Academic literature on the topic 'P13K signalling'

Previous investigations have demonstrated that protocatechuic acid (PCA) provides anti-tumour properties in different tumour cell types. It does, however, have an unknown cause on osteosarcoma cells. In this investigation, the underlying mechanism of the effect of PCA on osteosarcoma cells (MNNG or HOS) was investigated and established. The viability of the cell was assessed with the MTT test. Acridine orange/ethidium bromide staining and Western blot analysis were conducted for assessment of cell apoptosis. Western blot analysis was identified for cell cycle progression. In addition to establishing the above findings, the Western blot analysis demonstrated that PCA mediated osteosarcoma cellular apoptosis by triggering the apoptotic pathway of Caspase-9. Additionally, we found that PCA considerably stimulated osteosarcoma cell apoptosis and arrest of cell cycle proliferation by controlling a pathway involving P13K/Akt/ROS signalling. In short, we observed that PCA prevented the advancement of osteosarcoma through the stimulation of apoptosis in osteosarcoma cells. The mechanism underlying this study also showed that PCA generated effective anti-tumour activity on osteosarcoma cells by controlling the signalling pathways of P13K/Akt/ROS.

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

Plant-derived flavonoids are considered natural nontoxic chemo-preventers and have been widely studied for cancer treatment in recent decades. Mostly all flavonoid compounds show significant anti-inflammatory, anticancer and antioxidant properties. Kaempferol (Kmp) is a well-studied compound and exhibits remarkable anticancer and antioxidant potential. Kmp can regulate various cancer-related processes and activities such as cell cycle, oxidative stress, apoptosis, proliferation, metastasis, and angiogenesis. The anti-cancer properties of Kmp primarily occur via modulation of apoptosis, MAPK/ERK1/2, P13K/Akt/mTOR, vascular endothelial growth factor (VEGF) signalling pathways. The anti-cancer property of Kmp has been recognized in several in-vivo and in-vitro studies which also includes numerous cell lines and animal models. This flavonoid possesses toxic activities against only cancer cells and have restricted toxicity on healthy cells. In this review, we present extensive research investigations about the therapeutic potential of Kmp in the management of different types of cancers. The anti-cancer properties of Kmp are discussed by concentration on its capability to target molecular-signalling pathway such as VEGF, STAT, p53, NF-κB and PI3K-AKT signalling pathways. The anti-cancer property of Kmf has gained a lot of attention, but the accurate action mechanism remains unclear. However, this natural compound has a great pharmacological capability and is now considered to be an alternative cancer treatment.

e22212 Background: PI3Kinase is an important target for new oncology therapies, since it is central to several cell signalling pathways associated with proliferation and survival. Many pharmaceutical companies have PI3K inhibitors in their drug development pipelines all of which are in early phase trials. Activating mutations in exons 9 and 20 of the PI3KCA oncogene have been observed in a number of important cancer types including: gastrointestinal, breast, liver, lung and genito-urinary cancers. The presence of such mutations may prove useful as companion diagnostic biomarkers for prediction of response to therapy. Methods: Source BioScience (Nottingham, UK) tested three different methods for the detection of PI3K mutations in archival tumour samples, in their CPA and GLP accredited laboratories. Eighty formalin-fixed paraffin-embedded (‘FFPE') samples from histologically confirmed breast cancer samples were tested by the new DxS P13K Mutation Test Kit using real-time PCR, by pyrosequencing (Qiagen), and by conventional bidirectional capillary sequencing. Tumour content was evaluated by pathology review. Results: The results of the comparison showed that the DxS test using rtPCR technology was easy to perform, convenient and robust. The DxS test had a high degree of sensitivity provided there was more than 20% tumour content in the test sample as determined by histology and pathology review. Conclusions: In conclusion, the DxS test is a valuable tool for detecting PI3K mutations in FFPE samples from breast cancer patients. No significant financial relationships to disclose.

p130 was originally identified as an Ins(1,4,5)P3-binding protein similar to phospholipase C-∆ but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P3-mediated Ca2+ signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1p130). In vitro, p130 bound Ins(1,4,5)P3 with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1p130 cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P3. Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca2+ signalling. When fura-2-loaded COS-1p130 cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca2+ concentration, observed in control cells, was inhibited in COS-1p130. This inhibition was not accompanied by the decreased production of Ins(1,4,5)P3; the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P3 could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P3-mediated Ca2+ signalling.

