Phosphorylation Of Protein S6 Research Articles

CLONING OF CDNA-GENE OF ARABIDOPSIS THALIANA RIBOSOMAL PROTEIN S6, ITS EXPRESSION IN ESCHERICHIA COLI AND PURIFICATION OF ATRPS6B1 RECOMBINANT PROTEIN

Living organisms must adapt their growth and development in response to external factors like stress and nutrient availability throughout their lifespan. Consequently, they have evolved various regulatory pathways to enhance environmental perception and facilitate necessary metabolic adjustments. These pathways involve key proteins that, under stress and nutrient limitation, initiate anabolic and catabolic cellular processes. One critical pathway present in all eukaryotes involves the protein kinase TOR (Target of rapamycin), a large kinase that governs numerous biological processes, including the activation of S6K kinase, which phosphorylates the S6 protein (RPS6). Crystal structures have revealed the precise location of RPS6 within the 40S ribosomal subunit, highlighting structural features such as the C-terminal helix containing multiple phosphorylation sites. RPS6 primarily regulates mechanisms controlling cell growth and division. In plants, eS6 is encoded by two conserved genes, and its activation and phosphorylation are closely linked to S6Ks. Recent studies emphasize the significance of S6 protein phosphorylation as a key event in signaling pathways related to cell growth and survival factors, crucially regulating translation initiation and protein synthesis. S6 protein can be phosphorylated on various serine and threonine residues by S6K kinase, and further research shows its interaction with other molecules and proteins, expanding its functional roles in cell signaling. Further exploration of ribosomal protein S6 could unveil new insights into its molecular interactions, roles in cellular physiology and pathophysiology, and potential applications in enhancing plant biomass and yield. This study involved cloning and site-directed mutagenesis of cDNA for the second isoform of AtRPS6 protein (AtRPS6B), followed by expression of natural and phosphomimetic forms of the protein in E. coli ArcticExpress (DE3) cells. The proteins were then purified using metal-chelate affinity chromatography (IMAC), and their presence and degree of purification were confirmed via Western blotting. These findings contribute to the broader research context surrounding ribosomal protein S6. RPS6, a key effector in the TOR signaling pathway, plays a crucial role in regulating ribosome biosynthesis by controlling transcription and translation processes. Phosphorylation of RPS6, indicative of S6K activity, is associated with actively dividing cells. However, the precise role of RPS6 in ribosome biosynthesis regulation remains a subject of ongoing investigation. The data obtained from the study on AtRPS6B expand our understanding of the molecular mechanisms underlying ribosomal biosynthesis regulation. These insights may significantly contribute to our knowledge of the interplay between signaling pathways, ribosomal proteins, cellular metabolism, and offer new perspectives for therapeutic interventions targeting cellular processes associated with ribosomes.

RGS10 inhibits proliferation and migration of pulmonary arterial smooth muscle cell in pulmonary hypertension via AKT/mTORC1 signaling

ABSTRACT Objective: Excessive proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is a core event of pulmonary hypertension (PH). Regulators of G protein signaling 10 (RGS10) can regulate cellular proliferation and cardiopulmonary diseases. We demonstrate whether RGS10 also serves as a regulator of PH. Methods: PASMC was challenged by hypoxia to induce proliferation and migration. Adenovirus carrying Rgs10 gene (Ad-Rgs10) was used for external expression of Rgs10. Hypoxia/SU5416 or MCT was used to induce PH. Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were used to validate the establishment of PH model. Results: RGS10 was downregulated in hypoxia-challenged PASMC. Ad-Rgs10 significantly suppressed proliferation and migration of PASMC after hypoxia stimulus, while silencing RGS10 showed contrary effect. Mechanistically, we observed that phosphorylation of S6 and 4E-Binding Protein 1 (4EBP1), the main downstream effectors of mammalian target of rapamycin complex 1 (mTORC1) as well as phosphorylation of AKT, the canonical upstream of mTORC1 in hypoxia-induced PASMC were negatively modulated by RGS10. Both recovering mTORC1 activity and restoring AKT activity abolished these effects of RGS10 on PASMC. More importantly, AKT activation also abolished the inhibitory role of RGS10 in mTORC1 activity in hypoxia-challenged PASMC. Finally, we also observed that overexpression of RGS10 in vivo ameliorated pulmonary vascular wall thickening and reducing RVSP and RVHI in mouse PH model. Conclusion: Our findings reveal the modulatory role of RGS10 in PASMC and PH via AKT/mTORC1 axis. Therefore, targeting RGS10 may serve as a novel potent method for the prevention against PH.”

