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Abstract

The insulin-like growth factor (IGF) system consists of growth factors, receptors and binding proteins, and overlaps significantly with insulin signalling pathways. IGF-1 and IGF-2 are essential for growth and development and have also been shown to play a key role in insulin sensitivity, obesity, endothelial dysfunction, angiogenesis and tumorigenesis. They are tightly regulated by a family of six binding proteins (IGFBP) which form complexes with IGF, with only 1% of IGF free in the circulation. Despite this apparently predominantly inhibitory role, IGFBPs have been shown to have a variety of effects on cell growth, proliferation, angiogenesis and metabolism, with both IGF dependent and independent effects observed. IGFBP-2 in particular has been shown in several studies to be linked to tumour metastasis and poorer outcomes in cancer, and has been shown to promote tube formation, proliferation, migration and angiogenesis in vitro, through a variety of pathways. Higher circulating levels of IGFBP-2 have also been shown to protect against obesity and improve insulin sensitivity. In this brief review we will look at some of these actions in more detail, and describe a potential future role for IGFBP-2 incorporation in cell based therapies.

Introduction

The insulin-like growth factor (IGF) system is a complex hierarchy of growth factors, receptors and binding proteins, and exhibits significant overlap with traditional insulin signalling pathways. IGF-I share common ancestry with insulin and have a nearly homologous structure [1]. They therefore not only interact with IGF receptors, but also insulin and hybrid receptors [1]. IGF synthesis is primarily mediated by growth hormone rather than blood glucose, though free IGF-1 does play a role in blood glucose homeostasis [2]. Through these interactions, they promote cell growth and survival and are indispensable in normal development and whole-body metabolism [3]. However, dysregulation of both IGF activity and IGF-1 receptor expression has also been implicated in tumorigenesis, cardiovascular disease and diabetes mellitus [4,5].

Less than 1% of IGF is unbound in plasma, with activity tightly modulated by the insulin-like growth factor binding proteins (IGFBPs). These are a family of six structurally similar proteins, acting primarily to regulate the activity of IGFs through the binding and transport of these proteins within the vasculature and into peripheral tissues [6]. Through this binding, they increase IGF half-life and inhibit receptor interactions. They may additionally stimulate IGF activity by transporting directly to the IGF-1 receptor [7], and certain IGFBPs have reduced affinity for IGF when they are bound to cellular membrane receptors, increasing IGF bioavailability in the pericellular environment [8]. Although each binding protein has complementary structure and binding domains, this apparent paradoxical activity relates to the differing nature of interactions between the IGFs and each binding protein [9].

Evidence is also emerging that the IGFBPs can both potentiate the action of IGF and act independently [10,11], and the dependent and independent effects of IGFBP-2 in particular will be explored further in this mini review.

IGFBP-2 is the second most abundant of the IGFBPs, and has been strongly associated with metastasis and angiogenesis in the context of malignancies [12]. It has been shown to promote glioma progression and invasion [13] and is positively associated with both prostate and breast cancer grade and metastasis [14,15].  Furthermore, knockdown of IGFBP-2 in mice has been shown to reduce breast cancer metastatic colonisation of the lungs as well as tumour vascular density[16]. However, IGFBP-2 has also been shown to have protective effects against diet-induced obesity and enhance insulin sensitivity, independent of IGF-1 activity [17]. 

Structure

IGFBP-2 is a 36kDa protein with three distinct structural regions in common with the other IGFBPs: a N-terminal cysteine rich region, C-terminal cysteine rich region and link region [18]. Both the N and C terminals bind IGFs, with affinity of each to IGF varying between the IGFBPs [18]. In addition, IGFBP-2 possesses a heparin binding domain (HBD) and integrin binding domain (RGD), which are found in the link region and C-terminal region respectively.  Both of these motifs are functional and mediate both IGF-dependent and -independent actions, with the RGD motif predominantly mediating cell membrane integrin interactions [19]. In contrast, the HBD motif in the link region interacts with components of the extra cellular matrix (ECM) and promotes cell proliferation and migration [20]. An additional HBD domain has been identified in the C-terminal region, although less is known about its interactions [21].

