IGF-I Receptor Research Articles

A Rare Cause of Recurrent Hypoglycemia - The “Big-IGF-II” Factor

Abstract Introduction: Hormone secreting tumors are a rare cause of hypoglycemia. Here we present an interesting case of paraneoplastic phenomenon resulting in a non-islet cell tumor causing hypoglycemia (NICTH) from a solitary fibrous tumor (SFT). Clinical Case: An 83-year-old female with a history of hypertension presented with recurrent episodes of lightheadedness and falls for one month. Review of systems was negative except for insignificant weight loss. Her vital signs and physical examination were unremarkable. Only significant finding on initial blood work was a blood glucose level of 30 mg/dL (n: >70mg/dL). Additional work-up of hypoglycemia revealed a low serum free insulin level 0.6 IU/mL (n:1.5 - 14.9 IU/mL), low proinsulin <4 pmol/L (n: <18.8 pmol/L), low C-peptide level 0.14 ng/mL (n: 0.80-3.85 ng/mL), low serum insulin-like growth factor-I (IGF-I) 33 ng/ml (n:34-246 ng/ml) and elevated IGF-II levels 370 ng/mL (n:47-350 ng/mL). Blood sulfonylurea and insulin antibodies screen were negative, ACTH and morning cortisol levels were within normal limits. A CT scan of chest, abdomen and pelvis for neoplastic workup demonstrated a large heterogeneous mass within the lower lobe of the right lung. A CT-guided biopsy disclosed spindle cell neoplasm, positive for signal transducer and activator of transcription-6 (STAT6) and CD34. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the NGFI-A binding protein 2 (NAB2) and STAT6 fusion which is specific for solitary fibrous tumor (SFT). She underwent surgical resection of the thoracic mass which resulted in complete resolution of her symptoms. Clinical Lessons: NICTH is a paraneoplastic syndrome associated with various benign and malignant tumors including SFT, hemangiopericytoma and metastatic hepatocellular carcinoma to name a few. Our case is an example of NICTH associated with SFT called Doege-Potter syndrome. The prevalence of hypoglycemia is seen in less than 5% of patients with SFT. It is often secondary to ectopic overexpression of unprocessed precursor ‘big’ IGF-II molecule, which binds to IGF-I receptor, resulting in tumorigenesis. An IGF-II/IGF-1 ratio is a sensitive marker and a value of more than 10 suggests excess production of IGF-II and is likely the cause of NICTH. Physicians should be cognizant of NICTH, especially in non-diabetic, otherwise healthy individuals in whom preliminary workup for hypoglycemia is negative and should look to identify any potential tumor as the likely cause. Surgical treatment is often the only curative option. Frequent meals, dextrose infusion, glucagon and growth hormone are bridging modalities until definitive surgical treatment.

Astrocytic IGF-IRs Induce Adenosine-Mediated Inhibitory Downregulation and Improve Sensory Discrimination.

Insulin-like growth factor-I (IGF-I) signaling plays a key role in learning and memory processes. While the effects of IGF-I on neurons have been studied extensively, the involvement of astrocytes in IGF-I signaling and the consequences on synaptic plasticity and animal behavior remain unknown. We have found that IGF-I induces long-term potentiation (LTPIGFI) of the postsynaptic potentials that is caused by a long-term depression of inhibitory synaptic transmission in mice. We have demonstrated that this long-lasting decrease in the inhibitory synaptic transmission is evoked by astrocytic activation through its IGF-I receptors (IGF-IRs). We show that LTPIGFI not only increases the output of pyramidal neurons, but also favors the NMDAR-dependent LTP, resulting in the crucial information processing at the barrel cortex since specific deletion of IGF-IR in cortical astrocytes impairs the whisker discrimination task. Our work reveals a novel mechanism and functional consequences of IGF-I signaling on cortical inhibitory synaptic plasticity and animal behavior, revealing that astrocytes are key elements in these processes.SIGNIFICANCE STATEMENT Insulin-like growth factor-I (IGF-I) signaling plays key regulatory roles in multiple processes of brain physiology, such as learning and memory. Yet, the underlying mechanisms remain largely undefined. Here we demonstrate that astrocytes respond to IGF-I signaling, elevating their intracellular Ca2+ and stimulating the release of ATP/adenosine, which triggers the LTD of cortical inhibitory synapses, thus regulating the behavioral task performance related to cortical sensory information processing. Therefore, the present work represents a major conceptual advance in our knowledge of the cellular basis of IGF-I signaling in brain function, by including for the first time astrocytes as key mediators of IGF-I actions on synaptic plasticity, cortical sensory information discrimination and animal behavior.

