Rosemary O'Connor

Defects in IGF-1 receptor, insulin receptor and IRS-1/2 in Alzheimer's disease indicate possible resistance to IGF-1 and insulin signalling

Insulin like growth factor-1 receptor (IGF-1R) and insulin receptor (IR) signalling control vital growth, survival and metabolic functions in the brain. Here we describe specific and significant alterations in IGF-1R, IR, and their key substrate adaptor proteins IRS-1 and IRS-2 in Alzheimers disease (AD). Western immunoblot analysis detected increased IGF-1R levels, and decreased levels of IGF-1-binding protein-2 (IGFBP-2), a major IGF-1-binding protein, in AD temporal cortex. Increased IGF-1R was observed surrounding and within amyloid-beta (Abeta)-containing plaques, also evident in an animal model of AD, and in astrocytes in AD. However, despite the overall increase in IGF-1R levels, a significantly lower number of neurons expressed IGF-1R in AD, and IGF-1R was aberrantly distributed in AD neurons especially evident in those with neurofibrillary tangles (NFTs). IR protein levels were similar in AD and control cases, however, the IR was concentrated intracellularly in AD neurons, unlike its distribution throughout the neuronal cell soma and in dendrites in control brain. Significant decreases in IRS-1 and IRS-2 levels were identified in AD neurons, in association with increased levels of inactivated phospho(Ser312)IRS-1 and phospho(Ser616)IRS-1, where increased levels of these phosphoserine epitopes colocalised strongly with NFTs. Our results show that IGF-1R and IR signalling is compromised in AD neurons and suggest that neurons that degenerate in AD may be resistant to IGF-1R/IR signalling.

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology

Studies suggest that activation of phosphoinositide 3‐kinase‐Akt may protect against neuronal cell death in Alzheimers disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho‐Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated‐Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated‐Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3βSer9, tauSer214, mTORSer2448, and decreased levels of the Akt target, p27kip1, were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated‐Akt and PTEN‐containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.

Identification of Domains of the Insulin-Like Growth Factor I Receptor That Are Required for Protection from Apoptosis

Using a series of insulin-like growth factor I (IGF-I) receptor mutants, we have attempted to define domains required for transmitting the antiapoptotic signal from the receptor and to compare these domains with those required for mitogenesis or transformation. In FL5.12 cells transfected with wild-type IGF-I receptors, IGF-I affords protection from interleukin 3 withdrawal but is not mitogenic. An IGF-I receptor lacking a functional ATP binding site provided no protection from apoptosis. However, receptors mutated at tyrosine residue 950 or in the tyrosine cluster (1131, 1135, and 1136) within the kinase domain remained capable of suppressing apoptosis, although such mutations are known to inactivate transforming and mitogenic functions. In the C terminus of the IGF-I receptor, two mutations, one at tyrosine 1251 and one which replaced residues histidine 1293 and lysine 1294, abolished the antiapoptotic function, whereas mutation of the four serines at 1280 to 1283 did not. Interestingly, receptors truncated at the C terminus had enhanced antiapoptotic function. In Rat-1/ c-MycER fibroblasts, the Y950F mutant and the tyrosine cluster mutant could still provide protection from c-Myc-induced apoptosis, whereas mutant Y1250/1251F could not. These studies demonstrate that the domains of the IGF-I receptor required for its antiapoptotic function are distinct from those required for its proliferation or transformation functions and suggest that domains of the receptor required for inhibition of apoptosis are necessary but not sufficient for transformation.

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

Regulation of Insulin-Like Growth Factor Type I (IGF-I) Receptor Kinase Activity by Protein Tyrosine Phosphatase 1B (PTP-1B) and Enhanced IGF-I-Mediated Suppression of Apoptosis and Motility in PTP-1B-Deficient Fibroblasts

ABSTRACT The insulin-like growth factor type I (IGF-I) receptor (IGF-IR), activated by its ligands IGF-I and IGF-II, can initiate several signal transduction pathways that mediate suppression of apoptosis, proliferation, differentiation, and transformation. Here we investigated the regulation of IGF-IR activation and function by protein tyrosine phosphatase 1B (PTP-1B). Coexpression of PTP-1B with a β-chain construct of the IGF-IR (βWT) inhibited IGF-IR kinase activity in fission yeast Schizosaccharomyces pombe, in COS cells, and in IGF-IR-deficient fibroblasts. In both spontaneously immortalized and simian virus 40 T antigen-transformed embryonic fibroblast cell lines derived from PTP-1B knockout mice, IGF-I induced higher levels of IGF-IR autophosphorylation and kinase activity than were induced in PTP-1B-expressing control cells. PTP-1B-deficient cells exhibited enhanced IGF-I-mediated protection from apoptosis in response to serum withdrawal or etoposide killing, as well as enhanced plating efficiency and IGF-I-mediated motility. Reexpression of PTP-1B in spontaneously immortalized fibroblasts resulted in decreased IGF-IR and AKT activation, as well as decreased protection from apoptosis and decreased motility. These findings demonstrate that PTP-1B can regulate IGF-IR kinase activity and function and that loss of PTP-1B can enhance IGF-I-mediated cell survival, growth, and motility in transformed cells.

