Quantitative analysis of insulin-like growth factor 2 receptor and insulin-like growth factor binding proteins to identify control mechanisms for insulin-like growth factor 1 receptor phosphorylation

Experimental results for the determination of internalization rates for IGF1-IGF1R, IGF2-IGF1R, IGF2-IGF2R. (PDF 162 kb

Transcriptional regulation of the IGF signaling pathway by amino acids and insulin-like growth factors during myogenesis in Atlantic salmon

The insulin-like growth factor signalling pathway is an important regulator of skeletal muscle growth. We examined the mRNA expression of components of the insulin-like growth factor (IGF) signalling pathway as well as Fibroblast Growth Factor 2 (FGF2) during maturation of myotubes in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The transcriptional regulation of IGFs and IGFBP expression by amino acids and insulin-like growth factors was also investigated. Proliferation of cells was 15% d(-1) at days 2 and 3 of the culture, increasing to 66% d(-1) at day 6. Three clusters of elevated gene expression were observed during the maturation of the culture associated with mono-nucleic cells (IGFBP5.1 and 5.2, IGFBP-6, IGFBP-rP1, IGFBP-2.2 and IGF-II), the initial proliferation phase (IGF-I, IGFBP-4, FGF2 and IGF-IRb) and terminal differentiation and myotube production (IGF2R, IGF-IRa). In cells starved of amino acids and serum for 72 h, IGF-I mRNA decreased 10-fold which was reversed by amino acid replacement. Addition of IGF-I and amino acids to starved cells resulted in an 18-fold increase in IGF-I mRNA indicating synergistic effects and the activation of additional pathway(s) leading to IGF-I production via a positive feedback mechanism. IGF-II, IGFBP-5.1 and IGFBP-5.2 expression was unchanged in starved cells, but increased with amino acid replacement. Synergistic increases in expression of IGFBP5.2 and IGFBP-4, but not IGFBP5.1 were observed with addition of IGF-I, IGF-II or insulin and amino acids to the medium. IGF-I and IGF-II directly stimulated IGFBP-6 expression, but not when amino acids were present. These findings indicate that amino acids alone are sufficient to stimulate myogenesis in myoblasts and that IGF-I production is controlled by both endocrine and paracrine pathways. A model depicting the transcriptional regulation of the IGF pathway in Atlantic salmon muscle following feeding is proposed.Publisher PDFPeer reviewe

Receptors for Insulin-Like Growth Factor-2 and Androgens as Therapeutic Targets in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) occurs in 10-15% of all breast cancer patients, yet it accounts for about half of all breast cancer deaths. There is an urgent need to identify new antitumor targets to provide additional treatment options for patients afflicted with this aggressive disease. Preclinical evidence suggests a critical role for insulin-like growth factor-2 (IGF2) and androgen receptor (AR) in regulating TNBC progression. To advance this work, a panel of TNBC cell lines was investigated with all cell lines showing significant expression of IGF2. Treatment with IGF2 stimulated cell proliferation in vitro (p < 0.05). Importantly, combination treatments with IGF1R inhibitors BMS-754807 and NVP-AEW541 elicited significant inhibition of TNBC cell proliferation (p < 0.001). Based on Annexin-V binding assays, BMS-754807, NVP-AEW541 and enzalutamide induced TNBC cell death (p < 0.005). Additionally, combination of enzalutamide with BMS-754807 or NVP-AEW541 exerted significant reductions in TNBC proliferation even in cells with low AR expression (p < 0.001). Notably, NVP-AEW541 and BMS-754807 reduced AR levels in BT549 TNBC cells. These results provide evidence that IGF2 promotes TNBC cell viability and proliferation, while inhibition of IGF1R/IR and AR pathways contribute to blockade of TNBC proliferation and promotion of apoptosis in vitro

DAF-16/FoxO in Caenorhabditis elegans and its role in metabolic remodeling

DAF-16, the only forkhead box transcription factors class O (FoxO) homolog in Caenorhabditis elegans, integrates signals from upstream pathways to elicit transcriptional changes in many genes involved in aging, development, stress, metabolism, and immunity. The major regulator of DAF-16 activity is the insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway, reduction of which leads to lifespan extension in worms, flies, mice, and humans. In C. elegans daf-2 mutants, reduced IIS leads to a heterochronic activation of a dauer survival program during adulthood. This program includes elevated antioxidant defense and a metabolic shift toward accumulation of carbohydrates (i.e., trehalose and glycogen) and triglycerides, and activation of the glyoxylate shunt, which could allow fat-to-carbohydrate conversion. The longevity of daf-2 mutants seems to be partially supported by endogenous trehalose, a nonreducing disaccharide that mammals cannot synthesize, which points toward considerable differences in downstream mechanisms by which IIS regulates aging in distinct groups

