3486

IGFBP3

insulin-like growth factor binding protein 3;IGFBP3;insulin-like growth factor binding protein 3

IBP-3|IGF-binding protein 3|IGFBP-3|acid stable subunit of the 140 K IGF complex|binding protein 29|binding protein 53|growth hormone-dependent binding protein|insulin-like growth factor-binding protein 3

7p12.3

protein-coding

Over the last few decades, a great deal of attention has been directed to the IGF system for its vital role in regulating cell and tissue survival, growth and differentiation. The insulin-like growth factor binding proteins (IGFBPs), a main constituent of this system, have been implicated in the tumorigenesis of colorectal cancer (CRC). In this study, we intended to shed more light on two essential members; IGFBP3 as representative for the six main IGFBPs and IGFBP7 to represent their related proteins (IGFBP-rps). Our experiments on silencing IGFBP3 or IGFBP7 in the two human CRC cell lines SW480, Caco2, and in the rat CRC cell line CC531 show reduced proliferation, colony formation, and for IGFBP3, also reduced migration. The expression of both genes in 68 human CRC samples was higher in UICC stages II and III than in stages I and IV. Additionally, IGFBP3 was negatively correlated with age (p = 0.05) and positively related to IGFBP7 expression (p = 0.0001). Further, in a liver metastasis experiment, the expression of both genes was drastically increased in response to early metastatic growth in vivo. Since these high levels returned gradually to normal thereafter, it could be assumed that the up-regulation of IGFBPs is vital during the process of homing into the liver and early metastatic dissemination. Our results indicate that IGFBP3 and 7 cannot be simply considered as tumor suppressors but have additional properties, which become evident only during cancer progression and metastasis formation.

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.

Insulin-like growth factor-binding protein-3 (IGFBP-3) expression is frequently suppressed in liver cancers and can be reactivated by histone deacetylase (HDAC) inhibition. This study examined the role of IGFBP-3 in mediating the effects of the HDAC inhibitor MS-275 in liver cancer cells and identified IGFBP-3-dependent proteins that regulate proliferation and migration. In HepG2 cells, MS-275 inhibited DNA synthesis, cell cycle activity, and cell viability concomitantly with increased binding of acetylated histone H3 to IGFBP-3 promoter sequences and induction of IGFBP-3 expression. IGFBP-3 down-regulation by siRNA significantly reversed the inhibition of cell viability and DNA synthesis by MS-275, indicating an intermediary role for IGFBP-3. Induction of the cyclin-dependent kinase inhibitor p21 by MS-275 was attenuated by IGFBP-3 down-regulation, providing an explanation for IGFBP-3-dependent effects of MS-275 on cell cycle activity. In contrast, MS-275 stimulated HepG2 cell migration, an effect also inhibited by IGFBP-3 down-regulation. Among genes whose induction by MS-275 was attenuated by IGFBP-3 down-regulation, LYVE1 and THBS2 (thrombospondin-2) were identified as mediators of IGFBP-3-dependent effects of MS-275. Silencing of either protein had no effect on the inhibition of HepG2 viability by MS-275 but reversed its stimulatory effect on cell migration. We conclude that among genes up-regulated by MS-275, IGFBP-3 is a key mediator of effects on hepatoma cell growth and migration, involving IGFBP-3-dependent proteins p21 (proliferation) and LYVE1 and THBS2 (migration). The enhanced cell motility that accompanies reactivation of IGFBP-3 expression in liver cancer by HDAC inhibition suggests the possibility of increased metastatic spread despite inhibited cell proliferation.

Previous studies from our laboratory have identified several endothelial cell-associated marker genes implicated in human melanoma metastasis via tumor vasculogenic mimicry. In this study, we used dual model systems composed of melanoma cell lines and clinical melanoma samples to validate the importance of insulin-like growth factor binding protein-3 (IGFBP-3) as a marker involved in disease progression. Gene expression analysis was done using a microarray approach for both primary and metastatic melanoma samples. The expression of IGFBP-3 was decreased using a small interfering RNA (siRNA) knockdown approach and quantified with real-time quantitative reverse transcription-PCR analysis. The expression of insulin-like growth factor binding protein 3 (IGFBP-3) was up-regulated by nearly 16-fold in WM266-4 compared with WM35 cells. A subsequent parallel analysis using freshly isolated primary and metastatic melanoma cell samples and melanoma tissue array confirmed the previous findings. The functional significance of IGFBP-3 in melanoma invasion was further investigated using a siRNA gene knockdown approach, with the expression of IGFBP-3 markedly reduced. Additionally, siRNA knockdown resulted in a significant reduction in cell motility, migration, and invasive capacity of WM266-4 cells in vitro. These results strongly suggest that IGFBP-3 expression may be a vital cell motility, migration, and proliferation factor necessary for melanoma metastasis and is an important biomarker in human melanoma.

