Regulation Of Akt Phosphorylation Research Articles

FOXS1, frequently inactivated by promoter methylation, inhibited colorectal cancer cell growth by promoting TGFBI degradation through autophagy-lysosome pathway.

Tumor suppressor gene (TSG) inactivation by epigenetic modifications contributes to the carcinogenesis and progression of colorectal cancer (CRC). Expression profiling and CpG methylomics revealed that a forkhead-box transcriptional factor, FOXS1, is downregulated and methylated in CRC. To assess the biological functions and underlying mechanisms of FOXS1 in colorectal cancer. Public databases, semi-quantitative RT-PCR, immunohistochemistry, MSP, and BGS were used to analyze FOXS1 expression and promoter methylation in CRC. Stable FOXS1-overexpressing or knockdown cell lines were established. Cell growth, colony formation, flow cytometry, GFP-LC3 puncta detection, Ad-mCherry-GFP-LC3B, qPCR, in vivo subcutaneous tumor model, RNA-seq, western blotting, immunofluorescence, Co-IP assays, and protein stability analysis were performed to investigate the underlying molecular mechanisms of FOXS1. In CRC, FOXS1 was frequently downregulated due to promoter CpG methylation, acting as an independent prognostic marker. Moreover, FOXS1 exerts inhibitory effects on the growth of CRC cells in vitro and in vivo, while concurrently promoting CRC cell autophagy. Intriguingly, we found that FOXS1 interacted with transforming growth factor beta induced (TGFBI) and FOXS1 promoted TGFBI degradation through the autophagy-lysosome pathway rather than the ubiquitin-proteasome system. FOXS1 was also found to facilitate the interaction between TGFBI and lysosomal associated membrane protein 2A (LAMP2A), leading to the translocation of TGFBI into lysosomes for degradation. Additionally, FOXS1 regulates AKT phosphorylation and FOXO3a nuclear translocation, promoting the transcription of autophagy-related genes downstream of FOXO3a. Restoration of TGFBI expression reversed the suppressive effect exerted by FOXS1 on the growth of colorectal cancer cells. FOXS1 functions as a tumor suppressor that is methylated in CRC and promotes the lysosomal degradation of TGFBI, regulates cell growth and promotes autophagy in CRC through the TGFBI/AKT/FOXO3a signaling pathway. These findings indicate that FOXS1 exhibits potential as a promising biomarker and therapeutic target for colorectal cancer.

Exploring the Mechanism of Sempervirine Inhibiting Glioblastoma Invasion Based on Network Pharmacology and Bioinformatics

Background: Invasion is an important characteristic of the malignancy of glioblastoma (GBM) and a significant prognostic factor. Sempervirine (SPV), a yohimbine-type alkaloid, has been proven to inhibit GBM cells proliferation in previous research and found to have a potential effect in anti-invasion, but its mechanism of anti-invasion is still unknown. Methods: To explore its pharmacodynamics in inhibiting GBM cell invasion in this study, we combined network pharmacology and bioinformatics to comprehensive exploratory analysis of SPV and verified the mechanism in vitro. Results: Firstly, targets of SPV and invasion-related genes were collected from public databases. Moreover, GBM samples were obtained to analyze differentially expressed genes (DEGs) from The Cancer Genome Atlas (TCGA). Then, the relevant targets of SPV inhibiting GBM invasion (SIGI) were obtained through the intersection of the three gene sets. Further, GO and KEGG analysis showed that the targets of SIGI were heavily enriched in the AKT signaling pathway. Subsequently, based on the method of machine learning, a clinical prognostic model of the relevant targets of SIGI was constructed using GBM samples from TCGA and the Gene Expression Omnibus (GEO). A four-genes model (DUSP6, BMP2, MMP2, and BMP2) was successfully constructed, and Vina Scores of MMP2 and MMP13 in molecular docking were higher, which may be the main targets of SIGI. Then, the effect of SIGI was confirmed via functional experiments on invasion, migration, and adhesion assay, and the effect involved changes in the expressions of p-AKT, MMP2 and MMP13. Finally, combined with AKT activator (SC79) and inhibitor (MK2206), we further confirmed that SPV inhibits GBM invasion through AKT phosphorylation. Conclusions: This study provides valuable and an expected point of view into the regulation of AKT phosphorylation and inhibition of GBM invasion by SPV.

