Cancer Relevance of Circulating Antibodies Against LINE-1 Antigens in Humans.

Long interspersed nuclear element-1 (LINE-1 or L1), the most abundant family of autonomous retrotransposons occupying over 17% of human DNA, is epigenetically silenced in normal tissues by the mechanisms involving p53 but is frequently derepressed in cancer, suggesting that L1-encoded proteins may act as tumor-associated antigens recognized by the immune system. In this study, we established an immunoassay to detect circulating autoantibodies against L1 proteins in human blood. Using this assay in >2,800 individuals with or without cancer, we observed significantly higher IgG titers against L1-encoded ORF1p and ORF2p in patients with lung, pancreatic, ovarian, esophageal, and liver cancers than in healthy individuals. Remarkably, elevated levels of anti-ORF1p-reactive IgG were observed in patients with cancer with disease stages 1 and 2, indicating that the immune response to L1 antigens can occur in the early phases of carcinogenesis. We concluded that the antibody response against L1 antigens could contribute to the diagnosis and determination of immunoreactivity of tumors among cancer types that frequently escape early detection. SIGNIFICANCE: The discovery of autoantibodies against antigens encoded by L1 retrotransposons in patients with five poorly curable cancer types has potential implications for the detection of an ongoing carcinogenic process and tumor immunoreactivity.

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.

While reinforcing cell cycle arrest, rapamycin and Torins suppress senescence in UVA-irradiated fibroblasts.

Sunlight predisposes to skin cancer and melanomas. Ultraviolet A (UVA), a long wave component of sunlight, can reach dermal fibroblasts. Here we studied UVA-induced senescence in human fibroblasts in vitro. It is known that senescence occurs, when cell cycle is arrested, but mTOR is still active, thus converting arrest to senescence (geroconversion). We showed that, while arresting cell cycle, UVA did not inhibit mTOR, enabling geroconversion. In UVA-treated cells, mTOR remained fully active. Rapamycin and Torins 1/ 2 prevented UVA-induced senescent phenotype, although they further re-enforced cell cycle arrest. Given that senescent stromal fibroblasts support tumorigenesis, we envision that mTOR inhibitors may potentially be used to prevent sunlight-caused tumors as well as skin photo-aging.

Protein Kinase Cα (PKCα) Is Resistant to Long Term Desensitization/Down-regulation by Prolonged Diacylglycerol Stimulation.

Sustained activation of PKCα is required for long term physiological responses, such as growth arrest and differentiation. However, studies with pharmacological agonists (e.g. phorbol 12-myristate 13-acetate (PMA)) indicate that prolonged stimulation leads to PKCα desensitization via dephosphorylation and/or degradation. The current study analyzed effects of chronic stimulation with the physiological agonist diacylglycerol. Repeated addition of 1,2-dioctanoyl-sn-glycerol (DiC8) resulted in sustained plasma membrane association of PKCα in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKCα, prolonged activation by DiC8 failed to engage known desensitization mechanisms, with the enzyme remaining membrane-associated and able to support sustained downstream signaling. DiC8-activated PKCα did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKCα desensitization. Although DiC8 efficiently down-regulated novel PKCs PKCδ and PKCϵ, differences in Ca(2+) sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKCα. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC8, targeted PKCα to detergent-resistant membranes, and disruption of these domains with cholesterol-binding agents demonstrated a role for differential membrane compartmentalization in selective agonist-induced degradation. Chronic DiC8 treatment failed to desensitize PKCα in several cell types and did not affect PKCßI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKCα is seen in self-renewing epithelium of the intestine, cervix, and skin. PKCα deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKCα from desensitization in vivo.

Gerosuppression by pan-mTOR inhibitors.

