A common variant in PIK3CG gene associated with the prognosis of heart failure.

Phosphoinositide 3-kinase γ (PI3Kγ) is G-protein-coupled receptor-activated lipid kinase with both kinase-dependent and kinase-independent activity. Plenty of evidence have demonstrated that PI3Kγ participated in TAC and I/R-induced myocardial remodelling and heart failure (HF). In this study, we tested the hypothesis that common variants in the PI3Kγ gene (PIK3CG) were associated with the prognosis of HF in the Chinese Han population. Through re-sequencing and genotyping, we finally identified a common variant in the 3'UTR of PIK3CG strongly associated with the prognosis of HF in two-stage population: adjusted p = 0.007, hazard ratio = 0.56 (0.36-0.85) in the first cohort and adjusted p = 0.024, hazard ratio = 0.39 (0.17-0.88) in the replicated cohort. A series of functional assays revealed that rs10215499-A allele suppressed PIK3CG translation by facilitating has-miR-133a-3p binding, but not the G allele. Subjects carrying the GG genotype showed higher mRNA and protein level than those with AA and AG genotype. Furthermore, overexpression of PIK3CG could protect AC16 from hypoxia/reoxygenation (H/R)-induced apoptosis, while the case was opposite for PIK3CG silencing. In conclusion, common variant rs10215499 in the 3'-UTR of PIK3CG might affect the prognosis of HF by interfering with miR-133a-3p binding and PIK3CG is a promising target for HF treatment in the future.

Chemical analogue based drug design for cancer treatment targeting PI3K: integrating machine learning and molecular modeling.

Cancer is a generic term for a group of disorders defined by uncontrolled cell growth and the potential to invade or spread to other parts of the body. Gene and epigenetic alterations disrupt normal cellular control, leading to abnormal cell proliferation, resistance to cell death, blood vessel development, and metastasis (spread to other organs). One of the several routes that play an important role in the development and progression of cancer is the phosphoinositide 3-kinase (PI3K) signaling pathway. Moreover, the gene PIK3CG encodes the catalytic subunit gamma (p110γ) of phosphoinositide 3-kinase (PI3Kγ), a member of the PI3K family. Therefore, in this study, PIK3CG was targeted to inhibit cancer by identifying a novel inhibitor through computational methods. The study screened 1015 chemical fragments against PIK3CG using machine learning-based binding estimation and docking to select the potential compounds. Later, the analogues were generated from the selected hits, and 414 analogues were selected, which were further screened, and as most potential candidates, three compounds were obtained: (a) 84,332, 190,213, and 885,387. The protein-ligand complex's stability and flexibility were then investigated by dynamic modeling. The 100 ns simulation revealed that 885,387 exhibited the steadiest deviation and constant creation of hydrogen bonds. Compared to the other compounds, 885,387 demonstrated a superior binding free energy (ΔG = -18.80 kcal/mol) with the protein when the MM/GBSA technique was used. The study determined that 885,387 showed significant therapeutic potential and justifies further experimental investigation as a possible inhibitor of the PIK3CG target implicated in cancer.

Overexpression of PIK3CG in Cancer Cells Promotes Lung Cancer Cell Migration and Metastasis Through Enhanced MMPs Expression and Neutrophil Recruitment and Activation.

Metastasis is a major cause of death in lung cancer. The aim of this study is to analyze the role and mechanism of PI3K catalytic subunit gamma (PIK3CG, also known as p110γ) in lung cancer cell migration and metastasis. Knockdown (KD) and overexpression (OE) of PIK3CG expression in lung cancer cell lines A549 and H1299 in vitro cultured was achieved. Two PIK3CG-specific inhibitors, Eganelisib and CAY10505, were used to treat A549 and H1299 cells. An experimental lung metastasis mouse model was constructed using tail vein injection of LLC cells. Finally, a co-culture system was established using Transwell chambers. Compared with the NC group, the number of cells that completed migration and the expression levels of matrix metalloproteinases (MMPs) were significantly reduced in the KD group and Eganelisib and CAY10505 treatment groups, while the number of cells that migrated successfully and the expression levels of MMPs were significantly increased in the OE group. Lung tissues of mice injected with PIK3CG-stabilized overexpressed LLC cells showed more pronounced lung cancer growth, lung metastatic nodules, neutrophil infiltration and MMPs expression. Co-culture with neutrophils, soluble extracts of neutrophils and cathepsin G all promoted the migration of lung cancer cells. PIK3CG overexpression in tumor cells significantly promoted the migration and metastasis of lung cancer cell.

Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK.

BACKGROUND: The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy. METHODS: Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas. RESULTS: We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474. CONCLUSIONS: Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.

Multigenic truncation of the semaphorin-plexin pathway by a germline chromothriptic rearrangement associated with Moebius syndrome.

Moebius syndrome (MBS) is a congenital disorder caused by paralysis of the facial and abducens nerves. Although a number of candidate genes have been suspected, so far only mutations in PLXND1 and REV3L are confirmed to cause MBS. Here, we fine mapped the breakpoints of a complex chromosomal rearrangement (CCR) 46,XY,t(7;8;11;13) in a patient with MBS, which revealed 41 clustered breakpoints with typical hallmarks of chromothripsis. Among 12 truncated protein-coding genes, SEMA3A is known to bind to the MBS-associated PLXND1. Intriguingly, the CCR also truncated PIK3CG, which in silico interacts with REVL3 encoded by the other known MBS-gene REV3L, and with the SEMA3A/PLXND1 complex via FLT1. Additional studies of other complex rearrangements may reveal whether the multiple breakpoints in germline chromothripsis may predispose to complex multigenic disorders.

MEF2A alters the proliferation, inflammation-related gene expression profiles and its silencing induces cellular senescence in human coronary endothelial cells.

BACKGROUND: Myocyte enhancer factor 2A (MEF2A) plays an important role in cell proliferation, differentiation and survival. Functional deletion or mutation in MEF2A predisposes individuals to cardiovascular disease mainly caused by vascular endothelial dysfunction. However, the effect of the inhibition of MEF2A expression on human coronary artery endothelial cells (HCAECs) is unclear. In this study, expression of MEF2A was inhibited by specific small interference RNA (siRNA), and changes in mRNA profiles in response to MEF2A knockdown were analyzed using an Agilent human mRNA array. RESULTS: Silencing of MEF2A in HCAECs accelerated cell senescence and suppressed cell proliferation. Microarray analysis identified 962 differentially expressed genes (DEGs) between the MEF2A knockdown group and the negative control group. Annotation clustering analysis showed that the DEGs were preferentially enriched in gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to proliferation, development, survival, and inflammation. Furthermore, 61 of the 578 downregulated DEGs have at least one potential MEF2A binding site in the proximal promoter and were mostly enriched in the GO terms "reproduction" and "cardiovascular." The protein-protein interaction network analyzed for the downregulated DEGs and the DEGs in the GO terms "cardiovascular" and "aging" revealed that PIK3CG, IL1B, IL8, and PRKCB were included in hot nodes, and the regulation of the longevity-associated gene PIK3CG by MEF2A has been verified at the protein level, suggesting that PIK3CG might play a key role in MEF2A knockdown induced HCAEC senescence. CONCLUSIONS: MEF2A knockdown accelerates HCAEC senescence, and the underlying molecular mechanism may be involved in down-regulation of the genes related with cell proliferation, development, inflammation and survival, in which PIK3CG may play a key role.

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs).

Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein-coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme's activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gßγ heterodimers, we mapped these regulatory interactions using hydrogen-deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gßγ heterodimers, leading to PI3Kγ activation. Mutating Gßγ-interaction sites of either p110γ or p101 ablates G-protein-coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen-deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gßγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gßγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gßγ heterodimers; and the ß-adrenergic receptor kinase.

miR-502 inhibits cell proliferation and tumor growth in hepatocellular carcinoma through suppressing phosphoinositide 3-kinase catalytic subunit gamma.

