From interaction to function: Phospholipase C beta 1 protects cells from stress-induced apoptosis

The phosphoinositide-dependent signal transduction pathway has been implicated in the control of a variety of biological processes, such as the regulation of cellular metabolism and omeostasis, cell proliferation and differentiation. One of the key player in the regulation of inositol lipid signaling is phospholipase C beta 1 (PI-PLCβ1), which hydrolyses PtIns(4,5)P2, giving rise to the second messengers IP3 and DAG. The complete mapping of the PI-PLCβ1 interactome was undertaken, to understand its diverse functions within the nuclear compartment and to determine its contribution to physiological and pathological processes. Affinity purification-mass spectrometry (AP-MS) allowed for the identification of 160 proteins present in association with PI-PLCβ1 in the nucleus of erythroleukemia cells. Co-immunoprecipitation analysis of selected proteins confirmed the data obtained from mass spectrometry. Of particular interest was the identification of proteins involved in nuclear trafficking, as well as factors involved in hematological malignancies and several anti-apoptotic proteins (Piazzi et al., 2013). PI-PLCβ1 has been associated with the regulation of several cellular functions, some of which are not yet fully understood. In particular, it has been reported that PI-PLCβ1 protects murine fibroblasts from oxidative stress-induced cell death, through signaling events which remain unclear. Reactive oxygen species (ROS) have been shown to regulate major epigenetic processes causing the silencing of tumor suppressors and enhancing the proliferation of leukemic cells under oxidative stress. Investigation of the role for ROS and their signaling mediators in the pathogenesis of leukemia might, therefore, outline innovative approaches for the improvement of pharmacological therapies to treat leukemia. We demonstrate that in acute lymphoid leukemia cells (pro-B cells), treated with 250 μM of hydrogen peroxide (H2O2), PI-PLCβ1b conferred resistance to cell death, promoting cell cycle progression and cell proliferation. Interestingly, we found that, upon H2O2 exposure, the expression of PI-PLCβ1b affects the activity of several protein kinases, in particular it completely abolished the phosphorylation of Erk1/2 MAP kinases, down-regulated PTEN and up-regulated the phosphorylation of Akt; thereby sustaining cellular proliferation

Ectopic Expression of Ankrd2 Affects Proliferation, Motility and Clonogenic Potential of Human Osteosarcoma Cells

Simple Summary Osteosarcoma is a rare malignancy of bone, primarily affecting children and young adults. The main objective of this study was to identify novel therapeutic targets to fight the progression of this insidious disease. To this aim, the role of Ankrd2, a stress- and mechano- sensor protein known for being mostly expressed in muscle fibers, was analyzed in the modulation of osteosarcoma progression. By subjecting human osteosarcoma cell lines expressing or silencing Ankrd2 to several functional assays, our results demonstrated that Ankrd2 is involved in the pathogenesis of this cancer. Nonetheless, due to observations obtained by other studies in other model systems, our findings also suggest that Ankrd2 might behave as a "double-faced" cancer driver gene. Ankrd2 is a protein known for being mainly expressed in muscle fibers, where it participates in the mechanical stress response. Since both myocytes and osteoblasts are mesenchymal-derived cells, we were interested in examining the role of Ankrd2 in the progression of osteosarcoma which features a mechano-stress component. Although having been identified in many tumor-derived cell lines and -tissues, no study has yet described nor hypothesized any involvement for this protein in osteosarcoma tumorigenesis. In this paper, we report that Ankrd2 is expressed in cell lines obtained from human osteosarcoma and demonstrate a contribution by this protein in the pathogenesis of this insidious disease. Ankrd2 involvement in osteosarcoma development was evaluated in clones of Saos2, U2OS, HOS and MG63 cells stably expressing Ankrd2, through the investigation of hallmark processes of cancer cells. Interestingly, we found that exogenous expression of Ankrd2 influenced cellular growth, migration and clonogenicity in a cell line-dependent manner, whereas it was able to improve the formation of 3D spheroids in three out of four cellular models and enhanced matrix metalloproteinase (MMP) activity in all tested cell lines. Conversely, downregulation of Ankrd2 expression remarkably reduced proliferation and clonogenic potential of parental cells. As a whole, our data present Ankrd2 as a novel player in osteosarcoma development, opening up new therapeutic perspectives

