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1.  IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment 
Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
PMCID: PMC3902230  PMID: 24482736
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
2.  Methylation status of insulin-like growth factor-binding protein 7 concurs with the malignance of oral tongue cancer 
Aberrant insulin-like growth factor-binding protein 7 (IGFBP-7) expression has been found in various cancers such as prostate, breast, and colon. IGFBP-7 induced the apoptosis of tumor and potentially predicted the clinical outcome in some cancers is further demonstrated. This study investigates the causes and underlying mechanisms of aberrant IGFBP-7 expression in unravelling head and neck squamous cell carcinoma (HNSCC).
A total of 47 oral tongue cancer patient samples were primarily analyzed for the methylation status in 5′ region of IGFBP-7 by methylation-specific PCR (MS-PCR). Subsequently the invasion, overexpression, and knockdown of IGFBP-7 in the HNSCC A253 invasive subpopulation were employed to examine the effect of IGFBP-7. The epithelial–mesenchymal transition (EMT) marker genes and AKT/GSK3β/β-catenin signaling were further evaluated by Western blot for the understanding the role of aberrant IGFBP-7 expression and thereof putative mechanism.
EMT expressed in the invasive subpopulation of HNSCC cell lines (A253 and RPMI 2650) was contemporary with the down-regulation of IGFBP-7. After treatment with 5-AZA-2′ deoxycytidine, the de-methylated CpG sites in the 5′ region of IGFBP-7 were observed and IGFBP-7 mRNA expression was also restored. Accordingly, re-expression IGFBP-7 in invasive subpopulation of A253 could induce the mesenchymal–epithelial transition (MET) and concurrently inhibited the cell invasion. Moreover, IGFBP-7 methylation status of 47 oral tongue tumors showed a positive correlation to invasive depth of the tumor, loco-regional recurrence, and cancer sequence.
IGFBP-7 can alter EMT relative marker genes and suppress cell invasion in A253 cell through AKT/GSK3β/β-catenin signaling. The epigenetic control of IGFBP-7 in the invasion and metastasis of HNSCC was reported, suggesting that IGFBP-7 could be a prognostic factor for the probability of invasion and a therapeutic remedy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-015-0138-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4355468  PMID: 25880247
IGFBP-7; Head and neck cancer; Invasion; EMT
3.  IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer 
Zhu, S | Zhang, J | Xu, F | Xu, E | Ruan, W | Ma, Y | Huang, Q | Lai, M
Cell Death & Disease  2015;6(3):e1695-.
Epithelial–mesenchymal transition (EMT) was initially recognized during organogenesis and has recently been reported to be involved in promoting cancer invasion and metastasis. Cooperation of transforming growth factor-β (TGF-β) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined. Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression. We revealed the inhibitory role of IGFBP-rP1 through analyses of clinical colorectal cancer samples and various EMT and metastasis models in vitro and in vivo. Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-β-mediated EMT through the Smad signaling cascade. These data establish that IGFBP-rP1 functions as a suppressor of EMT and metastasis in colorectal cancer.
PMCID: PMC4385937  PMID: 25789970
4.  IGFBP-3 is a Metastasis Suppression Gene in Prostate Cancer 
Cancer research  2011;71(15):5154-5163.
The insulin-like growth factor binding protein IGFBP-3 is a pro-apoptotic and anti-angiogenic protein in prostate cancer (CaP). Epidemiological studies suggest that low IGFBP-3 is associated with greater risk of aggressive, metastatic prostate cancers, but in vivo functional data are lacking. Here we show that mice that are genetically deficient in IGFBP-3 exhibit weaker growth of primary prostate tumors growth but higher incidence of metastatic disease. Prostates in IGFBP-3 knockout mice (IGFBP-3KO mice) failed to undergo apoptosis after castration. Spontaneous prostate tumors did not develop in IGFBP-3KO mice, but splenic lymphomas occured in 23% of female IGFBP-3KO mice by 80 weeks of age. To assess the effects of IGFBP-3 deficiency on prostate cancer development, we crossed IGFBP-3KO mice with a c-Myc-driven model of CaP that develops slow-growing, non-metastatic tumors. By 24 weeks of age, well-differentiated prostate cancers were observed in all mice regardless of IGFBP-3 status. However, by 80 weeks of age IGFBP-3KO mice tended to exhibit larger prostate tumors than control mice. More strikingly, lung metastases were observed at this time in 55% of the IGFBP-3KO mice but none of the control animals. Cell lines established from Myc:IGFBP-3KO tumors displayed more aggressive phenotypes in proliferation, invasion and colony formation assays, relative to control Myc tumor cell lines. In addition, Myc:IGFBP-3KO cells exhibited evidence of epithelial-mesenchymal transition (EMT). Our findings establish a function for IGFBP-3 in suppressing metastasis in prostate cancer, and they also offer the first reported transgenic model of spontaneous metastatic prostate cancer for studies of this advanced stage of disease.
