Akt-mTOR Signalling Research Articles

Functional activation of the AKT-mTOR signalling axis in a real-world metastatic breast cancer cohort.

Mutations of the PIK3CA/AKT/mTOR axis are common events in metastatic breast cancers (MBCs). This study was designed to evaluate the extent to which genetic alterations of the PIK3CA/AKT/mTOR can predict protein activation of this signalling axis in MBCs. Molecular profiles were generated by CLIA-certified laboratories from a real-world evidence cohort of 171 MBC patients. Genetic alterations of the PIK3CA pathway were measured using next-generation sequencing. Activation levels of AKT and downstream signalling molecules were quantified using two orthogonal proteomic methods. Protein activity was correlated with underlying genomic profiles and response to CDK4/6 inhibition in combination with endocrine treatment (ET). Oncogenic alterations of the PIK3CA/AKT/PTEN pathway were identified in 49.7% of cases. Genomic profiles emerged as poor predictors of protein activity (AUC:0.69), and AKT phosphorylation levels mimicked those of mutant lesions in 76.9% of wild-type tumours. High phosphorylation levels of the PI3K/AKT/mTOR downstream target p70S6 Kinase (T389) were associated with shorter PFS in patients treated with CDK4/6 inhibitors in combination with ET (HR:4.18 95%CI:1.19-14.63); this association was not seen when patients were classified by mutational status. Phosphoprotein-based measurements of drug targets and downstream substrates should be captured along with genomic information to identify MBCs driven by the PI3K/AKT/mTOR signalling.

NRP1 downregulation correlates with enhanced ILC2 responses during IL-33 challenge.

Group 2 innate lymphoid cells (ILC2s) play critical roles in driving the pathogenesis of allergic airway inflammation. The mechanisms underlying the regulation of ILC2s remain to be fully understood. Here, we identified neuropilin-1 (NRP1) as a surface marker of ILC2s in response to IL-33 stimulation. NRP1 was abundantly expressed in ILC2s from lung under steady state, which was significantly reduced upon IL-33 stimulation. ILC2s with high expression of NRP1 (NRP1high) displayed lower response to IL-33, as compared with NRP1low ILC2s. Transcriptional profiling and flow cytometric analysis showed that downregulation of AKT-mTOR signalling participated in the diminished functionality of NRP1high ILC2s. These observations revealed a potential role of NRP1 in ILC2s responses under allergic inflammatory condition.

Ras drives malignancy through stem cell crosstalk with the microenvironment

Squamous cell carcinomas are triggered by marked elevation of RAS–MAPK signalling and progression from benign papilloma to invasive malignancy1–4. At tumour–stromal interfaces, a subset of tumour-initiating progenitors, the cancer stem cells, obtain increased resistance to chemotherapy and immunotherapy along this pathway5,6. The distribution and changes in cancer stem cells during progression from a benign state to invasive squamous cell carcinoma remain unclear. Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression. The non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways that are often mutated in advanced metastatic squamous cell carcinomas with high mutational burden7. Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGFβ signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)–AKT–mTOR signalling during the benign-to-malignant transition. By functionally examining each step in this pathway, we reveal how dynamic temporal crosstalk with the microenvironment orchestrated by the stem cells profoundly fuels this path to malignancy. These insights suggest broad implications for cancer therapeutics.

Interaction of SERINC5 and IFITM1/2/3 regulates the autophagy-apoptosis-immune network under CSFV infection

ABSTRACT The host restriction factor serine incorporator 5 (SERINC5) plays a key role in inhibiting viral activity and has been shown to inhibit classical swine fever virus (CSFV) infection. However, the action of SERINC5 in the interaction between host cells and CSFV remains poorly understood. This study found that SERINC5 represses CSFV-induced autophagy through MAPK1/3-mTOR and AKT-mTOR signalling pathways. Further research showed that SERINC5 promotes apoptosis by repressing autophagy. Likewise, it was demonstrated that SERINC5 interacting proteins IFITM1/2/3 inhibit CSFV replication and regulate autophagy in a lysosomal-associated membrane protein LAMP1-dependent manner. In addition, IFITM1/2/3 interference promotes the NF-κB signalling pathway for potential immunoregulation by inhibiting autophagy. Finally, the functional silencing of IFITM1/2/3 genes was demonstrated to enhance the inhibitory effect of SERINC5 on autophagy. Taken together, These data uncover a novel mechanism through SERINC5 and its interacting proteins IFITM1/2/3, which mediates CSFV replication, and provides new avenues for controlling CSFV.

