Induced Phosphorylation Of Akt Research Articles

Acute Kaempferol Stimulation Induces AKT Phosphorylation in HepG2 Cells.

Type 2 diabetes mellitus (T2DM) stands as a prevalent global public health issue caused by deficiencies in the action of insulin and/or insulin production. In the liver, insulin plays an important role by inhibiting hepatic glucose production and stimulating glycogen storage, thereby contributing to blood glucose regulation. Kaempferitrin (KP) and kaempferol (KM), flavonoids found in Bauhinia forficata, exhibit insulin-mimetic properties, showing promise in managing T2DM. In this study, we aimed to assess the potential of these compounds in modulating the insulin signaling pathway and/or glucose metabolism. Cell viability assays confirmed the non-cytotoxic nature of both compounds toward HepG2 cells at the concentrations and times evaluated. Theoretical molecular docking studies revealed that KM had the best docking pose with the IR β subunit when compared to the KP. Moreover, Langmuir monolayer evaluation indicated molecular incorporation for both KM and KP. Specifically, KM exhibited the capability to increase AKT phosphorylation, a key kinase in insulin signaling, regardless of insulin receptor (IR) activation. Notably, KM showed an additional synergistic effect with insulin in activating AKT. In conclusion, our findings suggest the potential of KM as a promising compound for stimulating AKT activation, thereby influencing energy metabolism in T2DM.

ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque

IntroductionThe N-terminal domain of angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity. Its C-terminal fibrinogen-like (FBN) domain is a ligand of macrophage integrin αvβ3. ObjectivesANGPTL3 might home to plaque where it directly regulates macrophage function via integrin αvβ3 for atherosclerosis progression. MethodsLdlr-/- mice on a high-fat diet and ApoE-/- mice on a chow diet were received adeno-associated virus (AAV)-mediated Angptl3 gene transfer and followed up for 12 weeks. ApoE-/- mice were injected AAV containing FLAG-tagged Angptl3 cDNA for tracing. Atherosclerotic features were compared between Angptl3-/-ApoE-/- mice and ApoE-/- littermates. THP-1 cells were exposed to 0 or 50 μg/ml ANGPTL3 FBN domain for 24 h to evaluate Toll-like receptor (TLR)4 expression using western blot analysis and circulating cytokine and chemokine profiles by the MILLIPLEX MAP assay. Phospho-proteomic profile was established in ANGPTL3-treated macrophages. Integrin β3 deficient THP-1 cells were obtained by sgRNAs targeting RGD sequence using Lentivirus-Cas9 system. ResultsAngptl3 overexpression increased atherosclerotic progression and CD68+ macrophages in plaque (p < 0.05 for all). By immunostaining, FLAG+ cells were identified in plaque of gene transferred ApoE-/- mice. Fluorescent immunostaining detected co-localisation of Angptl3 and CD68 in plaque macrophages. Phospho-proteomic analysis revealed that Angptl3 induced phosphorylation of proteins that were involved in the IL-17 signalling pathway in THP-1 cells. In vitro, ANGPTL3 treatment increased the production of interleukin (IL)-1β and tumour necrosis factor-α in THP-1 cells (p < 0.05 for both). Exposure of ANGPTL3 to THP-1 cells induced Akt phosphorylation which was weakened in integrin β3 deficient ones. ANGPTL3 elevated TLR4 expression via Akt phosphorylation. In response to lipopolysaccharide, nuclear factor-κB activity was 2.2-fold higher in THP-1 cells pre-treated with ANGPTL3 than in untreated cells (p < 0.05). ConclusionsTargeting ANGPTL3 could yield a dual benefit of lowering lipid levels in the blood and suppressing macrophage activation in plaque.

Research Note: Preliminary functional analysis of EGF-like domains of Eimeria tenella microneme protein 7 identified in sporozoites and merozoites

Eimeria tenella, an obligate intracellular apicomplexan parasite, is the major causative agent of chicken coccidiosis. Some epidermal growth factor (EGF)-like domain-containing proteins of other members of apicomplexan parasites have been reported to contribute to parasite survival. To date, however, EGF-like domain-containing proteins of E. tenella are not well studied. In this study, a gene fragment that encodes 4 EGF-like domains of E. tenella microneme protein 7 (EGF-EtMIC7) was amplified and expressed using an Escherichia coli expression system. Following generation of polyclonal antibodies that recognize recombinant EGF-EtMIC7 (rEGF-EtMIC7), the expression of EtMIC7 in sporozoites and merozoites was examined. Moreover, its roles in cellular regulation were investigated. The native EtMIC7 in E. tenella sporozoites and merozoites was detected by using Western blot and indirect immunofluorescence assays. rEGF-EtMIC7 could activate Akt, whereas blockade of EGF receptor (EGFR) failed to induce Akt phosphorylation. Compared with the control group, LMH cells treated with rEGF-EtMIC7 showed increased cell proliferation and expressed higher levels of B cell leukemia/lymphoma 2 (BCL-2). These findings contribute to the better understanding of parasite-host interactions at the molecular level during E. tenella infection.

