p55PIK deficiency and its NH2-terminal derivative inhibit inflammation and emphysema in COPD mouse model.

Chronic obstructive pulmonary disease (COPD) is composed of chronic airway inflammation and emphysema. Recent studies show that Class IA phosphatidylinositol 3-kinases (PI3Ks) play an important role in the regulation of inflammation and emphysema. However, there are few studies on their regulatory subunits. p55PIK is a regulatory subunit of Class IA PI3Ks, and its unique NH2-terminal gives it special functions. p55PIK expression in the lungs of nonsmokers, smokers, and patients with COPD was examined. We established a fusion protein TAT-N15 from the NH2-terminal effector sequence of p55PIK and TAT (the transduction domain of HIV transactivator protein) and investigated the effects of silencing p55PIK or adding TAT-N15 on cigarette smoke exposure at the cellular and animal level. p55PIK expression was increased in patients with COPD. p55PIK deficiency and TAT-N15 significantly inhibited the cigarette smoke extract-induced IL-6, IL-8, and activation of the Akt and the NF-κB pathway in BEAS-2B. p55PIK deficiency and TAT-N15 intranasal administration prevented emphysema and the lung function decline in mice exposed to smoke for 6 mo. p55PIK deficiency and TAT-N15 significantly inhibited lung inflammatory infiltration, reduced levels of IL-6 and KC in mice lung homogenate, and inhibited activation of the Akt and the NF-κB signaling in COPD mice lungs. Our studies indicate that p55PIK is involved in the pathogenesis of COPD, and its NH2-terminal derivative TAT-N15 could be an effective drug in the treatment of COPD by inhibiting the activation of the Akt and the NF-κB pathway.

Reduced Mrp2 surface availability as PI3Kγ-mediated hepatocytic dysfunction reflecting a hallmark of cholestasis in sepsis.

Sepsis-associated liver dysfunction manifesting as cholestasis is common during multiple organ failure. Three hepatocytic dysfunctions are considered as major hallmarks of cholestasis in sepsis: impairments of microvilli covering canalicular membranes, disruptions of tight junctions sealing bile-collecting canaliculae and disruptions of Mrp2-mediated hepatobiliary transport. PI3Kγ loss-of-function was suggested as beneficial in early sepsis. Yet, the PI3Kγ-regulated cellular processes in hepatocytes remained largely unclear. We analysed all three sepsis hallmarks for responsiveness to massive PI3K/Akt signalling and PI3Kγ loss-of-function, respectively. Surprisingly, neither microvilli nor tight junctions were strongly modulated, as shown by electron microscopical studies of mouse liver samples. Instead, quantitative electron microscopy proved that solely Mrp2 surface availability, i.e. the third hallmark, responded strongly to PI3K/Akt signalling. Mrp2 plasma membrane levels were massively reduced upon PI3K/Akt signalling. Importantly, Mrp2 levels at the plasma membrane of PI3Kγ KO hepatocytes remained unaffected upon PI3K/Akt signalling stimulation. The effect explicitly relied on PI3Kγ's enzymatic ability, as shown by PI3Kγ kinase-dead mice. Keeping the surface availability of the biliary transporter Mrp2 therefore is a cell biological process that may underlie the observation that PI3Kγ loss-of-function protects from hepatic excretory dysfunction during early sepsis and Mrp2 should thus take center stage in pharmacological interventions.

PIKfyve Deficiency in Myeloid Cells Impairs Lysosomal Homeostasis in Macrophages and Promotes Systemic Inflammation in Mice.

Macrophages are professional phagocytes that are essential for host defense and tissue homeostasis. Proper membrane trafficking and degradative functions of the endolysosomal system are known to be critical for the function of these cells. We have found that PIKfyve, the kinase that synthesizes the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate, is an essential regulator of lysosomal biogenesis and degradative functions in macrophages. Genetically engineered mice lacking PIKfyve in their myeloid cells (PIKfyvefl/fl LysM-Cre) develop diffuse tissue infiltration of foamy macrophages, hepatosplenomegaly, and systemic inflammation. PIKfyve loss in macrophages causes enlarged endolysosomal compartments and impairs the lysosomal degradative function. Moreover, PIKfyve deficiency increases the cellular levels of lysosomal proteins. Although PIKfyve deficiency reduced the activation of mTORC1 pathway and was associated with increased cleavage of TFEB proteins, this does not translate into transcriptional activation of lysosomal genes, suggesting that PIKfyve modulates the abundance of lysosomal proteins by affecting the degradation of these proteins. Our study shows that PIKfyve modulation of lysosomal degradative activity and protein expression is essential to maintain lysosomal homeostasis in macrophages.