Abstract P2-02-07: Patients’ selection of daily timing of oral intake of adjuvant hormonotherapy (HT) and everolimus (EVE) for high risk early breast cancer in the UCBG-UNIRAD phase III trial

Abstract Background: Everolimus (EVE) addition to adjuvant hormonotherapy (HT) for high-risk early breast cancer (BC) did not improve 3-year disease-free survival (DFS) compared with ET alone in the randomized UNIRAD trial (NCT01805271) (1). Most patients (pts) withdrew from EVE for adverse events nearly midway before the expected treatment duration of 2 years. The main EVE target involves m-TOR-induced S6 protein phosphorylation, which is controlled by the molecular circadian clock in mice and cells. Toward the analysis of possible circadian time-dependencies in EVE toxicities and efficacy, we first determine the actual distribution of the daily times chosen by the patients for both oral EVE and HT intakes. Patients and methods: The registered pts were asked to record the clock hours they chose for both HT and EVE or placebo (PLAC) intakes within four possible daily 6-h slots, i.e. from 06:00 to 11:59 (morning), 12:00 to 17:59 (midtime), 18:00 to 23:59 (evening), or 24:00 to 05:59, (night) in a daily diary throughout their participation in the trial. Modifications in times of drugs intake were reported. Comparisons between groups involved Kruskal-Wallis sum test and Pearson’s Chi-squared and Fisher’s exact tests. Results: Out of 1,278 randomized patients, 1063 (83,1 %) recorded the times of EVE or PLAC intakes, 852 recorded those for HT intakes. Only 10 pts reported night EVE/PLAC intakes and were not considered here. Of the 1053 evaluated pts; 549 pts took EVE/PLAC in the morning, 82 pts at midtime and 422 in the evening. As compared with evening intakes, morning or midtime intakes of EVE or PLAC were significantly associated with an older age, and post-menopausal status, whilst younger and premenopausal women preferentially chose evening intakes (p< 0.001 for both). Consistently, HT with aromatase inhibitors (AI) were mostly taken in the morning or at midtime, whereas tamoxifene (TAM) was mostly taken the evening (p = 0.001). Tumor size, lymph nodes involvement, histological grade, hormonal receptors status, and EVE initial dose were similarly distributed among the three time slots (Table 1). A similar distribution of oral intake times as in the whole timing study population was found according to age, menopausal status and HT type in the EVE arm (N=508 pts), and in the HT population (+/- EVE or PLAC) (N=852 pts). Initial oral timing intake was modified for EVE by 50 of 508 pts (10%) and for HT by 10 of 852 pts (1%). Conclusion: To the best of our knowledge, it is the first time that the patients’ spontaneous selection of daily times for oral intakes of a targeted agent (EVE) and HT is reported in a large series of early breast cancer pts. Age and menopausal status were important determinants of patient-selected daily timing intakes of both EVE and HT, as well as HT types. These findings will be carefully considered in the analyses of possible EVE and HT timing effects on adverse events and efficacy of the current adjuvant regimens. 1) Bachelot T et al, J Clin Oncol. 2022 May 23 Citation Format: Sylvie Giacchetti, Anne-Sophie Hamy, Thomas Bachelot, Jérôme Lemonnier, Fabrice Andre, David A. Cameron, Judith Bliss, Sylvie Chabaud, Anne-Claire Hardy-Bessard, Magali Lacroix-Triki, Jean-Luc Canon, Hervé R. Bonnefoi, Mario Campone, Paul Cottu, Florence Dalenc, Annabelle Ballesta, Francis Lévi, Enora Laas. Patients’ selection of daily timing of oral intake of adjuvant hormonotherapy (HT) and everolimus (EVE) for high risk early breast cancer in the UCBG-UNIRAD phase III trial. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-02-07.

