Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer.

Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.

AKT2 reduces IFNß1 production to modulate antiviral responses and systemic lupus erythematosus.

Interferon regulatory factor 3 (IRF3)-induced type I interferon (I-IFN) production plays key roles in both antiviral and autoimmune responses. IRF3 phosphorylation, dimerization, and nuclear localization are needed for its activation and function, but the precise regulatory mechanisms remain to be explored. Here, we show that the serine/threonine kinase AKT2 interacts with IRF3 and phosphorylates it on Thr207, thereby attenuating IRF3 nuclear translocation in a 14-3-3ε-dependent manner and reducing I-IFN production. We further find that AKT2 expression is downregulated in viral-infected macrophages or in monocytes and tissue samples from systemic lupus erythematosus (SLE) patients and mouse models. Akt2-deficient mice exhibit increased I-IFN induction and reduced mortality in response to viral infection, but aggravated severity of SLE. Overexpression of AKT2 kinase-inactive or IRF3-T207A mutants in zebrafish supports that AKT2 negatively regulates I-IFN production and antiviral response in a kinase-dependent manner. This negative role of AKT2 in IRF3-induced I-IFN production suggests that AKT2 may be therapeutically targeted to differentially regulate antiviral infection and SLE.

Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells.

BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

Lactate accumulation induced by Akt2-PDK1 signaling promotes pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with an abnormal accumulation of fibrotic tissue in the lung parenchyma and elevated glycolysis level in associated cells without effective therapy options. Lactate accumulation in pulmonary fibrotic tissue is a significant factor aggravating IPF development, but the main mechanism regulating glycolysis needs further investigation. In this study, lung fibrosis model was induced by bleomycin (BLM) intratracheally in female C57BL/6 mice. The changes of lactate level and fibrotic markers were detected. For in vitro studies, cell lines of alveolar epithelial cell and lung fibroblast cell were stimulated with TGF-ß1 and BLM respectively, to detect changes in their fibrotic properties. The function of lactate accumulation on facilitating fibrosis was verified. We demonstrated that BLM-induced pulmonary fibrosis is accompanied by lactate accumulation owing to glycolysis upregulation. Significantly high PDK1 expression in lung fibrotic tissue promotes glycolysis. Moreover, PDK1 stimulated trans-differentiation of lung fibroblasts and epithelial-mesenchymal transition (EMT) of alveolar epithelial cells. Furthermore, phosphorylated Akt2 activated PDK1 to cause pulmonary fibrosis and inhibitors of Akt2 and PDK1 could suppress fibrotic process. This study is the first to consider PDK1 facilitated lactate accumulation through glycolysis as a vital factor in pulmonary fibrosis and could be initiated by Akt2. We concluded that the pro-fibrotic properties of PDK1 are associated with Akt2 phosphorylation and thus provide new potential therapeutic targets for pulmonary fibrosis.

High glucose-induced downregulation of PTEN-Long is sufficient for proximal tubular cell injury in diabetic kidney disease.

During the progression of diabetic kidney disease, proximal tubular epithelial cells respond to high glucose to induce hypertrophy and matrix expansion leading to renal fibrosis. Recently, a non-canonical PTEN has been shown to be translated from an upstream initiation codon CUG (leucine) to produce a longer protein called PTEN-Long (PTEN-L). Interestingly, the extended sequence present in PTEN-L contains cell secretion/penetration signal. Role of this non-canonical PTEN-L in diabetic renal tubular injury is not known. We show that high glucose decreases expression of PTEN-L. As a mechanism of its function, we find that reduced PTEN-L activates Akt-2, which phosphorylates and inactivate tuberin and PRAS40, resulting in activation of mTORC1 in tubular cells. Antibacterial agent acriflavine and antiviral agent ATA regulate translation from CUG codon. Acriflavine and ATA, respectively, decreased and increased expression of PTEN-L to altering Akt-2 and mTORC1 activation in the absence of change in expression of canonical PTEN. Consequently, acriflavine and ATA modulated high glucose-induced tubular cell hypertrophy and lamininγ1 expression. Importantly, expression of PTEN-L inhibited high glucose-stimulated Akt/mTORC1 activity to abrogate these processes. Since PTEN-L contains secretion/penetration signals, addition of conditioned medium containing PTEN-L blocked Akt-2/mTORC1 activity. Notably, in renal cortex of diabetic mice, we found reduced PTEN-L concomitant with Akt-2/mTORC1 activation, leading to renal hypertrophy and lamininγ1 expression. These results present first evidence for involvement of PTEN-L in diabetic kidney disease.