α7 Nicotinic Acetylcholine Receptor Agonists Regulate Inflammation and Growth Hormone Resistance in Sepsis.

During sepsis the normal induction of circulating insulin-like growth factor-I (IGF-I) by growth hormone (GH) action on liver is attenuated, a phenomenon called hepatic GH resistance. Hepatic GH resistance can be caused by cytokine-mediated activation of the NF-κB pathway which interferes with normal GH-signaling. The afferent and efferent fibers of the vagus nerve are integral to the cholinergic anti-inflammatory pathway (CAP) which attenuates hepatic TNFα production by activating the α7 nicotinic acetylcholine receptor (α7nAChR). We examined the effects of selective afferent vagotomy (SAV) and α7nAChR activation on sepsis-induced mortality, hepatic and systemic inflammation, the GH/IGF system and hepatic GH resistance using Sprague Dawley (SD) rats, C57BL/6 wild type (WT) mice, and α7nAChR knockout (KO) mice. Capsaicin was used to perform SAV and GTS-21 (α7nAChR agonist) was used to activate the α7nAChR. Sepsis-induced mortality, hepatic NF-κB activation, and plasma cytokine levels were increased in SAV rats and reduced in GTS-21-treated mice. The effects of sepsis on the GH/IGF-I system plasma IGF-I, IGF binding protein-1 (IGFBP-1), hepatic IGF-I, IGFBP-1, and GH receptor (GHR) mRNA and rhGH-responsiveness in mice were improved by GTS-21. Collectively these results confirm the protective effects of the anti-inflammatory CAP and α7nAChR activation in sepsis. They also provide evidence the CAP and α7nAChR activation could be used to attenuate hepatic GH resistance and anabolic failure in sepsis.

The Role of Nuclear Insulin and IGF1 Receptors in Metabolism and Cancer.

Insulin (InsR) and insulin-like growth factor-1 (IGF1R) receptors mediate the metabolic and growth-promoting actions of insulin and IGF1/IGF2, respectively. Evidence accumulated in recent years indicates that, in addition to their typical cell-surface localization pattern and ligand-activated mechanism of action, InsR and IGF1R are present in the cell nucleus of both normal and transformed cells. Nuclear translocation seems to involve interaction with a small, ubiquitin-like modifier protein (SUMO-1), although this modification is not always a prerequisite. Nuclear InsR and IGF1R exhibit a number of biological activities that classically fit within the definition of transcription factors. These nuclear activities include, among others, sequence-specific DNA binding and transcriptional control. Of particular interest, nuclear IGF1R was capable of binding and stimulating its cognate gene promoter. The physiological relevance of this autoregulatory mechanism needs to be further investigated. In addition to its nuclear localization, studies have identified IGF1R in the Golgi apparatus, and this particular distribution correlated with a migratory phenotype. In summary, the newly described roles of InsR and IGF1R as gene regulators, in concert with their atypical pattern of subcellular distribution, add a further layer of complexity to traditional models of cell signaling. Furthermore, and in view of the emerging role of IGF1R as a potential therapeutic target, a better understanding of the mechanisms responsible for nuclear IGF1R transport and identification of IGF1R interactors might help optimize target directed therapies in oncology.

Leptin Modulates the Response of Brown Adipose Tissue to Negative Energy Balance: Implication of the GH/IGF-I Axis.

