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Recently, it has been shown that mice deficient in the proapoptotic protein prostate apoptosis response 4 (Par-4) are specifically prone to develop endometrial carcinomas. Based on this, we have examined here the possible role of Par-4 as a tumor suppressor gene in human endometrial cancer. Using cDNA arrays, quantitative reverse transcription-PCR, and immunohistochemistry, we detected Par-4 down-regulation in ∼40% of endometrial carcinomas. This alteration was not associated with phosphatase and tensin homologue (PTEN), K-RAS, or β-catenin mutations, but was more frequent among tumors showing microsatellite instability (MSI) or among tumors that were estrogen receptor positive. Mutational analysis of the complete coding sequence of Par-4 in endometrial cancer cell lines (n = 6) and carcinomas (n = 69) detected a mutation in a single carcinoma, which was localized in exon 3 [Arg (CGA) 189 (TGA) Stop]. Interestingly, Par-4 promoter hypermethylation was detected in 32% of the tumors in association with low levels of Par-4 protein and was more common in MSI-positive carcinomas. Par-4 promoter hypermethylation and silencing was also detected in endometrial cancer cell lines SKUT1B and AN3CA, and reexpression was achieved by treatment with the demethylating agent 5′-aza-2′-deoxycytidine. Together, these data show that Par-4 is a relevant tumor suppressor gene in human endometrial carcinogenesis. [Cancer Res 2007;67(5):1927–34]

Supplementary Tables 1-3 from Inactivation of the Candidate Tumor Suppressor Par-4 in Endometrial Cancer

Senescent cells accumulate with obesity in the white adipose tissue of mice and humans. These senescent cells enhance the pro-inflammatory environment that, with time, contributes to the onset of glucose intolerance and type 2 diabetes. Glucose intolerance in mouse models of obesity has been successfully reversed by the elimination of senescent cells with the senolytic compounds navitoclax or the combination of dasatinib and quercetin (D/Q). In this work, we generated obese mice by high-fat diet feeding, and treated them with five consecutive cycles of navitoclax or D/Q during 16 weeks. We observed an efficient reduction in the white adipose tissue of the senescence markers senescence-associated β-galactosidase activity, Cdkn2a-p16 and Cdkn2a-p19 at the end of the 5 cycles. Mice treated with both navitoclax and D/Q showed an improvement of their insulin sensitivity and glucose tolerance during a short period of time (cycles 3 and 4), that disappeared at the fifth cycle. Also, these mice tended to increase the expression at their adipose tissue of the adipogenic genes Pparg and, Cebpa, as well as their plasma adiponectin levels. Together, our work shows that two different senolytic treatments, acting through independent pathways, are transiently effective in the treatment of obesity-induced metabolic disorders.

Phaeodactylum tricornutum is a rich source of fucoxanthin, a carotenoid with several health benefits. In the present study, high performance countercurrent chromatography (HPCCC) was used to isolate fucoxanthin from an extract of P. tricornutum. A multiple sequential injection HPCCC method was developed combining two elution modes (reverse phase and extrusion). The lower phase of a biphasic solvent system (n-heptane, ethyl acetate, ethanol and water, ratio 5/5/6/3, v/v/v/v) was used as the mobile phase, while the upper phase was the stationary phase. Ten consecutive sample injections (240 mg of extract each) were performed leading to the separation of 38 mg fucoxanthin with purity of 97% and a recovery of 98%. The process throughput was 0.189 g/h, while the efficiency per gram of fucoxanthin was 0.003 g/h. Environmental risk and general process evaluation factors were used for assessment of the developed separation method and compared with existing fucoxanthin liquid-liquid isolation methods. The isolated fucoxanthin retained its well-described ability to induce nuclear translocation of transcription factor FOXO3. Overall, the developed isolation method may represent a useful model to produce biologically active fucoxanthin from diatom biomass.

Background: Frailty is a major age-associated syndrome leading to disability. Oxidative damage plays a significant role in the promotion of frailty. The cellular antioxidant system relies on reduced nicotinamide adenine dinucleotide phosphate (NADPH) that is highly dependent on glucose 6-P dehydrogenase (G6PD). The G6PD-overexpressing mouse (G6PD-Tg) is protected against metabolic stresses. Our aim was to examine whether this protection delays frailty. Methods: Old wild-type (WT) and G6PD-Tg mice were evaluated longitudinally in terms of frailty. Indirect calorimetry, transcriptomic profile, and different skeletal muscle quality markers and muscle regenerative capacity were also investigated. Results: The percentage of frail mice was significantly lower in the G6PD-Tg than in the WT genotype, especially in 26-month-old mice where 50% of the WT were frail vs. only 13% of the Tg ones (P < 0.001). Skeletal muscle transcriptomic analysis showed an up-regulation of respiratory chain and oxidative phosphorylation (P = 0.009) as well as glutathione metabolism (P = 0.035) pathways in the G6PD-Tg mice. Accordingly, the Tg animals exhibited an increase in reduced glutathione (34.5%, P < 0.01) and a decrease on its oxidized form (-69%, P < 0.05) and in lipid peroxidation (4-HNE: -20.5%, P < 0.05). The G6PD-Tg mice also showed reduced apoptosis (BAX/Bcl2: -25.5%, P < 0.05; and Bcl-xL: -20.5%, P < 0.05), lower levels of the intramuscular adipocyte marker FABP4 (-54.7%, P < 0.05), and increased markers of mitochondrial content (COX IV: 89.7%, P < 0.05; Grp75: 37.8%, P < 0.05) and mitochondrial OXPHOS complexes (CII: 81.25%, P < 0.01; CIII: 52.5%, P < 0.01; and CV: 37.2%, P < 0.05). Energy expenditure (-4.29%, P < 0.001) and the respiratory exchange ratio were lower (-13.4%, P < 0.0001) while the locomotor activity was higher (43.4%, P < 0.0001) in the 20-month-old Tg, indicating a major energetic advantage in these mice. Short-term exercise training in young C57BL76J mice induced a robust activation of G6PD in skeletal muscle (203.4%, P < 0.05), similar to that achieved in the G6PD-Tg mice (142.3%, P < 0.01). Conclusions: Glucose 6-P dehydrogenase deficiency can be an underestimated risk factor for several human pathologies and even frailty. By overexpressing G6PD, we provide the first molecular model of robustness. Because G6PD is regulated by pharmacological and physiological interventions like exercise, our results provide molecular bases for interventions that by increasing G6PD will delay the onset of frailty. This work was supported by Instituto de Salud Carlos III CB16/10/00435 (CIBERFES) (PID2019-110906RB-I00/AEI/10.13039/501100011033) from the Spanish Ministry of Innovation and Science; 109_RESIFIT from Fundación General CSIC; PROMETEO/2019/097 de ‘Consellería, de Sanitat de la Generalitat Valenciana’ and EU Funded H2020-DIABFRAIL-LATAM (Ref: 825546); European Joint Programming Initiative ‘A Healthy Diet for a Healthy Life’ (JPI HDHL); and the ERA-NET Cofund ERA-HDHL (GA No. 696295 of the EU Horizon 2020 Research and Innovation Programme). Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014–2020). Work in the laboratory of P.J.F.-M. was funded by the FBBVA and the Ramón Areces Foundations. Work in the laboratory of P.M.-C. and A.L.S was funded by by MWRF and Fundació La Marató/TV3-80/19-202021 to P.M.C. and Fundació La Marató de TV3 (Project 202033 to A.L.S.), MINECO-Spain (RTI2018-096068), ERC-2016-AdG-741966, LaCaixa-HEALTH-HR17-00040, MDA, UPGRADE-H2020-825825, AFM, and DPP-Spain; María-de-Maeztu-Program for Units of Excellence to UPF (MDM-2014-0370); and Severo-Ochoa-Program for Centers of Excellence to CNIC (SEV-2015-0505). A.S.-P. was supported by a fellowship from the Spanish ‘Ministerio de Educación, Cultura y Deporte’, award FPU 14/00098.