metastasis and invasion occur in the majority of epithelial ovarian carcinoma at diagnosis. To delineate the molecular signature in ovarian cancer invasion, we established and characterized a human ovarian endometrioid carcinoma (EC) cell line OVTW59-P0 and its invasion-related sublines (P1-P4, in the order of increasing invasive activity). P4 showed faster migration and larger xenograft formation with metastasis than P0. By microarray analysis of different gene expression among P0-P4 sublines, one group of gene was found negatively correlated with cancer invasion. Among these genes, IGFBP-3 was identified as one of the most remarkably suppressed gene that showed lower gene expression in P4 than P0. Re-expression of IGFBP-3 in P4 effectively inhibited cell migration, invasion and metastasis, but did not affect cell proliferation. In 35 patients with EC tumors, low IGFBP-3 expression correlated clinically with higher tumor grade, advanced stage and poor survival. Our results provide evidence and indicate that IGFBP-3 plays an important role as an invasion-metastasis suppressor in ovarian EC.

We recently reported on higher expression of the insulin-like growth factor pathway genes IGF-II and IGFBP3 in serous ovarian/peritoneal carcinoma compared to malignant peritoneal mesothelioma. The present study analyzed the diagnostic and clinical role of these proteins in serous effusions. Effusions (n = 327), including 294 carcinomas (205 ovarian, 48 breast, 17 cervical/endometrial, 12 lung, 12 gastrointestinal/genitourinary) and 33 malignant mesotheliomas, were immunostained for insulin-like growth factor-II and insulin-like growth factor binding protein-3. Surgical ovarian carcinoma (n = 124) and peritoneal mesothelioma (n = 18) specimens were additionally studied. Insulin-like growth factor binding protein-3 levels were measured in 148 effusion supernatants (114 ovarian carcinomas, 18 breast carcinomas, 16 mesotheliomas) using enzyme-linked immunosorbent assay. Insulin-like growth factor binding protein-3 promoter methylation was analyzed in 11 ovarian carcinoma effusions. Insulin-like growth factor binding protein-3 (P = .002) and insulin-like growth factor-II (P < .001) expression by immunohistochemistry was significantly higher in carcinomas compared to mesotheliomas, with diagnostic sensitivity of 77% and 70% and specificity of 55% and 70%, respectively. In surgical specimens, insulin-like growth factor binding protein-3 expression was higher in ovarian carcinomas compared to peritoneal mesotheliomas (P = .007), whereas insulin-like growth factor-II expression was comparable (P = .505). Insulin-like growth factor binding protein-3 levels by enzyme-linked immunosorbent assay were comparable in the 3 analyzed cancer types. Insulin-like growth factor binding protein-3 promoter methylation was found in 6 of 11 effusions. High insulin-like growth factor binding protein-3 expression in prechemotherapy and high insulin-like growth factor-II expression in postchemotherapy ovarian carcinoma effusions correlated with poor overall survival (P = .031 and P = .024, respectively). Insulin-like growth factor-II expression in postchemotherapy effusions was an independent prognostic factor in Cox multivariate analysis (P = .04). In conclusion, insulin-like growth factor-II and insulin-like growth factor binding protein-3 are more frequently expressed in metastatic carcinomas compared to mesothelioma in effusions but are less specific than currently used markers. Insulin-like growth factor-II and insulin-like growth factor binding protein-3 may be novel prognostic markers in metastatic ovarian carcinoma.

