S6 Protein Research Articles

Overexpressed p-S6 associates with lymph node metastasis and predicts poor prognosis in non-small cell lung cancer

BackgroundRibosomal protein S6 (S6), a downstream effect media of the AKT/mTOR pathway, not only is a part of 40S small subunit of eukaryotic ribosome, but also involves in protein synthesis and cell proliferation during cancer development.MethodsIn present study, we explore the association between phosphorylated S6 (p-S6) protein expression and clinicopathological features as well as prognostic implications in NSCLC. P-S6 was detected in tissue microarrays (TMAs) containing 350 NSCLC, 53 non-cancerous lung tissues (Non-CLT), and 88 cases of matched metastatic lymph node lesions via immunohistochemistry (IHC). Transwell assays and wound healing assay were used to assess the effects of p-S6 inhibition on NSCLC cell metastasis.ResultsThe p-S6 expression in NSCLC was more evident than that in Non-CLT (p < 0.05). Compared to NSCLC patients who have no lymph node metastasis (LNM), those with LNM had higher p-S6 expression (p = 0.001). Regardless of lung squamous cell carcinoma (SCC) or adenocarcinoma (ADC), p-S6 was increased obviously in metastatic lymph nodes compared with matched primary cancers (p = 0.001, p = 0.022, respectively). Inhibition of p-S6 decreased the metastasis ability of NSCLC cells. In addition, p-S6 was an independent predicted marker for LNM in patients with NSCLC (p < 0.001). According to survival analysis, patients with highly expressed p-S6 had a lower survival rate compared with that with lower expression (p = 0.013). P-S6 is an unfavorable independent prognostic factor for NSCLC patients (p = 0.011).ConclusionIncreased expression of p-S6 is not only a novel predictive biomarker of LNM but also poor prognosis in NSCLC.

Pilot study evaluating everolimus molecular mechanisms in tuberous sclerosis complex and focal cortical dysplasia.

Tuberous sclerosis complex (TSC) and some focal cortical dysplasias (FCDs) are associated with dysfunctional mTOR signaling, resulting in increased cell growth and ribosomal S6 protein phosphorylation (phospho-S6). mTOR inhibitors can reduce TSC tumor growth and seizure frequency, and preclinical FCD studies indicate seizure suppression. This pilot study evaluated safety of mTOR inhibitor everolimus in treatment resistant (failure of >2 anti-seizure medications) TSC and FCD patients undergoing surgical resection and to assess mTOR signaling and molecular pathways. We evaluated everolimus in 14 treatment resistant epilepsy patients undergoing surgical resection (4.5 mg/m2 daily for 7 days; n = 4 Active, mean age 18.3 years, range 4-26; n = 10, Control, mean age 13.1, range 3-45). Everolimus was well tolerated. Mean plasma everolimus in Active participants were in target range (12.4 ng/ml). Brain phospho-S6 was similar in Active and Control participants with a lower trend in Active participants, with Ser235/236 1.19-fold (p = 0.67) and Ser240/244 1.15-fold lower (p = 0.66). Histologically, Ser235/236 was 1.56-fold (p = 0.37) and Ser240/244 was 5.55-fold lower (p = 0.22). Brain proteomics identified 11 proteins at <15% false discovery rate associated with coagulation system (p = 1.45x10-9) and acute phase response (p = 1.23x10-6) activation. A weighted gene correlation network analysis (WGCNA) of brain proteomics and phospho-S6 identified 5 significant modules. Higher phospho-S6 correlated negatively with cellular respiration and synaptic transmission and positively with organophosphate metabolic process, nuclear mRNA catabolic process, and neuron ensheathment. Brain metabolomics identified 14 increased features in Active participants, including N-acetylaspartylglutamic acid. Plasma proteomics and cytokine analyses revealed no differences. Short-term everolimus before epilepsy surgery in TSC and FCD resulted in no adverse events and trending lower mTOR signaling (phospho-S6). Future studies should evaluate implications of our findings, including coagulation system activation and everolimus efficacy in FCD, in larger studies with long-term treatment to better understand molecular and clinical effects. ClinicalTrials.gov NCT02451696.

Calorie Restriction-Regulated Molecular Pathways and Its Impact on Various Age Groups: An Overview.