CircLIFRSA/miR-1305/PTEN axis attenuates malignant cellular processes in non-small cell lung cancer by regulating AKT phosphorylation

BackgroundNon-small cell lung cancer (NSCLC) is typically diagnosed at advanced stages, which limits the effectiveness of therapeutic interventions. The present study aimed to explore the role of the newly identified circLIFRSA in the PTEN/AKT signaling pathway and its involvement in the malignant processes of NSCLC.MethodsCircLIFRSA expression was identified through microarray analysis, and its levels in NSCLC samples were quantified by RT-qPCR. The impact of circLIFRSA on cell growth, proliferation, apoptosis, and cell cycle were evaluated by MTT assay, colony formation assay, and flow cytometry. Additionally, Western blotting was employed to analyze the expression of PTEN and phosphorylated AKT (pAKT) in NSCLC cells.ResultsThe expression of circLIFRSA was found to be significantly reduced in NSCLC cells and tissues. This downregulation correlated with various clinicopathological characteristics and indicated its potential as an early diagnostic biomarker for NSCLC. Importantly, circLIFRSA was shown to inhibit cell growth and proliferation while promoting apoptosis in NSCLC cells. Mechanically, circLIFRSA was found to attenuate the malignant processes of NSCLC cells via the miR-1305/PTEN axis and the suppression of AKT phosphorylation.ConclusionsThese findings indicate that circLIFRSA/miR-1305/PTEN axis attenuates malignant processes by regulating AKT phosphorylation, and provide new insights into the potential of circLIFRSA as a biomarker for early diagnosis and as a promising therapeutic target in NSCLC.

Caffeine alleviate lipopolysaccharide-induced neuroinflammation and depression through regulating p-AKT and NF-κB

Caffeine, a nonselective adenosine receptor antagonist, is the major component of coffee and the most consumed psychostimulant at nontoxic doses in the world. It has been identified that caffeine consumption reduces the risk of several neurological diseases. However, the mechanisms by which it impacts the pathophysiology of neurological diseases remain to be elucidated. In this study, we investigated whether caffeine exerts anti-inflammatory effects on lipopolysaccharide (LPS)-induced inflammation and depression in vivo and explored the potential mechanism of caffeine through LPS-induced brain injury. Adult male Sprague-Dawley (SD) rats were intraperitoneal injected with various concentrations of LPS to induce the neuroinflammation and depressive-like behavior. Then SD rats were treated with caffeine in the presence or absence of LPS. Open-filed and closed-field tests were applied to detect the behaviors of SD rats, while western blot was performed to measure the phosphorylation level of protein kinase B (p-AKT) and nuclear factor κB (NF-κB) in the cortex after caffeine was orally administered. Our findings indicated that caffeine markedly improved the neuroinflammation and depressive-like behavior of LPS-treated SD rats. Mechanistic investigations demonstrated that caffeine down-regulated the expression of p-AKT and NF-κB in LPS-induced SD rats cortex. Taken together, these results indicated that caffeine, a potential agent for preventing inflammatory diseases, may suppress LPS-induced inflammatory and depressive responses by regulating AKT phosphorylation and NF-κB.

Daphnetin Alleviates Bleomycin-Induced Pulmonary Fibrosis through Inhibition of Epithelial-to-Mesenchymal Transition and IL-17A.

