Modifying IGF1 Activity: an Approach to Treat Endocrine Disorders, Atherosclerosis and Cancer

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Modifying IGF1 activity: an approach to treat endocrine disorders, atherosclerosis and cancer

David R. Clemmons Abstract | Insulin-like growth factor 1 (IGF1) is a polypeptide hormone that has a high degree of structural similarity to human proinsulin. Owing to its ubiquitous nature and its role in promoting cell growth, strategies to inhibit IGF1 actions are being pursued as potential adjunctive measures for treating diseases such as short stature, atherosclerosis and diabetes. In addition, most tumour cell types possess IGF1 receptors and conditions in the tumour microenvironment, such as hypoxia, can lead to enhanced responsiveness to IGF1. Therefore, inhibiting IGF1 action has been proposed as a specific mechanism for potentiating the effects of existing anticancer therapies or for directly inhibiting tumour cell growth.

Stromal cells Insulin-like growth factor 1 (IGF1), a small polypeptide IGF1 inhibition is being pursued as a potential adjunctive Connective tissue cells, (7,500 kDa) involved in cellular growth, and is a member measure for treating atherosclerosis. Inhibiting IGF1 primarily fibroblasts, that are of a family of structurally related peptides that also include action has been proposed as a specific treatment either present in nearly every organ. insulin-like growth factor 2 (IGF2) and human proinsulin. for potentiating the effects of other forms of anticancer IGF1 circulates in relatively high concentrations (150–400 therapies or for directly inhibiting tumour cell growth. ng per ml) in plasma, predominantly as the protein-bound This Review will encompass a discussion of the factors form, with the free active peptide representing only a small that regulate IGF1 synthesis and secretion, and focus percentage (less than 1%) of the total1. Despite structural on the strategies that have been used to modify IGF1 similarities between family members, each peptide binds actions in tissues principally for developing drugs for selectively to distinct cell surface receptors, which accounts the treatment of growth disorders, catabolism, diabetes, for much of the specificity of each peptide’s actions. The atherosclerosis and cancer. type 1 IGF receptor is a heterotetramer composed of two α subunits that contain the hormone binding domain, IGF1 synthesis and tissue growth which are linked to two β subunits that contain tyrosine IGF1 is synthesized in multiple tissues including liver, kinase catalytic activity domains by disulphide bonds2. skeletal muscle, bone and cartilage. The changes in Upon ligand occupancy the receptor undergoes a confor- blood concentrations of IGF1 reflect changes in its mational change that activates the tyrosine kinase activity, synthesis and secretion from the liver, which accounts which then activates downstream signalling molecules by for 80% of the total serum IGF1 in experimental protein phosphorylation. The regulation and synthesis of animals4. The remainder of the IGF1 is synthesized in the IGF1, IGF2 and insulin is quite distinct3. IGF1 synthesis is periphery, usually by connective tissue cell types, such controlled by several factors, including the human pitui- as stromal cells that are present in most tissues. IGF1 that tary growth hormone (GH, also known as somatotropin), is synthesized in the periphery can function to regulate whereas insulin concentrations are controlled primarily cell growth by autocrine and paracrine mechanisms3. by changes in blood glucose. IGF2 concentrations are Within these tissues, the newly synthesized and secreted high during fetal growth but are less GH-dependent IGF1 can bind to receptors that are present either on the Division of Endocrinology, in adult life compared with IGF1. These three peptides connective tissue cells themselves and stimulate growth Department of Medicine, have complementary roles in growth regulation. Because (autocrine), or it can bind to receptors on adjacent cell University of North Carolina, of its anabolic and insulin-like properties, strategies are types (often epithelial cell types) that do not actually Chapel Hill, North Carolina 27599‑7170, USA. being pursued for treating short stature, catabolism and synthesize IGF1 but are stimulated to grow by locally e-mail: [email protected] controlling blood glucose in diabetes. IGF1 also has an secreted IGF1 (paracrine) (FIG. 1). Several experimental doi:10.1038/nrd2359 important role in promoting cell growth and consequently animal model systems have been analysed to determine

