Best Practice & Research Clinical Endocrinology & Metabolism 29 (2015) 685e700

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Best Practice & Research Clinical

Endocrinology & Metabolism
journal homepage: www.elsevier.com/locate/beem

Physiology and pathophysiology of IGFBP-1 and

IGFBP-2 e Consensus and dissent on metabolic
control and malignant potential
Andreas Hoeich, Group Leader, PhD a, **,
Vincenzo C. Russo, Group Leader, PhD b, *
a
Institute for Genome Biology, Leibniz-Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2,
18196 Dummerstorf, Germany
b
Hormone Research, Murdoch Childrens Research Institute, Department of Paediatrics, University of
Melbourne, Royal Children's Hospital, Parkville, Victoria, Australia

a r t i c l e i n f o
IGFBP-1 and IGFBP-2 are suppressed by growth hormone and
Article history: therefore represent less prominent members of the IGFBP family
Available online 8 July 2015 when compared to IGFBP-3 that carries most of the IGFs during
circulation under normal conditions in humans in vivo. As soon as
Keywords: the GH signal is decreased expression of IGF-I and IGFBP-3 is
IGFBP-1 reduced. Under conditions of lowered suppression by GH the time
IGFBP-2 seems come for IGFBP-1 and IGFBP-2. Both IGFBPs are potent ef-
metabolism fectors of growth and metabolism. Secretion of IGFBP-1 and IGFBP-
diabetes 2 is further suppressed by insulin and diminished with increasing
obesity
obesity. Both IGFBP family members share the RGD sequence motif
cancer
that mediates binding to integrins and is linked to PTEN/PI3K
AKT
PTEN signalling. In mice, IGFBP-2 prevents age- and diet-dependent
steroids glucose insensitivity and blocks differentiation of preadipocytes.
The latter function is modulated by two distinct heparin-binding
domains of IGFBP-2 which are lacking in IGFBP-1. IGFBP-2 is
further regulated by leptin and has been demonstrated to affect
insulin sensitivity and glucose tolerance, further supporting a
particular role of IGFBP-2 in glucose and fat metabolism. Since
IGFBP-2 is controlled by sex steroids as well, we devised a scheme
to compare IGFBP effects in breast, ovarian and prostate cancer.
While a positive association does not seem to exist with IGFBP-1
and risk of cancers within these reproductive tissues, a

* Corresponding author. Hormone Research, Murdoch Childrens Research Institute, Flemington Road, Parkville, Victoria 3052,
Australia.
** Corresponding author. Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany.
E-mail addresses: hoeich@fbn-dummerstorf.de (A. Hoeich), vince.russo@mcri.edu.au (V.C. Russo).

http://dx.doi.org/10.1016/j.beem.2015.07.002
1521-690X/ 2015 Elsevier Ltd. All rights reserved.
686 A. Hoeich, V.C. Russo / Best Practice & Research Clinical Endocrinology & Metabolism 29 (2015) 685e700

relationship between IGFBP-2 and breast cancer, ovarian cancer

and prostate cancer does indeed appear to be present. To date, the
specic roles of IGFBP-2 in estrogen signalling are unclear, though
there is accumulating evidence for an effect of IGFBP-2 on PI3K
signalling via PTEN, particularly in breast cancer.
2015 Elsevier Ltd. All rights reserved.

The insulin-like growth factors binding proteins (IGFBPs)

The IGFBPs coordinate and regulate IGF biological activity in several ways: 1) transport of IGF in
plasma and control of its diffusion/efux from the vascular space; 2) increasing the half-life and
regulating clearance of IGFs; 3) providing specic binding sites for IGFs in the extracellular and
pericellular space; and 4) modulating interaction of IGFs with their receptors [1]. The IGFBPs bind
IGF-I and IGF-II but do not bind insulin. IGFBP-1, -3, and -4 bind IGF-I and IGF-II with similar afnity
while IGFBP-2, -5 and -6 have a higher binding afnity for IGF-II. The amino and carboxy terminal
sequence of the six IGFBPs are highly conserved and when the NH2-terminal of the IGFBPs is
compared, it is apparent that at least 33 out of 80 residues of exon 1 are identical among the six
IGFBPs, including the position of the 12 cysteines. An exception is IGFBP-6 which lacks two cysteines
in a G-C-G-C-C stretch, highly conserved in the other IGFBPs. The carboxyl terminal region also
maintains a high homology amongst the IGFBPs with identity of 18 out of 28 residues, including the
four cysteines. The middle region, linking the amino and the carboxyl terminus domains of the
IGFBPs, is not conserved across the IGFBPs. Nevertheless, the six IGFBPs are distinct proteins, each
having evolved [2] with exclusive functions and properties. The circulating (endocrine) form of IGFs
is almost entirely bound to IGFBPs (only ~1% free) and approximately 75% of IGF-I or eII in human
plasma circulates as a ternary 150 kDa complex [3], consisting of IGF-I or eII bound to IGFBP-3 and to
a liver-derived non-IGF binding component, called the acid labile subunit (ALS). IGFBP-5 can also
form a ternary complex with IGF-I and the ALS, but at a lower afnity than that formed by IGFBP-3
[4]. In addition to the ternary complex, two other pools of IGFs exist in serum, being free IGF and the
~50 kDa IGF pool [5]. The 50 kDa complex (~25%) consists of IGF bound to the other low molecular
weight serum IGFBPs. These are IGFBP-1, IGFBP-2 and IGFBP-4, present in sufcient concentrations to
alter IGF action. Two other IGFBPs (IGFBP-5, -6) are found in serum at the limit of detection such
that they are unlikely to play a critical regulatory role with the endocrine form of IGFs. In a variety of
cases, the effects of IGFBPs are further dened by the actions of specic IGFBP proteases, which cleave
the binding proteins, generating fragments with reduced or no binding afnity for IGFs [6]. Potential
roles of IGFBP-1 and IGFBP-2 for normal growth and metabolism were discussed already in 1995 by
Strasser-Vogel et al. [7] in human patients with type 2 diabetes. Circulating levels of IGFBPs are
regulated in various physiological states, including diurnal variation, exercise, pregnancy and aging
and by hormones that modulate the levels of some IGFBPs in serum and other biological uids [8]. In
addition to these physiological changes in IGFBP levels and IGF bioavailability, it has also been re-
ported that in pathophysiological conditions, the ratio of IGF bound in the ternary complex (IGF/
IGFBP-3/ALS) to that bound to the low molecular weight IGFBPs (IGF/IGFBPs) is greatly decreased,
resulting in a shortened half-life for the IGFs, translating to modied IGF biological activity [6].
IGFBPs control IGF action at the cellular level by either restricting access to their receptors and thus
inhibiting mitogenic responses, or by targeting/facilitating IGF receptor binding and consequently
potentiating the mitogenic stimuli. While in vivo, some IGFBPs generally perform a buffering/
inhibitory function (e.g. preventing IGF-induced hypoglycemia), in vitro, the same IGFBPs can exert a
stimulatory or inhibitory effect depending on the experimental parameters [1]. IGFBP biological
activity is also regulated by post-translational modications such as glycosylation and phosphory-
lation or differential localization of the IGFBPs in the pericellular and extracellular space (e.g. cell
surface or extracellular matrix). These modications may also have important effects on the ability of
IGFBPs to affect IGF action. Other studies have demonstrated that some IGFBPs can induce direct
cellular effects independent of the IGFs. These non-canonical actions of IGFBPs do not involve
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binding to IGFs and are elicited in the absence of IGFs or in the absence of IGF signaling [9] and will be
discussed later in more detail.

