Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody.

Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated ß-galactosidase as a collection of semiselective markers to monitor the presence of senescent cells. To identify novel biomarkers of senescence, we immunized BALB/c mice with senescent mouse lung fibroblasts and screened for antibodies that recognized senescence-associated cell-surface antigens by FACS analysis and a newly developed cell-based ELISA. The majority of antibodies that we isolated, cloned, and sequenced belonged to the IgM isotype of the innate immune system. In-depth characterization of one of these monoclonal, polyreactive natural antibodies, the IgM clone 9H4, revealed its ability to recognize the intermediate filament vimentin. By using 9H4, we observed that senescent primary human fibroblasts express vimentin on their cell surface, and MS analysis revealed a posttranslational modification on cysteine 328 (C328) by the oxidative adduct malondialdehyde (MDA). Moreover, elevated levels of secreted MDA-modified vimentin were detected in the plasma of aged senescence-accelerated mouse prone 8 mice, which are known to have deregulated reactive oxygen species metabolism and accelerated aging. Based on these findings, we hypothesize that humoral innate immunity may recognize senescent cells by the presence of membrane-bound MDA-vimentin, presumably as part of a senescence eradication mechanism that may become impaired with age and result in senescent cell accumulation.

Core circadian protein CLOCK is a positive regulator of NF-κB-mediated transcription.

The circadian clock controls many physiological parameters including immune response to infectious agents, which is mediated by activation of the transcription factor NF-κB. It is widely accepted that circadian regulation is based on periodic changes in gene expression that are triggered by transcriptional activity of the CLOCK/BMAL1 complex. Through the use of a mouse model system we show that daily variations in the intensity of the NF-κB response to a variety of immunomodulators are mediated by core circadian protein CLOCK, which can up-regulate NF-κB-mediated transcription in the absence of BMAL1; moreover, BMAL1 counteracts the CLOCK-dependent increase in the activation of NF-κB-responsive genes. Consistent with its regulatory function, CLOCK is found in protein complexes with the p65 subunit of NF-κB, and its overexpression correlates with an increase in specific phosphorylated and acetylated transcriptionally active forms of p65. In addition, activation of NF-κB in response to immunostimuli in mouse embryonic fibroblasts and primary hepatocytes isolated from Clock-deficient mice is significantly reduced compared with WT cells, whereas Clock-Δ19 mutation, which reduces the transactivation capacity of CLOCK on E-box-containing circadian promoters, has no effect on the ability of CLOCK to up-regulate NF-κB-responsive promoters. These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively.

Tissue-specific changes in molecular clocks during the transition from pregnancy to lactation in mice.

Circadian clocks regulate homeostasis and mediate responses to stressors. Lactation is one of the most energetically demanding periods of an adult female's life. Peripartum changes occur in almost every organ so the dam can support neonatal growth through milk production while homeostasis is maintained. How circadian clocks are involved in adaptation to lactation is currently unknown. The abundance and temporal pattern of core clock genes' expression were measured in suprachiasmatic nucleus, liver, and mammary from late pregnant and early lactation mice. Tissue-specific changes in molecular clocks occurred between physiological states. Amplitude and robustness of rhythms increased in suprachiasmatic nucleus and liver. Mammary rhythms of core molecular clock genes were suppressed. Attenuated rhythms appeared to be a physiological adaptation of mammary to lactation, because manipulation of timing of suckling resulting in significant differences in plasma prolactin and corticosterone had no effect on amplitude. Analysis of core clock proteins revealed that the stoichiometric relationship between positive (CLOCK) and negative (PER2) components remained 1:1 in liver but was increased to 4:1 in mammary during physiological transition. Induction of differentiation of mammary epithelial cell line HC11 with dexamethasone, insulin, and prolactin resulted in similar stoichiometric changes among positive and negative clock regulators, and prolactin induced phase shifts in HC11 Arntl expression rhythm. Data support that distinct mechanisms drive periparturient changes in mammary clock. Stoichiometric change in clock regulators occurs with gland differentiation. Suppression of mammary clock gene expression rhythms represents a physiological adaptation to suckling cues. Adaptations in mammary clock are likely needed in part to support suckling demands of neonates.

Pharmacological modulators of the circadian clock as potential therapeutic drugs: focus on genotoxic/anticancer therapy.