Iron controls T helper cell pathogenicity by promoting glucose metabolism in autoimmune myopathy.

BackgroundT helper cells in patients with autoimmune disease of idiopathic inflammatory myopathies (IIM) are characterized with the proinflammatory phenotypes. The underlying mechanisms remain unknown.MethodsRNA sequencing was performed for differential expression genes. Gene expression in CD4+ T‐cells was confirmed by quantitative real‐time PCR. CD4+ T‐cells from IIM patients or healthy controls were evaluated for metabolic activities by Seahorse assay. Glucose uptake, T‐cell proliferation and differentiation were evaluated and measured by flow cytometry. Human CD4+ T‐cells treated with iron chelators or Pfkfb4 siRNA were measured for glucose metabolism, proliferation and differentiation. Signalling pathway activation was evaluated by western blot and flow cytometry. Mouse model of experimental autoimmune myositis (EAM) were induced and treated with iron chelator or rapamycin. CD4+ T‐cell differentiation and muscle inflammation in the EAM mice were evaluated.ResultsRNA‐sequencing analysis revealed that iron was involved with glucose metabolism and CD4+ T‐cell differentiation. IIM patient‐derived CD4+ T‐cells showed enhanced glycolysis and mitochondrial respiration, which was inhibited by iron chelation. CD4+ T‐cells from patients with IIM was proinflammatory and iron chelation suppressed the differentiation of interferon gamma (IFNγ)‐ and interleukin (IL)‐17A‐producing CD4+ T‐cells, which resulted in an increased percentage of regulatory T (Treg) cells. Mechanistically, iron promoted glucose metabolism by an upregulation of PFKFB4 through AKT‐mTOR signalling pathway. Notably, the knockdown of Pfkfb4 decreased glucose influx and thus suppressed the differentiation of IFNγ‐ and IL‐17A‐producing CD4+ T‐cells. In vivo, iron chelation inhibited mTOR signalling pathway and reduced PFKFB4 expression in CD4+ T‐cells, resulting in reduced proinflammatory IFNγ‐ and IL‐17A‐producing CD4+ T‐cells and increased Foxp3+ Treg cells, leading to ameliorated muscle inflammation.ConclusionsIron directs CD4+ T‐cells into a proinflammatory phenotype by enhancing glucose metabolism. Therapeutic targeting of iron metabolism should have the potential to normalize glucose metabolism in CD4+ T‐cells and reverse their proinflammatory phenotype in IIM.

PBRM1 deficiency oncogenic addiction is associated with activated AKT-mTOR signalling and aerobic glycolysis in clear cell renal cell carcinoma cells.

The PBRM1 (PB1) gene which encodes the specific subunit BAF180 of the PBAF SWI/SNF complex, is highly mutated (~ 40%) in clear cell renal cell carcinoma (ccRCC). However, its functions and impact on cell signalling are still not fully understood. Aerobic glycolysis, also known as the ‘Warburg Effect’, is a hallmark of cancer, whether PB1 is involved in this metabolic shift in clear cell renal cell carcinoma remains unclear. Here, with established stable knockdown PB1 cell lines, we performed functional assays to access the effects on 786‐O and SN12C cells. Based on the RNA‐seq data, we selected some genes encoding key glycolytic enzymes, including PFKP, ENO1, PKM and LDHA, and examined the expression levels. The AKT–mTOR signalling pathway activity and expression of HIF1α were also analysed. Our data demonstrate that PB1 deficiency promotes the proliferation, migration, Xenograft growth of 786‐O and SN12C cells. Notably, knockdown of PB1 activates AKT–mTOR signalling and increases the expression of key glycolytic enzymes at both mRNA and protein levels. Furthermore, we provide evidence that deficient PB1 and hypoxic conditions exert a synergistic effect on HIF 1α expression and lactate production. Thus, our study provides novel insights into the roles of tumour suppressor PB1 and suggests that the AKT–mTOR signalling pathway, as well as glycolysis, is a potential drug target for ccRCC patients with deficient PB1.