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in birth weight and a 70% reduction in final adult size16. GH Blood vessel GH By contrast, selective deletion of IGF1 synthesis in the liver (which leaves peripheral synthesis intact), decreases serum IGF1 by 80% but results in less than a 10% GH reduction in adult size13. These results imply that IGF1 receptor produced in the periphery is the main determinant of somatic growth in mice, whereas hepatic synthesis is the IGF1 primary determinant of plasma concentrations3. Hepatic Paracrine IGF1 Autocrine synthesis of IGF1 is regulated by several hormones, action action IGF1 principally GH, but the ability of GH to stimulate IGF1 receptor is strongly influenced by nutritional status. Following GH administration there is a major increase in blood IGF1 concentrations17. This increase in blood IGF1 acts IGF1 Extracellular to suppress GH synthesis in the pituitary gland through matrix a process termed negative-feedback regulation18, which synthesis IGFBP5 reservoir represents an important homeostatic mechanism for maintaining normal plasma IGF1 concentrations. After 5 days of fasting, hepatic synthesis of IGF1 is relatively Extracellular matrix refractory to GH stimulation and plasma IGF1 declines IGF1 receptor GH receptor by 50% (ref. 19). Upon refeeding, GH sensitivity is restored within 72 hours. Other hormones, including Figure 1 | Autocrine and paracrine actions of IGF1. Insulin-like growth factor 1 thyroxine, cortisol, oestradiol and testosterone20 par- (IGF1) is synthesized in peripheral tissues by connective tissueNatur celle Re typesviews |such Drug asDisc overy ticipate with GH in regulating hepatic IGF1 synthesis. fibroblasts. These cells contain growth hormone receptors and can respond to growth hormone (GH) that enters the tissues from blood vessels. Newly synthesized IGF1 is Thyroxine enhances sensitivity to GH, whereas cortisol secreted and transported to adjacent cells (paracrine action), where it stimulates acts to inhibit IGF1 synthesis. High cortisol concentra- coordinated cellular growth. It can also be secreted and then rebind to the cell of tions can lead to growth attenuation by this mecha- origin, where it stimulates cell growth (autocrine action). Similarly, locally produced nism. Oestradiol inhibits IGF1 synthesis in the liver by IGF1 can bind to IGF binding proteins (IGFBPs) such as IGFBP5 localized in the inducing suppressor of cytokine signalling 3 (SOCS3), extracellular matrix where a reservoir of IGF1, which can be released following tissue which inhibits GH stimulated signal transduction21. injury or during repair, is created. Stimuli other than GH, such as platelet-derived Testosterone not only enhances hepatic IGF1 synthesis, growth factor, can increase IGF1 synthesis; these factors are important for initiating but also alters the sensitivity of the pituitary gland to the response of tissue repair after injury. negative-feedback regulation of GH secretion, thus leading to increases in GH and IGF1. IGF2 concentrations in blood, which are threefold greater than IGF1, are mini- the variables that regulate autocrine and paracrine secre- mally increased by GH22 and are significantly decreased tion of IGF1 and its actions5,6. Following tissue or cellular by prolonged fasting. injury there is a wave of IGF1 synthesis that stimulates Following either local secretion or transport through reparative cell types to replicate; this response has been the circulation to target tissues, IGF1 and IGF2 bind to shown to occur in injured blood vessels6, skeletal mus- the type 1 IGF1 receptor. IGF1 binds to the receptor with cle5, cartilage7 and in the brain8. Other growth factors sixfold to eightfold higher affinity than IGF2 and both that are involved in the repair process, such as platelet- peptides have affinities that are more than 100-fold greater derived growth factor (PDGF), fibroblast growth factor than insulin23. Conversely, insulin has a much higher (FGF) and epidermal growth factor (EGF), can stimulate affinity for its receptor. Following receptor activation, local synthesis of IGF1 (refs 9–11). When transplanted the tyrosine kinase autophosphorylates tyrosine residues into experimental animal models, tumour cell types pro- that serve as important docking sites for the signalling duce IGF1 and/or IGF2, which can stimulate tumour proteins SRC homology 2 domain-containing protein growth. Additionally the mesenchymal cells surrounding (SHC) and insulin receptor substrate family 1 (IRS1)24. the tumour also provide an important paracrine source Both of these signalling intermediates are important for of IGF112. Autocrine and paracrine IGF1 signalling is the activation of the phosphoinositide 3 kinase (PI3K) believed to be important in determining normal fetal and mitogen-activated protein (MAP) kinase pathways growth13, and fetal brain expression of IGF1 is thought that mediate the metabolic and growth promoting to be crucial for determining brain growth and head actions of IGF1 and IGF2 (REF. 25). circumference14. IGF binding proteins Control of IGF1 synthesis and secretion The concentrations of IGF1 and IGF2 in blood are also The connective tissue cell types that synthesize IGF1 determined indirectly by the levels of IGF binding proteins contain growth hormone receptors and increases in (IGFBPs). IGFBP3 is the most abundant IGFBP in blood SOCS3 pituitary GH secretion stimulate IGF1 synthesis15. This and has the highest affinity for IGF1 and IGF2, therefore SOCS proteins bind to signalling elements in cytokine stimulation of Igf1 synthesis by GH in peripheral tissues it accounts for 75–80% of the total carrying capacity. The signalling pathways and inhibit is an important determinant of somatic growth. In mice, IGF1–IGFBP3 complex binds to a third protein termed their function. germline Igf1 gene deletion results in a 50% reduction acid labile subunit (ALS). This tripartite complex has a

