Construction of an immune-related gene prognostic model with experimental validation and analysis of immune cell infiltration in lung adenocarcinoma.

There is a correlation between tumors and immunity with the degree of immune cell infiltration in tumors being closely related to tumor growth and progression. Therefore, the present study identified immune-related prognostic genes and evaluated the immune infiltration level in lung adenocarcinoma (LUAD). This study performed Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and Gene Set Enrichment Analysis (GSEA) enrichment analyses on differential immune-associated genes. A risk model was created and validated using six immune-related prognostic genes. Reverse transcription-quantitative PCR was used to assess the prognostic gene expression in non-small cell lung cancer cells. Immune cell infiltration in LUAD was analyzed using the CIBERSORT method. Single sample GSEA was used to compare Tumor Immune Dysfunction and Exclusion (TIDE) scores between high and low-risk groups and to assess the activation of thirteen immune-related pathways. Multifactor Cox proportional hazards model analysis identified six prognostic risk genes (S100A16, FURIN, FGF2, LGR4, TNFRSF11A and VIPR1) to construct a risk model. The survival and receiver operating characteristic curves indicated that patients with higher risk scores had lower overall survival rates. The expression levels of prognostic genes S100A16, FURIN, LGR4, TNFRSF11A and VIPR1 were significantly increased in LUAD. B cells naive, plasma cells, T cells CD4 memory activated, T cells follicular helper, T cells regulatory, NK cells activated, macrophages M1, macrophages M2, and Dendritic cells resting cells showed elevated expression in LUAD. The prognostic genes were differentially associated with individual immune cells. Immune-related function scores, such as those for antigen presenting cell (APC) co-stimulation, APC co-inhibition, check-point, Cytolytic-activity, chemokine receptor, parainflammation, major histocompatibility complex-class-I, type-I-IFN-reponse and T-cell-co-inhibition, were higher in the high-risk group compared with the low-risk group. Furthermore, the TIDE score of the high-risk group was significantly lower than the low-risk group. This immune-related gene prognostic model has the potential to predict the prognosis of LUAD patients, supporting the development of a personalized clinical diagnosis and treatment plan.

The FGFR1 Signaling Pathway Upregulates the Oncogenic Transcription Factor FOXQ1 to Promote Breast Cancer Cell Growth.

FGFR1 is a receptor tyrosine kinase deregulated in certain breast cancers (BCs) with a poor prognosis. Although FGFR1-activated phosphorylation cascades have been mapped, the key genes regulated by FGFR1 in BC are largely unclear. FOXQ1 is an oncogenic transcription factor. Although we found that activation of FGFR1 robustly upregulated FOXQ1 mRNA, how FGFR1 regulates FOXQ1 gene expression and whether FOXQ1 is essential for FGFR1-stimulated cell proliferation are unknown. Herein, we confirmed that activation of FGFR1 robustly upregulated FOXQ1 mRNA and protein in BC cells. Knockdown of FOXQ1 blocked the FGFR1 signaling-stimulated BC cell proliferation, colony formation, and xenograft tumor growth. Inhibition of MEK or ERK1/2 activities, or knockout of ERK2 but not ERK1 suppressed the FGFR1 signaling-promoted FOXQ1 gene expression. Inhibition of ERK2 in ERK1 knockout cells blocked, while ectopic expression of FOXQ1 in ERK2 knockout cells rescued the FGFR1-signaling-promoted cell growth. Mechanistically, c-FOS, an early response transcription factor upregulated by the FGFR1-MEK-ERK2 pathway, bound to the FOXQ1 promoter to mediate the FGFR1 signaling-promoted FOXQ1 expression. These results indicate that the FGFR1-ERK2-c-FOS-FOXQ1 regulatory axis plays an essential role in the FGFR1 signaling-promoted BC growth. Targeting ERK2 and FOXQ1 should block BC growth caused by a deregulated FGFR1 signaling.

Therapeutic effect and mechanism of polysaccharides from Anoectochilus Roxburghii (Wall.) Lindl. in diet-induced obesity.