PMCID: PMC3148406  PMID: 21697285
IGFBP-3; metastasis; knockout mouse; prostate cancer
5.  Regulation of insulin-like growth factor binding protein-5, four and a half lim-2, and a disintegrin and metalloprotease-9 expression in osteoblasts 
The roles of insulin-like growth factors (IGFs) in regulating growth and their modulation by six IGF binding proteins (IGFBP) are well established. IGFBP-5, the most abundant IGFBP stored in bone, is an important regulator of bone formation via IGF-dependent and -independent mechanisms. Two new proteins, four and a half lim (FHL)-2, a transcription modulator that interacts with IGFBP-5, and a disintegrin and metalloprotease (ADAM)-9, an IGFBP-5 protease, have been identified as potential regulators of IGFBP-5 action in bone. We tested the hypothesis that agents which modulate bone formation by regulating IGFBP-5 expression would also regulate FHL-2 and ADAM-9 expression in a coordinated manner. We evaluated the expression of IGFBP-5, FHL-2, and ADAM-9 by real-time reverse transcriptase (RT)-PCR during differentiation of mouse bone marrow stromal cells into osteoblasts and in response to treatment with bone formation modulators in the LSaOS human osteosarcoma cell line. IGFBP-5 and FHL-2 increased 4.3- and 3.0-fold (P ≤ 0.01), respectively, during osteoblast differentiation. Dexamethasone (Dex), an inhibitor of bone formation, decreased IGFBP-5 and FHL-2 and increased ADAM-9 in LSaOS cells (P ≤ 0.05). Bone morphogenic protein (BMP)-7, a stimulator of bone formation, increased IGFBP-5 and decreased ADAM-9 (P < 0.01). To determine if BMP-7 would eliminate Dex inhibition of IGFBP-5, cells were treated with Dex + BMP-7. The BMP-7-induced increase in IGFBP-5 was reduced, but not eliminated, in the presence of Dex (P ≤ 0.01), indicating that BMP-7 and Dex may regulate IGFBP-5 via different mechanisms. Transforming growth factor (TGF)-β, a stimulator of bone formation, increased IGFBP-5 and FHL-2 expression (P ≤ 0.01). IGF-I and TNF-α decreased expression of ADAM-9 (P < 0.05). In conclusion, our findings are consistent with the hypothesis that FHL-2 and ADAM-9 are important modulators of IGFBP-5 actions and are, in part, regulated in a coordinated manner in bone.
PMCID: PMC2904509  PMID: 16311053
IGFBP-5; FHL-2; ADAM-9; Bone
6.  A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts. 
Journal of Clinical Investigation  1991;88(4):1354-1361.
Insulin-like growth factor I and II (IGF-I and IGF-II) associate with specific IGF binding proteins (IGFBPs) present in plasma and extracellular fluids that can modulate the anabolic effects of these peptides. IGF-I has been shown to increase IGFBP concentrations in vivo and in vitro, but the mechanism and significance of this action are unknown. We examined these issues using normal and simian virus 40-transformed adult human fibroblasts (SV40-HF) in culture. Treatment with IGF-I markedly stimulated the appearance of IGFBP-3 (42/38 kD doublet), a 36 kD IGFBP, and 28-32 kD IGFBPs in the medium of these cells, as assessed by Western ligand blotting; IGF-I decreased levels of 24 kD IGFBP in normal HF cultures. The IGF-I-induced change in IGFBP levels was not a type I IGF receptor-mediated effect on IGFBP synthesis because (a) high concentrations of insulin did not mimic IGF-I's effect; (b) IGF-II and IGF-I analogues having reduced affinity for the IGF-I receptor were equipotent with IGF-I in increasing medium IGFBPs; (c) [QAYL]IGF-I, and IGF-I analogue having normal receptor affinity and decreased affinity for IGFBPs, had no effect; and (d) alpha IR-3, a monoclonal antibody specific for the type I IGF receptor, did not block IGF-I-stimulated increases in IGFBPs. In physiological studies, preincubation with 1 nM IGF-I had no effect on type I IGF receptor binding in normal HF and SV40-HF. In contrast, preincubation of cells with an equivalent concentration of [QAYL]IGF-I downregulated the receptors 40-50%. Changes in cell surface receptor number were reflected in cell responsiveness to IGF-I-stimulated [3H]thymidine incorporation and [3H]aminoisobutyric acid uptake. In conclusion, IGF-I regulates the availability of specific IGFBPs in cultured human fibroblasts by a novel receptor-independent mechanism. Rapid changes in levels of soluble IGFBPs as a direct response to extracellular IGF-I, in turn, modulate IGF-I peptide and receptor interaction, and may constitute an important level of control in IGF cellular physiology.
