Inactivation of Ymr1, Sjl2/3 phosphatases promotes stress resistance and longevity in wild type and Ras2G19V yeast.

In Saccharomyces cerevisiae, RAt Sarcoma (Ras) activity plays a central role in mediating the effect of glucose in decreasing stress resistance and longevity, with constitutive Ras activation mutations promoting cell growth and oncogenesis. Here, we used transposon mutagenesis in yeast to identify suppressors of the constitutively active Ras2G19V, orthologue of the KRASG12C mammalian oncogene. We identified mutations in Yeast Myotubularin Related (YMR1), SynaptoJanin-Like (SJL2) and SJL3 phosphatases, which target phosphatidylinositol phosphates, as the most potent suppressors of constitutive active Ras, able to reverse its effect on stress sensitization and sufficient to extend longevity. In sjl2 mutants, the staining of Ras-GTP switched from membrane-associated to a diffuse cytoplasmic staining, suggesting that it may block Ras activity by preventing its localization. Whereas expression of the Sjl2 PI 3,4,5 phosphatase mediated stress sensitization in both the Ras2G19V and wild type backgrounds, overexpression of the phosphatidylinositol 3 kinase VPS34 (Vacuolar Protein Sorting), promoted heat shock sensitization only in the Ras2G19V background, suggesting a complex relationship between different phosphatidylinositol and stress resistance. These results provide potential targets to inhibit the growth of cancer cells with constitutive Ras activity and link the glucose-dependent yeast pro-aging Ras signaling pathway to the well-established pro-aging PhosphoInositide 3-Kinase(PI3K) pathway in worms and other species raising the possibility that the conserved longevity effect of mutations in the PI3K-AKT (AK strain Transforming) pathway may involve inhibition of Ras signaling.

Fasting mimicking diet in mice delays cancer growth and reduces immunotherapy-associated cardiovascular and systemic side effects.

Immune checkpoint inhibitors cause side effects ranging from autoimmune endocrine disorders to severe cardiotoxicity. Periodic Fasting mimicking diet (FMD) cycles are emerging as promising enhancers of a wide range of cancer therapies including immunotherapy. Here, either FMD cycles alone or in combination with anti-OX40/anti-PD-L1 are much more effective than immune checkpoint inhibitors alone in delaying melanoma growth in mice. FMD cycles in combination with anti-OX40/anti-PD-L1 also show a trend for increased effects against a lung cancer model. As importantly, the cardiac fibrosis, necrosis and hypertrophy caused by immune checkpoint inhibitors are prevented/reversed by FMD treatment in both cancer models whereas immune infiltration of CD3+ and CD8+ cells in myocardial tissues and systemic and myocardial markers of oxidative stress and inflammation are reduced. These results indicate that FMD cycles in combination with immunotherapy can delay cancer growth while reducing side effects including cardiotoxicity.

The impact of a fasting mimicking diet on the metabolic health of a prospective cohort of patients with prostate cancer: a pilot implementation study.

BACKGROUND: This pilot prospective study investigated the effect of a periodic fasting mimicking diet (FMD) on metabolic health factors in patients with Prostate Cancer (PC). There is a well-documented association between PC and metabolic health. Impaired metabolic health is a significant risk factor for the development of PC, and a metabolic syndrome can be induced by hormonal therapies commonly required for its management. (ClinicalTrials.gov Identifier: NCT04292041). METHODS: We introduced a periodic 4-day FMD -low in calories, sugars, and proteins but high in unsaturated fats -to a cohort of PC patients and features of metabolic syndrome. 29/35 patients completed 3-monthly cycles of the 4-consecutive day packaged FMD. We compared the subjects' baseline weight, abdominal circumference (AC), blood pressure (BP) and selected laboratory results to the same measurements 3-months after completing the FMD cycles. RESULTS: Several important metabolic factors showed improvements post-intervention. On average patients' weights dropped by 3.79 kg (95% CI: -5.61, -1.97, p = 0.0002). AC was reduced on average by 4.57 cm, (95% CI: -2.27, -6.87, p = 0.0003). There was also a decrease in systolic and diastolic BP by 9.52 mmHg (95% CI: -16.16, -2.88, p = 0.0066) and 4.48 mmHg (95% CI: -8.85, -0.43, p = 0.0316) respectively. A sub-analysis indicates that FMD had more relevant effects in 'at-risk' patients than those with normal values of risk factors for metabolic syndrome. For example, subjects with baseline levels of systolic BP > 130 mmHg experienced a greater reduction in BP(-16.04 mmHg, p = 0.0001) than those with baseline systolic BP < 130 mmHg (-0.78 mmHg, p = 0.89). CONCLUSIONS: The FMD cycles were safely introduced to this small cohort of PC patients with little or no observed toxicity, and a high overall compliance of 83%. Analysis of the metabolic variables showed an overall decrease in weight, AC, and BP. Larger clinical trials focused on metabolic risk factors, PC quality of life and progression free survival are needed to assess the effect of the FMD on prostate cancer patients.