The growth hormone (GH)/insulin-like growth factor I (IGF-I) axis is involved in metabolic control. Malnutrition reduces IGF-I and modifies the thermogenic capacity of brown adipose tissue (BAT). Leptin has effects on the GH/IGF-I axis and the function of BAT, but its interaction with IGF-I and the mechanisms involved in the regulation of thermogenesis remains unknown. We studied the GH/IGF-I axis and activation of IGF-I-related signaling and metabolism related to BAT thermogenesis in chronic central leptin infused (L), pair-fed (PF), and control rats. Hypothalamic somatostatin mRNA levels were increased in PF and decreased in L, while pituitary GH mRNA was reduced in PF. Serum GH and IGF-I concentrations were decreased only in PF. In BAT, the association between suppressor of cytokine signaling 3 and the IGF-I receptor was reduced, and phosphorylation of the IGF-I receptor increased in the L group. Phosphorylation of Akt and cyclic AMP response element binding protein and glucose transporter 4 mRNA levels were increased in L and mRNA levels of uncoupling protein-1 (UCP-1) and enzymes involved in lipid anabolism reduced in PF. These results suggest that modifications in UCP-1 in BAT and changes in the GH/IGF-I axis induced by negative energy balance are dependent upon leptin levels.

Pre- and Postoperative Circulating IGF-I, IGFBP-3, and IGFBP-7 Levels in Relation to Endocrine Treatment and Breast Cancer Recurrence: A Nested Case-Control Study

Insulin-like growth factor-I (IGF-I) and its binding proteins (BPs) have been associated with breast cancer risk, especially high IGF-I concentrations and the biologically active fraction estimated as the IGF-I/IGFBP-3 molar ratio. The relation of circulating IGF-I and IGFBP-3 concentrations with risk of breast cancer recurrence has been less documented. In addition a new member to a sub-group of the IGFBP-superfamily was recently identified, the low affinity IGFBP-7. To date, the role of systemic IGFBP-7 in breast cancer progression has not been investigated. Our purpose was to establish whether circulating IGF-I, IGFBP-3, and IGFBP-7 levels are related to recurrence-risk in breast cancer. A case-control study was nested within the population-based BCBlood cohort of 853 breast cancer patients diagnosed 2002–2010 in Sweden and followed through 2012. In total, 95 patients with recurrence and 170 controls were matched on age and tumor characteristics. Plasma IGF analytes and tumor membrane IGF-I receptor (IGF-IRm) positivity were analyzed and recurrence-risk was evaluated with conditional logistic regression. Preoperative tertiles of IGF-I and IGFBP-3 were both positively associated with recurrence-risk, but not IGFBP-7. The trend was of borderline significance for IGF-I, T1:REF, T2 OR:1.6, T3 OR: 2.2 adjusted P trend=0.057 and significant for IGFBP-3 T1:REF, T2 OR:1.2, T3 OR: 2.1 adjusted P trend=0.042. The models were adjusted for age, anthropometric factors, smoking, and treatments. There was a significant interaction between IGFBP-7 and IGF-IRm positivity on recurrence, where the highest IGFBP-7 highest IGFBP-7 tertile conferred increased recurrence-risk in patients with IGF-IRm positive tumors but not in those with IGF-IRm negative tumors (P interaction=0.024). By the 1-year visit, age-adjusted IGF-I levels were reduced by 17% while IGFBP-3 and IGFBP-7 were stable. IGF-I levels were significantly reduced by radiotherapy in all patients and by tamoxifen in patients with ER+ tumors. Postoperative changes >10% (n=208) in IGF-I, IGFBP-3, IGFBP-7, or the IGF-I/IGFBP-3 ratio did not predict recurrence after adjustment for preoperative levels, age, anthropometric factors, smoking, and treatments. In conclusion, this study suggests that preoperative IGF-I and IGFBP-3 levels, but not postoperative changes, might provide independent prognostic information and influence breast cancer recurrence. The role of IGFBP-7 in breast cancer merits further study.

Development of a Sensitive Bioassay for the Analysis of IGF-Related Activation of AKT/mTOR Signaling in Biological Matrices