K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway

Phospholipase C β1 (PLCβ1) is known to play an important role in cell proliferation. Previous studies reported aninvolvement of PLCβ1 in G0-G1/S transition and G2/M progression in Friend murine erythroleukemia cells (FELC). However,little has been found about its role in human models. Here, we used K562 cell line as human homologous of FELC inorder to investigate the possible key regulatory role of PLCβ1 during cell proliferation of this humancell line. Our studies on the effects of the overexpression of both these isoforms showed a specific and positive connection between cyclinD3 and PLCβ1 in K562 cells, which led to a prolonged S phase of the cell cycle and a delay in cell proliferation. In order to shed light on this mechanism, we decided to study the possible involvement of protein kinases C (PKC), known to be direct targets of PLC signaling and important regulators of cell proliferation. Our data showed a peculiar decrease of PKCα levels in cells overexpressing PLCβ1. Moreover, when we silenced PKCα, by RNAi technique, in order to mimic the effects of PLCβ1, we caused the same upregulation of cyclin D3 levels and the same decrease of cell proliferation found in PLCβ1-overexpressing cells. The key features emerging from our studies in K562 cells is that PLCβ1 targets cyclin D3, likely through a PKCα-mediated-pathway, and that, as a downstream effect of its activity, K562 cells undergo an accumulation in the S phase of the cell cycle

Combined Treatment with PI3K Inhibitors BYL-719 and CAL-101 Is a Promising Antiproliferative Strategy in Human Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer arising from skeletal muscle myogenic progenitors. Recent studies have shown an important role for AKT signaling in RMS progression. Aberrant activation of the PI3K/AKT axis is one of the most frequent events occurring in human cancers and serves to disconnect the control of cell growth, survival, and metabolism from exogenous growth stimuli. In the study reported here, a panel of five compounds targeting the catalytic subunits of the four class I PI3K isoforms (p110α, BYL-719 inhibitor; p110β, TGX-221 inhibitor; p110γ, CZC24832; p110δ, CAL-101 inhibitor) and the dual p110α/p110δ, AZD8835 inhibitor, were tested on the RMS cell lines RD, A204, and SJCRH30. Cytotoxicity, cell cycle, apoptosis, and the activation of downstream targets were analyzed. Of the individual inhibitors, BYL-719 demonstrated the most anti-tumorgenic properties. BYL-719 treatment resulted in G1/G0 phase cell cycle arrest and apoptosis. When combined with CAL-101, BYL-719 decreased cell viability and induced apoptosis in a synergistic manner, equaling or surpassing results achieved with AZD8835. In conclusion, our findings indicate that BYL-719, either alone or in combination with the p110δ inhibitor, CAL-101, could represent an efficient treatment for human rhabdomyosarcoma presenting with aberrant upregulation of the PI3K signaling pathway

