Activation Of PI3K-AKT Pathway Research Articles

Hypoxic stabilization of RIPOR3 mRNA via METTL3-mediated m6A methylation drives breast cancer progression and metastasis.

Dysregulated N6-methyladenosine (m6A) modification has been associated with breast cancer pathogenesis. Hypoxia which characterizes solid tumors is known to reprogram the m6A epitranscriptome, but the underlying mechanisms of how this process contributes to breast cancer progression remain poorly understood. Through integrative analyses of m6A-RIP sequencing and RNA sequencing databases, we reveal a cluster of mRNAs with upregulated m6A methylation and expression under hypoxia, that are enriched by many oncogenic pathways, including PI3K-Akt signaling. Furthermore, we identify the mRNA, RIPOR3, as a target of METTL3-mediated m6A methylation in response to hypoxia. We find that m6A methylation stabilizes RIPOR3, increasing its protein expression in a METTL3 catalytic activity-dependent manner, and consequently driving breast tumor growth and metastasis. RIPOR3 is found to be overexpressed in breast cancer cell lines and tumor tissues from breast cancer patients, in whom elevated RIPOR3 is associated with a worse prognosis. Mechanistically, we show that RIPOR3 interacts with EGFR and is essential for the PI3K-Akt pathway activation. In conclusion, we identify RIPOR3 as a hypoxia-stabilized oncogenic driver via METTL3-mediated m6A methylation, thus provide a potential therapeutic target for breast cancer.

MiR-196a Promotes Lipid Deposition in Goat Intramuscular Preadipocytes by Targeting MAP3K1 and Activating PI3K-Akt Pathway.

Meat quality in goats is partly determined by the intramuscular fat (IMF) content, which is associated with the proliferation and differentiation of intramuscular preadipocytes. Emerging studies have suggested that miRNA plays a crucial role in adipocyte proliferation and differentiation. In our recent study, we observed the expression variations in miR-196a in the longissimus dorsi muscle of Jianzhou goats at different ages. However, the specific function and underlying mechanism of miR-196a in IMF deposition are still unclear. This study demonstrated that miR-196a significantly enhanced adipogenesis and apoptosis and reduced the proliferation of preadipocytes. Subsequently, RNA-seq was employed to determine genes regulated by miR-196a, and 677 differentially expressed genes were detected after miR-196a overexpression. The PI3K-Akt pathway was identified as activated in miR-196a regulating intramuscular adipogenesis via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and further verified via Western blot and rescue assays. Lastly, using RT-qPCR, Western blot, dual-luciferase, and rescue assays, we found that miR-196a promoted adipogenesis and suppressed the proliferation of intramuscular preadipocytes by the downregulation of MAP3K1. In summary, these results suggest that miR-196a regulates IMF deposition by targeting MAP3K1 and activating the PI3K-Akt pathway and provide a theoretical foundation for improving goat meat quality through molecular breeding.

Click method preserves but EDC method compromises the therapeutic activities of the peptide-activated hydrogels for critical ischemic vessel regeneration

Peptide-functionalized hydrogel is one of commonly used biomaterials to introduce hydrogel-induced vessel regeneration. Despite many reports about the discoveries of high-active peptides (or ligands) for regeneration, the study on the conjugating methods for the hydrogel functionalization with peptides is limited. Here, we compared the vasculogenic efficacy of the peptide-functionalized hydrogels prepared by two commonly used conjugating methods, 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) and Click methods, through cell models, organ-on-chips models, animal models, and RNA sequencing analysis. Two vascular-related cell types, the human umbilical vein endothelial cells (HUVECs) and the adipose-derived stem cells (ADSCs), have been cultured on the hydrogel surfaces prepared by EDC/Click methods. It showed that the hydrogels prepared by Click method supported the higher vasculogenic activities while the ones made by EDC method compromised the peptide activities on hydrogels. The vasculogenesis assays further revealed that hydrogels prepared by Click method promoted a better vascular network formation. In a critical ischemic hindlimb model, only the peptide-functionalized hydrogels prepared by Click method successfully salvaged the ischemic limb, significantly improved blood perfusion, and enhanced the functional recoveries (through gait analysis and animal behavior studies). RNA sequencing studies revealed that the hydrogels prepared by Click method significantly promoted the PI3K-AKT pathway activation compared to the hydrogels prepared by EDC method. All the results suggested that EDC method compromised the functions of the peptides, while Click method preserved the vascular regenerating capacities of the peptides on the hydrogels, illustrating the importance of the conjugating method during the preparation of the peptide-functionalized hydrogels.