2,4-Disubstituted quinazolines targeting breast cancer cells via EGFR-PI3K.

A series of novel 2,4-disubstituted quinazolines were synthesized and evaluated for their anti-tumor activity against five human cancer cells (MDA-MB-231, MCF-7, PC-3, HGC-27 and MGC-803) using MTT assay. Among them, compound 9n showed the most potent cytotoxicity against breast cancer cells. Compound 9n also significantly inhibited the colony formation and migration of MDA-MB-231 and MCF-7 cells. Meanwhile, compound 9n induced cell cycle arrest at G1 phase and cell apoptosis, as well as increased accumulation of intracellular ROS. Furthermore, compound 9n exerted anti-tumor effects in vitro via decreasing the expression of anti-apoptotic protein Bcl-2 and increasing the pro-apoptotic protein Bax and p53. Mechanistically, compound 9n markedly decreased p-EGFR and p-PI3K expression, which revealed that compound 9n targeted breast cancer cells via interfering with EGFR-PI3K signaling pathway. Molecular docking suggested that compound 9n could indeed bind into the active pocket of EGFR. All the findings suggest that compound 9n might be a valuable lead compound for anti-tumor agents targeting breast cancer cells.

Deregulated Gab2 phosphorylation mediates aberrant AKT and STAT3 signaling upon PIK3R1 loss in ovarian cancer.

Copy number loss of PIK3R1 (p85α) most commonly occurs in ovarian cancer among all cancer types. Here we report that ovarian cancer cells manifest a spectrum of tumorigenic phenotypes upon knockdown of PIK3R1. PIK3R1 loss activates AKT and p110-independent JAK2/STAT3 signaling through inducing changes in the phosphorylation of the docking protein Gab2, thereby relieving the negative inhibition on AKT and promoting the assembly of JAK2/STAT3 signalosome, respectively. Additional mechanisms leading to AKT activation include enhanced p110α kinase activity and a decrease in PTEN level. PIK3R1 loss renders ovarian cancer cells vulnerable to inhibition of AKT or JAK2/STAT3. The combination of AKT and STAT3 inhibitors significantly increases the anti-tumor effect compared to single-agent treatments. Together, our findings provide a rationale for mechanism-based therapeutic approach that targets tumors with loss of PIK3R1.

PI3K: A Crucial Piece in the RAS Signaling Puzzle.

RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance.

Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia.

BACKGROUND: Phosphoinositide 3-kinase γ (PI3Kγ) and PI3Kδ are second messenger-generating enzymes with key roles in proliferation, differentiation, survival, and function of leukocytes. Deficiency of the catalytic subunits p110γ and p110δ of PI3Kγ and PI3Kδ in p110γ/δ-/- mice leads to defective B- and T-cell homeostasis. Here we examined the role of p110γ and p110δ in the homeostasis of neutrophils by analyzing p110γ-/-, p110δ-/- and p110γ/δ-/- mice. METHODS: Neutrophils and T cells in leukocyte suspensions from the bone marrow (BM), blood, spleen and lung were analyzed by flow cytometry. Serum concentrations of IL-17, of the neutrophilic growth factor G-CSF, and of the neutrophil mobilizing CXC chemokines CXCL1/KC and CXCL2/MIP-2 were measured by Bio-Plex assay. Production of G-CSF and CXCL1/KC by IL-17-stimulated primary lung tissue cells were determined by ELISA, whereas IL-17-dependent signaling in lung tissue cells was analyzed by measuring Akt phosphorylation using immunoblot. RESULTS: We found that in contrast to single knock-out mice, p110γ/δ-/- mice exhibited significantly elevated neutrophil counts in blood, spleen, and lung. Increased granulocytic differentiation stages in the bone marrow of p110γ/δ-/- mice were paralleled by increased serum concentrations of G-CSF, CXCL1/KC, and CXCL2/MIP-2. As IL-17 induces neutrophilia via the induction of G-CSF and CXC chemokines, we measured IL-17 and IL-17-producing T cells. IL-17 serum concentrations and frequencies of IL-17+ splenic T cells were significantly increased in p110γ/δ-/- mice. Moreover, IFN-γ+, IL-4+, and IL-5+ T cell subsets were drastically increased in p110γ/δ-/- mice, suggesting that IL-17+ T cells were up-regulated in the context of a general percentage increase of other cytokine producing T cell subsets. CONCLUSIONS: We found that p110γ/δ deficiency in mice induces complex immunological changes, which might in concert contribute to neutrophilia. These findings emphasize a crucial but indirect role of both p110γ and p110δ in the regulation of neutrophil homeostasis.