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half-life of 16 hours. By contrast, the half-life of free IGF1 Stimulation of statural growth. Patients with severe IGF1 is less than 15 minutes3. As the concentration of free IGF1 deficiency have been treated with recombinant human in normal subjects is less than 1% of the total IGF1 con- IGF1. The largest amount of clinical data has been gained centration, formation of this ternary complex results in with children with short stature and mutations of the GH most of the IGF1 and IGF2 in blood being present in a receptor that resulted in resistance to GH, as manifested stable reservoir26. GH also stimulates IGFBP3 and ALS by a failure to grow in response to GH therapy. Three secretion and this functions to further stabilize IGF1 large clinical studies have shown that if IGF1 (80–120 levels27. During severe catabolism or conditions such as µg per kg) is administered systemically twice a day by diabetes, IGFBP3 undergoes proteolysis, resulting in a subcutaneous injection, serum IGF1 concentrations lowering of the total IGF binding capacity28. can be increased from low levels to levels that are 1.8 The second most abundant IGFBP is IGFBP2. standard deviations above the mean for age36. Growth IGFBP2 does not bind to ALS and the IGF1–IGFBP2 or rates during the first year of therapy have averaged 8–9 IGF2–IGFBP2 complexes have much shorter half-lives cm per year and between 6–7 cm per year in the second (~90 minutes). IGFBP2 in serum is unsaturated and year of therapy. This growth velocity can be maintained represents an excess reservoir of binding capacity3. for several years and these children generally achieve a A third IGFBP, IGFBP1, accounts for only a small final adult height that is close to the lower limit of the percentage of the IGF carrying capacity. Like IGFBP2, normal range. A few children have been able to achieve IGFBP1 is generally unsaturated and, therefore, rep- their predicted final adult height but this is unusual. Side resents a potential regulator of free IGF1 and IGF2. effects have been minimal but have included oedema, IGFBP1 is suppressed by insulin and there is a fourfold headaches, hypoglycaemia and pseudotumour cerebri37. to fivefold increase in the fasting state29. Following feed- There is some disproportionate organ growth, with ing the decrease in IGFBP1 results in a rapid reduction splenic and tonsilar enlargement exceeding the increase in the total IGF binding capacity making free IGF1 and in the size of other organs. These changes revert to IGF2 more available to peripheral tissues. normal when IGF1 is discontinued. IGF1 has also been IGFBP4, 5 and 6 are present in lower concentrations co-administered with IGFBP3. Treatment of children and appear to be less important for regulating free IGF with this complex results in more stable IGF1 levels concentrations in serum. IGFBPs are also synthesized by in serum. Growth rates that were attained during the peripheral tissues and in interstitial fluids there is usually first year of therapy were equivalent to those obtained an excess of IGF binding capacity3. As the affinity of the using IGF1 alone36. Whether this will result in a reduced IGFBPs is higher than that of the IGF1 receptor, they side-effect profile has not been determined. represent a potential reservoir of peptide that can bind Other rare mutations that result in an alteration of to cell surface receptors (FIG. 1). GH actions and dwarfism, such as signal transduction In summary, IGF1 is regulated by multiple mecha- and activator of transcription 5b (STAT5B) mutations, nisms that are known to regulate systemic growth. As IGF1 gene defects and IGF1 receptor mutations that a general growth stimulant, IGF1 promotes multiple result in only partial loss of sensitivity to IGF1, repre- metabolic actions that are required for growth, such as sent reasonable targets for this therapy and studies to protein synthesis, calcium accretion and fatty acid and prove that these patients will respond are ongoing38. glucose transport30. Furthermore, as IGF1 receptors are Currently a Phase II clinical trial is underway to deter- ubiquitously expressed, targeting IGF1 actions either by mine whether short children (without known GH reducing ligand concentration or by blocking IGF1 bind- receptor mutations) who have low IGF1 values and a ing to the IGF1 receptor results in changes in multiple suboptimal response to GH therapy will benefit from tissues and limits the ability to target one specific cell IGF1 therapy. Because IGF2 has a lower affinity for the type or organ. Despite this limitation, it may be possible IGF1 receptor and because of uncertainty regarding its to selectively target specific cell types by identifying safety, it has not been developed as a therapy for short co-receptors that modify IGF1 action. Alternatively, stature. In summary, IGF1 clearly has a role in treating Pseudotumour cerebri using drugs that target the IGF1 receptor in a setting short children with severe IGF1 deficiency. The extent to A condition in which there where other agents are used to achieve cell type specifi- which children who are relatively resistant to GH but do is increased intracranial city or in which blocking IGF1 action would provide an not have mutations in signalling proteins in the GH axis pressure but no tumour. additive effect on a particular cell-type that had been will respond to IGF1 is currently being investigated. Stat5b targeted by an alternative therapy could be exploited. A signalling protein that Both of these approaches have been tested in cells in Catabolic states. Several catabolic states result in relative following activation of a culture or in experimental animal models31–35. resistance to GH, which is mediated by increases in the growth hormone receptor concentrations of cytokines, such as tumour necrosis is phosphorylated and Activation of IGF1 receptor to treat dieases translocated to the factor (TNF) and interleukin 1 (IL1), both of which 39 nucleus where it induces The availability of recombinant human IGF1 has made inhibit IGF1 synthesis and block its actions in tissues . gene transcription. it possible to complete several clinical studies aiming to GH resistance has been demonstrated in patients with determine whether an increase in the plasma concentra- HIV wasting and various disorders related to inadequate HIV wasting tions of IGF1 results in stimulation of systemic growth, nutrient intake or absorption such as, cystic fibrosis, A syndrome in which patients with HIV become severely reversal of catabolic states or enhancement of insulin coeliac disease and anorexia nervosa. IGF1 has been catabolic despite a normal action. These studies were all based on a sound rationale administered for relatively short intervals (for example, calorific intake. (detailed below), but their results have been variable. for less than 3 weeks) to these patients as well as to patients

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with burns and patients with closed head trauma, who treating diabetes have been proposed. Administration are severely catabolic. In general, these catabolic patients of IGF1 to mice lacking the IGF1 receptor in skeletal respond to IGF1 with increases in protein synthesis muscle lowered their blood glucose, due to inhibition and a positive or overall anabolic response40. When the of renal gluconeogenesis50. Other studies suggest that IGF1–IGBP3 complex was administered to patients with IGF1 improves insulin sensitivity by inhibiting the burns it led to an increase of protein synthesis41. IGF1 is a secretion of GH, which can function as an insulin potent growth factor for osteoblasts, and two studies, one antagonist51. in elderly patients with hip fractures and one in younger Several clinical studies have been designed to test patients with anorexia nervosa, have been undertaken42,43. the role of IGF1 in type 1 and type 2 diabetes. In type 1 The patients with anorexia received IGF1 alone, whereas diabetes, the mechanism of enhanced insulin sensitivity the hip fracture patients received IGF1–IGFBP3. Both is partly due to suppression of GH secretion, which studies showed that the reduction in bone mineral den- results in enhancement of insulin action as GH is a sity that is present in these two groups of patients can direct antagonist of insulin in the liver52. Larger trials be improved with IGF1 therapy. The effects that were of IGF1 administration to type 1 diabetics have shown achieved with IGF1 were significant compared with a consistent maintenance of reduced insulin require- control groups that received a placebo, but they required ments over 4–8-week periods and significant (~1%) relatively high dosages. At present none of these small reductions in haemoglobin A1C53,54. Theoretically hae- studies has led to a large, randomized, controlled trial. moglobin A1C could be lowered by more intensive Studies have also been conducted in catabolic patients insulin administration, but none of these studies has with muscle wasting disease. One placebo-controlled definitively proved that an independent mechanism study in a small number of patients (n = 7) with myotonic that accounts for IGF1’s glucose lowering effect exists. dystrophy gave positive results. The authors showed that However, the empiric fact remains that some patients IGF1 given for 4 months stimulated protein synthesis and are easier to control with co-administration of IGF1 inhibited protein breakdown. Muscle mass and strength and insulin. This may be due in part to the fact that were also improved44. Two large randomized trials have increases in IGF1 are stable and are maintained for been performed in patients with amyotrophic lateral several hours; this offers no advantage over long-acting sclerosis but they gave conflicting results45,46. insulins, such as glargine insulin, which were not generally available at the time these trials were initiated. IGF1 in diabetes. The underlying rationale for using Therefore, whether IGF1 administration offers any IGF1 in patients with diabetes has a strong physiological advantage compared with long-acting insulin in type 1 basis. IGF1 and insulin are ancestrally linked hormones diabetics has not been definitively proved. that diverged in evolution. Before that time, a single Several large trials have been conducted in patients IGF1/insulin precursor was used by organisms to link with type 2 diabetes. When given as monotherapy nutrient intake and growth. When abundant food was — without insulin or oral hypoglycaemic agents sensed the IGF1/insulin precursor was synthesized by — IGF1 reduced haemoglobin A1C by 1.2% in patients the olfactory apparatus, and it stimulated cells to use the with type 2 diabetes55, which is a clinically significant ingested nutrient for stimulation of protein synthesis improvement. In general, these were subjects who were and tissue growth47. IGF1 synthesis remains linked to not well controlled as their mean haemoglobin A1C nutrient intake and IGF1 has retained some insulin-like was 8%. One study measured insulin sensitivity at the properties such as stimulation of glucose transport into end of a 6-week administration period and showed that skeletal muscle cells. Studies in humans have shown insulin sensitivity was enhanced 3.4-fold in patients that post-prandial glucose disposal is partly depend- with type 2 diabetes38. Serum IGF1 concentrations that ent upon IGF1 concentrations and that administering were above the upper limit of normal had to be achieved IGF1 to patients with either severe insulin resistance to obtain this degree of improvement in glycaemic or type 2 diabetes results in improved post-prandial control. Administration of lower doses did not result glucose usage48. This occurs primarily at the level of in significant improvement in haemoglobin A1C and skeletal muscle as deletion of the IGF1 receptor in skel- blood glucose levels56. Because of this relatively high etal muscle in mice results in glucose intolerance and dosage requirement, side effects were frequent and were impaired insulin action49 (FIG. 2). similar to those that are seen in clinical conditions asso- Several observations suggest that IGF1 can enhance ciated with excess GH secretion, for example, oedema, insulin action in other tissues. This could occur either arthralgias, headaches and myalgias. Occasionally, com- by crosstalk between the insulin and the IGF1 sig- plications including Bell’s palsy, and pseudotumour nal transduction pathways, particularly in terms of cerebri, tachycardia and hypoglycaemia have occured.

