 KD resulted in the inhibition of FAK signaling, and decreased motility and invasive behavior in both UMSCC-1 and UMSCC-10B cells. Mechanistic dissection of senescent response to MUC4 silencing indicated decreased acetylated histone enrichment at Cyclin-E/Cyclin-D1 promoters, leading to their downregulation. Further, both cell lines UMSCC-1 and UMSCC-10B underwent a P16 and P21 dependent cellular senescence in response to MUC4 KD that requires inactivation of Akt and ERK signaling. In conclusions, these findings suggest a novel role of MUC4 in regulating cellular senescence and provide evidence of the functional role of MUC4 in the proliferation, motility and invasion of HNSCC cells. Therefore, downregulation of MUC4 may be a promising therapeutic approach for Head and Neck cancer treatment. Citation Format: Muzafar A. Macha, Satyanarayana Rachagani, Priya Pai, Maneesh Jain, Williams M. Lydiatt, Russell B. Smith, Sonny L. Johansson, Subodh M. Lele, Sham S. Kakar, Farghaly H. Ibrahim, John H. Lee. MUC4 knockdown induces cellular senescence in head and neck cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4044. doi:10.1158/1538-7445.AM2013-4044

Abstract 4049: The gain-of-function properties of mutant p53 rescues cells from telomere dysfunction-induced crisis and promotes tumorigenesis.

Abstract Telomeres are nucleoprotein complexes that cap the ends of eukaryotic chromosomes and maintain chromosomal stability. Telomeres are protected by shelterin, a complex of six core proteins (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1) that prevents the chromosome ends from being mistakenly recognized as damaged DNA. Telomeres that can no longer exert end-protective functions are said to be dysfunctional. Telomere dysfunction can be caused by progressive telomere attrition (telomere shortening) or through disruption of the shelterin complex (telomere uncapping). To study whether chromosomal instability caused by telomere uncapping leads to tumorigenesis, we used the Cre-lox system to delete the telomere binding protein TRF2 in mouse embryonic fibroblasts (MEFs). In the presence of tumor suppressor protein p53, loss of TRF2 inhibited cell proliferation via induction of cellular senescence. In contrast, the absence of p53 allowed the TRF2-deficient cells to bypass senescence as these cells were negative for SA-β-galactosidase staining. However, these p53-deficient cells stopped proliferating ten days after TRF2 depletion and eventually entered telomere uncapping-induced crisis (indicated by the significant appearance of chromosomal end to end fusions, mitotic failure and induction of apoptosis). Our mechanistic studies revealed that these p53-deficient cells underwent a novel ATM-caspase 2-dependent apoptosis upon loss of TRF2. Accordingly, knocking out caspase 2 rescued these cells from apoptosis, and thus increased long-term survival of the cell colonies as well as promoted cell transformation. Mutations of p53 are predominantly missense mutations that result in accumulation of mutant p53 proteins with loss of wild-type p53 tumor-suppressive functions and gain of oncogenic activities. To study whether the gain-of-function properties of mutant p53 can rescue cells undergoing telomere crisis from cell death, we generated MEFs expressing mutant p53R172H that corresponds to the p53R175H “hot spot” mutation in human cancers. We observed that mutant p53R172H did not prevent TRF2 depletion-induced chromosomal instability as indicated by extensive chromosome fusions. However, expression of mutant p53R172H significantly rescued cells from TRF2 loss-initiated cell apoptosis and promoted cell transformation as determined by anchorage-independent soft agar assay. More importantly, mutant p53R172H promoted tumor development of TRF2-deficient MEFs in immunodeficient mice. Collectively, our findings provide compelling genetic evidence that telomere uncapping-driven chromosomal instability activates ATM-caspase 2-dependent apoptosis to suppress tumorigenesis. However, the gain-of-function properties of mutant p53 can rescue the cells undergoing telomere crisis from cell death and thus promote tumorigenesis. Citation Format: Li-Ju Chang, Fengxia Wu, Xiaolan Guo, Yibin Deng. The gain-of-function properties of mutant p53 rescues cells from telomere dysfunction-induced crisis and promotes tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4049. doi:10.1158/1538-7445.AM2013-4049

Abstract 4041: Wild-type p53-induced phosphatase 1 (Wip1) prevents cellular senescence at physiological oxygen levels by regulating DNA damage signaling during DNA replication.

