**1. Introduction**

38 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

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### **1.1 The insulin-like growth factor (IGF) system**

The insulin-like growth factor (IGF) system has been shown to have an integral role in normal growth and development, and in the pathophysiology of various cancers. The IGF system is comprised of a series of circulating ligands (IGF-1, IGF-2), transmembrane receptor tyrosine kinases (IGF-1R, IGF-2R, and the insulin receptor (IR), high affinity ligandbinding proteins (IGFBP1-6), IGFBP proteases, and several low affinity IGFBP-related proteins (IGFBP-rp1 to 10) that work in unison to regulate cell growth [1].

There are two key circulating ligands, IGF-1 and IGF-2, which share approximately 50% structural homology with insulin[2]. IGF-1 is produced primarily in the liver in response to circulating levels of growth hormone(GH) [3]. IGF-1 and IGF-2 are highly homologous small peptide hormones of approximately 7 kDa molecular mass, which are important mitogens that affect cell growth and metabolism [2]. IGFs interact with specific cell surface receptors, designated type I and type 2 IGF receptors, and can also interact with insulin receptor (IR).

The type I IGF receptor (IGF-1R) is a transmembrane heterotetramer consisting of 2 extracellular alpha subunits and two intracellular beta subunits linked by disulfide bonds (fig 1). The intracellular component of IGF-1R has intrinsic tyrosine kinase activity that requires ligand binding for activation [4]. The IGF-1R and the IR share approximately 60% homology which allows them to form hybrid receptors [5]. As a result of this homology, IGF-1R can be activated not only by IGF-1 but also IGF-2 and insulin, although the affinity of IGF-1R for IGF-2 and insulin is approximately 10 fold and 1000 fold lower than for IGF-1, respectively [6]. The type 2 IGF receptor (IGF-2R), which is identical to the cationindependent mannose-6-phosphate receptor, binds IGF-2 with 500 fold increased affinity over IGF-1[7]. IGF-2R does not bind insulin. Most of the biological activity of IGF-2 is thought to be mediated through binding IGF-1R[7]. IGF-2 is known to function primarily as a scavenger receptor, regulating circulating IGF-II levels through internalization and degradation [7].

Insulin-Like-Growth Factor-Binding-Protein 7: An Antagonist to Breast Cancer 41

Unlike insulin, IGFs circulate in biological fluids complexed to a family of structurally related binding proteins, called IGF-binding proteins (IGFBPs). The IGFBP superfamily can be subdivided into two groups: the high affinity IGFBPs (IGFBP1 to 6) and the low-affinity

There are, to date, six well characterized mammalian IGFBPs, designated IGFBP-1 through - 6. IGFBPs are capable of binding IGF-1 and IGF-2 with higher affinity than their interactions with the IGF-1R, but do not bind to insulin. Some IGFBPs compete for activity of IGFs at the receptor level and antagonize IGF signaling, while others (eg. IGFBP2 and IGFBP5) appear to amplify IGF signaling [17]. Therefore, IGFBPs function not only as carriers of IGFs, thereby prolonging the half-life of the IGFs, but also act as modulators of IGF availability and activity[18]. Apart from their ability to inhibit or enhance IGF actions, all the IGFBPs have been reported to exert distinct biological actions such as cell proliferation, differentiation, migration, angiogenesis and apoptosis through an IGF/IGF-1R-independent