Pancreatic endocrine neoplasms are neoplastic proliferations of islet cells or islet cell precursors and are capable of secreting a variety of synthetic products, including insulin, glucagon, gastrin, and vasoactive intestinal peptide. The biological behavior of pancreatic endocrine neoplasms is often unpredictable, and there are few reliable histopathologic criteria reliably correlating with metastatic ability. We have used the Affymetrix U133 GeneChip set (HG_U133 A and B; Affymetrix; Santa Clara, CA) representing approximately 33,000 characterized transcripts to examine global gene expression profiles from well-differentiated nonmetastatic (n=5) and metastatic (n=7) pancreatic endocrine neoplasms to determine molecular markers that predict disease progression. Microarray hybridization data were normalized using the GeneLogic GeneExpress Software System to identify differentially up- and down-regulated genes in metastatic versus nonmetastatic pancreatic endocrine neoplasms. Using a 3-fold change in gene expression as a threshold, we have identified 65 overexpressed and 57 underexpressed genes in metastatic pancreatic endocrine neoplasms as compared with nonmetastatic pancreatic endocrine neoplasms. Several classes of genes, including growth factors and growth factor-related molecules (IGFBP1, IGFBP3, and MET), developmental factors (TBX3 and MEIS2), cytoskeletal factors (beta 1 tubulin and ACTN2), cholesterol homeostasis mediators (LRP5, SLC27A2, and RXRG), intracellular signaling pathway mediators (DYRK1A, PKIB, and AK2), methyltransferases (MGMT and GAMT), and DNA repair and regulatory molecules (CHEK1 and ZNF198), were identified as differentially over- or underexpressed via this method. Immunohistochemical validation of microarray data were performed for two overexpressed genes, namely, the met proto-oncogene (MET) and insulin-like growth factor binding protein 3 (IGFBP3) with tissue microarrays of nonmetastatic (n=24) and metastatic (n=15) pancreatic endocrine neoplasms. Increased expression of IGFBP3 was confirmed in metastatic versus nonmetastatic pancreatic endocrine neoplasms (12 of 15, 80% versus 10 of 24, 42%), as well as in lymph node (6 of 7, 86%) and liver (9 of 9, 100%) metastases. Similarly, overexpression of MET was confirmed in metastatic versus nonmetastatic pancreatic endocrine neoplasms (5 of 15, 33% versus 4 of 24, 17%), as well as in lymph node metastases (4 of 7, 57%) and liver metastases (5 of 9, 56%). The majority of genes that demonstrated altered expression has not been previously identified as differentially expressed in metastatic pancreatic endocrine neoplasm lesions and may therefore represent newly identified molecules in the progression of these lesions.

BACKGROUND: Insulin-like growth factor I (IGF-I), a potent proximal tubule cell (PTC) mitogen, has been implicated in the progression of many human cancers. Our previous work on human renal tissues has suggested that IGF-I and several of its binding proteins (IGFBP-3 and -6) are up-regulated in clear cell renal cell carcinoma (RCC). METHODS: To further elucidate the role of IGF-I and IGFBPs in RCC growth, immunohistochemistry, thymidine incorporation, and Western analysis were performed in primary cultures of normal PTC (priPTC) and clear-cell RCC (priRCC), as well as in SN12K1 cells (a cell line derived from metastatic RCC). RESULTS: By immunohistochemistry, IGFBP-3 and IGF-I were prominently expressed in SN12K1 cells, and weakly expressed in priPTC and priRCC. Incubation with 100 ng/mL IGF-I significantly augmented DNA synthesis by priPTC (mean +/- SD 120.7%+/- 19.7% of controls, P < 0.05), priRCC (238.7%+/- 279.9% of controls, P < 0.01), and SN12K1(120.0%+/- 22.9% of controls, P < 0.05). Neutralizing antibodies to IGF-I and IGF-I receptor significantly suppressed SN12K1 growth (81.9%+/- 13.5% of control, P < 0.01 and 87.4%+/- 16.2% of control, P < 0.05, respectively). Removal of endogenous IGFBP-3 by an anti-IGFBP-3 increased SN12K1 DNA synthesis (243.9%+/- 35.3% of control, P < 0.001), which was partially abrogated by coincubation with exogenous IGFBP-3 (135.97%+/- 5.9% of controls, P < 0.001). Using Western analysis, IGFBP-3 expression was enhanced in IGF-I-stimulated SN12K1 cells exposed to exogenous IGF-I. Coincubation with anti-IGFBP-3 further enhanced IGF-I-induced DNA synthesis. CONCLUSION: RCC cells express IGF-I and IGFBP-3, and are responsive to exogenous IGF-I stimulation. Moreover, in SN12K1 cells (derived from metastatic RCC), autocrine IGF-I and IGFBP-3 actions, respectively, stimulated and inhibited growth. These results suggest that IGF-I and IGFBP-3 may be potential candidates for therapeutic manipulation in patients with advanced RCC.