Nuclear phosphoinositides and their roles in cell biology and disease.

Since the late 1980s, a growing body of evidence has documented that phosphoinositides and their metabolizing enzymes, which regulate a large variety of cellular functions both in the cytoplasm and at the plasma membrane, are present also within the nucleus, where they are involved in processes such as cell proliferation, differentiation, and survival. Remarkably, nuclear phosphoinositide metabolism operates independently from that present elsewhere in the cell. Although nuclear phosphoinositides generate second messengers such as diacylglycerol and inositol 1,4,5 trisphosphate, it is becoming increasingly clear that they may act by themselves to influence chromatin structure, gene expression, DNA repair, and mRNA export. The understanding of the biological roles played by phosphoinositides is supported by the recent acquisitions demonstrating the presence in the nuclear compartment of several proteins harboring phosphoinositide-binding domains. Some of these proteins have functional roles in RNA splicing/processing and chromatin assembly. Moreover, recent evidence shows that nuclear phospholipase Cß1 (a key phosphoinositide metabolizing enzyme) could somehow be involved in the myelodysplastic syndrome, i.e. a hematopoietic disorder that frequently evolves into an acute leukemia. This review aims to highlight the most significant and updated findings about phosphoinositide metabolism in the nucleus under both physiological and pathological conditions.

The emerging multiple roles of nuclear Akt.

Akt is a central player in the signal transduction pathways activated in response to many growth factors, hormones, cytokines, and nutrients and is thought to control a myriad of cellular functions including proliferation and survival, autophagy, metabolism, angiogenesis, motility, and exocytosis. Moreover, dysregulated Akt activity is being implicated in the pathogenesis of a growing number of disorders, including cancer. Evidence accumulated over the past 15 years has highlighted the presence of active Akt in the nucleus, where it acts as a fundamental component of key signaling pathways. For example, nuclear Akt counteracts apoptosis through a block of caspase-activated DNase: deoxyribonuclease and inhibition of chromatin condensation, and is also involved in cell cycle progression control, cell differentiation, mRNA: messenger RNA export, DNA repair, and tumorigenesis. In this review, we shall summarize the most relevant findings about nuclear Akt and its functions.

The emerging role of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in normal myelopoiesis and leukemogenesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway mediates diverse and important physiological cell functions which include proliferation, differentiation, survival, motility, autophagy, and metabolism. However, dysregulated PI3K/Akt/mTOR signaling has been documented in a wide range of neoplasias, including malignant hematological disorders. It is now emerging that this signaling network plays a key role during normal hematopoiesis, a tightly regulated process resulting in the formation of all blood lineages. Blood cell development encompasses a complex series of events which are mainly regulated by actions of cytokines, a family of extracellular ligands which stimulate many biological responses in a wide array of cell types. Hematopoiesis is strictly dependent on the correct function of the bone marrow microenvironment (BMM), as BMM cells secrete most of the cytokines. Several of these cytokines activate the PI3K/Akt/mTOR signaling network and regulate proliferation, survival, and differentiation events during hematopoiesis. Here, we review the evidence that links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of hematopoietic stem cells and the process of myelopoiesis, including lineage commitment. We then highlight the emerging role played by aberrant PI3K/Akt/mTOR signaling during leukemogenesis.

Preclinical testing of the Akt inhibitor triciribine in T-cell acute lymphoblastic leukemia.

Over the past 20 years, survival rates of T-cell acute lymphoblastic leukemia (T-ALL) patients have improved, mainly because of advances in polychemotherapy protocols. Despite these improvements, we still need novel and less toxic treatment strategies targeting aberrantly activated signaling networks which increase proliferation, survival, and drug resistance of T-ALL cells. One such network is represented by the phosphatidylinositol 3-kinase (PI3K)/Akt axis. PI3K inhibitors have displayed some promising effects in preclinical models of T-ALL. Here, we have analyzed the therapeutic potential of the Akt inhibitor, triciribine, in T-ALL cell lines. Triciribine caused cell cycle arrest and caspase-dependent apoptosis. Western blots demonstrated a dose-dependent dephosphorylation of Akt1/Akt2, and of mammalian target of rapamycin complex 1 downstream targets in response to triciribine. Triciribine induced autophagy, which could be interpreted as a defensive mechanism, because an autophagy inhibitor (chloroquine) increased triciribine-induced apoptosis. Triciribine synergized with vincristine, a chemotherapeutic drug employed for treating T-ALL patients, and targeted the side population of T-ALL cell lines, which might correspond to leukemia initiating cells. Our findings indicate that Akt inhibition, either alone or in combination with chemotherapeutic drugs, may serve as an efficient treatment towards T-ALL cells requiring upregulation of this signaling pathway for their proliferation and survival.