All six IGFBPs share approximately 35% sequence identity with each other. The primary structures of mammalian IGFBPs appear to contain three distinct domains of roughly similar sizes: the conserved N-terminal domain, the highly variable midregion, and the conserved C-terminal domain. Within their N-terminal domain, all IGFBPs share a common conserved cysteine-rich domain termed IGFBP motif (GCGCCXXC) (fig 2). The IGFBP motif is encoded by a single exon, has overall similar topology and is only present in vertebrates [19]. Ten to 12 of the 16-20 cysteines found in the prepeptides are located within this domain. In IGFBP1-5 these 12 cysteines are fully conserved, whereas 10 of the 12 cysteines are invariant in IGFBP6 [19]. The midregion is believed to act structurally as a hinge between the N and C terminal domains. Posttranslational modifications (glycosylation, phosphorylation) of the IGFBPs has been found only in the midregion so far. The C-termini of IGFBPs, like the N-terminal domain, are highly conserved, and contain the remaining 6 of the total 16-20 cysteines. The primary sequence of all members of the IGFBP family surrounding the last 5 cysteines is strikingly similar (~40%), implying that the tertiary structure of the C-terminal domain should be almost identical. Interestingly, the amino acid sequences embracing these last 5 cysteines share 37% similarity with the thyroglobulin-type-1 domain, a structural motif occasionally employed as an inhibitor of proteases [19,24]. It has been hypothesized that the N and C-terminal domains are capable of acting independently of each other based on the fact that the cysteines within each of the conserved regions are even numbered, and that proteolytic cleavage products of IGFBPs contain either the C or N-terminal regions. Indeed, disulphide linkages have been shown to form typically within each conserved domain, rather than between domains[25,26]. All the IGFBPs are encoded by 4 exons, except IGFBP3 which has an extra exon, exon 5, that is not translated. The striking observation is the correlation between these IGFBP exons and the three protein domains of IGFBPs. The N-terminal domain is encoded within exon 1 in all of the IGFBPs, as is the 5' untranslated region and a few amino acids of the midregion. Exon 2 encodes the nonconserved midregion. Both exons 3 and 4 encode for the conserved C-terminal domain. The containment of the N-terminal domain within one exon, combined with the ability to

bind IGFs, supports the concept of an IGFBP superfamily [27,19].

**2. The IGFBP superfamily** 

*High affinity binding proteins (IGFBPs)* 

manner [19,20,21,22,23].

IGFBPs (IGFBP7 to 10, and IGFBP-rP5 to 10). [16].

Fig. 1. Cell surface receptors for IGFs and insulin. Illustration of the different transmembrane receptors and ligands of the IGF system. Purple represents the alpha and beta subunit of IGF-1R; red represents the alpha and beta subunit of the IR-B; orange represents the alpha and beta subunit of the IR-A ; green represents the IGF-2R. The potential ligand(s) is shown above the respective receptor.

Two distinct insulin receptor isoforms have been identified and are known to hybridize with IGF-1R. The insulin receptor isoform A (IR-A), the IR fetal isoform, is generated by alternative splicing through the deletion of exon 11 of the insulin receptor gene whereas the insulin receptor isoform B (IR-B) retains exon 11 [8]. IR-A is the predominant isoform expressed in fetal tissues and cancers with ubiquitous expression, whereas IR-B appears in postnatal life within insulin-target tissues, such as muscle, adipose tissue and kidney [9,10,11]. Data obtained from murine 32D hemopoietic cells demonstrated that IR-A preferentially induces mitogenic and anti-apoptotic signals, whereas IR-B predominantly induces cell differentiation signals [12]. IR-A, but not IR-B, binds IGF-II with high affinity and operates as a second physiological receptor for this growth factor [13]. The two IR isoform half receptors (composed of one alpha and one beta subunit) can heterodimerize, resulting in the formation of either homologous IR-A/IR-A or IR-B/IR-B receptors as well as the hybrid IR-A/IR-B insulin receptors [14](fig 1). Heterodimers can also form between IGF-1R and IR, resulting in the hybrid IGF-1R/IR-A and hybrid IGF-1R/IR-B. Hybrid IGF-1R/IR receptors are believed to mostly bind IGF-1, although they can also bind insulin but with a much lower affinity [15]. The IGF system is also regulated by a group of at least six high affinity ligand-binding proteins, the insulin-like binding proteins (IGFBPs), as well as low affinity ligand-binding proteins (IGFBP-rp1 to 10).