IGFBP-1 protein structure

In the 1980s, Bohn and Kraus reported the rst biochemical characterisation of IGFBP-1, known as
placental protein 12 (PP12) [10]. The 234 residues of human IGFBP-1 yield a protein of predicted
molecular mass of ~25 kDa. In the IGFBP-1 mature protein, similarly to the other IGFBPs, three
structural regions can be identied: the N-terminal cysteine rich region; the link region; and the C-
terminal cysteine rich region. The N-terminal region contains 79 residues which are highly conserved
(~80%) in rat and bovine and to a lesser extent (~20%) in the other IGFBPs. This region is involved in IGF
binding activity as demonstrated by the ability of an N-terminal IGFBP-1 fragment to bind IGF peptides.
Site-directed mutagenesis of a number of the amino-terminal cysteines and deletion of 60 amino-
terminal residues [11] have further dened the ligand binding activity of this N-terminal region. The
second region, or N/C-terminal link domain, of IGFBP-1 shares only 40% homology with the bovine and
rat IGFBP-1 protein and no homology with the other IGFBPs. This region of human, rat and bovine
IGFBP-1 possess PEST sequences (Pro-Glu-Ser-Thr) usually found in proteins with rapid turnover (e.g. c-
fos, c-myc), suggesting that IGFBP-1 is a rapidly metabolized protein as also indicated by its dynamic
regulation in vivo, with serum levels varying up to 10-fold or more in relation to meals [12]. The C-
terminal of human IGFBP-1 spans residues 145e234, and shows a 68% homology with rat and bovine
IGFBP-1, but only 14% with the other IGFBPs. Similar to the N-terminal region, the C-terminal region
also participates in IGF binding [13]. An RGD sequence (Arg-Gly-Asp) is found located at residues
221e223 of the C-terminal region. These RGD sequences are present in components of the extracellular
matrix (bronectin, laminin, collagens, etc.) and are involved in binding to their cognate cell membrane
integrin receptors [1,14]. Jones and co-workers have demonstrated in vitro that the RGD sequence of
IGFBP-1 is functional and enables IGFBP-1 to bind to the a5/b3 integrin receptor. However, specic
intracellular signals generated by IGFBP-1 binding to the a5/b3 integrin receptor have not been directly
demonstrated. IGFBP-1 is glycosylated such that 4% of carbohydrates are thought to be O-linked at Ser
and Thr residues. The physiological signicance of IGFBP-1 glycosylation is unclear. Further post-
translational modication of IGFBP-1 is in the form of phosphorylation of the serine residues, lead-
ing to a strengthened binding afnity to IGF peptides and a consequent inhibitory action of IGFBP-1.
The non-phosphorylated form of IGFBP-1 has a lower afnity for IGF peptides and appears to poten-
tiate IGF action.

IGFBP-2 protein structure

Similar to IGFBP-1, three structural regions can be identied in the IGFBP-2 mature protein: the N-
terminal cysteine-rich region; the link region; and the C-terminal cysteine-rich region, with the N- and
C-terminal regions of IGFBP-2 involved in IGF-I/II binding [15,16]. IGFBP-2 is not glycosylated, and even
though IGFBP-2 has a phosphorylation site [17], it is mainly found in the non-phosphorylated form.

IGFBP-2 integrin-binding domain (RGD)

Similar to IGFBP-1, IGFBP-2 possesses an RGD sequence (Arg-Gly-Asp) at position 265e267 in

humans and 246e248 in rats and other species [2]. The IGFBP-2 RGD motif is functional and mediates
IGFBP-2 associations with cell membrane integrins, including the integrins aVb3 and a5b1 [18].

IGFBP-2 heparin-binding domain (HBD)

The middle region of IGFBP-2 features a heparin-binding domain (HBD) represented by the
sequence 179PKKLRP184 (mature protein). Using a site-directed mutagenesis approach, the authors have
demonstrated that the HBD motif is required for IGFBP-2 binding to components of the ECM, and
IGFBP-2-mediated cell proliferation and migration [19]. Recent work from Shen et al. has shown that
the HBD motif in IGFBP-2, reported as HBD1, inuences the binding of IGFBP-2 to the cell surface
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receptor protein tyrosine phosphatase b (RPTP b), an event leading to inhibition of RPTP b phosphatase
activity [20]. An additional pH-dependent heparin-binding site, reported as HBD2, has been located
within the carboxyl-terminal region and thyroglobulin type 1 domain of IGFBP-2 [21].

IGFBP-2 nuclear localization signal (NLS) motif

Nuclear entry of IGFBP-2 [22] has only been recently reported by the authors with the presence of a
nuclear localization signal (NLS) [23]. In contrast to IGFBP-3, -5, -6, which all possess bi-partite NLS
motifs in their C-terminal domains [24], IGFBP-2 has a classic mono-partite NLS sequence (X-K-K/R-X-
K/R-X), within its link/middle domain [23]. This NLS domain is required for interaction with Importin-a
and overlaps exactly with the HBD1 sequence at 179PKKLRP184 [19,23].

