Catalytic Subunit Of Phosphatidylinositol 3-kinase Research Articles

Protective effects of PIK3CG knockdown against OGD/R-induced neuronal damage via inhibition of autophagy through the AMPK/mTOR pathway

BackgroundIschemic stroke represents an urgent need for more efficacious therapies owing to modest effectiveness of current treatment. MethodsDownload data from stroke patients and collect blood samples from clinical patients to analyze phosphatidylinositol-3 kinase catalytic subunit γ (PIK3CG) expression. To establish a brain damage model, oxygen glucose deprivation/reperfusion (OGD/R) was applied to SH-SY5Y cells. Impact of PIK3CG on AMPK/mTOR autophagy pathway was verified treating cells with AMPK activator metformin. Proliferation and apoptosis were identified by CCK8 and flow cytometry. ResultsDifferential expression analysis and clinical testing show that PIK3CG is highly expressed in patients. Prolonged ODG/R exposure increased PIK3CG levels, supressed cell proliferation, and induced apoptosis. KEGG pathway analysis implicated PIK3CG in autophagy pathway. Knockdown of PIK3CG supressed OGD/R-induced reductions in cell proliferation and OGD/R-induced increases in apoptosis and expressions of Beclin 1 and LC3 II. Following OGD/R, AMPK phosphorylation was upregulated while mammalian target of rapamycin (mTOR) phosphorylation was downregulated, indicating AMPK/mTOR autophagy activation. Knockdown of PIK3CG opposed metformin-induced rises in Beclin 1, LC3 II and apoptosis along with decreases in proliferation. ConclusionPIK3CG knockdown protects neuronal cells by inhibiting AMPK/mTOR autophagy pathway and further inhibiting autophagy.

Potassium molybdate blocks APN-dependent coronavirus entry by degrading receptor via PIK3C3-mediated autophagy.

Swine enteric coronaviruses pose a significant challenge to the global pig industry, inflicting severe diarrhea and high mortality rates among piglets, and resulting in substantial economic losses. In our clinical practice, we observed that the addition of potassium molybdate (PM) to the feed could dramatically reduce diarrhea and diarrhea-related mortality in piglets. However, the underlying mechanisms remain elusive and merit further investigation. In this study, we revealed that PM effectively inhibited the infection of both aminopeptidase N (APN)-dependent coronaviruses, transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV), both in vitro and ex vivo. Specifically, PM was found to block TGEV and PRCV penetration by degrading the cell receptor APN through the upregulation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) expression. In addition, knockdown and knockout of PIK3C3 resulted in the attenuation of PM-induced autophagy, thereby rescuing APN expression and viral infection. Correspondingly, replenishment of PIK3C3 in PIK3C3-null ST cells restored PM-mediated APN degradation and successfully blocked viral entry. Furthermore, our findings demonstrated that PM promoted the assembly of the PIK3C3-BECN1-ATG14 complex, leading to induced autophagic degradation by upregulating PIK3C3 Ser249 phosphorylation. In vivo experiments further confirmed that PM-induced PIK3C3-mediated autophagic degradation of APN, thereby limiting the pathogenicity of TGEV. In summary, our study for the first time identified the mechanism by which PM blocked TGEV and PRCV internalization by degrading the cell receptor APN via PIK3C3-mediated autophagy. This study provides valuable insights and potential strategies for preventing APN-restricted coronavirus infection.IMPORTANCEAminopeptidase N (APN) is one of the most important host receptors of coronavirus. Modulating APN expression can represent a novel approach for controlling APN-dependent coronaviruses and their variants infection. Here we found that a chemical compound potassium molybdate (PM) negatively regulates APN expression by inducing phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3)-mediated autophagy against APN-dependent coronavirus internalization, including transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV). Furthermore, PM can promote PIK3C3-BECN1-ATG14 complex assembly to induce autophagic degradation of APN by upregulating PIK3C3 Ser249 phosphorylation. Lastly, results from pig experiments also confirmed that PM can trigger PIK3C3-mediated autophagic degradation of APN to restrict TGEV pathogenicity in vivo without toxicity. Our findings underscore the promising potential of PM as an effective agent against APN-dependent coronavirus and potentially emerging viral disease entry.

Subunit-Specific Developmental Roles of PI3K in SF1-Expressing Cells.