USP12 downregulation orchestrates a protumourigenic microenvironment and enhances lung tumour resistance to PD-1 blockade

Oncogenic activation of KRAS and its surrogates is essential for tumour cell proliferation and survival, as well as for the development of protumourigenic microenvironments. Here, we show that the deubiquitinase USP12 is commonly downregulated in the KrasG12D-driven mouse lung tumour and human non-small cell lung cancer owing to the activation of AKT-mTOR signalling. Downregulation of USP12 promotes lung tumour growth and fosters an immunosuppressive microenvironment with increased macrophage recruitment, hypervascularization, and reduced T cell activation. Mechanistically, USP12 downregulation creates a tumour-promoting secretome resulting from insufficient PPM1B deubiquitination that causes NF-κB hyperactivation in tumour cells. Furthermore, USP12 inhibition desensitizes mouse lung tumour cells to anti-PD-1 immunotherapy. Thus, our findings propose a critical component downstream of the oncogenic signalling pathways in the modulation of tumour-immune cell interactions and tumour response to immune checkpoint blockade therapy.

AIM2 in regulatory T cells restrains autoimmune diseases.

The inflammasome initiates innate defense and inflammatory response by activating caspase-1 and pyroptotic cell death in myeloid cells1,2. It is comprised of an innate immune receptor/sensor, pro-caspase-1, and a common adaptor molecule, ASC (apoptotic speck-containing protein with a CARD). Consistent with their pro-inflammatory function, caspase-1, ASC and NLRP3 exacerbate autoimmunity during experimental autoimmune encephalomyelitis (EAE) by enhancing IL-1β and IL-18 secretion in myeloid cells3–6. Here we reveal an unexpected function of a DNA-binding inflammasome receptor, AIM2 (Absent in Melanoma 2)7–10, in T regulatory cells (Tregs) to restrain two models of autoimmunity (experimental autoimmune encephalomyelitis and T cell-mediated colitis) by studying whole-body and Treg-specific Aim2–/– mice. AIM2 is highly expressed by human and mouse Tregs, with its expression induced by TGF-β and its promoter occupied by transcription factors associated with Tregs, including Runx1, Ets1, Bcl11b and CREB. RNA-seq, biochemical and metabolic analyses revealed that AIM2 attenuates Akt-phosphorylation, mTOR, c-Myc and glycolysis, but promotes lipid oxidative phosphorylation in Tregs. Mechanistically, AIM2 interacts with the RACK1/PP2A-phosphatase complex to restrain Akt-phosphorylation. Lineage tracing demonstrates that AIM2 promotes the stability of Tregs during inflammation. While AIM2 is generally accepted as an inflammasome effector in myeloid cells, this report reveals a T cell-intrinsic role of AIM2 in restraining autoimmunity by diminishing Akt-mTOR signaling and altering immune-metabolism to enhance Treg stability.

High mobility group box 1 regulates gastric cancer cell proliferation and migration via RAGE-mTOR/ERK feedback loop.

Gastric cancer (GC) is a common malignancy tumour in China. Despite various therapeutic approaches to improve the survival rate of GC patients, the effectiveness of currently available treatments remains unsatisfactory. High mobility group box 1 (HMGB1) is reported to play a role in tumour development. However, the molecular mechanisms involved in HMGB1-mediated regulation of proliferation and migration of GC cells remain unclear. In the present study, we demonstrated that HMGB1 is highly expressed in GC cells and tissue. In HGC-27 GC cells, HMGB1 overexpression or HMGB1 RNA interference both demonstrated that HMGB1 could promote GC cell proliferation and migration. Investigation of the underlying molecular mechanisms revealed that HMGB1 enhanced cyclins expression, induced epithelial-to-mesenchymal transition and matrix metalloproteinase (MMPs) expression and promoted RAGE expression as well as RAGE-mediated activation of Akt/mTOR/P70S6K and ERK/P90RSK/CREB signalling pathways. We also found that inhibition of ERK and mTOR using specific inhibitors reduced recombinant human HMGB1-induced RAGE expression, suggesting that the RAGE-mTOR/ERK positive feedback loop is involved in HMGB1-induced GC cell proliferation and migration. Our study highlights a novel mechanism by which HMGB1 promotes GC cell proliferation and migration via RAGE-mediated Akt-mTOR and ERK-CREB signalling pathways which also involves the RAGE-mTOR/ERK feedback loop. These findings indicate that HMGB1 is a potential therapeutic target for GC.