Haemoglobin A1C stimulating PI3K activation, which is a key enzyme for One trial included a group of type 2 diabetics who were A form of haemoglobin that regulating insulin-stimulated glucose transport or by treated with the combination of insulin and IGF1. IGF1 is sensitive to non-enzymatic other, as yet undefined, mechanisms. IGF1 has 100- improved haemoglobin A1C by an additional 0.7% com- glycosylation, which therefore times less affinity than insulin for binding to the insulin pared with control subjects receiving insulin alone, sug- reflects long-term changes receptor, so it is unlikely that the free IGF1 levels that gesting that IGF1 enhanced insulin action, thus, leading in blood glucose. are required to stimulate insulin receptor activation are to a further improvement in the hyperglycaemic state57. Arthralgias ever reached. However, other mechanisms that provide Administration of IGFBP3 with IGF1 appears to be Joint pains. a basis for understanding how IGF1 might be of use in equally effective in terms of improving hyperglycaemia.

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GH Optimization of insulin sensitivity. A large body of hormone literature supports the concept that increased insulin resistance, even when normal or mildly elevated glucose concentrations are present, results in increased cardiovascular risk67. As IGF1 lowers insulin resistance in type 2 diabetes, it has been proposed that low serum IGF1 concentrations could contribute to the increased ↑Gluconeogenesis insulin resistance that is noted in certain clinical conditions such as cardiovascular disease. Epidemiological studies of individuals between the ages of 40 and 70 years support this concept. Individuals with IGF1 levels Liver in the lower quartile of the normal range and IGFBP3 ↓Gluconeogenesis in the highest quartile (therefore with the lowest free Kidney IGF1 concentrations) had a 4.23-fold increase in risk of ↑Lipolysis Pancreas developing cardiovascular disease over a 7-year period68. By contrast, individuals with IGF1 in the highest quartile and IGFBP3 in the lowest quartile had a reduced risk. Adipose The relative risk was calculated such that for each 40 ↑Glucose ↓Lipolysis Insulin transport IGF1 ↓Gluconeogenesis ng per ml decrease in serum IGF1, there was a twofold increase in the risk of developing coronary artery disease. Epidemiological studies have shown that a subset Muscle of the Dutch/Caucasian population (11% of the population) have a polymorphism in the number of CA repeats Figure 2 | Growth hormone and IGF1 actions on glucose homeostasis. In addition 69 to growth stimulation, growth hormone (GH) and insulin-likeNatur egrowth Reviews factor | Drug 1 Disc (IGF1)ove ry near the promoter region of the IGF1 gene . They are have many important metabolic actions. GH acts directly on the liver to antagonize the termed non-carriers of the two most common genotypes. ability of insulin, which is secreted by the pancreas, to inhibit gluconeogenesis. GH Interestingly, these non-carriers have a 32% reduction in acts directly on fat cells to enhance lypolysis, which functions to elevate blood glucose, serum IGF1 concentrations, their birth length is lower again antagonizing the effects of insulin. IGF1 can directly lower blood glucose by than unaffected siblings and their final adult height is inhibiting renal gluconeogenesis. IGF1 can also act indirectly, through the IGF1 3.2 cm less than the mean for the general population. It receptor in skeletal muscle, to enhance insulin action on glucose transport. IGF1 has been presumed that these individuals have to secrete inhibits GH secretion by the pituitary gland, thus, IGF1 indirectly blocks the ability of more GH to maintain a normal IGF1 and that this GH GH to antagonize insulin action; this indirect effect improves glucose homeostasis. hypersecretion worsens the insulin resistance. Consistent with this hypothesis is the observation that in a group of these individuals over the age of 60 the risk of developing type 2 diabetes was increased 2.2-fold and their risk of Administration of this combination to 52 diabetics for 2 myocardial infarction was increased 3.4-fold69. Whether weeks resulted in decreases in fasting blood glucose of these individuals will benefit from a normalization of 35–40% with a marked reduction in insulin require- IGF1 has not yet been determined. ments averaging 66% (REF. 58). Therefore, IGF1 is a potent insulin sensitizer, even when it’s given with its Inhibition of IGF1 to modify disease activity binding protein. Whether administration of IGFBP3 IGF1 and atherosclerosis. As outlined in the previous with IGF1 will result in a reduction in side effects has section, a reduction in systemic IGF1 concentration due not yet been determined. No long-term Phase III trial to gene polymorphisms or other variables may worsen with IGF1 in type 2 diabetes has been completed. insulin insensitivity, which is clearly a risk factor for the Overproduction of IGF2 by several different types development of atherosclerosis. Despite the known effect of tumours has been shown to lead to hypoglycaemia59. of circulating IGF1 on insulin sensitivity, IGF1 that is Normally, these forms of circulating IGF2 are precur- synthesized locally in blood vessels can function in an sor forms and have aberrant glycosylation patterns 22. autocrine or paracrine manner to stimulate the progres- Despite the ability of IGF2 to lower blood glucose levels, sion of atherosclerotic lesions (FIG. 3). Evidence obtained no human studies have determined whether IGF2 has from animal models shows that IGF1 could be involved clinical use in type 2 diabetes. in stimulating atherogenesis70,71. IGF1 receptors are Strategies to stimulate the IGF1 receptor by tech- abundant in vascular smooth muscle cells (SMCs) and nologies other than by administering the ligand and, factors that stimulate atherosclerosis, such as angiotensin therefore, enhance insulin sensitivity, are the subject of II, upregulate IGF1 receptor expression72. Activated ongoing experimental efforts but none has reached the macrophages that are deposited in blood vessels have point of clinical testing. These include the development increased expression of IGF1 (REF.73). Vascular injury that of IGF1-like peptide agonists that have undergone struc- is induced by balloon denudation or hypercholesterolae- tural modifications60–63. Another approach has been to mia increases IGF1 synthesis. IGF1 then acts to stimulate CA repeats Cytosine–adenine repeats administer peptides or small molecules that inhibit IGF1 SMC division and migration leading to an acceleration 70,73 that occur as polymorphisms binding to IGFBPs and therefore increase free IGF1 levels, of atherosclerosis . In hypertensive animals there is in genes. which results in enhanced insulin action64–66. increased IGF1 mRNA and protein expression74. A variety