Oncogenic ß-catenin stimulation of AKT2-CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer.

CTNNB1, encoding ß-catenin protein, is the most frequently altered proto-oncogene in hepatic neoplasms. In this study, we studied the significance and pathological mechanism of CTNNB1 gain-of-function mutations in hepatocarcinogenesis. Activated ß-catenin not only triggered hepatic tumorigenesis but also exacerbated Tp53 deletion or hepatitis B virus infection-mediated liver cancer development in mouse models. Using untargeted metabolomic profiling, we identified boosted de novo pyrimidine synthesis as the major metabolic aberration in ß-catenin mutant cell lines and livers. Oncogenic ß-catenin transcriptionally stimulated AKT2, which then phosphorylated the rate-limiting de novo pyrimidine synthesis enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase) on S1406 and S1859 to potentiate nucleotide synthesis. Moreover, inhibition of ß-catenin/AKT2-stimulated pyrimidine synthesis axis preferentially repressed ß-catenin mutant cell proliferation and tumor formation. Therefore, ß-catenin active mutations are oncogenic in various preclinical liver cancer models. Stimulation of ß-catenin/AKT2/CAD signaling cascade on pyrimidine synthesis is an essential and druggable vulnerability for ß-catenin mutant liver cancer.

In Silico Investigation of AKT2 Gene and Protein Abnormalities Reveals Potential Association with Insulin Resistance and Type 2 Diabetes.

Type 2 diabetes (T2D) develops from insulin resistance (IR) and the dysfunction of pancreatic beta cells. The AKT2 protein is very important for the protein signaling pathway, and the non-synonymous SNP (nsSNPs) in AKT2 gene may be associated with T2D. nsSNPs can result in alterations in protein stability, enzymatic activity, or binding specificity. The objective of this study was to investigate the effect of nsSNPs on the AKT2 protein structure and function that may result in the induction of IR and T2D. The study identified 20 variants that were considered to be the most deleterious based on a range of analytical tools included (SIFT, PolyPhen2, Mut-pred, SNAP2, PANTHER, PhD-SNP, SNP&Go, MUpro, Cosurf, and I-Mut). Two mutations, p.A179T and p.L183Q, were selected for further investigation based on their location within the protein as determined by PyMol. The results indicated that mutations, p.A179T and p.L183Q alter the protein stability and functional characteristics, which could potentially affect its function. In order to conduct a more in-depth analysis of these effects, a molecular dynamics simulation was performed for wildtype AKT2 and the two mutants (p.A179T and p.L183Q). The simulation evaluated various parameters, including temperature, pressure, density, RMSD, RMSF, SASA, and Region, over a period of 100 ps. According to the simulation results, the wildtype AKT2 protein demonstrated higher stability in comparison to the mutant variants. The mutations p.A179T and p.L183Q were found to cause a reduction in both protein stability and functionality. These findings underscore the significance of the effects of nsSNPs (mutations p.A179T and p.L183Q) on the structure and function of AKT2 that may lead to IR and T2D. Nevertheless, they require further verifications in future protein functional, protein-protein interaction, and large-scale case-control studies. When verified, these results will help in the identification and stratification of individuals who are at risk of IR and T2D for the purpose of prevention and treatment.

miR-126 regulates the proliferation, migration, invasion, and apoptosis of non-small lung cancer cells via AKT2/HK2 axis.