IGFBP-1 gene organization and expression

A single gene for IGFBP-1 has been localized to the human chromosomal region 7p14-p12 [25] and
found to be contiguous to the IGFBP-3 gene. A region 50 to the mRNA cap site contains TATA and CCAAT
promoter elements, thus functioning as a gene promoter [26]. Four exons span 5.2 kb of chromosomal
DNA of the IGFBP-1 gene. Exon 1 spans 514 bp and includes the entire 50 untranslated region and the
rst 91 amino acids of the N-terminal of the IGFBP-1 protein. Exon 2 spans 170 bp, which encodes most
of the amino acid sequence of the link domain (middle region). The C-terminal of IGFBP-1 is encoded
by exon 3 (rst half) and exon 4 (second half and 30 untranslated sequence). The IGFBP-1 transcription
start site is located 165 bp upstream from the ATG translation start site and the IGFBP-1 transcript is
~1.55 kb [27]. The 30 untranslated region contains ATTTA motifs that are characteristic of mRNA species
with a very short half-life. IGFBP-1 mRNA is expressed in fetal liver and in post-natal tissues, primarily
in the secretory endometrium, pregnancy decidua and liver [28], but is not found in human fetal and
post-natal kidney. IGFBP-1 mRNA and protein expression has also been described in human osteo-
blastic cells and appears to be triggered by stimulation through glucocorticoids yet inhibited by insulin
as aforementioned in hepatocytes, suggesting that IGFBP-1 produced by osteoblasts in vivo can
modulate the local actions of IGF on bone formation in response to changes in glucocorticoid and
insulin concentrations [29].

IGFBP-2 gene organization and expression

Somatic cell hybrid analysis and in situ hybridization have demonstrated that the human IGFBP-2
gene is localized to chromosome 2 region, q33-q34 [30]. IGFBP-2 is a single copy gene and spans
more than 32 kilobases of genomic sequence. It is organized in four exons with sizes of ~568, 220, 141,
and 496 nucleotides and three introns of lengths 27.0, 1.0, and 1.9 kilobase-pairs. A single transcrip-
tional start site is located at nucleotides 113 2, 50 of the ATG start codon of h-IGFBP-2 translation.
Similar to the IGF-I receptor, this region lacks TATA or CAAT consensus sequence motifs [30]. In a study
by Boisclair et al. [31], it was shown that efcient transcription of the TATA-less promoter of the rat
IGFBP-2 gene only occurs in the presence of three clustered Sp1 sites, which are potential response
elements for estradiol-17 beta (E2) and progesterone (P4). In fact, IGFBP-2 expression is regulated by E2
and P4 in the hypothalamus and by E2 in the pituitary gland [32]. Furthermore, there is evidence that
the transcription of the IGFBP-2 gene is also regulated by the basic helix-loop-helix transcription factor,
AP-4 [33], suggesting that bHLH proteins via binding to E-box elements may affect cell proliferation
and differentiation through induction of IGFBP-2 synthesis. A silencer element, which has a negative
effect on transcription, has been identied in the 50 anking region of the rat IGFBP-2 gene [34]. The
activity of the silencer is reduced and disappears with high cell density, resulting in elevated synthesis
of IGFBP-2. Analysis of the silencer sequence by Kutoh et al. [34] revealed that it contains the target
sequence for the pRb (retinoblastoma) tumour suppressor gene, referred to as the retinoblastoma
control element (RCE), frequently found in the regulatory element of cellular oncogenes and growth
factors. The presence of RCE suggests that the IGFBP-2 gene may be regulated by the pRb tumour
suppressor gene [34]. Further examination of the IGFBP-2 gene promoter sequence has pointed to four
 A. Hoeich, V.C. Russo / Best Practice & Research Clinical Endocrinology & Metabolism 29 (2015) 685e700 689

potential binding sites for transcription factors of the NF-kB/Rel family (NF-kB is a pleiotropic tran-
scription factor that controls the expression of many genes) [35].

Metabolic effects of IGFBP-1

Early work from Cotterill et al. [36] in Hep G2 cells provided initial evidence for metabolic regu-
lation of IGFBP-1. These studies indicated that conditioned media levels of IGFBP-1 were affected by
physiological variation of glucose or insulin concentrations, with low glucose stimulating secretion of
IGFBP-1 while insulin suppressed IGFBP-1 secretion (Fig. 1). In contrast, glucagon and glucacon-like
peptide-1 (GLP-1) stimulated IGFBP-1 secretion in Hep G2 cells and also increased IGFBP-1 serum
concentrations in healthy subjects and human patients [37]. Circulating levels of IGFBP-1, which vary
during the day in humans, are extremely sensitive to insulin secretion [38], with IGFBP-1 levels
abnormally high in diabetes. Variations in fasting IGFBP-1 levels are also documented during pubertal
development with IGFBP-1 levels lowering as puberty advances [38], an effect reecting the rising
concentrations of fasting insulin during pubertal advancement. Levels of IGFBP-1 are dramatically
reduced in excessively obese subjects, and despite normal glucose tolerance, IGFBP-1 decrease is
inversely correlated with elevated fasting insulin [39]. An important implication for chronically low
IGFBP-1 levels is increased IGF bioavailability (low IGFBP-1), which in turn can lead to further

