The type 1 insulin-like growth factor receptor signalling system and targeted tyrosine kinase inhibition in cancer

Abstract

Type 1 insulin-like growth factor receptor (IGF1R) signalling plays a critical role in normal cell growth, and in cancer development and progression. IGF1R and the insulin-like growth factors 1 and 2 (IGF1 and IGF2) are involved in various aspects of the malignant phenotype, suggesting that IGF1R is a potential target for cancer therapy. IGF1R is particularly important in the establishment and maintenance of the transformed phenotype, in mediating proliferation, and for the survival of tumour cells with anchorage-independent growth. IGF1R also exerts antiapoptotic activity and has a substantial influence on the control of the cell and body size. This property enables transformed cells to form macroscopic tumours and to survive the process of detachment required for metastasis. Pharmaceutical companies are investigating molecules that target IGF1R, including specific low molecular weight tyrosine kinase inhibitors and monoclonal antibodies, both of which possess various advantages and display different activity profiles. This review article focuses on the preclinical and clinical development of low molecular weight IGF1R tyrosine kinase inhibitors. It is critical to pursue a thorough molecular analysis of the metabolic activity of IGF1R to avoid possible side-effects of its inhibition.

Keywords

IGF1 IGF1R tyrosine kinase inhibitor cancer

Introduction

Cell growth and proliferation are regulated by complex signalling systems involving intrinsic factors and external stimuli. Insulin-like growth factors (IGFs) and their receptors comprise a signalling system required for G₁/S phase cell-cycle progression and cell division.¹ The IGF system includes three ligands – IGF1, IGF2 and insulin – that interact with type 1 insulin-like growth factor receptor (IGF1R),² IGFR2 and the insulin receptor (IR).³ IGF1R is an attractive target for cancer therapy as its abnormal expression is associated with tumourigenesis, metastasis and treatment resistance.⁴ Various strategies have been used to target IGFR1 in animal and human tumour cell lines and animal models, some of which may be advancing to clinical use.⁵ The three main approaches are receptor blockade with monoclonal antibodies, tyrosine kinase inhibition, and ligand neutralization via monoclonal antibodies targeted to ligand or recombinant IGF binding proteins (IGFBPs).⁶ Monoclonal antibodies directed against IGF1R cannot block the binding of both IGF1 and IGF2, nor can they downregulate both IGF1R homoreceptor and hybrid receptor pairs.⁷ Tyrosine kinase inhibitors indiscriminately inhibit the kinase domains of all IGF system receptors, however.⁸ This review will focus on the role of the IGF1R signalling pathway in cancer, and the preclinical tyrosine kinase inhibitor data that will likely guide the future development of such therapy.

IGF1R signalling pathway

System components

Homoreceptors

The tetrameric transmembrane receptor tyrosine kinase IGF1R was identified in 1986.² In humans, it is encoded by the IGF1R gene, which contains an open reading frame of 4101 nucleotides encoding a 1367 amino acid protein.⁹ IGF1R is synthesized as a single chain pre-propeptide, with a 30-amino acid signal peptide that is cleaved after translation of the nascent polypeptide chain. The propeptide is then glycosylated, dimerized, and transported to the Golgi where it is processed at a furin cleavage site, yielding α- and β-subunits.^2,10 The mature membrane-bound IGF1R is composed of two extracellular 130 kDa α-chains responsible for ligand binding, and two 98 kDa β-chains, comprising a transmembrane domain and a cytoplasmic tyrosine kinase domain, formed of several α–α and α–β disulphide bridges.¹¹ The α- and β-subunits contain 11 and five potential N-linked glycosylation sites (required for IGF1R translocation to the cell surface), respectively.¹² IGF1R is transported to the cell membrane as a fully assembled dimer.¹³

The intracellular domain of the β-subunit includes a binding site for phosphorylated substrates at Tyr⁹⁵⁰ with a role in signal transmission, a tyrosine kinase domain with an ATP-binding site at lysine¹⁰⁰³, three critical tyrosines at positions 1131, 1135 and 1136, and a C-terminal domain containing several phosphorylated tyrosine and serine residues (such as Tyr¹²⁵⁰, Tyr¹²⁵¹, Tyr¹³¹⁶ and Ser^1280–1283) that play roles in IGF1R signalling.¹⁴