The circadian clock is an evolutionary conserved intrinsic timekeeping mechanism that controls daily variations in multiple biological processes. One important process that is modulated by the circadian clock is an organism's response to genotoxic stress, such as that induced by anticancer drug and radiation treatments. Numerous observations made in animal models have convincingly demonstrated that drug-induced toxicity displays prominent daily variations; therefore, undesirable side effects could be significantly reduced by administration of drugs at specific times when they are better tolerated. In some cases, these critical times of the day coincide with increased sensitivity of tumor cells allowing for a greater therapeutic index. Despite encouraging results of chronomodulated therapies, our knowledge of molecular mechanisms underlying these observations remains sketchy. Here we review recent progress in deciphering mechanistic links between circadian and stress response pathways with a focus on how these findings could be applied to anticancer clinical practice. We discuss the potential for using high-throughput screens to identify small molecules that can modulate basic parameters of the entire circadian machinery as well as functional activity of its individual components. We also describe the discovery of several small molecules that can pharmacologically modulate clock and that have a potential to be developed into therapeutic drugs. We believe that translational applications of clock-targeting pharmaceuticals are twofold: they may be developed into drugs to treat circadian-related disorders or used in combination with existing therapeutic strategies to improve therapeutic index of a given genotoxic treatment via the intrinsic clock mechanism.

Circadian proteins and genotoxic stress response.

The circadian clock is an evolutionarily conserved time-keeping system that coordinates the physiology of the organism with daily changes in the environment. A growing body of evidence gradually leads to the conception that virtually all aspects of the biochemical, physiological, and behavioral functions of the animal are linked to circadian regulation. Moreover, proper synchronization of various processes through the activity of circadian components is important for the well-being of many organisms, including humans. The focus of this review is the circadian control of an organism's response to genotoxic stress, which is a major contributor to life-threatening human pathologies such as cancer and cardiovascular disease.

Circadian Rhythm Disruption Increases Tumor Growth Rate and Accumulation of Myeloid-Derived Suppressor Cells.

Circadian rhythm disruption is implicated in the initiation and progression of many diseases, including cancer. External stimuli, such as sunlight, serve to synchronize physiological processes and cellular functions to a 24-h cycle. The immune system is controlled by circadian rhythms, and perturbation of these rhythms can potentially alter the immune response to infections and tumors. The effect of circadian rhythm disruption on the immune response to tumors remains unclear. Specifically, the effects of circadian disruption (CD) on immunosuppressive cell types within the tumor, such as myeloid-derived suppressor cells (MDSCs), are unknown. In this study, a shifting lighting schedule is used to disrupt the circadian rhythm of mice. After acclimation to lighting schedules, mice are inoculated with 4T1 or B16-F10 tumors. Tumor growth is increased in mice housed under circadian disrupting lighting conditions compared to standard lighting conditions. Analysis of immune populations within the spleen and tumor shows an increased accumulation of MDSCs within these tissues, suggesting that MDSC mediated immunosuppression plays a role in the enhanced tumor growth caused by circadian disruption. This paves the way for future studies of the effects of CD on immunosuppression in cancer.

Unsupervised learning of aging principles from longitudinal data.

Age is the leading risk factor for prevalent diseases and death. However, the relation between age-related physiological changes and lifespan is poorly understood. We combined analytical and machine learning tools to describe the aging process in large sets of longitudinal measurements. Assuming that aging results from a dynamic instability of the organism state, we designed a deep artificial neural network, including auto-encoder and auto-regression (AR) components. The AR model tied the dynamics of physiological state with the stochastic evolution of a single variable, the "dynamic frailty indicator" (dFI). In a subset of blood tests from the Mouse Phenome Database, dFI increased exponentially and predicted the remaining lifespan. The observation of the limiting dFI was consistent with the late-life mortality deceleration. dFI changed along with hallmarks of aging, including frailty index, molecular markers of inflammation, senescent cell accumulation, and responded to life-shortening (high-fat diet) and life-extending (rapamycin) treatments.

Rapamycin extends maximal lifespan in cancer-prone mice.

Aging is associated with obesity and cancer. Calorie restriction both slows down aging and delays cancer. Evidence has emerged that the nutrient-sensing mammalian target of rapamycin (mTOR) pathway is involved in cellular and organismal aging. Here we show that the mTOR inhibitor rapamycin prevents age-related weight gain, decreases rate of aging, increases lifespan, and suppresses carcinogenesis in transgenic HER-2/neu cancer-prone mice. Rapamycin dramatically delayed tumor onset as well as decreased the number of tumors per animal and tumor size. We suggest that, by slowing down organismal aging, rapamycin delays cancer.

Superior cancer preventive efficacy of low versus high dose of mTOR inhibitor in a mouse model of prostate cancer.