Fig. 1. Hormonal control, postranslational modication and cellular actions of IGFBP-1 and IGFBP-2. Both IGFBPs bind to the cell
surface via the RGD sequence motif, which may have an effect on phosphorylation of phosphatase, tensin homolog (PTEN) and
activation of protein kinase B (AKT) but also for acute regulation of blood glucose via translocation of GLUT4. IGFBP-2 further
contains heparin binding domains, which might also regulate PTEN phosphorylation or nuclear import and gene expression of
vascular endothelial growth factor (VEGF).
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deterioration of metabolic functions and provide chronic mitogenic stimuli of relevance for neoplastic
transformation in target cells and tissue. Conversely, plasma IGFBP-1 levels are increased following
food deprivation, and similarly elevated in patients with anorexia nervosa [40], with IGFBP-1 returning
to nearly normal levels with re-feeding, as also reported through studies involving intensive care
patients [41]. Circulating levels of IGFBP-1 in normal subjects decreases after food ingestion or glucose
administration. Although this decrease appears, most likely, to involve insulin-mediated effects, it
could be instead a direct effect of glucose on IGFBP-1. This possibility was investigated by Snyder et al.
[42], where nutrients/energy sources that activate glycolysis but not insulin secretion were employed
to determine whether suppression of circulating levels of IGFBP-1 are directly controlled by glucose
levels. These studies suggest that while glucose dramatically subdues the level of circulating IGFBP-1,
equivalent concentrations of fructose, activating glycolysis but not insulin secretion, only minimally
affect IGFBP-1 levels [42]. Interestingly, the plasma levels of C-peptide are seen to rise during glucose
infusion, whereas they were only minutely modied by fructose, indicative of insulin-mediated effects
(e.g. stimulation of metabolic pathways, glucose uptake) being essential occurrences regulating the
suppression of circulating IGFBP-1 after food ingestion or glucose administration. In agreement is the
work by Lewitt and Baxter [43] showing that inhibitors of glucose uptake, presumably insulin-
mediated, stimulate the production IGFBP-1 by human fetal liver. In another study by Lewitt et al.
[44], in Wistar rats, it was established that IGFBP-1 infusion caused an elevation in plasma glucose
levels and this increase was maintained even when IGF-I was co-infused with IGFBP-1. These studies
suggested a functional role for circulating IGFBP-1 which, modulated by metabolic status as mentioned
earlier, might provide an effective mechanism for modulation of hypoglycaemic activity of unbound
IGFs in circulation. In addition to insulin modulation of IGFBP-1 levels, comparative investigations by
Norrelund et al. [45] have provided support for a direct suppressive effect of growth hormone (GH) on
serum IGFBP-1 that does not involve modulation of insulin levels by GH. Although early studies
indicated that GH does not exert any direct regulation of IGFBP-1 (e.g. no temporal association of
circadian patterns of GH and IGFBP-1), it appears that the suppressive effect of GH on IGFBP-1 is
unmasked in the presence of low or suppressed insulin levels. These studies also allude to an unrec-
ognized physiological regulation of IGF-1 bioactivity/bioavailability via GH-mediated suppression of
IGFBP-1. Involvement of GH in regulation of IGFBP-1 levels is also indicated by the higher serum levels
of IGFBP-1 seen in untreated GH-decient adult subjects and, conversely, by the suppressed levels of
IGFBP-1 seen in acromegaly [46]. As previously mentioned, IGFBP-1 regulates the bioactive fraction of
the circulating IGF-I pool, and low circulating IGFBP-1 levels are usually associated with conditions of
increased IGF-I activity. Therefore, it is possible to speculate that the suppressive effect of GH on IGFBP-
1 is one of the mechanisms by which GH anabolic effects are mediated (e.g. increased IGF-I bioavail-
ability). Of relevance to metabolic regulation of IGFBP-1 is a study by Allen et al. [47], looking at the
effects of a plant-based (vegan) diet compared with a meat-eating or lacto-ovo-vegetarian diet on
circulating levels of IGF and IGFBPs. Serum IGF-I was reduced by 13% while IGFBP-1 and IGFBP-2 levels
were up by 20e40 % in vegan subjects compared to meat-eaters and vegetarians [47]. Intriguingly,
there were no changes in IGFBP-3, C-peptide, or sex hormone-binding globulin concentrations be-
tween the different diet groups. Whether the elevated IGFBP-1 and IGFBP-2 in vegan subjects, along
with the lowered serum IGF-I, might also provide potential benecial anti-hyperglycaemic and insulin
sensitization effects, although likely, was not discussed. In this regard, Wheatcroft et al. [48] have noted
that in fact, uctuating levels of IGFBP-1 in response to changes in plasma insulin levels clearly point to
a role for IGFBP-1 in glucoregulation. Fasting levels of IGFBP-1 might be predictive of insulin sensitivity,
also inuenced by IGFBP-2.