Phosphatidylinositol 3-kinase (PI3K) regulates cellular development and energy homeostasis. However, the roles of its subunits in organ development remain largely unknown. We explored the roles of PI3K catalytic subunits in steroidogenic factor 1 (SF1)-expressing cells through knockout (KO) of the p110α and p110β subunits. We examined mice with a double KO of p110α and p110β in SF1-expressing cells (p110αβ KOSF1). Although these animals exhibited no significant changes in the development of the ventromedial hypothalamus, we noted pronounced hypotrophy in the adrenal cortex, testis, and ovary. Additionally, corticosterone and aldosterone levels were significantly reduced. The absence of these subunits also resulted in decreased body weight and survival rate, along with impaired glucose homeostasis, in p110αβ KOSF1 mice. The data demonstrate the specific roles of PI3K catalytic subunits in the development and function of SF1-expressing organs.

Single-cell sequencing of facial adipose tissue unveils FKBP5 as a therapeutic target for facial infiltrating lipomatosis

BackgroundFacial infiltrating lipomatosis is characterized by excessive growth of adipose tissue. Its etiology is associated with somatic phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) variants, but the specific mechanisms are not yet fully understood.MethodsWe collected facial adipose tissue from both FIL patients and non-FIL individuals, isolated the stromal vascular fraction (SVF) and performed single-cell transcriptome sequencing on these samples.ResultsWe mapped out the cellular landscape within the SVF, with a specific focus on a deeper analysis of fibro-adipogenic precursor cells (FAPs). Our analysis revealed that FAPs from FIL patients (FIL-FAPs) significantly overexpressed FK506 binding protein 51 (FKBP5) compared to FAPs from individuals without FIL. Further experiments indicated that FKBP5 is regulated by the PI3K-AKT signaling pathway. The overactivation of this pathway led to an increase in FKBP5 expression. In vitro experiments demonstrated that FKBP5 promoted adipogenic differentiation of FAPs, a process that could be hindered by FKBP5 knockdown or inhibition. Additionally, in vivo assessments confirmed FKBP5’s role in adipogenesis.ConclusionsThese insights into the pathogenesis of FIL underscore FKBP5 as a promising target for developing non-surgical interventions to manage the excessive adipose tissue growth in FIL.

Transcriptomic analysis reveals novel hub genes associated with astrocyte autophagy in intracerebral hemorrhage.

Neuroinflammation serves as a critical local defense mechanism against secondary brain injury following intracerebral hemorrhage (ICH), and astrocytes play a prominent role in this process. In this study, we investigated astrocytic changes during the inflammatory state after ICH to identify new targets for improving the inflammatory response. We stimulated mouse astrocytes with lipopolysaccharide (LPS) in vitro and analyzed their transcriptomes via ribonucleic acid sequencing. We created an ICH model in living organisms by injecting autologous blood. RNA sequencing revealed that 2,717 genes were differentially expressed in the LPS group compared to those in the saline group, with notable enrichment of the autophagic pathway. By intersecting the 2,717 differentially expressed genes (DEGs) with autophagy-related genes, we identified 36 autophagy-related DEGs and seven hub genes. Previous studies and quantitative reverse transcription-polymerase chain reaction results confirmed the increased expression of phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3), AKT serine/threonine kinase 1 (Akt1), and unc-51 like autophagy activating kinase 2 (Ulk2) in astrocytes after ICH. Transcription factors and target miRNAs were identified for the final three DEGs, and 3-methyladenine and leupeptin were identified as potential therapeutic agents for ICH. Our findings suggest that astrocyte autophagy plays a critical role in ICH complexity, and that Pik3c3, Akt1, and Ulk2 may be potential therapeutic targets.

Prevalence of somatic PIK3CA mutations in hormone receptor-positive metastatic breast cancer: The Indian experience.

e13047 Background: The phosphatidylinositol 3-kinase (PI3K) inhibitors have been found to improve survival outcomes in hormone receptor-positive metastatic breast cancer (MBC) and are FDA-approved in this setting after progression on hormonal therapy. However, real-world data from India on the prevalence of PIK3CA mutations is scarce. Methods: This is a retrospective study including patients with hormone receptor-positive breast cancer registered at our center between January 2017- and December 2023 who developed metastatic disease on follow-up after getting appropriate treatment (adjuvant /neoadjuvant) as per institutional protocol. The PIK3CA mutation testing was done on the biopsy specimen. FDA-approved therascreen PIL3CA RGQ PCR kit from Qiagen was used and the assay is a real-time qualitative PCR test for the detection of 11 mutations phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene (Exon 7: C420R; Exon 9: E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y) using genomic DNA (gDNA) extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue. The clinical demographics of patients with PIK3CA mutations were extracted from the hospital database and represented descriptively. Results: A total of 80 consecutive patients with hormone-positive breast cancer at relapse were screened and 26 were found to have PIK3CA mutations (32.5%) of which 24 were female (92.3%). The median age of patients with mutated PIK3CA cases was 50 years (range: 31 to 72 years). The clinical stage of disease at presentation was: stage II in 11.5%, IIIA in 23.1%, and IIIB in 65.4% of cases. The median time of relapse was 21 months from diagnosis (range: 10 to 42 months). The disease sites at relapse were multiple in 23 patients (88.46%) [including central nervous system (CNS) relapse in 3 patients) and CNS (brain) only in 3 patients (11.54%). The mutations detected included H1047R (3140A>G at exon 20) in 8 cases, E545K (1633 G>A at exon 9) in 5 cases, Q546R (1637 A>G at exon 9) in 4 cases, E542K (1624 G>A at exon7) in 3 cases and synchronous mutations (E542K/E545K at Exon 7) in 3 cases and (H1047R/H1047L at exon 20 ) in 3 cases. Only eight patients received alpelisib with fulvestrant due to financial constraints. Conclusions: This is the first Indian study that describes the prevalence of PIK3CA mutations in hormone-positive breast cancer at relapse. Around one-third of patients harbor this mutation and 23.1% of cases have CNS involvement at relapse. The most common PIK3CA mutation detected was H1047R (exon 20) followed by E545K (exon 9).