Idiopathic pulmonary fibrosis (IPF) is a chronic and refractory interstitial lung disease. Although there is no cure for IPF, the development of drugs with improved efficacy in the treatment of IPF is required. Daphnetin, a natural coumarin derivative, has immunosuppressive, anti-inflammatory, and antioxidant activities. However, its antifibrotic effects have not yet been elucidated. In this study, we investigated the antifibrotic effects of daphnetin on pulmonary fibrosis and the associated molecular mechanism. We examined the effects of daphnetin on splenocytes cultured in Th17 conditions, lung epithelial cells, and a mouse model of bleomycin (BLM)-induced pulmonary fibrosis. We identified that daphnetin inhibited IL-17A production in developing Th17 cells. We also found that daphnetin suppressed epithelial-to-mesenchymal transition (EMT) in TGF-β-treated BEAS2B cells through the regulation of AKT phosphorylation. In BLM-treated mice, the oral administration of daphnetin attenuated lung histopathology and improved lung mechanical functions. Our findings clearly demonstrated that daphnetin inhibited IL-17A and EMT both in vitro and in vivo, thereby protecting against BLM-induced pulmonary fibrosis. Taken together, these results suggest that daphnetin has potent therapeutic effects on lung fibrosis by modulating both Th17 differentiation and the TGF-β signaling pathway, and we thus expect daphnetin to be a drug candidate for the treatment of IPF.

Antidiabetic and antihyperlipidemic activities of Phyllanthus emblica L. extract in vitro and the regulation of Akt phosphorylation, gluconeogenesis, and peroxisome proliferator-activated receptor α in streptozotocin-induced diabetic mice.

The fruits of Phyllanthus emblica L. are high in nutrients and have excellent health care function and developmental value. There are many management strategies available for diabetes and hyperlipidemia. Nevertheless, there is a lack of an effective and nontoxic drug. The present study was designed to first screen four extracts of P. emblica L. on insulin signaling target gene expression levels, including glucose transporter 4 (GLUT4) and p-Akt/t-Akt. The ethyl acetate extract of P. emblica L. (EPE) exhibited the most efficient activity among the four extracts and was thus chosen to explore the antidiabetic and antihyperlipidemic activities in streptozotocin (STZ)-induced type 1 diabetic mice. All mice (in addition to one control (CON) group) were administered STZ injections (intraperitoneal) for 5 consecutive days, and then STZ-induced mice were administered EPE (at 100, 200, or 400 mg/kg body weight), fenofibrate (Feno) (at 250 mg/kg body weight), glibenclamide (Glib) (at 10 mg/kg body weight), or vehicle by oral gavage once daily for 4 weeks. Finally, histological examination, blood biochemical parameters, and target gene mRNA expression levels were measured, and liver tissue was analyzed for the levels of malondialdehyde (MDA), a maker of lipid peroxidation. EPE treatment resulted in decreased levels of blood glucose, HbA1C, triglycerides (TGs), and total cholesterol and increased levels of insulin compared with the vehicle-treated STZ group. EPE treatment decreased blood levels of HbA1C and MDA but increased glutathione levels in liver tissue, implying that EPE exerts antioxidant activity and could prevent oxidative stress and diabetes. The EPE-treated STZ mice displayed an improvement in the sizes and numbers of insulin-expressing β cells. EPE treatment increased the membrane expression levels of skeletal muscular GLUT4, and also reduced hepatic mRNA levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase thereby inhibiting hepatic gluconeogenesis. This resulted in a net glucose lowering effect in EPE-treated STZ mice. Furthermore, EPE increased the expression levels of p-AMPK/t-AMPK in both the skeletal muscle and liver tissue compared with vehicle-treated STZ mice. EPE-treated STZ mice showed enhanced expression levels of fatty acid oxidation enzymes, including peroxisome proliferator-activated receptor α (PPARα), but reduced expression levels of lipogenic genes including fatty acid synthase, as well as decreased mRNA levels of sterol regulatory element binding protein 1c (SREBP1c), apolipoprotein-CIII (apo-CIII), and diacylglycerol acyltransferase-2 (DGAT2). This resulted in a reduction in plasma TG levels. EPE-treated STZ mice also showed reduced expression levels of PPAR γ. This resulted in decreased adipogenesis, fatty acid synthesis, and lipid accumulation within liver tissue, and consequently, lower TG levels in liver tissue and blood. Furthermore, EPE treatment not only displayed an increase in the Akt activation in liver tissue, but also in C2C12 myotube in the absence of insulin. These results implied that EPE acts as an activator of AMPK and /or as a regulator of the insulin (Akt) pathway. Taken together, EPE treatment exhibited amelioration of the diabetic and hyperlipidemic state in STZ-induced diabetic mice.