N-acetylcysteine protects neonatal mice from ventricular hypertrophy induced by maternal obesity in a sex-specific manner

BackgroundMaternal obesity induces adverse cardiac programming in offspring, and effective interventions are needed to prevent cardiovascular ill-health. Herein we hypothesized that exposure to maternal obesogenic diet-induced obesity in mice results in left ventricular remodelling and hypertrophy in early childhood, and that maternal N-acetylcysteine (NAC) treatment alleviates these effects in a sex-dependent manner. Methods and ResultsThe maternal obesity was induced in mice by the consumption of a Western diet accompanied by a 20 % sucrose solution. To determine the effect of NAC on the cardiac outcomes induced by maternal obesity, obese dams were continuously exposed to the obesogenic diet, with or without the oral NAC treatment during pregnancy. Left ventricular remodelling and hypertrophy occurred as early as 7 days after birth in the male offspring of obese dams (O-OB) compared with controls (O-CO). An over-expression of key genes and markers related to cardiac fibrosis accompanied by more disorganized myofibrils was observed in the hearts of neonatal male O-OB mice. When we next evaluated the level of oxidative stress in the hearts of neonatal mice, the activity of enzymatic antioxidants declined and expression of NOX enzyme complex was up-regulated in O-OB offspring hearts, but was normal in the offspring of NAC treated mice (O-OB/NAC). Maternal obesity also activated cardiac Akt and mammalian target of rapamycin (mTOR) signalling in offspring, and NAC treatment restored offspring cardiac Akt-mTOR signalling to normal irrespective of sex. NAC treatment did not prevent cardiomyocyte hypertrophy but did alleviate increased heart weight, interventricular septal thickness, and collagen content in male O-OB/NAC pups. ConclusionsCollectively, our results indicated that NAC blunted cardiac fibrosis and related ventricular hypertrophy of male neonatal offspring in the setting of maternal obesity, potentially acting by reducing oxidative stress. The present study provides a basis for investigating the role of NAC in nutrition-related cardiac programming.

Glucagon-like peptide 1 infusions overcome anabolic resistance to feeding in older human muscle.

BackgroundDespite its known insulin‐independent effects, glucagon‐like peptide‐1 (GLP‐1) role in muscle protein turnover has not been explored under fed‐state conditions or in the context of older age, when declines in insulin sensitivity and protein anabolism, as well as losses of muscle mass and function, occur.MethodsEight older‐aged men (71 ± 1 year, mean ± SEM) were studied in a crossover trial. Baseline measures were taken over 3 hr, prior to a 3 hr postprandial insulin (~30 mIU ml−1) and glucose (7–7.5 mM) clamp, alongside I.V. infusions of octreotide and Vamin 14 (±infusions of GLP‐1). Four muscle biopsies were taken, and muscle protein turnover was quantified via incorporation of 13C6 phenylalanine and arteriovenous balance kinetics, using mass spectrometry. Leg macro‐ and microvascular flow was assessed via ultrasound and anabolic signalling by immunoblotting. GLP‐1 and insulin were measured by ELISA.ResultsGLP‐1 augmented muscle protein synthesis (MPS; fasted: 0.058 ± 0.004% hr−1 vs. postprandial: 0.102 ± 0.005% hr−1, p < 0.01), in comparison with non‐GLP‐1 trials. Muscle protein breakdown (MPB) was reduced throughout clamp period, while net protein balance across the leg became positive in both groups. Total femoral leg blood flow was unchanged by the clamp; however, muscle microvascular blood flow (MBF) was significantly elevated in both groups, and to a significantly greater extent in the GLP‐1 group (MBF: 5 ± 2 vs. 1.9 ± 1 fold change +GLP‐1 and −GLP‐1, respectively, p < 0.01). Activation of the Akt‐mTOR signalling was similar across both trials.ConclusionGLP‐1 infusion markedly enhanced postprandial microvascular perfusion and further stimulated muscle protein metabolism, primarily through increased MPS, during a postprandial insulin hyperaminoacidaemic clamp.

Fasting-mimicking diet and hormone therapy induce breast cancer regression.

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models ofhormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of afasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels ofinsulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of afasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.

Tetramethylpyrazine ameliorates hepatic fibrosis through autophagy-mediated inflammation.