Abstract 3142: Pik3c3 Safeguards Smooth Muscle Identity By Promoting Lysosome-mediated Yap1 Degradation

Background: Mature vascular smooth muscle cells (VSMCs) are quiescent and geared for maintaining vascular homeostasis by expressing a unique repertoire of contractile genes. However, key factors in controlling the contractile phenotype of VSMCs are poorly defined. Class III PI3K, PIK3C3 (Phosphatidylinositol 3-Kinase Catalytic Subunit Type 3) is a critical regulator of vesicular trafficking. Although PIK3C3 is known to play an essential role in cardiac homeostasis, its role in VSMCs remains elusive. Methods: To elucidate the role of Pik3c3 in regulating SMC fate and plasticity, we generated smooth muscle-specific Pik3c3 inducible knock out (KO) mice with SMC-lineage tracing. Proteomic mass spectrometry, next-generation RNA sequencing, SMC fate mapping coupled with single-cell RNA sequencing were applied to determine the phenotype and trajectories of SMCs after Pik3c3 ablation. Results: Specific ablation of Pik3c3 in SMCs results in severe gastrointestinal pseudo-obstruction and premature death. Analysis of aorta from the KO mice revealed dramatic vascular remodeling including dilatation, thickening of the vascular wall, spontaneous neointimal formation that was largely contributed by cells of SMC origin, and complete loss of contractility. Proteomics, bulk RNA-seq, and scRNA-seq studies of SMC-restricted Pik3c3 KO mouse aorta showed near complete loss of smooth muscle identity and the acquisition of an intermediate smooth muscle progenitor-like cell phenotype that underwent re-programming into multiple cell types including macrophages, T cells and osteoblasts. Mechanistically, we found that Pik3c3 deletion in VSMCs induces the accumulation of YAP1 (Yes associated protein 1) and its downstream target genes; this elevated YAP1 program appears to result from a blockade of autophagic lysosome-mediated degradation of YAP1, leading to phenotypic modulation of SMCs. Importantly, pharmacological inhibition of YAP by Verteporfin or genetic deletion of Yap1 rescues the vascular phenotype of Pik3c3 SM-specific KO mice. Conclusion: Our study reveals PIK3C3 as a master regulator safeguarding VSMC identity, and perturbation of the PIK3C3-YAP axis may be a novel mechanism underlying VSMC-driven vascular diseases in humans.

Unveiling potential drug targets for hyperparathyroidism through genetic insights via Mendelian randomization and colocalization analyses

Hyperparathyroidism (HPT) manifests as a complex condition with a substantial disease burden. While advances have been made in surgical interventions and non-surgical pharmacotherapy for the management of hyperparathyroidism, radical options to halt underlying disease progression remain lacking. Identifying putative genetic drivers and exploring novel drug targets that can impede HPT progression remain critical unmet needs. A Mendelian randomization (MR) analysis was performed to uncover putative therapeutic targets implicated in hyperparathyroidism pathology. Cis-expression quantitative trait loci (cis-eQTL) data serving as genetic instrumental variables were obtained from the eQTLGen Consortium and Genotype-Tissue Expression (GTEx) portal. Hyperparathyroidism summary statistics for single nucleotide polymorphism (SNP) associations were sourced from the FinnGen study (5590 cases; 361,988 controls). Colocalization analysis was performed to determine the probability of shared causal variants underlying SNP-hyperparathyroidism and SNP-eQTL links. Five drug targets (CMKLR1, FSTL1, IGSF11, PIK3C3 and SLC40A1) showed significant causation with hyperparathyroidism in both eQTLGen and GTEx cohorts by MR analysis. Specifically, phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) and solute carrier family 40 member 1 (SLC40A1) showed strong evidence of colocalization with HPT. Multivariable MR and Phenome-Wide Association Study analyses indicated these two targets were not associated with other traits. Additionally, drug prediction analysis implies the potential of these two targets for future clinical applications. This study identifies PIK3C3 and SLC40A1 as potential genetically proxied druggable genes and promising therapeutic targets for hyperparathyroidism. Targeting PIK3C3 and SLC40A1 may offer effective novel pharmacotherapies for impeding hyperparathyroidism progression and reducing disease risk. These findings provide preliminary genetic insight into underlying drivers amenable to therapeutic manipulation, though further investigation is imperative to validate translational potential from preclinical models through clinical applications.