Paroxetine alleviates dendritic cell and T lymphocyte activation via GRK2-mediated PI3K-AKT signaling in rheumatoid arthritis.

G protein-coupled receptor kinase 2 (GRK2) could participate in the regulation of diverse cells via interacting with non-G-protein-coupled receptors. In the present work, we explored how paroxetine, a GRK2 inhibitor, modulates the differentiation and activation of immune cells in rheumatoid arthritis (RA). The blood samples of healthy individuals and RA patients were collected between July 2021 and March 2022 from the First Affiliated Hospital of Anhui Medical University. C57BL/6 mice were used to induce the collagen-induced arthritis (CIA) model. Flow cytometry analysis was used to characterize the differentiation and function of dendritic cells (DCs)/T cells. Co-immunoprecipitation was used to explore the specific molecular mechanism. In patients with RA, high expression of GRK2 in peripheral blood lymphocytes, accompanied by the increases of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In animal model, a decrease in regulatory T cells (Tregs), an increase in the cluster of differentiation 8 positive (CD8+) T cells, and maturation of DCs were observed. Paroxetine, when used in vitro and in CIA mice, restrained the maturation of DCs and the differentiation of CD8+ T cells, and induced the proportion of Tregs. Paroxetine inhibited the secretion of pro-inflammatory cytokines, the expression of C-C motif chemokine receptor 7 in DCs and T cells. Simultaneously, paroxetine upregulated the expression of programmed death ligand 1, and anti-inflammatory cytokines. Additionally, paroxetine inhibited the PI3K-AKT-mTOR metabolic pathway in both DCs and T cells. This was associated with a reduction in mitochondrial membrane potential and changes in the utilization of glucose and lipids, particularly in DCs. Paroxetine reversed PI3K-AKT pathway activation induced by 740 Y-P (a PI3K agonist) through inhibiting the interaction between GRK2 and PI3K in DCs and T cells. Paroxetine exerts an immunosuppressive effect by targeting GRK2, which subsequently inhibits the metabolism-related PI3K-AKT-mTOR pathway of DCs and T cell in RA.

Dysfunction of astrocytic glycophagy exacerbates reperfusion injury in ischemic stroke

Glycophagy has evolved from an alternative glycogen degradation pathway into a multifaceted pivot to regulate cellular metabolic hemostasis in peripheral tissues. However, the pattern of glycophagy in the brain and its potential therapeutic impact on ischemic stroke remain unknown. Here, we observed that the dysfunction of astrocytic glycophagy was caused by the downregulation of the GABA type A receptor-associated protein like 1 (GABARAPL1) during reperfusion in ischemic stroke patients and mice. PI3K-Akt pathway activation is involved in driving GABARAPL1 downregulation during cerebral reperfusion. Moreover, glycophagy dysfunction-induced glucosamine deficiency suppresses the nuclear translocation of specificity protein 1 and TATA binding protein, the transcription factors for GABARAPL1, by decreasing their O-GlcNAcylation levels, and accordingly feedback inhibits GABARAPL1 in astrocytes during reperfusion. Restoring astrocytic glycophagy by overexpressing GABARAPL1 decreases DNA damage and oxidative injury in astrocytes and improves the survival of surrounding neurons during reperfusion. In addition, a hypocaloric diet in the acute phase after cerebral reperfusion can enhance astrocytic glycophagic flux and accelerate neurological recovery. In summary, glycophagy in the brain links autophagy, metabolism, and epigenetics together, and glycophagy dysfunction exacerbates reperfusion injury after ischemic stroke.

Mrgprb2-mediated mast cell activation exacerbates Modic changes by regulating immune niches