The mechanistic target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation and survival in response to the availability of energy sources and growth factors. Cancer development and progression is often associated with constitutive activation of the mTOR pathway, thus justifying mTOR inhibition as a promising approach to cancer treatment and prevention. However, development of previous rapamycin analogues has been complicated by their induction of adverse side effects and variable efficacy. Since mTOR pathway regulation involves multiple feedback mechanisms that may be differentially activated depending on the degree of mTOR inhibition, we investigated whether rapamycin dosing could be adjusted to achieve chemopreventive efficacy without side effects. Thus, we tested the efficacy of two doses of a novel, highly bioavailable nanoformulation of rapamycin, Rapatar, in a mouse prostate cancer model (male mice with prostate epithelium-specific Pten-knockout). We found that the highest efficacy was achieved by the lowest dose of Rapatar used in the study. While both doses tested were equally effective in suppressing proliferation of prostate epithelial cells, higher dose resulted in activation of feedback circuits that reduced the drug's tumor preventive efficacy. These results demonstrate that low doses of highly bioavailable mTOR inhibitor, Rapatar, may provide safe and effective cancer prevention.

Immune checkpoint protein VSIG4 as a biomarker of aging in murine adipose tissue.

Adipose tissue is recognized as a major source of systemic inflammation with age, driving age-related tissue dysfunction and pathogenesis. Macrophages (Mφ) are central to these changes yet adipose tissue Mφ (ATMs) from aged mice remain poorly characterized. To identify biomarkers underlying changes in aged adipose tissue, we performed an unbiased RNA-seq analysis of ATMs from young (8-week-old) and healthy aged (80-week-old) mice. One of the genes identified, V-set immunoglobulin-domain-containing 4 (VSIG4/CRIg), encodes a Mφ-associated complement receptor and B7 family-related immune checkpoint protein. Here, we demonstrate that Vsig4 expression is highly upregulated with age in perigonadal white adipose tissue (gWAT) in two mouse strains (inbred C57BL/6J and outbred NIH Swiss) independent of gender. The accumulation of VSIG4 was mainly attributed to a fourfold increase in the proportion of VSIG4+ ATMs (13%-52%). In a longitudinal study, VSIG4 expression in gWAT showed a strong correlation with age within a cohort of male and female mice and correlated strongly with physiological frailty index (PFI, a multi-parameter assessment of health) in male mice. Our results indicate that VSIG4 is a novel biomarker of aged murine ATMs. VSIG4 expression was also found to be elevated in other aging tissues (e.g., thymus) and was strongly induced in tumor-adjacent stroma in cases of spontaneous and xenograft lung cancer models. VSIG4 expression was recently associated with cancer and several inflammatory diseases with diagnostic and prognostic potential in both mice and humans. Further investigation is required to determine whether VSIG4-positive Mφ contribute to immunosenescence and/or systemic age-related deficits.

Daily Time of Radiation Treatment Is Associated with Subsequent Oral Mucositis Severity during Radiotherapy in Head and Neck Cancer Patients.

BACKGROUND: Limited treatment options are available for oral mucositis, a common, debilitating complication of cancer therapy. We examined the association between daily delivery time of radiotherapy and the severity of oral mucositis in patients with head and neck cancer. METHODS: We used electronic medical records of 190 patients with head and neck squamous cell carcinoma who completed radiotherapy, with or without concurrent chemotherapy, at Roswell Park Comprehensive Cancer Center (Buffalo, NY) between 2015 and 2017. Throughout a 7-week treatment course, patient mouth and throat soreness (MTS) was self-reported weekly using a validated oral mucositis questionnaire, with responses 0 (no) to 4 (extreme). Average treatment times from day 1 until the day before each mucositis survey were categorized into seven groups. Multivariable-adjusted marginal average scores (LSmeans) were estimated for the repeated- and maximum-MTS, using a linear-mixed model and generalized-linear model, respectively. RESULTS: Radiation treatment time was significantly associated with oral mucositis severity using both repeated-MTS (n = 1,156; P = 0.02) and maximum-MTS (n = 190; P = 0.04), with consistent patterns. The severity was lowest for patients treated during 8:30 to <9:30 am (LSmeans for maximum-MTS = 2.24; SE = 0.15), increased at later treatment times and peaked at early afternoon (11:30 am to <3:00 pm, LSmeans = 2.66-2.71; SEs = 0.16/0.17), and then decreased substantially after 3 pm. CONCLUSIONS: We report a significant association between radiation treatment time and oral mucositis severity in patients with head and neck cancer. IMPACT: Although additional studies are needed, these data suggest a potential simple treatment time solution to limit severity of oral mucositis during radiotherapy without increasing cost.

OT-82, a novel anticancer drug candidate that targets the strong dependence of hematological malignancies on NAD biosynthesis.