Tetrandrine Alleviates Silica-induced Pulmonary Fibrosis Through PI3K/AKT Pathway: Network Pharmacology Investigation and Experimental Validation.

Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid derived from Stephania tetrandra S. Moor, known for its potential use in attenuating the progression of silicosis. However, the precise effects and underlying mechanisms of TET remain controversial. In this study, we aimed to elucidate the pharmacological mechanism of TET using a network pharmacology approach, while also evaluating its effect on silica-induced lung fibrosis in mice and TGF-β1-stimulated pulmonary fibroblasts in vitro. We employed network pharmacology to unravel the biological mechanisms through which TET may exert its therapeutic effects on pulmonary fibrosis and silicosis. In a silica-induced mouse model of lung fibrosis, TET was administered orally either during the early or late stage of fibrotic progression. Additionally, we examined the effects of TET on fibroblasts stimulated by TGF-β1 in vitro. Through the analysis, we identified a total of 101 targets of TET, 7,851 genes associated with pulmonary fibrosis, and 80 overlapping genes. These genes were primarily associated with key pathways such as epidermal growth factor receptor tyrosine kinase inhibitor resistance, the vascular endothelial growth factor signaling pathway, and the phosphatidylinositol 3 kinase (PI3K)-protein kinase B (PKB or AKT) signaling pathway. Furthermore, molecular docking analysis revealed the binding of TET to AKT1, the catalytic subunit of phosphatidylinositol-3 kinase, and KDR. In vivo experiments demonstrated that TET significantly alleviated silica-induced pulmonary fibrosis and reduced the expression of fibrotic markers. Moreover, TET exhibited inhibitory effects on the migration, proliferation, and differentiation of TGF-β1-induced lung fibroblasts in vitro. Notably, TET mitigated silica-induced pulmonary fibrosis by suppressing the PI3K/AKT pathway. In conclusion, our findings suggest that TET possesses the ability to suppress silica-induced pulmonary fibrosis by targeting the PI3K/AKT signaling pathway. These results provide valuable insights into the therapeutic potential of TET in the treatment of pulmonary fibrosis and silicosis.

Inhibition of phosphatidylinositol 3-kinase catalytic subunit alpha by miR-203a-3p reduces hypertrophic scar formation via phosphatidylinositol 3-kinase/AKT/mTOR signaling pathway.

Hypertrophic scar (HS) is a common fibroproliferative skin disease that currently has no truly effective therapy. Given the importance of phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) in hypertrophic scar formation, the development of therapeutic strategies for endogenous inhibitors against PIK3CA is of great interest. Here, we explored the molecular mechanisms underlying the protective effects of miR-203a-3p (PIK3CA inhibitor) against excessive scar. Bioinformatic analysis, immunohistochemistry, immunofluorescence, miRNA screening and fluorescence in situ hybridization assays were used to identify the possible pathways and target molecules mediating HS formation. A series of in vitro and in vivo experiments were used to clarify the role of PIK3CA and miR-203a-3p in HS. Mechanistically, transcriptomic sequencing, immunoblotting, dual-luciferase assay and rescue experiments were executed. Herein, we found that PIK3CA and the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway were upregulated in scar tissues and positively correlated with fibrosis. We then identified miR-203a-3p as the most suitable endogenous inhibitor of PIK3CA. miR-203a-3p suppressed the proliferation, migration, collagen synthesis and contractility as well as the transdifferentiation of fibroblasts into myofibroblasts in vitro, and improved the morphology and histology of scars in vivo. Mechanistically, miR-203a-3p attenuated fibrosis by inactivating the PI3K/AKT/mTOR pathway by directly targeting PIK3CA. PIK3CA and the PI3K/AKT/mTOR pathway are actively involved in scar fibrosis and miR-203a-3p might serve as a potential strategy for hypertrophic scar therapy through targeting PIK3CA and inactivating the PI3K/AKT/mTOR pathway.

