Laron Syndrome Research Paves the Way for New Insights in Oncological Investigation

Abstract

Laron syndrome (LS) is a rare genetic endocrinopathy that results from mutation of the growth hormone receptor (GH-R) gene and is typically associated with dwarfism and obesity. LS is the best characterized entity under the spectrum of the congenital insulin-like growth factor-1 (IGF1) deficiencies. Epidemiological analyses have shown that LS patients do not develop cancer, whereas heterozygous family members have a cancer prevalence similar to the general population. To identify genes and signaling pathways differentially represented in LS that may help delineate a biochemical and molecular basis for cancer protection, we have recently conducted a genome-wide profiling of LS patients. Studies were based on our collection of Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines derived from LS patients, relatives and healthy controls. Bioinformatic analyses identified differences in gene expression in several pathways, including apoptosis, metabolic control, cytokine biology, Jak-STAT and PI3K-AKT signaling, etc. Genes involved in the control of cell cycle, motility, growth and oncogenic transformation are, in general, down-regulated in LS. These genetic events seem to have a major impact on the biological properties of LS cells, including proliferation, apoptosis, response to oxidative stress, etc. Furthermore, genomic analyses allowed us to identify novel IGF1 downstream target genes that have not been previously linked to the IGF1 signaling pathway. In summary, by 'mining' genomic data from LS patients, we were able to generate clinically-relevant information in oncology and, potentially, related disciplines.

Keywords: Laron syndrome; cancer protection; growth hormone; growth hormone receptor; insulin-like growth factor-1 (IGF1).

Figure 1

The growth hormone-IGF1 axis in normal and pathological growth. Pituitary-produced growth hormone (GH) stimulates insulin-like growth factor-1 (IGF1) secretion from the liver, leading to bone elongation and longitudinal growth (left panel). Laron syndrome (LS) is associated with mutation of the GHR gene, as a result of which the liver (and, most probably, additional extrahepatic tissue) is no longer able to produce IGF1 at physiological levels in response to GH stimulation (right panel). Abrogation of IGF1 biosynthesis leads to impaired growth and concomitant relaxation of negative feed-back regulation of GH production at the pituitary gland. Loss of inhibitory control results in very high circulating GH levels. In addition to short stature, LS is associated with obesity and protection from cancer. Enhanced longevity has been demonstrated in animal models of LS (‘Laron’ mouse).

Figure 2

Genomic analysis of Laron Syndrome patients. (A) Hierarchical cluster analysis of differentially expressed genes in lymphoblastoids derived from four female LS patients and four controls of the same age range (LS, 44.25 ± 6.08 yr; controls, 51.75 ± 11.3 y) and ethnic (Yemen, Irak, Iran) origin. The figure depicts a cluster of 39 differentially expressed genes (FC > 2 or < than −2 and p-value < 0.05). Up-regulated genes are shown in red and down-regulated genes are shown in blue. FC, fold change. This panel was obtained from [67]. (B) Functional analysis of differentially represented signaling pathways in LS. Bioinformatic analyses were conducted using the David and WebGestalt platforms. The % values in each category correspond to the percentage of the total number of differentially expressed genes.

Figure 3

Analysis of biological functions in LS cells. (A) Cell proliferation. LS- and control-derived lymphoblastoid cells were maintained in a serum-free, IGF1-free medium for three days, after which proliferation was assessed using an XTT colorimetric kit. The statistical significance of differences between groups was assessed by a Student’s t-test. *, significantly different versus control (p < 0.05). (B) Cell cycle. Lymphoblastoids were exposed to etoposide (a DNA-damaging agent) for 24 h, after which cells were stained with propidium iodide and analyzed with a FACScalibur system to determine the percentage of cells in G0-G1, S, and G2-M phases. The graph presents the percentage of cells within each cell cycle phase. (C) Apoptosis/Necrosis. Basal apoptosis and necrosis were measured by flow cytometry analysis after staining cells with an Annexin-V antibody and propidium iodide. Necrotic cells were stained with propidium iodide and Annexin V, whereas apoptotic cells were stained only with Annexin V. (D) Oxidative stress. Cells were grown to confluence, after which the medium was changed to a fresh full medium in the presence of increasing doses of paraquat dichloride. Paraquat generates superoxide anion, which leads to the formation of toxic reactive oxygen species, such as hydrogen peroxide and hydroxyl radical, and the oxidation of cellular NADPH. Proliferation in response to oxidative damage was measured using an XTT kit. The graph depicts a pair of LS-derived and control cells, normalized for age and ethnic origin. A value of 100% was given to the cell number at time zero. Data described here were originally reported in [67].

Figure 4

Regulation of thioredoxin-interacting protein (TXNIP) expression and action by IGF1. (A) Expression of TXNIP in LS cells. Total RNA was extracted from LS-derived and control lymphoblastoid cells and TXNIP mRNA levels were measured by RT-QPCR. (B) Effect of IGF1/insulin on TXNIP levels. HEK293 cells were starved for 24 h, after which they were treated with IGF1 or insulin (50 ng/mL) for 5 or 12 h. TXNIP levels were then measured by Western blots. (C) Effect of oxidative stress on TXNIP levels. Serum-starved HEK293 cells were treated with H₂O₂ (100 mM) or IGF1 or both for 2 h, after which TXNIP levels were measured by Western blots. (D) Effect of oxidative stress on TXNIP levels in LS-derived and control lymphoblastoid cell lines. Four individual LS-derived and three control lymphoblastoid cell lines were treated with 300 mM of H₂O₂ or left unstimulated. Cells were harvested after 2 h and levels of TXNIP mRNA were measured by RT-QPCR. A value of 1 was given to TXNIP mRNA levels in untreated cells (green bars). (E) Schematic representation of the regulation of TXNIP expression by IGF1. TXNIP is a metabolic gene that functions as an oxidative and glucose sensor. TXNIP mRNA levels were more than two-fold higher in LS-derived lymphoblastoid cells than in healthy control cells. IGF1 was shown to exert its anti-apoptotic effect via downregulation of TXNIP expression. Part of the data shown here was originally reported in [81].

Figure 5

QRT-PCR analysis of IGF-binding protein (IGFBP) mRNA levels in LS cells. Lymphoblastoid cell lines derived from four LS patients (red bars) and four controls (blue bars) of the same age range, gender, and ethnic origin were harvested, after which total RNA was prepared and IGFBPs’ mRNA levels were measured by QRT-PCR. Primers employed are described in [84]. For each IGFBP mRNA, a value of 1 was given to the level exhibited by control cells. Bars denote mean ± SD (n = 4). *, p < 0.05 versus respective control. Data shown here were originally reported in [84].

Figure 6

Schematic diagram of the regulation of IGF1R gene expression by wild-type and mutant p53. The mechanism of action of wild-type p53 involves transcriptional suppression of the IGF1R gene (left panel). Reduced IGF1R levels favor a post-mitotic, terminally-differentiated phenotype. Mutation of the p53 gene in tumor cells disrupts its inhibitory activity, generating oncogenic molecules capable of transactivating the IGF1R gene (right panel). The abundance and activity of p53 itself is regulated by IGF1, which induces p53 degradation in an Mdm2-dependent fashion.