Overexpression Of IGFBP2 Research Articles

Glucose intolerance in aging male IGFBP-3 transgenic mice: differential effects of human IGFBP-3 and its mutant IGFBP-3 devoid of IGF binding ability.

We have reported a reduction of insulin secretion and glucose intolerance in young mice overexpressing human IGFBP-3 (phosphoglycerate kinase [PGK]BP3) or its mutant Gly56/Gly80/Gly81-IGFBP-3 (PGKmutBP3) under the PGK promoter. Here, we investigated changes in glucose and lipid homeostasis with age in PGKBP3 and PGKmutBP3 mice compared with wild-type mice. Body weight, glucose tolerance, insulin tolerance, visceral fat, interscapular brown adipose tissue (BAT), serum lipids, and pancreas histology were examined at age 3, 6, and 12 months. Murine IGFBP-3 was similar in all mouse genotypes and decreased with age in parallel with total IGF-1. Visceral fat and BAT masses increased in PGKmutBP3 mice, but not in PGKBP3 mice. Glucose tolerance was impaired in both PGKBP3 and PGKmutBP3 mice. However, PGKBP3 mice had increased expression of uncoupling protein-1 in BAT and reduced adiposity, and continued to have smaller pancreatic β-cell mass and reduced insulin secretion through age 12 months. In contrast, PGKmutBP3 mice developed insulin resistance with age in association with pancreatic β-cell hyperplasia, impaired expression of uncoupling protein-1 in BAT, and increased adiposity. In addition, both PGKBP3 and PGKmutBP3 mice had elevated glycerol in the circulation, but only PGKBP3 mice had elevated free fatty acids and only PGKmutBP3 mice had elevated triglycerides. Estimated free IGF-1 did not increase with age in transgenic mice, as it did in wild-type mice. Thus, overexpression of human IGFBP-3 or its mutant devoid of IGF binding ability leads to glucose intolerance with, however, different effects on insulin secretion, insulin sensitivity, and lipid homeostasis in aging mice.

IGFBP-2 inhibits adipogenesis and lipogenesis in human visceral, but not subcutaneous, adipocytes.

IGF-binding protein (IGFBP)-2 is the principal IGFBP produced by white adipocytes during adipogenesis, and circulating levels are reduced in obesity. Overexpression of IGFBP-2 in transgenic mice prevents obesity, but depot-specific effects of IGFBP-2 on adipo/lipogenesis are unknown. The present study aimed to investigate whether IGFBP-2 affects adipo/lipogenesis in a depot-specific manner and explore potential mechanisms. Following adipocyte characterisation, IGFBP-2 levels were measured from human subcutaneous and visceral preadipocytes, and IGFBP-2 dose-responses were then undertaken with exogenous IGFBP-2 in an in vitro IGF-I-free system to examine adipo/lipogenesis. Following this, both types of adipocytes were transfected with human siRNA IGFBP-2 to assess auto-/para-/intra-crine effects, with and without additional add-back IGFBP-2. To elucidate the potential mechanisms, visceral preadipocytes were treated with either wild-type or Heparin Binding Domain (HBD)-mutant IGFBP-2 (which is unable to bind to cell-surface components), and experiments were also undertaken using Echistatin (an integrin receptor blocker). Outcomes included gene expression profiles, protein levels and phosphorylation and lipid staining. Human visceral adipocytes produced significantly more IGFBP-2 than subcutaneous adipocytes. Subsequent dose-responses to IGFBP-2 demonstrated significant reductions in adipo/lipogenesis in visceral, but not subcutaneous, adipocytes in response to increasing IGFBP-2. Silencing IGFBP-2 resulted in exaggerated adipo/lipogenesis in visceral, but not subcutaneous, adipocytes, an effect completely inhibited by add-back IGFBP-2. These effects occurred in the absence of changes in IGF-I levels. HBD-mutant IGFBP-2 had reduced effects compared with wild-type IGFBP-2. Wild-type IGFBP-2 increased phosphorylation of focal adhesion kinase (FAK) and decreased phosphatase and tensin homolog (PTEN) levels, suggestive of integrin-mediated signalling. Blockade of this signalling, using Echistatin, completely negated the effects of IGFBP-2 on visceral adipo/lipogenesis. IGFBP-2 inhibits both adipogenesis and lipogenesis in visceral, but not subcutaneous, adipocytes. This depot-specific impairment appears to be independent of IGF-I and involves cell-surface association of IGFBP-2 and activation of integrin signalling pathways.