BACKGROUND: Recent studies have shown that polysaccharides from Anoectochilus roxburghii (Wall.) Lindl. (ARPs) can reduce blood glucose levels, ameliorate oxidative stress and inflammation. However, whether ARPs have a beneficial effect on diet-induced obesity remain to be determined. PURPOSE: This study aims to investigate the effect and mechanism of ARPs in improving obesity and metabolic disorders induced by high-fat diet (HFD). METHODS: In this study, 6-week-old male mice were fed with HFD or chow diet for 13 weeks, and a dietary supplementation with ARPs was carried out. Glucose tolerance test and insulin tolerance test were performed to measure the glucose tolerance and insulin sensitivity. Adipose tissue and liver were isolated for analysis by qRT-PCR, Western blotting, hematoxylin-eosin staining and immunostaining. RESULTS: At week 13, body weight and fat mass were significantly increased by HFD, but ARPs supplementation abolished these phenotypes. Compared with HFD group, thermogenic genes including Ucp-1, Pgc-1α, Prdm16 and Dio2 in adipose tissue were up-regulated in ARPs-treated mice. In addition, ARPs decreased liver lipid accumulation by reducing lipid synthesis and increasing oxidation. Meanwhile, dyslipidemia and insulin resistance induced by HFD were improved by ARPs. Mechanistically, ARPs can promote fat thermogenesis via AMPK/SIRT1/PGC-1α signaling pathway. CONCLUSION: Dietary supplementation of ARPs can protect mice against diet-induced obesity, fatty liver and insulin resistance. Our study reveals a potential therapeutic effect for ARPs in regulating energy homeostasis.

Effect of LncPVT1/miR-20a-5p on Lipid Metabolism and Insulin Resistance in NAFLD.

PURPOSE: Nonalcoholic fatty liver disease (NAFLD) is closely related to lipid metabolism and insulin resistance. The current research mainly attempted to verify the clinical value of LncRNA plasmacytoma variant translocation 1 (PVT1), and whether microRNA regulates lipid metabolism and insulin resistance to participate in NAFLD. PATIENTS AND METHODS: 81 patients with NAFLD and 78 healthy individuals were enrolled in this study. In addition, C57BL/6 mice were fed a high-fat diet to establish NAFLD model in vivo. Serum PVT1 and miR-20a-5p expression in NAFLD patients and mice were assessed by RT-qPCR. ROC curves determine the diagnostic value of PVT1 and miR-20a-5p. NAFLD mice were subjected to IPGTT to detect changes in insulin sensitivity, and the common indicators of lipid metabolism and insulin resistance were also evaluated. Dual-luciferase reporter assay verified the regulation mechanism of PVT1 and miR-20a-5p. RESULTS: PVT1 was upregulated in NAFLD patients and mice, while miR-20a-5p was decreased. Their expression trends were similar in patients with HOMA-IR ≥2.5. What's more, miR-20a-5p, FBG, ALT, and HOMA-IR were independently correlated with PVT1. And PVT1 and miR-20a-5p show high clinical diagnostic value. Bodyweight, insulin sensitivity, lipid metabolism inductors were increased in NAFLD mice, but these increases were attenuated by PVT1 elimination. Finally, miR-20a-5p might function as the possible miRNA target of PVT1 via the binding sites at 3'-UTR and negatively regulated by it. CONCLUSION: PVT1 and miR-20a-5p are potential clinical biomarkers of NAFLD, and PVT1 promotes the occurrence of NAFLD by regulating insulin sensitivity and lipid metabolism, which may be achieved by targeting miR-20a-5p.

Kava root extracts hinder prostate cancer development and tumorigenesis by involvement of dual inhibition of MAO-A and LSD1.

AIM: Here, we aim to evaluate the chemopreventive efficacy of kava root extracts (KRE) in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and investigate potential molecular targets of kavalactones, the main components of kava. METHODS: TRAMP mice were administrated with KRE formulated food for different periods of time, and then the incidences of high-grade prostatic intraepithelial neoplasia (HG-PIN) and adenocarcinomas and tumor burdens were compared between vehicle control and KRE food fed groups. In addition, the inhibitory effect of the KRE and kavalactones on monoamine oxidase A (MAO-A) and lysine-specific demethylase 1 (LSD1) enzyme activities were examined by commercially available inhibitor screening kits. Histone H3 lysine 9 dimethylation was also evaluated in prostate cancer cells and tumor tissues using Western blotting analysis. RESULTS: Dietary feeding of 0.3% and 0.6% KRE to TRAMP mice from ages of 6 weeks to 12 weeks inhibited HG-PIN by 43.5% and 59.7%, respectively, and prostate adenocarcinoma by 53.5% and 66.4%, respectively. In addition, 0.6% KRE fed TRAMP mice from ages of 6 weeks to 24 weeks exhibited a significant reduction of genitourinary weight (a surrogate of tumor burden) by 54.5% and reduced body weight gain. Furthermore, the KRE and kavalactones showed a significant inhibition of LSD1 and MAO-A enzyme activities. CONCLUSION: Our results suggest that consumption of kava products through diet can delay prostate cancer development and progression and that kavalactones may be a new structure model for developing a potent dual inhibitor of LSD1 and MAO-A.