PMCID: PMC295606  PMID: 1717510
7.  Identification and characterization of novel IGFBP5 interacting protein: evidence IGFBP5-IP is a potential regulator of osteoblast cell proliferation 
Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells.
PMCID: PMC2902994  PMID: 16269403
alternate splicing; yeast two-hybrid screen; small interfering RNA
8.  Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies. 
Journal of Clinical Investigation  1993;91(3):1129-1137.
Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4 degrees C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.
PMCID: PMC288069  PMID: 7680662
9.  Immunohistochemical expression of insulin-like growth factor binding protein-3 in invasive breast cancers and ductal carcinoma in situ: implications for clinicopathology and patient outcome 
Breast Cancer Research  2004;7(1):R119-R129.
Insulin-like growth factor binding protein-3 (IGFBP-3) differentially modulates breast epithelial cell growth through insulin-like growth factor (IGF)-dependent and IGF-independent pathways and is a direct (IGF-independent) growth inhibitor as well as a mitogen that potentiates EGF (epidermal growth factor) and interacts with HER-2. Previously, high IGFBP-3 levels in breast cancers have been determined by enzyme-linked immunosorbent assay and immunoradiometric assay methods. In vitro, IGFBP-3's mechanisms of action may involve cell membrane binding and nuclear translocation. To evaluate tumour-specific IGFBP-3 expression and its subcellular localisation, this study examined immunohistochemical IGFBP-3 expression in a series of invasive ductal breast cancers (IDCs) with synchronous ductal carcinomas in situ (DCIS) in relation to clinicopathological variables and patient outcome.
Immunohistochemical expression of IGFBP-3 was evaluated with the sheep polyclonal antiserum (developed in house) with staining performed as described previously.
IGFBP-3 was evaluable in 101 patients with a variable pattern of cytoplasmic expression (positivity of 1+/2+ score) in 85% of invasive and 90% of DCIS components. Strong (2+) IGFBP-3 expression was evident in 32 IDCs and 40 cases of DCIS. A minority of invasive tumours (15%) and DCIS (10%) lacked IGFBP-3 expression. Nuclear IGFBP-3 expression was not detectable in either invasive cancers or DCIS, with a consistent similarity in IGFBP-3 immunoreactivity in IDCs and DCIS. Positive IGFBP-3 expression showed a possible trend in association with increased proliferation (P = 0.096), oestrogen receptor (ER) negativity (P = 0.06) and HER-2 overexpression (P = 0.065) in invasive tumours and a strong association with ER negativity (P = 0.037) in DCIS. Although IGFBP-3 expression was not an independent prognosticator, IGFBP-3-positive breast cancers may have shorter disease-free and overall survivals, although these did not reach statistical significance.
Increased breast epithelial IGFBP-3 expression is a feature of tumorigenesis with cytoplasmic immunoreactivity in the absence of significant nuclear localisation in IDCs and DCIS. There are trends between high levels of IGFBP-3 and poor prognostic features, suggesting that IGFBP-3 is a potential mitogen. IGFBP-3 is not an independent prognosticator for overall survival or disease-free survival, to reflect its dual effects on breast cancer growth regulated by complex pathways in vivo that may relate to its interactions with other growth factors.
PMCID: PMC1064109  PMID: 15642160
clinicopathology; ductal carcinoma in situ; IGFBP-3; immunohistochemistry; prognosis
10.  Purification and characterization of native human insulin-like growth factor binding protein-6 
Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 (IGFBP-6) from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3.
Electronic supplementary material
The online version of this article (doi:10.1007/s12079-011-0126-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3245759  PMID: 21484185
IGF-binding protein; Purification; Chromatography; Cellular uptake
11.  Purification and characterization of native human insulin-like growth factor binding protein-6 
Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 (IGFBP-6) from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3.
Electronic supplementary material
The online version of this article (doi:10.1007/s12079-011-0126-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3245759  PMID: 21484185
IGF-binding protein; Purification; Chromatography; Cellular uptake
12.  Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I 
The Journal of Cell Biology  1993;121(3):679-687.
Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-II, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF- I on fibroblasts. When IGFBP-5 was present only in the medium, it was degraded to a 22-kD fragment and had no effect on IGF-I-stimulated growth. We conclude that IGFBP-5 is present in fibroblast ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular growth response to these growth factors.
PMCID: PMC2119570  PMID: 7683690
13.  IGFBP-3 Regulates Esophageal Tumor Growth Through IGF-Dependent and Independent Mechanisms 
Cancer biology & therapy  2007;6(4):534-540.
Insulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP-3 in esophageal tumor biology remains elusive. To delineate the functional consequences of IGFBP-3 overexpression, we stably transduced Ha-RasV12-transformed human esophageal cells with either wild-type or mutant IGFBP-3, the latter incapable of binding Insulin-like growth factor (IGFs) as a result of substitution of amino-terminal Ile56, Leu80, and Leu81 residues with Glycine residues. Wild-type, but not mutant, IGFBP-3 prevented IGF-I from activating the IGF-1 receptor and AKT, and suppressed anchorage-independent cell growth. When xenografted in nude mice, in vivo bioluminescence imaging demonstrated that wild-type, but not mutant IGFBP-3, abrogated tumor formation by the Ras-transformed cells with concurrent induction of apoptosis, implying a prosurvival effect of IGF in cancer cell adaptation to the microenvironment. Moreover, there was more aggressive tumor growth by mutant IGFBP-3 overexpressing cells than control cell tumors, without detectable caspase-3 cleavage in tumor tissues, indicating an IGF-independent growth stimulatory effect of mutant IGFBP-3. In aggregate, these data suggest that IGFBP-3 contributes to esophageal tumor development and progression through IGF-dependent and independent mechanisms.
PMCID: PMC2993006  PMID: 17457048
IGFBP-3; IGF; Ras; esophageal cancer; in vivo bioluminescence
14.  Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3 
Cancer research  2004;64(21):7711-7723.
Epidermal growth factor receptor (EGFR) is frequently overexpressed in esophageal carcinoma and its precursor lesions. To gain insights into how EGFR overexpression affects cellular functions in primary human esophageal cells, we performed gene expression profiling and identified insulin-like growth factor-binding protein (IGFBP)-3 as the most up-regulated gene. IGFBP-3 regulates cell proliferation through both insulin-like growth factor-dependent and independent mechanisms. We found that IGFBP-3 mRNA and protein expression was increased in EGFR-overexpressing primary and immortalized human esophageal cells. IGFBP-3 was also up-regulated in EGFR-overexpressing cells in organotypic culture and in EGFR transgenic mice. Furthermore, IGFBP-3 mRNA was overexpressed in 80% of primary esophageal squamous cell carcinomas and 60% of primary esophageal adenocarcinomas. Concomitant up-regulation of EGFR and IGFBP-3 was observed in 60% of primary esophageal squamous cell carcinomas. Immunohistochemistry revealed cytoplasmic localization of IGFBP-3 in the preponderance of preneoplastic and neoplastic esophageal lesions. IGFBP-3 was also overexpressed in esophageal cancer cell lines at both mRNA (60%) and protein (40%) levels. IGFBP-3 secreted by cancer cells was capable of binding to insulin-like growth factor I. Functionally, epidermal growth factor appeared to regulate IGFBP-3 expression in esophageal cancer cell lines. Finally, suppression of IGFBP-3 by small interfering RNA augmented cell proliferation, suggesting that IGFBP-3 may inhibit tumor cell proliferation as a negative feedback mechanism. In aggregate, we have identified for the first time that IGFBP-3 is an aberrantly regulated gene through the EGFR signaling pathway and it may modulate EGFR effects during carcinogenesis.
PMCID: PMC4140096  PMID: 15520175
15.  The Pro-Fibrotic Factor IGFBP-5 Induces Lung Fibroblast and Mononuclear Cell Migration 
We have previously shown that insulin-like growth factor–binding protein-5 (IGFBP-5) is overexpressed in fibrotic lung tissues and that it induces production of extracellular matrix components such as collagen and fibronectin both in vitro and in vivo. We recently observed mononuclear cell infiltration in lung tissues of mice expressing IGFBP-5. We therefore examined the role of IGFBP-5 on the migration of immune cells. Migration assays demonstrated that IGFBP-5 induced migration of peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Preferential migration of monocytes/macrophages, natural killer cells, and T cells was observed. Moreover, the CD4/CD8 ratio of migrating cells was significantly higher in vitro and in vivo in response to IGFBP-5. IGFBP-5 resulted in preferential migration of activated CD4+ T cells and monocytes. Interestingly, IGFBP-5 also induced migration of primary human lung fibroblasts. Exogenous administration of IGFBP-5 induced activation of mitogen-activated protein kinase (MAPK) signaling cascade but not PI3K in PBMCs. IGFBP-5–induced migration was blocked by the MEK1/2 inhibitor U0126, suggesting that IGFBP-5–induced migration occurs via MAPK activation. Furthermore, monocytes treated with recombinant IGFBP-5 expressed the mesenchymal markers α–smooth muscle actin and fibronectin in vitro and in vivo, suggesting that IGFBP-5 can induce the transformation of monocytes into mesenchymal cells. Collectively, our results suggest that IGFBP-5 induces cell migration via MAPK-dependent and IGF-I–independent mechanisms.
PMCID: PMC2715907  PMID: 19131643
IGFBP-5; mononuclear cells; migration; MAPK; fibrosis
16.  Insulin-Like Growth Factor Factor Binding Protein-2 is a Novel Mediator of p53 Inhibition of Insulin-Like Growth Factor Signaling 
Cancer biology & therapy  2006;5(10):1408-1414.