Modic changes are radiographic features associated with microfracture, low-virulence organism infection and chronic inflammation with inflammatory cell infiltration in the vertebral endplate region. Mast cells, as innate immune cells similar to macrophages, are present in painful degenerated intervertebral discs. However, the involvement and mechanisms of mast cells in the development of Modic changes remain unclear. Herein, we found increased mast cell infiltration in samples from patients with Modic changes and in mouse models of Modic changes. To clarify the role of mast cells in the progression of Modic changes, we used mast cell-deficient (KITW-SH/W-SH) mice to construct a model of Modic changes and found that the severity of Modic changes in KITW-SH/W-SH mice was significantly lower than that in WT mice. These findings were further supported by the use of a mast cell-specific activator (compound 48/80) and a stabilizer (cromolyn). Furthermore, we found that mast cells were not activated via the classic IgE pathway in the Modic change models and that Mrgprb2 is the specific receptor for mast cell activation reported in recent studies. Then, we utilized Mrgprb2 knockout mice to demonstrate that Mrgprb2 knockout inhibited mast cell activation and thus reduced the degree of Modic changes. Transcriptomic sequencing revealed aberrant PI3K-AKT and MAPK pathway activation in the Mrgprb2-deficient mast cells. Additionally, Mrgpbrb2-activated mast cells regulate immune niches by recruiting macrophages, promoting M1 polarization and reducing M2 polarization, thereby promoting the progression of Modic changes. These findings suggest that mast cells may serve as a novel therapeutic target for addressing Modic changes.

WITHDRAWN: Recombinant osteopontin stabilizes the blood brain barrier following subarachnoid hemorrhage in rats

Background and PurposeRecombinant osteopontin (rOPN) decreases neuronal cell death and brain edema and improves the neurological scores following subarachnoid hemorrhage (SAH) in rats. The molecular mechanisms of the preservation of blood-brain barrier of rOPN following SAH in rats were examined in this study. MethodsAdult rats were divided into 3 groups: sham, vehicle, and treatment groups. Each animal in the SAH group underwent a surgical procedure to induce SAH, and the brains was harvested at 24 hours for analysis. For the mechanism part of the study two additional groups were operated and treated with Fib-14 and Wortmannin additional to rOPN. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 hours after SAH induction. The blood brain barrier (BBB) permeability was evaluated using Evans Blue dye extravasation. The expressions of tight junction proteins claudin-3, claudin-5, occludin and the phosphorylation of β-catenin were examined using Western blot analysis. ResultsrOPN improved neurological scores and reduced brain edema at 24 hours after SAH. The BBB preservation action of rOPN is mediated in part through stabilizing β-catenin and increasing expression of tight junction proteins, claudin-3, claudin-5, and occludin. rOPN is thought to exert its effects through FAK activated PI3K-Akt pathway. ConclusionSince BBB disruption is one of the major pathophysiology of EBI after SAH, rOPN may be a promising drug to maintain BBB integrity and functions in SAH treatment.

The M2 Macrophages Derived Migrasomes From the Surface of Titania Nanotubes Array as a New Concept for Enhancing Osteogenesis.

As newly discovered substrate anchored extracellular vesicles, migrasomes (Migs) may bring a new opportunity for manipulating target cells bioactivities. In this study, the M2 macrophages derived Migs are obtained by titania nanotubes surface (NTs). Due to the benefits of nanostructuring, the NTs surface is not only able to induce RAW264.7 for M2 polarization but also to generate more Migs formation, which can be internalized by following seeded mesenchymal stem cells (MSCs). Then, the NTs surface induced Migs are collected by density-gradient centrifugation for MSCs treatment. As indicated by immunofluorescence staining, alkaline phosphatase activity, and alizarin red staining, the osteogenic differentiation capacity of MSCs is significantly enhanced by Migs treatment, in line with the dosage. By RNA-sequence analysis, the enhancement of osteogenic differentiation is correlated with PI3K-AKT pathway activation that may originate from the M2 polarization state of donor cells. Finally, the Migs are coated onto Ti surface for therapeutic application. Both the in vitro and in vivo analysis reveal that the Migs coated Ti implant shows significant enhancement of osteogenesis. In conclusion, this study suggests that the nanosurface may be a favorable platform for Migs production, which may bring a new concept for tissue regeneration.

Abstract 3795: Possible role of miR-375-3p in cervical lymph node metastasis of oral squamous cell carcinoma