NUAK1 coordinates growth factor-dependent activation of mTORC2 and Akt signaling

BackgroundmTORC2 is a critical regulator of cytoskeleton organization, cell proliferation, and cancer cell survival. Activated mTORC2 induces maximal activation of Akt by phosphorylation of Ser-473, but regulation of Akt activity and signaling crosstalk upon growth factor stimulation are still unclear.ResultsWe identified that NUAK1 regulates growth factor-dependent activation of Akt by two mechanisms. NUAK1 interacts with mTORC2 components and regulates mTORC2-dependent activation of Akt by controlling lysosome positioning and mTOR association with this organelle. A second mechanism involves NUAK1 directly phosphorylating Akt at Ser-473. The effect of NUAK1 correlated with a growth factor-dependent activation of specific Akt substrates. NUAK1 induced the Akt-dependent phosphorylation of FOXO1/3a (Thr-24/Thr-32) but not of TSC2 (Thr-1462). According to a subcellular compartmentalization that could explain NUAK1’s differential effect on the Akt substrates, we found that NUAK1 is associated with early endosomes but not with plasma membrane, late endosomes, or lysosomes. NUAK1 was required for the Akt/FOXO1/3a axis, regulating p21CIP1, p27KIP1, and FoxM1 expression and cancer cell survival upon EGFR stimulation. Pharmacological inhibition of NUAK1 potentiated the cell death effect induced by Akt or mTOR pharmacological blockage. Analysis of human tissue data revealed that NUAK1 expression positively correlates with EGFR expression and Akt Ser-473 phosphorylation in several human cancers.ConclusionsOur results showed that NUAK1 kinase controls mTOR subcellular localization and induces Akt phosphorylation, demonstrating that NUAK1 regulates the growth factor-dependent activation of Akt signaling. Therefore, targeting NUAK1, or co-targeting it with Akt or mTOR inhibitors, may be effective in cancers with hyperactivated Akt signaling.

Platelet-Derived Apoptotic Vesicles Promote Bone Regeneration via Golgi Phosphoprotein 2 (GOLPH2)-AKT Signaling Axis.

Apoptotic vesicles (apoVs) are apoptotic-cell-derived nanosized vesicles that take on dominant roles in regulating bone homeostasis. We have demonstrated that mesenchymal stem cell (MSC)-derived apoVs are promising therapeutic agents for bone regeneration. However, clinical translation of MSC-derived apoVs has been hindered due to cell expansion and nuclear substance. As another appealing source for apoV therapy, blood cells could potentially eliminate these limitations. However, whether blood cells can release apoVs during apoptosis is uncertain, and the detailed characteristics and biological properties of respective apoVs are not elucidated. In this study, we showed that platelets (PLTs) could rapidly release abundant apoVs during apoptosis in a short time. To recognize the different protein expressions between PLT-derived apoVs and PLTs, we established their precise protein landscape. Furthermore, we identified six proteins specifically enriched in PLT-derived apoVs, which could be considered as specific biomarkers. More importantly, PLT-derived apoVs promoted osteogenesis of MSCs and rescued bone loss via Golgi phosphoprotein 2 (GOLPH2)-induced AKT phosphorylation, therefore, leading to the emergence of their potential in bone regeneration. In summary, we comprehensively determined characteristics of PLT-derived apoVs and confirmed their roles in bone metabolism through previously unrecognized GOPLH2-dependent AKT signaling, providing more understanding for exploring apoV-based therapy in bone tissue engineering.

Abstract C119: Combining forces: Integrating engineered bacterial therapeutics with targeted therapies for advanced-stage non-small cell lung cancer