The p53 tumor suppressor induces cellular growth arrest and apoptosis in response to DNA damage by transcriptionally activating or repressing target genes and also through protein-protein interactions and direct mitochondrial activities. In 1995, insulin-like growth factor binding protein (IGFBP)-3 was identified as one of the genes transcriptionally activated by p53. IGFBP-3 is one of six closely related IGFBP’s, with additional IGFBP-related proteins belonging to the IGFBP superfamily. Here we show that IGFBP-2 is also a p53 target. Like IGFBP-3, IGFBP-2 secretion is reduced when p53+/+ lung cancer cells are transfected with human papillomavirus E6, which targets p53 for degradation. IGFBP-2 mRNA is induced by irradiation in vivo in a p53-dependent manner. p53 protein binds IGFBP-2 intronic sequences in an electrophoretic mobility shift assay, and activates transcription in a luciferase assay. Loss of IGFBP-2 inhibits the ability of p53 to inhibit the activation of extracellular signal-regulated kinase (ERK)1 by IGF-I. Thus, p53 effects on the IGF axis are more complex than previously appreciated, and overall transform the axis from IGF-mediated mitogenesis to growth inhibition and apoptosis. This has significant implications for how growth hormone and IGF-I can induce growth without also inducing cancer.
PMCID: PMC1906874  PMID: 17102589
insulin like growth factor binding protein-2; p53; insulin-like growth factor-I; E6; extracellular signal-regulated kinase; transcription; prostate cancer
17.  EGF-mediated regulation of IGFBP-3 determines esophageal epithelial cellular response to IGF-I 
IGF and EGF regulate various physiological and pathological processes. IGF binding protein (IGFBP)-3 regulates cell proliferation in IGF-dependent and -independent fashions. Recently, we identified IGFBP-3 as a novel EGF receptor (EGFR) downstream target molecule in primary and immortalized human esophageal epithelial cells, suggesting an interplay between the EGF and IGF signaling pathways. However, the regulatory mechanisms for IGFBP-3 expression and its functional role in esophageal cell proliferation remain to be elucidated. Herein, we report that IGFBP-3 mRNA and protein were induced upon growth factor deprivation in primary and immortalized human esophageal cells through mechanisms requiring p53-independent de novo mRNA transcription and protein synthesis. This occurred in the face of the activated phosphatidylinositol 3-OH-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. Secreted IGFBP-3 neutralized IGFs and prevented IGF-I receptor (IGF-IR) activation. In contrast, EGF suppressed IGFBP-3 mRNA and protein expression through activation of MAPK in an EGFR-tyrosine kinase-dependent manner to restore the cellular response to IGF-I. When stably overexpressed, wild-type IGFBP-3 but not I56G/L80G/L81G (GGG) mutant IGFBP-3, which has a reduced affinity to IGFs, prevented IGF-I from activating IGF-IR and Akt as well as stimulating cell proliferation. However, unlike other cell types where IGFBP-3 exerts antiproliferative effects, neither wild-type nor GGG mutant IGFBP-3 alone affected cell proliferation or EGFR activity. These results indicate that IGF signaling is subject to negative regulation through IGFBP-3 and positive regulation by EGF, the latter of which suppresses IGFBP-3. This provides a platform for understanding the novel cross talk between EGF- and IGF-mediated pathways.
PMCID: PMC2996094  PMID: 16210470
insulin-like growth factor binding protein-3; epidermal growth factor receptor; mammalian target of rapamycin; esophageal epithelial cells
18.  Signalling pathways involved in antiproliferative effects of IGFBP-3: a review 
Molecular Pathology  2001;54(3):145-148.
Insulin-like growth factor binding protein-3 (IGFBP-3), the major circulating carrier protein for IGFs, is also active in the cellular environment as a potent antiproliferative agent. It appears to function both by cell cycle blockade and the induction of apoptosis. Transfection of p53 negative T47D breast cancer cells to express IGFBP-3 leads to induction of the apoptotic protein bax and an increase in sensitivity to ionising radiation. IGFBP-3 can be transported to the nucleus by an importin β mediated mechanism, where it has been shown to interact with the retinoid X receptor α and possibly other nuclear elements. Expression of oncogenic ras is associated with resistance to exogenous IGFBP-3, the effect being reversible by inhibition of mitogen activated protein (MAP) kinase phosphorylation. IGFBP-3 antiproliferative signalling appears to require an active transforming growth factor β (TGF-β) signalling pathway, and IGFBP-3 stimulates phosphorylation of the TGF-β signalling intermediates Smad2 and Smad3. These recent findings all point to a complex intracellular mode of action of IGFBP-3, which will need to be better understood if anti-cancer treatments are to take advantage of the antiproliferative activity of IGFBP-3.