Aging leads to a gradual decline in physical activity and disrupted energy homeostasis. The NAD+-dependent SIRT6 deacylase regulates aging and metabolism through mechanisms that largely remain unknown. Here, we show that SIRT6 overexpression leads to a reduction in frailty and lifespan extension in both male and female B6 mice. A combination of physiological assays, in vivo multi-omics analyses and 13C lactate tracing identified an age-dependent decline in glucose homeostasis and hepatic glucose output in wild type mice. In contrast, aged SIRT6-transgenic mice preserve hepatic glucose output and glucose homeostasis through an improvement in the utilization of two major gluconeogenic precursors, lactate and glycerol. To mediate these changes, mechanistically, SIRT6 increases hepatic gluconeogenic gene expression, de novo NAD+ synthesis, and systemically enhances glycerol release from adipose tissue. These findings show that SIRT6 optimizes energy homeostasis in old age to delay frailty and preserve healthy aging., Aging is associated with increased frailty and disrupted energy homeostasis. Here, the authors show that SIRT6 overexpression extends the lifespan of male and female mice and demonstrate that SIRT6 optimizes energy homeostasis in old age, which delays frailty and preserves healthy aging.

Sirtuins are a family of protein deacylases and ADP-ribosyl-transferases, homologs to the yeast SIR2 protein. Seven sirtuin paralogs have been described in mammals, with different subcellular locations, targets, enzymatic activities, and regulatory mechanisms. All sirtuins share NAD(+) as substrate, placing them as central metabolic hubs with strong relevance in lifespan, metabolism, and cancer development. Much effort has been devoted to studying the roles of sirtuins in cancer, providing a wealth of data on sirtuins roles in mouse models and humans. Also, extensive data are available on the effects of pharmacological modulation of sirtuins in cancer development. Here, we present a comprehensive and organized resume of all the existing evidence linking every sirtuin with cancer development. From our analysis, we conclude that sirtuin modulation after tumor initiation results in unpredictable outcomes in most tumor types. On the contrary, all genetic and pharmacological models indicate that sirtuins activation prior to tumor initiation can constitute a powerful preventive strategy.

Fasting exerts beneficial effects in mice and humans, including protection from chemotherapy toxicity. To explore the involved mechanisms, we collect blood from humans and mice before and after 36 or 24 hours of fasting, respectively, and measure lipid composition of erythrocyte membranes, circulating micro RNAs (miRNAs), and RNA expression at peripheral blood mononuclear cells (PBMCs). Fasting coordinately affects the proportion of polyunsaturated versus saturated and monounsaturated fatty acids at the erythrocyte membrane; and reduces the expression of insulin signaling-related genes in PBMCs. When fasted for 24 hours before and 24 hours after administration of oxaliplatin or doxorubicin, mice show a strong protection from toxicity in several tissues. Erythrocyte membrane lipids and PBMC gene expression define two separate groups of individuals that accurately predict a differential protection from chemotherapy toxicity, with important clinical implications. Our results reveal a mechanism of fasting associated with lipid homeostasis, and provide biomarkers of fasting to predict fasting-mediated protection from chemotherapy toxicity. General: We thank Prof. Jose Maria. Ordovas for his kind suggestions; nutritionists Helena Marcos-Pasero, Elena Aguilar-Aguilar and Isabel Espinosa-Salinas for their help with volunteers management; Rosa Serrano for her help with animal experiments; Susana Molina for her advice with PBMC isolation; Luisa Mariscal, Domingo Fernandez, Lola Martinez, Diego Megias, Patricia Gonzalez, Fernando Pelaez, Anabel Sanz, Carolina Pola, Celia de la Calle, Ana Ortega, Ana Sagrera, Jose Miguel Frade, Elena Lopez-Guadamillas, Maribel Munoz, Susana Llanos, Andres Fernandez, Aranzazu Sierra, Andres Lopez, Noemi Haro and Ildefonso Rodriguez for their excellent technical and scientific support. Work at the laboratory of P.J.F.M. is funded by the Ramon Areces Foundation, (CIVP18A3891), Asociacion Espanola contra el Cancer-AECC (SIRTBIO-LABAE18008FERN), a Ramon y Cajal Award from the Spanish Ministry of Science, Innovation and Universities (MICINN) (RYC-2017-22335), RETOS projects Program of MICINN (SAF2017-85766-R) and the Portuguese Foundation for Science and Technology (FCT-MCTES, SFRH/BD/124022/2016). Work at the laboratory of ARM was funded by the MICINN (PID2019-110183RB-C21), Regional Government of Community of Madrid (P2018/BAA-4343-ALIBIRD2020-CM) and the Ramon Areces Foundation. Work at the laboratory of A.D.R. Funded by the Comunidad de Madrid-Talento Grant 2018-T1/BMD-11966 and the MICINN PID-2019-106893RA-100. Work at the laboratory of L.D. is funded by projects from the Health Research Fund (ISCIII FIS PI14/01374 and FISPI17/00508) and from a Manuel de Oya research fellowship from the Beer and Health Foundation. Work at the laboratory of A.E. is funded by a Ramon y Cajal Award from MICINN (RYC-2013-13546) and RETOS projects Program of the MICINN, co-funded by the European Regional Development Fund (ERDF) (SAF2015-67538-R). Work in the laboratory of M.S. was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Council (ERC-2014-AdG/669622), and the "laCaixa" Foundation. Sí