The bioactivity of the IGF system is not a function of isolated hormone concentrations in a given biological matrix. Instead, the biological activities of IGFs are regulated by IGFBPs, IGFBP proteases, and inhibitors of IGFBP proteases. Therefore, assays based on IGF-related bioactivity may describe functions of the complete IGF system in a given biological matrix. Of particular interest are the IGF system effects on the AKT/mTOR pathway, as a dominant system for controlling growth, metabolism, and aging. In order to improve the sensitivity of IGF-dependent bioactivity, we made use of the known short-term and enhancing effects of IGFBP2 on the intracellular PI3K pathway. As a specific readout of this pathway, and further as a marker of the mTOR pathway, we assessed the phosphorylation of AKT-Ser473. Preincubation using IGFBP2 enhanced IGF1-dependent AKT-Ser473 phosphorylation in our experimental system. The assay’s specificity was demonstrated by inhibition of IGF1 receptors outside or inside the cell, using antiserum or small molecule inhibitors, which reduced AKT phosphorylation in response to exogenous IGF1 (p < 0.05). The maximal response of AKT phosphorylation was recorded 15 to 60 min after the addition of IGF1 to cell monolayers (p < 0.001). In our cellular system, insulin induced AKT phosphorylation only at supra-physiological concentrations (µM). Using this novel assay, we identified the differential biological activity of the IGF system in AKT-Ser473 phosphorylation in serum (mouse, naked mole rat, and human), in cerebrospinal fluid (human), and in colostrum or mature milk samples (dairy cow). We have developed a sensitive and robust bioassay to assess the IGF-related activation of the AKT/mTOR pathway. The assay works efficiently and does not require expensive cell culture systems. By using capillary immuno-electrophoresis, the readout of IGF-related bioactivity is substantially accelerated, requiring a minimum of hands-on time. Importantly, the assay system is useful for studying IGF-related activity in the AKT/mTOR pathway in a broad range of biological matrices.

Sex Hormones, Insulin, and Insulin-like Growth Factors in Recurrence of High-Stage Endometrial Cancer.

The influence of sex hormone and insulin/insulin-like growth factor (IGF) axis signaling on endometrial cancer recurrence is unknown. We evaluated these pathways in a prospective cohort of Gynecologic Oncology Group (GOG)0210 trial endometrial adenocarcinoma patients. Stage II-IV patients (N = 816) were included in this study. Pretreatment specimens were tested for tumor mRNA and protein expression of IGF1, IGF2, IGF-binding proteins (IGFBP)-1 and -3, insulin (IR) and IGF-I receptors (IGF1R), phosphorylated IR/IGF1R (pIGF1R/pIR), and estrogen (ER) and progesterone receptors (PR) using qPCR and IHC. Serum concentrations of insulin, IGF-I, IGFBP-3, estradiol, estrone, and sex hormone binding globulin were measured. HRs and 95% confidence intervals (CI) for progression-free survival were calculated from Cox models adjusting for age, stage, and grade. Recurrence occurred in 280 (34%) cases during a median of 4.6 years of follow-up. ER positivity (HR, 0.67; 95% CI, 0.47-0.95), IR positivity (HR, 0.53; 95% CI, 0.29-0.98), and circulating IGF-I (highest vs. lowest quartile: HR, 0.66; 95% CI, 0.47-0.92) were inversely associated with recurrence risk. Circulating estradiol (highest vs. lowest tertile: HR, 1.55; 95% CI, 1.02-2.36) and pIGF1R/pIR positivity (HR, 1.40; 95% CI, 1.02-1.92) were associated with increased recurrence risk. Circulating estradiol and tumor tissue phosphorylated (activated) IGR1R/IR were independently associated with higher risk of recurrence in patients with endometrial cancer. This study may inform future clinical trials of endocrine-targeted adjuvant therapies in patients with endometrial cancer that could include baseline assessment of serum and tissue biomarkers of estradiol and insulin signaling pathways.

Lessons Learned from Targeting IGF-I Receptor in Thyroid-Associated Ophthalmopathy.

Complex immunological mechanisms underlie the pathogenesis of thyroid-associated ophthalmopathy (TAO). Historical models of Graves’ disease and TAO have focused almost entirely on autoimmune reactivity directed against the thyrotropin receptor (TSHR). The insulin-like growth factor-I receptor (IGF-IR) has been proposed as a second participating antigen in TAO by virtue of its interactions with IGFs and anti-IGF-IR antibodies generated in Graves’ disease. Furthermore, the IGF-IR forms with TSHR a physical and functional complex which is involved in signaling downstream from both receptors. Inhibition of IGF-IR activity results in attenuation of signaling initiated at either receptor. Based on the aggregate of findings implicating IGF-IR in TAO, the receptor has become an attractive therapeutic target. Recently, teprotumumab, a human monoclonal antibody IGF-IR inhibitor was evaluated in two clinical trials of patients with moderate to severe, active TAO. Those studies revealed that teprotumumab was safe and highly effective in reducing disease activity and severity. Targeting IGF-IR with specific biologic agents may result in a paradigm shift in the therapy of TAO.