Phase Ib and pharmacokinetics study of alpelisib, a PIK3CA inhibitor, and capecitabine in patients with advanced solid tumors.

This phase Ib study was performed to determine the safety of combination capecitabine with alpleisib (phosphatidylinositol 3-kinase catalytic subunit p110α blockade) and determine the maximal tolerated dose (MTD) and recommended phase ll dose (RP2D) of this combination regimen in patients with advanced solid tumors refractory to standard therapy. The synergistic anti-tumor activity and pharmacokinetics (PK) were investigated. Dose escalation phases were conducted in patients with advanced solid cancers who were refractory to standard therapy regardless of PIK3CA mutation. Patients were administered orally once daily alpelisib (200mg and 300mg) and twice daily capecitabine (850mg, 1000mg, 1250mg orally, days 1-14) every 3 weeks. Standard "3+3" dose escalation was used to define the MTD. The effect of alpelisib on the PK of capecitabine was assessed. Patients with 6 colorectal cancer (three PIK3CA mutation) and 6 breast cancer (all PIK3CA mutation) were enrolled. The first three patients in dose level 0 (alpelisib 200mg daily, capecitabine 1,000 mg/m2 twice daily) had no dose-limiting toxicities (DLTs). In dose level 1 (alpelisib increased to 300 mg daily, capecitabine 1,000mg twice daily), one of six patients had DLT (grade (Gr) 3 hyperglycemia). When dose level 2 (alpelisib 300mg daily, capecitabine 1,250 mg/m2 twice daily) was expanded to 3 patients, no patients had DLTs. The combination of alpelisib 300mg daily and capecitabine 1,250 mg/m2 twice daily was declared as the MTD/RP2D in patients with advanced solid tumors. The most common AEs were Gr 1-3 hyperglycemia (75.0%). Frequent all-grade, treatment-related AEs included Gr 2-3 nausea (75.0%), Gr 1-2 diarrhea (50.0%), Gr 1-2 hand-foot syndrome (41.7%), Gr 1-2 anorexia (41.7%), Gr 2 mucositis (33.3%). Antitumor activity was observed in patients with PIK3CA mutant breast cancer (3 partial response and 3 stable disease of total 6 patients). Alpelisib exposure (Cmax and AUC0-12) was unaffected by concomitant capecitabine. There were no clinically relevant drug-drug interactions observed between alpelisib and capecitabine. The combination of alpelisib and capecitabine is generally tolerated, without pharmacokinetic interactions, and shows antitumor activity in patients with PIK3CA mutant advanced cancers.

LTN1 promotes RLR degradation to inhibit immune response to RNA virus through the ESCRT pathway

ABSTRACT The excessive activation of immune responses will trigger autoimmune diseases or inflammatory injury. The endosomal sorting complexes required for transport (ESCRT) system can capture and mediate ubiquitinated protein degradation, which timely terminates signaling pathway hyperactivation. However, whether the ESCRT system participates in regulating RIGI-like receptor (RLR)-mediated antiviral responses remains unknown. In this study, we show that LTN1/listerin, a major component of RQC, can recruit E3 ubiquitin ligase TRIM27 to trigger K63-linked polyubiquitination of RIGI and IFIH1/MDA5. This K63-linked polyubiquitination facilitates the sorting and degradation of RIGI and IFIH1 proteins through the ESCRT-dependent pathway. Concordantly, LTN1 deficiency enhances the innate antiviral response to infection with RNA viruses. Thus, our work uncovers a new mechanism for RIGI and IFIH1 degradation and identifies the role of LTN1 in negatively regulating RLR-mediated antiviral innate immunity, which may provide new targets for the intervention of viral infection. Abbreviation: 5'-pppRNA: 5’ triphosphate double stranded RNA; ATG5: autophagy related 5; ATG7: autophagy related 7; BafA1: bafilomycin A1; ESCRT: endosomal sorting complexes required for transport; CHX: cycloheximide; IFIH1/MDA5: interferon induced with helicase C domain 1; IFN: interferon; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptors; RQC: ribosome-associated protein quality control; SeV: Sendai virus; TRIM27: tripartite motif-containing 27; VSV: vesicular stomatitis virus; VPS4: vacuolar protein sorting 4.