Abstract Wip1 (PPM1D) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Upon repair of damage, Wip1 phosphatase activity dampens stress signaling and facilitates the return to homeostasis, thus, regulating recovery from DDR activation. Wip1 is amplified in several types of human primary cancer and has been shown to promote tumorigenesis by inhibiting several tumor suppressors, including p53, therefore its oncogenic potential is now recognized. Interestingly, while Wip1 knockout (Ppm1d–/–) mice are resistant to tumorigenesis, they exhibit reproductive and immune system defects, increased stress sensitivity and a moderately reduced lifespan. Furthermore, Ppm1d–/– Mouse Embryonic Fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions. Little is known, however, about the mechanism by which the deletion of Ppm1d induces premature senescence. Therefore, we have investigated the role of Wip1 in the regulation of cellular senescence in MEFs. We found that at physiological oxygen tension (3% oxygen), early passage Ppm1d–/– MEFs underwent increased cellular senescence as compared with wild–type (Wt) MEFs, as shown by reduced proliferation rate, increased number of flattened enlarged cells, increased SA–β–Gal positive cells, and reduced BrdU incorporation. Moreover, under 3% oxygen, early passage Ppm1d–/– MEFs exhibited more γ–H2AX positive foci, increased activation of p53 and increased levels of p21 than Wt MEFs. Interestingly, we found that the increased levels of γ–H2AX in Ppm1d–/– MEFs were primarily associated with S–phase cells. Given that S–phase cells are more susceptible to oxidative damage, the increased levels of γ–H2AX may result from increased production of reactive oxygen species (ROS) in Ppm1d–/– MEFs. However, primary passage Wt and Ppm1d–/– MEFs cultured under 3% oxygen exhibited no significant difference in intracellular–ROS levels. Ppm1d–/– MEFs did show sustained activation of DDR signaling (γ–H2AX, p–p53Ser15, p21) after H2O2 treatment compared with Wt MEFs. Furthermore, Ppm1d–/– MEFs exhibited increased activation of Chk1, indicating that there is increased replicative stress signaling. These findings suggest that Wip1 prevents the induction of cellular senescence by regulating both oxidative stress– and replicative stress–induced DNA damage signaling during DNA replication. Citation Format: Hiroyasu Sakai, Christophe E. Redon, William M. Bonner, Ettore Appella, Sharlyn Mazur. Wild-type p53-induced phosphatase 1 (Wip1) prevents cellular senescence at physiological oxygen levels by regulating DNA damage signaling during DNA replication. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4041. doi:10.1158/1538-7445.AM2013-4041

Toll-like receptor 4 activity protects against hepatocellular tumorigenesis and progression by regulating expression of DNA repair protein Ku70 in mice