Abstract Currently there are about 400 single-agent targeted therapies in various stages of clinical trials for non-small cell lung cancer (NSCLC) worldwide. However, less than 10% of them have been utilized in combination treatment to improve efficacy of advanced-stage NSCLC due to safety issues. As a result, emerging and safe treatment modalities such as engineered bacteria therapy, commonly known as “bugs as drugs”, in ongoing clinical trials (NCT00004988, NCT04589234, NCT05038150) for advanced-NSCLCs - have not been combined with current standards of care. We hypothesized that If advanced-stage NSCLC tumors are dependent on altered growth signaling upon single-agent bacteria therapy, blocking those signaling with current targeted therapy may provide a promising combination treatment strategy to achieve better efficacy. To test this, we first treated NSCLC-derived spheroids with engineered bacteria producing Theta toxin after localizing in the hypoxic core and eliminating spheroids from inside. Second, we characterized the signaling pathways of bacteria-treated NSCLC spheroids by RNA-seq followed by Gene Set Enrichment Analysis. Third, we blocked altered signaling with current targeted therapies to achieve better efficacy. Finally, we designed an optimum combination treatment and validated it in vivo in xenografted NSCLCs. We found that bacterially secreted Theta toxin significantly altered PI3K/AKT/mTOR signaling, cell cycle checkpoint and DNA repair pathways (normalized p-value &amp;lt; 0.5 and falls discovery rate q-value &amp;lt; 0.5) in multiple NSCLC spheroids, including one established from a chemotherapy and radiation-resistant tumor. We chose current targeted inhibitors of AKT (MK2206), CHK (AZD7762), WNT (IWR-1), ERK (SCH772984), MEK (Selumetinib), and 2 chemotherapy drugs (paclitaxel, pemetrexed) to treat these bacterially-treated NSCLC spheroids. We found that blocking AKT or CHK signaling in combination with bacteria therapy significantly decreased (p&amp;lt;0.002) NSCLC spheroid viability. Honing in on AKT signaling, we validated bacterially secreted Theta toxin induced AKT phosphorylation in NSCLC cells in vitro that we were able to block with MK2206 treatment. Finally, we tested this combination of bacterially secreted Theta toxin and MK2206 in xenografted NSCLC tumors. The combination treatment significantly improved efficacy in all tumors (p&amp;lt;0.001) compared to single-agent therapy (either engineered bacteria or MK2206 alone) without any additional toxicity suggesting a promising translational treatment strategy. We conclude that our modular experimental and analytical pipeline can be exploited to characterize engineered bacteria therapies and combine them with specific targeted therapy to expedite the translation of an untapped area of drug development with biological therapeutic agents for advanced-stage NSCLC. Citation Format: Dhruba Deb, Tal Danino. Combining forces: Integrating engineered bacterial therapeutics with targeted therapies for advanced-stage non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C119.

Chicken muscle antibody array reveals the regulations of LDHA on myoblast differentiation through energy metabolism

Myoblast proliferation and differentiation are highly dynamic and regulated processes in skeletal muscle development. Given that proteins serve as the executors for the majority of biological processes, exploring key regulatory factors and mechanisms at the protein level offers substantial opportunities for understanding the skeletal muscle development. In this study, a total of 607 differentially expressed proteins between proliferation and differentiation in myoblasts were screened out using our chicken muscle antibody array. Biological function analysis revealed the importance of energy production processes and compound metabolic processes in myogenesis. Our antibody array specifically identified an upregulation of LDHA during differentiation, which was associated with the energy metabolism. Subsequent investigation demonstrated that LDHA promoted the glycolysis and TCA cycle, thereby enhancing myoblasts differentiation. Mechanistically, LDHA promotes the glycolysis and TCA cycle but inhibits the ETC oxidative phosphorylation through enhancing the NADH cycle, providing the intermediate metabolites that improve the myoblasts differentiation. Additionally, increased glycolytic ATP by LDHA induces Akt phosphorylation and activate the PI3K-Akt pathway, which might also contribute to the promotion of myoblasts differentiation. Our studies not only present a powerful tool for exploring myogenic regulatory factors in chicken muscle, but also identify a novel role for LDHA in modulating myoblast differentiation through its regulation of cellular NAD+ levels and subsequent downstream effects on mitochondrial function.

Abstract 2553: Elucidation of the downstream signaling of PIK3CA genomic alterations in serous ovarian cancer

Abstract Genomic aberrations of PIK3CA, which encodes the catalytic subunit p110α of the class IA phosphatidylinositol 3-kinase (PI3K), is frequently observed in cancers. In ovarian cancer of the serous subtype (the most common histotype of the disease), PIK3CA amplification is dominant over single nucleotide mutations in multiple patient cohorts, suggesting the possibility that PIK3CA amplification is an alternative to PIK3CA “hot spot” mutation for serous ovarian tumorigenesis. However, whether PIK3CA amplification and mutation exert the same functional consequences on serous ovarian tumorigenesis remains uncertain. This study aims to explore and compare the functional roles between the two types of PIK3CA gene aberrations on tumorigenicity, cellular signaling, and therapeutic responses in serous ovarian cancer. We confirmed that both PIK3CA amplification and E545K mutation could induce multiple tumorigenic phenotypes in CRISPR/Cas9-mediated E545K knock-in or wild-type PIK3CA overexpressing serous ovarian cancer cell lines. Signaling pathway analyses showed that E545K knock-in cells, but not PIK3CA-overexpressing cells, had the MAPK signaling pathway activated and were sensitive to MAPK pathway inhibition. An induction of AKT phosphorylation and PDK1 phosphorylation was observed in both E545K knock-in cells and wild-type PIK3CA-overexpressing cells. Accordingly, these cells displayed higher sensitivity towards p110α or AKT inhibitors. Intriguingly, in line with previous report which demonstrated YAP as AKT substrate, the activation of AKT associated with an increase in YAP S127 phosphorylation in the knock-in or PIK3CA-overexpressing cells. AKT inhibition suppressed YAP phosphorylation. Corroborating with the role of S127 in nuclear exclusion of YAP, YAP accumulated in the cytoplasm of these cells, leading to reduced transcriptional activity of YAP. While the functional significance of YAP cytoplasmic accumulation is yet to be investigated, our results reveal the common and distinct downstream pathways between PIK3CA amplification and mutation. These findings may be applicable to p110α-targeting therapies in serous ovarian cancer. Citation Format: Shibo Zhang, Hei Ip Hong, Victor CY Mak, Yuan Zhou, Panpan Wang, Lydia Wai Ting Cheung. Elucidation of the downstream signaling of PIK3CA genomic alterations in serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2553.

