Tyrosine kinase signalling in breast cancer: Insulin-like growth factors and their receptors in breast cancer

The insulin-like growth factor (IGF) system exerts pleiotropic effects on mammalian cells. This review focuses on type I IGF receptor (IGF1R)-mediated signal transduction and its relevance in breast cancer. Upon activation by the IGFs, IGF1R, a transmembrane tyrosine kinase receptor, undergoes autophosphorylation, and then binds and phosphorylates additional signaling molecules. These intermediates initiate a series of downstream signaling events that are involved in multiple physiologic processes for cells. Recent data demonstrate that the IGF receptor system actively interacts with the estrogen receptor and integrin receptor systems. Cross-talk among these pathways regulates breast cancer proliferation, protection from cell death, and metastasis. Better understanding of IGF biochemical signaling pathways is of utmost importance for developing therapies for breast cancer.


Introduction
The IGF system is composed of IGF ligands, receptors, and binding proteins. These system components form a highly regulated network of interactions both among themselves and between other biologic signaling pathways.
The two well-characterized ligands, IGF-I and IGF-II, are mitogenic peptides that are highly homologous to each other and to insulin [1]. Whereas insulin is composed of two chains (A and B) of 21 and 30 amino acids, respectively, the IGFs are single-chain molecules that retain the equivalent of the connecting (C)-peptide of proinsulin between the A and B domains. IGF-I and IGF-II are thought to have autocrine, endocrine, and paracrine roles in normal mammary development and in the etiology of breast cancer [2][3][4][5].
Unlike insulin, circulating IGFs are found complexed to high-affinity binding proteins known as IGF-binding proteins (IGFBPs). Six distinct species have been cloned. An additional family of structurally homologous proteins has been identified and named IGFBP-related proteins, because their affinity for the IGFs is significantly lower than that of the IGFBPs [6,7]. Cleavage of IGFBPs by specific proteases modulates levels of free IGFs and IGFBPs, and thereby their actions. In addition, IGFBPs may also have effects that are completely independent of their role in modulating the action of IGF [8].
The cellular actions of IGFs are mediated by type I and type II receptors, insulin receptor, and insulin receptor-IGF1R hybrids. The type II IGF receptor (IGF2R) is a multifunctional nontyrosine kinase receptor [9][10][11] that is also known as the cation-independent mannose-6-phosphate receptor, and its function in regulating the action of IGF-II has been controversial. IGF1R is a glycosylated heterotetramer that is composed of two extracellular α-subunits and two transmembrane β-subunits that have intrinsic tyrosine kinase activity [12,13]. This review focuses on IGF1Rmediated signaling and its relevance in breast cancer.

Type I insulin-like growth factor receptor signaling
Activation of the IGF1R by IGFs results in its oligomerization, autophosphorylation, and activation of the intrinsic tyrosine kinase [12][13][14][15] IRS-1 is a well-characterized IGF1R-signaling molecule that has multiple sites for tyrosine phosphorylation and acts as a 'docking protein' for other signaling molecules [26,27]. Upon activation of IGF1R, IRS-1 binds and becomes rapidly tyrosine phosphorylated, allowing docking sites for SH2 domain-containing proteins. IRS-1 phosphorylation results in the activation of many downstream signaling pathways; many of these pathways are implicated in mitogenesis and protection from apoptosis. For instance, the following are all known to be stimulated through IRS-1: PI-3K pathway through the association with the p85 regulatory subunit of PI-3K Additional pathways may be affected by IGF1R activation. For example, the cytoplasmic tyrosine kinase c-Src can phosphorylate IGF1R on the same sites as the IGFinduced autophosphorylation sites [33]. CSK, a negative regulator of Src activity, associates with activated IGF1R, and therefore may play a role in the decrease in Src activity after IGF-I stimulation. Other substrates of Src are almost exclusively proteins that regulate actin cytoskeleton dynamics, such as FAK, p130 Crk-associated substrate, cortactin, and p190RhoGAP. IGF-I through its receptors has been shown to positively or negatively modulate tyrosine phosphorylation of focal adhesion proteins such as FAK, p130 Crk-associated substrate, and paxillin [24 • ,34,35]. Thus, activation of IGF1R, via its interaction with Src, could influence aspects of cytoskeletal organization and cell adhesion.