Targeted next-generation sequencing for detection of PIK3CA mutations in archival tissues from patients with Klippel–Trenaunay syndrome in an Asian population

BackgroundKlippel–Trenaunay syndrome (KTS) is a rare slow-flow combined vascular malformation with limb hypertrophy. KTS is thought to lie on the PIK3CA-related overgrowth spectrum, but reports are limited. PIK3CA encodes p110α, a catalytic subunit of phosphatidylinositol 3-kinase (PI3K) that plays an essential role in the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway. We aimed to demonstrate the clinical utility of targeted next-generation sequencing (NGS) in identifying PIK3CA mosaicism in archival formalin-fixed paraffin-embedded (FFPE) tissues from patients with KTS.ResultsParticipants were 9 female and 5 male patients with KTS diagnosed as capillaro-venous malformation (CVM) or capillaro-lymphatico-venous malformation (CLVM). Median age at resection was 14 years (range, 5–57 years). Median archival period before DNA extraction from FFPE tissues was 5.4 years (range, 3–7 years). NGS-based sequencing of PIK3CA achieved an amplicon mean coverage of 119,000x. PIK3CA missense mutations were found in 12 of 14 patients (85.7%; 6/8 CVM and 6/6 CLVM), with 8 patients showing the hotspot variants E542K, E545K, H1047R, and H1047L. The non-hotspot PIK3CA variants C420R, Q546K, and Q546R were identified in 4 patients. Overall, the mean variant allele frequency for identified PIK3CA variants was 6.9% (range, 1.6–17.4%). All patients with geographic capillary malformation, histopathological lymphatic malformation or macrodactyly of the foot had PIK3CA variants. No genotype–phenotype association between hotspot and non-hotspot PIK3CA variants was found. Histologically, the vessels and adipose tissues of the lesions showed phosphorylation of the proteins in the PI3K/AKT/mTOR signaling pathway, including p-AKT, p-mTOR, and p-4EBP1.ConclusionsThe PI3K/AKT/mTOR pathway in mesenchymal tissues was activated in patients with KTS. Amplicon-based targeted NGS could identify low-level mosaicism from low-input DNA extracted from FFPE tissues, potentially providing a diagnostic option for personalized medicine with inhibitors of the PI3K/AKT/mTOR signaling pathway.

MiR-506-3p induces autophagy of renal tubular epithelial cells in sepsis through targeting PI3K pathway.

To explore the effect of micro ribonucleic acid (miR)-506-3p on autophagy of renal tubular epithelial cells in sepsis and its mechanism. It was found through bioinformatics analysis that phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) was expressed at a low level in sepsis, and miR-506-3p had a targeted regulatory effect on PIK3CA. 40 8-week-old male C57BL/6 mice were randomly divided into control miR-506-3p NC group, control miR-506-3p OE group, sepsis miR-506-3p NC group, sepsis miR-506-3p OE group and sepsis miR-506-3p KD group. The pathological changes in kidney tissues of mice in each group were observed by hematoxylin-eosin (HE) staining and TUNEL staining, and mitochondria and autophagosomes were visualized by transmission electron microscopy. CCK8 assay was performed to detect the effect of miR-506-3p on the proliferation capacity of renal tubular epithelial cells. The changes in the expression of PI3K-Akt pathway proteins, mTOR and autophagy proteins were tested by Western blotting. The injury and apoptotic positive cells were suppressed and decreased in miR-506-3p OE mice vs. NC group. miR-506-3p could increase the number of mitochondria and autophagosomes in kidney tissues. After introduction of exogenous miR-506-3p OE into renal tubular epithelial cells, the expressions of PI3K pathway proteins were significantly inhibited, while the expressions of autophagy proteins were significantly enhanced. After 740Y-P was added, the expressions of associated proteins had no significant changes in each group. Overexpression of miR-506-3p can enhance the autophagy of renal tubular epithelial cells in sepsis through inhibiting the PI3K signaling pathway.

The WW domain of IQGAP1 binds directly to the p110α catalytic subunit of PI 3-kinase.

IQGAP1 is a multi-domain cancer-associated protein that serves as a scaffold protein for multiple signaling pathways. Numerous binding partners have been found for the calponin homology, IQ and GAP-related domains in IQGAP1. Identification of a binding partner for its WW domain has proven elusive, however, even though a cell-penetrating peptide derived from this domain has marked anti-tumor activity. Here, using in vitrobinding assays with human proteins and co-precipitation from human cells, we show that the WW domain of human IQGAP1 binds directly to the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K). In contrast, the WW domain does not bind to ERK1/2, MEK1/2, or the p85α regulatory subunit of PI3K when p85α is expressed alone. However, the WW domain is able to bind to the p110α/p85α heterodimer when both subunits are co-expressed, as well as to the mutationally activated p110α/p65α heterodimer. We present a model of the structure of the IQGAP1 WW domain, and experimentally identify key residues in the hydrophobic core and beta strands of the WW domain that are required for binding to p110α. These findings contribute to a more precise understanding of IQGAP1-mediated scaffolding, and of how IQGAP1-derived therapeutic peptides might inhibit tumorigenesis.