Insulin-Like Growth Factor I Controls a Mutually Exclusive Association of RACK1 with Protein Phosphatase 2A and β1 Integrin To Promote Cell Migration

ABSTRACT The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit β1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and β1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either β1 integrin or PP2A in a complex at the IGF-IR.

Fluorescence-Based Cell Viability Screening Assays Using Water-Soluble Oxygen Probes

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 μM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (105 to 107 cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability. (Journal of Biomolecular Screening 2003:264-272)

Phosphorylation of RACK1 on Tyrosine 52 by c-Abl Is Required for Insulin-like Growth Factor I-mediated Regulation of Focal Adhesion Kinase

Focal Adhesion Kinase (FAK) activity is controlled by growth factors and adhesion signals in tumor cells. The scaffolding protein RACK1 (receptor for activated C kinases) integrates insulin-like growth factor I (IGF-I) and integrin signaling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phos pho ryl a tion and dephos pho ryl a tion in response to IGF-I. Suppression of RACK1 by small interfering RNA ablates FAK phos pho ryl a tion and reduces cell adhesion, cell spreading, and clonogenic growth. Peptide array and mutagenesis studies localize the FAK binding interface to blades I-III of the RACK1 β-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg-47, Tyr-52, Arg-57, Arg-60, Phe-65, Lys-127, and Lys-130) as key determinants for association with FAK. Mutation of tyrosine 52 alone is sufficient to disrupt interaction of RACK1 with FAK in cells where endogenous RACK1 is suppressed by small interfering RNA. Cells expressing a Y52F mutant RACK1 are impaired in adhesion, growth, and foci formation. Comparative analyses of homology models and crystal structures for RACK1 orthologues suggest a role for Tyr-52 as a site for phos pho ryl a tion that induces conformational change in RACK1, switching the protein into a FAK binding state. Tyrosine 52 is further shown to be phos pho ryl a ted by c-Abl kinase, and the c-Abl inhibitor STI571 disrupts FAK interaction with RACK1. We conclude that FAK association with RACK1 is regulated by phos pho ryl a tion of Tyr-52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signaling.

PBK/TOPK promotes tumour cell proliferation through p38 MAPK activity and regulation of the DNA damage response.

The contribution of the insulin-like growth-factor-I receptor (IGF-IR) to tumour progression is well documented. To identify new mediators of IGF-IR function in cancer, we recently isolated genes differentially expressed in cells overexpressing the IGF-IR. Among these was the serine/threonine kinase PBK/TOPK (PDZ-binding kinase/T-LAK cell-originated protein kinase), previously associated with highly proliferative cells and tissues. Here, we show that PBK is expressed at high levels in tumour cell lines compared with non-transformed cells. IGF-I could induce PBK expression only in transformed cells, whereas epidermal growth factor could induce PBK in non-transformed MCF-10A breast epithelial cells. Suppression of PBK expression using small interfering RNA did not prevent progression through the cell cycle, but caused decreased proliferation over time in culture, and reduced clonogenic growth in soft agarose. PBK knockdown impaired p38 activation after long-term stimulation with different growth factors and reduced DU145 cells motility. Suppressed PBK expression also resulted in an impaired response to DNA damage that was evident by the decreased generation of γ-H2AX, increased DNA damage and decreased cell survival. Taken together, the data indicate that PBK is necessary for appropriate activation and function of the p38 pathway by growth factors. Thus, enhanced expression of PBK may facilitate tumour growth by mediating p38 activation and by helping cells to overcome DNA damage.

Tyrosine 302 in RACK1 Is Essential for Insulin-like Growth Factor-I-mediated Competitive Binding of PP2A and β1 Integrin and for Tumor Cell Proliferation and Migration

Insulin-like growth factor (IGF)-I regulates a mutually exclusive interaction of PP2A and β1 integrin with the WD repeat scaffolding protein RACK1. This interaction is required for the integration of IGF-I receptor (IGF-IR) and adhesion signaling. Here we investigated the nature of the binding site for PP2A and β1 integrin in RACK1. A WD7 deletion mutant of RACK1 did not associate with PP2A but retained some interaction with β1 integrin, whereas a WD6/WD7 mutant lost the ability to bind to both PP2A and β1 integrin. Using immobilized peptide arrays representing the entire RACK1 protein, we identified a common cluster of amino acids (FAGY) at positions 299–302 within WD7 of RACK1 which were essential for binding of both PP2A and β1 integrin to RACK1. PP2A showed a higher level of association with a peptide in which Tyr-302 was phosphorylated compared with an unphosphorylated peptide, whereas β1 integrin binding was not affected by phosphorylation. RACK1 mutants in which either the FAGY cluster or Tyr-302 were mutated to AAAF, or Phe, respectively, did not interact with either PP2A or β1 integrin. These mutants were unable to rescue the decrease in PP2A activity caused by suppression of RACK1 in MCF-7 cells with small interfering RNA. MCF-7 cells and R+ (IGF-IR-overexpressing fibroblasts) expressing these mutants exhibited decreased proliferation and migration, whereas R– cells (IGF-IR null fibroblasts) were unaffected. Taken together, the data demonstrate that Tyr-302 in RACK1 is required for interaction with PP2A and β1 integrin, for regulation of PP2A activity, and for IGF-I-mediated cell migration and proliferation.