Metabolic effects of IGFBP-2

There is increasing evidence for IGFBP-2 regulation by metabolism and in metabolic disorders,
while there are also signs of IGFBP-2's contribution to regulation of normal metabolism [49]. In
obesity, circulating levels of IGFBP-2 are suppressed, and low levels of IGFBP-2 are associated with
type 2 diabetes and metabolic syndrome [50]. At younger ages, overexpression of IGFBP-2 in trans-
genic mice increased adipose fat mass [51], suggesting age-dependent and/or pleiotropic effects of
IGFBP-2. Also, in the absence of a heparin binding domain present in the link-domain of the human
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IGFBP-2 molecule, juvenile obesity is observed [52]. As the authors have demonstrated in different
IGFBP-2 transgenic models, muscle mass is beyond the most sensitive target for the negative growth
effects of IGFBP-2 [53]. Reduced muscle mass at younger ages may inuence body composition and
potentially explain the obese phenotype. On the other hand, IGFBP-2 over expression protects against
obesity and diabetes [54] through mechanisms involving IGFBP-2 mediated inhibition of adipogenesis
and modulation of insulin sensitivity. In experiments using obese mice and men by Li and Picard [55],
it was seen that IGFBP-2 mRNA levels were signicantly lower in visceral white adipose tissue (WAT)
than in subcutaneous WAT. This differs from ndings in individuals with anorexia nervosa, who have
high IGFBP-2 levels and signicantly reduced levels of total percentage fat and visceral adipose tissue
[40]. What may be suggested here is that the IGFBP-2 gene could be modulated in a depot-specic
fashion. Furthermore, previous work by the authors [49] has demonstrated that increased con-
sumption of dietary fat or sugar during early life differentially impacts adiposity (increased) and in-
sulin sensitivity (decreased). These changes were associated with alterations in IGFBP-2 RNA
expression in muscle and fat, with monounsaturated fatty acids affecting IGFBP-2 mRNA in vitro
cultures [49]. A direct correlation between low circulating levels of IGFBP-2 and obesity has been
described by Nam et al. [56] in obese males. IGFBP-2 levels were found to be suppressed and inversely
related to levels of free IGF-I, which were elevated and positively correlated with increased fasting
serum insulin concentrations. However, in obese subjects, the levels of total IGF-I and those of IGFBP-
3, despite suppressed GH levels, were not signicantly different between obese and control subjects.
These ndings are indicative of a potential role for IGFBP-2 in modulation of IGF-I bioavailability and
action (e.g. abnormal low IGFBP-2 leading to abnormal elevated free IGF-I) and in turn, IGFBP-2
contribution to maintenance of metabolic functions. A study by Frystyk et al. [57], investigated po-
tential alterations in the GH/IGF axis following overnight fasting in matched groups of lean subjects,
obese subjects with or without T2DM, and subjects with T1DM. In obese subjects without T2DM,
IGFBP-2 levels were reduced and became further reduced in those patients that also had T2DM. As a
point of interest, in T1DM subjects, IGFBP-2 was actually increased [57], as also reported by others in
T1DM [58] and diabetic retinopathy [59], suggesting that T1DM and T2DM each have a unique impact
on IGFBP-2 levels, perhaps related to differences in insulin sensitivity. Low circulating levels of IGFBP-
2 have also been reported in lifestyle-related childhood obesity (with normal growth in spite of
reduced GH secretion) and pre-pubertal obese subjects [60]. With regards to childhood obesity, the
reduced IGFBP-2 serum levels, coupled with a lower ratio of IGFBP-2/IGF-I, imply an increase of tissue
IGF-I bioavailability that in turn may be contributing to the observed accelerated growth seen during
the pre-pubertal years [60]. Furthermore, in intra-uterine growth retardation (IUGR) [61], cord serum
levels of IGFBP-2 are elevated and negatively correlated with both birth length and weight [61]. On the
contrary, circulating levels of IGFBP-2 are low in pre-pubertal and pubertal subjects that were born
small relative to gestational age (IUGR/SGA) [62,63]. In both studies, growth pattern was assessed in
relation to insulin resistance, body composition and IGFBP-2 concentrations (potential modulator of
insulin sensitivity) in individuals born SGA (pre-pubertal and pubertal subjects). IGFBP-2 levels were
inversely correlated with BMI, body fat mass, percent body fat, insulin and leptin levels in both pre-
pubertal and pubertal groups, while being positively correlated with insulin sensitivity. In young
adults who were born SGA, reduced serum IGFBP-2 levels are also associated with increased car-
diovascular risk markers. Both studies thus indicate that IGFBP-2 could be a potential marker for early
recognition of insulin resistance, particularly in SGA children. Increasing evidence has suggested that
leptin, the product of the obesity gene (Ob), is a major metabolic regulator of circulating IGFBP-2
[54,64]. Obesity is linked to leptin resistance and increased circulating levels of leptin (a regulator
of energy balance produced from white adipose tissue) [65]. Investigations in rodents have shown that
leptin regulates hepatic IGFBP-2 expression [64] and that IGFBP-2 levels are closely related to body
weight and glucose metabolism [49]. In more recent work from the authors [66], leptin increases in
skeletal muscle IGFBP-2 by directly activating leptin signalling pathways in the periphery, as well as
activating central mechanisms which communicate to peripheral tissues via the sympathetic nervous
system (SNS) through sympathetic activation of b-adrenergic receptors [66], has been detected. The
authors have also shown that in sheep, central infusion of leptin improves glucose tolerance and
reduces circulating insulin levels in vivo, while silencing of IGFBP-2 in human skeletal muscle reduces
insulin signalling, as well as leptin- and insulin-stimulated glucose uptake [66]. The direct and indirect
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regulation of skeletal muscle IGFBP-2 by leptin, mediated via the STAT-3 and PI3K/AKT signalling
pathways, suggest that leptin appears to regulate peripheral insulin sensitivity through IGFBP-2 [66].
These ndings also infer that leptin may improve insulin sensitivity by directly and indirectly regu-
lating peripheral IGFBP-2 expression. As mentioned earlier, low circulating levels of IGFBP-2 are
associated with adiposity [49] and particularly with visceral adiposity rather than subcutaneous
adiposity [55]. In contrast, high IGFBP-2 levels by overexpressing IGFBP-2 in mice appears to prevent
diet-induced obesity and insulin resistance, with IGFBP-2 also preventing murine adipocyte differ-
entiation in vitro [49,54]. Interestingly, recent work by Xi et al. [67] has shown that IGFBP-2-based
peptides containing HBD motifs efciently inhibited preadipocyte differentiation in male mice, sug-
gesting that IGFBP-2 cell surface interactions via these motifs could be implicated in the modulation of
adipogenesis. Recent studies from our laboratory [68], investigating the effects of IGFBP-2 on visceral
versus subcutaneous adipocytes, have demonstrated that exogenously-added human IGFBP-2
signicantly reduced adipo/lipogenesis in visceral adipocytes, but not in subcutaneous adipocytes.
In fact, silencing IGFBP-2 resulted in exaggerated adipo/lipogenesis in visceral adipocytes, but not in
subcutaneous adipocytes, an effect eliminated when IGFBP-2 was added back. In agreement with Xi
et al. [67], the author's own HBD-1 mutant IGFBP-2 had reduced inhibitory effects on adipogenesis
when compared to wild-type IGFBP-2 [68]. Wild-type IGFBP-2 increased phosphorylation of focal
adhesion kinase (FAK) and decreased phosphatase and tensin homolog (PTEN) levels, suggestive of
integrin-mediated signalling. In fact, overexpression of IGFBP-2 further decelerates glucose clearance
after oral glucose tolerance tests and this is mediated by the RGD-motif present in IGFBP-2 (Reyer et al.
in press). The effect is related to altered FAK/ILK signaling and GLUT4 trafc but not to AKT signaling.
Blockade of this signalling, using Echistatin, completely negated the effects of IGFBP-2 on visceral
adipo/lipogenesis. These studies have thus provided strong support for the idea that IGFBP-2 inhibits
both adipogenesis and lipogenesis in visceral, but not subcutaneous, adipocytes. This depot-specic
impairment appears to be independent of IGF-I and involves cell surface association of IGFBP-2 and
activation of integrin signalling pathways.