PMCID: PMC1187052  PMID: 11376125
insulin-like growth factor binding protein-3; signalling pathway; apoptosis; transforming growth factor β
19.  Ras-Association Domain Family 1 Protein, RASSF1C, Is an IGFBP-5 Binding Partner and a Potential Regulator of Osteoblast Cell Proliferation 
The goal of this study was to identify downstream signaling molecules involved in mediating the IGF-independent effects of IGFBP-5 in osteoblasts. We identified RASSF1C, a member of the RASSF1 gene products, as a IGFBP-5 binding partner and as a potential mediator of IGFBP-5 effects on ERK phosphorylation and cell proliferation.
It has been predicted that the intrinsic growth factor action of insulin-like growth factor binding protein (IGFBP)-5 involves either the binding of IGFBP-5 to a putative receptor to induce downstream signaling pathways and/or intracellular translocation of IGFBP-5 to bind to potential signaling molecules involved in osteoblast cell regulation. This study reports the characterization of isoform C of the Ras association family 1 (RASSF1C) gene as an interacting partner of IGFBP-5.
Materials and Methods
IGFBP-5 was used as bait in a yeast two-hybrid screen of a human osteosarcoma cDNA library. Expression levels of RASSF1C were measured by RT-PCR and/or Northern blot. IGFBP-5 effects on ERK phosphorylation were evaluated by immunoblot analysis. The effect of RASSF1C siRNA on cell proliferation was measured by the AlamarBlue assay.
One of the clones that interacted strongly with the bait under high stringency conditions corresponded to RASSF1C. The interaction between RASSF1C and IGFBP-5 was confirmed by in vitro coimmunoprecipitation studies. Northern blot and RT-PCR analysis showed that RASSF1C was expressed in a variety of osteoblast cell types that produce IGFBP-5. Addition of synthetic RASSF1C-specific small interfering (si) RNA duplex or use of a RASSF1C-specific si-hairpin plasmid caused a decrease in cell number and abolished IGFBP-5–induced extracellular signal-regulated kinase (ERK)-1/2 phosphorylation but had no effect on IGFBP-5–induced increases in alkaline phosphatase (ALP) activity.
We have shown a novel interaction between IGFBP-5 and RASSF1C. Our findings that silencing of RASSF1C results in the reduction of osteoblast cell proliferation and that IGFBP-5 treatment increases phosphorylation of ERK-1/2 raise the possibility that RASSF1C, a Ras effector, could, in part, contribute to mediating the effects of IGFBP-5 on ERK phosphorylation and, consequently, cell proliferation.
PMCID: PMC2897826  PMID: 16007340
protein–protein interaction; yeast two-hybrid screen; small interfering RNA; extracellular signal-regulated kinase-1/2 phosphorylation; insulin-like growth factor–independent mechanism
20.  Circulating Insulin-like Growth Factor Binding Protein-4 (IGFBP-4) is not Regulated by Parathyroid Hormone and Vitamin D in vivo: Evidence from Children with Rickets 
Objective: Insulin-like growth factor binding protein-4 (IGFBP-4), inhibits IGF actions under a variety of experimental conditions. Parathyroid hormone (PTH), 1.25-hydroxy(OH)vitamin D, IGF-I, IGF-II and transforming growth factor (TGF)-b are the major regulators of IGFBP-4 production in vitro. However, little is known about the in vivo regulation of circulating IGFBP-4 in humans.
Methods: We measured serum concentrations of calcium (Ca), phosphorus (P), alkaline phosphatase (ALP), PTH, vitamin D, IGF-I, IGFBP-3, and IGFBP-4 in infants (n=22) with nutritional rickets before and after treatment of rickets with vitamin D (300 000 U single dose po).
Results: The mean±SD age of the patients was 1.3±1.6 years (range 0.2-3). Serum Ca and P increased, whereas ALP and PTH decreased after treatment (Ca from 6.6±1.4 to 9.5±1.6 mg/dL, P from 3.9±1.4 to 5.4±0.8 mg/dL, ALP from 2590±2630 to 1072±776 IU/mL and PTH from 407±248 to 27.4±20.8 ng/dL, respectively). Vitamin D levels were low (7.8±2.5 ng/mL) and increased after treatment (18.1±4.0 ng/mL, p<0.001). Serum IGF-I and IGFBP-3 levels both increased after treatment (IGF-I: 13.5±12.2 vs. 23.7±14.2 ng/mL, p<0.001 and IGFBP-3: 1108±544 vs. 1652±424 ng/mL, p<0.001). However, serum IGFBP-4 levels did not change significantly after treatment (18.8±8.0 vs. 21.5±4.8 ng/mL). No correlation between PTH and IGF-I, IGFBP-3 or IGFBP-4 was detected. Significant correlations were observed between PTH and ALP (r=0.53, p<0.05), and between IGF-I and IGFBP-3 (r=0.46, p<0.05).
Conclusion: The results demonstrate that contrary to in vivo studies, circulating IGFBP-4 levels are not influenced by secondary hyperparathyroidism in vitamin D deficiency rickets since IGFBP-4 levels did not change after normalization of PTH with vitamin D treatment.