Abstract 14007: Sodium-Dependent Glucose Transporters 2 Inhibition Ameliorates Obesity-Induced Metabolic Changes and Cardiomyopathy

Introduction: Inhibition of renal sodium-glucose cotransporter-2 (SGLT2) reduces urinary glucose reabsorption to decrease glycemia. Recent large-scale trials have shown that SGLT2 inhibitors provide cardio protection beyond lowering glucose. Hypothesis: Treating SGLT2 inhibitor (SGLT2i) reduces the cardiovascular risk in type 2 diabetes that indicates the potential correlation of SGLT2i in cardiomyopathy mediating metabolic effects. Methods: Male 8 weeks old C57/BL6 mice were fed high fat diet (&gt; 60% of fat) for 4 months, and treated SGLT2i, canagliflozin (CANA) in the third and the fourth month. Measured its blood glucose, body weight and echocardiology every month. Moreover, analyzed the lipid contents in its plasma, the adipose tissue, liver and heart, and also examine the cardiomyopathy markers and its potential signaling pathways. Furthermore, palmitate acid (PA) was treated to H9c2 cells to mimic a high lipid environment, and validate its potential signaling pathways. Results: Treatments of high fat diet-induced obese mice with the CANA reduced blood glucose, body weight and adiposity. Moreover, CANA reversed the obesity-induced cardiomyopathy including heart weight ratio, echocardiology and cardiomyopathy markers ( Col1 , Col4 , Icam1 and Myh7 ). Furthermore, CANA increased AKT phosphorylation which is the one of the key pathways, and reduces apoptosis in cardiomyopathy. To investigate the potential mechanisms of metabolic changes in cardiomyopathy by CANA, we searched the hepatic RNA expression from high fat diet induced obesity mice by GEO profiles online. FGF21, a hepatokine increased in CANA action by hepatic and adipose tissue lipolysis, next circulated to the heart, and induced AKT phosphorylation to reduce apoptosis. CANA increased plasma and hepatic FGF21 in obese mice. Further treating recombinant FGF21 in H9c2 differentiated cardiomyocyte like cells with palmitic acid, FGF21 reduced cardiomyocyte apoptosis via AKT phosphorylation. Conclusions: These data demonstrated that SGLT2 inhibition ameliorates metabolic changes and apoptosis via FGF21-AKT pathway in cardiomyopathy.

Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury.

Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G+ polymorphonuclear neutrophils, depletion of CD11c+SiglecF+ alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1-/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury.

Abstract P3127: Wnt1-inducible Signalling Pathway Protein 1 Contributes To Vascular Fibrosis By Promoting Fibroblasts Phenotypic Switch And Extracellular Matrix Deposition