Dlk2 interacts with Syap1 to activate Akt signaling pathway during osteoclast formation

Excessive osteoclast formation and bone resorption are related to osteolytic diseases. Delta drosophila homolog-like 2 (Dlk2), a member of the epidermal growth factor (EGF)-like superfamily, reportedly regulates adipocyte differentiation, but its roles in bone homeostasis are unclear. In this study, we demonstrated that Dlk2 deletion in osteoclasts significantly inhibited osteoclast formation in vitro and contributed to a high-bone-mass phenotype in vivo. Importantly, Dlk2 was shown to interact with synapse-associated protein 1 (Syap1), which regulates Akt phosphorylation at Ser473. Dlk2 deletion inhibited Syap1-mediated activation of the AktSer473, ERK1/2 and p38 signaling cascades. Additionally, Dlk2 deficiency exhibits increased bone mass in ovariectomized mice. Our results reveal the important roles of the Dlk2-Syap1 signaling pathway in osteoclast differentiation and osteoclast-related bone disorders.

Butein inhibits corticosterone-induced apoptosis of Neuro2A cells by maintaining MEK-ERK signaling.

Stress-induced overactivation of glucocorticoid signaling may contribute to mental illness by inducing neuronal death and dysfunction. We previously reported that pretreatment with the plant flavonoid butein inhibits corticosterone (CORT)-induced apoptosis of Neuro2A (N2A) cells. In the current study, we examined whether MEK-ERK and PI3K-AKT signaling pathways are involved in neuroprotection by butein. N2A cells were pre-incubated with serum-free DMEM containing 0.5μM butein for 30min, and then incubated with serum-free DMEM containing 0.5µM butein, 50µM CORT, 50µM LY294002, or 50µM PD98059 as indicated for 24h. We subsequently performed the MTT assay and the western blot analysis. As expected, CORT considerably reduced N2A cell viability and increased relative expression of the apoptosis effector cleaved caspase-3, whereas pretreatment with butein blocked these cytotoxic effects. Treatment with CORT alone also decreased both AKT and ERK protein phosphorylation. Butein pretreatment had no effect on AKT phosphorylation, and only partially reversed the reduction in phosphorylated ERK. However, cotreatment with butein and the PI3K inhibitor LY294002 during CORT exposure enhanced ERK phosphorylation, whereas cotreatment with butein and the ERK phosphorylation/activation inhibitor PD98059 enhanced AKT phosphorylation, suggesting that MEK-ERK negatively regulates AKT phosphorylation. Moreover, the protective efficacy of butein was blocked by PD98059 cotreatment but not LY294002 cotreatment. These findings suggest that butein protects neurons against glucocorticoid-induced apoptosis by sustaining ERK phosphorylation and downstream signaling.

GDF15 negatively regulates chemosensitivity via TGFBR2-AKT pathway-dependent metabolism in esophageal squamous cell carcinoma.

Treating patients with esophageal squamous cell carcinoma (ESCC) is challenging due to the high chemoresistance. Growth differentiation factor 15 (GDF15) is crucial in the development of various types of tumors and negatively related to the prognosis of ESCC patients according to our previous research. In this study, the link between GDF15 and chemotherapy resistance in ESCC was further explored. The relationship between GDF15 and the chemotherapy response was investigated through in vitro and in vivo studies. ESCC patients with high levels of GDF15 expression showed an inferior chemotherapeutic response. GDF15 improved the tolerance of ESCC cell lines to low-dose cisplatin by regulating AKT phosphorylation via TGFBR2. Through an in vivo study, we further validated that the anti-GDF15 antibody improved the tumor inhibition effect of cisplatin. Metabolomics showed that GDF15 could alter cellular metabolism and enhance the expression of UGT1A. AKT and TGFBR2 inhibition resulted in the reversal of the GDF15-induced expression of UGT1A, indicating that TGFBR2-AKT pathway-dependent metabolic pathways were involved in the resistance of ESCC cells to cisplatin. The present investigation suggests that a high level of GDF15 expression leads to ESCC chemoresistance and that GDF15 can be targeted during chemotherapy, resulting in beneficial therapeutic outcomes.

