Derek LeRoith

Insulin-like growth factor system and cancer

The insulin-like growth factor (IGF) family of ligands, binding proteins and receptors is an important growth factor system involved in both the development of the organism and the maintenance of normal function of many cells of the body. The system also has powerful anti-apoptotic effects. More recently, evidence has accrued to demonstrate that the IGFs play an important role in cancer. Individuals with serum IGF-II levels in the upper quartile of the normal range (and IGF binding protein-3 levels in the lower quartiles) have a relative risk for developing breast, prostate, colon and lung cancer. IGF-II is commonly expressed by tumor cells and may act as an autocrine growth factor; occasionally even reaching target tissues and causing tumor-induced hypoglycemia. The IGF-I receptor is commonly (though not always) overexpressed in many cancers, and many recent studies have identified new signaling pathways emanating from the IGF-I receptor that affect cancer cell proliferation, adhesion, migration and cell death; functions that are critical for cancer cell survival and metastases. In this review, many aspects of the IGF system and its relationship to cancer will be discussed.

Circulating levels of IGF-1 directly regulate bone growth and density

IGF-1 is a growth-promoting polypeptide that is essential for normal growth and development. In serum, the majority of the IGFs exist in a 150-kDa complex including the IGF molecule, IGF binding protein 3 (IGFBP-3), and the acid labile subunit (ALS). This complex prolongs the half-life of serum IGFs and facilitates their endocrine actions. Liver IGF-1-deficient (LID) mice and ALS knockout (ALSKO) mice exhibited relatively normal growth and development, despite having 75% and 65% reductions in serum IGF-1 levels, respectively. Double gene disrupted mice were generated by crossing LID+ALSKO mice. These mice exhibited further reductions in serum IGF-1 levels and a significant reduction in linear growth. The proximal growth plates of the tibiae of LID+ALSKO mice were smaller in total height as well as in the height of the proliferative and hypertrophic zones of chondrocytes. There was also a 10% decrease in bone mineral density and a greater than 35% decrease in periosteal circumference and cortical thickness in these mice. IGF-1 treatment for 4 weeks restored the total height of the proximal growth plate of the tibia. Thus, the double gene disruption LID+ALSKO mouse model demonstrates that a threshold concentration of circulating IGF-1 is necessary for normal bone growth and suggests that IGF-1, IGFBP-3, and ALS play a prominent role in the pathophysiology of osteoporosis.

A Molecular Basis for Insulin Resistance ELEVATED SERINE/THREONINE PHOSPHORYLATION OF IRS-1 AND IRS-2 INHIBITS THEIR BINDING TO THE JUXTAMEMBRANE REGION OF THE INSULIN RECEPTOR AND IMPAIRS THEIR ABILITY TO UNDERGO INSULIN-INDUCED TYROSINE PHOSPHORYLATION

Tumor necrosis factor α (TNFα) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943–984) but not with the carboxyl-terminal region (amino acids 1245–1331) of IR expressed in bacteria as His6fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFα, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFα for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20–60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFα effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFα on insulin action.

Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: Direct correlation with clinical retinopathy of prematurity

Retinopathy of prematurity is a blinding disease, initiated by lack of retinal vascular growth after premature birth. We show that lack of insulin-like growth factor I (IGF-I) in knockout mice prevents normal retinal vascular growth, despite the presence of vascular endothelial growth factor, important to vessel development. In vitro, low levels of IGF-I prevent vascular endothelial growth factor-induced activation of protein kinase B (Akt), a kinase critical for endothelial cell survival. Our results from studies in premature infants suggest that if the IGF-I level is sufficient after birth, normal vessel development occurs and retinopathy of prematurity does not develop. When IGF-I is persistently low, vessels cease to grow, maturing avascular retina becomes hypoxic and vascular endothelial growth factor accumulates in the vitreous. As IGF-I increases to a critical level, retinal neovascularization is triggered. These data indicate that serum IGF-I levels in premature infants can predict which infants will develop retinopathy of prematurity and further suggests that early restoration of IGF-I in premature infants to normal levels could prevent this disease.

Cellular pattern of type-I insulin-like growth factor receptor gene expression during maturation of the rat brain : comparison with insulin-like growth factors I and II