Signalling pathways and gene expression profiles in prostate cancer

In general, cancer, encompassing prostate cancer (PCa), is a disease that utilises signalling pathways to progress through the uncontrolled proliferation of cancerous cells. Although the mechanisms of how the cells evade intrinsic or extrinsic signals of death and keep on dividing is not completely understood, there is a plethora of evidence that point to certain signalling molecules that are crucial conveyors of the fine tuning that slightly differs in cancer in comparison to control states. The present chapter provides a detailed description of the key regulators of PCa cell life and unveils their closely communicating proteins that aid in the fine tuning of the cancerous state

RNA microarray analysis in prenatal mouse cochlea reveals novel IGF-I target genes: implication of MEF2 and FOXM1 transcription factors

Background: Insulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness. Methodology/Principal Findings: A combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1(-/-) null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1(-/-) and Igf1(+/+) transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1(+/+) mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27(Kip1). Conclusions/Significance: We have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I

Role of insulin receptor isoform A in breast cancer

Insulin like growth factor II (IGF-II) binds to Insulin Receptor isoform A (IR-A) to sustain cell growth and proliferation. This autocrine loop exists in many human carcinomas and provides an additional proliferation and survival pathway to the type 1 insulin-like growth factor (IGF-1R) signalling pathway. In addition, activation of the IGF-II/IR-A autocrine loop provides a mechanism of resistance drugs target the type 1 insulin-like growth factor (IGF- 1R). However, the mechanism of how IGF-II/IR-A differentiates itself from the IGF-II/IGF- 1R pathway is still unclear. Additionally, a novel phosphorylation site Thr¹¹⁴⁸ (numbered in IR-A) of IR was found in response to insulin stimulation and its phosphorylation in the absence of any Tyr¹¹⁴⁶, Tyr¹¹⁵⁰, and Tyr¹¹⁵¹ phosphorylation. We thus hypothesis that the The phosphorylation of Thr¹¹⁴⁸ inhibits the phosphorylation of Tyr¹¹⁴⁶, Tyr¹¹⁵⁰, and Tyr¹¹⁵¹ of the activation loop, which is possibly due to steric hindrance and electrostatic repulsion of the phosphate group on Thr¹¹⁴⁸ preventing interaction of the activation loop with the tyrosine kinase. The key areas of investigation included: 1. Investigate the role of Thr¹¹⁴⁸ phosphorylation in IR-A by overexpress the wild-type IR-A and its mutants Thr¹¹⁴⁸Ala and Thr¹¹⁴⁸Asp in R⁻ (Mouse fibroblast cells with IGF- 1R KO). 2. Knock down the IR and IGF-1R separately in MDA-MB-231 and MCF-7 cells using inducible expressed miR30 shRNA and measure their characteristics (proliferation, migration, epithelial–mesenchymal transition) in response to insulin, IGF-I, and IGFII stimulation separately. 3. Quantitative phosphoproteomics was conducted to compare the insulin/IR-A and IGFII/ IR-A signalling pathway with MDA MB 231 IGF-1RKD cell. The key findings from this work included: included: 1. Thr¹¹⁴⁸Ala and Thr¹¹⁴⁸Asp inhibit the insulin sensitivity of IR-A, which indicate the Thr¹¹⁴⁸ phosphorylation inhibits the IR activation. 2. IRKD has no effect on proliferation of MCF-7 cells and the migration of MDA-MB- 231 cells, but increases the proliferation of MDA-MB-231 cells. 3. IGF-1RKD inhibits the proliferation of both MCF-7 cells and MDA-MB-231 cells, but increases the migration of MDA-MB-231 cells. 4. IGF-1R increases the insulin sensitivity of MDA-MB-231 cells, shown by the Akt activation. 5. Insulin and IGF-II are confirmed to induce different signalling pathways confirmed by quantitative phosphoproteomics study. IGF-II is preferentially regulates the migration and stemness of MDA-MB-231 IGF-1RKD cells. Many markers are identified but verifications are still needed. Drug developed on the verified markers can be used to treat the patients who are resistant to anti-IGF-1R inhibitor.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

