The cell biology behind the oncogenic PIP3 lipids.

The different mechanisms of phosphoinositide 3-kinase (PI3K) activation in cancer as well as the events that result in PI3K pathway reactivation after patient treatment with PI3K inhibitors was discussed on October 15-17th, 2018, in the medieval town of Baeza (Universidad Internacional de Andalucía, Spain) at the workshop entitled 'The cell biology behind the oncogenic PIP3 lipids'. These topics and the data presented regarding cellular functions altered by PI3K deregulation, the cooperation of PI3K/PTEN mutations with other tumor drivers, and the lessons learned for PI3K-targeted therapy, are discussed below.

Functions of Nuclear Polyphosphoinositides.

Despite interest in phosphoinositide (PtdIns) kinases, such as PtdIns 3 kinases (PI3K), as targets for controlling plasma membrane PtdIns levels in disease, the PtdIns have another less well-known site of action in the cell nucleus.Recent studies show that PtdIns use a variety of strategies to alter DNA responses. Here, we provide an overview of these newly identified forms of gene expression control, which should be considered when studying the therapeutic use of PtdIns-directed compounds. As PI3K is one of the most important clinical targets in recent years, we will focus on two polyphosphoinositides, the PI3K substrate PtdIns(4,5)di-phosphate (PI4,5P2) and its product PtdIns(3,4,5)tri-phosphate (PI3,4,5P3).

Persistent regulatory T-cell response 2 years after 3 years of grass tablet SLIT: Links to reduced eosinophil counts, sIgE levels, and clinical benefit.

BACKGROUND: In the first 2 years of grass tablet sublingual immunotherapy treatment, we have previously demonstrated a progressive development of a regulatory T-cell response, which was preceded by an early decrease in the frequency of both IL-4+ cells and sIgE levels. A progressive increase in sIgG4 levels and FAB blockage were also found. METHODS: By monitoring immunological kinetics during 3 years of active treatment + 2 years of follow-up, we aimed to identify key immunological parameters that could explain sustained clinical benefit of grass tablet sublingual immunotherapy. RESULTS: Thirty patients completed the 5-year clinical trial protocol. Although individual responses were heterogeneous, reduction in both sIgE and circulating IL-4+ cells compared to the initial 1- to 4-month peak was maintained throughout the 3-year treatment period and for 2 years after discontinuation. Meanwhile, after a 2-year increase in sIgG4, the levels were stabilized during the third year and decreased post-therapy. FAB inhibition remained significantly inhibited throughout the study compared to preimmunotherapy in 83% of patients. A sustained regulatory T-cell response, after IT cessation, occurs in two-thirds of the patients. There was a statistical association between this regulatory response, the maintenance of lower eosinophil counts during grass pollen seasons, and sIgE titers lower than before immunotherapy treatment, and the latter were significantly associated with clinical response. CONCLUSION: Our results suggest that the immunological mechanisms underlying the sustained response after 2 years of cessation of immunotherapy (3-year treatment period) are linked to the acquisition and maintenance of a regulatory T-cell response.

Inhibition of PI3Kδ reduces kidney infiltration by macrophages and ameliorates systemic lupus in the mouse.

Systemic lupus erythematosus (SLE) is a human chronic inflammatory disease generated and maintained throughout life by autoreactive T and B cells. Class I phosphoinositide 3-kinases (PI3K) are heterodimers composed of a regulatory and a catalytic subunit that catalyze phosphoinositide-3,4,5-P3 formation and regulate cell survival, migration, and division. Activity of the PI3Kδ isoform is enhanced in human SLE patient PBLs. In this study, we analyzed the effect of inhibiting PI3Kδ in MRL/lpr mice, a model of human SLE. We found that PI3Kδ inhibition ameliorated lupus progression. Treatment of these mice with a PI3Kδ inhibitor reduced the excessive numbers of CD4(+) effector/memory cells and B cells. In addition, this treatment reduced serum TNF-α levels and the number of macrophages infiltrating the kidney. Expression of inactive PI3Kδ, but not deletion of the other hematopoietic isoform PI3Kγ, reduced the ability of macrophages to cross the basement membrane, a process required to infiltrate the kidney, explaining MRL/lpr mice improvement by pharmacologic inhibition of PI3Kδ. The observations that p110δ inhibitor prolonged mouse life span, reduced disease symptoms, and showed no obvious secondary effects indicates that PI3Kδ is a promising target for SLE.