Effective treatment of some types of cancer can be achieved by modulating cell lineage-specific rather than tumor-specific targets. We conducted a systematic search for novel agents selectively toxic to cells of hematopoietic origin. Chemical library screenings followed by hit-to-lead optimization identified OT-82, a small molecule with strong efficacy against hematopoietic malignancies including acute myeloblastic and lymphoblastic adult and pediatric leukemias, erythroleukemia, multiple myeloma, and Burkitt's lymphoma in vitro and in mouse xenograft models. OT-82 was also more toxic towards patients-derived leukemic cells versus healthy bone marrow-derived hematopoietic precursors. OT-82 was shown to induce cell death by inhibiting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway of NAD synthesis. In mice, optimization of OT-82 dosing and dietary niacin further expanded the compound's therapeutic index. In toxicological studies conducted in mice and nonhuman primates, OT-82 showed no cardiac, neurological or retinal toxicities observed with other NAMPT inhibitors and had no effect on mouse aging or longevity. Hematopoietic and lymphoid organs were identified as the primary targets for dose limiting toxicity of OT-82 in both species. These results reveal strong dependence of neoplastic cells of hematopoietic origin on NAMPT and introduce OT-82 as a promising candidate for the treatment of hematological malignancies.

Pharmacological modulators of the circadian clock as potential therapeutic drugs.

Circadian clocks are molecular time-keeping systems that underlie daily fluctuations in multiple physiological and biochemical processes. It is well recognized now that dysfunctions of the circadian system (both genetically and environmentally induced) are associated with the development of various pathological conditions. Here we describe the application of high throughput screening approach designed to search for small molecules capable of pharmacological modulation of the molecular clock. We provide evidence for the feasibility and value of this approach for both scientific and therapeutic purposes.

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation.

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.

Evidence for circadian regulation of activating transcription factor 5 but not tyrosine hydroxylase by the chromaffin cell clock.

In mammals, adrenal medulla chromaffin cells constitute a fundamental component of the sympathetic nervous system outflow, producing most of the circulating adrenaline. We recently found that the rhesus monkey adrenal gland expresses several genes in a 24-h rhythmic pattern, including TH (the rate-limiting enzyme in catecholamine synthesis) and Atf5 (a transcription factor involved in apoptosis and neural cell differentiation) together with the core-clock genes. To examine whether these core-clock genes play a role in adrenal circadian function, we exposed rat pheochromocytoma PC12 cells to a serum shock and found that it triggered rhythmic oscillation of the clock genes rBmal1, rPer1, rRev-erbalpha, and rCry1 and induced the circadian expression of Atf5 but not TH. Furthermore, we found that the CLOCK/brain and muscle Arnt-like protein-1 (BMAL1) heterodimer could regulate Atf5 expression by binding to an E-box motif and repressing activity of its promoter. The physiological relevance of this interaction was evident in Bmal1 -/- mice, in which blunted circadian rhythm of Atf5 mRNA was observed in the liver, together with significantly higher expression levels in both liver and adrenal glands. Although we found no compelling evidence for rhythmic expression of TH in chromaffin cells being regulated by an intrinsic molecular clock mechanism, the Atf5 results raise the possibility that other aspects of chromaffin cell physiology, such as cell survival and cell differentiation, may well be intrinsically regulated.

Dual role of the CLOCK/BMAL1 circadian complex in transcriptional regulation.

The basic helix-loop-helix (bHLH) -PAS domain containing transcription factors CLOCK and BMAL1 are two major components of the circadian molecular oscillator. It is known that the CLOCK/BMAL1 complex positively regulates the activity of E-box containing promoters. Here we demonstrate that the CLOCK/BMAL1 complex can also suppress the activity of some promoters upon its interaction with CRYPTOCHROME (CRY). Such a dual function of the circadian transcriptional complex provides a mechanistic explanation for the unpredicted pattern of circadian gene expression in the tissues of Bmal1 null mice. We speculate that the switch from transcriptional activation to transcriptional repression may provide a highly efficient mechanism for circadian control of gene expression. We also show that CLOCK/BMAL1 can interfere with promoter regulation by other, non-circadian, transcription factors including N-MYC and ETS, leading to attenuation or abrogation of transcription of CLOCK/BMAL1-controlled stress-induced genes. We propose that, based upon these results, both circadian repression and activation of the transcription of different target genes are required for circadian responses to various external stimuli, including genotoxic stress induced by anticancer treatment.

Physiological frailty index (PFI): quantitative in-life estimate of individual biological age in mice.