PI3Kα-selective inhibitor alpelisib (BYL719), may be effective as anticancer agents in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer that arises from the skeletal muscle. Recent studies have identified an important role of AKT signaling in RMS progression. This suggests targeting components of the PI3K/Akt pathway may be an effective therapeutic strategy. Here, we investigated the in vitro activity of the class I PI3K inhibitors [1] in human rhabdomyosarcoma cell lines (embryonal rhabdomyosarcoma RD and A204, alveolar rhabdomyosarcoma SJCRH30). We used a panel of four compounds which specifically target PI3K isoforms including the PI3Kα-selective (p110α ) inhibitor alpelisib BYL719, currently in clinical development by Novartis Oncology, the p110β TGX-221 inhibitor, the p110γ CZC24832, the p110δ CAL-101inhibitor and the dual p110α/p110δ inhibitor AZD8835. The effects of single drugs and of several drug combinations were analyzed to assess cytotoxicity by MTT assays, cell cycle by flow cytometry , apoptosis by caspase 3/7 assay and Western blot, as well as the phosphorylation status of the pathway. BYL719 treatment resulted in G1 phase cell cycle arrest and apoptosis. BYL719 administered in combination with CAL-101, for 48 h and 72h, decreased cell viability and induced apoptosis in a marked synergistic manner. Taken together, our findings indicate that BYL719, either alone or in combination with p110δ CAL-101inhibitor, may be an efficient treatment for human rhabdomyosarcoma cells that have aberrant upregulation of the PI3K signaling pathway for their proliferation and survival

The Cytotoxic Effect of Curcumin in Rhabdomyosarcoma Is Associated with the Modulation of AMPK, AKT/mTOR, STAT, and p53 Signaling

Approximately 7% of cancers arising in children and 1% of those arising in adults are soft tissue sarcomas (STS). Of these malignancies, rhabdomyosarcoma (RMS) is the most common. RMS survival rates using current therapeutic protocols have remained largely unchanged in the past decade. Thus, it is imperative that the main molecular drivers in RMS tumorigenesis are defined so that more precise, effective, and less toxic therapies can be designed. Curcumin, a common herbal supplement derived from plants of the Curcuma longa species, has an exceptionally low dietary biotoxicity profile and has demonstrated anti-tumorigenic benefits in vitro. In this study, the anti-tumorigenic activity of curcumin was assessed in rhabdomyosarcoma cell lines and used to identify the major pathways responsible for curcumin’s anti-tumorigenic effects. Curcumin treatment resulted in cell cycle arrest, inhibited cell migration and colony forming potential, and induced apoptotic cell death. Proteome profiler array analysis demonstrated that curcumin treatment primarily influenced flux through the AKT-mammalian target of rapamycin (mTOR), signal transducer and activator of transcription (STAT), AMP-dependent kinase (AMPK), and p53 associated pathways in a rhabdomyosarcoma subtype-specific manner. Thus, the strategic, combinational therapeutic targeting of these pathways may present the best option to treat this group of tumors

Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia

Nuclear phospholipase Cβ1 interactome: a morphological and proteomic approach

Inositide-dependent phospholipase Cβ1 (PI-PLCβ1b) has two isoforms generated by alternative splicing (PI-PLCβ1a and PI-PLCβ1b). In murine erythroleukemia (MEL) cells both the isoforms are present within the nucleus, but PI-PLCβ1b is exclusively nuclear. Our group has demonstrated that PI-PLCβ1 nuclear localisation is crucial for its function, although the mechanism by which PI-PLCβ1 is imported into the nucleus has never been carefully investigated. The purpose of the present study was to get more insights on the protein interactome of PI-PLCβ1b, namely the proteins present in the nucleus. Immuno-affinity purification coupled with tandem mass spectrometry analysis have been used to purify and identify PI-PLCβ1b interaction binding partners from Friend’s erythroleukemia isolated nuclei. Gene ontology and protein-protein interaction network were performed to analyze data. Some interactions were already characterized, such as the binding with the splicing factor SRp20 and the lamin B. Among the proteins identified, the binding of eEF1A and prohibitin 2 with PI-PLCβ1b was confirmed by western blot analysis. Of particular interest was the identification of importin a, importin b1 and Ran, which interact with PI-PLCβ1b. These proteins are believed to be involved in the import mechanism from the cytoplasm to the nucleus. Further analysis by overexpressing both wild type and cytoplasmatic mutant of PI-PLCβ1, suggests that importin b1 is responsible for the localisation of PI-PLCβ1b in the nucleus, giving new insight into the mechanism of trafficking of this signaling molecule