This study tended to clarify the role of miR-126 in non-small cell lung cancer (NSCLC) cell biological behaviors in vitro, containing cell proliferation, migration, invasion, and apoptosis. miRNA expression microarray related to NSCLC was accessed from gene expression omnibus (GEO) database and subjected to differential analysis using the "limma" package. Real-time quantitative PCR was conducted to assess the expression of miR-126 in NSCLC cell lines. wIn vitro experiments including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), wound healing assay, Transwell, and flow cytometry assay were used for evaluating the effect of miR-126 on cell proliferation, migration, invasion, and apoptosis. Additionally, target mRNA for miR-126 was predicted and further validated by bioinformatics analysis and dual-luciferase reporter assay, respectively. It suggested that miR-126 was significantly down-regulated in NSCLS based on the expression microarray, and similar expression trend was exhibited in cancer cell lines. In the meantime, overexpression of miR-126 was found to result in inhibition of cell proliferation, migration, and invasion while promotion of cell apoptosis, with reductions in protein expression of AKT2 and phosphorylated HK2 (p-HK2) as well. AKT2, identified to be a direct target of miR-126 in NSCLC as judged by dual-luciferase reporter assay. Additionally, overexpression of AKT2 was observed to have the ability of elevating p-HK2 protein expression and reversing the effect of miR-126 on NSCLC cell proliferation, migration, and invasion. Given the above findings, we can see that miR-126 exerts its role in NSCLC cell proliferation, migration, invasion, and apoptosis with the aid of AKT2/HK2 axis.

AKT Isoforms in the Immune Response in Cancer.

AKT is a protein kinase that exists in three isoforms: AKT1, AKT2, and AKT3. Though similar in structure, these isoforms display different effects. AKT is activated downstream of PI3K, and together, this signaling pathway helps regulate cellular processes including cell growth, proliferation, metabolism, survival, and apoptosis. Disruption in these pathways has been associated with disorders including cardiovascular diseases, developmental disorders, inflammatory responses, autoimmune diseases, neurologic disorders, type 2 diabetes, and several cancers. In cancer, deregulation in the PI3K/AKT pathway can be manifested as tumorigenesis, pathological angiogenesis, and metastasis. Increased activity has been correlated with tumor progression and resistance to cancer treatments. Recent studies have suggested that inhibition of the PI3K/AKT pathway plays a significant role in the development, expansion, and proliferation of cells of the immune system. Additionally, AKT has been found to play an important role in differentiating regulatory T cells, activating B cells, and augmenting tumor immunosurveillance. This emphasizes AKT as a potential target for inhibition in cancer therapy. This chapter reviews AKT structure and regulation, its different isoforms, its role in immune cells, and its modulation in oncotherapy.

Phosphatase and tensin homologue determine inflammatory status by differentially regulating the expression of Akt1 and Akt2 in macrophage alternative polarization of periodontitis.

AIM: Macrophages are closely involved in periodontitis. However, the molecular mechanism by which macrophages influence periodontitis is not well understood. We investigated the effects of phosphatase and tensin homologue (PTEN) on macrophage polarization, the underlying mechanism and the regulatory roles in periodontium regeneration. MATERIALS AND METHODS: PTEN expression in periodontitis macrophages was detected ex vivo. The effects of PTEN on macrophage polarization and the underlying mechanisms were investigated in vitro. We also analysed the ability of PTEN inhibitors to repair periodontitis in vivo in a ligature-induced mouse model of periodontitis. RESULTS: Macrophage PTEN expression in periodontitis patients was significantly higher than that of controls. PTEN inhibition in macrophages induced alternative macrophage polarization, whereas PTEN overexpression facilitated classical polarization. PTEN inhibition facilitated activation of Akt1 while inhibiting expression of Akt2. Furthermore, Akt2 overexpression could rescue the effects of PTEN inhibition on NF-κB. Treatment with a PTEN inhibitor significantly attenuated the local inflammatory status and prevented alveolar bone resorption in the mouse model. CONCLUSIONS: Our findings suggest that PTEN inhibition could induce alternative macrophage polarization by differentially regulating Akt1 and Akt2. This also changed a pro-inflammatory microenvironment to an anti-inflammatory environment by subsequently regulating the expression of NF-κB, thereby attenuating inflammatory alveolar bone resorption induced by ligature.

CircRNA Circ_0000118 Regulates Malignancy of Cervical Cancer Cells by Regulating miR-211-5p/miR-377-3p/AKT2 Axis.