Abstract Latent cervical lymph node metastasis (LNM) is the most important prognostic factor in early oral squamous cell carcinoma (OSCC). However, no clinically useful predictors of LNM in early OSCC are available. In this study, we focused on the microRNAs (miRNAs) involved in the expression of numerous genes and explored those associated with latent cervical LNM in OSCC. Microarray analysis of miRNA expression showed that the expression levels of miR-375-3p were significantly reduced in primary OSCC tissues with latent cervical LNM. Subsequently, we quantified the expression of miR-375-3p by RT-qPCR and digital PCR. Similar to the results of miRNA microarray analysis, miR-375-3p was significantly downregulated in primary OSCC tissues with latent cervical LNM. The results of digital PCR showed a more significant difference than those of RT-qPCR. When the reference value for predicting latent cervical LNM was set to less than 57.85 copies based on the receiver operating characteristic curve, the sensitivity, specificity, accuracy, and area under the curve were 80%, 100%, 90%, and 0.942, respectively. Next, we examined the effects of miR-375-3p mimics on the growth and migration of four human OSCC cell lines (SAS-L1, HSC2, HSC3, and Ca9-22) that do not express miR-375-3p. When an miR-375-3p mimic was introduced into these cells, cell proliferation and migration were significantly suppressed. Total RNA was extracted from these cells reverse-transfected with miR-375-3p mimic for 48 hours. Microarray analysis and Ingenuity Pathway Analysis (IPA) miRNA target filter showed that miR-375-3p overexpression reduced the expression of 266 genes, of which 37 genes had the target sequences of miR-375-3p in their 3’-untranslated region. Among these target genes, CEPT1 and TIMM8A knockdown significantly inhibited the migration of all human OSCC cells, similar to miR-375-3p mimics. Furthermore, IPA core analysis revealed a decrease in expression of genes involved in the activation of PI3K-AKT pathways by miR-375-3p overexpression. These results indicated that downregulating miR-375-3p expression could promote cervical LNM by enhancing cell proliferation and migration in OSCC. We also found that CEPT1 and TIMM8A as novel target genes of miR-375-3p regulated the migration of human OSCC cells. The expression levels of miR-375-3p in primary OSCC tissues appear to be a useful biomarker for predicting latent cervical LNM. Citation Format: Masato Saika, Koh-ichi Nakashiro, Norihiko Tokuzen, Hiroyuki Shirai, Daisuke Uchida. Possible role of miR-375-3p in cervical lymph node metastasis of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3795.

Abstract 3064: Unmasking the deadly tango: PKA and lipid genes in prostate cancer's bone invasion

Abstract About 88% of metastatic prostate cancer (PCa) cases involve bone metastasis. When PCa cells spread to the bone, a metabolic adaptation to the new environment takes place, partly influenced by factors secreted by the homing organ. Thus, we hypothesize that metabolic reprogramming, both in the tumor and bone niche drive PCa bone metastasis. In this work, we carried out an integrative in vitro, in vivo, and bioinformatics approach to identify key regulators governing the metabolic behavior of PCa cells. We performed an indirect co-culture between PCa (PC3) and bone progenitor (MC3T3 or Raw264.7) cells. PCa cells displayed a strong transcriptional (RNA-seq) activation of lipid metabolism, including PPAR-signaling, fat digestion, and PI3K-Akt pathway. Accordingly, treatment with the conditioned media (CM) of the co-culture promoted lipid accumulation in PC3 cells (Bodipy 493/503 staining). To assess the clinical relevance of the in vitro model, we performed an unsupervised clustering analysis using transcriptomics data from human PCa and bone metastatic samples (GSE74685). Results showed that the metabolic genes deregulated in PC3 by the co-culture, could accurately cluster samples in primary tumor or bone metastasis. We next performed a multivariable Cox regression analysis and built a prognostic model, identifying a novel signature of 5 lipid-associated genes, PPARA, VDR, SLC16A1, PAPSS2 and GPX1, associated with a 23-fold higher risk of death in PCa patients (SU2C-PCF dataset). The expression of this signature was validated in a PDX pre-clinical model (MDA PCa PDX series) when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously. In order to identify the central hubs regulating this transcriptional phenotype, we performed an Ingenuity Pathway Analysis (QIAGEN), showcasing that the lipid-related signature deregulated by the bone niche is indeed modulated by the Protein Kinase A (PKA). Accordingly, the expression of these signature was downregulated by PKA inhibition (H89; PKA inhibitor). Moreover, secretome analyses (ESI MS/MS) revealed that bone secreted type I collagen (Col1a1) and fibronectin (Fn1) regulate PKA activity in PCa cells. Further, we investigated whether PKA could modulate critical mechanisms for bone progression, focusing on osteopontin (SPP1), a secreted protein involved in the adhesion and invasion of PCa cells, whose regulatory mechanism in tumor cells is yet to be elucidated. Strikingly, we observed not only that the activation of PKA using Forskolin in PCa cells activated SPP1 expression, but also that PKA inhibition led to a significant impairment of the SPP1 induction triggered by bone soluble factors secreted during the PC3/MC3T3 co-culture. Altogether, we showcase PKA as a central hub of PCa energetic metabolism during bone progression and outline a lipid-associated gene signature that can be targeted to halt metastatic PCa. Citation Format: Pablo Sanchis, Nicolas Anselmino, Estefania Labanca, Agustina Sabater, Juan Bizzotto, Gaston Pascual, Rocio Seniuk, Sofia Lage-Vickers, Antonina Mitrofanova, Pia Valacco, Ayelen Toro, Javier Cotignola, Nora Navone, Elba Vazquez, Geraldine Gueron. Unmasking the deadly tango: PKA and lipid genes in prostate cancer's bone invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3064.