Fasting-induced differential stress sensitization in cancer treatment.

Most tumors are generated and evolve under high-nutrient conditions, yet therapy does not include dietary changes generating a hostile environment for cancer cells. Because fasting promotes the most drastic change in the levels of plasma macro- and micro-nutrients, and consequently in glucose and growth factors, it has the potential to maximize cancer cell sensitization.

Fasting vs dietary restriction in cellular protection and cancer treatment: from model organisms to patients.

The dietary recommendation for cancer patients receiving chemotherapy, as described by the American Cancer Society, is to increase calorie and protein intake. Yet, in simple organisms, mice, and humans, fasting--no calorie intake--induces a wide range of changes associated with cellular protection, which would be difficult to achieve even with a cocktail of potent drugs. In mammals, the protective effect of fasting is mediated, in part, by an over 50% reduction in glucose and insulin-like growth factor 1 (IGF-I) levels. Because proto-oncogenes function as key negative regulators of the protective changes induced by fasting, cells expressing oncogenes, and therefore the great majority of cancer cells, should not respond to the protective signals generated by fasting, promoting the differential protection (differential stress resistance) of normal and cancer cells. Preliminary reports indicate that fasting for up to 5 days followed by a normal diet, may also protect patients against chemotherapy without causing chronic weight loss. By contrast, the long-term 20 to 40% restriction in calorie intake (dietary restriction, DR), whose effects on cancer progression have been studied extensively for decades, requires weeks-months to be effective, causes much more modest changes in glucose and/or IGF-I levels, and promotes chronic weight loss in both rodents and humans. In this study, we review the basic as well as clinical studies on fasting, cellular protection and chemotherapy resistance, and compare them to those on DR and cancer treatment. Although additional pre-clinical and clinical studies are necessary, fasting has the potential to be translated into effective clinical interventions for the protection of patients and the improvement of therapeutic index.

A simple model system for age-dependent DNA damage and cancer.

Aging is the major risk factor for many human cancers. However, the mechanisms responsible for the effect of aging on tumor incidence are poorly understood, in part because few model systems are available to study age-dependent genomic instability. Furthermore, the role of DNA mutations in "normal aging" and "life span extension" is unclear. Our laboratory has developed a novel method to study aging in yeast based on the survival of non-dividing populations (chronological life span). Two major pathways have been identified that control chronological aging: the Ras/PKA/Msn2/4 and the Sch9 pathways. The downregulation of either of them promotes life span extension. Importantly, similar pathways (insulin/IGF-I-like), regulate longevity in higher eukaryotes suggesting a common evolutionary origin for the life span-regulatory mechanisms. Moreover, both Ras and Sch9 are functional homologs of two major mammalian oncogenes (Ras and Akt), which underlines the close link between cancer and aging. By combining chronological life span with simple assays for the detection of DNA mutations and dedifferentiation we have developed a powerful system to identify genes that regulate genomic instability and understand the fundamental mechanisms that may be responsible for age-dependent DNA mutations and cancer in mammals. Here, we describe the use of this system to monitor the age-dependent accumulation of different types of DNA mutations including base substitutions, frame-shift mutations, and gross chromosomal rearrangements (GCRs).

Chronological aging-independent replicative life span regulation by Msn2/Msn4 and Sod2 in Saccharomyces cerevisiae.

Mutations in RAS2, CYR1, and SCH9 extend the chronological life span in Saccharomyces cerevisiae by activating stress-resistance transcription factors and mitochondrial superoxide dismutase (Sod2). Here we show that mutations in CYR1 and SCH9 also extend the replicative life span of individual yeast mother cells. However, the triple deletion of stress-resistance genes MSN2/MSN4 and RIM15, which causes a major decrease in chronological life span, extends replicative life span. Similarly, the overexpression of superoxide dismutases, which extends chronological survival, shortens the replicative life span and prevents budding in 30-40% of virgin mother cells. These results suggest that stress-resistance transcription factors Msn2/Msn4 negatively regulate budding and the replicative life span in part by increasing SOD2 expression. The role of superoxide dismutases and of other stress-resistance proteins in extending the chronological life span of yeast, worms, and flies indicates that the negative effect of Sod2, Msn2/Msn4/Rim15 on the replicative life span of S. cerevisiae is independent of aging.