Deletion of PIKfyve alters alveolar macrophage populations and exacerbates allergic inflammation in mice.

Alveolar macrophages (AMs) are specialized tissue-resident macrophages that orchestrate the immune responses to inhaled pathogens and maintain organ homeostasis of the lung. Dysregulation of AMs is associated with allergic inflammation and asthma. Here, we examined the role of a phosphoinositide kinase PIKfyve in AM development and function. Mice with conditionally deleted PIKfyve in macrophages have altered AM populations. PIKfyve deficiency results in a loss of AKT activation in response to GM-CSF, a cytokine critical for AM development. Upon exposure to house dust mite extract, mutant mice display severe lung inflammation and allergic asthma accompanied by infiltration of eosinophils and lymphoid cells. Moreover, they have defects in production of retinoic acid and fail to support incorporation of Foxp3+ Treg cells in the lung, resulting in exacerbation of lung inflammation. Thus, PIKfyve plays a role in preventing excessive lung inflammation through regulating AM function.

Gene delivery to Nile tilapia cells for transgenesis and the role of PI3K-c2α in angiogenesis.

Microinjection is commonly performed to achieve fish transgenesis; however, due to difficulties associated with this technique, new strategies are being developed. Here we evaluate the potential of lentiviral particles to genetically modify Nile tilapia cells to achieve transgenesis using three different approaches: spermatogonial stem cell (SSC) genetic modification and transplantation (SC), in vivo transduction of gametes (GT), and fertilised egg transduction (ET). The SC protocol using larvae generates animals with sustained production of modified sperm (80% of animals with 77% maximum sperm fluorescence [MSF]), but is a time-consuming protocol (sexual maturity in Nile tilapia is achieved at 6 months of age). GT is a faster technique, but the modified gamete production is temporary (70% of animals with 52% MSF). ET is an easier way to obtain mosaic transgenic animals compared to microinjection of eggs, but non-site-directed integration in the fish genome can be a problem. In this study, PI3Kc2α gene disruption impaired development during the embryo stage and caused premature death. The manipulator should choose a technique based on the time available for transgenic obtainment and if this generation is required to be continuous or not.

PI3K p85 ß regulatory subunit deficiency does not affect NK cell differentiation and increases NKG2D-mediated activation.

Activation of NK cells depends on a balance between activating and inhibitory signals. Class Ia PI3K are heterodimeric proteins with a catalytic and a regulatory subunit and have a central role in cell signaling by associating with tyrosine kinase receptors to trigger signaling cascades. The regulatory p85 subunit participates in signaling through NKG2D, one of the main activating receptors on NK cells, via its interaction with the adaptor protein DAP10. Although the effects of inhibiting catalytic subunits or deleting the regulatory p85α subunit have been studied, little attention has focused on the role of the p85ß subunit in NK cells. Using p85ß knockout mice, we found that p85ß deficiency does not alter NK cell differentiation and maturation in spleen or bone marrow. NK cells from p85ß-/- mice nonetheless produced more IFN-γ and degranulated more effectively when stimulated with anti-NKG2D antibody. These cells also degranulated and killed NKG2D ligand-expressing target cells more efficiently. We show that p85ß deficiency impaired NKG2D internalization, which could contribute to the activated phenotype. Decreasing p85ß subunit protein levels might thus constitute a therapeutic target to promote NK cell activity toward NKG2D ligand-expressing cells.

Better Understanding of Phosphoinositide 3-Kinase (PI3K) Pathways in Vasculature: Towards Precision Therapy Targeting Angiogenesis and Tumor Blood Supply.

The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3Kα isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3Kα and other class I isoforms, i.e. PI3Kß, γ, δ, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3Kγ and PI3Kδ (Pik3cg(-/-)/Pik3cd(-/-)) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100 nM) and LY294002 (10 µM). These vasoconstrictions were also inhibited by the PI3Kα isoform inhibitors A66 (10 µM) and PI-103 (1 µM), but not by the PI3Kß isoform inhibitor TGX 221 (100 nM). Pik3cg(-/-)/Pik3cd(-/-)-arteries showed normal vasoconstriction. We conclude that PI3Kα is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via α1-adrenergic receptors. Our results highlight a regulatory role of PI3Kα in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3Kα in cancer cells and tumor angiogenesis and regional blood flow.