IGF-dependent actions

IGFBP-2 has been shown as a major regulator of angiogenesis in malignant melanomas. It appears to be induced by the expression of mda-9/syntenin, a protein associated with melanoma progression and metastasis [22]. In the context of human umbilical vein endothelial cells (HUVECs), addition of recombinant human IGFBP-2 augments cell proliferation and tube formation in vitro, and this effect is negated by an IGFBP-2 neutralising antibody [23]. Although this pro-angiogenic effect appears to be mediated by interaction with the integrin receptor aVβ3, IGF-1 receptor knockdown significantly attenuates the effects of IGFBP-2, suggesting an inter-dependent relationship between IGF-1 and IGF-1 receptor [23]. Interestingly, IGFBP-2 downregulates IGF-mediated proliferation of cells in the context of breast carcinoma through interactions with the same integrin receptor, suggesting that observed effects may be determined by environmental milieu [24].

Others have shown complementary findings, with IGFBP-2 shown to bind to receptor protein tyrosine phosphatase β (RPTPβ) via its HBD domain in the link region [25]. This caused inactivation of RPTPβ and inhibited transcription of the tumour suppressor gene PTEN. Inhibition of PTEN enables downstream activation of the PI3K/AKT pathway and promotes vascular smooth muscle cell (VSMC) proliferation. However, inhibition of IGF-1 receptor expression prevented RPTPβ inactivation, suggesting that effects of IGFBP-2 on PTEN phosphorylation require coordination of IGFBP-2, IGF-1 and its receptor [25].

The role of microRNA-126 in silencing IGFBP-2 induced metastatic angiogenesis has also been demonstrated in endothelial cells [16]. IGFBP-2 secreted by breast carcinoma cells positively modulated IGF-1 mediated activation of IGF-1 receptors on endothelial cells, promoting their recruitment. This interaction was suppressed by miRNA-126, and enhanced with miRNA-126 knockdown.

IGF-independent actions

The interaction of IGFBP-2 with integrins and ECM components is well established [26]. However, others have shown IGFBP-2 to localise to the nuclei of neuroblastoma cells, subsequently promoting angiogenesis through upregulation of VEGF mRNA transcription as well as upregulating activation of other protumorigenic genes [27]. This upregulation was only seen in the presence of intracellular IGFBP-2, with no role observed for IGF or its receptor. The same group later showed that nuclear translocation was mediated through a nuclear localisation signal sequence within the IGFBP-2 link domain, and that an IGFBP-2 mutant that could not enter the nucleus was unable to upregulate VEGF expression in the same fashion [28]. Nuclear translocation of IGFBP-2 was also seen in breast and prostate cancer cells in addition to neuroblastoma, indicating the potential for a similar role across several cancer types [28].

IGFBP-2 levels also appear to be regulated by hypoxia-inducible factor-1α (HIF-1α) [29], a growth factor strongly associated with the metastatic phenotype [30]. This association would be consistent with the role of IGFBP-2 in tumour angiogenesis.

A key role for IGBP-2 in the development and progression of lymphangioleiomyomatosis (LAM) has also been demonstrated [31]. This is an unusual disease affecting mainly young women, in which LAM cells – a histologically benign appearing smooth muscle-like cell - proliferate and metastasize to the lungs, causing progressive remodelling and emphysematous-like lung disease [32]. IGFBP-2 was found to accumulate in the nucleus of these cells independently of IGF-1, with nuclear translocation mediated by estrogen-receptor alpha (ER-α). Knockdown of IGFBP-2 by siRNA reduced LAM cell proliferation, migration and invasiveness, indicating the importance of IGFBP-2 in these tumorigenic actions. Additionally, IGFBP-2 knockdown abrogated mitogen-activated protein kinase (MAPK) phosphorylation, explaining a potential mechanism for its tumorigenic effects [31].

Summary

IGFBP-2 exhibits significant effects in a wide range of cell types, although the majority of studies thus far have assessed its role in the context of tumorigenesis. Effects appear to be both IGF-dependent and -independent, with function and activity affected by cell type as well as receptor activation and nuclear localisation. Its pro-angiogenic potential and upregulation in hypoxic conditions lends IGFBP-2 to be potentially reparative in disorders such as ischaemic heart disease or peripheral arterial disease. Nonetheless, further studies to scrutinise this potential are warranted.

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