Abstract 2269: Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer

Abstract PIK3CA mutations occur in ~15% of triple-negative breast cancers (TNBCs) and aberrantly activate the PI3K-AKT pathway, resulting in uncontrolled cell proliferation. Alpelisib is approved for hormone receptor-positive (HR+), HER2-negative BC patients with a PIK3CA mutation; however, not all PIK3CA-mutated (MUT), HR+ patients respond to alpelisib and others acquire resistance following exposure, suggesting adaptive mechanisms of PI3K activation. It has been proven that in TNBC, there exists a cohort of mutated genes which activate PI3K-AKT signaling. We hypothesize that testing alpelisib in combination with repurposed approved drugs, targeting alternative mechanisms of PI3K-AKT pathway activation, may be used to treat a cohort of TNBC patients. TNBC patient genetic data from the MSK (N=168), METABRIC (N=209), and TCGA (N=171) datasets were analyzed using cBioPortal to identify frequently occurring mutations. Differential expression analyses based on mutational status were performed on CCLE cell line (N=705) microarray and TCGA BC patient (N=981) RNA-sequencing data using R. Dysregulated genes were functionally analyzed using DAVID Ontology and those associated with poorer survival in TNBC were identified using KM-Plotter. STRING database was used to investigate protein interactions. The antiproliferative effects of targeted drugs tested alone and in combination in a panel of 6 TNBC cell lines were determined using acid phosphatase assays. Reverse-phase protein array analysis was carried out on protein lysates from cells following 24 hour treatment with drugs used alone and in combination.TP53 is the most frequently mutated gene in TNBC and mutations were associated with significant genetic dysregulation. A comprehensive analysis of upregulated genes in TP53-MUT TNBC led to the identification of 5 potential therapeutic targets - MET, S100A9, LCN2, CA9, TACSTD2, and KLK5 - based on their association with poorer survival in TNBC, protein interactions with TP53 and PIK3CA, and availability of targeted drugs. MET was selected for in vitro analysis based on its strong correlation with PI3K-AKT signaling. The presence of a TP53 mutation was associated with decreased antiproliferative effects in TNBC cell lines treated with alpelisib. Treatment with alpelisib in combination with a MET inhibitor, crizotinib, demonstrated greater antiproliferative effects in TP53-MUT/PIK3CA-MUT cell lines when compared to TP53 or PIK3CA MUT only models.TP53 mutations are associated with genetic dysregulation in TNBC and may confer resistance to alpelisib. Targeting upregulated proteins in a cohort of TP53-MUT/PIK3CA-MUT patients may improve sensitivity to alpelisib via dual inhibition of PI3K-AKT pathway activation. Protein analysis will uncover the mechanisms of synergy observed between alpelisib and crizotinib, and the therapeutic potential of the combination will be experimentally investigated in vivo. Citation Format: Grace Colley, Alexander Eustace, Dalal AlSultan. Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2269.

Abstract IA23: "NO Target for Cancer Stem Cells"