Hybrid receptors

Both IR and IGF1R evolved from the same receptor concerned with the regulation of longevity, metabolism and organism size.¹⁵ Since IGF1R is highly homologous to the IR, sharing 84% amino acid identity in the kinase domain and near absolute conservation in the ATP binding pocket,¹⁶ IR and IGFR proreceptors may heterodimerize to form insulin–IGF hybrid receptors, comprising one α-subunit and one β-subunit each of the IR and IGF1R.^17–20 Further complexity arises from the existence of two isoforms of the IR, IR-A and IR-B, each of which can associate with IGF1R to form IR-A/IGF1R and IR-B/IGF1R hybrids. IR-B/IGF1R hybrids have a higher affinity for IGF1, whereas IR-A/IGF1R hybrids have equal affinity for IGF2 and insulin.²⁰ These hybrid heterodimeric receptors could play a role in receptor signalling in normal and abnormal tissues.²¹ Research in the human breast cancer cell line MDA-MB157 showed that autophosphorylation of IR/IGF1R hybrid receptors in response to IGF1 exceeded that of IGF1R homoreceptors and led to increased cell proliferation, indicating that hybrid receptors were the major mediators of IGF signalling in these cells.²¹

Ligands

The mitogenic and antiapoptotic effects of IGF1 and IGF2 are mediated by IGF1R.²² IGF1 and IGF2 are produced by the liver and by extrahepatic sites including tumour cells and stromal fibroblasts.²³ IGFs are single chain polypeptides, comprising A-, B-, C- and D-domains, with high affinity binding to IGF1R being localized to the C-domain. The absence of Thr^A8, IleA¹⁰, His^B5 and Tyr^B16 contact points in the A- and B-domains of the IR may be responsible for the weak binding affinities of IGF1 and IGF2 for the insulin receptor.²⁴ IGF1 and IGF2 share a 62% homology in amino acid sequence, with a 40% homology between the IGFs and proinsulin.²⁵ The binding affinity for IGF1R varies under different experimental conditions and cell types.²⁶ Relative concentrations of IGF1 and IGF2 also vary, with IGF2 concentrations being five times higher than IGF1 levels in human foetuses, and 3.5 times higher in adult sera.²⁷

Also present in the cellular microenvironment are six IGF binding proteins (IGFBP1–6) that regulate the bioavailability of IGFs by competing with IGFRs, and IGFBP proteases that modulate the balance between IGFs and IGFBPs.²⁸ IGFBPs and IGFs comprise a major superfamily of protein hormones that regulate mitogenesis, differentiation, survival and other IGF-stimulated events in both normal and cancerous cells.^29,30 An in vivo study indicated that IGFBP3 inhibits the growth of HER2-overexpressing breast tumours and potentiates herceptin activity.³¹ Others have found, however, that high IGFBP2 expression was not associated with reduced cell proliferation in breast cancer, glioblastoma or prostate cancer.^32–34 These data suggest that IGFBPs can affect cell function in an independent manner, but their role in cancer is not yet clear.³⁵

IGF1R signal transduction

The binding of IGF1, IGF2 or insulin to IGF1R leads to the autophosphorylation of tyrosines 1131, 1135 and 1136 in the kinase domain on the intracellular portion of the β-subunits, inducing the phosphorylation of juxtamembrane tyrosines and carboxyl-terminal serines.³⁶ Subsequently, the juxtamembrane Tyr⁹⁵⁰ serves as a binding site for docking proteins including IR substrates 1–4 (IRS1–IRS4), and Src homology/collagen domain protein (SHC). These substrates initiate the PI3K/Akt (phosphatidylinositol 3-kinase/protein kinase B) and RAS/RAF/MEK/ERK signalling pathways, transmitting the IGF1R signal.³⁷

Phosphorylated IRS1 recruits the regulatory and catalytic subunits of PI3K, ultimately resulting in the downstream phosphorylation of Akt at Thr³⁰⁸.^38,39 It is well known that Akt plays a critical role in controlling survival and apoptosis.³⁹ Akt phosphorylation enhances the protein synthesis required for cell proliferation via the mammalian target of rapamycin complex, triggers the antiapoptotic effects of IGF1R through the phosphorylation and inactivation of Bcl-xL/Bcl-2 associated death promoter and the inactivation of caspase 9, and also stimulates cell growth through the inhibitory phosphorylation of growth factors (including forkhead box proteins, p21, p27, Chk1, and glycogen synthase kinase 3 [GSK3]).³⁸

In parallel to PI3K-driven signalling, the recruitment of growth factor receptor-bound protein 2 (Grb2)/SOS by either phosphorylated IRS1 or SHC leads to IGF1R signal transmission via the RAS/RAF/MEK/ERK pathway.⁴⁰ The Grb2/SOS complex activates the signalling pathway, resulting in the downstream phosphorylation and activation of MKK and ERK.⁴¹ Activated ERK induces cell proliferation by the phosphorylation of nuclear transcription factors such as Ets-like transcription factor-1 (Elk-1) and c-Fos.⁴² Activated MKK phosphorylates p38, and is responsible for inducing cell proliferation via the activation of c-Myc, JNK, and subsequently c-Jun.⁴³