Rapamycin slows organismal aging and delays age-related diseases, extending lifespan in numerous species. In cells, rapamycin and other rapalogs such as everolimus suppress geroconversion from quiescence to senescence. Rapamycin inhibits some, but not all, activities of mTOR. Recently we and others demonstrated that pan-mTOR inhibitors, known also as dual mTORC1/C2 inhibitors, suppress senescent phenotype. As a continuation of these studies, here we investigated in detail a panel of pan-mTOR inhibitors, to determine their optimal gerosuppressive concentrations. During geroconversion, cells become hypertrophic and flat, accumulate lysosomes (SA-beta-Gal staining) and lipids (Oil Red staining) and lose their re-proliferative potential (RPP). We determined optimal gerosuppressive concentrations: Torin1 (30 nM), Torin 2 (30 nM), AZD8055 (100 nM), PP242 (300 nM), both KU-006379 and GSK1059615 (1000 nM). These agents decreased senescence-associated hypertrophy with IC50s: 20, 18, 15, 200 and 400 nM, respectively. Preservation of RPP by pan-mTOR inhibitors was associated with inhibition of the pS6K/pS6 axis. Inhibition of rapamycin-insensitive functions of mTOR further contributed to anti-hypertrophic and cytostatic effects. Torin 1 and PP242 were more "rapamycin-like" than Torin 2 and AZD8055. Pan-mTOR inhibitors were superior to rapamycin in suppressing hypertrophy, senescent morphology, Oil Red O staining and in increasing so-called "chronological life span (CLS)". We suggest that, at doses lower than anti-cancer concentrations, pan-mTOR inhibitors can be developed as anti-aging drugs.

Dual mTORC1/C2 inhibitors suppress cellular geroconversion (a senescence program).

In proliferating cells, mTOR is active and promotes cell growth. When the cell cycle is arrested, then mTOR converts reversible arrest to senescence (geroconversion). Rapamycin and other rapalogs suppress geroconversion, maintaining quiescence instead. Here we showed that ATP-competitive kinase inhibitors (Torin1 and PP242), which inhibit both mTORC1 and TORC2, also suppressed geroconversion. Despite inhibition of proliferation (in proliferating cells), mTOR inhibitors preserved re-proliferative potential (RP) in arrested cells. In p21-arrested cells, Torin 1 and PP242 detectably suppressed geroconversion at concentrations as low as 1-3 nM and 10-30 nM, reaching maximal gerosuppression at 30 nM and 300 nM, respectively. Near-maximal gerosuppression coincided with inhibition of p-S6K(T389) and p-S6(S235/236). Dual mTOR inhibitors prevented senescent morphology and hypertrophy. Our study warrants investigation into whether low doses of dual mTOR inhibitors will prolong animal life span and delay age-related diseases. A new class of potential anti-aging drugs can be envisioned.

Comparison of rapamycin schedules in mice on high-fat diet.

At a wide range of doses, rapamycin extends life span in mice. It was shown that intraperitoneal injections (i.p.) of rapamycin prevent weight gain in mice on high-fat diet (HFD). We further investigated the effect of rapamycin on weight gain in female C57BL/6 mice on HFD started at the age of 7.5 months. By the age of 16 and 23 months, mice on HFD weighed significantly more (52 vs 33 g; p = 0.0001 and 70 vs 38 g; p < 0.0001, respectively) than mice on low fat diet (LFD). The i.p. administration of 1.5 mg/kg rapamycin, 3 times a week every other week, completely prevented weight gain, whereas administration of rapamycin by oral gavash did not. Rapamycin given in the drinking water slightly decreased weight gain by the age of 23 months. In addition, metabolic parameters were evaluated at the age of 16 and 23 months, 6 and 13 days after last rapamycin administration, respectively. Plasma leptin levels strongly correlated with body weight, (P < 0.0001, r=0.86), suggesting that the difference in weight was due to fat tissue mass. Levels of insulin, glucose, triglycerides and IGF1 were not statistically different in all groups, indicating that these courses of rapamycin treatment did not impair metabolic parameters at least after rapamycin discontinuation. Despite rapamycin discontinuation, cardiac levels of phospho-S6 and pAKT(S473) were low in the i.p.-treated group. This continuous effect of rapamycin can be explained by prevention of obesity in the i.p. group. We conclude that intermittent i.p. administration of rapamycin prevents weight gain without causing gross metabolic abnormalities. Intermittent gavash administration minimally affected weight gain. Potential clinical applications are discussed.