Calorie restriction (CR) if planned properly with regular exercise at different ages can result in healthy weight loss. CR can also have different beneficial effects on improving lifespan and decreasing the age-associated diseases by regulating physiological, biochemical, and molecular markers. The different pathways regulated by CR include:(1) AMP-activated protein kinase (AMPK), which involves PGC-1α, SIRT1, and SIRT3. AMPK also effects myocyte enhancer factor 2 (MEF2), peroxisome proliferator-activated receptor delta, and peroxisome proliferator-activated receptor alpha, which are involved in mitochondrial biogenesis and lipid oxidation; (2) Forkhead box transcription factor's signaling is related to the DNA repair, lipid metabolism, protection of protein structure, autophagy, and resistance to oxidative stress; (3) Mammalian target of rapamycin (mTOR) signaling, which involves key factors, such as S6 protein kinase-1 (S6K1), mTOR complex-1 (mTORC1), and 4E-binding protein (4E-BP). Under CR conditions, AMPK activation and mTOR inhibition helps in the activation of Ulk1 complex along with the acetyltransferase Mec-17, which is necessary for autophagy; (4) Insulin-like growth factor-1 (IGF-1) pathway downregulation protects against cancer and slows the aging process; (5) Nuclear factor kappa B pathway downregulation decreases the inflammation; and (6) c-Jun N-terminal kinase and p38 kinase regulation as a response to the stress. The acute and chronic CR both shows antidepression and anxiolytic action by effecting ghrelin/GHS-R1a signaling. CR also regulates GSK3β kinase and protects against age-related brain atrophy. CR at young age may show many deleterious effects by effecting different mechanisms. Parental CR before or during conception will also affect the health and development of the offspring by causing many epigenetic modifications that show transgenerational transmission. Maternal CR is associated with intrauterine growth retardation effecting the offspring in their adulthood by developing different metabolic syndromes. The epigenetic changes with response to paternal food supply also linked to offspring health. CR at middle and old age provides a significant preventive impact against the development of age-associated diseases.

Effects of maternal branched-chain amino acid and alanine supplementation on growth and biomarkers of protein metabolism in dams fed a low-protein diet and their offspring.

A considerable number of studies have reported that maternal protein restriction may disturb fetal growth and organ development due to a lower availability of amino acids. Leucine, one of branched-chain amino acid (BCAA) promotes protein synthesis through mechanistic target of rapamycin signaling. Here, we investigated the effects of BCAA supplementation in the dams fed a low-protein diet on serum and hepatic biochemical parameters of protein metabolism of dams and their offspring. Female ICR mice were fed a control (20% casein), a low-protein (10% casein), a low-protein with 2% BCAAs or a low-protein with 2% alanine diet for 2 weeks before mating and then throughout pregnancy and lactation. Alanine was used as an amino nitrogen control for the BCAA. Dams and their male offspring were sacrificed at postnatal day 21. There were no changes in body weight and fat mass in low-protein fed dams; however, BCAA supplementation significantly increased fat mass and serum leptin levels. Low-protein diet consumption reduced maternal protein synthesis based on biochemical analysis of serum albumin and hepatic protein levels and immunoblotting of S6 protein, which were increased by BCAA and alanine supplementation. Offspring from dams fed a low-protein diet exhibited lower body and organ weights. Body weight and hepatic protein levels of the offspring were increased by alanine supplementation. However, the decreased serum biochemical parameters, including glucose, triglyceride, total protein and albumin levels in the low-protein offspring group were not changed in response to BCAA or alanine supplementation. A reduced density of the hepatic vessel system in the offspring from dams fed a low-protein diet was restored in the offspring from dams fed either BCAA and alanine-supplemented diet. These results suggest that supplementation of amino nitrogen per se may be responsible for inducing hepatic protein synthesis in the dams fed a low-protein diet and alleviating the distorted growth and liver development of their offspring.