Dysferlin in a hyperCKaemic patient with caveolin 3 mutation and in C2C12 cells after p38 MAP kinase inhibition.

Dysferlin is a plasma membrane protein of skeletal muscle whose deficiency causes Miyoshi myopathy, limb girdle muscular dystrophy 2B and distal anterior compartment myopathy. Recent studies have reported that dysferlin is implicated in membrane repair mechanism and coimmunoprecipitates with caveolin 3 in human skeletal muscle. Caveolin 3 is a principal structural protein of caveolae membrane domains in striated muscle cells and cardiac myocytes. Mutations of caveolin 3 gene (CAV3) cause different diseases and where caveolin 3 expression is defective, dysferlin localization is abnormal. We describe the alteration of dysferlin expression and localization in skeletal muscle from a patient with raised serum creatine kinase (hyperCKaemia), whose reduction of caveolin 3 is caused by a CAV3 P28L mutation. Moreover, we performed a study on dysferlin interaction with caveolin 3 in C2C12 cells. We show the association of dysferlin to cellular membrane of C2C12 myotubes and the low affinity link between dysferlin and caveolin 3 by immunoprecipitation techniques. We also reproduced caveolinopathy conditions in C2C12 cells by a selective p38 MAP kinase inhibition with SB203580, which blocks the expression of caveolin 3. In this model, myoblasts do not fuse into myotubes and we found that dysferlin expression is reduced. These results underline the importance of dysferlin-caveolin 3 relationship for skeletal muscle integrity and propose a cellular model to clarify the dysferlin alteration mechanisms in caveolinopathies.

Targeting the liver kinase B1/AMP-activated protein kinase pathway as a therapeutic strategy for hematological malignancies.

INTRODUCTION: Despite considerable advances, several hematological malignancies remain incurable with standard treatments. Therefore, there is a need for novel targeted and less toxic therapies, particularly for patients who develop resistance to traditional chemotherapeutic drugs. The liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling pathway has recently emerged as a tumor suppressor axis. A critical point is that the LKB1/AMPK network remains functional in a wide range of cancers and could be stimulated by drugs, such as N,N-dimethylimidodicarbonimidic diamide (metformin) or 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR). AREAS COVERED: The literature data show that drugs activating LKB1/AMPK signaling induced cell cycle arrest, caspase-dependent apoptosis or autophagy in hematopoietic tumors. Moreover, metformin effectively inhibited mammalian target of rapamycin complex 1 (mTORC1)-controlled oncogenetic protein translation, which does not occur with allosteric mTORC1 inhibitors, such as rapamycin and its derivatives. Metformin was also capable of targeting leukemic stem cells, the most relevant target for leukemia eradication. EXPERT OPINION: Data emerging from preclinical settings suggest that the LKB1/AMPK pathway is critically involved in regulating proliferation and survival of malignant hematopoietic cells. Thus, it is proposed that drugs activating the LKB1/AMPK axis may offer a novel and less toxic treatment option for some types of hematological malignancies.

A combination of temsirolimus, an allosteric mTOR inhibitor, with clofarabine as a new therapeutic option for patients with acute myeloid leukemia.

Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiating cells (LICs) function. In this study, we assessed the therapeutic potential of a combination consisting of temsirolimus [an allosteric mTOR complex 1 (mTORC1) inhibitor] with clofarabine, a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. The drug combination (CLO-TOR) displayed synergistic cytotoxic effects against a panel of AML cell lines and primary cells from AML patients. Treatment with CLO-TOR induced a G0/G1-phase cell cycle arrest, apoptosis, and autophagy. CLO-TOR was pro-apoptotic in an AML patient blast subset (CD34⁺/CD38⁻/CD123⁺), which is enriched in putative leukemia initiating cells (LICs). In summary, the CLO-TOR combination could represent a novel valuable treatment for AML patients, also in light of its efficacy against LICs.