Genome-wide expression analysis in fibroblast cell lines from probands with Pallister Killian syndrome.

Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated) differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2.

Effects of Human IGFBP3 Overexpression on Mouse Brown Adipocyte Proliferation and Differentiation In Vitro

Effects of Human IGFBP3 Overexpression on Mouse Brown Adipocyte Proliferation and Differentiation In Vitro

Genome-wide association and functional studies identify a role for IGFBP3 in hip osteoarthritis

ObjectivesTo identify genetic associations with hip osteoarthritis (HOA), we performed a meta-analysis of genome-wide association studies (GWAS) of HOA.MethodsThe GWAS meta-analysis included approximately 2.5 million imputed HapMap single nucleotide polymorphisms...

STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma.

Insulin-like growth factor binding proteins (IGFBPs) have been implicated in the pathogenesis of glioma. In a previous study the authors demonstrated that IGFBP-3 is a novel glioblastoma biomarker associated with poor survival. Since signal transducer and activator of transcription 1 (STAT-1) has been shown to be regulated by IGFBP-3 during chondrogenesis and is a prosurvival and radioresistant molecule in different tumors, the aim in the present study was to explore the functional significance of IGFBP-3 in malignant glioma cells, to determine if STAT-1 is indeed regulated by IGFBP-3, and to study the potential of STAT-1 as a biomarker in glioblastoma. The functional significance of IGFBP-3 was investigated using the short hairpin (sh)RNA gene knockdown approach on U251MG cells. STAT-1 regulation by IGFBP-3 was tested on U251MG and U87MG cells by shRNA gene knockdown and exogenous treatment with recombinant IGFBP-3 protein. Subsequently, the expression of STAT-1 was analyzed with real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in glioblastoma and control brain tissues. Survival analyses were done on a uniformly treated prospective cohort of adults with newly diagnosed glioblastoma (136 patients) using Kaplan-Meier and Cox regression models. IGFBP-3 knockdown significantly impaired proliferation, motility, migration, and invasive capacity of U251MG cells in vitro (p < 0.005). Exogenous overexpression of IGFBP-3 in U251MG and U87MG cells demonstrated STAT-1 regulation. The mean transcript levels (by real-time RT-PCR) and the mean labeling index of STAT-1 (by IHC) were significantly higher in glioblastoma than in control brain tissues (p = 0.0239 and p < 0.001, respectively). Multivariate survival analysis revealed that STAT-1 protein expression (HR 1.015, p = 0.033, 95% CI 1.001-1.029) along with patient age (HR 1.025, p = 0.005, 95% CI 1.008-1.042) were significant predictors of shorter survival in patients with glioblastoma. IGFBP-3 influences tumor cell proliferation, migration, and invasion and regulates STAT-1 expression in malignant glioma cells. STAT-1 is overexpressed in human glioblastoma tissues and emerges as a novel prognostic biomarker.

Identification of head and neck squamous cell carcinoma biomarker candidates through proteomic analysis of cancer cell secretome

Protein biomarker discovery for early detection of head and neck squamous cell carcinoma (HNSCC) is a crucial unmet need to improve patient outcomes. Mass spectrometry-based proteomics has emerged as a promising tool for identification of biomarkers in different cancer types. Proteins secreted from cancer cells can serve as potential biomarkers for early diagnosis. In the current study, we have used isobaric tag for relative and absolute quantitation (iTRAQ) labeling methodology coupled with high resolution mass spectrometry to identify and quantitate secreted proteins from a panel of head and neck carcinoma cell lines. In all, we identified 2,472 proteins, of which 225 proteins were secreted at higher or lower abundance in HNSCC-derived cell lines. Of these, 148 were present in higher abundance and 77 were present in lower abundance in the cancer-cell derived secretome. We detected a higher abundance of some previously known markers for HNSCC including insulin like growth factor binding protein 3, IGFBP3 (11-fold) and opioid growth factor receptor, OGFR (10-fold) demonstrating the validity of our approach. We also identified several novel secreted proteins in HNSCC including olfactomedin-4, OLFM4 (12-fold) and hepatocyte growth factor activator, HGFA (5-fold). IHC-based validation was conducted in HNSCC using tissue microarrays which revealed overexpression of IGFBP3 and OLFM4 in 70% and 75% of the tested cases, respectively. Our study illustrates quantitative proteomics of secretome as a robust approach for identification of potential HNSCC biomarkers. This article is part of a Special Issue entitled: An Updated Secretome.

Tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone suppresses a newly identified anti-tumor IGFBP-3/IGFBP-3R system in lung cancer cells

IGFBP-3 is a tumor suppressor whose expression is frequently suppressed in lung cancer. NNK, the most potent tobacco carcinogen, enhanced cell proliferation of BEAS-2B normal lung epithelial cells and concomitantly suppressed IGFBP-3 expression through DNA methylation. Decreased IGFBP-3 expression and elevated levels of phospho-Akt, phospho-p65-NF-κB, and cyclin D1 were detected in tobacco carcinogen-induced tumorigenic derivatives of BEAS-2B. Overexpression of IGFBP-3 in NNKA, one of the derivatives, suppressed NF-κB activity and induced apoptosis, which was hindered by knocking-down of endogenous IGFBP-3R, an IGFBP-3 specific receptor. These results suggest that NNK inhibits IGFBP-3 expression to abrogate anti-tumor actions of the IGFBP-3/IGFBP-3R system in smoking-induced lung cancer.

Insulin-like growth factor binding protein-4 and -5 modulate ligand-dependent estrogen receptor-α activation in breast cancer cells in an IGF-independent manner

Insulin-like growth factor binding proteins (IGFBPs) are modulators of numerous cellular processes including cell proliferation. Although IGFBPs classically act by sequestration of extracellular insulin-like growth factors (IGFs), thereby contributing to the fine-tuning of growth factor signals, IGF-independent actions of IGFBPs have also been described. In the breast, growth factor signaling in association with estradiol (E2)-stimulated estrogen receptor function is organized in a complex cross-talk. The importance of phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) pathway components for the E2-induced activation of estrogen receptor-alpha (ERα) is well accepted. Here we show that in the absence of IGFs, IGFBP-4 or IGFBP-5, either overexpressed in MCF-7 breast cancer cells or added exogenously, decreased the capability of E2 to induce ERα transcriptional activity. In addition, overexpression or addition of recombinant IGFBP-4 or IGFBP-5 resulted in reduction of E2-induced phosphorylation of Akt/PKB, GSK-3α/β and ERα in MCF-7 cells. The activation of the Akt/PKB-pathway describes a non-genomic effect of E2, which did not involve activation/phosphorylation of the IGF-I receptor (IGF-IR). Furthermore, knockdown of the IGF-IR did not affect the inhibition of E2-induced ERα phosphorylation by IGFBP-4 and 5. Moreover, IGFBP-4 and IGFBP-5 strongly decreased E2-triggered growth of MCF-7 cells. Our data suggest that IGFBPs interfere with the E2-induced activation of the Akt/PKB-pathway and prevent full hormone-dependent activation of ERα and breast cancer cell growth in an IGF- and IGF-IR-independent manner.

Regulation of retinal endothelial cell apoptosis through activation of the IGFBP-3 receptor

The goal of this study was to investigate whether insulin-like growth factor binding protein-3 receptor (IGFBP-3 receptor) is required for IGFBP-3 to inhibit retinal endothelial cell (REC) apoptosis. REC were grown in normal glucose (5 mM) or high glucose medium (25 mM) for 3 days. Once cells reached confluence, they were transfected with an endothelial- specific IGFBP-3 plasmid DNA (non-IGF binding; IGFBP-3 NB) at 1 μg/ml for 24 h. Cell proteins were extracted and analyzed for IGFBP-3 receptor expression by Western blotting or use in coimmunoprecipitation or co-localization experiments for detection of IGFBP-3 and IGFBP-3 receptor binding. REC were also transfected with or without IGFBP-3 receptor siRNA before IGFBP-3NB plasmid DNA transfection. Cell lysates were processed for a cell death ELISA, a cleaved caspase 3 ELISA, and Western blotting to measure key pro- and anti-apoptotic markers: Bcl-xL, Bax, Cytochrome C and Akt. The IGFBP-3 receptor is present on REC. Overexpression of IGFBP-3 in REC significantly increased protein levels of IGFBP-3 receptor (p < 0.05). Significant increases in cell death were found in cells transfected with IGFBP-3 receptor siRNA versus not treated samples (p < 0.05). Data suggest that IGFBP-3 inhibits retinal endothelial cell death through activation of an IGFBP-3 receptor in a hyperglycemic environment. This is the first demonstration of the involvement of IGFBP-3 receptor in inhibition of REC cell death. Future studies will investigate the mechanism by which IGFBP-3 receptor may inhibit retinal endothelial cell death.