Excessive Aurantiochytrium acetophilum docosahexaenoic acid supplementation decreases growth performance and breast muscle mass of broiler chickens

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid with health-promoting potential. This study was to investigate effects of supplemental DHA from Aurantiochytrium acetophilum on growth performance, health status, meat quality, and protein synthesis signaling of broiler chickens. Day-old male chicks were housed in an environmental control room (6 cages/treatment, 8 chicks/cage), and fed a corn-soybean meal basal diet supplemented with the DHA-rich A. acetophilum biomass (Heliae, Gilbert, AZ) at 0, 1, 2, and 4% (0, 1.7, 3.4 and 6.8 g DHA/kg diet) for 6 weeks. Growth performance was measured weekly. Blood samples were collected at weeks 3 and 6 (2 chicks/cage). Four tissues were sampled (2 chicks/cage) for biochemical and meat quality analyses. Data were analyzed by one-way ANOVA and regression. Compared with the control, the 4% A. acetophilum diet decreased (p < 0.05) body weight gain (19%) and gain to feed ratio (19%) during weeks 4–6. The A. acetophilum supplementation dose-dependently decreased (p < 0.05, R2 = 0.21–0.54) plasma alanine amino transferase activity and glucose concentrations, but had little effect on plasma activity of alkaline phosphatase or concentrations of inorganic phosphorus and uric acid at weeks 3 and 6. Compared with the control, the 4% A. acetophilum diet decreased (p < 0.05) breast muscle weight by 21%, and down-regulated (p < 0.05) mRNA levels of mammalian target of rapamycin and ribosomal s6 protein (S6), and protein levels of phosphorylated S6 to S6 and phosphorylated S6 kinase beta 1 to S6 kinase beta 1. The A. acetophilum supplementation linearly increased (p < 0.01) lipid peroxidation (R2 = 0.62–0.90) and hardness and chewiness (R2 = 0.34–0.44) of breast and thigh muscles. In conclusion, supplemental 4% (6.8 g DHA/kg), but not 1 or 2%, of A. acetophilum impaired growth performance, breast muscle mass accumulation, and(or) protein synthesis signaling of broilers.

Pulsed EMF stimulation increased BDNF and activated S6 levels in the hippocampus of senescent rats

Abstract Purpose Low-frequency electromagnetic field (EMF) exposure in rat has positive effects on neuronal processes in vitro. Moreover, EMF improves learning-memory and psychomotor activity during advanced ageing, but the underlying molecular mechanisms are not known in the brain. In the present study we aimed to investigate the molecular effects of chronic EMF stimulation in the hippocampus of senescent rats in vivo. Materials/Methods Thirty months old rats were treated for six weeks with different EMF doses of 45, 95, and 1,250 µT. After sacrifice the levels of Brain Derived Neurotrophic Factor (BDNF) and activated ribosomal protein S6 as measures for protein synthesis intensity in the hippocampus were determined by Western blot analysis. Results The results showed that chronic EMF exposure dose dependently increased BDNF and the amount of phosphorylated S6 protein at the highest dose. The effects on the two proteins positively correlated at individual level. The results indicate that EMF exposure may enhance neurotrophic processes indicated by increased BDNF expression in the hippocampus of senescent rats. Increased phosphorylated S6 protein suggests coupling to support molecular regulation of protein synthesis. Conclusions In a broader perspective, these findings may support EMF as a beneficial alternative form of passive exercise in active, exercise-limited, aged individuals.

NPRL3 loss alters neuronal morphology, mTOR localization, cortical lamination and seizure threshold.

Mutations in nitrogen permease regulator-like 3 (NPRL3), a component of the GATOR1 complex within the mTOR pathway, are associated with epilepsy and malformations of cortical development. Little is known about the effects of NPRL3 loss on neuronal mTOR signalling and morphology, or cerebral cortical development and seizure susceptibility. We report the clinical phenotypic spectrum of a founder NPRL3 pedigree (c.349delG, p.Glu117LysFS; n = 133) among Old Order Mennonites dating to 1727. Next, as a strategy to define the role of NPRL3 in cortical development, CRISPR/Cas9 Nprl3 knockout in Neuro2a cells in vitro and in foetal mouse brain in vivo was used to assess the effects of Nprl3 knockout on mTOR activation, subcellular mTOR localization, nutrient signalling, cell morphology and aggregation, cerebral cortical cytoarchitecture and network integrity. The NPRL3 pedigree exhibited an epilepsy penetrance of 28% and heterogeneous clinical phenotypes with a range of epilepsy semiologies, i.e. focal or generalized onset, brain imaging abnormalities, i.e. polymicrogyria, focal cortical dysplasia or normal imaging, and EEG findings, e.g. focal, multi-focal or generalized spikes, focal or generalized slowing. Whole exome analysis comparing a seizure-free group (n = 37) to those with epilepsy (n = 24) to search for gene modifiers for epilepsy did not identify a unique genetic modifier that explained the variability in seizure penetrance in this cohort. Nprl3 knockout in vitro caused mTOR pathway hyperactivation, cell soma enlargement and the formation of cellular aggregates seen in time-lapse videos that were prevented with the mTOR inhibitors rapamycin or torin1. In Nprl3 knockout cells, mTOR remained localized on the lysosome in a constitutively active conformation, as evidenced by phosphorylation of ribosomal S6 and 4E-BP1 proteins, even under nutrient starvation (amino acid-free) conditions, demonstrating that Nprl3 loss decouples mTOR activation from neuronal metabolic state. To model human malformations of cortical development associated with NPRL3 variants, we created a focal Nprl3 knockout in foetal mouse cortex by in utero electroporation and found altered cortical lamination and white matter heterotopic neurons, effects which were prevented with rapamycin treatment. EEG recordings showed network hyperexcitability and reduced seizure threshold to pentylenetetrazol treatment. NPRL3 variants are linked to a highly variable clinical phenotype which we propose results from mTOR-dependent effects on cell structure, cortical development and network organization.