Analysis of genetic selection at insulin receptor substrate-2 gene loci.

Type 2 diabetes mellitus (T2DM) is highly heritable and exhibits significant variability in prevalence between different populations. Prevalence of T2DM is higher in Asian and African relative to European populations. During evolution, traditional feast-famine cycles likely led to significant natural selection impacting metabolic genes. Human adaptation to environmental changes (food supply, lifestyle, climate, and geography) likely influenced differential selection of T2DM-associated genes. Together, insulin receptor substrate-1 and -2 (IRS1 and IRS2) genes encode the major ligands of insulin and IGF1 receptors. Irs2-deficient mice exhibit a T2DM phenotype with severe insulin resistance, and a common IRS2 polymorphism is associated with T2DM. Therefore, the present study sought evidence of natural selection at IRS2 loci. Data were sourced from the HapMap and 1000 Genomes projects, comprising four different populations with distinct ancestries: European, Yoruba, Han Chinese, and Japanese. A three-step method was applied to detect IRS2 locus selection. The long-range haplotype (LRH) test detected unusual extended haplotypes, the integrated haplotype score (iHS) detected selection, and Wright's F-statistics (particularly Wright's fixation index: FST) were calculated as a measure of population differentiation. The African population exhibited highly significant LRH findings (percentile >99.9, p = 0.005-0.0009), while both the European and African populations exhibited extreme positive iHS test scores ([iHS] >2.5). These findings indicate that genetic selection has occurred at the IRS2 locus, warranting further research into the adaptive evolution of metabolic disorder-associated genes. The online version contains supplementary material available at 10.1007/s40200-021-00745-y.

Gonadal Cycle-Dependent Expression of Genes Encoding Peptide-, Growth Factor-, and Orphan G-Protein-Coupled Receptors in Gonadotropin- Releasing Hormone Neurons of Mice.

Rising serum estradiol triggers the surge release of gonadotropin-releasing hormone (GnRH) at late proestrus leading to ovulation. We hypothesized that proestrus evokes alterations in peptidergic signaling onto GnRH neurons inducing a differential expression of neuropeptide-, growth factor-, and orphan G-protein-coupled receptor (GPCR) genes. Thus, we analyzed the transcriptome of GnRH neurons collected from intact, proestrous and metestrous GnRH-green fluorescent protein (GnRH-GFP) transgenic mice using Affymetrix microarray technique. Proestrus resulted in a differential expression of genes coding for peptide/neuropeptide receptors including Adipor1, Prokr1, Ednrb, Rtn4r, Nmbr, Acvr2b, Sctr, Npr3, Nmur1, Mc3r, Cckbr, and Amhr2. In this gene cluster, Adipor1 mRNA expression was upregulated and the others were downregulated. Expression of growth factor receptors and their related proteins was also altered showing upregulation of Fgfr1, Igf1r, Grb2, Grb10, and Ngfrap1 and downregulation of Egfr and Tgfbr2 genes. Gpr107, an orphan GPCR, was upregulated during proestrus, while others were significantly downregulated (Gpr1, Gpr87, Gpr18, Gpr62, Gpr125, Gpr183, Gpr4, and Gpr88). Further affected receptors included vomeronasal receptors (Vmn1r172, Vmn2r-ps54, and Vmn1r148) and platelet-activating factor receptor (Ptafr), all with marked downregulation. Patch-clamp recordings from mouse GnRH-GFP neurons carried out at metestrus confirmed that the differentially expressed IGF-1, secretin, and GPR107 receptors were operational, as their activation by specific ligands evoked an increase in the frequency of miniature postsynaptic currents (mPSCs). These findings show the contribution of certain novel peptides, growth factors, and ligands of orphan GPCRs to regulation of GnRH neurons and their preparation for the surge release.

Growth Hormone Signaling Pathway Leading to the Induction of DNA Synthesis and Proliferation in Primary Cultured Hepatocytes of Adult Rats.

We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams' medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10-6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10-6 M), RTK (AG538, 10-6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10-6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10-6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

GTPases and phosphatidylinositol 3-kinase are critical for insulin-like growth factor-I-mediated Schwann cell motility.