Role of insulin-like growth factor binding protein-3 in glucose and lipid metabolism.

Insulin-like growth factor binding protein (IGFBP)-3 has roles in modulating the effect of IGFs by binding to IGFs and inhibiting cell proliferation in an IGF-independent manner. Although recent studies have been reported that IGFBP-3 has also roles in metabolic regulation, their exact roles in adipose tissue are poorly understood. In this review, we summarized the studies about the biological roles in glucose and lipid metabolism. IGFBP-3 overexpression in transgenic mice suggested that IGFBP-3 results in glucose intolerance, and insulin resistance. IGFBP-3 knockout (KO) mice exhibited normal insulin level and glucose response after glucose challenge. More recent study in IGFBP-3 KO mice with a high-fat diet demonstrated that IGFBP-3 KO mice exhibited elevated fasting glucose and insulin, but normal response to glucose challenge, suggesting that IGFBP-3 KO mice may induce insulin resistance even though preserved insulin sensitivity. In vitro and in vivo studies using 3T3-L1 adipocytes and rat, IGFBP-3 induced insulin resistance by inhibiting glucose uptake. In contrast, the reduced levels of IGFBP-3 in obesity might induce insulin resistance by suppression of IGFBP-3's anti-inflammatory function, suggesting IGFBP-3 has a protective effect on insulin resistance. Also, proteolysis of IGFBP-3 might contribute to the insulin resistance in obesity and type 2 diabetes mellitus. In addition, IGFBP-3 inhibited adipocyte differentiation, suggesting IGFBP-3 may contribute to the insulin insensitivity. Taken together, it is not yet certain that IGFBP-3 has a protective effect or enhancing effect on insulin resistance, and more studies will be needed to clarify the roles of IGFBP-3 in metabolic regulation.

IGFBP-3 sensitizes antiestrogen-resistant breast cancer cells through interaction with GRP78

IGFBP-3 is known to possess intrinsic biological activities such as anti-tumor property in addition to its IGF/IGF-R axis-dependent actions in a variety of human cancers including breast cancer. To investigate the molecular mechanisms underlying the intrinsic biological actions of IGFBP-3 on breast cancer cells, we performed yeast two-hybrid screening and found GRP78, known to cause drug-resistance, as a binding partner of IGFBP-3. Overexpression of IGFBP-3 in antiestrogen-resistant LCC9 cells showed that IGFBP-3 interacted with GRP78, resulting in disruption of the GRP78-caspase-7 complex, thereby activating caspase-7, and further inducing apoptosis. Combination of overexpression of IGFBP-3 and application of siRNAs against GRP78 led to decrease in cell viability upon ICI 182,780 treatment. These data suggest that IGFBP-3 could sensitize antiestrogen-resistant breast cancer cells to ICI 182,780 by preventing the anti-apoptotic function of GRP78.

Abstract LB-466: Genomic Loss of microRNA 491 contributes to IGFBP2 overexpression and stemness in GBM

Abstract Insulin-like growth factor-binding protein 2 (IGFBP2) is overexpressed in 80% of glioblastoma multiforme (GBM, WHO grade IV). Our mouse model experiments demonstrated that IGFBP2 is a glioma oncogene. However, the underlying mechanism for overexpression of IGFBP2 remains unknown. We examined a total of 388 GBM samples from the Cancer Genome Atlas (TCGA) and found that miR-491, which is located on chr9p21.3, is deleted in 57% of cases. TargetScan analysis revealed that there is a binding site of miR-491 on the 3′-UTR of the IGFBP2 gene. The miR-491 expression levels in GBM patients are inversely correlated with that of IGFBP2. We validated this regulatory relationship by overexpression of miR-491 and observed down-regulation of IGFBP-2 in both protein level and mRNA level in U251 and T98G glioma cell lines. Conversely, miR-491 inhibitors increased IGFBP2 in LN229 cells which express low level of endogenous IGFBP2. Luciferase reporter assay further proved that IGFBP2 is a direct target of miR-491. Moreover, ectopic expression of miR-491 inhibited cell migration and invasion of U251 and T98G as efficiently as knockdown of IGFBP2 and such inhibitory effects can be partially reversed by overexpression of IGFBP2-EGFP in U251, supporting the notion that IGFBP2 plays an important role in miR-491's functions. To investigate the role of miR-491 in stemness of GBM, we introduced miR-491 into 3 patient-derived glioma stem cell lines and found that miR-49 downregulated CDK6 and Bcl-xL and inhibited neurosphere formation and cell proliferation. These findings suggest that miR-491 acts as a tumor suppressor miR by coordinately attenuating multiple oncogenes in GBM. Our results provide new insights into the mechanism for oncogenic IGFBP2 overexpression and may provide new opportunities for therapeutic strategies for GBM treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-466. doi:1538-7445.AM2012-LB-466