Background: Vascular fibrosis is linked with many diseases, including hypertension. Upon pathophysiological stimulation, fibroblasts undergo phenotypic switch and increase extracellular matrix (ECM) synthesis and deposition. Wnt1-inducible signalling pathway protein 1 (WISP-1) is a growth factor implicated in fibrotic remodelling. We tested the hypothesis that WISP-1 induces cardiac fibroblast activation and increases ECM deposition, thereby contributing to vascular fibrosis in hypertension. Methods and Results: Primary human cardiac fibroblasts (HCFs) were treated with recombinant WISP-1. Western blotting analysis showed that WISP-1 treatment for 30 minutes activated Akt pathway (phospho-Akt:total-Akt, fold change: 1.33±0.14 vs. 1.00±0, n=9, P &lt;0.05). WISP-1 induced Akt phosphorylation was inhibited by an integrin β1 blocking antibody (phospho-Akt:total-Akt, fold change, 0.52±0.12 vs. 1.00±0, n=5, P &lt;0.05). WISP-1 treatment for 24 hours induced collagen 1 maturation in conditioned media and coincided with decreased meprin α metalloproteinase protein levels (fold change: 0.44±0.14 vs. 1.00±0, n=5, P &lt;0.05). The collagen maturation was inhibited by a zinc-mediated proteinase inhibitor (GM6001) or an Akt inhibitor. Additionally, immunocytochemistry analysis illustrated that 24-hour WISP-1 treatment increased the percentage of α-smooth muscle actin-positive HCFs compared to the control (1.89±0.37 vs. 1.00±0, n=8, P &lt;0.05). In vivo , WISP-1 knockout mice exhibited reduced collagen 1 content (0.39±0.09 vs. 1.19±0.25, n=5-7, P &lt;0.05) and proteoglycan content (2.20±0.37 vs. 6.22±1.10, n=5-7, P &lt;0.05,) in the perivascular area compared to their wild-type control after 28 days angiotensin II (1000ng/kg/min) infusion. Conclusions: WISP-1 activates cardiac fibroblasts via integrin β1-Akt pathway and meprin α metalloproteinase, thereby inducing fibroblast phenotypic switch and promoting collagen deposition. Deletion of WISP-1 alleviated hypertension induced vascular fibrosis in vivo . Modulation of WISP-1 levels may be beneficial for attenuating the effects of hypertension on vascular fibrosis, and could provide potential therapeutic targets in clinical treatment.

Abstract 12104: Treatment With Exogenous Atrial Natriuretic Peptide Ameliorates Myocardial Insulin Resistance and Protects Against Ischemia-Reperfusion Injury in Diet-Induced Obesity

Increasing evidence suggests natriuretic peptide (NP) coordinate inter-organ metabolic crosstalk in addition to its classical actions on hemodynamic regulation. We recently reported exogenous atrial NP (ANP) treatment ameliorated systemic insulin resistance by inducing adipose tissue browning and by attenuating hepatic steatosis in diet-induced obesity. We herein hypothesized that exogenous ANP treatment also ameliorates insulin resistance of the myocardium in which endogenous NP is produced, leading to the cardioprotection during ischemia-reperfusion injury (IRI) in diet-induced obesity. The mice fed a high fat diet (HFD) or normal fat diet (NFD) for 13 weeks were treated with or without ANP infusion subcutaneously via osmotic pump for another 3 weeks (0.5 μg/kg/min). Intraperitoneal administration of insulin induced Akt phosphorylation in NFD hearts, which was reduced in HFD. ANP treatment restored the impaired insulin-induced Akt phosphorylation in HFD hearts, suggesting that exogenous ANP ameliorated myocardial insulin resistance. To test the functional significance of this, hearts were exposed to IRI using Langendorff model. After IRI, HFD resulted in an impaired left ventricular developed pressure (LVDP) recovery with corresponding increase in infarct size. However, ANP treatment improved functional recovery and reduced injury along with restoration of the impaired IRI-induced Akt phosphorylation in HFD hearts. (% LVDP recovery of baseline: NFD 62.9%±3.6 &lt;n=8&gt;, NFD+ANP 64.5%±4.2% &lt;n=8&gt;, HFD 20.8%±3.3 &lt;n=9&gt;, HFD+ANP 31.4%±2.8% &lt;n=9&gt;, p&lt;0.05) Myocardial ultrastructural analyses by electron microscopy showed number of peri-mitochondrial lipid droplets was increased in HFD treated with ANP, suggesting that ANP protects mitochondria from lipid overload. Of note, ANP treatment substantially attenuated mitochondria cristae disruption after IRI in HFD hearts. In conclusion, exogenous ANP treatment ameliorates myocardial insulin resistance and protects against IRI associated with restoration of disrupted mitochondrial structure in diet-induced obesity. The findings that exogenous NP has significant impact on the NP-rich failing myocardium provide new insight into the biological properties and dynamics of NP.

Bee Venom Prevents Mucin 5AC Production through Inhibition of AKT and SPDEF Activation in Airway Epithelia Cells.

IL-13 induces mucus metaplasia, which causes airway obstruction in asthma. Bee venom (BV) and its components have shown anti-inflammatory effects in allergic diseases such as atopic dermatitis and asthma. In this study, we investigated the effect of BV on IL-13-induced mucus metaplasia through activation of the signal transducer and activator of transcription (STAT6), and regulation of SAM-pointed domain containing Ets-like factor (SPDEF) and forkhead box A2 (FOXA2) in the airway epithelia cell line A549. In A549 cells, BV (1.0 µg/mL) inhibited IL-13 (10 ng/mL)-induced AKT phosphorylation, increase in SPDEF protein expression, and decrease in FOXA2 protein expression—but not STAT6 phosphorylation. BV also prevented the IL-13-induced increase in mucin 5AC (MUC5AC) mRNA and protein expression. Moreover, we observed that inhibition of phosphoinositide 3 kinase (PI3K)/AKT using LY294002 (50 µM) could reverse the alterations in FOXA2 and MUC5AC expression -by IL-13 and BV. However, LY294002 did not affect IL-13- and BV-induced changes in SPDEF expression. These findings indicate that BV inhibits MUC5AC production through the regulation of SPDEF and FOXA2. The inhibition of MUC5AC production through FOXA2 is mediated via the suppression of PI3K/AKT activation by BV. BV may be helpful in the prevention of mucus metaplasia in asthma.