Phosphoinositide 3-kinase beta controls replication factor C assembly and function.

Genomic integrity is preserved by the action of protein complexes that control DNA homeostasis. These include the sliding clamps, trimeric protein rings that are arranged around DNA by clamp loaders. Replication factor C (RFC) is the clamp loader for proliferating cell nuclear antigen, which acts on DNA replication. Other processes that require mobile contact of proteins with DNA use alternative RFC complexes that exchange RFC1 for CTF18 or RAD17. Phosphoinositide 3-kinases (PI3K) are lipid kinases that generate 3-poly-phosphorylated-phosphoinositides at the plasma membrane following receptor stimulation. The two ubiquitous isoforms, PI3Kalpha and PI3Kbeta, have been extensively studied due to their involvement in cancer and nuclear PI3Kbeta has been found to regulate DNA replication and repair, processes controlled by molecular clamps. We studied here whether PI3Kbeta directly controls the process of molecular clamps loading. We show that PI3Kbeta associated with RFC1 and RFC1-like subunits. Only when in complex with PI3Kbeta, RFC1 bound to Ran GTPase and localized to the nucleus, suggesting that PI3Kbeta regulates RFC1 nuclear import. PI3Kbeta controlled not only RFC1- and RFC-RAD17 complexes, but also RFC-CTF18, in turn affecting CTF18-mediated chromatid cohesion. PI3Kbeta thus has a general function in genomic stability by controlling the localization and function of RFC complexes.

p85ß phosphoinositide 3-kinase subunit regulates tumor progression.

PIK3R2 encodes a ubiquitous regulatory subunit (p85ß) of PI3K, an enzyme that generates 3-polyphosphoinositides at the plasma membrane. PI3K activation triggers cell survival and migration. We found that p85ß expression is elevated in breast and colon carcinomas and that its increased expression correlates with PI3K pathway activation and tumor progression. p85ß expression induced moderate PIP(3) generation at the cell membrane and enhanced cell invasion. In accordance, genetic alteration of pik3r2 expression levels modulated tumor progression in vivo. Increased p85ß expression thus represents a cellular strategy in cancer progression.

Grass tablet sublingual immunotherapy downregulates the TH2 cytokine response followed by regulatory T-cell generation.

BACKGROUND: Sublingual administration of Phleum pratense allergen immunotherapy (SLIT) tablets is a clinically efficient treatment for grass pollen-induced rhinoconjunctivitis. This immunotherapy downregulates TH2 immune responses, induces tolerogenic pathways, and increases regulatory T cells. However, associated immune response markers of allergen desensitization remain undefined. OBJECTIVE: We sought to characterize the kinetics of individual changes in the immunologic response to grass tablet SLIT. METHODS: We evaluated the systemic effects of SLIT in a longitudinal analysis of humoral and cellular immune parameters in peripheral blood samples. RESULTS: Grass tablet SLIT administration induced a 2-phase systemic humoral and cellular response. The TH2 response was initially exacerbated and detected as increased allergen-specific IgE (sIgE) and IgG4 (sIgG4) levels and an increase in IL-4-producing cells, followed by downregulation of the TH2 response with a shift toward a TH1 cytokine profile. T cells with a regulatory phenotype were also elicited. Statistical correlations between immunologic measurements for each patient throughout therapy indicated that TH2 response downregulation and reduction of the immediate SLIT-induced IgE response were associated with increased allergen-specific IgG4 synthesis early in therapy. TH2 response downregulation by month 4 correlated with increased frequency of CD4(+) T cells with a regulatory phenotype by 12 months. CONCLUSION: Changes in sIgE levels after therapy were linked to a specific IgG4 response, and production of blocking antibodies correlated with TH2 response downregulation. Reduced IL-4(+) cell frequency was linked to an increase in the frequency of CD4(+) T cells with a regulatory phenotype. Changes in sIgE levels and reduced IL-4 and blocking antibody levels could thus be used as indicators of a patient's immune response to therapy.