Abstract Developing novel therapies that target cancer metastasis, chemoresistant cancer stem cells, and augment the efficacy of systemic chemo/radiotherapies is crucial to reducing cancer-associated mortality. A protein within the tumor microenvironment that has gained interest as a promising therapeutic target is nitric oxide synthase (NOS). Our previous findings have shown that increased inducible nitric oxide synthase (iNOS) expression is a poor prognostic indicator and associated with worse overall survival in triple negative breast cancer (TNBC) patients. Nitric oxide (NO) is a unique molecule in its ability to target multiple oncogenic pathways in a spatial and temporal manner, such as PI3K, extracellular signal-regulated kinase (ERK), β-catenin pathway, transforming growth factor beta (TGFβ) signaling, and hypoxia-inducible factor (HIF). Growing evidence has also suggested that hyperactivation of the phosphoinositide-3-kinase (PI3K) survival signaling pathway may be one of the most common oncogenic aberrations in certain TNBC subsets, in particular metaplastic breast cancer (MpBC). We will summarize the role of NO in PI3K-Akt pathway activation in human TNBC, as well as data from The Cancer Genome Atlas of the role of iNOS and PI3K-Akt interaction. This review discusses the current knowledge of the roles of nitric oxide (NO) in tumor progression and cancer metastasis, and promising preclinical studies that evaluated NOS inhibitors as an anti-cancer therapy. Lastly, we will offer a perspective on utilizing NOS inhibitors in the clinical setting, highlighting the prospects and outstanding challenges. Citation Format: Jenny C. Chang. "NO Target for Cancer Stem Cells" [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA23.

Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.

Polycystic ovarian syndrome (PCOS) and recurrent spontaneous abortion (RSA) are associated with the PI3K-AKT pathway activation.

We aimed to elucidate the mechanism leading to polycystic ovarian syndrome (PCOS) and recurrent spontaneous abortion (RSA). PCOS is an endocrine disorder. Patients with RSA also have a high incidence rate of PCOS, implying that PCOS and RSA may share the same pathological mechanism. The single-cell RNA-seq datasets of PCOS (GSE168404 and GSE193123) and RSA GSE113790 and GSE178535) were downloaded from the Gene Expression Omnibus (GEO) database. Datasets of PSCO and RSA patients were retrieved from the Gene Expression Omnibus (GEO) database. The "WGCNA" package was used to determine the module eigengenes associated with the PCOS and RSA phenotypes and the gene functions were analyzed using the "DAVID" database. The GSEA analysis was performed in "clusterProfiler" package, and key genes in the activated pathways were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Real-time quantitative PCR (RT-qPCR) was conducted to determine the mRNA level. Cell viability and apoptosis were measured by cell counting kit-8 (CCK-8) and flow cytometry, respectively. The modules related to PCOS and RSA were sectioned by weighted gene co-expression network analysis (WGCNA) and positive correlation modules of PCOS and RSA were all enriched in angiogenesis and Wnt pathways. The GSEA further revealed that these biological processes of angiogenesis, Wnt and regulation of cell cycle were significantly positively correlated with the PCOS and RSA phenotypes. The intersection of the positive correlation modules of PCOS and RSA contained 80 key genes, which were mainly enriched in kinase-related signal pathways and were significant high-expressed in the disease samples. Subsequently, visualization of these genes including PDGFC, GHR, PRLR and ITGA3 showed that these genes were associated with the PI3K-AKT signal pathway. Moreover, the experimental results showed that PRLR had a higher expression in KGN cells, and that knocking PRLR down suppressed cell viability and promoted apoptosis of KGN cells. This study revealed the common pathological mechanisms between PCOS and RSA and explored the role of the PI3K-AKT signaling pathway in the two diseases, providing a new direction for the clinical treatment of PCOS and RSA.

Enhancer-mediated FOXO3 expression promotes MSC adipogenic differentiation by activating autophagy

BackgroundMesenchymal stem cells (MSCs) are pluripotent stem cells capable of differentiating into osteocytes, adipocytes and chondrocytes. However, in osteoporosis, the balance of differentiation is tipped toward adipogenesis and the key mechanism is controversial. Researches have shown that, as upstream regulatory elements of gene expression, enhancers ar involved in the expression of identity genes. In this study, we identified enhancers-mediated gene FOXO3 promoting MSC adipogenic differentiation by activating autophagy. MethodsWe integrated data of RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and ATAC-sequencing (ATAC-seq) to find the identity gene FOXO3. The expression of FOXO3 protein, adipogenic transcription factors and the substrate of autophagy were measured by western blotting. The Oil Red O (ORO) staining was used to visualize the adipogenesis of MSCs. Immunohistochemistry was used to visualize the FOXO3 expression in adipocytes in bone marrow. Immunofluorescence was used to detect the expression of PPARγ and LC3B. ResultsDuring adipogenesis, enhancers redistribute to genes associated with adipogenic differentiation, among which we identified the pivotal identity gene FOXO3. FOXO3 could promote the expression of the adipogenic transcription factors PPARγ, CEBPα, and CEBPβ during adipogenic differentiation, while PPARγ, CEBPα, and CEBPβ could in turn bind to FOXO3 and continue to promote FOXO3 expression to form a positive feedback loop. Consistently elevated FOXO3 expression promotes autophagy by activating the PI3K-AKT pathway which mediates adipogenic differentiation. ConclusionsPivotal identity gene FOXO3 promotes autophagy by activating PI3K-AKT pathway, which provokes adipogenic differentiation of MSCs. Enhancer-regulated adipogenic identity gene FOXO3 could be an attractive treatment for osteoporosis.