CircRNAs are implicated in the development of several cancers. Nevertheless, the involvement of circ_0000118 in the development of cervical cancer (CC) remains unclear. Circ_0000118 levels in tumor tissues and cells were examined by qRT-PCR. The function of circ_0000118 in regulating the malignancy of CC cells was investigated using functional assays, including CCK-8, colony formation, transwell, and tube formation experiments. The functional interaction between circ_0000118 and microRNAs were validated by dual-luciferase activity assay and RNA precipitation experiments. In vivo mouse model was employed to assess the effect of circ_0000118 in the tumorigenesis of CC cells. Circ_0000118 was overexpressed in CC cells and tissues. Loss-of-function experiments demonstrated that circ_0000118 knockdown impaired the proliferation and tumor sphere formation, as well as the angiogenic potential of CC cells. RNA interaction experiments confirmed that circ_0000118 sponged miR-211-5p and miR-377-3p. AKT2 was found to be a target gene negatively modulated by miR-211-5p and miR-377-3p. AKT2 overexpression rescued the inhibition of circ_0000118 downregulation on CC cells. Our study suggested that circ_0000118 functions as an oncogenic factor in progression of CC by maintaining AKT2 level through targeting miR-211-5p and miR-377-3p as a ceRNA (competitive endogenous RNA), which provides novel therapeutic target in the management of CC.

Expression of genes in the AKT signalling pathway in human oocytes from patients with polycystic ovaries.

Polycystic ovary syndrome is an endocrine disorder commonly found among females of reproductive age. Different factors have been correlated with this syndrome, although the aetiology of the disease is still unrecognized with both environmental and hereditary factors leading to the progression. Hormonal effects of the AKT pathway have made it an interesting study unit for PCOS cases. The aim of this study was to investigate the expression patterns of genes involved in the AKT pathway, including IRS1, IRS2, AKT1 and AKT2. In total, 13 human oocytes were collected for this study at the meiosis II stage, in which seven of them were collected from individuals with polycystic ovaries and the rest formed the control group of individuals with no signs of polycystic ovaries. RNA was extracted from oocytes and then the RNA was converted into cDNA for the real-time PCR process. Expression levels of four genes in the AKT pathway, in addition to housekeeping gene (ACTB), were evaluated. Expression levels of each gene were quantified using real-time PCR and statistical analysis was performed. The results of this study showed that there was no significant correlation between the expression of genes in oocyte samples obtained from patients with polycystic ovaries and the control group. This study is the first to evaluate the expression levels of genes involved in the AKT pathway in human oocyte samples. Therefore, it provides crucial information to form the basis of further studies.

Impaired Insulin Signaling Mediated by the Small GTPase Rac1 in Skeletal Muscle of the Leptin-Deficient Obese Mouse.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4. The small GTPase Rac1 acts as a switch of signal transduction that regulates GLUT4 translocation to the plasma membrane following insulin stimulation. However, it remains obscure whether signaling cascades upstream and downstream of Rac1 in skeletal muscle are impaired by obesity that causes insulin resistance and type 2 diabetes. In an attempt to clarify this point, we investigated Rac1 signaling in the leptin-deficient (Lepob/ob) mouse model. Here, we show that insulin-stimulated GLUT4 translocation and Rac1 activation are almost completely abolished in Lepob/ob mouse skeletal muscle. Phosphorylation of the protein kinase Akt2 and plasma membrane translocation of the guanine nucleotide exchange factor FLJ00068 following insulin stimulation were also diminished in Lepob/ob mice. On the other hand, the activation of another small GTPase RalA, which acts downstream of Rac1, by the constitutively activated form of Akt2, FLJ00068, or Rac1, was partially abrogated in Lepob/ob mice. Taken together, we conclude that insulin-stimulated glucose uptake is impaired by two mechanisms in Lepob/ob mouse skeletal muscle: one is the complete inhibition of Akt2-mediated activation of Rac1, and the other is the partial inhibition of RalA activation downstream of Rac1.

AKT2 identified as a potential target of mir-29a-3p via microRNA profiling of patients with high proliferation lacrimal gland adenoid cystic carcinoma.