Fasting levels of hepatic p-S6 are increased in old mice.

TOR is involved in aging in a wide range of species from yeast to mammals. Here we show that, after overnight fasting, mTOR activity is higher in the livers of 28 months old female mice compared with middle-aged mice. Taken together with previous reports, our data predict that the life-extending effect of calorie restriction (CR) may be diminished, if CR is started in very old age. In contrast, rapamycin is known to be effective, even when started late in life.

Contact inhibition and high cell density deactivate the mammalian target of rapamycin pathway, thus suppressing the senescence program.

During cell cycle arrest caused by contact inhibition (CI), cells do not undergo senescence, thus resuming proliferation after replating. The mechanism of senescence avoidance during CI is unknown. Recently, it was demonstrated that the senescence program, namely conversion from cell cycle arrest to senescence (i.e., geroconversion), requires mammalian target of rapamycin (mTOR). Geroconversion can be suppressed by serum starvation, rapamycin, and hypoxia, which all inhibit mTOR. Here we demonstrate that CI, as evidenced by p27 induction in normal cells, was associated with inhibition of the mTOR pathway. Furthermore, CI antagonized senescence caused by CDK inhibitors. Stimulation of mTOR in contact-inhibited cells favored senescence. In cancer cells lacking p27 induction and CI, mTOR was still inhibited in confluent culture as a result of conditioning of the medium. This inhibition of mTOR suppressed p21-induced senescence. Also, trapping of malignant cells among contact-inhibited normal cells antagonized p21-induced senescence. Thus, we identified two nonmutually exclusive mechanisms of mTOR inhibition in high cell density: (i) CI associated with p27 induction in normal cells and (ii) conditioning of the medium, especially in cancer cells. Both mechanisms can coincide in various proportions in various cells. Our work explains why CI is reversible and, most importantly, why cells avoid senescence in vivo, given that cells are contact-inhibited in the organism.

Weekly administration of rapamycin improves survival and biomarkers in obese male mice on high-fat diet.

Recent discoveries have revealed the key role of mTOR (target of rapamycin) in aging. Furthermore, rapamycin extends lifespan in mice, especially in female mice. Here, we treated obese male mice on high-fat diet with rapamycin given intermittently: either weekly (once a week) or alternating bi-weekly (three injections every other week). While only marginally reducing obesity, intermittent administration of rapamycin significantly extended lifespan. Significance was achieved for weekly treated group and for the three rapamycin-received groups combined. In weekly treatment group, 100% mice were alive by the age of 2 years, whereas 60% of mice died in untreated group by this age. The effect of weekly treatment on survival was highly significant and cannot be fully explained by partial reduction in obesity. Alternating bi-weekly treatments seem to be less effective than weekly treatment, although effects of additional factors (see ) may not be excluded. After one year of treatment, all survived mice were sacrificed 8 days after the last administration of rapamycin to avoid its direct interference with parameters examined. Fasting levels of cardiac and hepatic p-S6, a marker of mTORC1 activity, were lower in weekly treatment group compared with control mice. In contrast, levels of p-Akt (S473), glucose, triglycerides and insulin were unchanged, whereas leptin and IGF-1 tended to be lower. Thus, weekly treatment with rapamycin may slow down aging in obese male mice on high-fat diet.

M(o)TOR of pseudo-hypoxic state in aging: rapamycin to the rescue.