SGLT2 inhibitors mitigate kidney tubular metabolic and mTORC1 perturbations in youth-onset type 2 diabetes.

The molecular mechanisms of sodium-glucose cotransporter-2 (SGLT2) inhibitors (SGLT2i) remain incompletely understood. Single-cell RNA sequencing and morphometric data were collected from research kidney biopsies donated by young persons with type 2 diabetes (T2D), aged 12 to 21 years, and healthy controls (HCs). Participants with T2D were obese and had higher estimated glomerular filtration rates and mesangial and glomerular volumes than HCs. Ten T2D participants had been prescribed SGLT2i (T2Di[+]) and 6 not (T2Di[-]). Transcriptional profiles showed SGLT2 expression exclusively in the proximal tubular (PT) cluster with highest expression in T2Di(-) patients. However, transcriptional alterations with SGLT2i treatment were seen across nephron segments, particularly in the distal nephron. SGLT2i treatment was associated with suppression of transcripts in the glycolysis, gluconeogenesis, and tricarboxylic acid cycle pathways in PT, but had the opposite effect in thick ascending limb. Transcripts in the energy-sensitive mTORC1-signaling pathway returned toward HC levels in all tubular segments in T2Di(+), consistent with a diabetes mouse model treated with SGLT2i. Decreased levels of phosphorylated S6 protein in proximal and distal tubules in T2Di(+) patients confirmed changes in mTORC1 pathway activity. We propose that SGLT2i treatment benefits the kidneys by mitigating diabetes-induced metabolic perturbations via suppression of mTORC1 signaling in kidney tubules.

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.

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.

Abstract 1104: Repotrectinib increases effectiveness of MEK inhibitors in KRAS mutant cancer models

Abstract KRAS is the most frequently mutated oncogene in cancers, accounting for approximately 25% of non small cell lung cancer (NSCLC), 45% of colorectal cancer (CRC), and 75% of pancreatic cancer. KRAS G12D and G12V mutations account for a large percent of mutant KRAS cancers (36% of NSCLC; 57% of CRC; 71% of pancreatic). MEK1/2 are critical downstream effectors of KRAS signaling and preclinical studies in KRAS mutant models show sensitivity to MEK inhibitors (MEKi). However, clinical studies of single agent MEKi or combinations with docetaxel in mutant KRAS NSCLC patients were associated with low response rates. Preclinical studies show that effectiveness of MEK inhibition can be limited by multiple resistance mechanisms such as compensatory upregulation of PI3K/AKT via SRC/FAK signaling, or activation of the JAK2/STAT3 pathway. Repotrectinib is a next-generation ROS1/TRK inhibitor with SRC/FAK/JAK2 inhibitory potencies which may suppress adaptive resistance to MEK inhibitors. In the current study, repotrectinib combinations with KRAS signaling network inhibitors including MEK (trametinib, selumetinib), MEK/RAF (VS-6766), ERK (LY3214996), SHP2 (TNO155) were explored. Repotrectinib/trametinib and repotrectinib/VS-6766 combinations yielded significant effects on cancer cell viability in NSCLC and pancreatic patient-derived spheroid models harboring KRASG12D and KRASG12V mutations. The repotrectinib/trametinib combination in A427 cells (NSCLC KRASG12D) exhibited increased inhibition of pAKT and pS6 compared to single agent treatments. This coincided with greater upregulation of p27 and elevated PARP cleavage, resulting in enhanced induction of apopotosis. To assess acquired MEKi resistance, several KRAS mutant cancer models were cultured under trametinib selection. Evaluation of trametinib-resistant cancer cells revealed activation of STAT3, FAK, and AKT signaling along with elevated S6 protein phosphorylation, which could be suppressed and resensitized to trametinib by combining with repotrectinib. Repotrectinib/trametinib combination in a syngeneic GEMM KRASG12D lung model had greater tumor growth inhibition than either single agent treatment. A similar combination efficacy benefit was observed in a HCT116 (CRC KRASG13D) xenograft model concomitant with suppression of SRC/FAK/STAT3/ERK activation. Taken together, repotrectinib combinations with MEK inhibitors demonstrated enhanced efficacy in both in vitro and in vivo preclinical models. Repotrectinib was shown to suppress molecular mechanisms of adaptive resistance mechanisms to MEK inhibition in preclinical models. These results suggest that the combination of repotrectinib with MEKi can repress the mutant KRAS signaling network to achieve more potent and durable anti-tumor activity and warrants clinical investigation in patients with KRASG12D and KRASG12V mutant cancers. Citation Format: Nathan V. Lee, Wei Deng, Dayong Zhai, Laura Rodon, Ana Parra, Jessica Cowell, Afsheen Banisadr, Xin Zhang, Brion W. Murray. Repotrectinib increases effectiveness of MEK inhibitors in KRAS mutant cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1104.