Rapamycin Attenuated Zinc-Induced Tau Phosphorylation and Oxidative Stress in Rats: Involvement of Dual mTOR/p70S6K and Nrf2/HO-1 Pathways.

Alzheimer’s disease is pathologically characterized by abnormal accumulation of amyloid-beta plaques, neurofibrillary tangles, oxidative stress, neuroinflammation, and neurodegeneration. Metal dysregulation, including excessive zinc released by presynaptic neurons, plays an important role in tau pathology and oxidase activation. The activities of mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (p70S6K) are elevated in the brains of patients with Alzheimer’s disease. Zinc induces tau hyperphosphorylation via mTOR/P70S6K activation in vitro. However, the involvement of the mTOR/P70S6K pathway in zinc-induced oxidative stress, tau degeneration, and synaptic and cognitive impairment has not been fully elucidated in vivo. Here, we assessed the effect of pathological zinc concentrations in SH-SY5Y cells by using biochemical assays and immunofluorescence staining. Rats (n = 18, male) were laterally ventricularly injected with zinc, treated with rapamycin (intraperitoneal injection) for 1 week, and assessed using the Morris water maze. Evaluation of oxidative stress, tau phosphorylation, and synaptic impairment was performed using the hippocampal tissue of the rats by biochemical assays and immunofluorescence staining. The results from the Morris water maze showed that the capacity of spatial memory was impaired in zinc-treated rats. Zinc sulfate significantly increased the levels of P-mTOR Ser2448, P-p70S6K Thr389, and P-tau Ser356 and decreased the levels of nuclear factor erythroid 2-related factor-2 (Nrf2) and heme oxygenase-1 (HO-1) in SH-SY5Y cells and in zinc-treated rats compared with the control groups. Increased expression of reactive oxygen species was observed in zinc sulfate-induced SH-SY5Y cells and in the hippocampus of zinc-injected rats. Rapamycin, an inhibitor of mTOR, rescued zinc-induced increases in mTOR/p70S6K activation, tau phosphorylation, and oxidative stress, and Nrf2/HO-1 inactivation, cognitive impairment, and synaptic impairment reduced the expression of synapse-related proteins in zinc-injected rats. In conclusion, our findings imply that rapamycin prevents zinc-induced cognitive impairment and protects neurons from tau pathology, oxidative stress, and synaptic impairment by decreasing mTOR/p70S6K hyperactivity and increasing Nrf2/HO-1 activity.

Effects of replacing fish meal with corn gluten meal on growth performance, intestinal microbiota, mTOR pathway and immune response of abalone Haliotis discus hannai