Enhanced VEGF secretion and blood-brain barrier disruption: Radiation-mediated inhibition of astrocyte autophagy via PI3K-AKT pathway activation.

Radiation-induced damage to the blood-brain barrier (BBB) is the recognized pathological basis of radiation-induced brain injury (RBI), a side effect of head and neck cancer treatments. There is currently a lack of therapeutic approaches for RBI due to the ambiguity of its underlying mechanisms. Therefore, it is essential to identify these mechanisms in order to prevent RBI or provide early interventions. One crucial factor contributing to BBB disruption is the radiation-induced activation of astrocytes and oversecretion of vascular endothelial growth factor (VEGF). Mechanistically, the PI3K-AKT pathway can inhibit cellular autophagy, leading to pathological cell aggregation. Moreover, it acts as an upstream pathway of VEGF. In this study, we observed the upregulation of the PI3K-AKT pathway in irradiated cultured astrocytes through bioinformatics analysis, we then validated these findings in animal brains and in vitro astrocytes following radiation exposure. Additionally, we also found the inhibition of autophagy and the oversecretion of VEGF in irradiated astrocytes. By inhibiting the PI3K-AKT pathway or promoting cellular autophagy, we observed a significant amelioration of the inhibitory effect on autophagy, leading to reductions in VEGF oversecretion and BBB disruption. In conclusion, our study suggests that radiation can inhibit autophagy and promote VEGF oversecretion by upregulating the PI3K-AKT pathway in astrocytes. Blocking the PI3K pathway can alleviate both of these effects, thereby mitigating damage to the BBB in patients undergoing radiation treatment.

Antioxidant-Engineered Milk-Derived Extracellular Vesicles for Accelerating Wound Healing via Regulation of the PI3K-AKT Signaling Pathway.

Inspired by the experience of relieving inflammation in infants with milk, antioxidant-engineered milk-derived extracellular vesicles (MEVs) are developed to evaluate their potential for accelerating wound healing. In this work, MEVs with polydopamines (PDA) are engineered using the co-extrusion method. Subsequently, the authors incorporated them into a Schiff-based crosslink hydrogel, forming a skin dosage form that could facilitate the wound healing process. The antioxidant properties of PDA assist in the anti-inflammatory function of engineered MEVs, while the gel provides better skin residency. The PDA@MEVs+GEL formulation exhibits excellent biocompatibility, pro-angiogenic capacity, and antioxidant ability in vitro. Furthermore, in vivo experiments demonstrate its efficacy in wound repair and inflammation inhibition. Mechanistically, PDA@MEVs+GEL simultaneously promotes the growth, migration, and anti-inflammation of 3T3 cells by activating PI3K-AKT pathway. Moreover, PDA@MEVs+GEL exhibits enhanced functionality in promoting wound healing in vivo, attributed to its ability to inhibit inflammation, stimulate angiogenesis, and promote collagen synthesis. In conclusion, this study delves into the mechanism of MEVs and underscores the improved efficacy of engineered extracellular vesicles. Additionally, the feasibility and prospect of engineered MEVs in treating skin wounds are verified, suggesting that antioxidant-engineered MEVs could be a promising therapeutic agent for wound healing applications.

Longitudinal Profiling of Tumor RNA Expression Signatures Reveal Key Biological Features Associated with Response to Neoadjuvant Stereotactic Body Radiation Therapy in High-Risk Prostate Cancer