MicroRNA-31 inhibits lung adenocarcinoma stem-like cells via down-regulation of MET-PI3K-Akt signaling pathway.

Accumulated evidences suggested that microRNAs (miRs) play an important role in non-small cell lung cancer (NSCLC). However, how miRs perform their functions in lung adenocarcinoma cancer stem cells (CSCs) remains unknown. Notably, most studies pay more attention to the effects of miRNAs on the metastasis traits whereas the growth activities of CSCs are rather undervalued. In our report, using A549CD133+cells, we examined the inhibitory effects and the underlying mechanisms of microRNA-31 (miR-31) on the growth of lung adenocarcinoma CSC-like cells. Initially, we determined the level of miR-31 in A549 and A549CD133+ cells. Over-expression of miR-31 was found in A549CD133+ cells by microarray and real-time quantitative PCR (RTqPCR) assays. Experiments in multiple NSCLC cell lines in vitro and A549CD133+ cells xenograft models in vivo confirmed that down regulation of miR-31 resulted in increase of A549CD133+ cells growth, whereas overexpression of miR-31 led to the inhibition of adenocarcinoma cell proliferation. Also, MET proto-oncogene has been determined to be a direct target of miR-31 by dual luciferase report, RT-qPCR and western blot analysis. Down regulation of MET inhibited viability of A549CD133+ cells. The levels of PI3Kinase, Akt and p-Akt as well as downstream proteins were consequently decreased. These results suggest that miR-31 might inhibit the growth of lung adenocarcinoma cancer stem-like cells via down regulation of the MET-PI3K-Akt signaling pathway.

Knockdown of AKT3 and PI3KCA by RNA interference changes the expression of the genes that are related to apoptosis and autophagy in T98G glioblastoma multiforme cells.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor and it is characterized by a poor prognosis and short survival time. The PI3K/AKT/PTEN signaling pathway plays a crucial role in GBM development and it is connected with the regulation of apoptosis and autophagy. Akt is involved in various aspects of cancer cell biology such as cell survival, in addition to both apoptosis and autophagy. The current study was undertaken to examine the effect of the siRNAs that target AKT3 and PI3KCA genes on the apoptosis and autophagy of T98G cells. METHODS: T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. Alterations in the mRNA expression of apoptosis- and autophagy-related genes were analyzed using QRT-PCR. LC3IIA protein-positive cells were identified using flow cytometry with specific antibodies. RESULTS: Our findings demonstrate for the first time that the siRNAs that target AKT3 and PI3KCA change the expression of the genes that are related to apoptosis and autophagy and change the expression of the LC3IIA protein in T98G cells. CONCLUSIONS: Thus, there is a high probability that the knockdown of these genes induces apoptosis and autophagy in T98G cells, but further studies are necessary in order to clarify and check whether autophagy induction is a positive phenomenon for the treatment of GBM.

PI3Kα is essential for the recovery from Cre/tamoxifen cardiotoxicity and in myocardial insulin signalling but is not required for normal myocardial contractility in the adult heart.

AIMS: Genetic mouse models have yielded conflicting conclusions about the role of PI3Kα in heart physiology: specifically, the question of whether PI3Kα has a direct role in regulating myocardial contractility. This has led to concerns that PI3K inhibitors currently in clinical trials for cancer may potentiate cardiotoxicity. Here we seek to clarify the role of PI3Kα in normal heart physiology and investigate changes in related signalling pathways. METHODS AND RESULTS: Targeted deletion of PI3Kα and PI3Kß in the heart with a tamoxifen-dependent Cre recombinase transgene caused transient heart dysfunction in all genotypes, but only PI3Kα deletion prevented functional recovery. Reduction in tamoxifen dosing allowed for maintained gene deletion without any cardiomyopathy, possibly through activation of survival signalling through the related ERK pathway. Similarly, mice with PI3Kα deletion induced by constitutively active Cre recombinase had normal heart function. Insulin-mediated activation of Akt, a marker of PI3Kα activity, was impaired with increased ERK1/2 activation in PI3Kα mutant hearts. Pharmacological inhibition of PI3Kα with BYL-719 also caused impaired insulin signalling in murine and human cardiomyocytes as well as in vivo in mice, with increased fasting blood glucose levels, but did not affect myocardial contractility as determined by echocardiography and invasive pressure-volume loop analysis. CONCLUSION: Our results show that PI3Kα does not directly regulate myocardial contractility, but is required for recovery from tamoxifen/Cre toxicity. The important role for PI3Kα in insulin signalling and recovery from tamoxifen/Cre toxicity justifies caution when using PI3Kα inhibitors in combination with other cardiovascular comorbidities and cardiotoxic compounds in cancer patients.