Cell-cycle progression is positively regulated by IGF1R signalling through cycle checkpoints in several phases. It increases ribosome activity through activation of p70S6K and S6 to facilitate the transition from G₀ to G₁,⁴⁴ increases the expression of cyclin D₁ to facilitate the G₁/S transition⁴⁵ and may increase cyclins A and B and cdc2 synthesis to promote the G₂/M transition.⁴⁶ The major direct effect of the IGF1R mediated signal is most likely exerted at the G₁/S interface, however.⁴⁷

IGF1R: A target for cancer therapy

Emerging evidence implicates IGF1R signalling in the development and progression of cancer. IGF1R, IGF1 and IGF2 are involved in various aspects of the malignant phenotype, suggesting that IGF1R may be a target for cancer therapy.

IGF1R signalling in normal physiology

It is known that IGF1R protects cells from apoptosis, while promoting proliferation and cell growth. Newborn mice lacking IGF1 or IGF2 were found to be significantly smaller (60% of normal birth weight) than wild-type mice, and IGF1R knockout mice were even smaller (45% of normal size), confirming that IGF and its receptor play a role in normal growth.⁴⁸ Mice with double IR/IGF1R deficiency in both cardiac and skeletal muscle die early from heart failure, due to the co-ordinated effects of IR/IGF1R on the downregulation of electron transport and mitochondrial fatty acid β-oxidation.⁴⁹ IGF signalling from these receptors is therefore essential for normal cardiac metabolism and function. IGF1R also plays a role in embryonic bone development.⁵⁰ Studies of IGF1R signalling in the nervous system have shown that IGF signalling promotes differentiation and proliferation, and sustains survival by preventing apoptosis of neuronal and brain derived cells in vitro.⁵¹ Other studies have shown that IGF1R regulates cell lifespan, adhesion and motility, and can induce differentiation in specific cellular contexts.⁵² The role of IGF1R in normal physiology appears to be more complex than was thought when this protein was initially identified.

IGF-IR signalling in malignancy

It has been shown that IGF1R, its ligands and IGFBPs are highly expressed in prostate,⁵³ breast,⁵⁴ colorectal⁵⁵ and pancreatic cancers,⁵⁶ melanoma,⁵⁷ multiple myeloma,⁵⁸ mesothelioma,⁵⁹ glioblastoma⁶⁰ and childhood malignancies.²⁷ IGFs and IGF1R regulate all aspects of the malignant phenotype.⁶¹

Studies show a correlation between circulating IGF1 levels and cancer risk in some malignancies (premenopausal breast, prostate and colorectal carcinomas, as well as lung, endometrial and bladder cancers).²⁸ Individuals with high serum IGF1 concentrations and/or lower levels of IGFBPs had more than twice the risk of developing cancer than those at the low end of the normal range.⁶¹ Other studies have failed to indicate consistent correlations, for example circulating IGF1, IGFBP1, IGFBP3 and growth hormone levels were found to have no association with breast cancer risk in a large cohort of premenopausal women.⁶²

Type 1 insulin-like growth factor receptor plays an important role in the establishment and maintenance of the transformed phenotype. It is not unique in driving tumour cell proliferation, but is essential in the mediation of proliferation and survival of tumour cells with anchorage-independent growth.⁶³ IGF1R also exerts antiapoptotic activity and has a significant influence on the control of cell and body size.⁶³ This property enables transformed cells to form macroscopic tumours, and to survive the process of detachment that is required for metastasis.⁶⁴ Data from in vitro and in vivo studies have confirmed the importance of the IGF1R system in cancer growth and metastasis. Treatment of synovial sarcoma cell lines with the IGF1R antagonist NVP-AEW541 led to increased apoptosis, and impaired growth, mitotic activity and cell migration.⁶⁵ IGF1R did not enhance motility in LCC6-DN cells (a breast cancer cell line with dominant negative IGF1R), in contrast to increased motility in wild-type LCC6 cells, suggesting that metastasis is inhibited by truncated IGF1R.⁶⁶ The injection of mice with LCC6, but not LCC6-DN, cells resulted in the formation of lung metastases, indicating that IGF1R regulates metastasis independently from tumour growth.⁶⁶ Using a similar approach, human KM12L4 colon cancer xenograft tumours induced in nude mice were significantly smaller when the KM12L4 cells were transfected with a truncated dominant-negative form of IGF1R than when using wild-type cells.⁶⁷ In addition, the colon cancer cells with dominant negative IGF1R failed to produce liver metastases after splenic injection.⁶⁷ These findings suggest that IGF1R plays an important role in the metastasis of cancer cells.