A novel family of signalling regulators, Tribbles (Trb), have been recently identified and characterised. They possess a single kinase like domain but are catalytically inactive. Numerous interacting protein partners of Tribbles have been identified; they have been highlighted to play a role in many cellular and disease processes. Gab (Grb2 associated binder) proteins play a role in signalling events induced by receptor tyrosine kinases (RTKs). Gabl is a prototypic member of this family, which has been shown to have an important function in the mechanism ofPI3K/Akt activation. I demonstrated that Gab I and Trb2 interact, a protein-protein interaction that has not been reported previously. Based on the role of Gabl and Trb proteins in the regulation of P13K/Akt signalling, I examined the Gabl-Trb2 interaction in the wider context ofP13K and AktlPKB signalling. Subsequently, I demonstrated the Gabl-Trb2 protein interaction is regulated by PI3K1 Akt signalling. The cellular actin cytoskleton, functions in the control of cell motility and proliferation, morphology, and also cell-cell communication. Thus, it is inevitable that abnormalities in its regulation result in disease processes. A role for Gab 1 has been demonstrated previously in regulation of the actin cytoskeleton and membrane ruffles in response to RTK activation. The role of Gab! and Trb2, both individually and together in the regulation of membrane ruffling and actin cytoskeleton arrangement was investigated. Novel findings are presented that implicate a role for Trb2 in cellular morphology, and initial investigations were conducted to begin elucidating the potential signaling pathways involved. Overall this study demonstrates a role for Gab 1- Trb2 interaction in PI3K1 Akt signalling and cellular morphology. P13K signalling and actin cytoskeleton dynamics play a central role in many vascular disease processes. Thus, it is possible that investigations into Trb2's role in such processes may result in the identification of novel potential therapeutic strategies. xx

Abstract Adhesive interactions between cells and extracellular matrix proteins play a vital role in biological processes such as cell proliferation, differentiation and survival. Integrins comprise a major family of cell surface receptors that mediate these interactions. Integrin engagement triggers adhesion-dependent intracellular signalling cascades that include the phosphorylation of tyrosines in intracellular signalling proteins. Integrin-dependent signals act in concert with signals from growth factors and other signalling receptors. The objective of this thesis was to study how cell adhesion and growth factors interact with intracellular components to regulate cell behavior in normal and transformed cells. One of the main proteins phosphorylated following integrin ligation in several different cell types is the docking protein p130Cas (Cas), which is tyrosine phosphorylated after stimulation of cells with low concentrations of epidermal growth factor (EGF). Tyrosine-phosphorylated Cas associates with an adapter protein c-Crk, the main binding protein for Cas, suggesting a novel role for EGF in Cas signalling. The interaction of cells with a variety of agonists such as growth factors and integrin ligation results in stimulation of mitogen-activated protein kinases (MAPKs), which control the expression of genes important for many cell functions. Expression of Cas and Crk induces activation of C-Jun N-terminal kinases (JNKs), which are members of MAPK family. JNK activation induced by integrin ligand binding is blocked by the expression of a dominant-negative mutant of Cas or Crk demonstrating an important role for the Cas-Crk complex in integrin-mediated JNK activation. The proto-oncogene product p120Cbl (Cbl) was identified as the main tyrosine-phosphorylated protein following integrin ligation in hematopoietic cells of myeloid lineage. Tyrosine-phosphorylated Cbl interacts with and activates other signalling proteins, such as Src tyrosine kinase and phosphatidylinositol 3"-kinase (PI 3-kinase), thereby mediating adhesion-dependent signals in hematopoietic cells. Unlike the cellular Cbl, the transforming mutants of Cbl were tyrosine-phosphorylated in an adhesion-independent manner and interacted with and activated signalling molecules both in suspended and in adherent cells. Further, the oncogenic forms of Cbl induced anchorage-independent but serum-dependent proliferation of cells. These results support the view that transformation by Cbl results from constitutive activation of integrin-dependent rather than growth factor-dependent signalling events.

Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008.
Introduction: Skeletal muscle atrophy is a mitigating complication that is characterized by a reduction in muscle fibre cross-sectional area as well as protein content, reduced force, elevated fatigability and insulin resistance. It seems to be a highly ordered and regulated process and signs of this condition are often seen in inflammatory conditions such as cancer, AIDS, diabetes and chronic heart failure (CHF). It has long been understood that an imbalance between protein degradation (increase) and protein synthesis (decrease) both contribute to the overall loss of muscle protein. Although the triggers that cause atrophy are different, the loss of muscle mass in each case involves a common phenomenon that induces muscle proteolysis. It is becoming evident that interactions among known proteolytic systems (ubiquitin-proteosome) are actively involved in muscle proteolysis during atrophy. Factors such as TNF-α and ROS are elevated in a wide variety of chronic inflammatory diseases in which skeletal muscle proteolysis presents a lethal threat. There is an increasing body of evidence that implies TNF-α may play a critical role in skeletal muscle atrophy in a number of clinical settings but the mechanisms mediating its effects are not completely understood. It is also now apparent that the transcription factor NF-κB is a key intracellular signal transducer in muscle catabolic conditions. This study investigated the various proposed signalling pathways that are modulated by increasing levels of TNF-α in a skeletal muscle cell line, in order to synthesize our current understanding of the molecular regulation of muscle atrophy. Materials and Methods: L6 (rat skeletal muscle) cells were cultured under standard conditions where after reaching ± 60-65% confluency levels, differentiation was induced for a maximum of 8 days. During the last 2 days, myotubes were incubated with increasing concentrations of recombinant TNF-α (1, 3, 6 and 10 ng/ml) for a period of 40 minutes, 24 and 48 hours. The effects of TNF-α on proliferation and cell viability were measured by MTT assay and Trypan Blue exclusion technique. Morphological assessment of cell death was conducted using the Hoechst 33342 and Propidium Iodide staining method. Detection of apoptosis was assessed by DNA isolation and fragmentation assay. The HE stain was used for the measurement of cell size. In order to determine the source and amount of ROS production, MitoTracker Red CM-H2 X ROS was utilised. Ubiquitin expression was assessed by immunohistochemistry. PI3-K activity was calculated by using an ELISA assay and the expression of signalling proteins was analysed by Western Blotting using phospho-specific and total antibodies. Additionally, the antioxidant Oxiprovin was used to investigate the quantity of ROS production in TNF-α-induced muscle atrophy. Results and Discussion: Incubation of L6 myotubes with increasing concentrations of recombinant TNF-α revealed that the lower concentrations of TNF-α used were not toxic to the cells but data analysis of cell death showed that 10 ng/ml TNF-α induced apoptosis and necrosis. Long-term treatment with TNF-α resulted in an increase in the upregulation of TNF- α receptors, specifically TNF-R1. The transcription factors NF-κB and FKHR were rapidly activated thus resulting in the induction of the ubiquitin-proteosome pathway. Activation of this pathway produced significant increases in the expression of E3 ubiquitin ligases MuRF-1 and MAFbx. Muscle fibre diameter appeared to have decreased with increasing TNF-α concentrations in part due to the suppressed activity of the PI3-K/Akt pathway as well as significant reductions in differentiation markers. Western blot analysis also showed that certain MAPKs are activated in response to TNF-α. No profound changes were observed with ROS production. Finally, the use Oxiprovin significantly lowered cell viability and ROS production. These findings suggest that TNF-α may elicit strong catabolic effects on L6 myotubes in a dose and time dependent manner. Conclusion: These observations suggest that TNF-α might have beneficial effects in skeletal muscle in certain circumstances. This beneficial effect however is limited by several aspects which include the concentration of TNF-α, cell type, time of exposure, culture conditions, state of the cell (disturbed or normal) and the cells stage of differentiation. The effect of TNF-α can be positive or negative depending on the concentration and time points analysed. This action is mediated by various signal transduction pathways that are thought to cooperate with each other. More understanding of these pathways as well as their subsequent upstream and downstream constituents is obligatory to clarify the central mechanism/s that control physiological and pathophysiological effects of TNF-α in skeletal muscle.

The B cell receptor (BCR) is a major regulator of B cell development, activation, and cell death. The misregulation of these processes results in autoimmunity and B cell lymphoma. Engagement of the BCR activates multiple signalling pathways that are essential for normal B cell responses. However, the roles of individual signalling pathways in mediating these responses is not completely understood. Activation of the phosphatidylinositol-3-kinase (PI3K) pathway is important for the proper regulation of B cell survival and development. In this thesis I have investigated the regulation and targets of the PI3K pathway in B cells. A putative target of the PI3K pathway is β-catenin, a transcriptional activator with important roles in early development. I found that the BCR regulates β-catenin levels via the activation of the phospholipase-C/protein kinase C/glycogen synthase kinase-3 pathway and is partially dependent on PI3K. Signalling by the BCR also activates the Rap GTPases, putative antagonists of Ras-mediated signalling. Ras can activate both the Raf-1/ERK 1/21/2 pathway and the PI3K/Akt pathway, pathways that can promote cell survival. I investigated whether Rap activation limits the activation of either of these pathways. I found that Rap activation had no effect on the BCR-induced activation of the Raf-1/ERK1/21/2 pathway. However, endogenous Rap limited the BCRinduced activation of the POK/Akt pathway, opposed the Akt-mediated inhibition of the FKHR/p27[sup Kip1] pro-apoptotic module, and enhanced cell death. Therefore, Rap can oppose the pro-survival role of the PI3K pathway. BCR-induced activation of the PI3K effector Akt leads to changes in gene transcription through the Akt-mediated activation or inhibition of transcription factors which can promote cell survival. I used cDNA microarray technology to identify novel gene targets of Akt in B cells. I found that Akt activation resulted in changes in expression of genes involved in the regulation of cell cycle progression, cell adhesion and apoptosis. In summary, I have identified novel targets and novel mechanisms of regulating the PI3K/Akt pathway in B cells. This work will hopefully contribute to the overall understanding of how the PI3K pathway affects the regulation of B cell development, activation, and survival.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