Stereotactic body radiation therapy (SBRT) is a safe and effective treatment for localized prostate cancer (PCa). PREPARE-SBRT is a clinical trial testing the safety of neoadjuvant MRI-guided SBRT for men with high-risk localized PCa. We leveraged paired samples from pre-treatment biopsy and irradiated prostatectomy (RP) specimens to evaluate transcriptomic changes in irradiated tumors at acute time points following neoadjuvant SBRT. Tumor RNA expression profiles were generated using Decipher GRID by Veracyte on 12 subjects with paired pre- and post-SBRT tissues (n = 24). Descriptive statistics using gene expression profiles describing key biological features [DNA damage & repair (DDR), tumor proliferation, suppressed immune, activated immune, tumor microenvironment (TME)] and an exploratory analysis of RT sensitivity score with binary classification as sensitive or resistant were reported. A control cohort of transcriptomic profiles of 803 matched untreated biopsy and matching RP samples from the same subjects were used to control for signature differences attributable to sample type. Key tumor biology signatures most frequently observed were DDR (15/24), TME (11/24) and tumor proliferation (11/24). Signatures associated with tumor proliferation were disproportionately represented in pre-treatment samples (10/11) whereas TME-associated signatures were enriched predominantly in irradiated RP samples (8/11). Collectively, immune-related immune signatures skewed towards immune activation. All 3 samples annotated with suppressed immune signatures were from pre-treatment specimens whereas 75% (6/8) of samples annotated with activated immune status were from irradiated specimens. Additionally, conversion from suppressed to activated immune status was observed in 2 of 3 subjects (66%). In total, 42% of specimens (10/24) were designated as radio-resistant by RT sensitivity score. Among 8 baseline specimens annotated with RT resistant status, 75% of subjects (6/8) converted to RT sensitive status after neoadjuvant SBRT. Interestingly, in the two subjects with persistent radio-resistant status in pre- and post-samples there was associated with upregulation of TGF-β or PI3K-AKT pathway activation signatures. Pre- and post-SBRT transcriptomic signatures were heterogeneous and dynamic in a cohort of 12 patients with high-risk localized PCa highlighting the importance of studying longitudinal changes in individual patients. These data highlight an opportunity to leverage tumor RNA expression profiles to personalize patient and treatment selection and augment radiation response assessment. gov ID (NCT03663218).

IGF1R Contributes to Cell Proliferation in ALK-Mutated Neuroblastoma with Preference for Activating the PI3K-AKT Signaling Pathway.

Aberrant activation of anaplastic lymphoma kinase (ALK) by activating point mutation or amplification drives 5-12% of neuroblastoma (NB). Previous work has identified the involvement of the insulin-like growth factor 1 receptor (IGF1R) receptor tyrosine kinase (RTK) in a wide range of cancers. We show here that many NB cell lines exhibit IGF1R activity, and that IGF1R inhibition led to decreased cell proliferation to varying degrees in ALK-driven NB cells. Furthermore, combined inhibition of ALK and IGF1R resulted in synergistic anti-proliferation effects, in particular in ALK-mutated NB cells. Mechanistically, both ALK and IGF1R contribute significantly to the activation of downstream PI3K-AKT and RAS-MAPK signaling pathways in ALK-mutated NB cells. However, these two RTKs employ a differential repertoire of adaptor proteins to mediate downstream signaling effects. We show here that ALK signaling led to activation of the RAS-MAPK pathway by preferentially phosphorylating the adaptor proteins GAB1, GAB2, and FRS2, while IGF1R signaling preferentially phosphorylated IRS2, promoting activation of the PI3K-AKT pathway. Together, these findings reveal a potentially important role of the IGF1R RTK in ALK-mutated NB and that co-targeting of ALK and IGF1R may be advantageous in clinical treatment of ALK-mutated NB patients.

Downregulation of PIK3IP1 in retinal microglia promotes retinal pathological neovascularization via PI3K-AKT pathway activation

Retinal pathological neovascularization involves endothelial cells, pericytes, photoreceptor cells, ganglion cells, and glial cells, whose roles remain unclear. Using the Scissor algorithm, we found that microglia are associated with formation of fibrovascular membranes and can promote pathological neovascularization. GO and KEGG results showed that PI3K-AKT pathway activation in retinal microglia was associated with pathological neovascularization, and PIK3IP1 was associated with retinal microglia activation. Then we used PCR, Western blot and Elisa techniques to confirm that the expression of VEGFA, FGF2, HGFα and MMP9 was increased in microglia after Lipopolysaccharide (LPS) induction. We also used cell flow cytometry and OIR models to verify the role of PI3K-AKT pathway and PIK3IP1 in microglia. Targeting of PIK3IP1 regulated the activation of the PI3K-AKT pathway in microglia, microglia function activation, and pro-angiogenic effects. These findings reveal the role of M1-type microglia in pathological neovascularization and suggests that targeting the PI3K-AKT pathway in microglia may be a new strategy for treating retinal pathological neovascularization.