Development and validation of an immune gene-set based prognostic signature for soft tissue sarcoma.

BACKGROUND: Sarcomas is a group of heterogeneous malignant tumors originated from mesenchymal tissue and different types of sarcomas have disparate outcomes. The present study aims to identify the prognostic value of immune-related genes (IRGs) in sarcoma and establish a prognostic signature based on IRGs. METHODS: We collected the expression profile and clinical information of 255 soft tissue sarcoma samples from The Cancer Genome Atlas (TCGA) database and 2498 IRGs from the ImmPort database. The LASSO algorithm and Cox regression analysis were used to identify the best candidate genes and construct a signature. The prognostic ability of the signature was evaluated by ROC curves and Kaplan-Meier survival curves and validated in an independent cohort. Besides, a nomogram based on the IRGs and independent prognostic clinical variables was developed. RESULTS: A total of 19 IRGs were incorporated into the signature. In the training cohort, the AUC values of signature at 1-, 2-, and 3-years were 0.938, 0.937 and 0.935, respectively. The Kaplan-Meier survival curve indicated that high-risk patients were significantly worse prognosis (P < 0.001). In the validation cohort, the AUC values of signature at 1-, 2-, and 3-years were 0.730, 0.717 and 0.647, respectively. The Kaplan-Meier survival curve also showed significant distinct survival outcome between two risk groups. Furthermore, a nomogram based on the signature and four prognostic variables showed great accuracy in whole sarcoma patients and subgroup analyses. More importantly, the results of the TF regulatory network and immune infiltration analysis revealed the potential molecular mechanism of IRGs. CONCLUSIONS: In general, we identified and validated an IRG-based signature, which can be used as an independent prognostic signature in evaluating the prognosis of sarcoma patients and provide potential novel immunotherapy targets.

Genome-wide identification and characterization of cytokinin oxidase/dehydrogenase family genes in Medicago truncatula.

Cytokinin oxidase/dehydrogenases (CKXs) play a key role in the irreversible degradation of phytohormone cytokinin that is necessary for various plant growth and development processes. However, thus far, detailed investigations of the CKX gene family in the model legume Medicago truncatula are limited. In this study, we identified 9 putative CKX homologues with conserved FAD- and cytokinin-binding domains in the M. truncatula genome. We analyzed their phylogenetic relationship, gene structure, conserved domain, expression pattern, protein subcellular locations and other properties. The tissue-specific expression profiles of the MtCKX genes are different among different members and these MtCKXs also displayed different patterns in response to synthetic cytokinin 6-benzylaminopurine (6-BA) and indole-3-acetic acid (IAA), suggesting their diverse roles in M. truncatula development. To further understand the biological function of MtCKXs, we identified and characterized mutants of each MtCKX by taking advantage of the Tnt1 mutant population in M. truncatula. Results indicated that M. truncatula plants harboring Tnt1 insertions in each single MtCKX genes showed no morphological changes in aerial parts, suggesting functional redundancy of MtCKXs in M. truncatula shoot development. However, disruption of Medtr4g126160, which is predominantly expressed in roots, leads to an obvious reduced primary root length and increased lateral root number, indicating the specific roles of cytokinin in regulating root architecture. We systematically analyzed the MtCKX gene family at the genome-wide level and revealed their possible roles in M. truncatula shoot and root development, which shed lights on understanding the biological function of CKX family genes in related legume plants.

Slow slip source characterized by lithological and geometric heterogeneity.

Slow slip events (SSEs) accommodate a significant proportion of tectonic plate motion at subduction zones, yet little is known about the faults that actually host them. The shallow depth (<2 km) of well-documented SSEs at the Hikurangi subduction zone offshore New Zealand offers a unique opportunity to link geophysical imaging of the subduction zone with direct access to incoming material that represents the megathrust fault rocks hosting slow slip. Two recent International Ocean Discovery Program Expeditions sampled this incoming material before it is entrained immediately down-dip along the shallow plate interface. Drilling results, tied to regional seismic reflection images, reveal heterogeneous lithologies with highly variable physical properties entering the SSE source region. These observations suggest that SSEs and associated slow earthquake phenomena are promoted by lithological, mechanical, and frictional heterogeneity within the fault zone, enhanced by geometric complexity associated with subduction of rough crust.

