Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer

Epigenetics refers to heritable changes in gene expression that are independent of alterations in DNA sequence. It is now accepted that disruption of epigenetic mechanisms plays a key role in the pathogenesis of cancer: culminating in altered gene function and malignant cellular transformation. DNA methylation and histone modifications are the most widely studied changes but non-coding RNAs such as miRNAs are also considered part of the epigenetic machinery. The insulin-like growth factor (IGF) axis is composed of two ligands, IGF-I and –II, their receptors and six high affinity IGF binding proteins (IGFBPs). The IGF axis plays a key role in cancer development and progression. As IGFBP genes have consistently been identified among the most common to be aberrantly altered in tumours, this review will focus on epigenetic regulation of IGFBP-3 in cancer for which the majority of evidence has been obtained.     

Single nucleotide polymorphism in Egyptian cattle insulin-like growth factor binding protein-3 gene

Insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene responsible for the multiple influences of insulin-like growth factors (IGFs) system. It is considered as a candidate gene for growth and production traits. In the present study, we aimed to determine the genetic polymorphism of Egyptian cattle IGFBP-3 gene.The amplified fragment of cattle IGFBP-3 gene at 651-bp was digested with three different endonucleases; HaeIII, MspI and TaqI. The digestion of the PCR products with MspI and TaqI endonucleases revealed similar restriction patterns in all tested animals.Digestion of the PCR product with HaeIII restriction enzyme revealed three different genotypes in Egyptian cattle due to the presence of two alleles; allele A with 7 digested fragments resulting from the presence of 6 restriction sites and allele C with 8 digested fragments resulting from the presence of 7 restriction sites; six sites as allele A in the addition of another restriction site at position 298^299 as a result of SNP (A → C) in C allele at position 299. The restriction patterns of IGFBP-3/HaeIII showed that forty-six examined animals are genotyped as AA, CC and AC with frequencies of 21.74%, 21.74% and 56.52%, respectively.It is concluded that the IGFBP-3/HaeIII polymorphism may be utilized as a good marker for genetic differentiation between cattle animals for different body functions such as growth, metabolism, reproduction, immunity and energy balance. The nucleotide sequences of Egyptian cattle IGFBP-3 A and C alleles were submitted to GenBank with the accession numbers KF899893 and KF899894, respectively.     

Matrix metalloproteinase-7 degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability

Proteolytic modification of insulin-like growth factor binding proteins (IGFBPs) plays an important physiological role in regulating insulin-like growth factor (IGF) bioavailability. Recently, we demonstrated that matrix metalloproteinase-7 (MMP-7)/Matrilysin produced by various cancer cells catalyzes the proteolysis of IGFBP-3 in vitro and regulates IGF bioavailability, resulting in an anti-apoptotic effect against anchorage-independent culture. In the present study, we investigated whether MMP-7 contributes to proteolysis of the other five IGFBPs, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6, and whether this results in phosphorylation of the IGF type 1 receptor (IGF-1R). MMP-7 cleaved all six IGFBPs, resulting in IGF-mediated IGF-1R phosphorylation, which was inhibited by EDTA treatment. These results suggest that MMP-7 derived from cancer cells can regulate IGF bioavailability in the microenvironment surrounding the tumor, where various kinds of IGF/IGFBP complexes are found, thereby favoring cancer cell growth and survival during the processes of invasion and metastasis.     

Interaction of insulin-like growth factor binding protein-3 with latent transforming growth factor-beta binding protein-1

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits the replication and promotes apoptosis in various cell lines in an IGF-independent manner. We utilized a yeast two-hybrid system to identify binding partners for IGFBP-3 in a mouse embryo cDNA library. A partial cDNA encoding mouse latent transforming growth factor beta (TGF-) binding protein-1 (LTBP-1) was identified. This cDNA encoded a mouse LTBP-1 mRNA fragment corresponding to amino acid residues 1160–1712. Analysis of C-terminal deleted mutants indicated that the IGFBP-3 interacting domain resides in the 552 residue C-terminal fragment encoding amino acids 831–1383. The interaction of IGFBP-3 with recombinant human LTBP-1 immobilized on nitrocellulose was also demonstrated. Neither binding of IGF-I to IGFBP-3 nor binding of latency associated protein (LAP) with LTBP-1 inhibited the interaction of IGFBP-3 with LTBP-1. Furthermore the large latent complex, ¹²⁵I-TGF-/LAP/LTBP-1 was able to bind to immobilized IGFBP-3. These data demonstrate that IGFBP-3 can bind to LTBP-1 and provide a potential mechanism whereby IGFBP-3 can interact with the TGF- system.     

Enhanced expression of insulin-like growth factor binding protein-3 sensitizes the growth inhibitory effect of anticancer drugs in gastric cancer cells

Insulin-like growth factor (IGF)-I and -II are potent mitogens and their mitogenic effects are modulated by IGF binding proteins (IGFBPs). In this study, we evaluated whether the enhanced expression of IGFBP-3 may increase the sensitivity of human gastric cancer cells to the anticancer drugs. We further investigated the potential mechanism for the growth inhibitory effect of anticancer drug induced-IGFBP-3 expression. These IGFBP-3-expressing gastric cancer cells showed a lower proliferation rate than IGFBP-3-non-expressing cells. Treatment with anticancer drugs resulted in up-regulation of IGFBP-3 expression in IGFBP-3-expressing cells. Interestingly the anticancer drug-induced-growth inhibition was more evident in IGFBP-3-expressing cells causing the IGFBP-3 expressing cells but not the IGFBP-3 non-expressing cells to accumulate in the G1/G0 phase and induce apoptosis. The exogenous addition of IGFBP-3 inhibited the growth of IGFBP-3-non-expressing cells, causing them to undergo apoptosis. Our data suggest that IGFBP-3 may have an important role in the biology of gastric cancer cell growth and provides a potential marker for predicting the responsiveness to anticancer drugs.     

The amino-terminal region of insulin-like growth factor binding protein-3, (1-95)IGFBP-3, induces apoptosis of MCF-7 breast carcinoma cells

In an earlier study, we reported that an N-terminal proteolytic fragment ((1-95)IGFBP-3) corresponding to the first 95 residues of human insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation in a variety of fibroblasts. With a view to investigating its cytostatic capacity in carcinoma cells, we transiently transfected MCF-7 breast adenocarcinoma cells with an expression vector containing (1-95)IGFBP-3 cDNA. The transfected cells secreted a hyper-glycosylated form of (1-95)IGFBP-3. Twenty-four hours after transfection, cell morphology and viability were similar in control and (1-95)IGFBP-3-secreting cells. However, after 48 h, (1-95)IGFBP-3-secreting cells were apoptotic, with marked cytoplasmic vacuolation and increased free histones in the cytoplasm. Culture media conditioned by (1-95)IGFBP-3-secreting cells also induced morphological changes and apoptosis in wild-type MCF-7 cells, indicating that (1-95)IGFBP-3 was responsible for the effects observed. These results provide further evidence that the N-terminal proteolytic fragment of IGFBP-3 has a functional role.     

PTEN-induction in U251 glioma cells decreases the expression of insulin-like growth factor binding protein-2

PTEN is a tumor suppressor gene whose loss of function is observed in approximately 40-50% of human cancers. Although insulin-like growth factor binding protein-2 (IGFBP-2) was classically described as a growth inhibitor, multiple recent reports have shown an association of overexpression and/or high serum levels of IGFBP-2 with poor prognosis of several malignancies, including gliomas. Using an inducible PTEN expression system in the PTEN-null glioma cell line U251, we demonstrate that PTEN-induction is associated with reduced proliferation, increased apoptosis, and a substantial reduction of the high levels of IGFBP-2 expression. The PTEN-induced decrease in IGFBP-2 expression could be mimicked with the PI3-kinase inhibitor LY294002, indicating that the lipid phosphatase activity of PTEN is responsible for the observed effect. However, the rapamycin analog CCI-779 did not affect IGFBP-2 expression, suggesting that the PTEN-induced decrease in IGFBP-2 expression is not attributable to decreased mTOR signalling. Recombinant human IGFBP-2 was unable to rescue U251-PTEN cells from the antiproliferative effects of PTEN, and IGFBP-2 siRNA did not affect the IGF-dependent or -independent growth of this cell line. These results suggest that the clinical data linking IGFBP-2 expression to poor prognosis may arise, at least in part, because high levels of IGFBP-2 expression correlate with loss of function of PTEN, which is well known to lead to aggressive behavior of gliomas. Our results motivate translational research regarding the relationship between IGFBP-2 expression and loss of function of PTEN.     

Growth inhibition of breast epithelial cells by celecoxib is associated with upregulation of insulin-like growth factor binding protein-3 expression

Several experimental and epidemiological studies have suggested a role for the use of cyclooxygenase (COX)-2 inhibitors in the prevention of breast cancer. The relative lack of toxicity associated with these compounds favors their use as chemopreventive agents, but the underlying mechanism of their chemopreventive effect remains unclear. We have observed that the COX-2 inhibitor celecoxib inhibits growth and induces apoptosis in the immortalized breast epithelial cell line 184htert. Microarray gene expression analysis of 184htert cells treated with 50 microM celecoxib for 6h revealed the modulation of several genes of interest, including a significant induction of expression of the mRNA encoding insulin-like growth factor binding protein-3 (IGFBP-3). IGFBP-3 is a potent pro-apoptotic protein and growth inhibitor of breast cancer cells, which acts mainly by inhibiting the access of the mitogens IGF-I and IGF-II to their cell surface receptor, but also via IGF-independent effects. Quantitative real-time RT PCR demonstrated that 50 microM celecoxib induced a approximately 3-fold increase in expression of IGFBP-3 mRNA after 6h. Furthermore, ligand blot analysis revealed that celecoxib treatment was associated with the upregulation of IGFBP-3 at the protein level. IGFBP-3 (500 ng/ml) treatment of 184htert cells inhibited IGF-I and serum-induced proliferation, but had no effect on cell growth under serum-free conditions, indicating that IGF-independent effects of IGFBP-3 are not observed in this system. Our results suggest that celecoxib may decrease IGF-I-associated breast cancer risk by a mechanism involving induction of expression of IGFBP-3 and subsequent reduced proliferation of at-risk breast epithelial cells.     

Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M_r 150,000 (peak I kinase) and M_r 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC₅₀ = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC₅₀ = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-³²P]ATP lowered the incorporation of ³²P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-³²P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases. © 1996 Wiley-Liss, Inc.     

Insulin-like growth factor binding protein-3 (IGFBP-3): Novel ligands mediate unexpected functions

In addition to its important role in the regulation of somatic growth by acting as the major circulating transport protein for the insulin-like growth factors (IGFs), IGF binding protein-3 (IGFBP-3) has a variety of intracellular ligands that point to its function within major signaling pathways. The discovery of its interaction with the retinoid X receptor has led to the elucidation of roles in regulating the function of several nuclear hormone receptors including retinoic acid receptor-α, Nur77 and vitamin D receptor. Its interaction with the nuclear hormone receptor peroxisome proliferator-activated receptor-γ is believed to be involved in regulating adipocyte differentiation, which is also modulated by IGFBP-3 through an interaction with TGFβ/Smad signaling. IGFBP-3 can induce apoptosis alone or in conjunction with other agents, and in different systems can activate caspases −8 and −9. At least two unrelated proteins (LRP1 and TMEM219) have been designated as receptors for IGFBP-3, the latter with a demonstrated role in inducing caspase-8-dependent apoptosis. In contrast, IGFBP-3 also has demonstrated roles in survival-related functions, including the repair of DNA double-strand breaks through interaction with the epidermal growth factor receptor and DNA-dependent protein kinase, and the induction of autophagy through interaction with GRP78. The ability of IGFBP-3 to modulate the balance between pro-apoptotic and pro-survival sphingolipids by regulating sphingosine kinase 1 and sphingomyelinases may be integral to its role at the crossroads between cell death and survival in response to a variety of stimuli. The pleiotropic nature of IGFBP-3 activity supports the idea that IGFBP-3 itself, or pathways with which it interacts, should be investigated as targets of therapy for a variety of diseases.     

Decreased intracellular degradation of insulin-like growth factor binding protein-3 in cathepsin L-deficient fibroblasts

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) is the major mechanism of releasing IGFs from their IGFBP complexes. Analysis of fibroblasts deficient for the lysosomal cysteine protease cathepsin L (CTSL) revealed an accumulation of IGFBP-3 in the medium which was due neither to alterations in IGFBP-3 mRNA expression nor to extracellular IGFBP-3 protease activity. Incubation of CTSL-deficient fibroblasts with radiolabeled IGFBP-3 followed by subcellular fractionation indicates that both intact and fragmented IGFBP-3 accumulate transiently in endosomal and lysosomal fractions of CTSL-deficient cells. This suggests the involvement of CTSL in the intracellular degradation of IGFBP-3 representing a new mechanism to regulate the extracellular concentration of IGFBP-3.     

Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3.

In the circulation, most of the insulin-like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF-binding protein (IGFBP)-3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF-binding fraction separated from human plasma. IGF ligand blotting, cross-linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP-3 showed a 5-fold higher affinity for IGF-II than IGFBP-3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP-3 functions.     

Attenuation of insulin secretion by insulin-like growth factor binding protein-1 in pancreatic beta-cells

IGFBP-1 is involved in glucohomeostasis, but the direct action of IGFBP-1 on the beta-cell remains unclear. Incubation of dispersed mouse beta-cells with IGFBP-1 for 30min inhibited insulin secretion stimulated by glucose, glucagon-like peptide 1 (GLP-1) or tolbutamide without changes in basal release of insulin and in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and NAD(P)H evoked by glucose. In contrast, IGFBP-1 augmented glucose-stimulated insulin secretion in intact islets, associated with a reduced somatostatin secretion. These results suggest a suppressive action of IGFBP-1 on insulin secretion in isolated beta-cells through a mechanism distal to energy generating steps and not involving regulation of [Ca(2+)](i). In contrast, IGFBP-1 amplifies glucose-stimulated insulin secretion in intact islets, possibly by suppressing somatostatin secretion. These direct modulatory influences of IGFBP-1 on insulin secretion may imply an important regulatory role of IGFBP-1 in vivo and in the pathogenesis of type 2 diabetes, in which loss of insulin release is an early pathogenetic event.     

Orchestrating a biomarker panel with lncRNAs and mRNAs for predicting survival in pancreatic ductal adenocarcinoma

The low survival of patients with pancreatic ductal adenocarcinoma (PDAC) makes the treatment of this disease one of the most challenging task in modern medicine. Here, by mining a large‐scale cancer genome atlas data set of pancreatic cancer tissues, we identified 21 long noncoding RNAs (lncRNAs) that significantly associated with overall survival in patients with PDAC (P < .01). Further analysis revealed that 8 lncRNAs turned out to be independently correlated with patients’ overall survival, and the risk score could be calculated based on their expression. To obtain a better predicting power, we integrated lncRNA data with a total of 410 differently expressed messenger RNAs (mRNAs) screened from PDAC and normal tissues in gene expression omnibus (GEO) database. The integration resulted in a much better panel including 8 lncRNAs (RP3.470B24.5, CTA.941F9.9, RP11.557H15.3, LINC00960, AP000479.1, LINC00635, LINC00636, and AC073133.1) and 8 mRNAs (DHRS9, ONECUT1, OR8D4, MT1M, TCN1, MMP9, DPYSL3, and TTN) to predict prognosis. A functional evaluation showed that these lncRNAs might play roles in pancreatic secretion, cell adhesion, and proteolysis. Using normal and pancreatic cancer cell lines, we confirmed that a majority of identified lncRNAs and mRNAs showed altered expressions in pancreatic cancer cells. Especially, LINC01589, LINC00960, TCN1, and MT1M showed a profoundly increased expression in pancreatic cancer cells, which suggests their potentially important role in pancreatic cancer. The results of our work indicate that lncRNAs have vital roles in PADC and provide new insights to integrate multiple kinds of markers in clinical practices.     

Serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, and the pubertal growth spurt in the female rhesus monkey

While there is good evidence suggesting IGF-I links to pubertal development and crown-rump length growth among rhesus monkeys, linkages between IGF-I and other measures of morphological growth have not been established. In this study, the pubertal growth spurt in a number of morphological characteristics of female rhesus monkeys is related to serum endocrine status of insulin-like growth factor-I (IGF-I) and its binding protein, insulin-like growth factor binding protein-3 (IGFBP-3), to test the hypothesis that elevations in IGF-I and IGFBP-3 coincide with the time of greatest growth rate of different morphological characteristics. A longitudinal study of pubertal growth among four female rhesus monkeys was carried out across a 3-year period. Morphometric measurements included weight, crown-rump length, foot-length, and skinfolds at five sites (biceps, triceps, abdominal, subscapular, and suprailiac). These measures were taken as being representative of total mass, skeletal growth of the trunk and head, limb length, and body fatness, respectively. Measurements were carried out as closely as possible to 3-monthly, with interpolations being performed to standardise the data to exactly 3-monthly intervals for all individuals. Blood samples were taken at time of morphometry. Elevations in serum IGF-I and IGFBP-3 took place in a manner similar to that of humans, and across the period associated with onset of puberty. Mean 3-monthly gain in crown-rump length and foot length showed significant peaks across the measurement period, while mean 3-monthly gains in weight and sum of five skinfolds did not. Greatest foot length gain occurred on average between 3-3.5 years of age, while crown-rump length gain was greatest between 3.75-4 years of age. Periods of greatest gain in crown-rump length and foot length took place across the period of elevated serum IGF-I levels, which was between 3-4.5 years of age. Significant elevations in IGF-I and IGFBP-3 were not coincident with greatest gains in foot length or crown-rump length. Thus the hypothesis does not hold true for the two measures showing significant peaks in 3-monthly gain across the measurement period. The nature of the endocrine impact on macaque morphology remains unclear, although this may be fundamental to the understanding of the variation in the pubertal growth spurt and its influence on morphology at maturity both within and across primate species.