Suppressor of cytokine signaling 1 blocks mitosis in human melanoma cells.

Hypermethylation of SOCS genes is associated with many human cancers, suggesting a role as tumor suppressors. As adaptor molecules for ubiquitin ligases, SOCS proteins modulate turnover of numerous target proteins. Few SOCS targets identified so far have a direct role in cell cycle progression; the mechanism by which SOCS regulate the cell cycle thus remains largely unknown. Here we show that SOCS1 overexpression inhibits in vitro and in vivo expansion of human melanoma cells, and that SOCS1 associates specifically with Cdh1, triggering its degradation by the proteasome. Cells therefore show a G1/S transition defect, as well as a secondary blockade in mitosis and accumulation of cells in metaphase. SOCS1 expression correlated with a reduction in cyclin D/E levels and an increase in the tumor suppressor p19, as well as the CDK inhibitor p53, explaining the G1/S transition defect. As a result of Cdh1 degradation, SOCS1-expressing cells accumulated cyclin B1 and securin, as well as apparently inactive Cdc20, in mitosis. Levels of the late mitotic Cdh1 substrate Aurora A did not change. These observations comprise a hitherto unreported mechanism of SOCS1 tumor suppression, suggesting this molecule as a candidate for the design of new therapeutic strategies for human melanoma.

Enhanced phosphoinositide 3-kinase δ activity is a frequent event in systemic lupus erythematosus that confers resistance to activation-induced T cell death.

Systemic lupus erythematosus (SLE) is a human chronic inflammatory disease caused by the action of autoreactive T and B cells. Class I phosphoinositide-3-kinases (PI3K) are enzymes that trigger formation of 3-poly-phosphoinositides that induce cell survival. Enhanced PI3K activation is a frequent event in human cancer. Nonetheless, in a genetic model with enhanced activation of class I(A) PI3K in T cells, mice show a greater tumor index but die of a lupus-like disease. In this study, we studied the potential PI3K involvement in human SLE. The PI3K pathway was frequently activated in SLE patient PBMC and T cells (∼70% of cases), more markedly in active disease phases. We examined the mechanism for PI3K pathway activation and found enhanced activation of PI3Kδ in SLE peripheral blood T cells. The magnitude of PI3K pathway activation in patients paralleled activated/memory T cell accumulation. We examined potential tolerance mechanisms affected by increased PI3K activity; SLE patients showed reduced activation-induced cell death of activated/memory T cells. Moreover, the defective activation-induced cell death in SLE T cells was corrected after reduction of PI3Kδ activity, suggesting that PI3Kδ contributes to induction of enhanced SLE memory T cell survival. These observations point to PI3Kδ as a target of clinical interest for SLE.

Nuclear phosphoinositide 3-kinase beta controls double-strand break DNA repair.

Class I phosphoinositide 3-kinases are enzymes that generate 3-poly-phosphoinositides at the cell membrane following transmembrane receptor stimulation. Expression of the phosphoinositide 3-kinase beta (PI3Kbeta) isoform, but not its activity, is essential for early embryonic development. Nonetheless, the specific function of PI3Kbeta in the cell remains elusive. Double-strand breaks (DSB) are among the most deleterious lesions for genomic integrity; their repair is required for development. We show that PI3Kbeta is necessary for DSB sensing, as PI3Kbeta regulates binding of the Nbs1 sensor protein to damaged DNA. Indeed, Nbs1 did not bind to DSB in PI3Kbeta-deficient cells, which showed a general defect in subsequent ATM and ATR activation, resulting in genomic instability. Inhibition of PI3Kbeta also retarded the DNA repair but the defect was less marked than that induced by PI3Kbeta deletion, supporting a kinase-independent function for PI3Kbeta in DNA repair. These results point at class I PI3Kbeta as a critical sensor of genomic integrity.

PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell-mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) gamma, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kgamma may be a useful target in the treatment of chronic inflammation.

Specific function of phosphoinositide 3-kinase beta in the control of DNA replication.