Insulin-like growth factors have a number of potent trophic effects on cultured neural tissue and most if not all of these effects appear to be mediated by the type-I insulin-like growth factor receptor. In order to establish the identity of cell types expressing this receptor in the rat central nervous system during development and maturity, we have used in situ hybridization to map sites of type-I insulin-like growth factor receptor mRNA synthesis in the developing and adult rat brain. In order to identify possible local sources of peptide ligands for this receptor, we have also mapped the sites of insulin-like growth factors I and II mRNA synthesis in parallel brain sections. From early development onward, there is a uniform and stable pattern of type-I insulin-like growth factor receptor gene expression in all neuroepithelial cell lineages, in which regional variations reflect primarily differences in cell density. In addition to this generalized pattern, during late postnatal development, high levels of type-I insulin-like growth factor receptor gene expression are found in specific sets of sensory and cerebellar projection neurons in conjunction with abundant insulin-like growth factor-I gene expression in these same neurons. While insulin-like growth factor-I expression is confined to the principal neurons in each system, receptor mRNA is also found in local interneurons. In the cerebral cortex and hippocampal formation, type-I insulin-like growth factor receptor mRNA and insulin-like growth factor-I are concentrated in different cell populations: receptor mRNA is abundant in pyramidal cells in Ammons horn, in granule cells in the dentate gyrus, and in pyramidal cells in lamina VI of the cerebral cortex. Insulin-like growth factor-I mRNA is found in isolated medium- to large-sized cells which are rather irregularly distributed throughout the hippocampus and isocortex. In the hypothalamus, receptor mRNA is concentrated in the suprachiasmatic nucleus but is in low abundance elsewhere, including the median eminence, while insulin-like growth factor-I mRNA is not detected in this region at all. Type-I insulin-like growth factor receptor and insulin-like growth factor-II mRNAs are both abundant in choroid plexus, meninges and vascular sheaths from early development to maturity, but insulin-like growth factor-II mRNA is not detected in cells of neuroepithelial origin at any stage of development. This study provides evidence for two fundamentally different patterns of gene expression for the brain type-I insulin-like growth factor receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

The Role of the Insulin-like Growth Factor System in Human Cancer

Publisher Summary This chapter focuses on the role of the insulin-like growth factor system in human cancer. The normal process of growth and differentiation results from the genetically programmed action of a number of different cellular and extracellular factors. Derangement in the function of one or more of those agents can result in a pathologic phenotype, including neoplastic growth. A family of growth factors shown to be intimately involved in the regulation of cell growth as well as in cellular transformation is the insulin-like growth factor (IGF) family. IGF-I and IGF-II are mitogenic polypeptides produced in the largest amounts by the liver and secreted into the circulation, where they mediate the effects of growth hormone (GH) on longitudinal growth. In addition to ligands and receptors, the IGF system comprises a third category of molecules, which bind IGFs in the circulation and in extracellular compartments. Six IGF-binding proteins (IGFBPs) have been characterized to date. Binding of IGFs to the IGF-I receptor induces receptor autophosphorylation. The cell cycle consists of four major phases: (1) the presynthetic phase, G; (2) the phase of DNA synthesis, S; (3) the premitotic phase, G; and (4) mitosis, M. IGF-I, IGF-II, and insulin are chemotactic agents for the human melanoma cell line A2058, as assayed in a modified Boyden chamber. The chapter also gives selected examples of IGF involvement in human cancer. The wealth of information generated in the IGF field, as well as continued research efforts, both basic and clinic, promise to produce rational therapeutic approaches for those cancers in which the IGF system is involved.

Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo.

The transcription factor, signal transducer and activator of transcription-3 (STAT-3) contributes to various physiological processes. Here we show that mice with liver-specific deficiency in STAT-3, achieved using the Cre-loxP system, show insulin resistance associated with increased hepatic expression of gluconeogenic genes. Restoration of hepatic STAT-3 expression in these mice, using adenovirus-mediated gene transfer, corrected the metabolic abnormalities and the alterations in hepatic expression of gluconeogenic genes. Overexpression of STAT-3 in cultured hepatocytes inhibited gluconeogenic gene expression independently of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), an upstream regulator of gluconeogenic genes. Liver-specific expression of a constitutively active form of STAT-3, achieved by infection with an adenovirus vector, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. Hepatic STAT-3 signaling is thus essential for normal glucose homeostasis and may provide new therapeutic targets for diabetes mellitus.