Type 1 insulin-like growth factor mediates proliferation, motility and apoptosis protection in cancer, and represents a promising target for cancer therapy. Clinical trials are required to examine this treatment potential fully.

Targeting the IGF system for cancer therapy

The critical role of IGF signalling in initiating and promoting tumour progression has resulted in it becoming an attractive target for cancer therapy. Various strategies have been used to target components of this system both in vitro and in vivo, some of which have advanced to clinical use.⁴ The general aim of these approaches is to interfere with the function of IGF system components by methods including small interfering RNA, antisense oligonucleotides, antisense RNA, triple helix-forming oligodeoxynucleotides, specific kinase inhibitors, single chain antibodies and fully humanized anti-IGF1R monoclonal antibodies.⁴ The neutralization of natural ligands can also inhibit IGF1R activation. There are three ways to neutralize IGF action: the overexpression of IGF2R⁶⁸ and IGFBP1⁶⁹; the use of soluble IGF1R (e.g. IGF1R⁹³³);⁷⁰ and antibodies against IGF2 and IGF2 (such as the rat monoclonal antibody, KM1468).⁷¹ These studies demonstrated that functional interference of IGF1R leads to the inhibition of cancer cell proliferation, survival, anchorage-independent growth in vitro, tumour growth and metastasis, and sensitizes cancer cells to various kinds of radiation and chemotherapeutic regimens in vivo.

IGF1R tyrosine kinase inhibitors

The two most thoroughly investigated strategies for IGF1R inhibition are small-molecule tyrosine kinase inhibitors and monoclonal antibodies, both of which have various advantages and display different activity profiles. Studies have concentrated on modulating IGF1R tyrosine kinase activity by targeting its intracellular kinase domain. The high sequence homology of the kinase domains of IGF1R and IR² has complicated the design process for an IGF1R specific low-molecular weight tyrosine kinase inhibitor. Table 1 lists nine IGFR1 kinase-specific inhibitors at different stages of development.⁷²

Table 1.

Class and trial status of type 1 insulin-like growth factor receptor (IGFR1) tyrosine kinase inhibitors currently under developmen.

Drug name	Company	Class	Trial status
INSM-18	Insmed UCSF	Reversible ATP-competitive	Phase I/II
OSI-906	OSI Pharmaceuticals	Oral small molecule Reversible ATP-competitive	Phase I
XL-228	Exelixis	Oral small molecule	Phase I
NVP-ADW742	Novartis	Reversible ATP-competitive	Preclinical
NVP-AEW541	Novartis	Reversible ATP-competitive	Preclinical
AG-1024	Merck	Non ATP-competitive	Preclinical
BMS-536924	Bristol-Myers Squibb	ATP-competitive	Preclinical
BMS-554417	Bristol-Myers Squibb	Oral small molecule Reversible ATP-competitive	Preclinical
BVP-51004 (Cyclolignan PPP)	Karolinska Cancer Institute Biovitrum	Oral small molecule Non ATP-competitive	Preclinical

Small-molecule tyrosine kinase inhibitors can be given orally and are able to cross the blood–brain barrier, but may result in drug–drug interactions or overlapping toxicities when used in combination with other agents.⁷³ In addition, small-molecule tyrosine kinase inhibitors may be associated with more serious metabolic adverse effects than monoclonal antibodies, as they can affect both IR and IGF1R signalling.⁷⁴ An advantage of small-molecule IGF1R tyrosine kinase inhibitors over antibodies is their wider range of inhibition due to the frequent expression of IR in tumours, hyperinsulinism in aggressive cancers, and IR-mediated resistance to IGF1R-targeted therapy, all of which may enhance antineoplastic activity.⁷⁵

Combination therapy with either cytotoxic (chemotherapy or radiation) or cytostatic treatments and tyrosine kinase inhibitors has been shown to increase the effect of inhibitors, as IGF1R is closely associated with apoptosis, cell survival and radiation, and chemotherapy resistance. For example, combination treatment with the IGFR1 tyrosine kinase inhibitor NVP-AEW541 and cytostatics such as doxorubicin or fluvastatin resulted in additive antineoplastic effects in gastrointestinal neuroendocrine tumour (NET) cells.⁷⁶ In addition, NVP-AEW541 in combination with IGFBP3 abrogated IGF1 responses at 10-fold lower doses than either compound alone in human head and neck squamous cell carcinoma (HNSCC).⁷⁷ These findings suggest that combination treatment may be a promising approach in IGF1R targeted therapy, but this requires further investigation.