Corn gluten meal (CGM) is used as a protein source in aquafeeds. However, the potential for replacing fish meal (FM) with CGM in abalone feeds has not been adequately evaluated. A 110-day feeding trial was conducted to evaluate effects of replacing FM with CGM on abalone Haliotis discus hannai (initial weight: 21.83 ± 0.25 g). The control diet (CGM0) was designed to contain 20% of FM. The other four isonitrogenous (about 30% of crude protein) and isolipidic (about 4% of crude lipid) experimental diets were formulated with CGM protein replacing 25%, 50%, 75% and 100% of dietary FM protein, respectively. The five experimental diets were named as CGM0, CGM25, CGM50, CGM75 and CGM100, respectively. Results showed that no significant differences were found in survival rate among all the groups. Weight gain rate (WGR) of abalone in the CGM100 group was significantly lower than that in the control group. There were no significant differences in WGR among the CGM25, CGM50, CGM75 and the control groups. The increasing dietary CGM inclusion level elevated the operational taxonomic units, ACE estimator and Chao1 estimator, and changed the intestinal microbiota composition in abalone. The activities of trypsin, alanine transaminase, aspartate transaminase and Na+, K+-ATPase in digestive gland were significantly decreased when replacement level over 50%. The increasing dietary CGM inclusion level significantly downregulated the expressions of ribosomal S6 protein kinase (s6k), mammalian target of rapamycin (mtor) and eukaryotic translation initiation factor 4E (eif4e) in digestive gland. The CGM75 and CGM100 groups had significant lower activities of catalase and lysozyme in cell-free hemolymph and digestive gland. In conclusion, CGM could replace 75% of dietary FM (equivalent to 16.2% of CGM inclusion level in the diet) without significantly affecting the growth of abalone. However, high inclusion levels of CGM in diets (> 10.8%) had negative effects on intestinal microbiota, mTOR pathway and immunity in abalone.

Evolutionary paths to macrolide resistance in a Neisseria commensal converge on ribosomal genes through short sequence duplications.

Neisseria commensals are an indisputable source of resistance for their pathogenic relatives. However, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (≥ 2 μg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n = 7/16), we find mutations that reduce susceptibility to azithromycin converge on the locus encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through short tandem duplication events. Interestingly, one of the laboratory evolved mutations in rpmH is identical (7LKRTYQ12), and two nearly identical, to those recently reported to contribute to high-level azithromycin resistance in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Ultimately, studies like this will be critical for predicting commensal alleles that could rapidly disseminate into pathogen populations via allelic exchange across recombinogenic microbial genera.

Trichodysplasia spinulosa polyomavirus small T antigen synergistically modulates S6 protein translation and DNA damage response pathways to shape host cell environment.

TSPyV is a viral agent linked to Trichodysplasia spinulosa, a disfiguring human skin disease which presents with hyperkeratotic spicule eruption in immunocompromised hosts. This proliferative disease state requires extensive modulation of the host cell environment. While the small T (sT) antigen of TSPyV has been postulated to cause widespread cellular perturbation, its specific substrates and their mechanistic connection are unclear. To identify the cellular substrates and pathways perturbed by TSPyV sT and propose a nuanced model that reconciles the multiple arms of TSPyV pathogenesis, changes in expression of several proteins and phospho-proteins in TSPyV sT expressing and TSPyV sT deletion mutant-expressing cell lysates were interrogated using Western blot assays. TSPyV sT expression exploits the DNA damage response pathway, by inducing hyperphosphorylation of ATM and 53BP1 and upregulation of BMI-1. Concurrently, sT dysregulates the S6 protein translation pathway via hyperphosphorylation of CDC2, p70 S6 kinase, S6, and PP1α. The S6S244/247 and p-PP1αT320 phospho-forms are points of overlap between the DDR and S6 networks. We propose a mechanistic rationale for previous reports positioning sT antigen as the key driver of TSPyV pathogenesis. We illuminate novel targets in the S6 and DDR pathways and recognize a potential synergy between these pathways. TSPyV may sensitize the cell to both unrestricted translation and genomic instability. This multi-pronged infection model may inform future therapeutic modalities against TSPyV and possibly other viruses with overlapping host substrates.

Formimidoyltransferase cyclodeaminase prevents the starvation-induced liver hepatomegaly and dysfunction through downregulating mTORC1.

The liver is a crucial center in the regulation of energy homeostasis under starvation. Although downregulation of mammalian target of rapamycin complex 1 (mTORC1) has been reported to play pivotal roles in the starvation responses, the underpinning mechanisms in particular upstream factors that downregulate mTORC1 remain largely unknown. To identify genetic variants that cause liver energy disorders during starvation, we conduct a zebrafish forward genetic screen. We identify a liver hulk (lvh) mutant with normal liver under feeding, but exhibiting liver hypertrophy under fasting. The hepatomegaly in lvh is caused by enlarged hepatocyte size and leads to liver dysfunction as well as limited tolerance to starvation. Positional cloning reveals that lvh phenotypes are caused by mutation in the ftcd gene, which encodes the formimidoyltransferase cyclodeaminase (FTCD). Further studies show that in response to starvation, the phosphorylated ribosomal S6 protein (p-RS6), a downstream effector of mTORC1, becomes downregulated in the wild-type liver, but remains at high level in lvh. Inhibition of mTORC1 by rapamycin rescues the hepatomegaly and liver dysfunction of lvh. Thus, we characterize the roles of FTCD in starvation response, which acts as an important upstream factor to downregulate mTORC1, thus preventing liver hypertrophy and dysfunction.