Overexpression of insulin like growth factor binding protein 5 reduces liver fibrosis in chronic cholangiopathy

The ATP-binding cassette, sub-family B member 4 knock-out mouse (Abcb4−/−) is a relevant model for chronic cholangiopathy in man. Due to the lack of this P-glycoprotein in the canalicular membrane of hepatocytes, the secretion of phospholipids into bile is absent, resulting in increased bile toxicity. Expression of insulin like growth factor binding protein 5 (Igfbp5) increases in time in the livers of these mice. It is unclear whether this induction is a consequence of or plays a role in the progression of liver pathology. The aim of this study was therefore to investigate the effect of IGFBP5 induction on the progression of liver fibrosis caused by chronic cholangiopathy. IGFBP5 and, as a control, green fluorescent protein were overexpressed in the hepatocytes of Abcb4−/− mice, using an adeno-associated viral vector (AAV). Progression of liver fibrosis was studied 3, 6, and 12weeks after vector injection by analyzing serum parameters, collagen deposition, expression of pro-fibrotic genes, inflammation and oxidative stress. A single administration of the AAV vectors provided prolonged expression of IGFBP5 and GFP in the livers of Abcb4−/− mice. Compared to GFP control, fractional liver weight, extracellular matrix deposition and amount of activated hepatic stellate cells significantly decreased in IGFBP5 overexpressing mice even 12weeks after treatment. This effect was not due to a change in bile composition, but driven by reduced inflammation, oxidative stress, and proliferation. Overexpression of IGFBP5 seems to have a protective effect on liver pathology in this model for chronic cholangiopathy.

Insulin-like growth factor binding protein-3 inhibits migration of endometrial cancer cells

Cell migration and invasion leading to metastasis is a major cause of death from endometrial cancer (EC). We have shown that the rate of EC cell migration is inversely related to the level of insulin-like growth factor protein-3 (IGFBP-3). Down-regulation of IGFBP-3 by siRNA in EC cells accelerated migration without affecting proliferation and cells displayed a more migratory phenotype, with co-localization of migration-associated markers at the leading edge of cell membranes. Opposite effects were seen with either the addition of recombinant IGFBP-3 or overexpression of IGFBP-3. Cells with mutated PTEN had the highest IGFBP-3 expression and the slowest migration rates. This study demonstrates that endogenous IGFBP-3 modulates adhesion–migration dynamics in EC cells, implying that it may be important in regulating metastasis in this disease.

IGFBP-2 expression, angiogenesis and pseudopalisades in glioblastoma

Abstract Glioblastoma is the most frequent, the most malignant and the best vascularised primary brain tumor. Substantial evidence suggests that IGFBP-2 (insulin-like growth factor binding protein) may play a significant role in the development and progression of various types of cancer, including the anaplastic progression of astroglial tumors. The correlation of IGFBP-2 and VEGF over-expression in diffuse gliomas and strong IGFBP-2 immunostaining and co-expression with VEGF in the cells of pseudopalisading necrosis suggests the induction of IGFBP-2 expression by hypoxia and/or possible involvement of its protein product in angiogenesis. The aim of this study was to analyze the expression of IGFBP-2 and the relationship between the IGFBP-2 expression, extent of bizarre angiogenesis and the presence of pseudopalisades. The prognostic impact of these variables was estimated, as well. Our results revealed that most glioblastoma (75%) express IGFBP2 and that IGFBP2 expression was associated with the presence of pseudopalisading necrosis as well as extensive bizarre angiogenesis. IGFBP-2 immunoreactivity in more than 5% of tumor cells and the presence of pseudopalisading necrosis were prognostically significant in univariate analysis (p=0.034) while in multivariate analysis only a patient’s age and the presence of pseudopalisades remained statistically significant (p=0.004). The results of our study showed the association of IGFBP-2 expression in glioblastoma with pseudopalisades and bizarre angiogenesis thus confirming on morphological grounds presumed induction of this gene under hypoxic conditions and its possible role in angiogenesis.