Class I(A) phosphoinositide 3-kinase (PI3K) are enzymes comprised of a p85 regulatory and a p110 catalytic subunit that induce formation of 3-polyphosphoinositides, which activate numerous downstream targets. PI3K controls cell division. Of the 2 ubiquitous PI3K isoforms, alpha has selective action in cell growth and cell cycle entry, but no specific function in cell division has been described for beta. We report here a unique function for PI3Kbeta in the control of DNA replication. PI3Kbeta regulated DNA replication through kinase-dependent and kinase-independent mechanisms. PI3Kbeta was found in the nucleus, where it associated PKB. Modulation of PI3Kbeta activity altered the DNA replication rate by controlling proliferating cell nuclear antigen (PCNA) binding to chromatin and to DNA polymerase delta. PI3Kbeta exerted this action by regulating the nuclear activation of PKB in S phase, and in turn phosphorylation of PCNA negative regulator p21(Cip). Also, p110beta associated with PCNA and controlled PCNA loading onto chromatin in a kinase-independent manner. These results show a selective function of PI3Kbeta in the control of DNA replication.

Targeting PTEN Regulation by Post Translational Modifications.

Phosphatidylinositol-3,4,5-triphosphate (PIP3) is a lipidic second messenger present at very low concentrations in resting normal cells. PIP3 levels, though, increase quickly and transiently after growth factor addition, upon activation of phosphatidylinositol 3-kinase (PI3-kinase). PIP3 is required for the activation of intracellular signaling pathways that induce cell proliferation, cell migration, and survival. Given the critical role of this second messenger for cellular responses, PIP3 levels must be tightly regulated. The lipid phosphatase PTEN (phosphatase and tensin-homolog in chromosome 10) is the phosphatase responsible for PIP3 dephosphorylation to PIP2. PTEN tumor suppressor is frequently inactivated in endometrium and prostate carcinomas, and also in glioblastoma, illustrating the contribution of elevated PIP3 levels for cancer development. PTEN biological activity can be modulated by heterozygous gene loss, gene mutation, and epigenetic or transcriptional alterations. In addition, PTEN can also be regulated by post-translational modifications. Acetylation, oxidation, phosphorylation, sumoylation, and ubiquitination can alter PTEN stability, cellular localization, or activity, highlighting the complexity of PTEN regulation. While current strategies to treat tumors exhibiting a deregulated PI3-kinase/PTEN axis have focused on PI3-kinase inhibition, a better understanding of PTEN post-translational modifications could provide new therapeutic strategies to restore PTEN action in PIP3-dependent tumors.

PI3Kß-regulated ß-catenin mediates EZH2 removal from promoters controlling primed human ESC stemness and primitive streak gene expression.

The mechanism governing the transition of human embryonic stem cells (hESCs) toward differentiated cells is only partially understood. To explore this transition, the activity and expression of the ubiquitous phosphatidylinositol 3-kinase (PI3Kα and PI3Kß) were modulated in primed hESCs. The study reports a pathway that dismantles the restraint imposed by the EZH2 polycomb repressor on an essential stemness gene, NODAL, and on transcription factors required to trigger primitive streak formation. The primitive streak is the site where gastrulation begins to give rise to the three embryonic cell layers from which all human tissues derive. The pathway involves a PI3Kß non-catalytic action that controls nuclear/active RAC1 levels, activation of JNK (Jun N-terminal kinase) and nuclear ß-catenin accumulation. ß-Catenin deposition at promoters triggers release of the EZH2 repressor, permitting stemness maintenance (through control of NODAL) and correct differentiation by allowing primitive streak master gene expression. PI3Kß epigenetic control of EZH2/ß-catenin might be modulated to direct stem cell differentiation.

Phosphatidylinositol 3-kinase gamma inhibition ameliorates inflammation and tumor growth in a model of colitis-associated cancer.