The lacrimal gland adenoid cystic carcinoma (LACC) is a major orbital malignancy. The recurrence rate and mortality rate are higher in high proliferation LACC(HP-LACC) compared with low proliferation LACC(LP-LACC). In this study, miRNA microarray was used to explore the differentially expressed miRNAs profiling between HP-LACC and LP-LACC and its potential signaling pathway. Tissues from 17 patients with LACC were collected and made into tissue microarrays. Patients were divided into a high proliferation group and a low proliferation group based on Ki-67 value. HE, immunofluorescence (IF), and Immunohistochemistry (IHC) were performed on the tissue microarrays. Eight LACC tissues(4 HP-LACC and 4 LP-LACC) were made into miRNA microarrays and analyzed for miRNA profiles. Differentially expressed miRNAs were analyzed by volcano plot and heat map. Target gene were predicted using the miRWalk and miRDB for these differentially expressed miRNAs, the intersection of the results are used as targets for further gene ontology and KEGG pathway analysis.The four differentially expressed miRNAs were validated by qRT-PCR, the miRNAs with statistically significant differences validated by dual luciferase reporter and qRT-PCR. Finally, IHC was used for their downstream signaling pathway proteins.HE staining showed the presence of tubular, cribriform, and basaloid structures in LACC. IF showed the presence of CK7,P63 fluorescence expression in all three structures.Patients were divided into HP-LACC and LP-LACC based on Ki-67 median value of 11%. IHC and survival analysis showed with the increase of KI-67 ratio, the proportion of P63 decreased, and the expression of P53 increased. The disease-free survival and overall survival of the patients decreased. IHC and survival analysis showed as Ki-67 expression increased, P63 expression decreased, P53 expression elevated, with prognosis worse. Heat map and volcano plot yielded 15 differentially expressed miRNAs between HP-LACC and LP-LACC.The 15 differential miRNAs were used to predict target genes in miRWalk and miRDB databases respectively, and there were 559 target genes after intersection.559 predicted target genes obtained. Go and KEGG analysis showed that these target genes exerted important biological functions through multiple signaling pathways. Among the 15 differentially expressed miRNAs, miR-29a-3p was verified to be significant by qRT-PCR. Dual luciferase reporter and tissue microarray immunohistochemical assays validated that AKT2 was a direct target gene of miR-29a-3p. Current studies have identified differentially expressed miRNAs associated with LACCs of variable proliferation ability, and found that AKT2 is a direct target gene of miR-29a-3p, which will contribute to target gene therapy in patients with high proliferation LACC in the future.

PHLPP isoforms differentially regulate Akt isoforms and AS160 affecting neuronal insulin signaling and insulin resistance via Scribble.

BACKGROUND: The aim of the present study was to determine the role of individual PHLPP isoforms in insulin signaling and insulin resistance in neuronal cells. METHODS: PHLPP isoforms were either silenced or overexpressed individually, and the effects were observed on individual Akt isoforms, AS160 and on neuronal glucose uptake, under insulin sensitive and resistant conditions. To determine PHLPP regulation itself, we tested effect of scaffold protein, Scribble, on PHLPP isoforms and neuronal glucose uptake. RESULTS: We observed elevated expression of both PHLPP1 and PHLPP2 in insulin resistant neuronal cells (Neuro-2A, mouse neuroblastoma; SHSY-5Y, human neuroblastoma) as well as in the whole brain lysates of high-fat-diet mediated diabetic mice. In insulin sensitive condition, PHLPP isoforms differentially affected activation of all Akt isoforms, wherein PHLPP1 regulated serine phosphorylation of Akt2 and Akt3, while PHLPP2 regulated Akt1 and Akt3. This PHLPP mediated Akt isoform specific regulation activated AS160 affecting glucose uptake. Under insulin resistant condition, a similar trend of results were observed in Akt isoforms, AS160 and glucose uptake. Over-expressed PHLPP isoforms combined with elevated endogenous expression under insulin resistant condition drastically affected downstream signaling, reducing neuronal glucose uptake. No compensation was observed amongst PHLPP isoforms under all conditions tested, indicating independent roles and pointing towards possible scaffolding interactions behind isoform specificity. Silencing of Scribble, a scaffolding protein known to interact with PHLPP, affected cellular localization of both PHLPP1 and PHLPP2, and caused increase in glucose uptake. CONCLUSIONS: PHLPP isoforms play independent roles via Scribble in regulating Akt isoforms differentially, affecting AS160 and neuronal glucose uptake. Video abstract.