A groundbreaking publication by Sinclair and coworkers has illuminated the pseudo-hypoxic state in aging and its reversibility. Remarkably, these data also fit the mTOR-centered model of aging. Here we discuss that the mTOR pathway can cause cellular pseudo-hypoxic state, manifested by HIF-1 expression and lactate production under normoxia. We found that rapamycin decreased HIF-1 and lactate levels in proliferating and senescent cells in vitro. This reduction was independent from mitochondrial respiration: rapamycin decreased lactate production in normoxia, hypoxia, and in the presence of the OXPHOS inhibitor oligomycin. We suggest that pseudo-hypoxic state is not necessarily caused by mitochondrial dysfunction, but instead mitochondrial dysfunction may be secondary to mTOR-driven hyperfunctions. Clinical applications of rapamycin for reversing pseudo-hypoxic state and lactate acidosis are discussed.

Gerosuppression in confluent cells.

The most physiological type of cell cycle arrest - namely, contact inhibition in dense culture - is the least densely studied. Despite cell cycle arrest, confluent cells do not become senescent. We recently described that mTOR (target of rapamycin) is inactive in contact-inhibited cells. Therefore, conversion from reversible arrest to senescence (geroconversion) is suppressed. I this Perspective, we further extended the gerosuppression model. While causing senescence in regular cell density, etoposide failed to cause senescence in contact-inhibited cells. A transient reactivation of mTOR favored geroconversion in etoposide-treated confluent cells. Like p21, p16 did not cause senescence in high cell density. We discuss that suppression of geroconversion in confluent and contact-inhibited cultures mimics gerosuppression in the organism. We confirmed that levels of p-S6 were low in murine tissues in the organism compared with mouse embryonic fibroblasts in cell culture, whereas p-Akt was reciprocally high in the organism.

Tumor promoter-induced cellular senescence: cell cycle arrest followed by geroconversion.

Phorbol ester (PMA or TPA), a tumor promoter, can cause either proliferation or cell cycle arrest, depending on cellular context. For example, in SKBr3 breast cancer cells, PMA hyper-activates the MEK/MAPK pathway, thus inducing p21 and cell cycle arrest. Here we showed that PMA-induced arrest was followed by conversion to cellular senescence (geroconversion). Geroconversion was associated with active mTOR and S6 kinase (S6K). Rapamycin suppressed geroconversion, maintaining quiescence instead. In this model, PMA induced arrest (step one of a senescence program), whereas constitutively active mTOR drove geroconversion (step two). Without affecting Akt phosphorylation, PMA increased phosphorylation of S6K (T389) and S6 (S240/244), and that was completely prevented by rapamycin. Yet, T421/S424 and S235/236 (p-S6K and p-S6, respectively) phosphorylation became rapamycin-insensitive in the presence of PMA. Either MEK or mTOR was sufficient to phosphorylate these PMA-induced rapamycin-resistant sites because co-treatment with U0126 and rapamycin was required to abrogate them. We next tested whether activation of rapamycin-insensitive pathways would shift quiescence towards senescence. In HT-p21 cells, cell cycle arrest was caused by IPTG-inducible p21 and was spontaneously followed by mTOR-dependent geroconversion. Rapamycin suppressed geroconversion, whereas PMA partially counteracted the effect of rapamycin, revealing the involvement of rapamycin-insensitive gerogenic pathways. In normal RPE cells arrested by serum withdrawal, the mTOR/pS6 pathway was inhibited and cells remained quiescent. PMA transiently activated mTOR, enabling partial geroconversion. We conclude that PMA can initiate a senescent program by either inducing arrest or fostering geroconversion or both. Rapamycin can decrease gero-conversion by PMA, without preventing PMA-induced arrest. The tumor promoter PMA is a gero-promoter, which may be useful to study aging in mammals.

Dysregulation of the mTOR pathway in p53-deficient mice.