BACKGROUND & AIMS: A large body of evidence supports a correlation between inflammation and cancer, although the molecular mechanisms that govern this process are incompletely understood. Phosphatidylinositol 3-kinase (PI3K) is an enzyme that regulates the immune response and contributes to cell transformation in several tumor types. Here, we address the role of the PI3Kgamma isoform in inflammatory bowel disease and in the development of colitis-associated cancer. METHODS: PI3Kgamma(-/-) and control mice were repeatedly treated with dextran sulfate sodium to induce chronic colitis and colitis-associated cancer. Colorectal tumor burden and colon inflammation were evaluated in these mice. Leukocyte populations in colon were characterized by flow cytometry analysis. RESULTS: PI3Kgamma-deficient mice had a lower incidence of colitis-associated tumors, as well as reduced tumor multiplicity and smaller tumor size compared with controls. Reduced tumor development paralleled less colon inflammation in PI3Kgamma-deficient mice. Analysis of leukocyte populations in the colon of PI3Kgamma-deficient mice showed defective activation and infiltration of myeloid cells and defective recruitment of T cells to the colon compared with controls. CONCLUSIONS: PI3Kgamma regulates the innate immune response in a murine model of ulcerative colitis, thereby controlling colon inflammation and tumor formation.

p85beta phosphoinositide 3-kinase regulates CD28 coreceptor function.

CD28 is a receptor expressed on T cells that regulates their differentiation after antigen stimulation to long-term-survival memory T cells. CD28 enhances T-cell receptor signals and reduces expression of CBL ubiquitin ligases, which negatively control T-cell activation. In the absence of CD28 ligation during the primary stimulation, CBL levels remain high and T cells fail to mount an efficient secondary response. CD28 associates with p85alpha, one of the regulatory subunits of phosphoinositide-3-kinase (PI3K), but the relevance of this interaction is debated. We examined here the contribution of the other ubiquitous PI3K regulatory subunit, p85beta, in CD28 function. We describe that p85beta bound to CD28 and to CBL with greater affinity than p85alpha. Moreover, deletion of p85beta impaired CD28-induced intracellular events, including c-CBL and CBL-b down-regulation as well as PI3K pathway activation. This resulted in defective differentiation of activated T cells, which failed to exhibit an efficient secondary immune response. Considering that p85beta-deficient T cells fail in recall responses and that p85beta binds to and regulates CD28 signals, the presented observations suggest the involvement of p85beta in CD28-mediated activation and differentiation of antigen-stimulated T cells.

Fluctuations in AKT and PTEN Activity Are Linked by the E3 Ubiquitin Ligase cCBL.

3-Poly-phosphoinositides (PIP3) regulate cell survival, division, and migration. Both PI3-kinase (phosphoinositide-3-kinase) and PTEN (phosphatase and tensin-homolog in chromosome 10) control PIP3 levels, but the mechanisms connecting PI3-kinase and PTEN are unknown. Using non-transformed cells, the activation kinetics of PTEN and of the PIP3-effector AKT were examined after the addition of growth factors. Both epidermal growth factor and serum induced the early activation of AKT and the simultaneous inactivation of PTEN (at ~5 min). This PIP3/AKT peak was followed by a general reduction in AKT activity coincident with the recovery of PTEN phosphatase activity (at ~10-15 min). Subsequent AKT peaks and troughs followed. The fluctuation in AKT activity was linked to that of PTEN; PTEN reconstitution in PTEN-null cells restored AKT fluctuations, while PTEN depletion in control cells abrogated them. The analysis of PTEN activity fluctuations after the addition of growth factors showed its inactivation at ~5 min to be simultaneous with its transient ubiquitination, which was regulated by the ubiquitin E3 ligase cCBL (casitas B-lineage lymphoma proto-oncogene). Protein-protein interaction analysis revealed cCBL to be brought into the proximity of PTEN in a PI3-kinase-dependent manner. These results reveal a mechanism for PI3-kinase/PTEN crosstalk and suggest that cCBL could be new target in strategies designed to modulate PTEN activity in cancer.

Blocking of HIV-1 infection by targeting CD4 to nonraft membrane domains.

Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered. We report that all CD4 mutants that retain raft partitioning mediate HIV-1 entry and CD4-induced Lck activation independently of their transmembrane and cytoplasmic domains. Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt. In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4(+) cell line. These results not only indicate that HIV-1 exploits host membrane raft domains as cell entry sites, but also suggest new strategies for preventing HIV-1 infection.

PI3Kgamma activation by CXCL12 regulates tumor cell adhesion and invasion.

Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3Kgamma regulates tumor cell adhesion through mechanisms different from those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.

Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis.

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol-anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide-differentiated HL60 cells in a pertussis toxin-sensitive, actin-dependent manner. A transmembrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase gamma in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge.