Genome-Wide Profiling of Laron Syndrome Patients Identifies Novel Cancer Protection Pathways

Abstract

Laron syndrome (LS), or primary growth hormone resistance, is a prototypical congenital insulin-like growth factor 1 (IGF1) deficiency. The recent epidemiological finding that LS patients do not develop cancer is of major scientific and clinical relevance. Epidemiological data suggest that congenital IGF1 deficiency confers protection against the development of malignancies. This 'experiment of nature' reflects the critical role of IGF1 in tumor biology. The present review article provides an overview of recently conducted genome-wide profiling analyses aimed at identifying mechanisms and signaling pathways that are directly responsible for the link between life-time low IGF1 levels and protection from tumor development. The review underscores the concept that 'data mining' an orphan disease might translate into new developments in oncology.

Keywords: IGF1 receptor (IGF1R); Laron syndrome; cancer protection; growth hormone receptor (GH-R); insulin-like growth factor 1 (IGF1); thioredoxin-interacting protein (TXNIP).

Figure 1

Schematic representation of the GH-IGF1 axis in health and in Laron syndrome (LS) patients. Pituitary-produced GH leads to IGF1 secretion from the liver, with ensuing bone elongation and longitudinal growth (left panel). As a result of a GH-R mutation in LS patients, the liver (and, probably, additional extrahepatic tissues) is no longer able to produce physiological levels of IGF1 (right panel). Abrogation of IGF1 production leads to impaired growth and defective negative feed-back at the pituitary gland level, leading to high circulating GH levels.

Figure 2

Genome-wide profiling of LS patients. (a) Cluster analysis of differentially expressed genes in Epstein-Bar virus (EBV)-immortalized lymphoblastoids derived from four LS patients (four bottom rows, blue color) and four age-, gender-, and ethnicity-matched controls (four upper rows, red color). The figure depicts a cluster of 39 differentially expressed genes (fold change (FC) > 2 or < −2 and p value < 0.05). The names of the genes are presented in the x-axis. Up-regulated genes are shown in red and down-regulated genes are shown in blue. (b) Principal component analysis (PCA) display of four LS and four control arrays. Hierarchical cluster analysis was performed using Partek Genomics Suite software with Pearson’s dissimilarity correlation and average linkage methods. Data analysis was followed by one-way ANOVA. Blue circles: LS patients; red circles: controls. The figure was adapted from [55].

Figure 3

Signaling pathways altered in LS. The pie chart illustrates the signaling pathways that were differentially represented in LS cells as a percentage of the total number of differentially expressed genes.

Figure 4

Analysis of signaling pathways associated with cancer protection in LS. (A) Western blot analysis of Sp1 and pTEN levels in LS-derived and control lymphoblastoids. Lymphoblastoid cell lines of four LS patients and four controls were lysed and extracts were electrophoresed through SDS-PAGE. Blots were incubated with antibodies against Sp1 and pTEN. The lanes correspond to individual controls and patients. (B) Western blot analysis of downstream mediators of IGF1 action in LS. Cell extracts were resolved on SDS-PAGE and membranes were incubated with antibodies against phospho- and total-IGF1 receptor (IGF1R), phospho- and total-AKT and phospho- and total-ERK. Tubulin levels were measured as a loading control. (C) Cell proliferation of LS and control cells. Proliferation of LS- and control-derived lymphoblastoid cells was assessed using an XTT colorimetric kit. The statistical significance of differences between groups was assessed by Student’s t-test. Legend: *, significantly different versus control (p < 0.05); red bars, LS; blue bars, controls. (D) Basal apoptosis and necrosis of LS and control cells. Apoptosis and necrosis were measured by flow cytometry analysis after staining cells with an annexin-V antibody and propidium iodide (PI). Necrotic cells were stained with PI as well as annexin V; apoptotic cells were stained only with annexin V. The figure was adapted from [55].

Figure 5

Regulation of thioredoxin-interacting protein (TXNIP) expression by IGF1. The processes of cell survival and homeostasis are tightly controlled by IGF1 action from early ontogenetic stages throughout adulthood. Left panel: normal physiological stress conditions, including starvation and oxidative and glucose stress, might lead to upregulation of TXNIP. Augmented TXNIP levels initiate apoptosis by interacting with thioredoxin and translocating to mitochondria. Cellular stress in the absence of IGF1 (e.g., Laron syndrome) may lead to cell death. Right panel: IGF1 significantly downregulates oxidative and glucose stress-induced TXNIP upregulation and controls glucose uptake in order to improve the energy balance of the cell. Cellular stress in the presence of IGF1 might lead to deregulated cell growth, including cancer.

Figure 6

Expression of IGF-binding protein (IGFBP) mRNA in Laron syndrome. Total RNA was prepared from lymphoblastoid cell lines derived from four LS patients (gray bars) and four controls (closed bars) of the same age range, gender, and ethnic origin. Levels of IGFBP-2, -3, -4, -5, and -6 mRNAs were measured by RQ-PCR. For each IGFBP mRNA, a value of 1 was given to the level displayed by controls. Bars denote mean ± SD (n = 4). Legend: *, p < 0.05 versus respective control. Results indicate that mRNA levels of IGFBPs usually regarded as pro-mitogenic (IGFBP-2, -5, and -6) were reduced in LS, whereas IGFBP-3 (a pro-apoptotic protein) levels were increased under this condition. The figure was adapted from [71].