Terminal Uridylyltransferases TUT4/7 Regulate microRNA and mRNA Homeostasis.

The terminal nucleotidyltransferases TUT4 and TUT7 (TUT4/7) regulate miRNA and mRNA stability by 3' end uridylation. In humans, TUT4/7 polyuridylates both mRNA and pre-miRNA, leading to degradation by the U-specific exonuclease DIS3L2. We investigate the role of uridylation-dependent decay in maintaining the transcriptome by transcriptionally profiling TUT4/7 deleted cells. We found that while the disruption of TUT4/7 expression increases the abundance of a variety of miRNAs, the let-7 family of miRNAs is the most impacted. Eight let-7 family miRNAs were increased in abundance in TUT4/7 deleted cells, and many let-7 mRNA targets are decreased in abundance. The mRNAs with increased abundance in the deletion strain are potential direct targets of TUT4/7, with transcripts coding for proteins involved in cellular stress response, rRNA processing, ribonucleoprotein complex biogenesis, cell-cell signaling, and regulation of metabolic processes most affected in the TUT4/7 knockout cells. We found that TUT4/7 indirectly control oncogenic signaling via the miRNA let-7a, which regulates AKT phosphorylation status. Finally, we find that, similar to fission yeast, the disruption of uridylation-dependent decay leads to major rearrangements of the transcriptome and reduces cell proliferation and adhesion.

METTL3-IGF2BP3-axis mediates the proliferation and migration of pancreatic cancer by regulating spermine synthase m6A modification.

Spermine synthase (SMS) is an enzyme participating in polyamine synthesis; however, its function and role in pancreatic cancer remains elusive. Here we report that SMS is upregulated in pancreatic cancer and predicts a worse overall survival and significantly promotes the proliferation and migration of pancreatic cancer cells. Excessive SMS reduces the accumulation of spermidine by converting spermidine into spermine, which activates the phosphorylation of serine/threonine kinase (AKT) and epithelial-mesenchymal transition (EMT) signaling pathway, thereby inhibiting pancreatic cancer cell proliferation and invasion. Moreover, SMS was identified as the direct target of both methyltransferase like 3 (METTL3) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly bind to the m6A modification sites of SMS and inhibit mRNA degradation. Knockdown of METTL3 or IGF2BP3 significantly reduced the SMS protein expression and inhibited the migration of pancreatic cancer. We propose a novel regulatory mechanism in which the METTL3-IGF2BP3 axis mediates the mRNA degradation of SMS in an m6A-dependent manner to regulate spermine/spermidine conversion, which regulates AKT phosphorylation and EMT activation, thereby inducing tumor progression and migration in pancreatic cancer.

Reproductive toxicity of ritonavir in male: Insight into mouse sperm capacitation

Since COVID-19 began in 2019, therapeutic agents are being developed for its treatment. Among the numerous potential therapeutic agents, ritonavir (RTV), an anti-viral agent, has recently been identified as an important element of the COVID-19 treatment. Moreover, RTV has also been applied in the drug repurposing of cancer cells. However, previous studies have shown that RTV has toxic effects on various cell types. In addition, RTV regulates AKT phosphorylation within cancer cells, and AKT is known to control sperm functions (motility, capacitation, and so on). Although deleterious effects of RTV have been reported, it is not known whether RTV has male reproduction toxicity. Therefore, in this study, we aimed to investigate the effects of RTV on sperm function and male fertility. In the present study, sperm collected from the cauda epididymis of mice were incubated with various concentrations of RTV (0, 0.1, 1, 10, and 100 μM). The expression levels of AKT, phospho-AKT (Thr308 and Ser473), and phospho-tyrosine proteins, sperm motility, motion kinematics, capacitation status, and cell viability were assessed after capacitation. The results revealed that AKT phosphorylation at Thr308 and Ser473 was significantly increased, and the levels of tyrosine-phosphorylated proteins (at approximately 25 and 100 kDa) were significantly increased in a dose-dependent manner. In addition, RTV adversely affected sperm motility, motion kinematics, and cell viability. Taken together, RTV may have negative effects on sperm function through an abnormal increase in tyrosine phosphorylation and phospho-AKT levels. Therefore, individuals taking or prescribing RTV should be aware of its reproductive toxicity.