Role of Akt isoforms in neuronal insulin signaling and resistance.

The aim of the present study was to determine the role of Akt isoforms in insulin signaling and resistance in neuronal cells. By silencing Akt isoforms individually and in pairs, in Neuro-2a and HT22 cells we observed that, in insulin-sensitive condition, Akt isoforms differentially reduced activation of AS160 and glucose uptake with Akt2 playing the major role. Under insulin-resistant condition, phosphorylation of all isoforms and glucose uptake were severely affected. Over-expression of individual isoforms in insulin-sensitive and resistant cells differentially reversed AS160 phosphorylation with concomitant reversal in glucose uptake indicating a compensatory role of Akt isoforms in controlling neuronal insulin signaling. Post-insulin stimulation Akt2 translocated to the membrane the most followed by Akt3 and Akt1, decreasing glucose uptake in the similar order in insulin-sensitive cells. None of the Akt isoforms translocated in insulin-resistant cells or high-fat-diet mediated diabetic mice brain cells. Based on our data, insulin-dependent differential translocation of Akt isoforms to the plasma membrane turns out to be the key factor in determining Akt isoform specificity. Thus, isoforms play parallel with predominant role by Akt2, and compensatory yet novel role by Akt1 and Akt3 to regulate neuronal insulin signaling, glucose uptake, and insulin-resistance.

JIB-04, a Pan-Inhibitor of Histone Demethylases, Targets Histone-Lysine-Demethylase-Dependent AKT Pathway, Leading to Cell Cycle Arrest and Inhibition of Cancer Stem-Like Cell Properties in Hepatocellular Carcinoma Cells.

JIB-04, a pan-histone lysine demethylase (KDM) inhibitor, targets drug-resistant cells, along with colorectal cancer stem cells (CSCs), which are crucial for cancer recurrence and metastasis. Despite the advances in CSC biology, the effect of JIB-04 on liver CSCs (LCSCs) and the malignancy of hepatocellular carcinoma (HCC) has not been elucidated yet. Here, we showed that JIB-04 targeted KDMs, leading to the growth inhibition and cell cycle arrest of HCC, and abolished the viability of LCSCs. JIB-04 significantly attenuated CSC tumorsphere formation, growth, relapse, migration, and invasion in vitro. Among KDMs, the deficiency of KDM4B, KDM4D, and KDM6B reduced the viability of the tumorspheres, suggesting their roles in the function of LCSCs. RNA sequencing revealed that JIB-04 affected various cancer-related pathways, especially the PI3K/AKT pathway, which is crucial for HCC malignancy and the maintenance of LCSCs. Our results revealed KDM6B-dependent AKT2 expression and the downregulation of E2F-regulated genes via JIB-04-induced inhibition of the AKT2/FOXO3a/p21/RB axis. A ChIP assay demonstrated JIB-04-induced reduction in H3K27me3 at the AKT2 promoter and the enrichment of KDM6B within this promoter. Overall, our results strongly suggest that the inhibitory effect of JIB-04 on HCC malignancy and the maintenance of LCSCs is mediated via targeting the KDM6B-AKT2 pathway, indicating the therapeutic potential of JIB-04.

Cardiac metallothionein overexpression rescues diabetic cardiomyopathy in Akt2-knockout mice.