Previously, we reported insulin-like growth factor-I (IGF-I) promotes motility and focal adhesion kinase (FAK) activation in neuronal cells. In the current study, we examined the role of IGF-I in Schwann cell (SC) motility. IGF-I increases SC process extension and motility. In parallel, IGF-I activates IGF-I receptor, insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3 (PI-3)-kinase, and FAK. LY294002, a PI-3 kinase inhibitor, blocks IGF-I-induced motility and FAK phosphorylation. The Rho family of GTPases is important in the regulation of the cytoskeleton. Overexpression of constitutively active Leu-61 Cdc42 and Val-12 Rac1 enhances SC motility which is unaffected by LY294002. In parallel, stable transfection of SC with dominant negative Asn-17 Rac1 blocks IGF-I-mediated SC motility and FAK phosphorylation, implying Rac is an upstream regulator of FAK. Collectively our results suggest that IGF-I regulates SC motility by reorganization of the actin cytoskeleton via the downstream activation of a PI-3 kinase, small GTPase, and FAK pathway.

Proteomic identification and validation of novel interactions of the putative tumor suppressor PRELP with membrane proteins including IGFI-R and p75NTR

Proline and arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich repeat proteoglycans (SLRPs) family. Levels of PRELP mRNA are downregulated in many types of cancer, and PRELP has been reported to have suppressive effects on tumor cell growth, although the molecular mechanism has yet to be fully elucidated. Given that other SLRPs regulate signaling pathways through interactions with various membrane proteins, we reasoned that PRELP likely interacts with membrane proteins to maintain cellular homeostasis. To identify membrane proteins that interact with PRELP, we carried out coimmunoprecipitation coupled with mass spectrometry (CoIP-MS). We prepared membrane fractions from Expi293 cells transfected to overexpress FLAG-tagged PRELP or control cells and analyzed samples precipitated with anti-FLAG antibody by mass spectrometry. Comparison of membrane proteins in each sample identified several that seem to interact with PRELP; among them, we noted two growth factor receptors, insulin-like growth factor I receptor (IGFI-R) and low-affinity nerve growth factor receptor (p75NTR), interactions with which might help to explain PRELP’s links to cancer. We demonstrated that PRELP directly binds to extracellular domains of these two growth factor receptors with low micromolar affinities by surface plasmon resonance analysis using recombinant proteins. Furthermore, cell-based analysis using recombinant PRELP protein showed that PRELP suppressed cell growth and affected cell morphology of A549 lung carcinoma cells, also at micromolar concentration. These results suggest that PRELP regulates cellular functions through interactions with IGFI-R and p75NTR and provide a broader set of candidate partners for further exploration.

Abstract PO-003: Adaptation of colorectal cancer cells to the brain microenvironment: The role of IRS2

Abstract Brain metastases (BMs) from colorectal cancer (CRC) are currently the fourths leading cause of BMs, and their incidence is on the rise. Yet, mechanisms mediating the formation of BMs from CRC are unknown. We aimed to explore genomic drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence was noted in the insulin receptor substrate 2 (IRS2) gene, which was significantly amplified in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. Next, we created an in-vitro system mimicking the brain microenvironment using human astrocytes or their conditioned media (CM). Under these conditions, IRS2-overexpressed CRC cells survived better and formed larger 3D spheres. IRS2-silenced CRC cells showed a mirror image. Furthermore, IRS2-overexpressed CRC cells generated larger brain lesions in an intracranial CRC BMs mice model. Interestingly, a transcriptomic analysis revealed significant enrichment of the AKT and β-catenin pathways by IRS2. Indeed, IRS2-amplified cells showed enhanced AKT phosphorylation and inhibition of PI3K or AKT using Alpelisib or Akt1/2 kinase inhibitor, respectively, effectively decreased their proliferation in CM. Importantly, IRS2-amplified cells had increased expression and transcriptional activity of the β-catenin, and AKT pathway inhibitors or IRS2 inhibitor (NT-219) had a more pronounced effect on the activity of β-catenin on those cells. These data indicate, for the first time, the unique genomic profile of CRC BMs and imply the IRS2 role in promoting CRC BMs. These effects may be mediated, at least in part, by modulation of the AKT and β-catenin pathway. These findings may pave the way for the development of novel therapeutic strategies to prevent BMs formation. Citation Format: Inbal Greenberg, Anat Klein, Rachel Grossman, Ethan Sokol, Eilam Yeini, Paula Ofek, Ronit Satchi-Fainaro, Hadas Reuveni, Tami Rubinek, Ido Wolf. Adaptation of colorectal cancer cells to the brain microenvironment: The role of IRS2 [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-003.