CircSEMA4B inhibits the progression of breast cancer by encoding a novel protein SEMA4B-211aa and regulating AKT phosphorylation

PI3K/AKT signaling pathway plays an important role in regulating the tumorigenesis, recurrence, and metastasis of breast cancer (BC). In this study, we discovered a circRNA with protein-coding potential, which we named circSEMA4B. CircSEMA4B could encode a novel protein, SEMA4B-211aa. Both circSEMA4B and SEMA4B-211aa were remarkably downregulated in BC tissues and cell lines. Low expression of circSEMA4B was positively associated with TNM stage, tumor size, lymph node metastasis, and distant metastasis of BC patients. The functional investigation showed that circSEMA4B and SEMA4B-211aa could significantly inhibit the proliferation and migration of BC in vivo and in vitro. Of note, SEMA4B-211aa inhibited the generation of PIP3 by binding to p85, thereby inhibiting the phosphorylation of AKT (Thr308). CircSEMA4B inhibited the phosphorylation of AKT (Ser473) through miR-330-3p/PDCD4 axis. Taken together, circSEMA4B is a novel negative regulator of PI3K/AKT signaling pathway, providing novel mechanistic insights into the underlying mechanisms of BC.

P10.11.B Re-sensitizing cancer cells to Tumor Treating Fields (TTFields) through PI3K/AKT/mTOR pathway inhibition

Abstract Background Tumor Treating Fields (TTFields) are alternating electric fields disrupting cellular processes critical for cancer cell replication and tumor progression. TTFields therapy is approved for treatment of newly diagnosed glioblastoma (GBM), recurrent GBM, and unresectable malignant pleural mesothelioma, and is currently under clinical investigation for treating other solid tumors, such as ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The research herein aimed to identify potential treatment escape mechanisms and explore the possibility of targeted inhibition of these pathways for re-sensitizing the cells to TTFields. Material and Methods GBM U-87 MG, ovarian A2780, and NSCLC H1299 cells were treated with TTFields (1.7 V/cm RMS, 200 or 150 kHz) for continuous long-term application (7 or 13 days, specific conditions depending on the cell line). Changes in signaling pathways in these cells relative to cells exposed to short term TTFields application (3 or 7 days, depending on cell line) were examined by Luminex multiplex assay. Specific pathway markers were examined by immunohistochemistry of tumor sections from sham or TTFields-treated rats bearing N1S1 HCC tumors. TTFields were then co-applied with relevant pathway inhibitors, followed by cell count measurements and western blot examinations for specific pathway markers. The concomitant application of TTFields with a selected inhibitor was tested in mice inoculated orthotopically with MOSE-L firefly luciferase (FFL) ovarian cancer cells. Tumor volume was measured at study end by luciferin signal detection using the In Vivo Imaging System (IVIS). Results Cancer cells exposed to long-term application of TTFields displayed decreased sensitivity to TTFields. The PI3K/AKT/mTOR pathway was activated in these cells, with significant increases in AKT and RPS6 phosphorylation levels also observed in HCC tumors from rats treated with TTFields. PI3K inhibitors re-sensitized the cells to TTFields cytotoxicity, with associated down regulation of AKT phosphorylation. Application of TTFields concomitant with the PI3K inhibitor alpelisib resulted in enhanced efficacy in the ovarian cancer mouse model. Conclusions The current research demonstrated that PI3K/AKT/mTOR signaling pathway activation was involved in the response to long-term application of TTFields, with increased phosphorylation of key proteins observed both in vitro and in vivo. Concomitant treatment with PI3K inhibitors and TTFields re-sensitized the cells to TTFields, as it alleviated TTFields-induced AKT activation.