IGFBP-2 Enhances VEGF Gene Promoter Activity and Consequent Promotion of Angiogenesis by Neuroblastoma Cells

IGF binding protein (IGFBP)-2 is one of the most significant genes in the signature of major aggressive cancers. Previously, we have shown that IGFBP-2 enhances proliferation and invasion of neuroblastoma cells, suggesting that IGFBP-2 activates a protumorigenic gene expression program in these cells. Gene expression profiling in human neuroblastoma SK-N-SHEP (SHEP)-BP-2 cells indicated that IGFBP-2 overexpression activated a gene expression program consistent with enhancement of tumorigenesis. Regulation was significant for genes involved in proliferation/survival, migration/adhesion, and angiogenesis, including the up-regulation of vascular endothelial growth factor (VEGF) mRNA (>2-fold). Specific transcriptional activation of the VEGF gene by IGFBP-2 overexpression was demonstrated via cotransfection of a VEGF promoter Luciferase construct in SHEP-BP-2. Cotransfection of VEGF promoter Luciferase construct with IGFBP-2 protein in wild-type SHEP cells indicated that transactivation of VEGF promoter only occurs in the presence of intracellular IGFBP-2. Cell fractionation and immunofluorescence in SHEP-BP-2 cells demonstrated nuclear localization of IGFBP-2. These findings suggest that transcriptional activation of VEGF promoter is likely to be mediated by nuclear IGFBP-2. The levels of secreted VEGF (up to 400 pg/10(6) cells) suggested that VEGF might elicit angiogenic activity. Hence, SHEP-BP-2 cells and control clones cultured in collagen sponge were xenografted onto chick embryo chorioallantoic membrane. Neomicrovascularization was observed by 72 h, solely in the SHEP-BP-2 cell xenografts. In conclusion, our data indicate that IGFBP-2 is an activator of aggressive behavior in cancer cells, involving nuclear entry and activation of a protumorigenic gene expression program, including transcriptional regulation of the VEGF gene and consequent proangiogenic activity of NB cell xenografts in vivo.

Functional characterization of IGFBP5 in the inhibition of osteosarcoma tumorgenicity.

10038 Background: Osteosarcoma (OS) is the most common primary malignancy of bone. At presentation, the majority of patients have metastatic disease. Therefore, there is a clinical need to identify molecular markers that provide insight into the pathogenesis OS. The goal of this investigation is to functionally characterize the novel marker, IGFBP5, in osteosarcoma tumor progression. Methods: We created adenoviral constructs expressing the unique domains of IGFBP5 (N-term, C-term, linker), full length IGFBP5, and siIGFBP5. We conducted in vitro and in vivo assays on adenovirally infected OS cell lines MG63, MG63.2, and 143B. Western blot and immunohistochemical (IHC) staining were used to assay the apoptotic marker Caspase 3. Cell migration and invasion were measured using Wound Healing and Matrigel assays. Osteoblastic differentiation was measured using alkaline phosphastase activity and IHC staining for the late osteogenic markers osteocalcin and osteopontin. Finally, clinical patient samples were stained for IGFBP5 expression in patient matched primary tumors and metastatic pulmonary lesions. Results: IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and osteogenic differentiation. Analysis of the three functional domains indicated that the N-terminal domain was primarily responsible for inhibiting cell proliferation and inducing apoptosis, while the C-terminal domain inhibited cell migration and invasiveness. In an orthotopic animal model, overexpression of IGFBP5 inhibited OS tumor growth and lung metastasis, while siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and lung metastasis. IHC staining of clinical OS samples confirmed that primary OS tumors expressed significantly lower levels of IGFBP5 than their corresponding pulmonary metastases. Conclusions: These results suggest that IGFBP5 is a novel marker in OS pathogenesis, and the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS. Furthermore, while the N-terminal domain of IGFB5 is responsible for inhibition of cell proliferation, the C-terminal domain is responsible for inhibiting cell invasion and migration.