Regulation of longevity and stress resistance: a molecular strategy conserved from yeast to humans?

Recent studies implicate similar proteins in the regulation of longevity in organisms ranging from yeast to mice. Studies in yeast and worms suggest that inactivation of glucose or insulin/insulin-like growth factor-l (IGF-1) signaling pathways extends longevity by causing a shift from a reproductive phase to a non-reproductive maintenance phase involving the expression of many genes. These stress resistance pathways appear to have evolved to induce maintenance systems and promote longevity during periods of starvation. In yeast, mutations that decrease the activity of glucose signaling pathways extend longevity by activating stress resistance transcription factors that regulate the expression of genes involved in antioxidant and heat protection, glycogen storage, protein degradation, DNA repair, and metabolism. A remarkably similar set of proteins regulated by growth factors that control glucose metabolism is implicated in life span extension in worms, and possibly in flies and mice. Studies in worms and flies point to secondary hormones as mediators of the effect of insulin/ IGF-1 signaling on longevity, whereas studies in yeast and mammalian cells indicate that glucose or insulin/ IGF-1 may decrease longevity by directly down-regulating stress resistance genes. In yeast, longevity mutations postpone superoxide toxicity and mitochondrial damage. However, the small life span extension caused by the overexpression of superoxide dismutases and catalase in yeast and flies indicates that increased antioxidant protection alone cannot be responsible for the major life span extension caused by signal transduction mutations. Although we are only beginning to understand the molecular mechanisms that mediate life span extension, the similarities between longevity regulatory pathways in organisms ranging from yeast to mice suggest that insulin/ IGF-1 signaling pathways may also regulate cell damage and longevity in humans.

Regulation of longevity and stress resistance by Sch9 in yeast.

The protein kinase Akt/protein kinase B (PKB) is implicated in insulin signaling in mammals and functions in a pathway that regulates longevity and stress resistance in Caenorhabditis elegans. We screened for long-lived mutants in nondividing yeast Saccharomyces cerevisiae and identified mutations in adenylate cyclase and SCH9, which is homologous to Akt/PKB, that increase resistance to oxidants and extend life-span by up to threefold. Stress-resistance transcription factors Msn2/Msn4 and protein kinase Rim15 were required for this life-span extension. These results indicate that longevity is associated with increased investment in maintenance and show that highly conserved genes play similar roles in life-span regulation in S. cerevisiae and higher eukaryotes.

Reversible inactivation of superoxide-sensitive aconitase in Abeta1-42-treated neuronal cell lines.

The activity of the superoxide-sensitive enzyme aconitase was monitored to evaluate the generation of superoxide in neuronal cell lines treated with beta-amyloid (Abeta) peptide 1-42. Treatment of differentiated and undifferentiated rat PC12 and human neuroblastoma SK-N-SH cells with soluble Abeta1-42 (Abeta-derived diffusible ligands) or fibrillar Abeta1-42 caused a 35% reversible inactivation of aconitase, which preceded loss of viability and was correlated with altered cellular function. Aconitase was reactivated upon incubation of cellular extracts with iron and sulfur, suggesting that Abeta causes the release of iron from 4Fe-4S clusters. Abeta neurotoxicity was partially blocked by the iron chelator deferoxamine. These data suggest that increased superoxide generation and the release of iron from 4Fe-4S clusters are early events in Abeta1-42 neurotoxicity.

Mitochondrial superoxide decreases yeast survival in stationary phase.