IGFBP-1 and breast cancer

The role of IGFBP-1 in breast cancer remains controversial. Early experiments e.g. by Clemmons
et al. [69], demonstrated that ER negative cell lines secrete IGFBP-1 while ER positive do not. These
ndings were also substantiated by the studies of Lonning et al. [70], showing that in breast cancer
patients, Tamoxifen, an antagonist of the estrogen receptor, produced an elevation of plasma IGFBP-1,
an outcome associated with suppression of plasma IGF-I, as also shown by others. Conversely, studies
by Pekonen et al. [71], looking at IGFBPs mRNA expression in human breast cancer tissue, indicated no
correlation between ER content and IGFBP-1expression. Despite the ER content having no relationship
with the expression of specic IGFBP, Pekonen et al. clearly demonstrated that IGFBP expression was
higher in breast cancer tissue than the adjacent normal tissue, suggesting a role for some of these
IGFBPs in malignant transformation in breast tissue. An interesting study by Figueroa et al. [72]
explored the effects of exogenously-added IGFBP-1 on growth of the ER MCF-7 cell line in the
presence or absence of serum, IGF-I or estrogen. MCF-7 proliferation, in the presence of IGF-I, estrogen-
depleted serum or estrogen, was blunted by elevated IGFBP-1 (80 nM), with IGFBP-1 at this concen-
tration also abolishing ligand-dependent IGF-IR phosphorylation. Since MCF-7 cells exposure to serum
or estrogen (E2) did not elicit phosphorylation of the IGF-IR, it was argued that while IGBP-1 inhibits
IGF-dependent phosphorylation of IGF-IR, inhibition of serum- or E2-induced MCF-7 cell growth by
IGFBP-1 most likely involves different mechanisms. These might include transduction of IGFBP-1 ac-
tivity via integrin signalling as previously described e.g. by Perks et al. [73], for IGFBP-2 in breast cancer
cells. A direct inhibitory role for IGFBP-1 of IGF-dependent phosphorylation of IGF-IR is further sup-
ported by Yee et al.'s studies [74] demonstrating that while IGF-IR could not be phosphorylated by IGF-I
in cells expressing IGFBP-1, an IGF-I analogue (Arg-3-IGF-I), unable to complex with IGFBPs, stimulated
IGF-IR phosphorylation in MCF-7 cells.
Epidemiological studies are also discordant on the role of IGFBP-1 in breast cancer, evincing that
while circulating levels of IGFBP-1 inversely correlated to insulin levels might be of prognostic value
[75], Schernhammer et al. [76] have indicated that circulating IGFBP-1 and free IGF-I are not associated
 A. Hoeich, V.C. Russo / Best Practice & Research Clinical Endocrinology & Metabolism 29 (2015) 685e700 693

with breast cancer risk. Furthermore, haplotype-based association studies of IGFBP1 with breast cancer
risk (in a multiethnic cohort) by Cheng et al. [77] has indicated that common genetic variation in the
IGFBP-1 gene does not substantially inuence breast cancer susceptibility. Based on these reports, a
causative role for IGFBP-1 in breast cancer is unlikely, yet it does appear that IGFBP-1 exerts tumor
suppressive activity via sequestration of circulating and local IGFs and therefore blunting IGFs mito-
genic actions in these tumours.

IGFBP-2 and breast cancer

IGFBP-2 is one of the most commonly and abundantly expressed IGFBPs in a broad range of human
cancers [78], often reaching the same concentrations seen in fetal life. IGFBP-2 expression is linked
with tumour aggressiveness and the expression of known tumor markers. IGFBP-2 is consistently
pronouncedly expressed in breast cancer tissue, of most types and grades, when compared with
benign lesions as shown in breast carcinoma tissue by microarray analysis [79]. Expression levels of
IGFBP-2 are strongly correlated with the grade of malignancy, with increased cytoplasm levels and cell
surface association of IGFBP-2 a distinctive feature of carcinoma in situ and of inltrating/invasive
breast carcinomas. Although the precise mechanisms of IGFBP-2 action in these tumors are not fully
understood, it can be proposed that local, tumor-derived IGFBP-2 might inuence autocrine and/or
paracrine growth modulation of the tumour and promote the invasive phenotype of these breast
carcinomas. Whether these potential activities of IGFBP-2 are exerted independently of IGFs, as
described by Frommer et al. [80] in the human breast cancer cell, line Hs578T, via modulation of genes
controlling proliferation, cell adhesion, cell migration or apoptosis remains to be determined. As for
prostate development and prostate cancer, breast development is regulated by hormones such as
estrogens, which has a growth stimulatory action on breast cancer [81]. Anti-estrogen treatment,
targeting the estrogen receptor, is therefore used in breast cancer therapy to counteract estrogen
action. However, these tumours, with time, acquire resistance to anti-estrogen treatment, such that
management of these hormone-resistant tumors becomes a major clinical challenge. IGFBP-2
expression is found at elevated levels in breast cancer cells that exhibit resistance to various anti-
estrogens (e.g. Tamoxifen) [81]. Even though IGFBP-2 does not appear to be causatively involved in
the development of anti-estrogen resistance, and is not a major contributor to the growth of anti-
estrogen resistant breast cancer cells, it is a useful predictive marker for an anti-estrogen treatment
response. IGFBP-2 expression, associated with estrogen receptor status, is in fact an independent
predictor of overall survival. Studies examining IGFBP-2 association with hormonal factors and
obesity, suggest that the prognostic relevance of IGFBP-2 protein expression during breast cancer may
also depend on hormonal context and body weight. A study by Sohn et al. [82], using functional
proteomics in a cohort of residual triple receptor negative breast cancers (TNBC), has identied IGFBP-
2 as a potential indicator of recurrence-free survival (RSF) in TNBC. In this study, IGFBP-2 was not
prognostic in patients with ER-positive disease, but was associated with worse breast cancer disease-
specic survival in patients with ER-negative disease. IGFBP-2 plays a key role in the proliferation and
survival of breast epithelial cells, and silencing of IGFBP-2 mRNA in the MCF-7 breast cancer cell line
results in growth inhibition and increases apoptosis, inferring a crucial action of IGFBP-2 in mitogenic
and survival capacity of these cells [83]. These IGFBP-2 protective activities in MCF-7 cells (e.g. pro-
tective against chemotherapeutic agents induced cell death), are mediated via the integrin receptors
[84], involving downregulation of PTEN and dependent on the presence of a functional estrogen re-
ceptor. With this, IGFBP-2 expression in MCF-7 cells is modulated by the PI3K/Akt/mTOR pathway
[85], of which its activity is inuenced by the levels of PTEN.
In a recent immunohistological study by Dean et al. [86], it was reported that loss of PTEN frequently
takes place in TBNC and is associated with increased IGFBP-2 expression. In agreement with this
nding is that activation of the PI3K/Akt pathway, presumably because of the loss of PTEN, is predictive
of relapse-free survival (RFS) risk in patients with TNBC disease [82], suggesting that this pathway and
its downstream effector IGFBP-2 might be deployed as potential novel therapeutic targets for patients
with residual TNBCs. In these aggressive breast cancers, IGFBP-2 is a potential predictive biomarker for
clinical and biological risk, resistance and relapse [79].
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IGFBP-1 and ovarian cancer