Aging and cancer are two interrelated processes, with aging being a major risk factor for the development of cancer. Parallel epigenetic alterations have been described for both, although differences, especially within the DNA hypomethylation scenario, have also been recently reported. Although many of these observations arise from the use of mouse models, there is a lack of systematic comparisons of human and mouse epigenetic patterns in the context of disease. However, such comparisons are significant as they allow to establish the extent to which some of the observed similarities or differences arise from pre-existing species-specific epigenetic traits. Here, we have used reduced representation bisulfite sequencing to profile the brain methylomes of young and old, tumoral and nontumoral brain samples from human and mouse. We first characterized the baseline epigenomic patterns of the species and subsequently focused on the DNA methylation alterations associated with cancer and aging. Next, we described the functional genomic and epigenomic context associated with the alterations, and finally, we integrated our data to study interspecies DNA methylation levels at orthologous CpG sites. Globally, we found considerable differences between the characteristics of DNA methylation alterations in cancer and aging in both species. Moreover, we describe robust evidence for the conservation of the specific cancer and aging epigenomic signatures in human and mouse. Our observations point toward the preservation of the functional consequences of these alterations at multiple levels of genomic regulation. Finally, our analyses reveal a role for the genomic context in explaining disease- and species-specific epigenetic traits., This work was supported by the Spanish Association Against Cancer (PROYE18061FERN to M.F.F.), the Asturias Government (PCTI) cofunding 2018-2022/FEDER (IDI/2018/146 to M.F.F.), Fundación General CSIC (0348_CIE_6_E to M.F.F.), and the Health Institute Carlos III (Plan Nacional de I + D+I) cofunding FEDER (PI15/00892 and PI18/01527 to M.F.F and A.F.F.). They also acknowledge support from the Ramón Areces Foundation (CIVP18A3891 to P.J.F.M.), the AECC (SIRTBIO to P.J.F.M.), the MICINN (SAF2017-85766-R to P.J.F.M.), a Ramón y Cajal fellowship (MICINN, RYC-2017-22335 to P.J.F.M.), and the European Commission ATTRACT project (777222 to A.P.C.). J.R.T. is supported by a Juan de la Cierva fellowship from the Spanish Ministry of Science and Innovation (FJCI-2015-26965). R.F.P. and P.S.O. are supported by the Severo Ochoa program (BP17-114 and BP17-165, respectively). R.G.U. is supported by the Centro de Investigación Biomédica en Red de Enfermedades Raras (Health Institute Carlos III). L.V. was supported by the UAB Predoctoral training programme (PIF predoctoral fellowships). They also acknowledge support from the IMDEA Food Institute and IUOPA-ISPA-FINBA (the IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain).

Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases., Senolytic compounds have the ability to eliminate senescent cells from tissues and have been shown to be beneficial in various animal models of age-related diseases. Here the authors show that cardiac glycosides commonly used for heart diseases have senolytic properties in humanized mouse models of tumorigenesis and lung fibrosis.

This article belongs to the Special Issue Dietary Patterns, Dietary Indices for Colorectal Cancer, Mortality and the Related Disease., Nowadays, obesity and its associated metabolic disorders, including diabetes, metabolic syndrome, cardiovascular disease, or cancer, continue to be a health epidemic in westernized societies, and there is an increased necessity to explore anti-obesity therapies including pharmaceutical and nutraceutical compounds. Considerable attention has been placed on the identification of bioactive compounds from natural sources to manage the metabolic stress associated with obesity. In a previous work, we have demonstrated that a CO2 supercritical fluid extract from yarrow (Yarrow SFE), downregulates the expression of the lipogenic master regulator SREBF1 and its downstream molecular targets FASN and SCD in a tumoral context. Since obesity and diabetes are strongly considered high-risk factors for cancer development, herein, we aimed to investigate the potential therapeutic role of Yarrow SFE in the metabolic stress induced after a high-fat diet in mice. For this purpose, 32 C57BL/6 mice were distributed in four groups according to their diets: standard diet (SD); SD supplemented with Yarrow SFE (SD + Yarrow); high-fat diet (HFD); and HFD supplemented with Yarrow SFE (HFD + Yarrow). Fasting glycemia, insulin levels, homeostasis model assessment for insulin resistance (HOMA-IR), lipid profile, gene expression, and lipid content of liver and adipose tissues were analyzed after three months of treatment. Results indicate improved fasting glucose levels in plasma, enhanced insulin sensitivity, and diminished hypercholesterolemia in the HFD + Yarrow group compared to the HFD group. Mechanistically, Yarrow SFE protects liver from steatosis after the HFD challenge by augmenting the adipose tissue buffering capacity of the circulating plasma glucose., This work was supported by the Spanish Ministry of Science (Plan Nacional I + D + i AGL2016-76736-C3), Regional Government of Community of Madrid (P2013/ABI-2728, ALIBIRD-CM; P2018/BAA-4343-ALIBIRD2020-CM), the Ramón Areces Foundation, and the EU Structural Funds.