There are two categories of IGF1R tyrosine kinase inhibitor. The most common are ATP antagonists (INSM-18, OSI-906, NVP-ADW742, NVP-AEW541, BMS-536924 and BMS-554417), with non-ATP antagonists also under development (BVP-51004 [PPP] and AG-1024). In vitro pharmacological studies have found variations in the selectivity of these molecules for IGF1R over IR. OSI-906, INSM-18, picropodophylin and NVP-AEW541 appear to be more selective for IGF1R, and BMS-554417 has equivalent potency towards both receptors. The different selectivity for each receptor defines a specific therapeutic opportunity for each drug.

INSM-18

The orally available molecule INSM-18 directly inhibits the human epidermal growth factor receptor c-erbB2/HER2/neu and IGF1R tyrosine kinase.⁷² INSM-18 was discovered by the University of California, San Francisco and is currently being developed with Insmed (Monmouth Junction, NJ, USA). Its IGF1R inhibitory mechanism is unclear. INSM-18 may also induce apoptosis, inhibit arachidonic acid 5-lipoxygenase and prevent the release of reactive oxygen species.⁷² The antitumour activity of INSM-18 has been demonstrated in breast, lung, pancreatic and prostate tumour preclinical studies.⁷² INSM-18 had a good safety profile and was well tolerated in two single-dose phase I clinical studies in healthy volunteers.⁷² A dose-escalating phase I/II clinical study has been completed by the University of California, the results of which will be used to design a phase II clinical study.⁷²

NVP-AEW541

The low molecular weight molecule NVP-AEW541 is a pyrrolo[2,3-d]pyrimidine derived kinase inhibitor with a higher selectivity for IGF1R (50% inhibitory concentration [IC₅₀] 0.086 µM) than IR (IC₅₀ 2.3 µM) in cellular assays.⁷⁸ Preclinical studies are ongoing, but its antitumour activity has been demonstrated in musculoskeletal tumours,⁷⁹ hepatocellular carcinoma,⁸⁰ NET,⁷⁶ malignant mesothelioma,⁸¹ HNSCC,⁷⁷ colorectal cancer,⁸² neuroblastoma,⁸³ Ewing's sarcoma,⁸⁴ multiple myeloma,⁸⁵ pancreatic cancer,⁸⁶ gastrointestinal stromal tumour,⁸⁷ gastrointestinal cancer,⁸⁸ breast cancer⁸⁹ and synovial sarcoma.⁶⁵ As expected for a specific IGF1R kinase inhibitor, NVP-AEW541 abrogated IGF1-mediated survival, inhibited the proliferation of cultured tumour cell lines by inducing apoptosis and cell cycle arrest in vitro, and significantly inhibited the growth of tumour xenografts in vivo.^{65,76,77,79–89} Accumulating evidence suggests that NVP-AEW541 represents a potential therapeutic strategy for the treatment of tumour types in which IGF1R-mediated signalling is required for driving/survival.

BVP-51004

The non ATP antagonist BVP-51004 (cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo [1,5-a]pyrazin-8-ylamine, also known as PPP and PQIP) was originally identified at the Karolinska Cancer Institute (Sweden) and is currently being developed by Swedish Orphan Biovitrum (Stockholm, Sweden). BVP-51004 inhibits IGF1R autophosphorylation with 14-fold selectivity relative to IR, via inhibition of the phosphorylation of Tyr¹¹³⁶ in the kinase activation loop, stabilizing the activation loop conformation.^90,91 It has been demonstrated that BVP-51004 preferentially inhibits the PI3K-Akt pathway, and blocks the growth of a variety of tumours, both in vitro and in vivo.⁹² A study in human colorectal cancer cells indicated that BVP-51004 is a selective IGF1R kinase inhibitor that is highly effective in IGF2-driven tumours.⁵⁹

Conclusions

Multiple lines of evidence indicate that the IGFR1 signalling system plays a critical role in carcinogenesis, and high concentrations of IGF1R and its ligands are predictive of increased cancer risk. Pharmaceutical companies are investigating candidate drugs that target IGF1R, and early clinical data are encouraging. Ongoing trials will determine the relative safety and efficacy of IGF1R tyrosine kinase inhibitors and monoclonal antibodies. Research is required to identify the most rational combination therapies, which may also provide information for future clinical trial designs. It is critical to pursue a thorough molecular analysis of the metabolic activity of IGF1R in order to avoid possible side-effects of its inhibition.

Footnotes

Declaration of conflicting interest

The author declares that there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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