The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling network and the control of normal myelopoiesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a central role in cell growth, proliferation, differentiation, and survival under physiological conditions. Aberrant regulation of the PI3K/Akt/mTOR signal transduction network has been observed in a wide range of neoplasias, including malignant hematological disorders. This observation suggests that this signaling cascade could also play a critical role during normal hematopoiesis, a highly regulated process which results in the formation of all blood lineages. The development of blood cells comprises a complex series of events which are mainly regulated through the actions of cytokines, a large family of extracellular ligands than can stimulate many biological responses in a wide array of cell types. Several of these cytokines are known to activate the PI3K/Akt/mTOR signal transduction network and thus regulate proliferation, survival, and differentiation events during hematopoiesis. Moreover, hematopoiesis is strictly dependent on the correct functions of the bone marrow microenvironment. Here, we review the evidence which links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of hematopoietic stem cells and the process of lineage commitment, which then gives rise to myeloid lineage-restricted cells. We then further highlight the key role played by the PI3K/Akt/mTOR network during erythropoiesis, megakaryocytopoiesis, and granulo-cytopoiesis/monocytopoiesis.

Identification of a functional nuclear export sequence in diacylglycerol kinase-zeta.

Diacylglycerol kinases (DGKs) are key regulators of diacylglycerol-dependent signaling pathways. Among the 10 DGK isoforms, DGK-zeta is the only nuclear form that contains a nuclear localization signal. Here, by site-directed mutagenesis, we showed that DGK-zeta also displays a functional independent nuclear export signal (NES) sequence between the amino acid residues 362-370. Indeed, the NES mutant forms of DGK-zeta accumulated in the nucleus to a much greater extent than wildtype DGK-zeta. Moreover, treatment with leptomycin B, an inhibitor of leucine-rich type NES, resulted in accumulation of both endogenous and ectopically expressed DGK-zeta in the nucleus, demonstrating that nuclear export of DGK-zeta is chromosome regional maintenance protein 1 (CRM1)-dependent. Previously, we reported that nuclear DGK-zeta is a negative regulator of cell cycle progression in C2C12 mouse myoblasts. In this paper, we documented that enhancement of DGK-zeta nuclear localization by NES sequence mutation, increases G(0)/G(1) block in C2C12 cells. Overall, our data demonstrate that DGK-zeta export from nucleus to cytoplasm is regulated by a leucine-rich NES through the exportin CRM1 and suggest that the nuclear localization of DGK-zeta could finely tune its function as a regulator of G(1)/S cell cycle transition.

Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against T-cell acute lymphoblastic leukemia.

Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival, and drug resistance. These observations lend compelling weight to the application of PI3K/Akt/mTOR inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. NVP-BEZ235 was cytotoxic to a panel of T-ALL cell lines as determined by MTT assays. NVP-BEZ235 treatment resulted in cell cycle arrest and apoptosis. Western blots showed a dose- and time-dependent dephosphorylation of Akt and mTORC1 downstream targets in response to NVP-BEZ235. Remarkably, NVP-BEZ235 targeted the side population of both T-ALL cell lines and patient lymphoblasts, which might correspond to leukemia-initiating cells, and synergized with chemotherapeutic agents (cyclophosphamide, cytarabine, dexamethasone) currently used for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic to T-ALL patient lymphoblasts displaying pathway activation, where the drug dephosphorylated eukaryotic initiation factor 4E-binding protein 1, at variance with rapamycin. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment of those T-ALLs that have aberrant upregulation of this signaling pathway for their proliferation and survival.

The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycin induce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage: implications for therapy of drug resistant breast cancers.