Diabetes refers to a group of metabolic diseases characterized by impaired insulin signalling and high blood glucose. A growing body of epidemiological evidence links diabetes to several types of cancer but the underlying molecular mechanisms are poorly understood. The signalling cascade connecting insulin and FOXO proteins provides a compelling example for a conserved pathway at the interface between insulin signalling and cancer. FOXOs are transcription factors that orchestrate programs of gene expression known to control a variety of processes in response to cellular stress. Genes regulated by this family of proteins are involved in the regulation of cellular energy production, oxidative stress resistance and cell viability and proliferation. Accordingly, FOXO factors have been shown to play an important role in the suppression of tumour growth and in the regulation of metabolic homeostasis. There is emerging evidence that deregulation of FOXO factors might account for the association between insulin resistance-related metabolic disorders and cancer.

Flow cytometry is a powerful multiparametric technology, widely used for the identification, quantification, and isolation of defined populations of cells based on the expression of target proteins. It also allows for the use of surrogate reporters, either enzymatic or fluorescent, to indirectly monitor the expression of these target proteins. In this work, we optimised the dissociation protocol for the detection of the enzymatic reporter LacZ using the FACS-Gal detection system with the fluorogenic substrate FDG to compare cis- versus trans-positioned reporters efficiency. Particularly, for the FACS-Gal optimization, we studied lung and haematopoietic tissues, focusing on cell recovery, viability, FDG loading conditions and distribution of cellular populations. Reporter genes such as LacZ can be placed together with the gene of interest in the same polycistronic mRNA (in cis), or in independent alleles (in trans), which can strongly affect the correlation with the reporter readout. To address this issue, we generated a mouse model containing both types of reporters for the same gene, and compared them. Our results clearly indicate that trans-positioned reporters can be misleading, and that using a reporter gene in cis rather than trans is a much more specific method to sort for cells undergoing Cre-mediated recombination. © 2017 International Society for Advancement of Cytometry.

Partial inhibition of PI3K is one of the best-validated and evolutionary conserved manipulations to extend longevity. The best known health beneficial effects of reduced PI3K are related to metabolism and include increased energy expenditure, reduced nutrient storage, and protection from obesity. We have previously shown that a dual chemical inhibitor of the alpha and delta PI3K isoforms (CNIO-PI3Ki) reduces obesity in mice and monkeys, without evident toxic effects after long-term treatment. Here, we dissect the role of the alpha and delta PI3K isoforms by making use of selective inhibitors against PI3Kɑ (BYL-719 also known as alpelisib) or PI3Kδ (GS-9820 also known as acalisib). Treatment of mice with the above mentioned inhibitors indicated that BYL-719 increases energy expenditure in normal mice and efficiently reduces body weight in obese (ob/ob) mice, whereas these effects were not observed with GS-9820. Of note, the dose of BYL-719 required to reduce obesity was 10-times higher than the equivalent dose of CNIO-PI3Ki, which could suggest that simultaneous inhibition of PI3K alpha and delta is more beneficial than single inhibition of the alpha isoform. In summary, we conclude that inhibition of PI3Kɑ is sufficient to increase energy expenditure and reduce obesity, and suggest that concomitant PI3Kδ inhibition could play an auxiliary role.

Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases. We thank Matthias Drosten, Alejo Efeyan and Sean Morrison for plasmids. F.T-M. is a postdoctoral fellow from CONACYT (cvu 268632); P.P. is a predoctoral fellow from Xunta de Galicia; M.C. is a "Miguel Servet II" investigator (CPII16/00015). P.P.-R. receives support from a program by the Deputacion de Coruna (BINV-CS/2019). Work in the laboratory of M.C. is funded by grant RTI2018-095818-B-100 (MCIU/AEI/FEDER, UE). P.J.F.-M. is funded by the IMDEA Food Institute, the Ramon Areces Foundation, (CIVP18A3891), and a Ramon y Cajal Award (MICINN) (RYC-2017-22335). M.P.I. is funded by Talento Modalidad-1 Program Grant, Madrid Regional Government (#2018-T1/BIO-11262). F.P. was funded by a Long Term EMBO Fellowship (ALTF-358-2017) and F.H-G. was funded by the PhD4MD Programme of the IRB, Hospital Clinic and IDIBAPS. Work in the laboratory of M.S. was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Council (ERC-2014-AdG/669622), and "laCaixa" Foundation. Sí

Devil facial tumor disease (DFTD) and its lack of available therapies are propelling the Tasmanian devil population toward extinction. This study demonstrates that cholesterol homeostasis and carbohydrate energy metabolism sustain the proliferation of DFTD cells in a cell-type-dependent manner. In addition, we show that the liver-X nuclear receptor-β (LXRβ), a major cholesterol cellular sensor, and its natural ligand 24S-hydroxycholesterol promote the proliferation of DFTD cells via a metabolic switch toward aerobic glycolysis. As a proof of concept of the role of cholesterol homeostasis on DFTD proliferation, we show that atorvastatin, an FDA-approved statin-drug subtype used against human cardiovascular diseases that inhibits cholesterol synthesis, shuts down DFTD energy metabolism and prevents tumor growth in an in vivo DFTD-xenograft model. In conclusion, we show that intervention against cholesterol homeostasis and carbohydrate-dependent energy metabolism by atorvastatin constitutes a feasible biochemical treatment against DFTD, which may assist in the conservation of the Tasmanian devil.

FOXO proteins are transcription factors with important roles in the regulation of the expression of genes involved in cell growth, proliferation, differentiation, and longevity. FOXO proteins are active in the nucleus but, upon post-translational modification they form a docking site for 14-3-3 proteins and are translocated to the cytoplasm where they are inactive.We make use of this regulatory mechanism of FOXO proteins to develop an image-based high-throughput screening platform to detect compounds that regulate FOXO3 subcellular localization. This system has proven a powerful tool to isolate inhibitors of proteins upstream of FOXO, such as PI3K inhibitors.