A breakthrough-like effect of metformin reduces peripheral resistance to triiodothyronine in euthyroid, non-insulin-resistant, type 2 diabetic patients.

BackgroundType 2 diabetes is characterized, beyond the insulin resistance, by polyhormonal resistance. Thyroid hormonal resistance has not yet been described in this population of patients. Metformin is used to decrease insulin resistance, and at present, it is assumed to influence the effect of triiodothyronine, as well.MethodsIn this open-label, pilot, hypothesis-generating, follow-up study, 21 patients were included; all of them were euthyroid with drug naïve, newly diagnosed type 2 diabetes. Before and after 4 weeks of metformin therapy, fructosamine, homeostasis model assessment for insulin resistance (HOMA-IR), thyroid hormones, T3/T4 ratio, and TSH, as well as blood pressure and heart rate using ambulatory blood pressure monitor were measured. We also conducted an in vitro study to investigate the possible mechanisms of T3 resistance, assessing T3-induced Akt phosphorylation among normal (5 mM) and high (25 mM) glucose levels with or without metformin treatment in a human embryonal kidney cell line.ResultsMetformin decreased the level of T3 (P < 0.001), the ratio of T3/T4 (P = 0.038), fructosamine (P = 0.008) and HOMA-IR (P = 0.022). All these changes were accompanied by an unchanged TSH, T4, triglyceride, plasma glucose, bodyweight, blood pressure, and heart rate. In our in vitro study, T3-induced Akt phosphorylation decreased in cells grown in 25 mM glucose medium compared to those in 5 mM. Metformin could not reverse this effect.ConclusionMetformin seems to improve T3 sensitivity in the cardiovascular system in euthyroid, type 2 diabetic patients, the mechanism of which may be supracellular.

55-OR: Targeted Deletion of Insulin Receptor in Vascular Smooth Muscle Cells Promoted Atherosclerosis and Unstable Plaque

Risks of unstable arterial plaques are increased in diabetes or insulin resistance. We hypothesized that insulin resistance in VSMCs contributes to reduced plaque stability since we have reported that insulin receptors (IR) on VSMCs are potent regulators of its proliferation and apoptosis. Further, insulin induced Akt phosphorylation was decreased in the aorta of diabetic mice. Lipid deposition were increased in the aortas of ApoE-/- and VSMCs IR double knockout (SMIRKO/ApoE-/-) mice vs. control mice. Pathological analysis of the atherosclerotic plaques of SMIRKO/ApoE-/- mice demonstrated features of unstable plaques, including reduced VSMCs proliferation and reduced cap thickness and collagen, but increased VSMCs apoptosis and more macrophages and necrosis area. VSMCs from SMIRKO/ApoE-/- mice exhibited less insulin induced proliferation and more apoptosis which were not inhibited by insulin compared to VSMCs from ApoE-/- mice. Artery relaxation induced by sodium nitroprusside (SNP) (NO donor) was impaired in artery from SMIRKO/ApoE-/- mice vs. control mice. Insulin and SNP induced VASP phosphorylation, a downstream signaling protein of NO in VSMCs, were reduced in SMIRKO/ApoE-/- mice vs. control mice. Expressions of MMP2, THBS1, sFRP3, and COMP were increased in the aorta of SMIRKO/ApoE-/- mice vs. ApoE-/- mice, which were also observed in the aorta of diabetic mice fed with high fat diet or diabetes induced by STZ. Insulin induced Akt and Foxo1 phosphorylation were impaired in cultured VSMCs from SMIRKO/ApoE-/- mice vs. control mice. Overexpression of constitutively activated Akt increased VASP phosphorylation and downregulated THBS1 expression in cultured VSMCs, whereas overexpression of FoxO1 increased THBS1 expression. This is the first report that selective inhibition of IR activation of pAkt in VSMCs is partially causing the elevated risk of unstable atherosclerotic plaques found in insulin resistance or diabetes. Disclosure Q. Li: None. J. Fu: None. K. Park: None. I. Wu: None. G. L. King: Consultant; Self; Agios, Inc., Medtronic, Other Relationship; Self; Janssen Pharmaceuticals, Inc. Funding National Institutes of Health (R01DK053105)

LncRNA lnc-ISG20 promotes renal fibrosis in diabetic nephropathy by inducing AKT phosphorylation through miR-486-5p/NFAT5.