From membrane to nucleus: New roles and functions of SUMOylated IGF-1R and EGFR

Cell surface receptor tyrosine kinases (RTKs) role in cell signaling have been studied for decades and their role in cancer progression are undisputable. The insulin-like growth factor 1 receptor, IGF-1R, has been demonstrated to play a critical part in tumorigenesis; downregulation of the IGF-1R in tumor xenografts results in complete tumor regression. Previously, RTK research has focused on the canonical signaling pathways activated by ligand binding at the plasma membrane. However, strong evidence keeps emerging that several RTKs have a second functionally mechanism, inside the cell nucleus, where the receptors reside after ligand stimulation. The aim of this thesis was to elucidate the function of recently discovered nuclear IGF-1R as well as to investigate its nuclear translocation pathway. Since it was previously shown that SUMOylation of the IGF-1R is essential for its nuclear translocation we also set out to investigate SUMO modification of the epidermal growth factor receptor (EGFR). In paper I, we present a functional role for nuclear IGF-1R in gene transcription. Inside the nucleus, IGF-1R functions as a co-activator to LEF-1/TCF transcription factor. Nuclear IGF-1R enhances transcription of cyclin D1 and axin2, and we show that it is enriched in the cyclin D1 promoter region. In the following study, paper II, we propose a pathway by which IGF-1R is transported into the nucleus. IGF-1R is transported along microtubules via the dynactin transportation complex, to the nuclear pore where it is transferred to importin-β which guides the receptor to the nuclear pore complex protein RanBP2, which further assists the receptor into the cell nucleus in a RanGTPase dependent manner. Inhibition or obstruction of any of these components results in a reduction in nuclear IGF-1R. Further, we suggest that RanBP2 is the SUMO E3 ligase in IGF-1R SUMOylation and we show that SUMO-1 modification of the receptor is also important for its stability. In paper III, we demonstrate that the EGFR is SUMOylated and propose five lysine residues as SUMO-1 targets which were identified by two different mass spectrometry strategies. One of these residues, lysine 37, came up as a suggested target in both mass spectrometry methods. EGFR mutated in this site – EGFR-K37R – causes a decrease in protein levels as well as transcriptional activity of cyclin D1 and c-myc, two target genes of nuclear EGFR. To summarize, our data shows (I) a pathway by which nuclear IGF-1R is being transported and the functional importance of nuclear IGF-1R as a co-activator in transcription and (II) that the EGFR is also SUMOylated and might play a role in its transcriptional activity. Together these results may unravel new mechanisms for IGF-1R and EGFR that have implications in carcinogenesis

EMPLOYING QUANTITATIVE SYSTEMS PHARMACOLOGY TO CHARACTERIZE DIFFERENCES IN IGF1 AND INSULIN SIGNALING PATHWAYS IN BREAST CANCER

Insulin and insulin-like growth factor I (IGF1) have been shown to influence cancer risk and progression through poorly understood mechanisms. Here, new insights on the mechanisms of differential MAPK and Akt activation are revealed by an iterative quantitative systems pharmacology approach. In the first iteration, I combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array of 21 breast cancer cell lines treated with a time course of IGF1 and insulin, I constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. Both of the knock-down perturbations caused phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro. In the second iteration, mechanistic representation of IGF1 and insulin dual signaling cascades by a set of ODEs is generated by rule-based modeling. The mechanistic network modeling provided a framework to elucidate experimental targets downstream of two receptors, which were treated as indistinguishable in previous models. The model included cascades of both mitogen-activated protein kinase (MAPK) and Akt signaling, as well as the crosstalk and feedback loops in between. The parameter perturbation scanning employed for seven different models of seven cell lines yielded new experimental hypotheses on how differential responses of MAPK and Akt originate. Complementary to the first iteration, the results in this part suggested that regulation of insulin receptor substrate 1 (IRS1) is critical in inducing differential MAPK or Akt activation. Compensation and activating feedback mechanisms collectively depressed the efficacy of anti-IGF1R/InsR therapies. With the quantitative systems pharmacologic approach, the networks of signal transduction constructed in this thesis are aimed to discern novel downstream components of the IGF1R/InsR system, and to direct patients with suitable tumor subclasses to efficient personalized clinical interventions