Mammalian or mechanistic target of rapamycin (mTOR) is involved in growth, aging, and age-related diseases including cancer. There is an extensive cross talk between p53 and mTOR. In cell culture, p53 inhibits the mTOR pathway in a cell type-dependent manner. p53-deficient mice develop pro-inflammation and cancer. We have shown that rapamycin delayed cancer and extended lifespan, thus partially substituting for p53. Here we show that a marker of mTOR activity, phosphorylated S6 (p-S6), is increased in the hearts of p53-deficient mice. Furthermore, cardiac p-S6 correlated with body weight. Also, p53(-/-) mice were slightly hyperinsulinemic with a tendency to elevated IGF-1. Radiation exacerbated the difference between IGF-1 levels in normal and p53(-/-) mice. Noteworthy, radiation induced Thr-308 Akt phosphorylation in the livers (but not in the hearts) of both p53(+/+) and p53(-/-) mice. Simultaneously, radiation decreased p-S6 in the livers of normal mice, consistent with the negative effect of p53 on mTOR. Our data indicate that the activity of mTOR is increased in some but not all tissues of p53(-/-) mice, associated with the tendency to increased insulin and IGF-1 levels. Therefore, the absence of p53 may create oncophilic microenvironment, favoring cancer.

S6K in geroconversion.

Markers of cellular senescence depend in part on the MTOR (mechanistic target of rapamycin) pathway. MTOR participates in geroconversion, a conversion from reversible cell cycle arrest to irreversible senescence. Recently we demonstrated that hyper-induction of cyclin D1 during geroconversion was mostly dependent on MEK, whereas rapamycin only partially inhibited cyclin D1 accumulation. Here we show that, while not affecting cyclin D1, siRNA for p70S6K partially prevented loss of RP (replicative/regenerative potential) during p21-induced cell cycle arrest. Similarly, an inhibitor of p70 S6 kinase (PF-4708671) partially inhibited phosphorylation of S6 and preserved RP, while only marginally prevented cyclin D1 induction. Thus S6K and MEK play different roles in geroconversion.

CDK4/6-inhibiting drug substitutes for p21 and p16 in senescence: duration of cell cycle arrest and MTOR activity determine geroconversion.

CDKN1A (p21) and CDKN2A (p16) inhibit CDK4/6, initiating senescence. According to our view on senescence, the role of p21 and p16 is to cause cell cycle arrest, whereas MTOR (mechanistic target of rapamycin) drives geroconversion to senescence. Recently we demonstrated that one of the markers of p21- and p16-initiated senescence is MEK-dependent hyper-elevation of cyclin D1. We noticed that a synthetic inhibitor of CDK 4/6 (PD0332991) also induced cyclin D1-positive senescence. We demonstrated that PD0332991 and p21 caused almost identical senescence phenotypes. p21, p16, and PD0332991 do not inhibit MTOR, and rapamycin decelerates geroconversion caused by all 3 molecules. Like p21, PD0332991 initiated senescence at any concentration that inhibited cell proliferation. This confirms the notion that a mere arrest in the presence of active MTOR may lead to senescence.

Hyper-mitogenic drive coexists with mitotic incompetence in senescent cells.

When the cell cycle is arrested, even though growth-promoting pathways such as mTOR are still active, then cells senesce. For example, induction of either p21 or p16 arrests the cell cycle without inhibiting mTOR, which, in turn, converts p21/p16-induced arrest into senescence (geroconversion). Here we show that geroconversion is accompanied by dramatic accumulation of cyclin D1 followed by cyclin E and replicative stress. When p21 was switched off, senescent cells (despite their loss of proliferative potential) progressed through S phase, and levels of cyclins D1 and E dropped. Most cells entered mitosis and then died, either during mitotic arrest or after mitotic slippage, or underwent endoreduplication. Next, we investigated whether inhibition of mTOR would prevent accumulation of cyclins and loss of mitotic competence in p21-arrested cells. Both nutlin-3, which inhibits mTOR in these cells, and rapamycin suppressed geroconversion during p21-induced arrest, decelerated accumulation of cyclins D1 and E and decreased replicative stress. When p21 was switched off, cells successfully progressed through both S phase and mitosis. Also, senescent mouse embryonic fibroblasts (MEFs) overexpressed cyclin D1. After release from cell cycle arrest, senescent MEFs entered S phase but could not undergo mitosis and did not proliferate. We conclude that cellular senescence is characterized by futile hyper-mitogenic drive associated with mTOR-dependent mitotic incompetence.