Long non‐coding RNA (lncRNA) lnc‐ISG20 has been found aberrantly up‐regulated in the glomerular in the patients with diabetic nephropathy (DN). We aimed to elucidate the function and regulatory mechanism of lncRNA lnc‐ISG20 on DN‐induced renal fibrosis. Expression patterns of lnc‐ISG20 in kidney tissues of DN patients were determined by RT‐qPCR. Mouse models of DN were constructed, while MCs were cultured under normal glucose (NG)/high glucose (HG) conditions. The expression patterns of fibrosis marker proteins collagen IV, fibronectin and TGF‐β1 were measured with Western blot assay. In addition, the relationship among lnc‐ISG20, miR‐486‐5p, NFAT5 and AKT were analysed using dual‐luciferase reporter assay and RNA immunoprecipitation. The effect of lnc‐ISG20 and miR‐486/NFAT5/p‐AKT axis on DN‐associated renal fibrosis was also verified by means of rescue experiments. The expression levels of lnc‐ISG20 were increased in DN patients, DN mouse kidney tissues and HG‐treated MCs. Lnc‐ISG20 silencing alleviated HG‐induced fibrosis in MCs and delayed renal fibrosis in DN mice. Mechanistically, miR‐486‐5p was found to be a downstream miRNA of lnc‐ISG20, while miR‐486‐5p inhibited the expression of NFAT5 by binding to its 3'UTR. NFAT5 overexpression aggravated HG‐induced fibrosis by stimulating AKT phosphorylation. However, NFAT5 silencing reversed the promotion of in vitro and in vivo fibrosis caused by lnc‐ISG20 overexpression. Our collective findings indicate that lnc‐ISG20 promotes the renal fibrosis process in DN by activating AKT through the miR‐486‐5p/NFAT5 axis. High‐expression levels of lnc‐ISG20 may be a useful indicator for DN.

Increased Extracellular Adenosine in Radiotherapy-Resistant Breast Cancer Cells Enhances Tumor Progression through A2AR-Akt-β-Catenin Signaling.

Simple SummaryIn our previous study, purinergic P2Y2 receptor (P2Y2R) activation by ATP was found to play an important role in tumor progression and metastasis by regulating various responses in cancer cells and modulating crosstalk between cancer cells and endothelial cells (ECs). Therefore, we expected that P2Y2R would play a critical role in radioresistance and enhanced tumor progression in radioresistant triple-negative breast cancer (RT-R-TNBC). However, interestingly, P2Y2R expression was slightly decreased in RT-R-TNBC cells, while the expression of A2AR was significantly increased both in RT-R-TNBC cells and in tumor tissues, especially triple negative breast cancer (TNBC) tissues of breast cancer (BC) patients. Thus, we aimed to investigate the role of adenosine A2A receptor (A2AR) and its signaling pathway in the progression of RT-R-TNBC. The results reveal for the first time the role of A2AR in the progression and metastasis of RT-R-BC cells and suggest that the adenosine (ADO)-activated intracellular A2AR signaling pathway is linked to the AKT-β-catenin pathway to regulate RT-R-BC cell invasiveness and metastasis.Recently, we found that the expressions of adenosine (ADO) receptors A2AR and A2BR and the ectonucleotidase CD73 which is needed for the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and the extracellular ADO level are increased in TNBC MDA-MB-231 cells and RT-R-MDA-MB-231 cells compared to normal cells or non-TNBC cells. The expression of A2AR, but not A2BR, is significantly upregulated in breast cancer tissues, especially TNBC tissues, compared to normal epithelial tissues. Therefore, we further investigated the role of ADO-activated A2AR and its signaling pathway in the progression of RT-R-TNBC. ADO treatment induced MDA-MB-231 cell proliferation, colony formation, and invasion, which were enhanced in RT-R-MDA-MB-231 cells in an A2AR-dependent manner. A2AR activation by ADO induced AKT phosphorylation and then β-catenin, Snail, and vimentin expression, and these effects were abolished by A2AR-siRNA transfection. In an in vivo animal study, compared to 4T1-injected mice, RT-R-4T1-injected mice exhibited significantly increased tumor growth and lung metastasis, which were decreased by A2AR-knockdown. The upregulation of phospho-AKT, β-catenin, Snail, and vimentin expression in mice injected with RT-R-4T1 cells was also attenuated in mice injected with RT-R-4T1-A2AR-shRNA cells. These results suggest that A2AR is significantly upregulated in BC tissues, especially TNBC tissues, and ADO-mediated A2AR activation is involved in RT-R-TNBC invasion and metastasis through the AKT-β-catenin pathway.