Insulin-like Growth Factors and Cancer

Dr. Derek LeRoith (Molecular and Cellular Physiology Section, National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], National Institutes of Health [NIH], Bethesda, Maryland): The insulin-like growth factor (IGF) system comprises a collection of ligands, receptors, and binding proteins Table 1 [1]. Insulin, IGF-I, and IGF-II are polypeptides that affect many tissues and result in diverse biological actions. The major role of insulin is controlling metabolic homeostasis. In contrast, IGF-I and IGF-II are vital for normal growth and development during fetal, neonatal, and pubertal stages [2]. In addition, IGFs have specialized functions in differentiated tissues, including the reproductive, cardiovascular, and neurologic systems. The biological functions of the IGFs are initiated by their interactions with cell-surface receptors, in particular the IGF-I receptor. When activated, this receptor initiates a cascade of events that begins with the activation of tyrosine kinase and results in divergent effects depending on specific cell types [3]. Table 1. The Insulin-like Growth Factor System* Circulating IGFs are synthesized primarily in the liver and serve an endocrine function, whereas locally produced IGFs act in an autocrine-paracrine mode. Both forms are bound by a family of binding proteins, six of which have been well characterized Table 1 [4]. Insulin-like growth factor-binding proteins are responsible for protecting IGFs in the circulation, prolonging their half-lives, and delivering them to their specific target tissues. At the local level, IGF-binding proteins may regulate the interaction of IGFs with their receptors by either inhibiting or augmenting the interaction. In addition, IGF-binding proteins may have some actions that are independent of interactions between IGF and IGF receptors. As could be predicted from the importance of IGFs, their binding proteins, and their receptors in normal cellular growth and development, it has become apparent over the past few years that IGFs are important mitogens in many types of malignancies [5]. Although these conclusions were initially derived from in vitro studies, IGFs may enhance in vivo tumor cell formation, growth, and even metastasis. Insulin-like growth factors may reach tumors either from the circulation (endocrine) or as a result of local production by the tumor itself (autocrine) or by adjacent stromal tissue (paracrine). Tumors also express many of the IGF-binding proteins, which modulate IGF action, and IGF receptors, which mediate the effects of IGFs on tumors. We highlight important aspects of IGFs in normal cell growth and their role in certain malignancies. The syndrome of hypoglycemia with non-islet cell tumors, although not covered in this review, deserves special mention because it was one of the earliest links of IGFs to tumors and was derived from studies done in the mid-1970s at the Diabetes Branch of the NIDDK. The clinical syndrome was described shortly after insulinomas were first described in the late 1920s. The advent of the radioimmunoassay for insulin in the early 1960s showed that insulinomas release insulin but that the nonislet tumors that produce hypoglycemia usually lack insulin, a finding that triggered intense speculation about the mechanism of hypoglycemia. In the mid-1970s, the NIH group devised a novel radioreceptor assay for IGF-II. Using this assay, they showed that in patients with this syndrome, IGF-II-like material is often present in elevated amounts in the circulation and in the tumors [6]. Of further interest is that the major clinical culprit may be a higher-molecular-weight precursor form of IGF-II; this IGF-II-like material probably binds to insulin receptors, activates them, and thereby produces hypoglycemia [7, 8]. The Role of the Insulin-like Growth Factor I Receptor in Cell Growth and Transformation Dr. Renato Baserga (Jefferson Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania): Mammalian cell growth in vitro and in vivo is regulated by factors that interact with specific cell-surface receptors. Most normal cells require at least two factors for optimal growth. Insulin-like growth factor I is often one of them and is required for the growth of such cells as fibroblasts, epithelial cells, bone marrow stem cells, and osteoblasts [9]. The other required growth factor varies depending on the cell type, but in fibroblasts, platelet-derived growth factor and epidermal growth factor act with IGF-I to stimulate cell proliferation. In culture, neither of these factors alone can sustain cell growth. The recent finding that mice in which the IGF-I and IGF-I receptor genes had been inactivated grow to only 30% of the size of normal littermates underscores the role of the IGF-I receptor in murine development [10, 11]. Further support for the role of the IGF-I receptor in growth and tumorigenesis has come from studies showing the transforming potential of transfected cells overexpressing the IGF-I receptor [12] and abrogation of this effect by specific mutations of the receptor [13]. On the basis of research using fibroblast cell lines derived from IGF-I receptor-deficient mice [R-cells], it has been possible to show that 1) IGF-I receptors are essential for the growth of cells in serum-free media supplemented with factors that support the growth of normal mouse cells [W cells that are fibroblasts derived from normal mice or 3T3 cells]; 2) IGF-I receptors are not necessary for growth in media containing 10% serum but are required for optimal growth; 3) IGF-I receptors are also required for platelet-derived growth factor-stimulated or epidermal growth factor-stimulated growth and transformation; and 4) IGF-I receptors stimulate both ras-dependent and ras-independent signaling pathways [14]. It has been shown that SV40 T antigen increases IGF-I expression, leading to transformation of BALB/c 3T3 cells [15]. The obligate role of the IGF-I receptor in T-antigen-mediated transformation was confirmed by the inability of T antigen to transform the R-cells described above. Further studies have shown that R- cells are also refractory to transformation by v-src and by bovine papilloma virus, both of which efficiently transform cells expressing IGF-I receptors; thus, some oncogenic viruses require IGF-I receptors to transform mouse embryo fibroblasts. To ascertain the role of IGF-I receptors in the growth and transformation of other cell types, C6 rat glioblastoma cells were rendered IGF-I receptor-deficient by expressing an antisense RNA that prevented efficient expression of the endogenous IGF-I receptor gene. These cells were then evaluated for their ability to form tumors when transplanted into syngeneic rats in comparison with wild-type C6 cells. In all 50 rats injected with wild-type C6 cells, the C6 cells grew well and formed large tumors. Insulin-like growth factor I receptor-deficient C6 cells did not grow in the 27 animals into which they were injected, and no tumors were formed [16]. These results suggest that IGF-I receptors are extremely important in establishing and maintaining the transformed phenotype and that they may represent a suitable target for the inhibition of cell proliferation in vivo. It is currently accepted that a major signal transduction pathway triggered by the IGF-I receptor and by other receptor tyrosine kinases such as the receptors for insulin, epidermal growth factor, and platelet-derived growth factor involves activation of ras, the protein kinase Raf-1, and the mitogen-activated protein kinase cascade [17]. It is interesting that overexpression of an activated ras or Raf-1 could not confer in IGF-I receptor-deficient R- cells the ability to grow in soft agar or in a serum-free medium supplemented with purified growth factors [14, 15, 18]. Thus, the IGF-I receptor must also use a ras (and Raf-1)-independent pathway to stimulate cell proliferation and transformation. Insulin-like Growth Factors and Breast Cancer Derek LeRoith (Section on Molecular and Cellular Physiology, Diabetes Branch, NIDDK, NIH): Breast cancer is a common malignancy that affects almost 1 in every 7 women and is the leading cause of death from cancer in women in North America. During normal development, estrogen is primarily involved in promoting the development of breast ducts, whereas progesterone promotes lobuloalveolar development. Many cancers, especially those developing in the postmenopausal period, express estrogen and progesterone receptors. The presence of these receptors and the likelihood that these cancers will respond to endocrine therapy are strongly correlated. Initial therapy for breast cancer is primarily surgical, but once metastatic disease has developed, endocrine therapy is appropriate. In premenopausal patients, lowering hormone levels by removing the ovaries is useful, whereas in postmenopausal patients, antiestrogens such as tamoxifen have proved useful [19]. In addition to classic hormones, several growth factors, including transforming growth factors, epidermal growth factors, and IGFs, have been shown to be involved in breast cancer. The cellular proto-oncogene products such as c-myc, c-fos, and c-jun are also involved Table 2 [20]. Table 2. Growth Factors and Oncogenes Involved in Breast Cancer Breast cancer cells in vivo express low levels of IGF-II [21], whereas the adjacent stromal tissue expresses IGF-I [22]. In addition, most breast cancer cells express insulin and IGF receptors [23]. Different cancers express different combinations of the IGF-binding proteins: In vitro, estrogen receptor-positive cancer cells synthesize IGF-binding proteins 2, 4, and 5, and estrogen receptor-negative cancer cells synthesize IGF-binding proteins 1, 3, 4, and 5 [24]. Examination of biopsy specimens of breast cancer cells has confirmed this specific pattern of IGF-binding protein expression [25]. Because it has been shown that the proliferation of breast cancer cell lines i