MicroRNAs (miRNAs) play a key role in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). In the present study, we demonstrated that miR-502 significantly inhibits HCC cell proliferation in vitro and tumor growth in vivo. G1/S cell cycle arrest and apoptosis of HCC cells were induced by miR-502. Phosphoinositide 3-kinase catalytic subunit gamma (PIK3CG) was identified as a direct downstream target of miR-502 in HCC cells. Notably, overexpression of PIK3CG reversed the inhibitory effects of miR-502 in HCC cells. Our findings suggest that miR-502 functions as a tumor suppressor in HCC via inhibition of PI3KCG, supporting its utility as a promising therapeutic gene target for this tumor type.

Therapeutic potential of anti-PIK3CG treatment for multiple myeloma via inhibiting c-Myc pathway.

Multiple myeloma (MM) is a malignant plasma cell disease. The activity of PIK3CG (PI3K catalytic subunit γ) is regulated directly by G-protein-coupled receptor and has been confirmed to be highly expressed in MM cells. This study aimed to determine the effect of pharmacological inhibition of PIK3CG on MM. We found that different concentrations of the PIK3CG inhibitor AS-605240 could suppress the growth of MM cell lines and the expression of c-Myc. The combination of PIK3CG inhibitor and the chemotherapy Melphalan could effectively inhibit the proliferation and migration of MM cells, promote the cell apoptosis, and decrease the ratio of Bcl-2/Bax and the expression of vimentin. The expression of proto-oncogene c-Myc was decreased and the sensitivity of cells to chemotherapeutic drugs was enhanced. Collectively, PIK3CG regulates growth of MM via c-Myc pathway, thus emerging as a promising molecular targeted therapy.

Identification of PIK3CG as a hub in septic myocardial injury using network pharmacology and weighted gene co-expression network analysis.

Sepsis causes multiple organ injuries, among which the heart is one most severely damaged organ. Melatonin (MEL) alleviates septic myocardial injury, although a systematic and comprehensive approach is still lacking to understand the precise protective machinery of MEL. This study aimed to examine the underlying mechanisms of MEL on improvement of septic myocardial injury at a systematic level. This study integrated three analytic modalities including database investigations, RNA-seq analysis, and weighted gene co-expression network analysis (WCGNA), in order to acquire a set of genes associated with the pathogenesis of sepsis. The Drugbank database was employed to predict genes that may serve as pharmacological targets for MEL-elicited benefits, if any. A pharmacological protein-protein interaction network was subsequently constructed, and 66 hub genes were captured which were enriched in a variety of immune response pathways. Notably, PIK3CG, one of the hub genes, displayed high topological characteristic values, strongly suggesting its promise as a novel target for MEL-evoked treatment of septic myocardial injury. Importantly, molecular docking simulation experiments as well as in vitro and in vivo studies supported an essential role for PIK3CG in MEL-elicited effect on septic myocardial injury. This study systematically clarified the mechanisms of MEL intervention in septic myocardial injury involved multiple targets and multiple pathways. Moreover, PIK3CG-governed signaling cascade plays an important role in the etiology of sepsis and septic myocardial injury. Findings from our study provide valuable information on novel intervention targets for the management of septic myocardial injury. More importantly, this study has indicated the utility of combining a series of techniques for disease target discovery and exploration of possible drug targets, which should shed some light on elucidation of experimental and clinical drug action mechanisms systematically.

PIK3CG Regulates NLRP3/GSDMD-Mediated Pyroptosis in Septic Myocardial Injury.