Phosphorylation of S6 Protein as a Potential Biomarker in Surgically Treated Refractory Epilepsy

The tuberous sclerosis complex (TSC), focal cortical dysplasia IIB (FCD IIB), and hemimegalencephaly (HME) exhibit similar molecular features that are dependent on the hyperactivation of the mTOR pathway. They are all associated with refractory epilepsy and the need for surgical resection with varying outcomes. The phosphorylated protein S6 (pS6) is a downstream target of mTOR, whose increased expression might indicate mTOR hyperactivation, but which is also present when there is no alteration in the pathway (such as in FCD type I). We have performed immunohistochemical marking and quantification of pS6 in resected brain specimens of 26 patients clinically and histologically diagnosed with TSC, FCD IIB, or HME and compared this data to a control group of 25 patients, to measure the extent of pS6 positivity and its correlation with clinical aspects. Our results suggest that pS6 may serve as a reliable biomarker in epilepsy and that a greater percentage of pS6 marking can relate to more severe forms of mTOR-dependent brain anomalies.

Abstract PO049: Compartment-specific profiling for evaluation of disrupted signaling in early stage, low-grade endometrial cancer

Abstract Understanding tumor microenvironment is critical to improving conservative management (non-surgical) strategies for early stage, low grade endometrial cancer (EC). This remains an unmet clinical need for women who wish to maintain fertility and those who cannot undergo surgery due to medical co-morbidities. Current regimens rely on progestin treatment, but resistance and early relapse require improved therapeutic strategies. We sought to evaluate alterations in gene expression in the epithelial and stromal compartment of EC compared to normal endometrium to identify targets for combination therapies. RNA was extracted from microdissected epithelial and stromal compartments of formalin fixed paraffin embedded early stage, low grade EC and normal endometrium (NE). Comprehensive transcriptome profiling was performed with Affymetrix Clariom D assay and pathway analysis was performed. Immunohistochemistry (IHC) was used for validation. Functional studies of differentially expressed genes were performed in Hec1A EC cell lines. Among differentially expressed genes in EC vs NE, multiple altered signaling pathways were identified. EIF2 signaling, eIF4 and p70S6K signaling, and PI3K/AKT/mTOR were the most significant pathways activated in both epithelial and stromal compartments. In the epithelia compartment, the most significant gene expression changes included reduced expression of RBPMS (RNA Binding Protein with Multiple Splicing), SORD (Sorbitol Dehydrogenase), DDX17 (DEAD-Box Helicase 17), and TPT1 (Translationally-Controlled Tumor Protein) in EC, while MTDH (Metadherin) was significantly increased in EC. Similar changes were observed in the stroma compartment, but with lower fold changes than that observed in gland. Together with decreased RBPMS expression, decreased RBPMS-interacting molecules IGF2, TCF7L2 (Transcription Factor 7 Like 2), FOS (Proto-Oncogene C-Fos) and increased GPS2 (G protein Pathway Suppressor 2) were also observed. Differential expression of proteins in gland and stroma was also confirmed by IHC. An inverse correlation of RBPMS expression and EC proliferation was also observed in EC at the protein level. Enhanced AKT and S6 protein phosphorylation were observed by RBPMS silencing in Hec1A endometrial cells. Reverse Phase Protein Array demonstrated activation of Caveolin 1, FOXO3 (Forkhead Box O3), MT-CO1 (Mitochondrially Encoded Cytochrome C Oxidase I), L1CAM (L1 Cell Adhesion Molecule) with RBPMS silencing in Hec1A cell. These molecules are involved in pathways such as molecular mechanisms of cancer, estrogen receptor signaling, and senescence pathway. While estrogen-regulated changes were prominent in early stage low-grade EC, the identification of additional signaling changes provide support for an ongoing trial of levonorgestrel IUD + everolimus (mTOR inhibitor) and can be probed further to identify new targets for combination conservative management strategies. Additionally, biomarkers from these pathways, such as RBPMS, can be studied to guide the selection of progestin-only therapy versus combination therapy. Citation Format: Qian Zhang, Xiaoping Su, Joseph Celestino, Ying Yuan, Samuel C. Mok, Karen H. Lu, Melinda S. Yates. Compartment-specific profiling for evaluation of disrupted signaling in early stage, low-grade endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO049.