Insulin-like growth factors and insulin-like growth factor receptor signaling in breast cancer
Expression of IGF-I and IGF-II has been measured in normal and breast tumor tissues by in situ hybridization and immunohistochemistry. IGF-I is found mainly in stromal cells that are adjacent to normal breast cells [2]. IGF-II is also mainly expressed in the stroma [5], but may occasionally be found in malignant epithelial cells [4]. Increased IGF-II expression is seen in stromal cells that are adjacent to malignant epithelial cells, whereas levels are lower in stroma that are adjacent to benign and normal breast epithelium [36,37]

Consequence of insulin-like growth factor activation in breast cancer
Activation of the IGF system is known to have substantial pleiotropic effects on mammalian cells. Mitogenesis, transformation, and antiapoptosis induced by IGF1R stimulation could account for many aspects of the malignant phenotype. Both IGF-I and IGF-II stimulate ER-positive breast cancer cell proliferation at picomolar to nanomolar concentrations [51]. Once IGFs interact with receptors, we found that IRS-1 is the predominant signaling molecule activated in ER-positive human breast cancer cells [52 • ].
There is also accumulating evidence that IGF action influences breast cancer cell responsiveness to estrogen. It is well established that estrogens stimulate the growth of ERpositive breast cancer cells. ER acts as a ligand-activated transcription factor. Two forms of ER have been cloned, ERα and ERβ [53-55]. Erα contains a hormone-binding domain, a DNA-binding domain, and two transcriptional activation domains (AF-1 and AF-2). Estradiol binding to ERα results in dimerization and subsequent binding of the hormone-receptor complex to specific DNA palindromic sequences (estrogen response elements) to initiate gene transcription, and therefore induce the expression of growth promoting genes. To date, a similar role for ERβ has not been found. Antiestrogens, such as tamoxifen, influence ERα function by blocking the initiation of transcription from estrogen response elements without interfering with the binding of ligand-receptor complex to DNA.  67], and inhibiting ligand-independent activation of the ER by IGF-I [68][69][70][71]. Thus, several members of the IGF family could be the growthpromoting genes that are regulated by estrogen.
On the other hand, IGF-I also directly increases the transcriptional activity of the ER and increases expression of estrogen-inducible genes, such as the progesterone receptor gene [71]. Furthermore, IGFBP-1, an inhibitor of IGF-1 action, not only inhibited IGF-mediated activation of the ER, but also had a significant inhibitory effect upon estrogen-mediated activation of the ER. Although the mechanisms that account for this cross-talk are not clear, it is obvious that both signaling pathways can positively influence each other, resulting in reinforcement of biologic effects for both estrogen and IGFs.
Many model systems have shown that IGF1R activation protects cells from programmed cell death. The PI-3K pathway and its substrate AKT1 probably mediate this effect. It has been reported [72] that AKT1 is highly expressed in several human breast carcinoma cell lines, and its activity in MCF-7 cells is modulated by estradiol and IGF-I. Overexpression and activation of AKT1 produces estrogen and IGF-I independent proliferation and controls an antiapoptotic pathway. IGF-I reduces apoptosis in doxorubicin-treated and paclitaxel-treated MCF-7 cells [73]. Detailed studies indicate that IGF-I rescue of MCF-7 cells from chemotherapy-induced cell death involves at least two mechanisms: inhibition of apoptosis through PI-3K and induction of proliferation through both PI-3K and MAPK cascades. In clinical specimens, high levels of IGF1R may protect cells from radiation therapyinduced apoptosis [74 • ].
Several reports have documented the interaction of IGF and integrin signaling pathways. The direct interaction of the two pathways was demonstrated through a physical association between α v β 3 integrin and IRS-1 [75 •• ]. Later reports [24 • ] also showed that FAK, a downstream signaling molecule of integrins, is a substrate for the insulin receptor and IGF1R. In vascular smooth muscle cells, ligand occupancy of α v β 3 integrin is required for full activation of the IGF1R β-subunit and IRS-1 by IGF-I stimulation [76,77 • ]. IGFs are chemoattractants for breast cancer cells, perhaps due to the ability of IGF to affect the integrins [78]. Activation of integrin signaling pathways have been reported [79] to inhibit the mitogenic effect of IGF-I in human breast cancer cell lines. Recently, IGF1R activation was shown to induce rapid and transient tyrosine dephosphorylation of FAK, p130 Crkassociated substrate, and paxillin in MCF-7 breast cancer epithelial cells [80]. Finally, IGFs may be involved in cell migration and invasion, because dominant-negative IGF1R constructs inhibit invasion and metastasis of MDA-435 breast cancer cells in vitro and in vivo [81 • ].

Conclusion
Breast cancer is a lethal disease because the transformed epithelial cells proliferate, metastasize, and are protected from programmed cell death. The pathways responsible for each of these phenotypes are only now becoming understood. Despite the multiple accumulated genetic abnormalities that cause malignant transformation, however, it is evident that some of the transformed cells can still respond to signals from their external environment. Notably, the inhibition of ER function has proven to be a powerful weapon in breast cancer treatment.
There now is a large body of evidence showing that IGF activation is involved in these malignant processes; clearly some fully transformed cells can still respond to these cues. It is also evident that the signaling pathways that are activated by the IGFs are not simple or linear. Multiple divergent and convergent biochemical signaling pathways are stimulated after receptor activation, which then impinge upon multiple other pathways that are known to be important in breast cancer biology. We are now just beginning to understand how the IGFs affect breast cancer biology. The next challenge will be to untangle the web of signal cascades initiated by these factors. By doing so, we will be better positioned to develop therapies based on interruption of the key signaling pathways that are responsible for the malignant phenotype.