FOXO proteins are transcription factors with important roles in the regulation of the expression of genes involved in cell growth, proliferation, differentiation, and longevity. FOXO proteins are active in the nucleus but, upon post-translational modification they form a docking site for 14-3-3 proteins and are translocated to the cytoplasm where they are inactive.We make use of this regulatory mechanism of FOXO proteins to develop an image-based high-throughput screening platform to detect compounds that regulate FOXO3 subcellular localization. This system has proven a powerful tool to isolate inhibitors of proteins upstream of FOXO, such as PI3K inhibitors.

Thermogenesis converts the chemical energy of nutrients into heat, and this is associated to improved metabolic performance. In a previous issue of Cell Metabolism, Berry et al. (2017) reported that the progenitors of thermogenic beige adipocytes lose functionality with aging, and this process can be reversed with strategies that eliminate senescent cells.

Colorectal cancer has the second highest cancer-related mortality rate, with an estimated 881,000 deaths worldwide in 2018. The urgent need to reduce the incidence and mortality rate requires innovative strategies to improve prevention, early diagnosis, prognostic biomarkers, and treatment effectiveness. Caloric restriction (CR) is known as the most robust nutritional intervention that extends lifespan and delays the progression of age-related diseases, with remarkable results for cancer protection. Other forms of energy restriction, such as periodic fasting, intermittent fasting, or fasting-mimicking diets, with or without reduction of total calorie intake, recapitulate the effects of chronic CR and confer a wide range of beneficial effects towards health and survival, including anti-cancer properties. In this review, the known molecular, cellular, and organismal effects of energy restriction in oncology will be discussed. Energy-restriction-based strategies implemented in colorectal models and clinical trials will be also revised. While energy restriction constitutes a promising intervention for the prevention and treatment of several malignant neoplasms, further investigations are essential to dissect the interplay between fundamental aspects of energy intake, such as feeding patterns, fasting length, or diet composition, with all of them influencing health and disease or cancer effects. Currently, effectiveness, safety, and practicability of different forms of fasting to fight cancer, particularly colorectal cancer, should still be contemplated with caution.

The NAD (+)‐dependent deacetylase SIRT1 can be oncogenic or tumor suppressive depending on the tissue. Little is known about the role of SIRT1 in non‐small cell lung carcinoma (NSCLC), one of the deadliest cancers, that is frequently associated with mutated K‐RAS. Therefore, we investigated the effect of SIRT1 on K‐RAS‐driven lung carcinogenesis. We report that SIRT1 protein levels are downregulated by oncogenic K‐RAS in a MEK and PI3K‐dependent manner in mouse embryo fibroblasts (MEFs), and in human lung adenocarcinoma cell lines. Furthermore, Sirt1 overexpression in mice delays the appearance of K‐Ras(G12V)‐driven lung adenocarcinomas, reducing the number and size of carcinomas at the time of death and extending survival. Consistently, lower levels of SIRT1 are associated with worse prognosis in human NSCLCs. Mechanistically, analysis of mouse Sirt1‐Tg pneumocytes, isolated shortly after K‐Ras(G12V) activation, reveals that Sirt1 overexpression alters pathways involved in tumor development: proliferation, apoptosis, or extracellular matrix organization. Our work demonstrates a tumor suppressive role of SIRT1 in the development of K‐RAS‐driven lung adenocarcinomas in mice and humans, suggesting that the SIRT1–K‐RAS axis could be a therapeutic target for NSCLCs.

Introduction Frailty is a major geriatric syndrome that has been associated to oxidative stress. The antioxidant system is largely based on the reducing power of NADPH, whose levels are mainly determined by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Material and methods Using old female Tg-mice overexpressing G6PD (18 to 26 months old), we measured frailty and different muscle parameters: oxidative stress, cross-sectional area (CSA), markers regulating protein synthesis, mitochondrial dynamics, and apoptosis. Results Our results show that 18–24 months old G6PD-Tg animals performed better in the motor coordination and grip strength test than the WT. We also found lower changes in body weight and improved performance in the running speed in the G6PD-Tg mice. Taking into account the five frailty components, we found that the percentage of mice considered as frail was higher in the WT than in the G6PD-Tg group. G6PD-Tg mice exhibited higher GSH over GSSG ratio, reduced apoptosis (Bax/Bcl2), lower intramuscular adipocyte markers (FABP4, PDGFR α) and a higher mitochondrial content (COXIV) than the WT. Conclusion. Our results show that the overexpression of G6PD prevents frailty in 18 to 26 months old mice modulating the oxidative stress and markers of muscle quality.

For cell reprogramming, context matters Differentiated cells in a culture dish can assume a new identity when manipulated to express four transcription factors. This “reprogramming” process has sparked interest because conceivably it could be harnessed as a therapeutic strategy for tissue regeneration. Mosteiro et al. used a mouse model to study the signals that promote cell reprogramming in vivo. They found that the factors that trigger reprogramming in vitro do the same in vivo; however, they also inflict cell damage. The damaged cells enter a state of senescence and begin secreting certain factors that promote reprogramming, including an inflammatory cytokine called interleukin-6. Thus, in the physiological setting, cell senescence may create a tissue context that favors reprogramming of neighboring cells. Science , this issue p. 10.1126/science.aaf4445

et al., Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the role of stress-responsive tumor suppressors in fasting. Here, we have examined the expression of several tumor suppressors upon fasting in mice. Interestingly, p21 mRNA is uniquely induced in all the tissues tested, particularly in liver and muscle (>10 fold), and this upregulation is independent of p53. Remarkably, in contrast to wild-type mice, p21-null mice become severely morbid after prolonged fasting. The defective adaptation to fasting of p21-null mice is associated to elevated energy expenditure, accelerated depletion of fat stores, and premature activation of protein catabolism in the muscle. Analysis of the liver transcriptome and cell-based assays revealed that the absence of p21 partially impairs the transcriptional program of PPARα, a key regulator of fasting metabolism. Finally, treatment of p21-null mice with a PPARα agonist substantially protects them from their accelerated loss of fat upon fasting. We conclude that p21 plays a relevant role in fasting adaptation through the positive regulation of PPARα., E.L.-G. was recipient of a predoctoral contract from the Spanish Ministry of Education. P.J.F.-M. has been funded by the Spanish Association Against Cancer (aecc). Work in the laboratory of M.S. is funded by the CNIO and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (SAF project), the European Research Council (ERC Advanced Grant), the Regional Government of Madrid co-funded by the European Social Fund (ReCaRe project), the Botin Foundation and Banco Santander (Santander Universities Global Division), the Ramon Areces Foundation, and the AXA Foundation.