Inactivation of PI3Kδ induces vascular injury and promotes aneurysm development by upregulating the AP-1/MMP-12 pathway in macrophages.

OBJECTIVE: An aneurysm is an inflammatory vascular condition. Phosphatidylinositol 3-kinases δ is highly expressed in leukocytes, and play a key role in innate immunity. However, the link between phosphatidylinositol 3-kinases δ and aneurysm development has not yet been elucidated. APPROACH AND RESULTS: Carotid ligation unexpectedly induced characteristic aneurysm formation beneath the ligation point in p110δ(D910A/D910A) mice (n=25; P<0.001 versus wild-type). Besides, p110δ inactivation exacerbated CaCl2-induced abdominal aortic aneurysms development. A reverse transcription polymerase chain reaction microarray revealed significant extracellular matrix components degradation and matrix metalloproteinases (MMPs) upregulation in the abdominal aorta of p110δ(D910A/D910A) mice. Similarly, the expression of both collagen I and IV was significantly decreased (n=10; P<0.05 versus wild-type) in carotid artery. Western blot assay confirmed that MMP-12 was significantly upregulated in arteries of p110δ(D910A/D910A) mice (n=10; P<0.01 versus wild-type). In vitro, p110δ inactivation marked increase peritoneal macrophages recruitment and synergistically enhance tumor necrosis factor-α-induced recruitment. A specific phosphatidylinositol 3-kinases δ inhibitor (IC87114) or genetic p110δ inactivation upregulated MMP-12 expression and c-Jun phosphorylation (n=6; P<0.05 versus wild-type macrophages). IC87114 also increased activator protein-1 DNA-binding activity (n=6; P<0.001 versus control) and enhanced the effect of tumor necrosis factor-α on activator protein-1-binding activity (n=5; P<0.01 versus tumor necrosis factor-α treatment groups). Knockdown of c-Jun suppressed the effect of the IC87114 and tumor necrosis factor-α on MMP-12 mRNA expression (n=5 in each group; P<0.01 versus scrRNA treatment groups). CONCLUSIONS: Our findings demonstrate that p110δ inactivation leads to extracellular matrix degradation in vessels and promotes aneurysm development by inducing macrophages migration and upregulating the activator protein-1/MMP-12 pathway in macrophages.

Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice.

PIKfyve is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and for the regulation of endolysosomal membrane dynamics in mammals. PtdIns(3,5)P2 deficiency causes neurodegeneration in mice and humans, but the role of PtdIns(3,5)P2 in non-neural tissues is poorly understood. Here we show that platelet-specific ablation of PIKfyve in mice leads to accelerated arterial thrombosis, and, unexpectedly, also to inappropriate inflammatory responses characterized by macrophage accumulation in multiple tissues. These multiorgan defects are attenuated by platelet depletion in vivo, confirming that they reflect a platelet-specific process. PIKfyve ablation in platelets induces defective maturation and excessive storage of lysosomal enzymes that are released upon platelet activation. Impairing lysosome secretion from PIKfyve-null platelets in vivo markedly attenuates the multiorgan defects, suggesting that platelet lysosome secretion contributes to pathogenesis. Our findings identify PIKfyve as an essential regulator for platelet lysosome homeostasis, and demonstrate the contributions of platelet lysosomes to inflammation, arterial thrombosis and macrophage biology.

Arginase activity in alternatively activated macrophages protects PI3Kp110δ deficient mice from dextran sodium sulfate induced intestinal inflammation.

Alternatively activated or M2 macrophages have been reported to protect mice from intestinal inflammation, but the mechanism of protection has not been elucidated. In this study, we demonstrate that mice deficient in the p110δ catalytic subunit activity of class I phosphatidylinositol 3-kinase (PI3Kp110δ) have increased clinical disease activity and histological damage during dextran sodium sulfate (DSS) induced colitis. Increased disease severity in PI3Kp110δ-deficient mice is dependent on professional phagocytes and correlates with reduced numbers of arginase I+ M2 macrophages in the colon and increased production of inflammatory nitric oxide. We further demonstrate that PI3Kp110δ-deficient macrophages are defective in their ability to induce arginase I when skewed to an M2 phenotype with IL-4. Importantly, adoptive transfer of IL-4-treated macrophages derived from WT mice, but not those from PI3Kp110δ-deficient mice, protects mice during DSS-induced colitis. Moreover, M2 macrophages mediated protection is lost when mice are cotreated with inhibitors that block arginase activity or during adoptive transfer of arginase I deficient M2 macrophages. Taken together, our data demonstrate that arginase I activity is required for M2 macrophages mediated protection during DSS-induced colitis in PI3Kp110δ-deficient mice.