The role of IGFBP-1 in ovarian cancer is also unclear. Early studies by Inaba et al. [87] detailed
relatively high levels of IGFBP-1 (PP12) expression in ovarian adenocarcinomas, including mucinous
and serous cystadenocarcinomas, with IGFBP-1 levels more commonly elevated in patients with
advanced disease. IGFBP-1 is also expressed specically in ovarian clear cell adenocarcinoma
(CCA) [88], indicating that IGFBP-1 could be used as an immunohistochemical marker for
these tumours. IGFBP-1 is found elevated in serum of patients with ovarian tumours [89], while
IGFBP-1 levels in uid from malignant ovarian cyst are low compared to the concentrations of
IGFBP-1 in benign ovarian cyst uid. A study by Lukanova et al. [90] has investigated the risk of
ovarian cancer in relation to prediagnostic levels of C-peptide and IGFBP-1 and -2. However, this
study, taking into account three prospective cohorts (USA, Sweden and Italy), indicated that C-
peptide does not have a direct etiological role in ovarian cancer, while IGFBP-1 and -2 levels appear
to confer a protective effect, correlated with clinical staging and therefore possibly having prog-
nostic value.

IGFBP-2 and ovarian cancer

The IGF system, including IGFBP-2, is signicantly expressed in primary and metastatic ovarian
cancer tissue, suggesting the presence of a local IGF autocrine loop regulating the growth of these
tumors [91]. Circulating levels of IGFBP-2 are elevated in women with invasive malignancy, but
these are lower than those measured for IGFBP-2 in the cyst uid of epithelial ovarian cancer [92].
Of note is that estradiol and IGF-I are also elevated in invasive malignant cyst [93], and their levels
together with those of IGFBP-2, which can be up to 30-fold of that seen in benign neoplasms, enable
discrimination between benign and malignant neoplasm. Despite evidence that the IGFBP2 gene
appears not to be amplied in these tumors, levels of IGFBP-2 mRNA are in fact correlated positively
with the aggressiveness and invasive properties of these tumors. IGFBP-2 is the major IGFBP present
in tissue extracts from these tumors and once again, higher levels are seen in the malignant types,
further supporting a potential key role for IGFBP-2 as a progressive factor in ovarian cancer. A
central part for IGFBP-2 in the development of high grade ovarian carcinoma is further corroborated
by tissue microarray analysis (tissue arrays including normal surface epithelium, benign serous
cysts, serous borderline tumors, low-grade and high-grade serous carcinomas and other ovarian
cancers of different histologic types) [94], demonstrating that IGFBP-2 is greatly overexpressed in
high-grade serous carcinomas compared to normal surface epithelium, benign serous cysts, serous
borderline tumors, or low-grade serous carcinoma. Similarly, an in vitro study by the Zhang group
[95] clearly showed that increased expression of IGFBP-2 contributes to the highly invasive nature of
ovarian cancer cells and that suppression of IGFBP-2 expression, by siRNA, in highly invasive PA-1
ovarian cancer cells dramatically decreased the invasiveness of these cells. Circulating levels of
IGFBP-2 are positively associated with the highly sensitive serum tumor marker, cancer antigen 125
(CA 125) [96], and the fact that IGFBP-2 is found overexpressed in tumors from patients not
responding to chemotherapy suggests that elevated circulating IGFBP-2 might be predictive of
chemotherapy response. This is also substantiated by a study from the Scott laboratory [97] that
indicated ovarian cancer patients presenting at diagnosis with elevated IGFBP-2 are more likely to
relapse and have a poorer outlook. Their work suggests that IGFBP-2 may be an important additional
early prognostic marker of this disease that could enable identication at diagnosis for those pa-
tients that would benet from more aggressive therapies. As has been described, IGFBP-2 may be a
potential biomarker for clinical and biological risk, resistance and relapse in aggressive ovarian
tumors.

IGFBP-1 and prostate cancer

Despite a considerable number of investigations, a causative role or involvement of IGFBP-1 in

prostate cancer is unlikely. While elevated IGF-I is associated with a higher risk of prostate cancer, data
on IGFBP-1 and prostate cancer risk are sparse with most studies indicating that higher levels of IGFBP-
 A. Hoeich, V.C. Russo / Best Practice & Research Clinical Endocrinology & Metabolism 29 (2015) 685e700 695

1 are related to a decreased risk of prostate cancer [98]. Low levels of IGFBP-1 cognate with high levels
of IGF-I appear to be sufcient in elevating the risk of prostate cancer. Haplotype-based association
studies of IGFBP-1 with prostate cancer risk in a multiethnic cohort have also indicated that common
genetic variation in the IGFBP1 gene do not substantially inuence prostate cancer susceptibility [77].