Imbalanced immune response and hepatic fibrosis are key factors related to the progression of chronic liver diseases. Tetramethylpyrazine (TMP), a natural alkaloid, has been widely used for treating liver injury. In this study, we explored the effect of TMP on hepatic fibrosis and the related mechanisms regulating autophagy. A rat model of hepatic fibrosis and a model using an hepatic stellate cell line (HSC-T6) were created using CCl4 and platelet-derived growth factor (PDGF). Staining with haematoxylin and eosin (HE), Masson's stain, and TUNEL were performed for pathological diagnosis. ELISA, Western blotting, and immunofluorescence analyses were conducted to determine the expression levels of the specific markers for fibrosis, autophagy, inflammation, and signalling pathways. TMP treatment significantly rescued pathological injury and hepatic fibrosis. It also alleviated imbalances in the immune system, accumulation of extracellular matrix, and autophagy signals in hepatic fibrosis. At the same time, we found that application of the autophagy inducer rapamycin enhanced the therapeutic effect of TMP, whereas the autophagy inhibitor 3-methyladenine, PI3K pathway inhibitor LY294002, and AKT pathway agonist SC79 did the opposite. TMP exerts therapeutic effects in hepatic fibrosis mainly through promoting autophagy to ameliorate inflammation by inhibiting the AKT-mTOR signalling pathway, providing a new perspective for the treatment of chronic liver diseases.

Aloin Inhibits the Proliferation and Migration of Gastric Cancer Cells by Regulating NOX2-ROS-Mediated Pro-Survival Signal Pathways.

BackgroundAloin has been reported to have many pharmacological effects including anti-inflammatory, anti-oxidant and anti-tumour activities. However, the precise molecular mechanisms underlying the anti-tumour properties of aloin are yet to be elucidated.MethodsHGC-27 and BGC-823 gastric cancer cells were treated with aloin. EdU and colony formation assays were used to detect the proliferation ability of cells. The migration of cells was detected using wound healing and transwell assays. Western blotting was used to detect the levels of cyclinD1, cyclin E1, MMPs, N-cadherin, E-cadherin and NOX2. The phosphorylation of Akt, mTOR, P70S6K, S6, Src, stat3 and IκBα were also detected by Western blotting. Flow cytometry was used to detect the cell cycle distribution.The location of p65 in cells was determined by using a confocal microscopy assay. The total amounts of ROS present in cells were measured using an ROS assay kit.ResultsHere, we found that aloin inhibited the proliferation and migration of HGC-27 and BGC-823 gastric cancer cells using a combination of EdU, colony formation, wound healing and transwell assays. Further investigations revealed that aloin decreased the protein expression levels of cyclin D1, N-cadherin, and the matrix metalloproteinases (MMP)-2 and MMP-9; increased E-cadherin expression in a dose-dependent manner; inhibited reactive oxygen species (ROS) generation; and mediated the activation of Akt-mTOR, signal transducer and activator of transcription-3 (Stat3), and NF-κB signalling pathways. Our results also indicated that aloin is able to attenuate the expression levels of the two regulatory proteins of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), p47phox and p22phox, but had no effect on the level of gp91phox. N-acetylcysteine treatment of gastric cancer cells inhibited ROS production and Akt-mTOR, Stat3, and IκBα phosphorylation. Taken together, our data suggest that aloin inhibits the proliferation and migration of gastric cancer cells by downregulating NOX2–ROS-mediated activation of the Akt-mTOR, Stat3, and NF-κB signalling pathways.ConclusionOur findings suggest a potential role for aloin in the prevention of gastric cancer cell proliferation and migration and provide novel insights into the anti-cancer properties of aloin.

WITHDRAWN: RNA binding protein QKI5 accelerates tenogenic differentiation and promotes tendon healing through AKT-mTOR signalling by stabilizing MALAT1

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Normalisation of circulating adiponectin levels in obese pregnant mice prevents cardiac dysfunction in adult offspring