Conflict of interest:None declared.
PMCID: PMC3005664  PMID: 21274331
Vitamin D; rickets; IGF-I; IGFBP-3; IGFBP-4; PTH; Bone
21.  Insulin-like growth factors and their binding proteins in human colonocytes: preferential degradation of insulin-like growth factor binding protein 2 in colonic cancers. 
British Journal of Cancer  1997;76(1):60-66.
We have compared the expression of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ten paired samples of normal and tumour colonic tissue with regard to both mRNA and protein. We have compared sensitivity of these tissues to IGF-I using primary cultures of epithelial cells of colonic mucosa, and we have examined the production of IGFs and IGFBPs by these cells. In the tissues, IGFBP-2 mRNA was expressed in all normal and cancer samples but other IGFBPs showed variable expression. mRNAs for IGF-I were expressed in all normal and cancer tissues but IGF-II mRNA was only detected in cancer tissue (3 out of 10). Immunostaining of sections of normal and cancer tissue was negative for IGF-I and IGF-II; IGFBP-2 was positive in 2 out of 10 cancer tissues and 7 out of 10 normal tissues; IGFBP-3 was positive in 7 out of 10 cancer tissues and 7 out of 10 normal tissues; and IGFBP-4 was positive in 5 out of 10 cancer tissues and 6 out of 10 normal tissues. In the cells in culture, cancer cells showed increased incorporation of [35S]methionine into protein and [3H]thymidine into DNA (P < 0.02) when treated with IGF-I. Western blotting of serum-free conditioned media from cells in culture showed that 8 out of 10 normal and 3 out of 10 cancer cultures produced a 32-kDa immunoreactive IGFBP-2. No IGFBP-3 was secreted by any culture but 24-kDa IGFBP-4 was found in 3 out of 10 normal and 5 out of 10 cancer tissues. Because of the discrepancy between mRNA and protein expression for IGFBP-2, degradation of native IGFBPs was assessed using tissue extracts. Colon cancer extracts were able to degrade exogenous IGFBP-2, IGFBP-3 and IGFBP-4, whereas normal tissue extracts were without effect on IGFBP-2. We conclude that IGFBPs are synthesized and secreted by cells of the colonic mucosa but that proteolysis of secreted IGFBP-2 occurs in colon cancer tissue. This selective degradation may confer a growth advantage.
PMCID: PMC2223789  PMID: 9218734
22.  Insulin-like Growth Factor Binding Protein-3 Expression in the Human Corneal Epithelium 
Experimental eye research  2007;85(4):492-501.
Insulin-like Growth Factor Binding Protein-3 (IGFBP3) is a high affinity binding protein shown to regulate cell growth, differentiation, and apoptosis in a variety of cellular systems. The primary aim of this study was to characterize IGFBP3 expression in the human corneal epithelium and in a corneal epithelial cell line and to establish a potential role for IGFBP3 mediated apoptotic signaling in corneal epithelial cells.
Using a telomerase-immortalized human corneal epithelial (hTCEpi) cell line cultured in serum-free media and fresh human eye bank donor tissue, expression and localization of IGFBP3 were established in situ and in vitro by indirect immunofluorescence and western blotting. Real time PCR was used to measure IGFBP3 mRNA levels following Trichostatin-A (TSA) treatment and as a function of confluence. IGFBP3 protein levels were assessed in resting human tears and in conditioned media by western blotting; as was the ability of recombinant human IGFBP3 protein to associate with the cell surface. Apoptotic signaling was assessed in vitro using TSA and recombinant human (rh)IGFBP3. Apoptosis was measured by Viability/Cytotoxicity, Annexin V, and TUNEL assays.
IGFBP3 localized to the plasma membrane of human corneal epithelial cells in situ and was upregulated in surface cells in the central cornea. IGFBP3 was secreted in conditioned media of growing cells, with a robust upregulation following confluence (P<0.001) and differentiation. IGFBP3 was undetectable in human tears. Addition of TSA to the culture media resulted in an upregulation of IGFBP3 mRNA (P<0.001) and protein. In addition, TSA treatment led to a significant increase in Annexin V positive cells at 18 and 24 hours (P<0.001) and TUNEL positive cells at 24 and 48 hours (P<0.001). The addition of rhIGFBP3 to the cell culture media appeared to induce occasional membrane blebbing, but cells failed to become positive with Annexin V or TUNEL.
Taken together, these results demonstrate that cell membrane-associated IGFBP3 is produced by corneal epithelial cells and associates with the plasma membrane of superficial cells in situ and in cultured cells, but is not present in human tears. The differential localization and effect(s) on apoptosis suggest that the effects of IGFBP3 are likely tissue compartment and receptor specific and may be regulated by glycosylation.