Protein Supplementation Enhances the Effects of Intermittent Loading on Skeletal Muscles by Activating the mTORC1 Signaling Pathway in a Rat Model of Disuse Atrophy.

Inactivity leads to skeletal muscle atrophy, whereas intermittent loading (IL) during hind limb unloading (HU) attenuates muscle atrophy. However, the combined effects of IL and protein supplementation on disuse muscle atrophy are unclear. Therefore, we investigated the effects of IL and a high-protein oral nutritional supplement (HP) during HU on skeletal muscle mass and protein synthesis/breakdown. Male F344 rats were assigned to the control (CON), 14-day HU (HU), IL during HU (HU + IL), and IL during HU followed by HP administration (2.6 g protein/kg/day; HU + IL + HP) groups. Soleus and gastrocnemius muscles were sampled 30 min after the last IL and HP supplementation. HU decreased relative soleus and gastrocnemius muscle masses. Relative muscle masses and p70 ribosomal protein S6 kinase/ribosomal protein S6 phosphorylation in soleus and gastrocnemius muscles were higher in the HU + IL group than the HU group and further higher in the HU + IL + HP group than the HU + IL group in gastrocnemius muscle. Therefore, protein administration plus IL effectively prevented skeletal muscle atrophy induced by disuse, potentially via enhanced activation of targets downstream of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway.

Abstract 4143: Identification of molecular targets for conservative management of early stage, low-grade endometrial cancer through compartment-specific transcriptome profiling

Abstract Understanding tumor microenvironment is critical to improving conservative management (non-surgical) strategies for early stage, low grade endometrial cancer (EC). This remains an unmet clinical need for women who wish to maintain fertility and those who cannot undergo surgery due to medical co-morbidities. Current regimens rely on progestin treatment, but resistance and early relapse require improved therapeutic strategies. We sought to evaluate alterations in gene expression in the epithelial and stromal compartment of EC compared to normal endometrium to identify targets for combination therapies. RNA was extracted from microdissected epithelial and stromal compartments of formalin fixed paraffin embedded early stage, low grade EC and normal endometrium (NE). Comprehensive transcriptome profiling was performed with Affymetrix Clariom D assay and pathway analysis was performed. Immunohistochemistry (IHC) was used for validation. Functional studies of differentially expressed genes were performed in EC cell lines, ECC-1 and Hec1A. Focusing on differentially expressed genes in the epithelial compartment of EC vs NE, multiple activated signaling pathways were identified, including eIF2 signaling, eIF4 and p70S6K signaling, PI3K/AKT/mTOR, and hormone signaling. Top gene expression changes with significantly lower expression in EC included RBPMS (RNA-binding protein with multiple splicing), SORD (sorbitol dehydrogenase), DDX17 (DEAD-box helicase 17), and TPT1 (translationally-controlled tumor protein) gene expression, while MTDH (metadherin) was significantly increased in EC. Differential expression of proteins coded by these genes was confirmed by IHC. Additionally, RBPMS silencing enhanced AKT and S6 protein phosphorylation in Hec1A cells and forced expression of RBPMS in ECC-1 cells attenuated Erk1/2 MAP kinase activation. Expression of RBPMS inhibited cell proliferation and knockdown of RBPMS increased proliferation. Treatment of EC cells with everolimus (mTOR inhibitor) resulted in differential inhibition of proliferation based on RBPMS expression. Communication between tumor and stroma is also being evaluated. While estrogen-regulated changes were prominent in early stage low-grade EC, the discovery of alterations in other signaling pathways suggest additional targets for conservative management of the disease. The identification of signaling nodes shared between hormone-regulated pathways and mTOR signaling provides further justification for the ongoing combination therapy trial (progestin plus everolimus). Additionally, biomarkers from these pathways, such as RBPMS, can be studied to guide the selection of progestin-only therapy versus combination therapy. Citation Format: Qian Zhang, Xiaoping Su, Joseph Celestino, Ying Yuan, Samuel C. Mok, Karen H. Lu, Melinda S. Yates. Identification of molecular targets for conservative management of early stage, low-grade endometrial cancer through compartment-specific transcriptome profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4143.