Rapamycin extends lifespan and delays tumorigenesis in heterozygous p53+/- mice.

TOR (Target of Rapamycin) pathway accelerates cellular and organismal aging. Similar to rapamycin, p53 can inhibit the mTOR pathway in some mammalian cells. Mice lacking one copy of p53 (p53+/- mice) have an increased cancer incidence and a shorter lifespan. We hypothesize that rapamycin can delay cancer in heterozygous p53+/- mice. Here we show that rapamycin (given in a drinking water) extended the mean lifespan of p53+/- mice by 10% and when treatment started early in life (at the age less than 5 months) by 28%. In addition, rapamycin decreased the incidence of spontaneous tumors. This observation may have applications in management of Li-Fraumeni syndrome patients characterized by heterozygous mutations in the p53 gene.

Hypoxia and gerosuppression: the mTOR saga continues.

Growth-promoting and nutrient/mitogen-sensing pathways such as mTOR convert p21- and p16-induced arrest into senescence (geroconversion). We have recently demonstrated that hypoxia, especially near-anoxia, suppresses geroconversion. This gerosuppressive effect of hypoxia correlated with inhibition of the mTOR/S6K pathway but not with modulation of the LKB1/AMPK/eEF2 pathway. Here we further show that mTOR inhibition is required for gerosuppression by hypoxia, at least in some cellular models, because depletion of TSC2 abolished mTOR inhibition and gerosupression by hypoxia. Also, in two cancer cell lines resistant to inhibition of mTOR by both p53 and hypoxia, hypoxia did not suppress geroconversion. Therefore, the effects of hypoxia on the oxygen-sensing mTOR pathway and geroconversion are cell type-specific. We also briefly discuss replicative senescence, organismal aging and free radical theory.

Hypoxia suppresses conversion from proliferative arrest to cellular senescence.

Unlike reversible quiescence, cellular senescence is characterized by a large flat cell morphology, ß-gal staining and irreversible loss of regenerative (i.e., replicative) potential. Conversion from proliferative arrest to irreversible senescence, a process named geroconversion, is driven in part by growth-promoting pathways such as mammalian target of rapamycin (mTOR). During cell cycle arrest, mTOR converts reversible arrest into senescence. Inhibitors of mTOR can suppress geroconversion, maintaining quiescence instead. It was shown that hypoxia inhibits mTOR. Therefore, we suggest that hypoxia may suppress geroconversion. Here we tested this hypothesis. In HT-p21-9 cells, expression of inducible p21 caused cell cycle arrest without inhibiting mTOR, leading to senescence. Hypoxia did not prevent p21 induction and proliferative arrest, but instead inhibited the mTOR pathway and geroconversion. Exposure to hypoxia during p21 induction prevented senescent morphology and loss of regenerative potential, thus maintaining reversible quiescence so cells could restart proliferation after switching p21 off. Suppression of geroconversion was p53- and HIF-1-independent, as hypoxia also suppressed geroconversion in cells lacking functional p53 and HIF-1α. Also, in normal fibroblasts and retinal cells, hypoxia inhibited the mTOR pathway and suppressed senescence caused by etoposide without affecting DNA damage response, p53/p21 induction and cell cycle arrest. Also hypoxia suppressed geroconversion in cells treated with nutlin-3a, a nongenotoxic inducer of p53, in cell lines susceptible to nutlin-3a-induced senescence (MEL-10, A172, and NKE). Thus, in normal and cancer cell lines, hypoxia suppresses geroconversion caused by diverse stimuli. Physiological and clinical implications of the present findings are discussed.