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway.

Sorafenib is currently the first-line treatment for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a significant challenge. Aberrant AKT signaling activation is a crucial mechanism driving sorafenib resistance in HCC. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a vital role in antitumor immune responses. In this study, we demonstrate that aberrant PCSK9 upregulation promotes cell proliferation and sorafenib resistance in HCC by inducing AKT-S473 phosphorylation. After palmitoylation at cysteine 600, the binding affinity between PCSK9 and tensin homolog (PTEN) is dramatically increased, inducing lysosome-mediated PTEN degradation and subsequent AKT activation. We identify zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) as a palmitoyltransferase that promotes PCSK9 palmitoylation at cysteine 600. We also develop a biologically active PCSK9-derived peptide that competitively inhibits PCSK9 palmitoylation, suppressing AKT phosphorylation and augmenting the antitumor effects of sorafenib in HCC.

Early Growth Response 1 Strengthens Pol-III-Directed Transcription and Transformed Cell Proliferation by Controlling PTEN/AKT Signalling Activity.

RNA polymerase III (Pol III) products play essential roles in ribosome assembly, protein synthesis, and cell survival. Deregulation of Pol-III-directed transcription is closely associated with tumorigenesis. However, the regulatory pathways or factors controlling Pol-III-directed transcription remain to be investigated. In this study, we identified a novel role of EGR1 in Pol-III-directed transcription. We found that Filamin A (FLNA) silencing stimulated EGR1 expression at both RNA and protein levels. EGR1 expression positively correlated with Pol III product levels and cell proliferation activity. Mechanistically, EGR1 downregulation dampened the occupancies of Pol III transcription machinery factors at the loci of Pol III target genes. Alteration of EGR1 expression did not affect the expression of p53, c-MYC, and Pol III general transcription factors. Instead, EGR1 activated RhoA expression and inhibited PTEN expression in several transformed cell lines. We found that PTEN silencing, rather than RhoA overexpression, could reverse the inhibition of Pol-III-dependent transcription and cell proliferation caused by EGR1 downregulation. EGR1 could positively regulate AKT phosphorylation levels and is required for the inhibition of Pol-III-directed transcription mediated by FLNA. The findings from this study indicate that EGR1 can promote Pol-III-directed transcription and cell proliferation by controlling the PTEN/AKT signalling pathway.

CaMKII and Kalirin, a Rac1-GEF, regulate Akt phosphorylation involved in contraction-induced glucose uptake in skeletal muscle cells

Rac1 plays an important role in contraction-stimulated muscle glucose uptake, but the mechanism is not fully elucidated. We previously identified Rac1-dependent activation of Akt played a partial role in contraction-stimulated GLUT4 translocation to the cell surface of C2C12 myotubes. Recognizing that contraction activates CaMKII in muscle and CaMKII is known to regulate Rac1 activity in other systems, here we investigated the relationship between CaMKII, Akt and contraction-stimulated glucose uptake. Expression of a constitutively-active mutant of CaMKIIδ stimulated Akt phosphorylation that was inhibited by Rac1 inhibitor II. C2C12 myotubes were contracted by electrical pulse stimulation (EPS). We observed the CaMKII inhibitor, KN-93 and CaMKIIδ siRNA-mediated knockdown, reduced EPS-induced Akt phosphorylation in C2C12 myotubes. ITX3, an inhibitor of the Rac-GTPase Kalirin and Kalirin siRNA-mediated knockdown reduced EPS-stimulated Akt phosphorylation in myotubes. In addition, the Akt inhibitor MK2206 partly reduced EPS-stimulated glucose uptake without simultaneously affecting CaMKII phosphorylation and Kalirin protein abundance. Our findings demonstrate EPS leads to Akt activation through a CaMKII-Kalirin-Rac1 signaling pathway and partly regulates contraction-stimulated glucose uptake in muscle cells.