Decorin inhibits the insulin-like growth factor I signaling in bone marrow mesenchymal stem cells of aged humans.

Aging impairs the IGF-I signaling of bone marrow mesenchymal stem cells (bmMSCs), but the mechanism is unclear. Here, we found that the ability to auto-phosphorylate IGF-I receptor (IGF-IR) in response to IGF-I was decreased in the bmMSCs of aged donors. Conversely, data showed that decorin (DCN) expression was prominently increased in aged bmMSCs, and that under IGF-I treatment, DCN knockdown in serum-starved aged bmMSCs potentiated their mitogenic activity and IGF-IR auto-phosphorylation, whereas DCN overexpression in serum-starved adult bmMSCs decreased both activities. Co-immunoprecipitation assays suggested that IGF-I and DCN bound to IGF-IR in a competitive manner. Online MethPrimer predicted 4 CpG islands (CGIs) in the introns of DCN gene. RT-qPCR and bisulfite sequencing showed that dimethyloxalylglycine, an inhibitor of DNA demethylation, increased DCN mRNA expression and CGI-I methylation in adult bmMSCs, whereas 5-aza-2’-deoxycytidine, a DNA methylation inhibitor, decreased DCN mRNA expression and CGI-I methylation in aged bmMSCs, and ultimately enhanced the proliferation of serum-starved aged bmMSCs under IGF-I stimulation. Thus, IGF-IR could be the prime target of aging in down-regulating the IGF-I signaling of bmMSCs, where DCN could be a critical mediator.

Autocrine secretion of insulin-like growth factor-I mediates growth hormone-stimulated DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

The intracellular signaling pathway of growth hormone (GH)-stimulated DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes. DNA synthesis and cell proliferation were detected in hepatocyte parenchymal cells grown in serum-free, defined medium containing GH (100 ng/ml). GH-stimulated hepatocyte DNA synthesis and proliferation were almost completely blocked by TG101209 (10−6 M), a selective Janus kinase (JAK)2 inhibitor, U-73122 (10−6 M), a selective phospholipase C (PLC) inhibitor, and a monoclonal antibody to insulin-like growth factor-I (IGF-I) receptor (100 ng/ml) or anti-secretion agents such as somatostatin (10−6 M) and BAPTA/AM (10−7 M). In addition, blocking monoclonal antibodies to IGF-I, but not transforming growth factor-α, completely inhibited GH-induced hepatocyte DNA synthesis and proliferation. IGF-I levels in the culture medium increased rapidly versus baseline levels within 5 min in response to GH (100 ng/ml), and the maximum IGF-I level (100 pg/ml) was reached 20 min after GH stimulation. Autocrine secretion of IGF-I into the culture medium was inhibited by a growth-inhibitory dose of TG101209, U-73122, somatostatin, or BAPTA/AM. These data indicate that the proliferative mechanism of action of GH is mediated mainly through a GH receptor/JAK2/PLC-stimulated increase in the autocrine secretion of IGF-I by primary cultured hepatocytes, followed by stimulation of the 95 kDa IGF-I receptor tyrosine kinase signaling pathway.