Abstract #1400018: Genetic Alterations in NRAS, TERT, PIK3CA and SPEN Genes Leading to an Aggressive Metastatic RAI Refractory Follicular Thyroid Cancer

Follicular thyroid cancer (FTC) is a well-differentiated thyroid tumor and the second most common type after papillary thyroid cancer (PTC) with higher incidence of distant metastases, more aggressive behavior, and poorer outcome than PTC Patient is 43-year-old female with 6.2 cm TR4 left thyroid nodule on Neck US. On Exam, Cricoid is deviated to the right with left thyroid firm nodule. FNA showed Follicular neoplasm. TSH level was 1.59 (0.45-4.70 mIU/L) and Free T4 level: 1.19 (0.78-2.20 ng/dl) CT scan of neck with contrast and MRI thoracic spine: Large, solid heterogenous mass in left thyroid lobe causing lateral displacement and mild narrowing of the trachea with retrosternal extension, Osteolytic lesion of T4 vertebral body and right sided pleura-based mass at the level of T7-T8 with No enlarged cervical lymph nodes. IR biopsy of vertebra: thyroid Origin Metastatic Carcinoma, Patient underwent total thyroidectomy with central neck dissection. Follicular thyroid cancer (STAGE II pT3A,pN1B,pM1) Mutational Testing: Negative BRAF and KRAS with Positive NRAS, PIK3CA, SPEN and TERT. Patient started on TSH suppression therapy. 1 month Later: Thyroglobulin: 2443.4 ng/ml (1.3-31.8), Thyroglobulin Ab< 0.9 (0.0-4.0 IU/ml), TSH 0.02 with Free T4 2.53 FDG PET: Multifocal osseous metastatic disease, predominantly in the axial skeleton Thyrogen stimulated WBS I-123 showed No evidence of residual or metastatic disease. Patient recieved 214 mCi of iodine-131. Post treatment MN scan showed accumulation of iodine in the metastatic bone lesions with small focal activity in the thyroidectomy bed. Post RAI treatment: TG trended to 1102.7 then 1307 Patient Completed 10 fractions of external beam radiation on T4 spine Ultrasound thyroid: No residual thyroid tissue or suspicious lymph nodes Oncology started Bone directed therapy with zoledronate with consideration of PIK3CA mutation targeted therapy with Everolimus or Temsirolimus FTC is more aggressive with poorer prognosis than PTC that is more likely to hematogenously disseminate, with mortality rate of 50%. The neoplastic proliferation of thyroid follicular cells develops by altering multiple molecular pathways such as the PI3K/AKT pathway involving the RAS, RAF, RET, and NTRK1 genes. RAS is a dual activator of the mitogen activated protein kinase and PI3K/AKT pathways TERT is a catalytic subunit of the telomerase complex. Reported in 11–17% of FTC. TERT promoter mutations associated with tumor aggressiveness and mortality PIK3CA encodes p110-alpha, is a catalytic subunit of phosphatidylinositol 3-kinase (PI3K) involves in thyroid cancer tumorigenesis and progression, reported in 7.7% of FTC with sensitivity to therapies targeting PI3K49-56, AKT57-58, or mTOR59-66. In our patient, pretreatment MN scan failed to show RAI avid lesions but PET showed increase FDG uptake and since receiving RAI treatment helped in lowering TG that indicate a possibility of mixture of RAI avid and non-RAI avid lesions existing at same time so RAI treatment is still recommended despite negative Diagnostic Scan for persistent elevated TG.

Multiple PIK3CA mutation clonality correlates with outcomes in taselisib + fulvestrant-treated ER+/HER2–, PIK3CA-mutated breast cancers

BackgroundMutations in the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), encoded by the PIK3CA gene, cause dysregulation of the PI3K pathway in 35–40% of patients with HR+/HER2– breast cancer. Preclinically, cancer cells harboring double or multiple PIK3CA mutations (mut) elicit hyperactivation of the PI3K pathway leading to enhanced sensitivity to p110α inhibitors.MethodsTo understand the role of multiple PIK3CAmut in predicting response to p110α inhibition, we estimated the clonality of multiple PIK3CAmut in circulating tumor DNA (ctDNA) from patients with HR+/HER2– metastatic breast cancer enrolled to a prospectively registered clinical trial of fulvestrant ± taselisib, and analyzed the subgroups against co-altered genes, pathways, and outcomes.ResultsctDNA samples with clonal multiple PIK3CAmut had fewer co-alterations in receptor tyrosine kinase (RTK) or non-PIK3CA PI3K pathway genes compared to samples with subclonal multiple PIK3CAmut indicating a strong reliance on the PI3K pathway. This was validated in an independent cohort of breast cancer tumor specimens that underwent comprehensive genomic profiling. Furthermore, patients whose ctDNA harbored clonal multiple PIK3CAmut exhibited a significantly higher response rate and longer progression-free survival vs subclonal multiple PIK3CAmut.ConclusionsOur study establishes clonal multiple PIK3CAmut as an important molecular determinant of response to p110α inhibition and provides rationale for further clinical investigation of p110α inhibitors alone or with rationally-selected therapies in breast cancer and potentially other solid tumor types.