Reactive oxygen species (ROS) are constantly generated by cells and ROS-derived damage contributes to ageing. Protection against oxidative damage largely relies on the reductive power of NAPDH, whose levels are mostly determined by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here, we report a transgenic mouse model with moderate overexpression of human G6PD under its endogenous promoter. Importantly, G6PD-Tg mice have higher levels of NADPH, lower levels of ROS-derived damage, and better protection from ageing-associated functional decline, including extended median lifespan in females. The G6PD transgene has no effect on tumour development, even after combining with various tumour-prone genetic alterations. We conclude that a modest increase in G6PD activity is beneficial for healthspan through increased NADPH levels and protection from the deleterious effects of ROS., The enzyme G6PD generates the reductive metabolite NADPH, which has antioxidant effects, but has also been linked to tumour growth. Here the authors generate mice that modestly overexpress G6PD and report increased lifespan in females, and no negative effects on tumour formation in various genetic models.

S.N.-P. and P.J.F.-M. have been funded by the Spanish Association Against Cancer(aecc). Work in the laboratory of M.S. is funded by the CNIO and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund(SAF project), the European Research Council (ERC Advanced Grant), the Regional Government of Madrid co-funded by the European Social Fund (ReCaRe project), the European Union (RISK-IR project), the Botin Foundation and Banco Santander(Santander Universities Global Division), the Ramon Areces Foundation, and the AXA Foundation. Work in the laboratory of J.V. was supported by grants SAF2013-44663-R,from the Spanish Ministry of Education and Science (MEC); ISCIII2012-RED-43-029from RETICEF; PROMETEO2014/056 from "Conselleria d’Educacio , Cultura i Esport de la Generalitat Valenciana"; RS2012-609 Intramural Grant from INCLIVA and EUFunded CM1001 andFRAILOMIC-HEALTH.2012.2.1.1-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Reactive oxygen species (ROS) are constantly generated by cells and ROS-derived damage contributes to ageing. Protection against oxidative damage largely relies on the reductive power of NAPDH, whose levels are mostly determined by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here, we report a transgenic mouse model with moderate overexpression of human G6PD under its endogenous promoter. Importantly, G6PD-Tg mice have higher levels of NADPH, lower levels of ROS-derived damage, and better protection from ageing-associated functional decline, including extended median lifespan in females. The G6PD transgene has no effect on tumour development, even after combining with various tumour-prone genetic alterations. We conclude that a modest increase in G6PD activity is beneficial for healthspan through increased NADPH levels and protection from the deleterious effects of ROS. Sí

Liver receptor homolog-1 (NR5A2) is a nuclear receptor originally identified in the liver and mostly known for its regulatory role in cholesterol and bile acid homeostasis. More recently, liver receptor homolog-1 has emerged as a key regulator of intestinal function, coordinating unanticipated actions, such as cell renewal and local immune function with important implications to common intestinal diseases, including colorectal cancer and inflammatory bowel disease. Unlike most of the other nuclear receptors, liver receptor homolog-1 acts as a constitutively active transcription factor to drive the transcription of its target genes. Liver receptor homolog-1 activity however is to a major extent regulated by different corepressors and posttranslational modifications, which may account for its tissue-specific functions. This review will provide an update on the molecular aspects of liver receptor homolog-1 action and focus on some emerging aspects of its function in normal and diseased gut. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

To assess the impact of lifelong overexpression of G6PD on reactive oxygen species (ROS)-derived damage and the prevention of frailty, we measured the levels of macromolecular oxidative damage in young and old mice and the we tested the neuromuscular fitness and the grip strength in old mice. Old G6PD-Tg male and female mice showed diminished accumulation of DNA oxidation (measured as 8-hydroxyguanosine or 8-OHdG) in liver and brain. Old females also showed reduced lipid oxidation (measured as malondialdehyde or MDA) in the liver. Old G6PD-Tg males, but not females, presented a small but significant increase in brain protein carbonylation. In accordance with these findings, liver from 2-year-old G6PD-Tg female mice presented an elevated reduced versus oxidized gluthatione ratio (GSH:GSSG) ratio, due to an elevation in reduced GSH.Old G6PD-Tg female mice at 1.5–2 years of age also showed a significant improvement in neuromuscular fitness (evaluated by the Rotarod test) and in the maximum and mean grip strength, both parameters related to frailty. Our data point out that G6PD overexpression in mice protects against ROS-derived damage at old age.

Prostate cancer is one of the most common neoplasias in men. The tumor suppressor Par-4 is an important negative regulator of the canonical NF-κB pathway and is highly expressed in prostate. Here we show that Par-4 expression is lost in a high percentage of human prostate carcinomas, and this occurs in association with phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss. Par-4 null mice, similar to PTEN-heterozygous mice, only develop benign prostate lesions, but, importantly, concomitant Par-4 ablation and PTEN-heterozygosity lead to invasive prostate carcinoma in mice. This strong tumorigenic cooperation is anticipated in the preneoplastic prostate epithelium by an additive increase in Akt activation and a synergistic stimulation of NF-κB. These results establish the cooperation between Par-4 and PTEN as relevant for the development of prostate cancer and implicate the NF-κB pathway as a critical event in prostate tumorigenesis.

Summary Telomerase confers limitless proliferative potential to most human cells through its ability to elongate telomeres, the natural ends of chromosomes, which otherwise would undergo progressive attrition and eventually compromise cell viability. However, the role of telomerase in organismal aging has remained unaddressed, in part because of the cancer-promoting activity of telomerase. To circumvent this problem, we have constitutively expressed telomerase reverse transcriptase (TERT), one of the components of telomerase, in mice engineered to be cancer resistant by means of enhanced expression of the tumor suppressors p53, p16, and p19ARF. In this context, TERT overexpression improves the fitness of epithelial barriers, particularly the skin and the intestine, and produces a systemic delay in aging accompanied by extension of the median life span. These results demonstrate that constitutive expression of Tert provides antiaging activity in the context of a mammalian organism.

International audience; NOTCH signaling suppresses tumor growth and proliferation in several types of stratified epithelia. Here, we show that missense mutations in NOTCH1 and NOTCH2 found in human bladder cancers result in loss of function. In murine models, genetic ablation of the NOTCH pathway accelerated bladder tumorigenesis and promoted the formation of squamous cell carcinomas, with areas of mesenchymal features. Using bladder cancer cells, we determined that the NOTCH pathway stabilizes the epithelial phenotype through its effector HES1 and, consequently, loss of NOTCH activity favors the process of epithelial-mesenchymal transition. Evaluation of human bladder cancer samples revealed that tumors with low levels of HES1 present mesenchymal features and are more aggressive. Together, our results indicate that NOTCH serves as a tumor suppressor in the bladder and that loss of this pathway promotes mesenchymal and invasive features.

Cellular senescence is a damage response characterized by a stable cell-cycle arrest and an intense secretion of cytokines. In this issue of Cell Metabolism, Wiley et al. (2016) report that, in the case of mitochondrial damage, senescence occurs with an atypical secretory phenotype, in both adipose tissue and skin.

Calo et al. (2010) recently reported that the absence of the transcriptional regulator pRb enhances differentiation of mesenchymal precursors toward the brown adipocyte lineage in detriment of osteoblast and white fat populations. These findings may have therapeutic implications for cancer and bone and metabolic disease.

The implication of the Notch pathway in cancer has been known since it was found hyperactivated in acute T-cell lymphoblastic leukemias (T-ALL) one decade ago. During this time, the link between the Notch pathway and cancer has been extended to many types of malignancies. A unique feature of the pathway is the fact that it can be oncogenic or tumor suppressive depending on the tumor type. For example, NOTCH receptors present gain-of-function mutations that make the receptor constitutively active in T-ALL, B-cell chronic lymphocytic leukemia, or lung adenocarcinoma. In contrast, NOTCH receptors undergo loss-of-function mutations in myeloid leukemias and in squamous cell carcinomas (SCCs) from esophagus, skin, lung or head and neck [1]. Two recent reports, from the group of Dr. Klinakis [2] and from us [3], have addressed the role of the Notch pathway in urinary bladder cancer demonstrating that it plays a relevant tumor suppressive role. The Klinakis group performed an extensive mutational analysis of up to nine components of the Notch pathway and copy number variations of the NOTCH1 gene in a cohort of human bladder cancers [2]. Remarkably, they found that up to 60% of bladder cancers have loss-of-function alterations in components of the Notch pathway (43% overall mutation incidence, being more than half concentrated in NOTCH1 and NOTCH2) and NOTCH1 gene copy losses. In support of this, we also showed that NOTCH1 and NOTCH2 mutations previously found in bladder cancers are functionally defective [3]. Of further relevance, both studies found a correlation between low activity of the Notch pathway, higher cancer aggressiveness and shorter patient survival [2,3]. To support these findings, the two groups used mouse genetics to demonstrate that inactivation of the canonical Notch pathway at different levels promotes the development of bladder cancer in mice [2,3]. As it was the case in human cancers, Notch-deficient murine bladder cancers were also highly infiltrating [2,3]. The two studies performed mechanistic analyses that shed light on different aspects of the biology of Notch in bladder cancer. We focused on the aggressive behavior of the cancers with loss of Notch function. By manipulating the Notch pathway in cultured cells, we found that inhibition of the Notch pathway results in the upregulation of key mediators and effectors of the epithelial-mesenchymal-transition (EMT), including SNAIL, SLUG, ZEB2 and VIMENTIN, and the concomitant downregulation of the epithelial marker E-CADHERIN [3]. The transcriptional repressor HES1 is one of the critical transcriptional targets of the Notch pathway, and we showed that reduced HES1 activity upon Notch inhibition is responsible for the de-repression of the EMT program in bladder cancer cells [3]. Therefore, we conclude that Notch represses EMT in bladder, where it acts as a tumor suppressive pathway. In this regard, it is interesting to note that a large body of evidence indicates that Notch induces EMT in those tissues where Notch is oncogenic. It appears that Notch acts in opposite directions in different tissues: it represses EMT in tissues where Notch is tumor suppressive; and it activates EMT in tissues where it is oncogenic. We will come back to this issue at the end of this commentary. A sizeable proportion of human bladder cancers have gain-of-function mutations in FGFR3, HRAS, KRAS or PIK3CA genes. These mutations potently activate the canonical MAPK cascade and, accordingly, are associated to high levels of phospho-ERK (pERK1/2). The Notch signaling pathway is not generally considered a potent activator of the MAPK cascade. Unexpectedly, Klinakis' group made the intriguing observation that Notch-deficient bladder cancers (with no other concomitant mutations in FGFR3 or RAS) present high levels of pERK1/2, even higher than those in FGFR3/RAS-mutant bladder cancers [2]. Mechanistic analyses in culture cells led the authors to conclude that the Notch pathway transcriptionally activates several members of the dual-specificity phosphatases (DUSPs), including DUSP1, by direct binding of the activated NOTCH receptors to their promoter regions [2]. The DUSP phosphatases are responsible for the dephosphorylation of pERK1/2 and, therefore, when the Notch pathway is not functional DUSP expression decreases and there is an increase of pERK1/2 in bladder cancer cells [2]. Of note, previous work from others and us in lung adenocarcinoma, where Notch is oncogenic, has shown that the Notch pathway, through HES1, represses the DUSP1 promoter [4,5]. Again, as in the case of EMT, it appears that Notch acts in opposite directions depending on the tissues: it activates DUSPs in tissues where it is tumor suppressive; and it represses DUSPs in tissues where it is oncogenic. Based on the above-described sets of mechanistic studies, it is tempting to speculate that the dual actions of the Notch pathway, oncogenic or tumor suppressive, reflect opposite actions on the same transcriptional targets. This situation is not new to researchers working on Notch in Drosophila, where it is well established that the Notch pathway can act positively and negatively on the same genes. Specifically, Notch-regulated genes often have binding sites for NOTCH/RBPJ (positive) and for the Notch target HES1 (negative), and this is thought to allow transient transcriptional effects [6]. In this context, alterations in the activation/repression equilibrium in different cell types may dramatically change the outcome of Notch activity. For example, this may well be the case of DUSP1: in those settings where the Notch pathway is tumor suppressive (as in bladder), the activation promoted by NOTCH1 is dominant; in contrast, in tissues where Notch is oncogenic (as in lung adenocarcinoma), the repressive action of HES1 is the dominant one. Conceivably, understanding the epigenetic status of critical Notch target genes could eventually explain the function, oncogenic or tumor suppressive, of the Notch pathway in cancer. In summary, a new tumor suppressive role has been found for the Notch pathway in the urinary bladder, and this has yielded important mechanistic insights about bladder cancer and the biology of the Notch pathway.

Mechanisms responsible for energy management in the cell and in the whole organism require a complex network of transcription factors and cofactors. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has emerged as a master regulator of mitochondrial biogenesis and function, thus becoming a crucial metabolic node. We present an overview of the mechanisms by which PGC-1α is regulated, including the transcriptional regulation of PGC-1α expression and the fine-tuning of its final activity via posttranslational modifications.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Sei1 is a positive regulator of proliferation that promotes the assembly of Cdk4-cyclin D complexes and enhances the transcriptional activity of E2f1. The potential oncogenic role of Sei1 is further suggested by its overexpression in various types of human cancers. To study the role of Sei1, we have generated a mouse line deficient for this gene. Sei1-null fibroblasts did not show abnormalities regarding proliferation or susceptibility to neoplastic transformation, nor did we observe defects on Cdk4 complexes or E2f activity. Sei1-null mice were viable, did not present overt pathologies, had a normal lifespan, and had a normal susceptibility to spontaneous and chemically-induced cancer. Pancreatic insulin-producing cells are known to be particularly sensitive to Cdk4-cyclin D and E2f activities, and we have observed that Sei1 is highly expressed in pancreatic islets compared to other tissues. Interestingly, Sei1-null mice present lower number of islets, decreased beta-cell area, impaired insulin secretion, and glucose intolerance. These defects were associated to nuclear accumulation of the cell-cycle inhibitors p21(Cip1) and p27(Kip1) in islet cells. We conclude that Sei1 plays an important role in pancreatic beta-cells, which supports a functional link between Sei1 and the core cell cycle regulators specifically in the context of the pancreas., Work in the laboratory of M.S. is funded by the Centro Nacional de Investigaciones Oncológicas (CNIO) and by grants from the Spanish Ministry of Science (SAF and CONSOLIDER), the Regional Government of Madrid (GsSTEM), the European Union (PROTEOMAGE), the European Research Council (ERC), and the ‘‘Marcelino Botin’’ Foundation. Work in the laboratory of A.M.V. was funded by the Ministry of Science (BFU2008-02420) and by the Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM).

14 páginas, 9 figuras, 1 tabla., Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16INK4a, p21WAF1, and p19ARF upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research., This study was supported by Fondo de Investigación Sanitario (FIS) project 04/0530 and Marato TV3 project 052130. M.E.L. is an FIS investigator (CP03/00101).

The atypical PKC-interacting protein, Par-4, inhibits cell survival and tumorigenesis in vitro, and its genetic inactivation in mice leads to reduced lifespan, enhanced benign tumour development and low-frequency carcinogenesis. Here, we demonstrate that Par-4 is highly expressed in normal lung but reduced in human lung cancer samples. We show, in a mouse model of lung tumours, that the lack of Par-4 dramatically enhances Ras-induced lung carcinoma formation in vivo, acting as a negative regulator of Akt activation. We also demonstrate in cell culture, in vivo, and in biochemical experiments that Akt regulation by Par-4 is mediated by PKCzeta, establishing a new paradigm for Akt regulation and, likely, for Ras-induced lung carcinogenesis, wherein Par-4 is a novel tumour suppressor.

Recently, it has been shown that mice deficient in the proapoptotic protein prostate apoptosis response 4 (Par-4) are specifically prone to develop endometrial carcinomas. Based on this, we have examined here the possible role of Par-4 as a tumor suppressor gene in human endometrial cancer. Using cDNA arrays, quantitative reverse transcription-PCR, and immunohistochemistry, we detected Par-4 down-regulation in ∼40% of endometrial carcinomas. This alteration was not associated with phosphatase and tensin homologue (PTEN), K-RAS, or β-catenin mutations, but was more frequent among tumors showing microsatellite instability (MSI) or among tumors that were estrogen receptor positive. Mutational analysis of the complete coding sequence of Par-4 in endometrial cancer cell lines (n = 6) and carcinomas (n = 69) detected a mutation in a single carcinoma, which was localized in exon 3 [Arg (CGA) 189 (TGA) Stop]. Interestingly, Par-4 promoter hypermethylation was detected in 32% of the tumors in association with low levels of Par-4 protein and was more common in MSI-positive carcinomas. Par-4 promoter hypermethylation and silencing was also detected in endometrial cancer cell lines SKUT1B and AN3CA, and reexpression was achieved by treatment with the demethylating agent 5′-aza-2′-deoxycytidine. Together, these data show that Par-4 is a relevant tumor suppressor gene in human endometrial carcinogenesis. [Cancer Res 2007;67(5):1927–34]