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Blood flow Monocyte

Activated monocyte

ECM IGF1 synthesis release

SMC SMC proliferation Migration of activated SMC Figure 3 | IGF1 and atherosclerosis. Insulin-like growth factor 1 (IGF1) is synthesized and released by activated monocytes when they penetrate the vascular wall and are activated by various stimuli, includingNature Re oxidizedviews | Drug low-density Discovery lipoprotein and advanced end glycosylation products (proteins that are abnormally glycosylated as a result of the high glucose concentrations that are present in diabetes). IGF1 that is secreted by monocytes can act directly on smooth muscle cells (SMCs) to stimulate their migration into the neointimal space or their proliferation. IGF1 can also stimulate extracellular matrix (ECM) synthesis. This expansion of the neointima leads to an enlargement of the developing atherosclerotic plaque, resulting in blood flow restriction.

of techniques have been used to block IGF1 action in in pigs using antagonists that block ligand occupancy animal models of atherosclerosis and this strategy suc- of αVβ3 showed that IGF1 signalling was inhibited cessfully blocks lesion progression. In one approach, and atherosclerotic lesion progression was blocked80. investigators used a protease resistant form of an IGFBP, Atherosclerotic lesion size was reduced by 48% following IGFBP4, which has been shown to inhibit IGF1 actions75. a 3-week infusion of αVβ3 antagonists, suggesting that Overexpression of this mutant IGFBP in mouse SMCs led by blocking IGF1 action in this cell type the proliferative to a decrease in the number of SMCs in blood vessels75. phase of atherosclerosis could be inhibited. Inhibitors of IGF1 synthesis, such as octreotide, prevent vascular SMC proliferation and neointimal proliferation IGF1 and cancer therapy. Because of its role as a general- in experimental animal models76. A stable decapeptide ized systemic growth factor and because many tumour analogue of IGF1 that inhibits IGF1 receptor activation cell types possess IGF1 receptors, targeting IGF1 has was shown to inhibit vascular SMC DNA synthesis after been extensively studied in the context of cancer rat carotid balloon injury77. Therefore, although inhib- research. Early experimental findings showed that breast iting IGF1 receptor activation has been proposed as a cancer cells expressing IGF1 receptors were responsive to strategy for inhibiting atherosclerosis, the IGF1 receptor this growth factor81. Furthermore, overexpression of the is expressed ubiquitously and it is possible that global IGF1 receptor in various cell types results in acquisition inhibition of IGF1 actions in non-vascular tissue could of the transformed phenotype82 (FIG. 4). This has been have deleterious effects. observed in cells grown in agar and in cells transplanted To circumvent the problem of inhibiting IGF1 recep- into experimental animals82,83. These types of experi- tor function ubiquitously investigators have searched ments have shown that when human tumour cell types for co-receptors that may be activated together with with increased expression of IGF1 receptors were trans- the IGF1 receptor during vascular injury. One such planted into nude mice they were often tumorogenic84. receptor is the αVβ3 integrin. As SMCs require attach- Deletion of the IGF1 receptor in several tumour cell ment to the substratum through integrins in order to models showed that other growth factors, such as PDGF, respond to IGF1, it has been proposed that by inhibit- were incapable of transformation without IGF1 recep- ing ligand occupancy of the αVβ3 integrin may inhibit tor expression85. These observations have been extended IGF1 actions selectively in cell types that express αVβ3. to tumorogenic viruses such as SV40. Therefore, it has Cyclin dependent kinase‑4 Ligand occupancy of αVβ3 integrin leads to the ability of been proposed that IGF1 has a critical role in allowing Following cyclin C1 binding, CDK4 phosphorylates critical SMCs to recruit the signalling protein SHC to the plasma tumour cells to maintain the malignant phenotype and 78 substrates for cell progression membrane . As SHC activation is required for IGF1 to respond inappropriately to viruses or peptide growth through the cell cycle. stimulate SMC migration and division, blocking ligand factors that have transforming activity. IGF1 induces occupancy of αVβ3 prevents the recruitment of SHC cell-cycle regulatory changes that may be important for Cyclin E to the membrane and, therefore, results in an inability acquisition of the malignant phenotype. Specifically it A member of the cyclin family (REF. 79) cyclin-dependent kinase 4 that binds to CDK1 and that to facilitate SHC responsiveness to IGF1 . αVβ3 increases cyclin D1 and (CDK4) is required for G1to S phase antagonists that block IGF1-stimulated SHC activation gene expression leading to retinoblastoma (RB) phos- transition. block IGF1 stimulated cell growth. An in vivo study phorylation and activation of cyclin E86. In addition, it