Hepatocellular carcinoma (HCC) is a devastating consequence of chronic inflammatory liver diseases. The goal of this study was to investigate whether Toll-like receptor 4 (TLR4) activity contributes to HCC initiation and progression in mice. A mouse model of diethylnitrosamine (DEN)-induced HCC was generated with wild-type and TLR4 mutant mice, and the development and progression of HCC and senescent responses were assessed using morphologic, immunological, and biochemical criteria. We found that genetic or pharmacologic blocking of TLR4 increased susceptibility to DEN-induced HCC carcinogenesis and progression, which was indicated by increases in number of tumor nodules, tumor volume, and animal death. The enhanced HCC was associated with a broad-spectrum reduction of immune response to DEN liver injury, as indicated by decreases in the liver-infiltrating F4/80+ macrophages, the apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase/NF-κB and IRF3 signaling activities, and the expression of inflammatory cytokines. Suppressed immune networks resulted in a halt of cellular senescence induction in TLR4 mutant liver tissue, which promoted proliferation and suppressed programmed cell death. Moreover, TLR4 mutation resulted in a suppressed capacity of DNA repair due to a decrease in TLR4-medicated expression of DNA repair proteins Ku70/80 in liver tissue and cells. Isotopic expression of Ku70 in TLR4 mutant mice restored senescence and interrupted the positive feedback loop of DNA damage and oxidative stress, which reversed TLR4 mutation-deteriorated HCC carcinogenesis and progression. TLR4 plays an integrated defense role against HCC carcinogenesis by enhancing the expression and function of DNA repair protein Ku70. Our studies provide novel insight into TLR4 activity in the regulation of HCC tumorigenesis, which may be useful for the prevention of HCC development.

Bexarotene Induces Cellular Senescence in MMTV-Neu Mouse Model of Mammary Carcinogenesis

Previous studies have shown that retinoids and rexinoids can prevent breast cancer in animal models and in women with increased risk of developing the disease. The cellular effects of these vitamin A analogues have been primarily associated with induction of differentiation and inhibition of proliferation. In this study, we tested the hypothesis that bexarotene (LGD1069, Targretin), a rexinoid, can not only inhibit cell proliferation but also induce cellular senescence in mammary epithelial cells, premalignant lesions, and tumors of the MMTV-Neu model of mammary carcinogenesis, which develops estrogen receptor-negative tumors. Mice with palpable mammary tumors were treated for 4 weeks with bexarotene at 80 or 40 mg/kg body weight, and senescent cells were determined by SA-β-Gal assay. Bexarotene decreased in a dose-dependent manner the multiplicity of premalignant lesions and tumors, and this was associated with inhibition of cell proliferation and induction of cellular senescence and apoptosis. By double labeling of senescent cells, first by SA-β-Gal and then by antibodies against genes related to cellular senescence, we found that p21, p16, and RARβ, but not p53, were upregulated by bexarotene in mammary tumors and in breast cancer cell lines, suggesting involvement of multiple signaling pathways in mediating the senescence program of rexinoids. These findings indicate that, in addition to cell proliferation and apoptosis, cellular senescence could be used as a potential biomarker of response in breast cancer prevention and therapy studies with rexinoids and possibly with other antitumor agents.

Noncanonical roles of the immune system in eliciting oncogene addiction

Cancer is highly complex. The magnitude of this complexity makes it highly surprising that even the brief suppression of an oncogene can sometimes result in rapid and sustained tumor regression, illustrating that cancers can be 'oncogene addicted' [1-10]. The essential implication is that oncogenes may not only fuel the initiation of tumorigenesis, but in some cases must be excessively activated to maintain a neoplastic state [11]. Oncogene suppression acutely restores normal physiological programs that effectively overrides secondary genetic events and a cancer collapses [12,13]. Oncogene addiction is the description of the dramatic and sustained regression of some cancers upon the specific inactivation of a single oncogene [1-13,14(••),15,16(••)], that can occur through tumor intrinsic [1,2,4,12], but also host immune mechanisms [17-23]. Notably, oncogene inactivation elicits a host immune response that involves specific immune effectors and cytokines that facilitate a remodeling of the tumor microenvironment including the shut down of angiogenesis and the induction of cellular senescence of tumor cells [16(••)]. Hence, immune effectors are not only critically involved in tumor prevention, initiation [17-19], and progression [20], but also appear to be essential to tumor regression upon oncogene inactivation [21,22(••),23(••)]. Understanding how the inactivation of an oncogene elicits a systemic signal in the host that prompts a deconstruction of a tumor could have important implications. The combination of oncogene-targeted therapy together with immunomodulatory therapy may be ideal for the development of both robust tumor intrinsic and immunological responses, effectively leading to sustained tumor regression.

Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters.

Activation of Lung p53 by Nutlin-3a Prevents and Reverses Experimental Pulmonary Hypertension

Induction of cellular senescence through activation of the p53 tumor suppressor protein is a new option for treating proliferative disorders. Nutlins prevent the ubiquitin ligase MDM2 (murine double minute 2), a negative p53 regulator, from interacting with p53. We hypothesized that cell senescence induced by Nutlin-3a exerted therapeutic effects in pulmonary hypertension (PH) by limiting the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). Nutlin-3a treatment of cultured human PA-SMCs resulted in cell growth arrest with the induction of senescence but not apoptosis; increased phosphorylated p53 protein levels; and expression of p53 target genes including p21, Bax, BTG2, and MDM2. Daily intraperitoneal Nutlin-3a treatment for 3 weeks dose-dependently reduced PH, right ventricular hypertrophy, and distal pulmonary artery muscularization in mice exposed to chronic hypoxia or SU5416/hypoxia. Nutlin-3a treatment also partially reversed PH in chronically hypoxic or transgenic mice overexpressing the serotonin-transporter in SMCs (SM22-5HTT+ mice). In these mouse models of PH, Nutlin-3a markedly increased senescent p21-stained PA-SMCs; lung p53, p21, and MDM2 protein levels; and p21, Bax, PUMA, BTG2, and MDM2 mRNA levels; but induced only minor changes in control mice without PH. Marked MDM2 immunostaining was seen in both mouse and human remodeled pulmonary vessels, supporting the use of Nutlins as a PH-targeted therapy. PH prevention or reversal by Nutlin-3a required lung p53 stabilization and increased p21 expression, as indicated by the absence of Nutlin-3a effects in hypoxia-exposed p53(-/-) and p21(-/-) mice. Nutlin-3a may hold promise as a prosenescence treatment targeting PA-SMCs in PH.

Roles and mechanisms of cellular senescence in regulation of tissue homeostasis

Cellular senescence is the state of irreversible cell cycle arrest that can be induced by a variety of potentially oncogenic stimuli and has therefore long been considered to suppress tumorigenesis, acting as a guardian of homeostasis. However, surprisingly, emerging evidence reveals that senescent cells also promote secretion of a series of inflammatory cytokines, chemokines, growth factors and matrix remodeling factors, which alter the local tissue environment and contribute to chronic inflammation and cancer. This newly identified senescence phenotype, termed the senescence-associated secretory phenotype (SASP) or the senescence-messaging secretome (SMS), is induced by DNA damage that promotes the induction of cellular senescence. All of these senescence-associated secreting factors are involved in homeostatic disorders such as cancer. Therefore, it is quite possible that accumulation of senescent cells during the aging process in vivo might contribute to age-related increases in homeostatic disorders. In this review, current knowledge of the molecular and cellular biology of cellular senescence is introduced, focusing on its positive and negative roles in controlling tissue homeostasis in vivo.

Impact of Cellular Senescence in Aging and Cancer

Cellular senescence is a response to nonlethal intrinsic or extrinsic stress that results in persistent growth arrest with a distinct morphological and biochemical phenotype. The engagement of senescence may represent a key component for therapeutic intervention in the eradication of cancer. Nevertheless, for many years, the role of senescence in opposing tumour growth in vivo had previously been underestimated. The potential role of cellular senescence in anti-cancer therapy may be particularly attractive in advanced age, because of the age-related changes occurring at the level of both tumor suppressor genes and immune functions. This review, which is focused on the impact of cellular senescence in aging and cancer, summarises the intrinsic pathways and the molecular and epigenetic changes involved in the induction of cellular senescence, and analyzes the changes occurring at the level of these pathways during aging and cancer.

A positive role for senescence in reproduction?

A positive role for senescence in reproduction?