PMCID: PMC2782519  PMID: 17709104
23.  IGF binding protein-6 expression in vascular endothelial cells is induced by hypoxia and plays a negative role in tumor angiogenesis 
Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here we report that hypoxia induced the expression of insulin-like growth factor binding protein-6 (IGFBP-6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a HIF-mediated mechanism. Addition of human IGFBP-6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP-6 mutant with at least 10,000-fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP-6 is IGF-independent. The functional relationship between IGFBP-6 and VEGF, a major hypoxia-inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro, co-incubation with IGFBP-6 abolished VEGF-stimulated angiogenesis. The in vivo role of IGFBP-6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP-6 mRNA reduced the number of embryonic inter-segmental blood vessels by ∼40%. This anti-angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP-6b had similar effects. To determine the anti-angiogenic effect of IGFBP-6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP-6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP-6-transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP-6 in VECs is up-regulated by hypoxia and IGFBP-6 inhibits angiogenesis in vitro and in vivo.
PMCID: PMC3259243  PMID: 21618524
24.  Decreased expression of insulin-like growth factor binding protein-5 during N-(4-hydroxyphenyl)retinamide-induced neuronal differentiation of ARPE-19 human retinal pigment epithelial cells: Regulation by CCAAT/enhancer-binding protein 
Journal of cellular physiology  2010;224(3):827-836.
Insulin-like growth factor (IGF)-binding protein -5 (IGFBP5), an important member of the IGF axis involved in regulating cell growth and differentiation, acts by modulating IGF signaling and also by IGF-independent mechanisms. We identified IGFBP5 by microarray analysis as a gene differentially regulated during N-(4-Hydroxyphenyl)-retinamide (4HPR)-induced neuronal differentiation of human retinal pigment epithelial (RPE) cells. IGFBP5 is expressed in human RPE cells, and its expression, mRNA as well as protein, is greatly decreased during the 4HPR-induced neuronal differentiation. Exogenous IGFBP5 does not block the neuronal differentiation indicating that IGFBP5 down-regulation may not be a prerequisite for the neuronal differentiation. IGFBP5 down-regulation, similar to neuronal differentiation, is mediated by the MAPK pathway since U0126, an inhibitor of MEK1/2, effectively blocked it. The overexpression of transcription factor CCAAT/enhancer binding protein-β (C/EBPβ) inhibited the 4HPR-induced down-regulation of IGFBP5 expression and the neuronal differentiation of RPE cells. The deletion of C/EBP response element from IGFBP5 promoter markedly decreased the basal promoter activity and abolished its responsiveness to 4HPR treatment in reporter assays, suggesting that the expression of IGFBP5 is regulated by C/EBP. Thus, our results clearly demonstrate that the IGFBP5 expression is down-regulated during 4HPR-induced neuronal differentiation of human RPE cells through a MAPK signal transduction pathway involving C/EBPβ.
PMCID: PMC2946194  PMID: 20583135
RPE; IGFBP5; MAPK; C/EBP; Retinoic acid
25.  Transforming Growth Factor-β1 Modulates Insulin-Like Growth Factor Binding Protein-4 Expression and Proteolysis in Cultured Periosteal Explants 
Periosteum is involved in bone growth and fracture healing and has been used as a cell source and tissue graft for tissue engineering and orthopedic reconstruction including joint resurfacing. Periosteum can be induced by transforming growth factor beta (TGF-β) or insulin-like growth factor-I (IGF-I) alone or in combination to form cartilage. However, little is known about the interaction between IGF and TGF-β signaling during periosteal chondrogenesis. The purpose of this study was to determine the effect of TGF-β1 on IGF binding protein-4 (IGFBP-4) and the IGFBP-4 protease pregnancy-associated plasma protein-A (PAPP-A) expression in cultured periosteal explants.
Periosteal explants from rabbits were cultured with or without TGF-β1. IGFBP-4 and PAPP-A mRNA levels were determined by real-time quantitative PCR. Conditioned medium was analyzed for IGFBP-4 and PAPP-A protein levels and IGFBP-4 protease activity.
TGF-β1-treated explants contained lower IGFBP-4 mRNA levels throughout the culture period with a maximum reduction of 70 % on day 5 of culture. Lower levels of IGFBP-4 protein were also detected in the conditioned medium from TGF-β1-treated explants. PAPP-A mRNA levels were increased 1.6 fold, PAPP-A protein levels were increased 3 fold, and IGFBP-4 protease activity was increased 8.5 fold between 7 and 10 days of culture (the onset of cartilage formation in this model) in conditioned medium from TGF-β1-treated explants.
This study demonstrates that TGF-β1 modulates the expression of IGFBP-4 and PAPP-A in cultured periosteal explants.
PMCID: PMC2844918  PMID: 19656700
Cartilage; Chondrocyte; Insulin Like Growth Factor I; Periosteum; Transforming Growth Factor Beta; insulin-like growth factor binding protein-4; pregnancy-associated plasma protein-A

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