To efficiently prevent diabetic cardiomyopathy (DCM), we have explored and confirmed that metallothionein (MT) prevents DCM by attenuating oxidative stress, and increasing expression of proteins associated with glucose metabolism. To determine whether Akt2 expression is critical to MT prevention of DCM, mice with either global Akt2 gene deletion (Akt2-KO), or cardiomyocyte-specific overexpressing MT gene (MT-TG) or both combined (MT-TG/Akt2-KO) were used. Akt2-KO mice exhibited symptoms of DCM (cardiac remodelling and dysfunction), and reduced expression of glycogen and glucose metabolism-related proteins, despite an increase in total Akt (t-Akt) phosphorylation. Cardiac MT overexpression in MT-TG/Akt2-KO mice prevented DCM and restored glucose metabolism-related proteins expression and baseline t-Akt phosphorylation. Furthermore, phosphorylation of ERK1/2 increased in the heart of MT-TG/Akt2-KO mice, compared with Akt2-KO mice. As ERK1/2 has been implicated in the regulation of glucose transport and metabolism this increase could potentially underlie MT protective effect in MT-TG/Akt2-KO mice. Therefore, these results show that although our previous work has shown that MT preserving Akt2 activity is sufficient to prevent DCM, in the absence of Akt2 MT may stimulate alternative or downstream pathways protecting from DCM in a type 2 model of diabetes, and that this protection may be associated with the ERK activation pathway.

Phosphatidic acid directly binds with rice potassium channel OsAKT2 to inhibit its activity.

The plant Shaker K+ channel AtAKT2 has been identified as a weakly rectifying channel that can stabilize membrane potentials to promote photoassimilate phloem loading and translocation. Thus, studies on functional characterization and regulatory mechanisms of AtAKT2-like channels in crops are highly important for improving crop production. Here, we identified the rice OsAKT2 as the ortholog of Arabidopsis AtAKT2, which is primarily expressed in the shoot phloem and localized at the plasma membrane. Using an electrophysiological assay, we found that OsAKT2 operated as a weakly rectifying K+ channel, preventing H+ /sucrose-symport-induced membrane depolarization. Three critical amino acid residues (K193, N206, and S326) are essential to the phosphorylation-mediated gating change of OsAKT2, consistent with the roles of the corresponding sites in AtAKT2. Disruption of OsAKT2 results in delayed growth of rice seedlings under short-day conditions. Interestingly, the lipid second messenger phosphatidic acid (PA) inhibits OsAKT2-mediated currents (both instantaneous and time-dependent components). Lipid dot-blot assay and liposome-protein binding analysis revealed that PA directly bound with two adjacent arginine residues in the ANK domain of OsAKT2, which is essential to PA-mediated inhibition of OsAKT2. Electrophysiological and phenotypic analyses also showed the PA-mediated inhibition of AtAKT2 and the negative correlation between intrinsic PA level and Arabidopsis growth, suggesting that PA may inhibit AKT2 function to affect plant growth and development. Our results functionally characterize the Shaker K+ channel OsAKT2 and reveal a direct link between phospholipid signaling and plant K+ channel modulation.

LncRNA HOTTIP inhibits cell pyroptosis by targeting miR-148a-3p/AKT2 axis in ovarian cancer.

Long noncoding RNA HOTTIP is a crucial regulator in multiple types of cancer, including ovarian cancer (OC). However, the biological roles and underlying mechanisms of HOTTIP in OC have rarely been studied. Hence, this study aimed to investigate the functional correlation between HOTTIP and pyroptosis in OC progression. The expression of HOTTIP in OC tissues and cell lines was characterized by quantitative real-time PCR. Cell proliferation was evaluated using Cell Counting Kit-8 and clone formation assays. Western blot was performed to quantify protein levels. A dual-luciferase reporter assay was used to analyze the molecular interaction among HOTTIP, miR-148a-3p, and AKT2. The expression of HOTTIP was significantly upregulated in OC tissue samples and cell lines. The silencing of HOTTIP led to the inhibition of cell proliferation and NLRP1 inflammasome-mediated pyroptosis. In addition, HOTTIP increased AKT2 expression by negatively regulating miR-148a-3p and then inhibited ASK1/JNK signaling. Further rescue experiments revealed that downregulation of miR-148a-3p and overexpression of AKT2 obviously diminished the effects of HOTTIP downregulation in OC cells. Thus, our study elucidated a novel pyroptosis-related mechanism by which HOTTIP participated in OC progression, which might provide a theoretical reference for clinical treatment.