Cardiac insufficiency is a common complication of sepsis with high mortality. Inflammatory programmed cell death (pyroptosis) executed by NLRP3/gasdermin D (GSDMD) is intrinsically correlated with septic myocardial injury. However, it remains unclear whether PIK3CG, a classical target of septic myocardial injury, can affect pyroptosis by regulating NLRP3/GSDMD signaling. In this study, a series of experimental methods were used to observe the effect of PIK3CG on NLRP3/GSDMD-mediated pyroptosis in Cecal ligation and puncture (CLP)-injured BALB/c mice and lipopolysaccharide (LPS)-injured HL-1 cardiomyocytes. Transcriptome analysis of CLP-injured myocardium revealed a regulatory relationship between PIK3CG and NLRP3/GSDMD signaling, which was further verified in clinical myocardium samples from GEO database. Both in vitro and in vivo experiments showed that the protein and mRNA levels of PIK3CG, GSDMD, NLRP3, IL-1ß, Caspase-1, and IL-18 were significantly increased. Importantly, PIK3CG siRNA was found to improve these changes, while PIK3CG overexpression worsened them. Notably, pyroptosis induced by CLP in the myocardium was reversed by the PIK3CG inhibitor (AS-604850). In conclusion, PIK3CG activates NLRP3 inflammasomes, thus promoting pyroptosis in septic myocardial injury.

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain.

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCß activated by the IgE receptor, and Gßγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gßγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCß helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCß phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCß-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.

Repressing phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma by microRNA-142-3p restrains the progression of hepatocellular carcinoma.

This paper probes the mechanisms underlying miR-142-3p's modulation of hepatocellular carcinoma (HCC) invasion and apoptosis. Quantitative real-time PCR and Western blot monitored the miR-142-3p profile in HCC tissues and non-tumor tissues. The correlation between miR-142-3p expression and HCC patients' clinicopathological indicators was analyzed. miR-142-3p overexpression and knockdown models were established in HCC cell lines. Cell proliferation was gauged by the colony formation assay and BrdU staining. For measuring apoptosis, flow cytometry and Western blot were implemented. Transwell assay tested cell migration and invasion. miR-142-3p mimics or inhibitors were transfected in Huh7 and HCCLM3 cells. The targeting association between miR-142-3p and PIK3CG was predicted through bioinformatics and further verified by related experiments. The influence of PIK3CG overexpression on miR-142-3p's role in HCC was assayed. A xenografted tumor model was built in mice to validate miR-142-3p knockdown's influence on HCC in vivo. As a result, miR-142-3p exhibited a decreased profile in HCC tissues and cells. Overexpressing miR-142-3p accelerated apoptosis and suppressed the PI3K/AKT/HIF-1α signal. Knocking down miR-142-3p presented opposite effects. PIK3CG overexpression dampened the anti-tumor effect of miR-142-3p. miR-142-3p repressed HCC invasion and intensified apoptosis to restrain HCC by abating the PIK3CG-mediated PI3K/AKT/HIF-1α pathway.

Global expression profiling of CD10 + /CD19 + pre-B lymphoblasts from Hispanic B-ALL patients correlates with comparative TARGET database analysis.

Mexico City has one of the highest incidences of acute lymphoblastic leukemia (ALL) globally, with patients showing low survival, and high relapse rates. To gain more insight into the molecular features of B-ALL in Mexican children, we isolated CD10 + /CD19 + precursor B lymphoblasts from four bone marrow and nine peripheral blood samples of B-ALL patients using a fluorescence-activated cell sorting protocol. The global gene expression profile (BM vs PB) revealed 136 differentially expressed genes; 62 were upregulated (45.6%) and 74 were downregulated (54.4%). Pearson's correlation coefficient was calculated to determine the similarity between pre-B lymphoblast populations. We selected 26 highly significant genes and validated 21 by RT-qPCR (CNN3, STON2, CALN1, RUNX2, GADD45A, CDC45, CDC20, PLK1, AIDA, HCK, LY86, GPR65, PIK3CG, LILRB2, IL7R, TCL1A, DOCK1, HIST1H3G, PTPN14, CD72, and NT5E). The gene set enrichment analysis of the total expression matrix and the ingenuity pathway analysis of the 136 differentially expressed genes showed that the cell cycle was altered in the bone marrow with four overexpressed genes (PLK1, CDC20, CDC45, and GADD45A) and a low expression of IL7R and PIK3CG, which are involved in B cell differentiation. A comparative bioinformatics analysis of 15 bone marrow and 10 peripheral blood samples from Hispanic B-ALL patients collected by the TARGET program, corroborated the genes observed, except for PIK3CG. We conclude the Mexican and the Hispanic B-ALL patients studied present common driver alterations and histotype-specific mutations that could facilitate risk stratification and diagnostic accuracy and serve as potential therapeutic targets.

Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases.

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

Proteogenomic analysis of melanoma brain metastases from distinct anatomical sites identifies pathways of metastatic progression.

Melanoma brain metastases (MBM) portend a grim prognosis and can occur in up to 40% of melanoma patients. Genomic characterization of brain metastases has been previously carried out to identify potential mutational drivers. However, to date a comprehensive multi-omics approach has yet to be used to analyze brain metastases. In this case report, we present an unbiased proteogenomics analyses of a patient's primary skin cancer and three brain metastases from distinct anatomic locations. We performed molecular profiling comprised of a targeted DNA panel and full transcriptome as well as proteomics using mass spectrometry. Phylogeny demonstrated that all MBMs shared a SMARCA4 mutation and deletion of 12q. Proteogenomics identified multiple pathways upregulated in the MBMs compared to the primary tumor. The protein, PIK3CG, was present in many of these pathways and had increased gene expression in metastatic melanoma tissue from the cancer genome atlas data. Proteomics demonstrated PIK3CG levels were significantly increased in all 3 MBMs and this finding was further validated by immunohistochemistry. In summary, this case report highlights the potential role of proteogenomics in identifying pathways involved in metastatic tumor progression. Furthermore, our multi-omics approach can be considered to aid in precision oncology efforts and provide avenues for therapeutic innovation.

Targeting PIK3CG in Combination with Paclitaxel as a Potential Therapeutic Regimen in Claudin-Low Breast Cancer.

PURPOSE: Molecular targeting is a powerful approach for aggressive claudin-low breast cancer (CLBC). Overexpression of PI3K catalytic subunit gamma (PIK3CG) in human CLBC is offering a promising opportunity for targeted therapies. We utilized a specific inhibitor of PIK3CG combined with paclitaxel (PTX) to treat CLBC cells in vitro and in vivo. PATIENTS AND METHODS: The tumor cells growth and apoptosis in vitro were analyzed by CCK8, plate clone formation assay, tumorsphere assay, Hoechst staining and flow cytometry. The invasion and metastasis ability of tumor cells in vitro were investigated by wound healing and transwell experiments. Critical gene expression levels were checked by qRT-PCR and Western blot. Xenograft models with CLBC cell lines in SCID mice were established to investigate the effect of combined drugs in vivo. RESULTS: We identified that PIK3CG was a potential therapeutic target for CLBC patients. Targeting PIK3CG potentiated CLBC cells growth inhibition in 2D and 3D cultures by PTX. Inhibition of PIK3CG activation could enhance CLBC cells apoptosis and migration suppression induced by PTX. Manipulating autophagy was a validated approach for the use of PIK3CG inhibitor. Using CLBC xenograft mice model, we found that CLBC tumors in vivo could be well treated by combined drugs of PIK3CG inhibitor and PTX. CONCLUSION: We demonstrated that PIK3CG was a potential target for the therapy of CLBC and inhibition of PIK3CG activation could reinforce the therapeutic effect of this aggressive disease by PTX. The combined use of PIK3CG inhibitor and PTX might be a potential regimen for treating this subtype of breast cancer.

PI3Kγ Regulatory Protein p84 Determines Mast Cell Sensitivity to Ras Inhibition-Moving Towards Cell Specific PI3K Targeting?

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ  responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.