Abstract 4321: A critical role for IGFBP2 in the progression and maintenance of glioma

Abstract Despite recent advances in detection methods and treatments for other tumor types, the standard of care for glioma patients remains relatively unchanged and survival rates remain dismal. Identification of key signaling nodes that function throughout glioma development will likely provide new targets for intervention. To elucidate the role of the critical oncogene, IGFBP2, in the progression and maintenance of glioma, we generated an inducible mouse model. This model utilizes the RCAS/Tv-a system, which places the Tv-a receptor for the avian RCAS virus under the control of the Nestin promoter to drive Tv-a expression in glial progenitors. The RCAS virus, expressing the desired oncogenes, infects Tv-a expressing cells and delivers the oncogene. By using RCAS to express a tetracycline-responsive IGFBP2 and a tetracycline transactivator, we can modify this system to generate an inducible IGFBP2 model. Using this model, we have tested the hypothesis that IGFBP2 is a key target for glioma therapy due to its important role in glioma maintenance. The studies described here aim to: (1) determine the effects of delayed IGFBP2 expression on the progression of PDGFB-induced, low-grade gliomas; and (2) determine whether continued expression of IGFBP2 is required to maintain the high-grade nature of gliomas induced by the co-expression of PDGFB and IGFBP2. Previously, our lab and others determined that over-expression of PDGFB in glial progenitors led to the development of low-grade glioma. Our lab also determined that IGFBP2 cooperates with PDGFB to promote progression to high-grade glioma. In the inducible model system, tetracycline-mediated IGFBP2 induction in vivo was confirmed by both quantitative RT-PCR of mouse brain tissue and serum ELISA, indicating that RCAS-delivered IGFBP2 is both expressed and secreted upon induction. IGFBP2 induction from birth increased the proportion of tumors that progressed to high-grade glioma, whereas delayed IGFBP2 induction led to later onset of high-grade tumor development. Significantly, there was a decrease in tumor-free survival upon IGFBP2 induction. This is consistent with analysis of transcriptome data from the Rembrandt and TCGA databases. These studies validate an important role for IGFBP2 in the progression of glioma. Ongoing studies using the inducible system are exploring the response of high-grade gliomas to sustained and brief inactivation of IGFBP2. Elucidating the roles of IGFPB2 in glioma progression and maintenance will provide critical information pertinent to the design and implementation of future therapies to target this molecule or associated signaling pathways, as well as how IGFBP2 might be useful as a marker for disease state. Further, these studies may have a more global impact on cancer research as IGFBP2 over-expression has also been associated with other high-grade tumors such as prostate and breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4321. doi:10.1158/1538-7445.AM2011-4321

Abstract 5004: Functional modulation of insulin-like growth factor binding protein-3 in melanoma

Abstract Insulin-like growth factor binding protein-3 (IGFBP3) is a member of the IGFBP family, which regulates the mitogenic and anti-apoptotic effects of insulin-like growth factors. Hypermethylation of the IGFBP3 promoter has been found to suppress its expression in different tumors. In the present study we evaluated the role of IGFBP3 in melanoma. Quantitative real time PCR (qRT-PCR) of 12 melanoma cell lines indicated a significant down-regulation of IGFBP3 expression as compared to a normal melanocyte cell line. qRT-PCR of human melanoma tissues showed significant downregulation of IGFBP3 as compared to nevus samples. Melanoma cell lines treated with the demethylating agent 5-AZA-2′deoxycytidine (5AZA) re-expressed IGFBP3 at the mRNA level, indicating a possible role for methylation in the suppression of its expression. We examined the methylation status of the IGFBP3 promoter in cell lines and in human tissue samples by bisulfite DNA sequencing. The promoter region of IGFBP3 in C8161.9 and DO4 melanoma cell lines was hypermethylated, which was completely reversed by treatment of these cells with 5AZA. The IGFBP3 promoter region was also highly methylated in human melanoma samples as compared to normal nevus samples. Overexpressing IGFBP3 in melanoma cells in vitro suppressed cell survival and colony formation and increased rate of apoptosis. Stable overexpression of IGFBP3 suppressed melanoma cell growth in vivo. Our results indicate that silencing of IGFBP3 in melanoma is due to the methylation of its promoter, and overexpression of IGFBP3 induces apoptosis and suppresses cell survival and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5004.