Resistance to multiple chemotherapeutic agents is a common clinical problem which can arise during cancer treatment. Drug resistance often involves overexpression of the multidrug resistance MDR1 gene, encoding P-glycoprotein (P-gp), a 170-kDa glycoprotein belonging to the ATP-binding cassette superfamily of membrane transporters. We have recently demonstrated apoptosis-induced, caspase-3-dependent P-gp cleavage in human T-lymphoblastoid CEM-R VBL100 cells. However, P-gp contain many aspartate residues which could be targeted by caspases other than caspase-3. To test whether other caspases could cleave P-gp in vivo, we investigated the fate of P-gp during roscovitine- and sangivamycin- induced apoptosis in MCF7 human breast cancer cells, as they lack functional caspase-3. MCF7 cells were stably transfected with human cDNA encoding P-gp. P-gp was cleaved in vitro by purified recombinant caspase-3, -6 and -7. However, P-gp cleavage was not detected in vivo in MCF7 cells induced to undergoing apoptosis by either roscovitine or sangivamycin, despite activation of both caspase-6 and -7. Interestingly, P-gp overexpressing MCF7 cells were more sensitive to either roscovitine or sangivamycin than wild-type cells, suggesting a novel potential therapeutic strategy against P-gp overexpressing cells. Taken together, our results support the concept that caspase-3 is the only caspase responsible for in vivo cleavage of P-gp and also highlight small molecules which could be effective in treating P-gp overexpressing cancers.

Ultrastructural defects of collagen VI filaments in an Ullrich syndrome patient with loss of the alpha3(VI) N10-N7 domains.

Ultrastructural alterations of collagen VI in cultured fibroblasts and reduced collagen VI immunostaining in the papillary dermis and endomysium were detected in a patient with a mild form of Ullrich congenital muscular dystrophy caused by a COL6A3 gene mutation. The patient had been previously demonstrated to express an alpha3(VI) chain shorter than normal due to skipping of the mutated exon. We show that collagen VI filaments are not organized in a normal network in the extracellular matrix secreted by patient's cultured fibroblasts. Moreover, we demonstrate that in this patient the alpha3(VI) chain is produced in lower amounts and it is almost exclusively represented by the shorter, alternatively spliced N6-C5 isoform. These results suggest that different alpha3(VI) chain isoforms, containing also domains of the N10-N7 region, are required for assembling a proper collagen VI network in the extracellular matrix.

Laminopathies: involvement of structural nuclear proteins in the pathogenesis of an increasing number of human diseases.

Just at the beginning of the millennium the neologism laminopathies has been introduced in the scientific vocabulary. An exponential increase of interest on the subject started concomitantly, so that a formerly quite neglected group of rare human diseases is now widely investigated. This review will cover the history of the identification of the molecular basis for fourteen (since now) hereditary diseases arising from defects in genes that encode nuclear envelope and nuclear lamina-associated proteins and will also consider the hypotheses that can account for the role of structural nuclear proteins in the pathogenesis of diseases affecting a wide spectrum of tissues.

Failure of lamin A/C to functionally assemble in R482L mutated familial partial lipodystrophy fibroblasts: altered intermolecular interaction with emerin and implications for gene transcription.

Familial partial lipodystrophy is an autosomal dominant disease caused by mutations of the LMNA gene encoding alternatively spliced lamins A and C. Abnormal distribution of body fat and insulin resistance characterize the clinical phenotype. In this study, we analyzed primary fibroblast cultures from a patient carrying an R482L lamin A/C mutation by a morphological and biochemical approach. Abnormalities were observed consisting of nuclear lamin A/C aggregates mostly localized close to the nuclear lamina. These aggregates were not bound to either DNA-containing structures or RNA splicing intranuclear compartments. In addition, emerin did not colocalize with nuclear lamin A/C aggregates. Interestingly, emerin failed to interact with lamin A in R482L mutated fibroblasts in vivo, while the interaction with lamin C was preserved in vitro, as determined by coimmunoprecipitation experiments. The presence of lamin A/C nuclear aggregates was restricted to actively transcribing cells, and it was increased in insulin-treated fibroblasts. In fibroblasts carrying lamin A/C nuclear aggregates, a reduced incorporation of bromouridine was observed, demonstrating that mutated lamin A/C in FPLD cells interferes with RNA transcription.