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Wilms’ tumour downregulates the transcriptional inhibitor, p27/KIP1 in angiogenesis. IGF1 has been shown to synergize with 87 Also termed nephroblastoma. leading to CDK4 activation . In addition, phosphatases, hypoxia inducible factor 1 (HIF1) in promoting tumour A tumour of kidney origin such as PTEN, that have been shown to be dysregulated cell replication102,103. IGF1 also functions with hypoxia usually occurring in young in cancer are important for restraining IGF1-mediated to mediate metastases of transplanted tumours, and children. cell-cycle progression88. hypoxia plus IGF1 can modulate the expression of Hypoxia inducible factor 1 Studies that are more directly linked to human malig- vascular endothelial growth factor (VEGF), a potent (HIF1). A transcription factor nancy have shown that there is increased expression of inducer of tumour angiogenesis104,105. IGF1 has been that is expressed in response IGF1 or the IGF1 receptor in breast, lung, thyroid, gastro shown to enhance expression of matrix metalloproteases to low oxygen. intestinal tract, prostate, glioblastoma, neuroblastoma, (MMPs), such as MMP2 and MMP9 that are important meningioma and rhabdomyosarcoma89. Epidemiological in tumour invasion and metastases106,107. Tumours that studies have shown that subjects with serum IGF1 levels are transplanted into experimental animal models that in the upper quartile of the normal range compared had reduced serum IGF1 levels have been shown to with subjects who have values in the lower quartile of have reduced metastatic potential. Animals with a 75% the normal range are at increased risk of pre-menopausal reduction in serum IGF1 levels following deletion of breast cancer and other cancers, such as prostate, lung, IGF1 synthesis in the liver showed minimal metastases colorectal, endometrial and bladder90–95. of cecal carcinoma compared with mice that had nor- Subjects with a loss of imprinting of the IGF2 gene mal serum IGF1 levels108. Moreover, nutrient depriva- have been shown to be at increased risk for adenoma- tion, which has been shown to reduce IGF1 synthesis, tous polyp formation and early cervical squamous cell reduced the metastatic potential of colon carcinoma109. changes that are considered pre-malignant96. Loss of Overexpression of a soluble form of the IGF1 recep- IGF2 imprinting has been noted in subjects who devel- tor that prevents IGF1 from binding to its receptor oped Wilms’ tumour and neuroblastoma97. IGF2 has also decreased the metastatic potential of a lung carcinoma been implicated in accelerating tumour metastasis. cell line110. In an animal model that develops early stages Overexpression of IGF2 in the primary tumour has of adenocarcinoma of the prostate, overexpression of been shown to be predictive of colorectal metastases98 the IGF1 receptor within the basal epithelial layers led and overexpression of the IGF1 receptor has been to a spontaneous, stepwise progression of prostate car- noted in highly metastasizing synovial carcinomas and cinoma111. Mice expressing a constitutively active form melanomas99–100. Furthermore, IGF2 overexpression has of the IGF1 receptor developed salivary and mammary been used to predict metastasis from adrenal cortical adenocarcinomas112. carcinomas101. One of the major actions of IGF1 in both normal and tumour cell types is to inhibit apoptosis. As cancer Potential mechanisms that alter IGF1 synthesis or action therapies, for example, radiation and chemotherapy, in tumours. In addition to direct effects on growth and/ often induce apoptosis, it is predicted that IGF1 should or metastases of tumour cells, the IGF1 axis is implicated confer radioresistance113,114 and resistance to induction of apoptosis that occurs in tumour cells following exposure to chemotherapeutic agents. This is indeed Tumour the case: the addition of IGF1 rescues breast cancer cells from doxorubicin-induced apoptosis115,116. Similarly, Mitosis of IGF1 upregulation was found to induce resistance to tumour cells cytosine arabinoside (Ara-C) in leukaemia117. Cells that Stromal cells IGF1 overexpress IGF1 are relatively resistant to both of these IGF1 treatments and IGF1 receptor transfection prevents receptor apoptosis after radiation damage. Thus, treatment with agents that block IGF1 actions when combined with Endocrine these other therapeutic approaches may enhance the secreted IGF1 Migration efficacy of these treatment modalities. and metastasis Factors that change the equilibrium between extra- Blood vessel cellular concentrations of IGF1 or IGF2 and the IGF1 receptor have been shown to alter tumour cell growth. Endothelial Some tumour cell types in prostate or colon carcinomas, break for example, have increased IGF1 receptor expression118– 121, and the affinity of IGF1 receptors for IGF1 is altered Figure 4 | IGF1 actions and mechanisms of tumour development. Insulin-like in some tumour cell types, such as breast carcinoma. growth factor 1 (IGF1) concentrations in the tumour microenvironment can be altered | Altered glycosylation of the IGF1 receptor-α subunit, by several factors. Tumours have increased numbers of bloodNatur vessels,e Revie whichws Drug provide Discove anry abundant source of IGF1. IGF1 can be synthesized by stromal cells that are adjacent to which results in equal affinity of this form of the recep-

the cancer cells and contained within the tumour mass. The tumour itself can at times tor for IGF1 and IGF2 (ref. 122), has been reported in synthesize excess IGF1. IGF1 can act directly on cancer cells that possess IGF receptors gliomas. Altered glycosylation of the β-chain of the to stimulate tumour cell growth. Likewise, tumour cell migration and metastasis can be IGF1 receptor has also been reported123. Leukaemia stimulated. IGF1 can alter metastases by changing the ability of tumour cells to cells expressing this form of the β-subunit were penetrate the vascular wall or by stimulating the production of extracellular matrix to shown to respond to low concentrations of IGF1 with form a nidus for attachment of the metastizing tumour cells. increased growth and receptor autophosphorylation124.