The Class IB phosphatidylinositol 3-kinase (PI3K) enzyme, PI3Kγ, is activated and recruited to the plasma membrane in response to G protein-coupled receptor stimulation. Upon activation, the lipid-kinase activity and downstream signalling cascades initiated by PI3Kγ lead to cytoskeletal rearrangements and the formation of a leading edge for the induction of directed cell migration. PI3Kγ consists of the catalytic subunit p110γ, which forms a mutually exclusive heterodimer with one of two regulatory adaptor subunits, p84 or p101. Although expressed by most cells in the organism, PI3Kγ subunits are expressed at highest levels in motile haematopoietic cells, where the regulation of PI3Kγ signalling is critical to controlling and maintaining coordinated cell migration during immune responses. Consistent with a central role in leukocyte chemotaxis, innate and adaptive immune cell subsets from p110γ-deficient mice have been shown to exhibit migration defects in vitro and in vivo. Furthermore, the aberrant expression of PI3Kγ subunits and dysregulation of PI3Kγ signalling pathways has been shown to contribute to pathologies such as cancer and autoimmunity where enhanced cell migration promotes disease progression. Despite this, the mechanistic basis for PI3Kγ signal regulation is not well understood, particularly with respect to the distinct contributions of the individual regulatory adaptor subunits, p84 and p101. Many PI3Kγ-dependent cell functions have been elucidated experimentally using p110γ- and p101-deficient genetically-modified mouse strains and the PI3Kγ-selective inhibitor, AS605240. However, detailed functional data regarding p84 is lacking due to the absence of a p84-deficient mouse strain and limited availability of high quality p84-specific reagents. Three major research goals were addressed in the present study to improve our understanding of the role of p84 in PI3Kγ lipid-kinase signalling and its implication in PI3Kγ-dependent cell migration. The first goal was to examine the phosphorylation status of p84 during PI3Kγ signalling and assess the role of identified regulatory phosphorylation sites for p84 function using the mammary epithelial carcinoma model cell line, MDA.MB.231. Data presented in this thesis demonstrate that in contrast to the p110γ and p101 subunits that promote the migration and metastasis of carcinoma cells, the p84 adaptor protein has tumour suppressor function in vitro and in vivo, which was determined to be dependent on a potential phosphorylation site within p84, Thr607. It was found that Thr607 was required for p84 to form an inducible heterodimer with p110γ (after initial PI3Kγ signal activation) in a complex sequestered from active signalling at the membrane. This Thr607-dependent p84/p110γ dimerisation may therefore represent a novel mechanism of negative PI3Kγ signal regulation that limits the migration and metastasis of cancer cells. Next, the contribution of p84 to PI3Kγ-dependent immune cell function was determined through the generation and characterisation of a novel p84-deficient mouse (Pik3r6⁻ʹ⁻) using CRISPR gene-editing technology. Pik3r6⁻ʹ⁻ mice were characterised in the context of immune cell development, activation and migration in a variety of haematopoietic cell subsets. It was shown that Pik3r6⁻ʹ⁻ mice develop normally with respect to lymphoid organ and circulating leukocyte populations at homeostasis. However upon stimulation, neutrophils from Pik3r6⁻ʹ⁻ mice display reduced migration in response to GPCR agonists in vitro and in a murine model of inflammatory autoimmunity (experimental autoimmune encephalomyelitis; EAE), it was found that activated Th lymphocytes display impaired trafficking and reduced infiltration to inflammatory sites. The final goal was to develop and optimise a proteomic platform to investigate and compare the proteomes of migratory CD4⁺ lymphocytes isolated from tissues at different stages of inflammatory disease progression using experimental autoimmune encephalomyelitis as a model. An isotope-coded protein-labelling (ICPL) approach was developed and optimised to assess the proteomes of CNS-infiltrating CD4⁺ lymphocytes during disease progression in two models of EAE; chronic MOG₃₅₋₅₅-induced EAE and relapsing-remitting PLP₁₃₉₋₁₅₁-induced EAE. This study identified differentially regulated proteins related to immune cell function and represented a initial feasibility study to verify the validity of ICPL as an approach to examine the differential proteomes of wildtype and p84-deficient migratory CD4⁺ lymphocytes during inflammatory disease. Collectively, the data presented in this thesis represent the identification and characterisation of novel roles for p84 within PI3Kγ lipid-kinase signalling during both the regulation of cell migration in carcinoma cells and in haematopoietic cells during immune responses. In addition to furthering the understanding of the unique roles for p84 within PI3Kγ signal regulation, the generation of a p84-deficient mouse strain constitutes an important tool to further experimental research in this area.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2015.

