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.

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.

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).

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.

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.

E-cadherin downregulation sensitizes PTEN-mutant tumors to PI3Kß silencing.

Alterations in phosphatidylinositol 3-kinase (PI3K) and in PTEN (phosphatase and tensin homolog), the negative regulator of the PI3K pathway, are found in nearly half of human tumors. As PI3Kß, the main isoform activated in PTEN-mutant tumors, has kinase-dependent and -independent activities, we compared the effects of depleting vs. drug-inhibiting PI3Kß kinase activity in a collection of diverse tumor types and in a set of bladder carcinoma cell lines grown as xenografts in mice. PI3Kß depletion (by intratumor injection of PIK3CB siRNA) induced apoptosis and triggered regression of PTEN-mutant tumors more efficiently than PI3Kß inhibition. A small proportion of these tumors was resistant to PI3Kß downregulation; we analyzed what determined resistance in these cases. Using add-back experiments, we show that both PTEN mutation and low E-cadherin expression are necessary for PI3Kß dependence. In bladder carcinoma, loss of E-cadherin expression coincides with N-cadherin upregulation. We found that PI3Kß associated with N-cadherin and that PIK3CB depletion selectively disrupted N-cadherin cell adhesions in PTEN-mutant bladder carcinoma. These results support the use of PIK3CB interfering RNA as a therapeutic approach for high-risk bladder cancers that show E-cadherin loss and express mutant PTEN.

Phosphoinositide 3-kinase beta protects nuclear envelope integrity by controlling RCC1 localization and Ran activity.

The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kß) showed an aberrant nuclear morphology, we studied the contribution of PI3Kß to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kß regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kß controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kß regulates the nuclear envelope through upstream regulation of RCC1 and Ran.

Phosphoinositide 3-kinase p85beta regulates invadopodium formation.

The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85ß, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85ß might facilitate cell invasion. We show that p85ß localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85ß induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85ß lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85ß function in invadopodium-like formation, p85ß levels increased in metastatic melanoma and p85ß depletion reduced invadopodium formation and invasion. These results show that p85ß enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85ß levels.

Cell activation-induced phosphoinositide 3-kinase alpha/beta dimerization regulates PTEN activity.

The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110ß at several points during the cell cycle. We studied the potential connections between p110α and p110ß activation, and we show that cell stimulation promotes p110α and p110ß association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P3 levels.

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.

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.

PI3K p110δ is expressed by gp38(-)CD31(+) and gp38(+)CD31(+) spleen stromal cells and regulates their CCL19, CCL21, and LTßR mRNA levels.

The role of p110δ PI3K in lymphoid cells has been studied extensively, showing its importance in immune cell differentiation, activation and development. Altered T cell localization in p110δ-deficient mouse spleen suggested a role for p110δ in non-hematopoietic stromal cells, which maintain hematopoietic cell segregation. We tested this hypothesis using p110δ(WT/WT) mouse bone marrow to reconstitute lethally irradiated p110δ(WT/WT) or p110δ(D910A/D910A) (which express catalytically inactive p110δ) recipients, and studied localization, number and percentage of hematopoietic cell subsets in spleen and lymph nodes, in homeostatic conditions and after antigen stimulation. These analyses showed diffuse T cell areas in p110δ(D910A/D910A) and in reconstituted p110δ(D910A/D910A) mice in homeostatic conditions. In these mice, spleen CD4(+) and CD8(+) T cell numbers did not increase in response to antigen, suggesting that a p110δ(D910A/D910A) stroma defect impedes correct T cell response. FACS analysis of spleen stromal cell populations showed a decrease in the percentage of gp38(-)CD31(+) cells in p110δ(D910A/D910A) mice. qRT-PCR studies detected p110δ mRNA expression in p110δ(WT/WT) spleen gp38(-)CD31(+) and gp38(+)CD31(+) subsets, which was reduced in p110δ(D910A/D910A) spleen. Lack of p110δ activity in these cell populations correlated with lower LTßR, CCL19 and CCL21 mRNA levels; these molecules participate in T cell localization to specific spleen areas. Our results could explain the lower T cell numbers and more diffuse T cell areas found in p110δ(D910A/D910A) mouse spleen, as well as the lower T cell expansion after antigen stimulation in p110δ(D910A/D910A) compared with p110δ(WT/WT) mice.

PI3K p110γ deletion attenuates murine atherosclerosis by reducing macrophage proliferation but not polarization or apoptosis in lesions.

Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice. Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR(-/-)p110γ(-/-) mice were smaller than in LDLR(-/-)p110γ(+/-) controls, which coincided with decreased macrophage proliferation in LDLR(-/-)p110γ(-/-) mouse lesions. This proliferation defect was also observed in p110γ(-/-) bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR(-/-)p110γ(-/-) mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR(-/-) mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.

CXCL12-mediated murine neural progenitor cell movement requires PI3Kß activation.

The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110ß in NPC, only p110ß participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110ß blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110ß is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110ß is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.

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.

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.

Phosphoinositide 3-kinase ß regulates chromosome segregation in mitosis.

Class I(A) phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP(3)). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110α and p110ß. Whereas p110α is activated at G1-phase entry and promotes protein synthesis and gene expression, p110ß activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110α and p110ß function in mitosis. p110α was activated at mitosis entry and regulated early mitotic events, such as PIP(3) generation, prometaphase progression, and spindle orientation. In contrast, p110ß was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110ß function in preserving genomic stability during mitosis.

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.