ATM is a key driver of NF-κB-dependent DNA-damage-induced senescence, stem cell dysfunction and aging.

NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1-deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1-/- MEFs. Ercc1-/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenetor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1-/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus represents a therapeutic target to slow the progression of aging.

Elevated Expression of miR-629 Predicts a Poor Prognosis and Promotes Cell Proliferation, Migration, and Invasion of Osteosarcoma.

PURPOSE: Osteosarcoma (OS) is an invasive bone tumor that primarily affects children and adolescents. MicroRNA-629 (miR-629) acts as an oncogene involved in the development of various cancers. This study aims to reveal the clinical significance and biological function of miR-629 in OS. PATIENTS AND METHODS: The levels of miR-629 expression in tissues and cells were detected through quantitative real-time polymerase chain reaction (qRT-PCR). Chi-square test was used to evaluate the relationship between miR-621 expression and clinical parameters in patients with OS. Survival analysis was performed by the Kaplan-Meier method. Cox regression analysis of the effect of miR-629 expression on the prognosis of OS patients. CCK-8 and Transwell experiments were used to demonstrate the effect of miR-629 on OS cell function. RESULTS: Compared with the controls, miR-629 levels were significantly elevated in patients with OS (P < 0.001), Furthermore, miR-629 upregulation showed significantly associated with clinical stage (P = 0.011), distant metastasis (P = 0.003) and poor survival (log rank test, P = 0.013) in OS patients. miR-629 might be a potential prognostic biomarker for OS (HR = 2.890, 95% CI = 1.126-7.416, P = 0.027). Cell function experiments proved that the high expression of miR-629 promoted cell proliferation, migration, and invasion of OS. CONCLUSION: All experimental results demonstrated that miR-629 as an oncogene promotes the tumor cell growth, migration and invasion of OS, and miR-629 may act as a novel prognostic biomarker and therapeutic target for patients with this malignant tumor.

The emerging role of cellular senescence in renal diseases.

Cellular senescence represents the state of irreversible cell cycle arrest during cell division. Cellular senescence not only plays a role in diverse biological events such as embryogenesis, tissue regeneration and repair, ageing and tumour occurrence prevention, but it is also involved in many cardiovascular, renal and liver diseases through the senescence-associated secretory phenotype (SASP). This review summarizes the molecular mechanisms underlying cellular senescence and its possible effects on a variety of renal diseases. We will also discuss the therapeutic approaches based on the regulation of senescent and SASP blockade, which is considered as a promising strategy for the management of renal diseases.

miR-342-5p inhibits expression of Bmp7 to regulate proliferation, differentiation and migration of osteoblasts.

BACKGROUND: Fracture healing is a complex process, and patients with fracture will undergo non-union or compromised regeneration. MicroRNA (miR)-342-5p is a Notch downstream molecule, and its roles in fracture healing remain unclear. We aimed to explore the functional roles of miR-342-5p in osteoblasts as well as the underlying mechanisms. METHODS: The expression of miR-342-5p in differentiation of MC3T3-E1 cells or hMSCs was examined by quantitative reverse transcription PCR (qRT-PCR). The effects of aberrantly expressed miR-342-5p on cell proliferation, apoptosis, migration, and expressions of proteins associated with proliferation and osteogenic differentiation were determined by Cell Counting Kit-8, trypan blue staining, flow cytometry, Transwell assay, Western blot and qRT-PCR assays, respectively. The downstream factor and the target genes of miR-342-5p as well as the involvements of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were finally assessed. RESULTS: miR-342-5p level was decreased during differentiation of MC3T3-E1 cells or hMSCs. After cell transfection, miR-342-5p overexpression significantly reduced cell viability, induced apoptosis, inhibited proliferation, migration and differentiation, and down-regulated Bmp7 expression. Subsequent experiments showed the effects of miR-342-5p inhibition on MC3T3-E1 cells were abrogated by Bmp7 knockdown. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. Finally, phosphorylated levels of MEK and ERK were increased by miR-342-5p inhibition via up-regulating Bmp7 expression. CONCLUSION: miR-342-5p inhibition promoted proliferation, migration and differentiation of osteoblasts via regulating Bmp7, along with activation of the MEK/ERK pathway.

Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation.

BACKGROUND: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. METHODS: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. RESULTS: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. CONCLUSION: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

TNFAIP3 is required for FGFR1 activation-promoted proliferation and tumorigenesis of premalignant DCIS.COM human mammary epithelial cells.

BACKGROUND: Although ductal carcinoma in situ (DCIS) is a non-invasive breast cancer, many DCIS lesions may progress to invasive cancer and the genes and pathways responsible for its progression are largely unknown. FGFR1 plays an important role in cell proliferation, differentiation and carcinogenesis. The purpose of this study is to examine the roles of FGFR1 signaling in gene expression, cell proliferation, tumor growth and progression in a non-invasive DCIS model. METHODS: DCIS.COM cells were transfected with an empty vector to generate DCIS-Ctrl cells. DCIS-iFGFR1 cells were transfected with an AP20187-inducible iFGFR1 vector to generate DCIS-iFGFR1 cells. iFGFR1 consists of the v-Src myristoylation membrane-targeting sequence, FGFR1 cytoplasmic domain and the AP20187-inducible FKBP12 dimerization domain, which simulates FGFR1 signaling. The CRISPR/Cas9 system was employed to knockout ERK1, ERK2 or TNFAIP3 in DCIS-iFGFR1 cells. Established cell lines were treated with/without AP20187 and with/without FGFR1, MEK, or ERK1/2 inhibitor. The effects of these treatments were determined by Western blot, RNA-Seq, real-time RT-PCR, cell proliferation, mammosphere growth, xenograft tumor growth, and tumor histopathological assays. RESULTS: Activation of iFGFR1 signaling in DCIS-iFGFR1 cells enhanced ERK1/2 activities, induced partial epithelial-to-mesenchymal transition (EMT) and increased cell proliferation. Activation of iFGFR1 signaling promoted DCIS growth and progression to invasive cancer derived from DCIS-iFGFR1 cells in mice. Activation of iFGFR1 signaling also altered expression levels of 946 genes involved in cell proliferation, migration, cancer pathways, and other molecular and cellular functions. TNFAIP3, a ubiquitin-editing enzyme, is upregulated by iFGFR1 signaling in a FGFR1 kinase activity and in an ERK2-dependent manner. Importantly, TNFAIP3 knockout not only inhibited the AP20187-induced proliferation and tumor growth of DCIS-iFGFR1 cells, but also further reduced baseline proliferation and tumor growth of DCIS-iFGFR1 cells without AP20187 treatment. CONCLUSIONS: Activation of iFGFR1 promotes ERK1/2 activity, EMT, cell proliferation, tumor growth, DCIS progression to invasive cancer, and altered the gene expression profile of DCIS-iFGFR1 cells. Activation of iFGFR1 upregulated TNFAIP3 in an ERK2-dependent manner and TNFAIP3 is required for iFGFR1 activation-promoted DCIS.COM cell proliferation, mammosphere growth, tumor growth and progression. These results suggest that TNFAIP3 may be a potential target for inhibiting DCIS growth and progression promoted by FGFR1 signaling.

Dysregulation of DAF-16/FOXO3A-mediated stress responses accelerates oxidative DNA damage induced aging.

DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF-16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation corresponded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep-1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself.

Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.

Lateral heterogeneity of soil physicochemical properties in riparian zones after agricultural abandonment.

The study aimed to identify the lateral heterogeneity of soil physicochemical properties in riparian zones, and its underlying drivers during natural restoration after agricultural abandonment. Abandoned farmlands, after 5-year natural restoration, within 500 m from the edges on both sides of Liaohe River were selected as the study area. Soil physicochemical properties of four lateral buffers (<10 m, 10~100 m, 100~300 m, and >300 m from river edge, respectively) along riparian zones were measured. The results showed that riparian soils were characterized by high sand content (78.88%~96.52%) and poor soil nutrients. Soil silt content, organic carbon (OC), cation exchange capacity (CEC), total nitrogen (TN), and available nitrogen (AN) increased laterally with increasing distance from river edge, while soil sand content decreased. Total phosphorus (TP) and available phosphorus (AP) are not spatially autocorrelated. Soil OC, TN, AN, and CEC along upstream and midstream reaches showed negative spatial autocorrelation along the lateral gradients, and positive along downstream reach. Altitude, distance from river edge and distance from nearest farmland were the pronounced factors affecting soil physicochemical properties in this study.