CHRONIC NEUROGENIC PAIN PROMOTES DEVELOPMENT AND GROWTH OF M1 SARCOMA CHANGING LOCAL LEVELS OF GROWTH FACTORS

Introduction. The influence of chronic neurogenic pain on local levels of growth factors in the lung and the risk of lung cancer development has been little studied.The purpose of the study was to analyze the levels of VEGF, TGF-β, IGF-I, IGF-II, FGF-21 and receptors of VEGFR2, TGF-βR2 in the lungs of white outbred rats with chronic neurogenic pain after intravenous injection of M1 sarcoma.Material and Methods. A total of 28 white outbred male rats weighing 200–250 g were divided into 4 groups: 1 – sham-operated animals (control group) (n=7); 2 – animals with chronic neurogenic pain (n=7); 3 – sham-operated animals with intravenous injection of M1 sarcoma (n=7); 4 – rats with intravenous injection of M1 sarcoma in presence of chronic neurogenic pain (n=7). Animals were decapitated, the lungs were harvested on ice; 10 % cytosolic fractions were prepared in 0.1 M potassium phosphate buffer pH 7.4 containing 0.1 % Tween-20 and 1 % BSA. Levels of VEGFR2, TGF-β and receptors of TGF-βR2, IGF-I, IGF-II (CUSABIO BIOTECH Co., Ltd., China) and FGF21 (BioVender, Czech Republic) were determined by ELISA. The data were statistically processed using Statistica 10.0 software.Results. Multifocal tumors of sarcoma in the lungs were determined only in the group of rats with chronic neurogenic pain after intravenous transplantation of M1 sarcoma. In the lungs of sham-operated animals, tumor foci were not detected after M1 sarcoma transplantation. Lung tissues with M1 growth and presence of chronic neurogenic pain demonstrated decreased VEGF-А levels with increased concentrations of TGFβ, IGF-I, IGF-II and FGF-21. Chronic neurogenic pain directly or indirectly influenced levels of some growth factors in the lung, and altered cell homeostasis making possible transplantation of M1 sarcoma into the lung.

Somatotropic-Testicular Axis: A crosstalk between GH/IGF-I and gonadal hormones during development, transition, and adult age.

The hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-somatotropic (HPS) axes are strongly interconnected. Interactions between these axes are complex and poorly understood. These interactions are characterized by redundancies in reciprocal influences at each level of regulation and the combination of endocrine and paracrine effects that change during development. To comprehensively review the crosstalk between the HPG and HPS axes and related pathological and clinical aspects during various life stages of male subjects. A thorough search of publications available in PubMed was performed using proper keywords. Molecular studies confirmed the expressions of growth hormone (GH) and insulin-like growth factor-I (IGF-I) receptors on the HPG axis and reproductive organs, indicating a possible interaction between HPS and HPG axes at various levels. Insulin growth factors participate in sexual differentiation during fetal development, indicating that normal HPS axis activity is required for proper testicular development. IGF-I contributes to correct testicular position during minipuberty, determines linear growth during childhood, and promotes puberty onset and pace through gonadotropin-releasing hormone activation. IGF-I levels are high during transition age, even when linear growth is almost complete, suggesting its role in reproductive tract maturation. Patients with GH deficiency (GHD) and insensitivity (GHI) exhibit delayed puberty and impaired genital development; replacement therapy in such patients induces proper pubertal development. In adults, few studies have suggested that lower IGF-I levels are associated with impaired sperm parameters. The role of GH-IGF-I in testicular development remains largely unexplored. However, it is important to evaluate gonadic development in children with GHD. Additionally, HPS axis function should be evaluated in children with urogenital malformation or gonadal development alterations. Correct diagnosis and prompt therapeutic intervention are needed for healthy puberty, attainment of complete gonadal development during transition age, and fertility potential in adulthood.

Genetic causes of growth hormone insensitivity beyond GHR.

Growth hormone insensitivity (GHI) syndrome, first described in 1966, is classically associated with monogenic defects in the GH receptor (GHR) gene which result in severe post-natal growth failure as consequences of insulin-like growth factor I (IGF-I) deficiency. Over the years, recognition of other monogenic defects downstream of GHR has greatly expanded understanding of primary causes of GHI and growth retardation, with either IGF-I deficiency or IGF-I insensitivity as clinical outcomes. Mutations in IGF1 and signaling component STAT5B disrupt IGF-I production, while defects in IGFALS and PAPPA2, disrupt transport and release of circulating IGF-I, respectively, affecting bioavailability of the growth-promoting IGF-I. Defects in IGF1R, cognate cell-surface receptor for IGF-I, disrupt not only IGF-I actions, but actions of the related IGF-II peptides. The importance of IGF-II for normal developmental growth is emphasized with recent identification of defects in the maternally imprinted IGF2 gene. Current application of next-generation genomic sequencing has expedited the pace of identifying new molecular defects in known genes or in new genes, thereby expanding the spectrum of GH and IGF insensitivity. This review discusses insights gained and future directions from patient-based molecular and functional studies.