Dynamic analysis of 4E-BP1 phosphorylation in neurons with Tsc2 or Depdc5 knockout

TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to mechanistic target of rapamycin (mTOR) hyperactivation evidenced by hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding protein 1 (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 phosphorylation in non-neural cells, but this has not been comprehensively investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging demonstrated that AAF media promoted movement of mTOR off the lysosome, functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating that AA levels modulate lysosomal mTOR localization and account, in part, for differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal mTOR signaling in mTOR-associated epilepsies may have distinct responses to mTOR inhibitors and to levels of cellular amino acids.

2120-P: The Protein Phosphatase M1K Connects Glucose Sensing to Protein Translation in Pancreatic Beta Cells

The protein phosphatase m1K (PPm1K) dephosphorylates and activates the rate limiting enzyme in the branched-chain amino acid (BCAA) catabolism pathway, branched chain a-keto acid dehydrogenase (BCKDH). A common single nucleotide polymorphism upstream of the gene for PPm1K is associated with elevated circulating levels of BCAA and increased risk for development of type 2 diabetes; however, the function of PPm1K in the pancreatic beta cell has not been assessed. In line with our previous observations in liver, we found that increasing glucose concentrations in INS1 cells results in elevated expression of the ChREBP β isoform and lower PPM1K mRNA expression. SiRNA-mediated knockdown (KD) of PPm1K to levels achieved by glucose in INS1 cells lead to robust phosphorylation of the ribosomal protein S6 on its regulatory phosphosites ser235/236 and ser240/s244. Notably, this occurred absent any effect on the phosphorylation of the upstream proteins mammalian target of rapamycin (mTOR) or p70 S6 Kinase (S6K) and could not be reversed by inhibiting BCKDH phosphorylation with the small molecule, BT2. Thus Ppm1K appears to regulate S6 independently of mTOR, S6K, and BCKDH. Surface sensing of translation (SUnSET) studies revealed that the increase in S6 phosphorylation in PPm1K KD INS1 cells is associated with a 2-fold increase in protein translation rate. Accordingly, PPm1K KD increased both insulin content and glucose stimulated insulin secretion in INS1 cells. In line with the higher translation rate, PPm1K KD cells displayed signs of endoplasmic reticulum stress including increased expression of spliced xbp1 and Bip mRNA. In conclusion, our data highlight a new role for PPm1K in pancreatic beta cells, as a mechanism connecting glucose sensing to protein translation via modulation of S6 phosphorylation. Disclosure Y. Deleye: None. J. Herring: None. K.H. Hess: None. B. Leininger: None. R.W. McGarrah: None. J.S. Tessem: None. P.J. White: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-16-INI-17 to P.J.W.)

Enhanced phosphorylation of S6 protein in mouse cortical layer V and subplate neurons.