Viral expression of constitutively active AKT3 induces CST axonal sprouting and regeneration, but also promotes seizures

Increasing the intrinsic growth potential of neurons after injury has repeatedly been shown to promote some level of axonal regeneration in rodent models. One of the most studied pathways involves the activation of the PI3K/AKT/mTOR pathways, primarily by reducing the levels of PTEN, a negative regulator of PI3K. Likewise, activation of signal transducer and activator of transcription 3 (STAT3) has previously been shown to boost axonal regeneration and sprouting within the injured nervous system. Here, we examined the regeneration of the corticospinal tract (CST) after cortical expression of constitutively active (ca) Akt3 and STAT3, both separately and in combination. Overexpression of caAkt3 induced regeneration of CST axons past the injury site independent of caSTAT3 overexpression. STAT3 demonstrated improved axon sprouting compared to controls and contributed to a synergistic improvement in effects when combined with Akt3 but failed to promote axonal regeneration as an individual therapy. Despite showing impressive axonal regeneration, animals expressing Akt3 failed to show any functional improvement and deteriorated with time. During this period, we observed progressive Akt3 dose-dependent increase in behavioral seizures. Histology revealed increased phosphorylation of ribosomal S6 protein within the unilateral cortex, increased neuronal size, microglia activation and hemispheric enlargement (hemimegalencephaly).

S-adenosylmethionine inhibits la ribonucleoprotein domain family member 1 in murine liver and human liver cancer cells.

Methionine adenosyltransferase 1A (MAT1A) is responsible for S-adenosylmethionine (SAMe) biosynthesis in the liver. Mice lacking Mat1a have hepatic SAMe depletion and develop NASH and HCC spontaneously. Several kinases are activated in Mat1a knockout (KO) mice livers. However, characterizing the phospho-proteome and determining whether they contribute to liver pathology remain open for study. Our study aimed to provide this knowledge. We performed phospho-proteomics in Mat1a KO mice livers with and without SAMe treatment to identify SAMe-dependent changes that may contribute to liver pathology. Our studies used Mat1a KO mice at different ages treated with and without SAMe, cell lines, in vitro translation and kinase assays, and human liver specimens. We found that the most striking change was hyperphosphorylation and increased content of La-related protein 1 (LARP1), which, in the unphosphorylated form, negatively regulates translation of 5'-terminal oligopyrimidine (TOP)-containing mRNAs. Consistently, multiple TOP proteins are induced in KO livers. Translation of TOP mRNAs ribosomal protein S3 and ribosomal protein L18 was enhanced by LARP1 overexpression in liver cancer cells. We identified LARP1-T449 as a SAMe-sensitive phospho-site of cyclin-dependent kinase 2 (CDK2). Knocking down CDK2 lowered LARP1 phosphorylation and prevented LARP1-overexpression-mediated increase in translation. LARP1-T449 phosphorylation induced global translation, cell growth, migration, invasion, and expression of oncogenic TOP-ribosomal proteins in HCC cells. LARP1 expression is increased in human NASH and HCC. Our results reveal a SAMe-sensitive mechanism of LARP1 phosphorylation that may be involved in the progression of NASH to HCC.

Abstract P123: Antitumor activity of tipifarnib and PI3K pathway inhibitors in HRAS-associated HNSCC