RAS interaction with PI3K p110α is required for tumor-induced angiogenesis.

Direct interaction of RAS with the PI3K p110α subunit mediates RAS-driven tumor development: however, it is not clear how p110α/RAS-dependant signaling mediates interactions between tumors and host tissues. Here, using a murine tumor cell transfer model, we demonstrated that disruption of the interaction between RAS and p110α within host tissue reduced tumor growth and tumor-induced angiogenesis, leading to improved survival of tumor-bearing mice, even when this interaction was intact in the transferred tumor. Furthermore, functional interaction of RAS with p110α in host tissue was required for efficient establishment and growth of metastatic tumors. Inhibition of RAS and p110α interaction prevented proper VEGF-A and FGF-2 signaling, which are required for efficient angiogenesis. Additionally, disruption of the RAS and p110α interaction altered the nature of tumor-associated macrophages, inducing expression of markers typical for macrophage populations with reduced tumor-promoting capacity. Together, these results indicate that a functional RAS interaction with PI3K p110α in host tissue is required for the establishment of a growth-permissive environment for the tumor, particularly for tumor-induced angiogenesis. Targeting the interaction of RAS with PI3K has the potential to impair tumor formation by altering the tumor-host relationship, in addition to previously described tumor cell-autonomous effects.

The PI3K regulatory subunits p55α and p50α regulate cell death in vivo.

The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately transcriptionally upregulated by signal transducer and activator of transcription 3 (Stat3) at the onset of mammary gland involution, a process that requires Stat3. Deletion of both p55α and p50α subunits in vivo abrogated mammary epithelial cell death during involution. This was associated also with reduced cytosolic levels and activity of the cysteine protease cathepsin L, which is implicated in lysosomal-mediated programmed cell death (LM-PCD) and is upregulated in involution. Furthermore, involution is delayed in cathepsin L-deficient mice suggesting that the p55α/p50α subunits mediate cell death in part by elevating the level of cathepsin L resulting in increased cytosolic activity. Surprisingly, we found that p55α/p50α localize to the nucleus where they bind to chromatin and regulate transcription of a subset of inflammatory/acute phase genes that are also Stat3 targets. Our findings reveal a novel role for these PI3K regulatory subunits as regulators of LM-PCD in vivo.

Phosphoinositide 3-kinases p110α and p110ß have differential roles in insulin-like growth factor-1-mediated Akt phosphorylation and platelet priming.

OBJECTIVE: Platelet hyperactivity is a contributing factor in the pathogenesis of cardiovascular disease and can be induced by elevated levels of circulating growth factors, such as insulin-like growth factor-1 (IGF-1). IGF-1 is a primer that cannot stimulate platelet activation by itself, but in combination with physiological stimuli can potentiate platelet functional responses via a phosphoinositide 3-kinase-dependent mechanism. In this study, we explored the role of the phosphoinositide 3-kinase p110α isoform in IGF-1-mediated enhancement of platelet function. APPROACH AND RESULTS: Using a platelet-specific p110α knockout murine model, we demonstrate that genetic deletion, similar to pharmacological inactivation of p110α, did not affect proteinase-activated receptor 4 signaling to Akt/protein kinase B but significantly reduced IGF-1-mediated Akt phosphorylation. The p110ß inhibitor TGX-221 abolished IGF-1-induced Akt phosphorylation in p110α-deficient platelets, demonstrating that both p110α and p110ß contribute to IGF-1-mediated Akt phosphorylation. Genetic deletion of p110α had no effect on IGF-1-mediated increases in thrombus formation on collagen and enhancement of proteinase-activated receptor 4-mediated integrin activation and α-granule secretion. In contrast, pharmacological inhibition of p110α blocked IGF-1-mediated potentiation of integrin activation and α-granule secretion. Functional enhancement by IGF-1 in p110α knockout samples was lost after TGX-221 treatment, suggesting that p110ß drives priming in the absence of the p110α isoform. CONCLUSIONS: Together, these results demonstrate that both p110α and p110ß are involved in Akt signaling by IGF-1, but that it is the p110α isoform that is responsible for IGF-1-mediated potentiation of platelet function.