Research Doctorate - Doctor of Philosophy (PhD)
Aberrant activation of survival-signaling pathways causes uncontrolled cell proliferation and resistance to apoptosis, and plays an important role in cancer development, progression, and resistance to treatment (Courtney et al., 2010; Ferte et al., 2010). In colorectal cancer (CRC), activation of the phosphatidylinositol 3-kinase (PI3K) pathway is of particular importance, in that many common genetic and epigenetic anomalies in the disease, such as amplification of epidermal growth factor (EGF) receptor, activating mutations in KRAS, and loss of phosphate and tensin homolog deleted on chromosome 10 (PTEN), converge on activation of PI3K signalling (Colakoglu et al., 2008). Moreover, activating mutations of PIK3CA, the gene encoding the catalytic subunit of PI3K, is found in up to 40% of colon cancers (Colakoglu et al., 2008; De Roock et al., 2011). In melanoma, identification of activating mutations in BRAF as the major cause of constitutive activation of the mitogen activated protein kinase (MAPK) pathway has led to successful development of mutant BRAF-specific inhibitors in the treatment of the disease (Chapman et al., 2011; Davies et al., 2002; Houslay, 2011; Ribas and Flaherty, 2011). However, primary and acquired resistance, which is commonly associated with activation of other survival pathways, in particular, the phosphatidylinositol 3-kinase (PI3K) signaling pathway, remains a major obstacle in the quest for curative treatment (Jiang et al., 2011; Karreth et al., 2011; Paraiso et al., 2011; Poulikakos and Rosen, 2011). Indeed, activation of PI3K signaling has been shown to cooperate with mutant BRAF in melanomagenesis using in vivo models (Cheung et al., 2008; Dankort et al., 2009). Activation of PI3K signaling is negatively regulated by three classes of inositol polyphosphate phosphatases (Fedele et al., 2010; Gewinner et al., 2009; Kisseleva et al., 2002). The inositol polyphosphate 3-phosphatase (3-phosphatase) PTEN dephosphorylates the 3-position of PI(3,4,5)P₃ to generate PI(4,5)P₂ (Carracedo et al., 2011; Ma et al., 2008), whereas 5-phosphatases, such as Src homology 2-containing inositol 5- phosphatase (SHIP) and phosphatidylinositol 4,5-Bisphosphate 5- Phosphatase (PIB5PA)/proline-rich inositol polyphosphate phosphatase (PIPP) dephosphorylate the 5-position to produce PI(3,4)P₂ (Ooms et al., 2006; Ye et al., 2013a). PI(3,4)P₂ is in turn subjected to dephosphorylation by inositol polyphosphate 4-phosphatase type I (INPP4A) and type II (INPP4B) at the 4-position to generate PI(3)P, thus terminating PI3K signaling (Fedele et al., 2010; Gewinner et al., 2009; Hodgson et al., 2011). Interestingly, despite INPP4B tumor suppressive role in some other tissues, in this study we found that inositol polyphosphate 4-phosphatase type II (INPP4B) functions as an oncogenic regulator in human colon cancer and melanoma. While INPP4B is upregulated in two cancers and its high expression is associated with poor patient survival, INPP4B knockdown blocks activation of PI3K downstream signaling, inhibits proliferation, undermines survival of colon cancer and melanoma cells, and retards cancer growth in a xenograft model. Conversely, overexpression of INPP4B causes increased proliferation and anchorage-independent growth in normal colon epithelial cells and melanocytes. However, INPP4B regulates PI3K signalling pathway in two cancers by different mechanisms. It plays an important role for maintaining cellular PI(3,4,5)P₃ and PI(3,4)P₂ levels in colon cancer whereas PI(3)P levels in melanoma cells. Also, the increase in INPP4B is primarily due to Ets-1-mediated transcriptional upregulation in colon cancer cells, whereas a posttranscriptional increase via reduction of miRNA-494 and miRNA-599 in melanoma.