Background/objectivesAdiponectin concentrations are low in obese pregnant women. Restoring normal adiponectin concentrations by infusion in obese pregnant mice prevents placental dysfunction, foetal overgrowth and metabolic syndrome in the offspring. We hypothesised that normalising maternal adiponectin in obese late pregnant dams prevents cardiac dysfunction in the adult offspring.Subjects/methodsPregnant female mice with diet-induced obesity were infused with adiponectin (0.62 μg g−1 day−1, n = 24) or saline (n = 22) over days 14.5–18.5 of pregnancy (term = day 19.5). Control dams ate standard chow and received saline (n = 22). Offspring were studied at 3 and 6 months of age.ResultsMaternal obesity impaired ventricular diastolic function, increased cardiomyocyte cross-sectional area and upregulated cardiac brain natriuretic peptide (Nppb) and α-skeletal actin (Acta1) gene expression in adult male offspring, compared to control offspring. In adult female offspring, maternal obesity increased Nppb expression, decreased end-diastolic volume and caused age-dependent diastolic dysfunction but not cardiomyocyte hypertrophy. Maternal obesity also activated cardiac Akt and mechanistic target of rapamycin (mTOR) signalling in male, but not in female, offspring and inhibited cardiac extracellular signal-regulated kinase 1/2 (ERK1/2) in both sexes. Normalising maternal circulating adiponectin concentrations by infusing obese dams with adiponectin prevented offspring diastolic dysfunction and ventricular dilation and normalised cardiac Akt-mTOR signalling irrespective of sex. Maternal adiponectin infusion also reduced cardiac Nppb expression and increased ERK1/2 signalling in offspring of obese dams. Adiponectin infusion did not prevent cardiomyocyte hypertrophy but reduced ventricular wall thickness in male offspring and increased collagen content in female offspring of obese dams, compared to controls.ConclusionsLow maternal adiponectin levels in obese mice in late pregnancy are mechanistically linked to in utero programming of cardiac dysfunction in their offspring. Interventions enhancing endogenous adiponectin secretion or signalling in obese pregnant women could prevent the development of cardiac dysfunction in their children.

Curcumin ameliorates PRMT5-MEP50 arginine methyltransferase expression by decreasing the Sp1 and NF-YA transcription factors in the A549 and MCF-7 cells.

The protein arginine methyltransferase 5 (PRMT5) and its catalytic partner methylosome protein MEP50 (WDR77) catalyse the mono- and symmetric di-methylation of selective arginines in various histones and non-histone target proteins. It has emerged as a crucial epigenetic regulator in cell proliferation and differentiation; which also reported to be overexpressed in many forms of cancers in humans. In this study, we aimed to assess the modulations in the expression of this enzyme upon exposure to the well-studied natural compound from the spice turmeric, curcumin. We exposed the lung and breast cancer cell lines(A549 and MCF-7) to curcumin (2 and 20μM) and observed a highly significant inhibitory effect on the expression of both PRMT5 and MEP50. The level of symmetrical dimethylarginine (SDMA) in multiple proteins, and more specifically, the H4R3me2s mark (which predominates in GC-rich motifs in nucleosomal DNA) was also diminished significantly. We also found that curcumin significantly reduced the level and enrichment of the transcription factors Sp1 and NF-YA which shares their binding sites within the GC-rich region of the PRMT5 proximal promoter. Furthermore, the involvement of both PKC-p38-ERK-cFos and AKT-mTOR signalling was observed in reducing the Sp1 and NF-YA expression by curcumin. Therefore, we propose curcumin decreased the expression of PRMT5 in these cells by affecting at least these two transcription factors. Altogether, we report a new molecular target of curcumin and further elucidation of this proposed mechanism through which curcumin affects the PRMT5-MEP50 methyltransferase expression might be explored for its therapeutic application.

Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes.

Klotho interacts with various membrane proteins, such as receptors for transforming growth factor (TGF)-β and insulin-like growth factor (IGF), to alter their function. Renal expression of klotho is diminished in diabetes. The present study examined whether exogenous klotho protein supplementation ameliorates kidney injury and renin-angiotensin system (RAS) in db/db mice. We investigated the effects of klotho supplementation on diabetic kidney injury and RAS. Recombinant human klotho protein (10μg/kg/d) was administered to db/db mice daily. Klotho protein supplementation reduced kidney weight, systolic blood pressure (SBP), albuminuria, glomerular filtration rate, and 8-epi-prostaglandin F2α excretion without affecting body weight. Although klotho supplementation did not alter glycated albumin, it reduced renal angiotensin II levels associated with reduced renal expression of angiotensinogen. Klotho supplementation improved renal expression of superoxide dismutase (SOD), and endogenous renal expression of klotho. Klotho supplementation reduced the levels of hypoxia-inducible factor, phosphorylated Akt, and phosphorylated mTOR and decreased the renal expression of TGF-β, tumour necrosis factor (TNF), and fibronectin. These data indicate that klotho supplementation reduces blood pressure and albuminuria along with ameliorating renal RAS activation in db/db mice. Furthermore, these results suggest that klotho inhibits IGF signalling, induces SOD expression to reduce oxidative stress, and suppresses Akt-mTOR signalling to inhibit abnormal kidney growth. Collectively, the results suggest that klotho inhibits TGF-β and TNF signalling, resulting in a decline in renal fibrosis.