Palbociclib blocks neutrophilic phosphatidylinositol 3-kinase activity to alleviate psoriasiform dermatitis.

Neutrophilic inflammation is a critical pathogenic factor in psoriasis. The therapeutic applicability of palbociclib, a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor clinically used to treat cancer, in the treatment of neutrophil-associated psoriasis remains undefined. In this study, we evaluated the therapeutic potential and pharmacological effect of palbociclib on neutrophil-associated psoriasiform dermatitis. The anti-inflammatory effects of palbociclib were determined in activated human neutrophils. The therapeutic feasibility of palbociclib in psoriasis was demonstrated in a mouse model of imiquimod-induced psoriasiform dermatitis. The in vitro enzymatic assays and in silico analyses were used to identify the underlying pharmacological mechanisms. This study found that palbociclib inhibited neutrophilic inflammation, including superoxide anion generation, reactive oxygen species (ROS) formation, elastase degranulation and chemotactic responses. The mechanistic studies identified that the anti-inflammatory effects of palbociclib involved the targeting of phosphatidylinositol 3-kinase (PI3K) but not CDK4/6 in human neutrophils. Palbociclib preferentially targeted the p110δ catalytic subunit of PI3K and thereby blocked signalling via the PI3K/protein kinase B (Akt) pathway. Furthermore, topical application of palbociclib significantly ameliorated imiquimod-induced psoriasiform dermatitis in mice, including psoriatic symptoms, neutrophil infiltration, Akt activation and cytokine up-regulation. This is the first study to demonstrate that palbociclib can potentially be used to treat neutrophil-associated psoriasiform dermatitis through the targeting of neutrophilic PI3K activity. Our findings prompt further research to explore the potential of palbociclib and PI3K in psoriasis and other inflammatory diseases.

P85α regulatory subunit isoform controls PI3-kinase and TRPC6 membrane translocation

Activation of phosphatidylinositol 3-kinase (PI3K) by lipid oxidation products, including lysophosphatidylcholine (lysoPC), increases the externalization of canonical transient receptor potential 6 (TRPC6) channels leading to a subsequent increase in intracellular calcium that contributes to cytoskeletal changes which inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo. The PI3K p110α and p110δ catalytic subunit isoforms regulate lysoPC-induced TRPC6 externalization in vitro, but have many other functions. The goal of the current study is to identify the PI3K regulatory subunit isoform involved in TRPC6 externalization to potentially identify a more specific treatment regimen to improve EC migration and arterial healing, while minimizing off-target effects. Decreasing the p85α regulatory subunit isoform protein levels, but not the p85β and p55γ regulatory subunit isoforms, with small interfering RNA inhibits lysoPC-induced translocation of the PI3K catalytic subunit to the plasma membrane, dramatically decreased phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production and TRPC6 externalization, and significantly improves EC migration in the presence of lysoPC. These results identify the important and specific role of p85α in controlling translocation of PI3K from the cytosol to the plasma membrane and PI3K-mediated TRPC externalization by oxidized lipids. Current PI3K inhibitors block the catalytic subunit, but our data suggest that the regulatory subunit is a novel therapeutic target to promote EC migration and healing after arterial injuries that occur with angioplasty.

Enzyme-Free Colorimetric Method for Fast Detection of PIK3CA Gene Mutation by Praseodymia Nanorods.

The frequently mutated phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene is associated with multiple tumors and endocytosis of viruses. Identification of muted nucleotides at the hotspot can help in finding the susceptible people who are vulnerable to cancers and viruses. Herein, a simple enzyme-free colorimetric method is developed for the quick detection of PIK3CA gene mutations. The main mechanism lies in the dissimilar interactions between praseodymia nanorods and different nucleotides, as well as the underlying oxidase-mimicking characteristics of praseodymia. With rational designs of probes and processes, this method has great potential for expanded applications in the screening of mutations in other genes of interest.