The mammalian neocortex is composed of six major layers of neurons. Each group of neurons in the cortical layers has distinct characteristics based on the expression of specific genes and connectivity patterns of neural circuits. Neuronal subtype transition and regional identity acquisition are established by temporal cues and interaction between several transcription factors during neurogenesis. The impairment of cortical lamination or neural circuits results in a wide range of neurodevelopmental disorders such as autism, schizophrenia, and certain forms of childhood epilepsy. Despite continuous efforts to classify neurons with the aid of genetic and epigenetic analyses, the neuron-specific properties associated with post-transcriptional modification remain unclear. In the present study, the distribution of phosphorylated S6-positive layers across the neocortex was examined using several layer markers. The development of pS6 S235/236 layers in layer V and the subplate was spatiotemporally regulated in the mouse brain. In addition, enhanced phosphorylation of ribosomal protein S6 in Ctip2-positive layer V neurons in vivo was sustained under in-vitro conditions using a culture of primary cortical neurons.

SNX-2112, an Hsp90 inhibitor, suppresses cervical cancer cells proliferation, migration, and invasion by inhibiting the Akt/mTOR signaling pathway

Patients with advanced-stage cervical cancer have a high rate of morbidity and mortality, predominantly due to the metastasis of cervical cancer cells. Therefore, the development of novel agents to prevent metastasis is required for improved cervical cancer therapeutics. SNX-2112, a potent and selective Hsp90 inhibitor, is an anticancer candidate in clinical trials for the treatment of some solid tumors and lymphomas. However, the effects of SNX-2112 on the migration and invasion of cervical cancer cells are unclear. This study aimed at exploring the effects of SNX-2112 on the migration and invasion of cervical cancer cells and revealing the underlying molecular mechanisms. We found that SNX-2112 significantly decreased the viability, colony formation ability, and migration of HeLa and U14 cells. The invasiveness of HeLa cells and the proteins involved in metastasis were markedly reduced after SNX-2112 treatment. SNX-2112 inactivated the focal adhesion kinase (FAK) and inhibited the expression levels of matrix metallopeptidase (MMP)-9, MMP-2, SLUG, SNAIL, β-catenin, and Vimentin. Furthermore, SNX-2112 inhibited the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway in HeLa cells by decreasing the phosphorylation of Akt, mTOR, S6, and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1). It also reduced the expression levels of the endoplasmic reticulum (ER)-localized molecular chaperones, Calnexin and BiP, and unfolded protein response (UPR)-related proteins IRE1α and PERK, suggesting that SNX-2112 can suppress ER stress and thus, inactivate the UPR in HeLa cells. Taken together, these findings indicate that SNX-2112 may efficiently suppress the proliferation, migration, and invasion of cervical cancer cells by inhibiting the Akt/mTOR pathway and could serve as a candidate drug for the treatment of human cervical cancer.

Hypomyelinating leukodystrophy-associated mutation of RARS leads it to the lysosome, inhibiting oligodendroglial morphological differentiation

Pelizaeus-Merzbacher disease (PMD) is a central nervous system (CNS) demyelinating disease in human, currently known as prototypic hypomyelinating leukodystrophy 1 (HLD1). The gene responsible for HLD1 encodes proteolipid protein 1 (PLP1), which is the major myelin protein produced by oligodendrocytes. HLD9 is an autosomal recessive disorder responsible for the gene differing from the plp1 gene. The hld9 gene encodes arginyl-tRNA synthetase (RARS), which belongs to a family of cytoplasmic aminoacyl-tRNA synthetases. Herein we show that HLD9-associated missense mutation of Ser456-to-Leu (S456L) localizes RARS proteins as aggregates into the lysosome but not into the endoplasmic reticulum (ER) and the Golgi body. In contrast, wild-type proteins indeed distribute throughout the cytoplasm. Expression of S456L mutant constructs in cells decreases lysosome-related signaling through ribosomal S6 protein phosphorylation, which is known to be required for myelin formation. Cells harboring the S456L mutant constructs fail to exhibit phenotypes with myelin web-like structures following differentiation in FBD-102b cells, as part of the mammalian oligodendroglial cell model, whereas parental cells exhibit them. Collectively, HLD9-associated RARS mutant proteins are specifically localized in the lysosome with downregulation of S6 phosphorylation involved in myelin formation, inhibiting differentiation in FBD-102b cells. These results present some of the molecular and cellular pathological mechanisms for defect in myelin formation underlying HLD9.