Effect of Eukarion-134 on Akt-mTOR signalling in the rat soleus during 7days of mechanical unloading.

What is the central question of this study? Translocation of nNOSμ initiates catabolic signalling via FoxO3a and skeletal muscle atrophy during mechanical unloading. Recent evidence suggests that unloading-induced muscle atrophy and FoxO3a activation are redox sensitive. Will a mimetic of superoxide dismutase and catalase (i.e. Eukarion-134) also mitigate suppression of the Akt-mTOR pathway? What is the main finding and its importance? Eukarion-134 rescued Akt-mTOR signalling and sarcolemmal nNOSμ, which were linked to protection against the unloading phenotype, muscle fibre atrophy and partial fibre-type shift from slow to fast twitch. The loss of nNOSμ from the sarcolemma appears crucial to Akt phosphorylation and is redox sensitive, although the mechanisms remain unresolved. Mechanical unloading stimulates rapid changes in skeletal muscle morphology, characterized by atrophy of muscle fibre cross-sectional area and a partial fibre-type shift from slow to fast twitch. Recent studies revealed that oxidative stress contributes to activation of forkhead box O3a (FoxO3a), proteolytic signalling and unloading-induced muscle atrophy via translocation of the μ-splice variant of neuronal nitric oxide synthase (nNOSμ) and activation of FoxO3a. There is limited understanding of the role of reactive oxygen species in the Akt-mammalian target of rapamycin (mTOR) pathway signalling during unloading. We hypothesized that Eukarion-134 (EUK-134), a mimetic of the antioxidant enzymes superoxide dismutase and catalase, would protect Akt-mTOR signalling in the unloaded rat soleus. Male Fischer 344 rats were separated into the following three study groups: ambulatory control (n=11); 7days of hindlimb unloading+ saline injections (HU, n=11); or 7days of HU + EUK-134; (HU+EUK-134, n=9). EUK-134 mitigated unloading-induced dephosphorylation of Akt, as well as FoxO3a, in the soleus. Phosphorylation of mTOR in the EUK-treated HU rats was not different from that in control animals. However, EUK-134 did not significantly rescue p70S6K phosphorylation. EUK-134 attenuated translocation of nNOSμ from the membrane to the cytosol, reduced nitration of tyrosine residues and suppressed upregulation of caveolin-3 and dysferlin. EUK-134 ameliorated HU-induced remodelling, atrophy of muscle fibres and the 12% increase in type II myosin heavy chain-positive fibres. Attenuation of the unloaded muscle phenotype was associated with decreased reactive oxygen species, as assessed by ethidium-positive nuclei. We conclude that oxidative stress affects Akt-mTOR signalling in unloaded skeletal muscle. Direct linkage of abrogation of nNOSμ translocation with Akt-mTOR signalling during unloading is the subject of future investigation.

Dual \u201cmAb\u201d HER family blockade in head and neck cancer human cell lines combined with photon therapy

Head and neck cancer stem cells (CSCs) are highly resistant to treatment. When EGFR is overexpressed in head and neck squamous cell carcinoma (HNSCC), HER2 and HER3 are also expressed. The aim of the present study was to investigate the effect of HER1/2/3 blockade through a combination of cetuximab and pertuzumab, with or without photon irradiation, on the proliferation and migration/invasion capabilities of an HNSCC chemo- and radioresistant human cell line (SQ20B) and its corresponding stem cell subpopulation. Cell proliferation, migration and invasion were studied after treatment with cetuximab +/− pertuzumab +/− 10 Gy photon irradiation. EGFR, phospho-EGFR, HER2 and HER3 protein expression levels were studied. Activation or inhibition of the RAS/MAPK and AKT-mTOR downstream signalling cascades was investigated through phospho-AKT and phospho-MEK1/2 expression. Cetuximab strongly inhibited SQ20B and FaDu cell proliferation, migration and invasion, whereas it had little effect on SQ20B-CSCs. Cetuximab–pertuzumab combined with radiation significantly inhibited SQ20B and FaDu cell and SQ20B-CSC proliferation, migration and invasion. Cetuximab–pertuzumab with 10 Gy photon irradiation switched off both phospho-AKT and phospho-MEK1/2 expression in the three populations. The triple therapy is therefore thought to inhibit SQ20B cells, SQ20B-CSCs and FaDu cells through an AKT-mTOR and Ras-MAPK downstream signalling blockade.