INSULIN-LIKE GROWTH FACTOR-I RECEPTOR SIGNAL TRANSDUCTION : AT THE INTERFACE BETWEEN PHYSIOLOGY AND CELL BIOLOGY

The insulin-like growth factor-I receptor (IGF-IR) mediates the biological actions of IGF-I and IGF-II. The IGFs play a critical role in promoting development, stimulating growth and organogenesis via mitogenic, antiapoptotic and chemotactic activity. Recent research has focused on the events that occur intracellularly upon receptor activation. Several pathways have been shown to be important. The insulin-receptor substrate (IRS), SHC, GRB2, CRKII and CRKL adaptor proteins have all been implicated in transmitting signals to the nucleus of the cell. This review outlines some of the signalling pathways believed to be important in converting IGF-IR activation into changes in cell behavior and metabolism.

Minireview: IGF, Insulin, and Cancer

In recent years, the influence of the IGF system and insulin on cancer growth has been widely studied. Observational human studies have reported increased cancer mortality in those with obesity and type 2 diabetes, which may be attributable to hyperinsulinemia, elevated IGF-I, or potentially both factors. Conversely, those with low insulin, IGF-I and IGF-II levels appear to be relatively protected from cancer development. Initial attention focused on the role of IGF-I in tumor development. The results of these investigations allowed for the development of therapies targeting the IGF-I receptor signaling pathway. However, after in vitro and in vivo studies demonstrating that insulin may also play a significant and independent role in tumorigenesis, insulin is now receiving more attention in this regard. Some studies suggest that targeting insulin receptor signaling may be an important alternative or adjunct to targeting IGF-I receptor signaling. In this minireview, we discuss some of the recent in vitro, animal, and clinical studies that have elaborated our understanding of the influence of IGF and insulin on tumorigenesis. These studies have shed more light on the interaction between insulin and IGF signaling in cancer cells. They have made possible the development of novel targeted therapies and highlighted some of the potential future directions for research and therapeutics.