MZT2A promotes NSCLC viability and invasion by increasing Akt phosphorylation via the MOZART2 domain.

Mitotic spindle organizing protein 2A (MZT2A) is localized at the centrosome and regulates microtubule nucleation activity in cells. This study assessed the role of MZT2A in non–small‐cell lung cancer (NSCLC). Differential MZT2A expression was bioinformatically assessed using TCGA database, the GEPIA database, and Kaplan‐Meier survival data to determine the association between MZT2A expression and NSCLC prognosis. Furthermore, NSCLC tissue specimens were evaluated by immunohistochemistry. MZT2A was overexpressed or knocked down in NSCLC cells using cDNA and siRNA, respectively. The cells were subjected to various assays and treated with the selective Akt inhibitor LY294002 or co‐transfected with galectin‐3‐binding protein (LGALS3BP) siRNA. MZT2A mRNA and protein levels were upregulated in NSCLC lesions and MTZ2A expression was associated with poor NSCLC prognosis. MZT2A protein was also highly expressed in NSCLC cells compared with the expression in normal bronchial cells. MZT2A expression promoted NSCLC cell viability and invasion, whereas MTZ2A siRNA had the opposite effect on NSCLC cells in vitro. At the protein level, MZT2A induced Akt phosphorylation, promoting NSCLC proliferation and invasion (but the selective Akt inhibitor blocked these effects) through upregulation of LGALS3BP via the MTZ2A MOZART2 domain, whereas LGALS3BP siRNA suppressed MTZ2A activity in NSCLC cells. The limited in vivo experiments confirmed the in vitro data. In conclusion, MZT2A exhibits oncogenic activity by activating LGALS3BP and Akt in NSCLC. Future studies will assess MTZ2A as a biomarker to predict NSCLC prognosis or as a target in the control of NSCLC progression.

PI3K activation promotes resistance to eribulin in HER2-negative breast cancer.

Eribulin is a microtubule-targeting agent approved for the treatment of advanced or metastatic breast cancer (BC) previously treated with anthracycline- and taxane-based regimens. PIK3CA mutation is associated with worse response to chemotherapy in oestrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic BC. We aimed to evaluate the role of phosphoinositide 3-kinase (PI3K)/AKT pathway mutations in eribulin resistance. Resistance to eribulin was evaluated in HER2- BC cell lines and patient-derived tumour xenografts, and correlated with a mutation in the PI3K/AKT pathway. Eleven out of 23 HER2- BC xenografts treated with eribulin exhibited disease progression. No correlation with ER status was detected. Among the resistant models, 64% carried mutations in PIK3CA, PIK3R1 or AKT1, but only 17% among the sensitive xenografts (P = 0.036). We observed that eribulin treatment induced AKT phosphorylation in vitro and in patient tumours. In agreement, the addition of PI3K inhibitors reversed primary and acquired resistance to eribulin in xenograft models, regardless of the genetic alterations in PI3K/AKT pathway or ER status. Mechanistically, PI3K blockade reduced p21 levels likely enabling apoptosis, thus sensitising to eribulin treatment. PI3K pathway activation induces primary resistance or early adaptation to eribulin, supporting the combination of PI3K inhibitors and eribulin for the treatment of HER2- BC patients.

Epiregulin confers EGFR-TKI resistance via EGFR/ErbB2 heterodimer in non-small cell lung cancer.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective against non-small cell lung cancer (NSCLC) with EGFR-activating mutations. The mechanisms underlying EGFR-TKI resistance are not fully understood. This study aimed to analyze the effects of seven EGFR ligands on EGFR-TKI sensitivity in NSCLC cells and patients. Cells with EGFR E746-A750del mutation were treated with recombinant EGFR ligands, and analyzed for cell viability, proliferation, and apoptosis. shRNA knockdown of endogenous Epiregulin (EREG) or overexpression of exogenous EREG and immunofluorescence experiments were carried out. Public gene expression datasets were used for tumor microenvironment and clinical assessment. Among the EGFR ligands, EREG significantly diminished cellular sensitivity to TKIs and was associated with decreased response to erlotinib in NSCLC patients. EREG induced AKT phosphorylation and attenuated TKI-induced cellular apoptosis in an ErbB2-dependent manner. EREG induced the formation of the EGFR/ErbB2 heterodimer regardless of gefitinib treatment. However, overexpression or knockdown of EREG in cancer cells had little impact on TKI sensitivity. Single-cell RNA sequencing data revealed that EREG was predominantly expressed in macrophages in the tumor microenvironment. In addition, EREG-enriched macrophage conditional medium induced EGFR-TKI resistance. These findings shed new light on the mechanism underlying EGFR-TKI resistance, and suggest macrophage-produced intratumoral EREG as a novel regulator and biomarker for EGFR-TKI therapy in NSCLC.