Transketolase promotes colorectal cancer metastasis through regulating AKT phosphorylation

Transketolase (TKT) which is an important metabolic enzyme in the pentose phosphate pathway (PPP) participates in maintaining ribose 5-phosphate levels. TKT is necessary for maintaining cell growth. However, we found that in addition to this, TKT can also affect tumor progression through other ways. Our previous study indicate that TKT could promote the development of liver cancer by affecting bile acid metabolism. And in this study, we discovered that TKT expression was remarkably upregulated in colorectal cancer, abnormal high expression of TKT is associated with poor prognosis of colorectal cancer. Additionally, TKT promoted colorectal cancer cell growth and metastasis. Further study demonstrated that TKT interacted with GRP78 and promoted colorectal cancer cell glycolysis through increasing AKT phosphorylation, thereby enhancing colorectal cancer cell metastasis. Thus, TKT is expected to become an indicator for judging the prognosis of colorectal cancer, and provide a theoretical basis for drug development of new treatment targets for colorectal cancer.

Rutin promotes activation and reduces apoptosis of primordial follicles by regulating Akt phosphorylation after in vitro culture of ovine ovarian tissue

The aims of this study were to analyze the effects of different concentrations of rutin on primordial follicle survival and development after invitro culture of sheep ovarian tissue, and to verify the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway in the rutin actions. Ovarian fragments were fixed for histological analysis (fresh control) or cultured in α-minimum essential medium alone (α-MEM+: control medium) or in α-MEM+supplemented with different concentrations of rutin (0.1; 1 or 10μg/mL) for 7 days. Inhibition of the PI3K activity was performed in fragments cultured with 50μM LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3 (apoptosis) and Akt phosphorylation (p-Akt). The results showed that 1μg/mL rutin has a greater percentage of normal follicles (P<0.05) than those of α-MEM+ and other rutin treatments. In addition, 1μg/mL rutin maintained the follicular apoptosis similar (P>0.05) to that of the fresh control and lower than α-MEM+ and 10μg/mL rutin. All rutin concentrations increased (P<0.05) follicular activation compared to fresh control and α-MEM+. Furthermore, follicular and oocyte diameters increased (P<0.05) only after culture with 1μg/mL rutin. After PI3K inhibition, there was a reduction (P<0.05) of rutin follicular effects. In conclusion, rutin at 1μg/mL reduces apoptosis, promotes activation and growth of sheep primordial follicles through the modulation of the PI3K/Akt signaling pathway after invitro culture of ovine ovarian tissue.

Circular RNA VRK1 facilitates pre-eclampsia progression via sponging miR-221-3P to regulate PTEN/Akt.

Pre‐eclampsia (PE) is a worldwide pregnancy‐related disorder. It is mainly characterized by defect migration and invasion of trophoblast cells. Recently, circular RNAs (circRNAs) have been believed to play a vital role in PE. The expression patterns and the biological functions of circRNAs in PE remain elusive. Here, we performed a circRNA microarray to identify putative PE‐related circRNAs. Bioinformatics analyses were used to screen the circRNAs which have potential relationships with pre‐eclampsia, and we identified a novel circRNA (circVRK1) that was up‐regulated in PE placenta tissues. By using HTR‐8/SVneo cells, circVRK1 knockdown significantly enhanced cell migration and invasion abilities, as well as epithelial‐mesenchymal transition (EMT). Mechanistically, we found that circVRK1 and PTEN could function as the ceRNAs to miR‐221‐3p. Overexpression of miR‐221‐3p promoted cell migration, invasion and EMT via regulating PTEN. The cotransfection of miR‐221‐3p inhibitor or PTEN reversed the effect from circVRK1 knockdown. Moreover, the circVRK1/miR‐221‐3p/PTEN axis greatly regulated Akt phosphorylation. In general, circVRK1 suppresses trophoblast cell migration, invasion and EMT, by acting as a ceRNA to miR‐221‐3p to regulate PTEN, and further inhibit PI3K/Akt activation. The purpose of this paper is to open wide insights to investigate the onset of PE and provide new potential therapeutic targets in PE.