Abstract HRAS-MAPK and PI3K-AKT-mTOR are important oncogenic pathways in head and neck squamous cell carcinoma (HNSCC) and other squamous cell carcinomas (SCCs). HRAS is mutated in ~5% and overexpressed in approximately 30% of HNSCC patients, raising the possibility that some HRAS wild-type (WT) HNSCCs may also display a degree of dependence on HRAS. RAS proteins undergo several post-translational modifications, including the addition of a farnesyl isoprenoid moiety by the enzyme farnesyltransferase (FT), which facilitate their attachment to membranes. This dependence prompted the development of selective inhibitors (FTIs) for the treatment of RAS-driven cancers. Tipifarnib is a potent and selective FTI that has demonstrated antitumor activity in recurrent/metastatic HNSCC carrying HRAS mutations. Based on these data, a pivotal study (NCT03719690) evaluating the efficacy of tipifarnib in HRAS mutant HNSCC (AIM-HN) is currently ongoing. PIK3CA (the catalytic subunit of PI3K), another prominent driver in HNSCC, is commonly activated either by gain of function mutations or gene amplification with some overlap between the two subsets. Multiple reports indicate that HRAS and PIK3CA pathways cooperate and crosstalk in driving tumor progression in SCCs and resistance to inhibitors of respective pathways. In this study, we explored whether combined inhibition of HRAS farnesylation (by tipifarnib) and inhibition of PI3K pathway signaling (with inhibitors of PI3K-a, AKT or mTORC1/2) would be more effective in CDX and PDX models of HRAS-associated SCCs relative to the monotherapy approaches. In a panel of HNSCC cell lines harboring HRAS and/or PIK3CA alterations (mutation, overexpression or amplification), tipifarnib reduced cell growth and, in combination with PI3K-a inhibitor alpelisib, induced cytotoxicity. Consistent with in vitro findings, robust inhibition of tumor growth was observed in majority of animals treated with the combination of tipifarnib and alpelisib. Similar activity was noted with the AKT inhibitor uprosertib and the mTORC1/2 inhibitor sapanisertib. Regressions with the FTI-PI3K-a inhibitor doublet were observed both in tumors that were HRAS mutant or HRAS overexpressed with or without mutation or amplification of the PIK3CA gene or carried the latter without alterations in HRAS, suggesting that concomitant blockade of both targets may have surprisingly broad and potent anti-tumor activity in HNSCC. In dose-scheduling experiments in PDX models, simultaneous blockade of both targets was superior to split intermittent dosing of the two drugs, underlining the cooperativity of the two pathways in these models. Mechanistically, tipifarnib repressed the compensatory MAPK pathway activation induced by alpelisib at the level of phosphorylated ribosomal S6 protein. Impact of tipifarnib and alpelisib combination on additional MAPK and AKT signaling mediators will be reported. Citation Format: Francis Burrows, Shivani Malik, Stacia Chan, Asako Mccloskey, Zhiyong Wang, Mara Gilard, Silvio Gutkind. Antitumor activity of tipifarnib and PI3K pathway inhibitors in HRAS-associated HNSCC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P123.

Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus.

Background and Aim:Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein.Materials and Methods:The S1-2 segment of the S gene of the PEDV Thai isolate (G2b) was amplified, cloned into the pBAD202/D-TOPO® vector (Invitrogen, Carlsbad, CA, USA), and expressed in Escherichia coli. The optimum concentration of arabinose and the optimum induction time for the expression of the recombinant S1-2 protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenic reactivity of the recombinant S1-2 protein was determined using Western blot analysis with rabbit polyclonal antibodies against the SM98 strain of PEDV (G1a).Results:The recombinant S1-2 segment of the S gene of the PEDV Thai isolate protein was cloned and the recombinant S1-2 protein was successfully expressed. The optimum concentration of arabinose and the optimum induction time for the induction of the recombinant S1-2 protein were 0.2% and 8 h, respectively. The recombinant S1-2 protein reacted specifically with both rabbit anti-histidine polyclonal antibodies and rabbit anti-PEDV polyclonal antibodies.Conclusion:The recombinant S1-2 protein reacted with rabbit anti-PEDV polyclonal antibodies induced by the different PEDV genogroup. Therefore, the recombinant S1-2 protein may be a useful tool for the development of a diagnostic test for PEDV or for a vaccine against PEDV.

The mechanism of rapamycin in promoting asthmatic regulatory T cell differentiation and function.

To investigate the mechanism of rapamycin in promoting asthmatic regulatory T cell differentiation . Asthma model was prepared by sensitization and challenge of ovalbumin in mice. Spleen CD4CD25 T cells were sorted from the asthmatic mice and normal mice by ultrahigh speed flow cytometer, and divided into three groups. Transforming growth factor-β and interleukin-2, or combined with rapamycin (final concentration of 500 nmol/L) were given in the model group or the rapamycin group. The levels of Treg cells and CD4CD25 T cells were detected by flow cytometry. The phosphorylation level of downstream proteins of S6 and Akt in the mTORC1/2 signaling pathway were examined by Western blotting. Compared with the model group, the differentiation level of Treg cells in the rapamycin group was significantly increased, the proliferation level of CD4CD25 T cells was decreased, and the phosphorylations of the mTORC1/2 substrates, S6 protein and Akt were decreased （all <0.05）. Rapamycin can promote the differentiation and function of Treg cells via inhibition of the mTORC1/2 signaling pathway.