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An alternative form of the insulin receptor that is enzymatic activity of the receptor. Last, overexpression expressed in normal fetal, but not adult, tissues can bind of inhibitory forms of IGFBPs has also been proposed to IGF1 and IGF2 with higher affinity due to alterna- as a potential therapeutic strategy. A list of studies that tive splicing; this form of the insulin receptor has been have used these approaches in animal models is shown found in breast, colon and lung carcinoma, resulting in in Tables 1, table 2. an enhanced mitogenic response to IGF2 (REF.125,126). Numerous studies have used monoclonal antibod- As there is three times as much IGF2 in serum com- ies to target the IGF1 receptor (Table 1). Most of these pared with IGF1, this may be an important tumorogenic antibodies have a high affinity for the hormone bind- mechanism. ing domain of the receptor and function by directly inhibiting IGF1 or IGF2 binding. However, in some A change in the extracellular concentrations of IGFBPs of these studies the antibodies have also been shown can alter the ligand–receptor equilibrium. Many to reduce the number of IGF1 receptors by enhanc- tumours synthesize IGFBPs. This is believed to keep ing the rate of receptor internalization. Most of these tumour growth in check as, generally, when the IGFBPs model systems used immunocompromised mice and are present in high concentrations in extracellular fluids human tumour cell lines to establish xenografts. The they can inhibit IGF1 and IGF2 actions127. Some tumour antibody was then injected over several weeks. The cell types such as non-small cell cancer and gastric adeno parameters that have been analysed include tumour carcinoma have been reported to synthesize less IGFBP size, cell growth, apoptosis, metastases and mouse following malignant transformation128,129. Under these survival. A wide range of tumour cell types have been conditions it is reasonable to postulate that the amount used. The initial studies136–139 were conducted using of free IGF1 may increase even if there is no change in mouse monoclonal antibodies, but more recently either IGF1 synthesis. Furthermore, animal experiments have humanized140 or fully human141 antibodies have been shown that administration of IGFBPs can attenuate prepared and tested. In some experiments, the authors IGF1 action and reduce tumour cell growth. Cleavage analysed whether administration of the antibody of IGFBPs by proteases can result in an increased release enhanced the effect of radiation142 or an established of free IGF1 to receptors and several proteases that have chemotherapeutic approach143,144, confirming the idea been shown to be secreted by tumour cell types cleave that inhibition of IGF1 actions enhances the effects IGFBP3 (ref. 130). Therefore, IGF1 release from IGFBPs of established antitumour therapies145. One group of has been proposed as a mechanism for altering cancer investigators developed a bispecific antibody by com- cell growth131–133. bining two previously identified antibodies, one against The altered expression of tumour suppressor genes the IGF1 receptor and one against the EGF receptor, alters the growth of many cancers. Two tumour sup- into a single antibody. This bispecific antibody bound pressor genes, p53 and Wilms’ tumour protein (WT1) both targets simultaneously and prevented IGF1 and have been shown to alter IGF1 expression or function. EGF from binding to their receptors146. This antibody Overexpression of p53 mutants (a mutation that occurs has in vivo tumour activity; specifically it was shown frequently in cancer) can result in reduced IGF1 recep- to inhibit the growth of BXPC3 or HD29 tumour cells tor ubiquitination, thus reducing the rate of degradation that were injected into mice141. of the IGF1 receptor134. IGF1 itself can increase murine In summary, multiple antibodies directed against the double minute 2 (MDM2) expression which leads to p53 human IGF receptor have potent activity in xenograft degradation. WT1 normally regulates the expression of models. Many of the antibodies are currently in Phase I the IGF1 receptor and IGF2 synthesis; however, when trials and the results of single-dose escalation studies, mutations are present (as occur in Wilms’ tumour) this as well as those from studies that combine chemothera- repression is lost135. peutic agents, are eagerly awaited. No Phase II human In summary, multiple mechanisms have been shown studies have been reported with any of these antibodies, to modulate tumour cell responsiveness to IGF1 and therefore, their degree of antitumour activity in human IGF2. These include increases in IGF1 or IGF2 synthesis, cancers remains to be determined. increased IGF1 receptor expression, release of proteases that cleave IGFBPs and mutations that alter the func- Tyrosine kinase inhibitors. The development of small- tion of tumour suppression genes. Strategies to deter- molecule inhibitors of the IGF1 receptor tyrosine kinase mine which of these changes have occurred in a specific has been a major challenge owing to cross-reactivity tumour type are likely to lead to rational strategies that with insulin receptors. The inhibitors that have been target IGF1 actions with greater specificity. developed and tested in animal models so far are shown in table 2. As for the monoclonal antibodies, these Targeting the IGF system for cancer therapy. Several studies have used immunocompromised mice and strategies have been outlined by which the IGF1 system human tumour xenografts to assess efficacy. In general, might be targeted as a form of cancer therapy. These changes in tumour growth have been the primary end include reduction in the number of IGF1 receptors by point, although changes in animal survival and the abil- antisense oligonucleotides or short-interfering RNA ity to enhance the effects of chemotherapeutic agents (siRNA), inhibition of IGF1 receptor function by have also been studied147–150. Most of these compounds inhibiting IGF1 binding using monoclonal antibodies have been designed to compete for the ATP binding or by using tyrosine kinase inhibitors that inhibit the site on the tyrosine kinase. However, one compound

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Table 1 | Anti-IGF1 receptor antibody efficacy in animal studies Antibody Name Species Tumour cell type Model Comment Refs αIR3 Mouse Breast cancer Athymic mice Decreased tumour size 136 αIR3 Mouse Rhabdomysarcoma Athymic mice Decreased tumour size 137 αIR3 Mouse Ewing sarcoma Athymic mice Decreased tumour size 138 αIR3 Mouse Non-small-cell Athymic mice Decreased tumour size 139 lung cancer SCFV/FC Mouse/human Breast cancer Athymic mice Decreased tumour size 164 chimera SCFV/FC Mouse/human Breast cancer Athymic mice IGF1 receptor 165 chimera downregulation SCF/FC Mouse/human Breast cancer Athymic mice Decreased tumour size, enhanced effect 143 chimera combined with tamoxifen EM/164 Mouse Pancreatic Athymic mice Decreased tumour growth 166 A‑12 Fully humanized Breast cancer Athymic mice Decreased tumour size, apoptosis 167 Bispecific Humanized anti-IGF1 Pancreatic/colon Athymic mice Induced receptor down regulation, 141 and EGF receptor inhibited tumour growth A‑12 Fully humanized Prostate cancer Athymic mice Decreased tumour size, apoptosis 168 19D12 Fully humanized Ovarian cancer Athymic mice Decreased tumour size 169 H7C10 Humanized Non-small-cell lung cancer Athymic mice Decreased tumour size, prolonged lifespan 140 CP751‑871 Fully humanized Breast cancer Athymic mice IGF1 receptor downregulation, decreased 170 tumour size KM1468 Mouse (Anti-IGF2) Colon cancer Athymic mice Blocked colon cancer metastases 171 SCFV/FC Mouse/human Breast cancer Athymic mice Downregulated IGF1 and insulin receptors 172 chimera A‑12 Fully humanized Prostate cancer Athymic mice Augmented doxytaxel-induced inhibition 144 of tumour growth A‑12 Fully humanized Non-small-cell lung cancer Athymic mice Enhanced radiation-induced tumour cell 142 apoptosis A‑12 Fully humanized Multiple myeloma SCID mice Decreased tumour growth and 163 vascularization EGF, epidermal growth factor; IGF, insulin-like growth factor; SCID, severe combined immunodeficiency.

that does not compete for this site has been tested151,152. Challenges and opportunities for IGF1 therapies Some of the ATP binding site inhibitors have activity There are several challenges and opportunities for using against the insulin receptor, as demonstrated by the IGF1 as an anabolic agent. Recent molecular studies have increase in blood glucose levels after a single injec- precisely defined the pathways that are used to augment tion151. None of these small molecule inhibitors has protein synthesis in response to IGF1. These pathways entered clinical trials. have both positive and negative regulatory elements156. Another approach has been to use antisense RNA How these elements are altered and function in conjunc- or siRNAs to inhibit tumour growth153. To date these tion with other important pathways, such as the pathways targeting strategies have used cells in culture and have that mediate the direct effects of leucine on protein syn- not been broadly applied to in vivo models of tumour thesis, or in conjunction with pathways that are stimulated activity. In general, these strategies have been effec- by other hormones and growth factors, such as oestrogens, tive in downregulating the number of IGF receptors androgens or transforming growth factor-β (TGFβ), will and, thereby, have increased the susceptibility of these provide important information for designing future stud- tumour cells to chemotherapeutic agents154. Likewise, ies and for the development of therapies to assess clinical increased cell death has been demonstrated with these responses157–159. As the defects that occur in these signal- approaches and this response did not require the co- ling pathways in catabolic states become better defined, it administration of other cytotoxic agents. In a pilot study will be possible to plan rational uses for the stimulation with patients who had gliomas using an IGF1 receptor of the IGF1 pathway; this information should prevent antisense oligonucleotide showed clinical or radiologi- significant off-target effects. Similarly, the ability to target cal improvement in 8 out of 12 patients. Two patients IGF1 to particular tissue compartments or cell types, such were classified as complete responders and four of them as the gastrointestinal epithelium in short bowel disorders, as partial responders155. has not been exploited and such local targeting could take

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Table 2 | IGF1 receptor tyrosine kinase inhibitor efficacy in animal studies Compound Tumour Type Model Comment Refs NVP-AEW541‑A Fibrosarcoma Athymic mice Inhibited tumour growth 173 BMS‑536,924 Salivary Gland Athymic mice Inhibited tumour growth 174 BMS‑554,417 Salivary gland Athymic mice Inhibited tumour growth 175 Cyclolignan Uveal melanoma Athymic mice Inhibited tumour growth 151 TAE226 Glioma Athymic mice Inhibited tumour growth, increased survival 176 NVP-AEW541 Neuroblastoma SCID mice Inhibited tumour invasiveness 177 Cyclolignan Myeloma cells Syngeneic mice Inhibited tumour growth, increased survival 152 NVPADW742 Myeloma cells SCID mice Inhibited tumour growth, metastases and increased 178 survival NVPAEW541 Ewing sarcoma Athymic mice Inhibited tumour growth and angiogenesis 179 PQ401 Breast cancer Syngeneic mice Inhibited tumour growth 180 NVP-AEW541 Ewing sarcoma Athymic mice Combined with vitronectin inhibited tumour growth 147 SCID, severe combined immunodeficiency.

advantage of IGF1’s known anabolic properties while avenue of research and Phase II trials that test this minimizing off-target effects — high concentrations of hypothesis in human subjects are likely to be carried out peptide outside the local gastrointestinal epithelial area in the near future. The generalized use of IGF1 recep- would not be required to achieve the desired therapeutic tor antagonists may require either targeted delivery or response. Likewise, in patients with burns it may be possi- the use of dosages that do not induce systemic toxicity. ble to apply IGF1 locally to improve tissue healing without As most chemotherapeutic agents induce generalized requiring high blood concentrations and systemic admin- catabolism, the effect of uniformly targeting the IGF1 istration. Co-administration of IGF1 and IGFBP3 may receptor could induce a further deleterious response. also obviate the side effects associated with high systemic This necessitates the use of either local delivery methods plasma IGF1 levels but allow sufficient IGF1 to equilibrate or the molecular identification of co-receptors that are with the extravascular space to achieve a therapeutically not affected by the primary chemotherapeatic approach useful effect160. Several small Phase II studies have been but whose activation is necessary for full IGF1 anti- completed using this combination and the results suggest apoptotic effects to be targeted. Bispecific antibodies that in catabolic states, such as myotonic dystrophy, there that target more than one growth factor receptor such may be efficacy160. as EGF and IGF1 receptors or EGF–IGF receptor Inhibition of the IGF1 axis may also be helpful in heterodimers provide a unique opportunity for developing modifying the activity of a number of disease states. agents that target both pathways simultaneously146. The Because of its potent anti-apoptotic activity, inhibiting extent that this augmentation is used by certain cancers IGF1 signalling is a major therapeutic goal in cancer may provide a selective therapeutic method that can research161,162. Recent studies that have combined the result in more inhibition without inducing generalized use of local radiotherapy and/or chemotherapy with systemic toxicity. Using a variety of these approaches, IGF1 inhibitors to take advantage of the fact that IGF1 future efforts are likely to be more focused and rely on can antagonize the pro-apoptotic effects of these treat- unique technologies that inhibit this signalling system ments142,145,163. This appears to be a particularly promising locally to improve efficacy and reduce side effects.

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