Yeast lacking mitochondrial superoxide dismutase (MnSOD) display shortened stationary-phase survival and provide a good model system for studying mitochondrial oxidative damage. We observed a marked decrease in respiratory function preceding stationary-phase death of yeast lacking MnSOD (sod2Delta). Agents (mitochondrial inhibitors) that are known to increase or decrease superoxide production in submitochondrial particles affected stationary-phase survival in a manner inversely correlated with their effects on superoxide production, implicating superoxide in this mitochondrial disfunction. Similar but less-dramatic effects were observed in wild-type yeast. The activities of certain mitochondrial enzymes were particularly affected. In sod2Delta yeast the activity of aconitase, a 4Fe-4S-cluster-containing enzyme located in the matrix, was greatly and progressively decreased as the cells established stationary phase. Succinate dehydrogenase activity also decreased in MnSOD mutants; cytochrome oxidase and ATPase activities did not. Aconitase could be reactivated by addition of materials required for cluster assembly (Fe3+ and a sulfur source), both in extracts and in vivo, indicating that inactivation of the enzyme was by disassembly of the cluster. Our results support the conclusion that superoxide is generated in the mitochondria in vivo and under physiological conditions and that MnSOD is the primary defense against this toxicity. When the balance between superoxide generation and MnSOD activity is disrupted, superoxide mediates iron release from mitochondrial iron-sulfur clusters, leading first to loss of mitochondrial function and then to death, independently of mtDNA damage. These results raise the possibility that similar processes may occur in higher eukaryotes.

Mutations in signal transduction proteins increase stress resistance and longevity in yeast, nematodes, fruit flies, and mammalian neuronal cells.

Mutations in Ras and other signal transduction proteins increase survival and resistance to oxidative stress and starvation in stationary phase yeast, nematodes, fruit flies, and in neuronal PC12 cells. The chronological life span of yeast, based on the survival of nondividing cells in stationary phase, has allowed the identification and characterization of long-lived strains with mutations in the G-protein Ras2. This paradigm was also used to identify the in vivo sources and targets of reactive oxygen species and to examine the role of antioxidant enzymes in the longevity of yeast. I will review this model system and discuss the striking phenotypic similarities between long-lived mutants ranging from yeast to mammalian neuronal cells. Taken together, the published studies suggest that survival may be regulated by similar fundamental mechanisms in many eukaryotes and that simple model systems will contribute to our understanding of the aging process in mammals.

Biodemographic trajectories of longevity.

Old-age survival has increased substantially since 1950. Death rates decelerate with age for insects, worms, and yeast, as well as humans. This evidence of extended postreproductive survival is puzzling. Three biodemographic insights--concerning the correlation of death rates across age, individual differences in survival chances, and induced alterations in age patterns of fertility and mortality--offer clues and suggest research on the failure of complicated systems, on new demographic equations for evolutionary theory, and on fertility-longevity interactions. Nongenetic changes account for increases in human life-spans to date. Explication of these causes and the genetic license for extended survival, as well as discovery of genes and other survival attributes affecting longevity, will lead to even longer lives.

Human Bcl-2 reverses survival defects in yeast lacking superoxide dismutase and delays death of wild-type yeast.

We expressed the human anti-apoptotic protein, Bcl-2, in Saccharomyces cerevisiae to investigate its effects on antioxidant protection and stationary phase survival. Yeast lacking copper-zinc superoxide dismutase (sod1Delta) show a profound defect in entry into and survival during stationary phase even under conditions optimal for survival of wild-type strains (incubation in water after stationary phase is reached). Expression of Bcl-2 in the sod1Delta strain caused a large improvement in viability at entry into stationary phase, as well as increased resistance to 100% oxygen and increased catalase activity. In addition, Bcl-2 expression reduced mutation frequency in both wild-type and sod1Delta strains. In another set of experiments, wild-type yeast incubated in expired minimal medium instead of water lost viability quickly; expression of Bcl-2 significantly delayed this stationary phase death. Our results demonstrate that Bcl-2 has activities in yeast that are similar to activities it is known to possess in mammalian cells: (a) stimulation of antioxidant protection and (b) delay of processes leading to cell death.

Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae. Mitochondrial production of toxic oxygen species in vivo.

Yeast lacking copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (SOD), catalase T, or metallothionein were studied using long-term stationary phase (10-45 days) as a simple model system to study the roles of antioxidant enzymes in aging. In well aerated cultures, the lack of either SOD resulted in dramatic loss of viability over the first few weeks of culture, with the CuZnSOD mutant showing the more severe defect. The double SOD mutant died within a few days. The severity reversed in low aeration; the CuZnSOD mutant remained viable longer than the manganese SOD mutant. To test whether reactive oxygen species generated during respiration play an important role in the observed cellular death, growth in nonfermentable carbon sources was measured. All strains grew under low aeration, indicating respiratory competence. High aeration caused much reduced growth in single SOD mutants, and the double mutant failed to grow. However, removal of respiration via another mutation dramatically increased short term survival and reversed the known air-dependent methionine and lysine auxotrophies. Our results suggest strongly that mitochondrial respiration is a major source of reactive oxygen species in vivo, as has been shown in vitro, and that these species are produced even under low aeration.