The development of healthspan-extending pharmaceuticals requires quantitative estimation of age-related progressive physiological decline. In humans, individual health status can be quantitatively assessed by means of a frailty index (FI), a parameter which reflects the scale of accumulation of age-related deficits. However, adaptation of this methodology to animal models is a challenging task since it includes multiple subjective parameters. Here we report a development of a quantitative non-invasive procedure to estimate biological age of an individual animal by creating physiological frailty index (PFI). We demonstrated the dynamics of PFI increase during chronological aging of male and female NIH Swiss mice. We also demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity and provide a tool for its quantitative assessment. Additionally, we showed that PFI could reveal anti-aging effect of mTOR inhibitor rapatar (bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors. Together, these data introduce PFI as a reliable, non-invasive, quantitative tool suitable for testing potential anti-aging pharmaceuticals in pre-clinical studies.

The orphan receptor Rev-erbalpha gene is a target of the circadian clock pacemaker.

Rev-erbalpha is a ubiquitously expressed orphan nuclear receptor which functions as a constitutive transcriptional repressor and is expressed in vertebrates according to a robust circadian rhythm. We report here that two Rev-erbalpha mRNA isoforms, namely Rev-erbalpha1 and Rev-erbalpha 2, are generated through alternative promoter usage and that both show a circadian expression pattern in an in vitro system using serum-shocked fibroblasts. Both promoter regions P1 (Rev-erbalpha1) and P2 (Rev-erbalpha2) contain several E-box DNA sequences which function as response elements for the core circadian-clock components: CLOCK and BMAL1. The CLOCK-BMAL1 heterodimer stimulates the activity of both P1 and P2 promoters in transient transfection assay by 3-6-fold. This activation was inhibited by the overexpression of CRY1, a component of the negative limb of the circadian transcriptional loop. Critical E-box elements were mapped within both promoters. This regulation is conserved in vertebrates since we found that the CLOCK-BMAL1 heterodimer also regulates the zebrafish Rev-erbalpha gene. In line with these data Rev-erbalpha circadian expression was strongly impaired in the livers of Clock mutant mice and in the pineal glands of zebrafish embryos treated with Clock and Bmal1 antisense oligonucleotides. Together these data demonstrate that CLOCK is a critical regulator of Rev-erbalpha circadian gene expression in evolutionarily distant vertebrates and suggest a role for Rev-erbalpha in the circadian clock output.

Daily rhythms are retained both in spontaneously developed sarcomas and in xenografts grown in immunocompromised SCID mice.

The circadian clock generates and regulates many daily physiological, metabolic and behavioral rhythms as well as acute responses to various types of stresses including those induced by anticancer treatment. It has been proposed that modulatory function of the clock may be used for improving the therapeutic efficacy of established anti-cancer treatments. In order to rationally exploit this mechanism, more information is needed to fully characterize the functional status of the molecular clock in tumors of different cellular origin; however, the data describing tumor clocks are still inconsistent. Here we tested the status of clock in two models of tumors derived from connective tissue: sarcomas spontaneously developed in p53-deficient mice and human fibrosarcoma cells grown as xenografts in immunocompromised severe combined immunodeficient (SCID) mice. We show that both types of tumors retain a functional clock, which is synchronized in phase with normal tissues. We also show that spontaneously developed tumors are not only oscillating in the context of an organism where they receive hormonal and metabolic signals but continue oscillating ex vivo in tissue explants demonstrating that tumors have functional clocks capable of timing all their functions. We also provide evidence that similar to liver, tumors can be synchronized by food availability independent of the central pacemaker in the suprachiasmatic nuclei (SCN). These data provide the basis for the design of anticancer therapies that take into account the circadian metabolic and physiological patterns of both the tumor and normal tissues.

Metabolic clock generates nutrient anticipation rhythms in mTOR signaling.

The mTOR signaling pathway modulates metabolic processes with respect to nutrient availability and other growth-related cues. According to the existing paradigm, mTOR complex 1 (mTORC1) activityin vivo is induced by food and gradually decreases during fasting. We found that mTORC1 activity is controlled by an internal clock mechanism different from the known light-entrainable circadian clock. We observed 24-hr rhythms in phosphorylation of mTORC1 downstream targets, which were entrained by food, persisted during fasting and could be uncoupled from oscillating expression of the canonical circadian clock genes. Furthermore, these rhythms were present in tissues of mice with disrupted light-entrainable circadian clock. We propose tissue-specific rhythms in the expression of tor and its negative regulator deptor as the molecular mechanism of the mTORC1 activity oscillation. Our data demonstrate the existence of at least two independent molecular circadian clocks: one providing metabolic adaptation to periodic light/darkness and the other - to feeding.