Identification of HSP90 inhibitors as a novel class of senolytics.

Aging is the main risk factor for many chronic degenerative diseases and cancer. Increased senescent cell burden in various tissues is a major contributor to aging and age-related diseases. Recently, a new class of drugs termed senolytics were demonstrated to extending healthspan, reducing frailty and improving stem cell function in multiple murine models of aging. To identify novel and more optimal senotherapeutic drugs and combinations, we established a senescence associated ß-galactosidase assay as a screening platform to rapidly identify drugs that specifically affect senescent cells. We used primary Ercc1 -/- murine embryonic fibroblasts with reduced DNA repair capacity, which senesce rapidly if grown at atmospheric oxygen. This platform was used to screen a small library of compounds that regulate autophagy, identifying two inhibitors of the HSP90 chaperone family as having significant senolytic activity in mouse and human cells. Treatment of Ercc1 -/∆ mice, a mouse model of a human progeroid syndrome, with the HSP90 inhibitor 17-DMAG extended healthspan, delayed the onset of several age-related symptoms and reduced p16INK4a expression. These results demonstrate the utility of our screening platform to identify senotherapeutic agents as well as identified HSP90 inhibitors as a promising new class of senolytic drugs.The accumulation of senescent cells is thought to contribute to the age-associated decline in tissue function. Here, the authors identify HSP90 inhibitors as a new class of senolytic compounds in an in vitro screening and show that administration of a HSP90 inhibitor reduces age-related symptoms in progeroid mice.

High Sensitivity of an Ha-RAS Transgenic Model of Superficial Bladder Cancer to Metformin Is Associated with ∼240-Fold Higher Drug Concentration in Urine than Serum.

While pharmacoepidemiologic and laboratory studies have supported the hypothesis that the antidiabetic drug metformin may be useful in treating or preventing cancer, there is limited evidence to suggest which specific cancer sites may be particularly sensitive. Sensitivity likely is determined both by features of tumor pathophysiology and by pharmacokinetic factors. We used UPII-mutant Ha-ras transgenic mice that develop hyperplasia and low-grade, papillary urothelial cell carcinoma to determine whether metformin has activity in a model of superficial bladder cancer. Metformin significantly improved survival, reduced urinary tract obstruction, reduced bladder weight (a surrogate for tumor volume), and led to clear activation of AMP α kinase and inhibition of mTOR signaling in neoplastic tissue. We investigated the basis of the unusual sensitivity of this model to metformin, and observed that following oral dosing, urothelium is exposed to drug concentrations via the urine that are approximately 240-fold higher than those in the circulation. In addition, we observed that bladder cancer cell lines (RT4, UMUC-3, and J82) with homozygous deletion of either TSC1 or PTEN are more sensitive to metformin than those (TEU2, TCCSUP, and HT1376) with wild-type TSC1 and PTEN genes. Our findings provide a strong rationale for clinical trials of oral metformin in treatment of superficial bladder cancer.

Flavokawain A induces deNEDDylation and Skp2 degradation leading to inhibition of tumorigenesis and cancer progression in the TRAMP transgenic mouse model.

S phase kinase-associated protein 2 (Skp2) has been shown to be required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb) deficient mice. Here we have demonstrated that flavokawain A (FKA), a novel chalcone from the kava plant, selectively inhibited the growth of pRb deficient cell lines and resulted in a proteasome-dependent and ubiquitination-mediated Skp2 degradation. Degradation of Skp2 by FKA was found to be involved in a functional Cullin1, but independent of Cdh1 expression. Further studies have demonstrated that FKA docked into the ATP binding pocket of the precursor cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) complex, inhibited NEDD8 conjugations to both Cullin1 and Ubc12 in PC3 cells and Ubc12 NEDDylation in an in vitro assay. Finally, dietary feeding of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with FKA inhibited the formation of high-grade prostatic intra-epithelial neoplasia lesions (HG-PIN) and prostate adenocarcinomas, reduced the tumor burden and completely abolished distant organ metastasis. Immunohistochemistry studies revealed that dietary FKA feeding resulted in marked anti-proliferative and apoptotic effects via down-regulation of Skp2 and NEDD8 and up-regulation of p27/Kip1 in the prostate of TRAMP mice. Our findings therefore provide evidence that FKA is a promising NEDDylation inhibitor for targeting Skp2 degradation in prostate cancer prevention and treatment.