Charles T. Roberts

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 mRNA levels are developmentally regulated in specific regions of the rat brain

The expression of mRNAs encoding insulin-like growth factor I (IGF-I) and the IGF-I receptor in the developing rat brain from embryonic day 16 to postnatal day 82 was analyzed using solution hybridization-RNase protection assays. Four distinct developmental patterns in the steady-state levels of IGF-I mRNA were seen. Specifically, the olfactory bulb showed a high perinatal level of IGF-I mRNA which declined dramatically by postnatal day 8. In contrast, cerebral cortex displayed maximal levels of IGF-I mRNA at postnatal day 8 and 13, which subsequently declined to adult levels (P82). A third developmental pattern was seen in the hypothalamus, where IGF-I mRNA increased from E16 up to postnatal day 3 and remained elevated thereafter. Finally, IGF-I mRNA levels in brainstem and cerebellum remained unchanged throughout the time period studied. We conclude that there are specific regional patterns of IGF-I gene expression in the developing rat brain. In contrast, IGF-I receptor gene expression did not exhibit any region-specific developmental changes. The developmental patterns of IGF-I gene expression seen in this study further substantiate the potential role of IGF-I in normal brain development.

Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.

Regulation of endometrial cancer cell growth by insulin-like growth factors and the luteinizing hormone-releasing hormone antagonist SB-75

The involvement of IGFs in growth regulation of the Ishikawa endometrial tumor cell line and the possible interference of LH-RH analogues with a potential autocrine or paracrine loop involving IGFs was evaluated. The mitogenic effects of IGF-I, IGF-II, and insulin were compared. IGF-I was found to be 3-fold more potent than IGF-II and 30-fold more potent than insulin, suggesting that the effects of these growth factors are mediated by the IGF-I receptor. Ishikawa endometrial cancer cells secrete IGF-II, but not IGF-I, and insulin (1 microM) stimulates IGF-II release. The LH-RH antagonist [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]-GnRH (SB-75, CETRORELIX) inhibited basal and IGF-induced growth. Moreover, this antagonist almost completely inhibited IGF-II release from Ishikawa cells, while having no significant effect on the number or affinity of IGF-I binding sites. Inhibition of IGF-II release occurred at a lower SB-75 concentration than that needed for a reduction in cell number. The ED50 of SB-75 for IGF-II release was 0.3 microM as compared to 1.5 microns concentration which is required for reduction in cell number, suggesting that inhibition of growth factor release precedes cell growth inhibition. We conclude that the LH-RH antagonist SB-75 can inhibit the growth of endometrial cancer cells by interfering with the autocrine action of IGF-II and also by directly inhibiting the growth-stimulatory effects of IGFs, probably through effects on a post-receptor mechanism.

Evolutionary aspects of the endocrine and nervous systems.

Publisher Summary This chapter illustrates the evolutionary aspects of the endocrine and nervous systems. In mammals and other vertebrates, the major systems of intercellular communication are the endocrine and nervous systems. Intercellular communication is not unique to organisms which possess endocrine and nervous systems; rather it is essential to all forms of life including microbes. In the classic concept of the endocrine system the chemical messenger molecule, the hormone, is produced in a localized region, released into the general circulation, and acts on a target cell at a distance. In the nervous system, on the other hand, the secretory cell is a neuron and the messenger molecule a neurotransmitter. Many of the messenger molecules assigned to the endocrine or nervous systems reach their target tissues by other systems of intercellular communication such as exocrine and paracrine systems. Furthermore, a number of nonhormonal peptides which act on a target tissue have structural similarities to classic hormones and act on the target tissues in a manner almost identical to hormones. Interestingly, the biochemical elements necessary for the intercellular communication, that is, messenger molecules, their receptors, as well as post receptor intracellular components, are present in unicellular organisms and show distinct structural and functional similarities to their counterparts in vertebrate tissues.

TPA-induced neurite formation in a neuroblastoma cell line (SH-SY5Y) is associated with increased IGF-I receptor mRNA and binding

The neuroblastoma cell line SH-SY5Y was cultured in the presence of TPA for three days. Increased neurite formation was noted as early as 24 hours after TPA was added. These changes were associated with an increase in IGF-I receptor binding as well as increased mRNA for the IGF-I receptor.

The Insulin-Like Growth Factor Family of Peptides, Binding Proteins and Receptors: Their Potential Role in Tissue Regeneration

The insulin-like growth factors (IGF-I and IGF-II) are mitogenic pep tides that are structurally related to insulin (Figure 1). Until recently, these growth factors were thought to be produced exclusively by the liver and to act solely in an endocrine manner.1-3 According to the “somatomedin hypothesis,” the synthesis of IGF-I by the liver and its secretion are regulated by growth hormone (GH) and, following its release into the circulation, IGF-I reaches its target tissues where it induces growth and development, thereby mediating the effect of GH during the growth period.

Evolution of Insulin and Insulin Receptors

Insulin traditionally has been considered to be a unique product of the vertebrate pancreas. More recent studies, however, have suggested that there may be extrapancreatic sources of insulin and that insulin may act on tissues other than its classic target tissues. Of specific interest to us has been the suggestion that nervous tissue may be an extrapancreatic source of insulin synthesis and, furthermore, that nervous tissue may be a specific target for insulin. To pursue these questions, we and others have chosen a phylogenetic approach in our studies. In this review we will first discuss the evolutionary origins of insulin-related molecules including their presence in non-vertebrate species. These findings strongly suggest that insulin or a related molecule can be produced by tissues other than a pancreas. In the second part, we will describe studies on the phylogenetic conservation of the brain insulin receptor. Finally, we discuss the phylogenetic and developmental conservation of the structurally unique brain insulin receptor.

Apoptosis in breast cancer

Abstract It has become increasingly clear that apoptosis plays a critical role in mammary gland development and is an important facet of mammary carcinogenesis. The critical role of apoptosis in breast cancer derives from: (1) the influence of BRCA and p53 mutations that result in continued proliferation and increased genetic instability in precancerous mammary epithelial cells that would otherwise be subject to growth arrest and/or apoptotic death: (2) the apoptosis-modulating effects of many hormones and growth factors that had independently been demonstrated to influence breast cancer development and progression; and (3) the potential diagnostic value of factors such as IGF-I and Bcl-2, whose predictive value may reflect their anti-apoptotic effects. The continuing elucidation of the molecular pathways involved in apoptosis and their regulatory mechanisms will provide new avenues for improved diagnosis and treatment of breast cancer.

Rat Brain/Hep G2 Glucose Transporter Gene Expression in Brain

Publisher Summary This chapter presents a methodology to quantitate Glut-1 mRNA and protein levels in the brain and in neuronal and astroglial cells in primary culture. Measurement of Glut-1 mRNA levels include isolation of total RNA, cDNA probe preparation, Northern blotting, and hybridization. In a study described in the chapter, a single ∼2.8-kb band that hybridized with Glut-1 cDNA probe was observed at each stage of brain development. The steady state levels of Glut-1 mRNA were relatively low at embryonic day 20 and at early postnatal stages. These levels increased around weaning time (postnatal day 21) and reached maximal values in the adult brain. Using scanning densitometry, it was found that Glut-1 mRNA levels in the adult brain were three- to fourfold higher than at birth. The chapter also discusses the measurement of Glut-1 protein by western blotting. It presents an autoradiogram of a representative experiment where Glut-1 protein levels have been measured in astroglial cells in response to the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA). TPA stimulation of [ 3 H]deoxy-D-glucose uptake in astrocytes is associated with an increase in the amount of Glut-1 protein.