Correlation of phosphorylated S6 protein expression in blood and brain tissue in mice and rats with kainic acid-induced seizure

To determine the correlation of phosphorylated ribosomal S6 protein (P-S6) content in blood and brain tissue in mice and rats with seizure. Seizure models were induced by intraperitoric injection of kainic acid (KA) in C57BL/mice and SD rats. Flow cytometry was used to detect the content of P-S6 in blood; Western blot was used to detect the expression of P-S6 in brain tissues. The correlation between P-S6 expression in blood and in brain tissue was examine by Pearson analysis, and the correlation between P-S6 expression in blood and the severity of seizure was also observed. Western blotting analysis showed that the expression of P-S6 was significantly increased in peripheral blood and brain tissue in mice 1 h after KA-induced seizure,and the expression levels increased to (1.49±0.45) times (P<0.05) and (2.55±0.66) times (P <0.01) of the control group, respectively. Flow cytometry showed that the positive percentage and average fluorescence intensity of P-S6 in the blood of mice increased significantly 1 h after KA-induced seizures (P<0.01), which was consistent with the expression of P-S6 in brain tissue (r=0.8474, P<0.01). Flow cytometry showed that the average fluorescence intensity of P-S6 in blood increased from 14.89±9.75 to 52.35±21.72 (P<0.01) in rats with seizure, which was consistent with the change of P-S6 in brain tissue (r=0.9385, P<0.01). Rats with higher levels of seizure were of higher levels of P-S6 in peripheral blood. Consistent correlation of P-S6 expression is demonstrated in peripheral blood and in brain tissue after KA-induced seizure, suggesting that the expression of P-S6 in blood can accurately reflect the changes of mTOR signaling pathway in brain tissue.

RagA, an mTORC1 activator, interacts with a hedgehog signaling protein, WDR35/IFT121.

Small Ras-like GTPases act as molecular switches for various signal transduction pathways. RagA, RagB/RagC and RagD are small Ras-like GTPases that play regulatory roles in mTORC1. Lack of proper activation of mTORC1 can lead to diseases, such as cancer and diabetes. In this study, we found an interaction between RagA and WDR35. Mutations of WDR35 may cause genetic diseases including Sensenbrenner syndrome. WDR35 seems to be a hedgehog signaling protein with a possible ciliary function and a possible upstream regulator of RagA. RagB is a homologue of RagA and is also associated with WDR35. WDR35 is present in the endoplasmic reticulum, but usually not in lysosomes, where Rag family proteins act as an mTORC1 switch. Over-expression of WDR35 results in decreased phosphorylation of ribosome S6 protein in a RagA-, RagB- and RagC-dependent manner. Thus, WDR35 is associated with RagA, RagB and RagC and might negatively influence mTORC1 activity.

TIM-3 Engagement Promotes Effector Memory T Cell Differentiation of Human Antigen-Specific CD8 T Cells by Activating mTORC1.

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.

Phosphorylated ribosomal protein S6 correlation with p21 expression and inverse association with tumor size in oral squamous cell carcinoma.

The purpose of this study was to investigate the clinical relevance of phosphorylated ribosomal protein S6 (p-S6), a surrogate marker of mammalian target of rapamycin (mTOR) activation, and p21 in a series of 125 patients with oral squamous cell carcinomas (OSCCs). Immunohistochemical analysis was performed to ascertain the phosphorylation status of p-S6 at Ser235/236 and Ser240/244, p21, and p53 protein expression. Expression of phosphorylated S6 protein on either serine 235/236 or serine 240/244 was detected in 83% and 88% tumors, respectively, and both of them were inversely and significantly correlated with the tumor size and local infiltration. Positive p21 expression was found in 91.5% of the cases, and was inversely correlated with tumor size. In OSCC, p21 expression correlates with p-S6 levels, a surrogate marker of mTOR activation, independently of p53 status. Expression of both p21 and p-S6 was found to inversely associate with tumor size but not survival outcomes in patients with OSCC.