IGFBP-2 and prostate cancer

IGFBP-2 is the main IGFBP produced by prostate epithelial cells, particularly prostate cancer cells
[99], and is found elevated in the serum of patients with prostate carcinoma [100], where its levels
positively correlate with tumour stage/grade. IGFBP-2 is greatly increased in serum from patients with
metastatic prostate cancer compared to healthy controls, and its levels are also increased in subjects
with elevated serum levels of prostate specic antigen (PSA) [15]. Studies by Tennant et al. [101] have
shown that, similar to the changes seen in IGFBP-2 serum levels reported in patients with prostate
cancer, IGFBP-2 levels also change locally in prostatic tissue. In those studies, the expression of IGFBP-2
was investigated in prostate tissue containing benign epithelium, high grade prostate intraepithelial
neoplasia (PIN), and adenocarcinoma. IGFBP-2 protein and mRNA expression were found signicantly
increased in PIN regions and further risen in malignant cells when compared to normal epithelium,
suggesting a potential local autocrine/paracrine tumorigenic action of IGFBP-2. In addition to IGFBP-2
expression being associated with advanced prostate cancer and bone metastasis [102], IGFBP-2 also
appears to be linked with the development of hormone refractory prostate cancer. As reported by
DeGraff et al. [103], androgen treatment in the LNCaP prostate cancer cell line (derived from metastatic
site left supraclavicular Lymph Node; androgen sensitive; AS), initially increases the levels of IGFBP-2,
but with extended androgen treatment levels, decreases via mechanisms likely to involve proteolysis of
IGFBP-2. Interestingly, when the androgen insensitive (AI) and metastatic prostate cancer cell lines
C4e2B4 and C4-2 (LNCaP derived) were investigated, it was found that these cells express a greater
amount of IGFBP-2 and exhibit an attenuated ability to proteolyse extracellular IGFBP-2. These data
suggest that reduced downregulation of IGFBP-2 and loss of the associated proteolytic fragments result
in increased IGFBP-2 levels and lead to enhanced metastatic behaviour of these cells in vivo. These data
also suggest that in AS prostate cancers, proteolysis of IGFBP-2, which generates fragments with
reduced binding afnity for IGFs, leads to an increase in local free IGF, activation IGF-I receptor and
cognate tumorigenic pathways. In other words, in AS prostate cancer, IGFBP-2-mediated tumour
progression appears to be primarily dependent on IGF. In AI prostate cancers, IGFBP-2 levels, not
affected by proteolysis, are higher and it is possible that some of the protumorigenic and metastatic
activities of IGFBP-2 in AI are elicited via IGF-independent mechanisms. These IGF-independent ac-
tivities of IGFBP-2 are likely to involve IGFBP-2 interactions with integrin receptors [104], other cell
surface receptors (e.g. proteoglycans, RPTPb) [16,20], or mediated via intracellular/nuclear action of
IGFBP-2 [22,23]. A number of studies have also explored the involvement of IGFBP-2 in a common
proliferative disorder of the prostate, namely benign prostatic hyperplasia (BPH). Although BPH is not a
feature of prostate cancer, in some individuals, the two pathologies might occur at the same time. As
well, though a selection of studies have reported an altered IGF axis in subjects with BPH, IGFBP-2
levels are consistent with those of normal individuals [15]. IGFBP-2, like IGFBP-1, is thus a potential
biomarker for clinical and biological risk, resistance and relapse, specically in the case of aggressive
prostate cancer [15].

Summary

Under normal conditions, IGFBP-1 and IGFBP-2 are suppressed by GH, but this suppression may be
released e.g. during critical illness [105]. As a result, it can be proposed that both IGFBPs have adaptive
functions in vivo. Very clearly, IGFBP-1 and IGFBP-2 share mechanistic regulation by glucose meta-
bolism, while IGFBP-2 is also linked to lipid metabolism via distinct heparin binding domains or leptin.
Furthermore, IGFBP-2 is controlled by a high number of steroids, most notably by estrogen, and it
seems that IGFBP-2 can also mediate gender-specic effects. In contrast to IGFBP-2, bioactivity of
IGFBP-1 is posttranslationally regulated by glycosylation and phosphorylation. Also, at the structural
level, both IGFBPs possess similarities while also having distinct features: both IGFBPs share the RGD
696 A. Hoeich, V.C. Russo / Best Practice & Research Clinical Endocrinology & Metabolism 29 (2015) 685e700

sequence motif, which seems to link IGFBP-1 and -2 to intracellular PI3K signalling; IGFBP-2 has
heparin binding domains, which at least for that present in the linker domain, connects it to PI3K/PTEN
signalling. The linker domain-resident heparin binding domain further contains a nuclear import
sequence required for nuclear effects of IGFBP-2 on gene expression. As well, a small selection of
reproductive tissues is sufcient to demonstrate that the malignant potentials of IGFBP-1 and -2 are
clearly distinct. It seems obvious that IGFB-2, which is regulated by sex steroids and by itself controls
estrogen receptor expression, has biomarker value for clinical risk in prostate, breast and ovary cancers.
This is in contrast to IGFBP-1 and may thus shed light on the intricate relationships of IGFBP-2 and sex
steroid actions in various malignancies.

Practice points

 IGFBP-1 and IGFBP-2 have tremendous potential for health monitoring (metabolic diseases,
growth disorders, malignant growth).
 In order to develop such potential, it is permissive to differentiate intact from fragmented
IGFBPs by adequate methodology.
 Quantitative Western ligand blotting might be considered as a routine technology for dif-
ferential diagnostics in human patients.

Research agenda

 Functional analysis of subcellular distribution of integrin and proteoglycan binding of IGFBP-

1 and IGFBP-2.
 Interaction with steroid signalling and dissection of sex-specific IGFBP effects.
 Functional analysis of post-translational IGFBP-1 modifications.
 IGFBP proteolysis as a challenge to clinical research.

Conict of interest statement

Andreas Hoeich is a consultant of Ligandis GbR. No other relations have to be disclosed by both
authors.

Acknowledgments

VCR was supported by the National Health and Medical Research Council (NHMRC) of Australia
(Project Grant, # 1008062). VCR also wishes to acknowledge the generous support received from the
Murdoch Childrens Research Institute and the Royal Children's Hospital Foundation. VCR thanks the
Victorian Government Operational Infrastructure Support Program. AH was supported by a grant from
the Deutsche Forschungsgemeinschaft (DFG HO2003/6-1).

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