Arginine Regulates TOR Signaling Pathway through SLC38A9 in Abalone Haliotis discus hannai.

Arginine plays an important role in the regulation of the target of the rapamycin (TOR) signaling pathway, and Solute Carrier Family 38 Member 9 (SLC38A9) was identified to participate in the amino acid-dependent activation of TOR in humans. However, the regulations of arginine on the TOR signaling pathway in abalone are still unclear. In this study, slc38a9 of abalone was cloned, and the slc38a9 was knocked down and overexpressed to explore its function in the regulation of the TOR signaling pathway. The results showed that knockdown of slc38a9 decreased the expression of tor, ribosomal s6 protein kinase (s6k) and eukaryotic translation initiation factor 4e (eif4e) and inhibited the activation of the TOR signaling pathway by arginine. Overexpression of slc38a9 up-regulated the expression of TOR-related genes. In addition, hemocytes of abalone were treated with 0, 0.2, 0.5, 1, 2 and 4 mmol/L of arginine, and abalones were fed diets with 1.17%, 1.68% and 3.43% of arginine, respectively, for 120 days. Supplementation of arginine (0.5–4 mmol/L) increased the expressions of slc38a9, tor, s6k and eif4e in hemocytes, and abalone fed with 1.68% of dietary arginine showed higher mRNA levels of slc38a9, tor, s6k and eif4e and phosphorylation levels of TOR, S6 and 4E-BP. In conclusion, the TOR signaling pathway of abalone can be regulated by arginine, and SLC38A9 plays an essential role in this regulation.

Discovery, Optimization, and Structure-Activity Relationship Study of Novel and Potent RSK4 Inhibitors as Promising Agents for the Treatment of Esophageal Squamous Cell Carcinoma.

Ribosomal S6 protein kinase 4 (RSK4) was identified to be a promising target for the treatment of esophageal squamous cell carcinoma (ESCC) in our previous research, whose current treatments are primarily chemotherapy and radiotherapy due to the lack of targeted therapy. However, few potent and specific RSK4 inhibitors are reported. In this study, a series of 1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-ones derivatives were designed and synthesized as novel and potent RSK4 inhibitors. Compound 14f was identified with potent RSK4 inhibitory activity both in vitro and in vivo. 14f significantly inhibited the proliferation and invasion of ESCC cells in vitro with IC50 values of 0.57 and 0.98 μM, respectively. It dose dependently inhibited the phosphorylation of RSK4 downstream substrates while exerting little effect on the substrates of RSK1-3 in ESCC cells. The markedly suppressed tumor growth and no observed toxicity to main organs in the ESCC xenograft mouse model suggested 14f to be a promising RSK4-targeting agent for ESCC treatment.

Transcranial Laser Photobiomodulation Improves Intracellular Signaling Linked to Cell Survival, Memory and Glucose Metabolism in the Aged Brain: A Preliminary Study.

Aging is often accompanied by exacerbated activation of cell death-related signaling pathways and decreased energy metabolism. We hypothesized that transcranial near-infrared laser may increase intracellular signaling pathways beneficial to aging brains, such as those that regulate brain cell proliferation, apoptosis, and energy metabolism. To test this hypothesis, we investigated the expression and activation of intracellular signaling proteins in the cerebral cortex and hippocampus of aged rats (20 months old) treated with the transcranial near-infrared laser for 58 consecutive days. As compared to sham controls, transcranial laser treatment increased intracellular signaling proteins related to cell proliferation and cell survival, such as signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB), also known as Akt that is linked to glucose metabolism. In addition, ERK is linked to memory, while ERK and JNK signaling pathways regulate glucose metabolism. Specifically, the laser treatment caused the activation of STAT3, ERK, and JNK signaling proteins in the cerebral cortex. In the hippocampus, the laser treatment increased the expression of p70S6K and STAT3 and the activation of Akt. Taken together, the data support the hypothesis that transcranial laser photobiomodulation improves intracellular signaling pathways linked to cell survival, memory, and glucose metabolism in the brain of aged rats.