346 Insulin-Like Growth Factors The Molecular Basis of IGF-I Receptor Gene Expression in Human Cancer Haim Werner Abstract T he insulin-like growth factor-I receptor (IGF-IR) has a central role in normal cellular proliferation as well as in transformation processes. Transcription factors have been identified that modulate the activity of the IGF-IR gene. Transcription factors with tumor suppressor activity, such as p53 and WT1, were shown to inhibit transcription of the IGF-IR gene. Loss-of-function mutation of these genes in certain malignancies results in transcriptional derepression of the IGF-IR gene, with ensuing increases in the levels of IGF-IR. Likewise, the mechanisms of action of many oncogenic agents depend on their ability to transactivate the IGF-IR promoter and/ or to phosphorylate the cytoplasmic domain of the receptor. The expression of the IGF-IR gene is, ultimately, the net result of complex interactions between positive and negative nuclear factors, as well as between stimulatory and inhibitory secreted factors. The proliferative status of the cell is a direct consequence of this level of expression. Introduction The central role of the IGF system in a variety of growth and differentiation processes has been well established. Similarly well accepted is the notion that the vast majority of the biological actions of both IGF-I and IGF-II are mediated via activation of the IGF-IR heterotetramer. The role of the IGF-IR in cell cycle progression and apoptosis, as well as the signal transduction pathways responsible for these activities, are described in large detail in other Chapters of this book. The present Chapter will focus on the molecular mechanisms that are responsible for the expression of the IGF-IR gene during normal development, on understanding the events and factors that govern its levels of expression (and therefore determine, to a large extent, the proliferative status of the cell) and, in particular, on analyzing the mechanisms that underlie the pathological expression of the IGF-IR in the transformed cell. Overexpression of the IGF-IR Gene as a Common Theme in Malignancy Most human cancers and transformed cell lines express increased levels of IGF-IR on their cell surface, as well as augmented levels of IGF-IR mRNA (Table 1). These tumors include ovarian, colon, thyroid, lung, pheochromocytoma, breast, glioblastoma, astrocytoma, hepatoma, gastric, renal, rhabdomyosarcoma, and others (for an extensive review see ref. 1). In addition, amplification of the IGF-IR locus at band 15q26 has been reported in a small number of breast and melanoma cases.2,3 It is generally assumed that the tumor IGF-IR is capable of responding to circulating/ endocrine IGFs, and to IGFs produced locally by neighboring (stromal) cells or by the cancer cells themselves. In view of the abundant expression of the IGF-IR gene in most cancers, it is relevant to ask what are the mechanisms employed by the cell in order to control the synthesis and function of this important receptor. To gain an understanding on these basic questions it may be helpful to briefly review the role and regulation of the IGF-IR gene during ontogenesis. Insulin-Like Growth Factors, edited by Derek LeRoith, Walter Zumkeller and Robert Baxter. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers. ©2003 Copyright Landes Bioscience. Not for Distribution. CHAPTER 21 The Molecular Basis of IGF-I Receptor Gene Expression in Human Cancer 347 Ovary Colon Thyroid Lung Pheochromocytoma Breast Pancreas Leukemia Astrocytoma Hepatoma Stomach Kidney Rhabdomyosarcoma Glioblastoma Endometrium Ewings IGF-IR have been characterized using competitive binding assays, affinity cross-linking, Northern blots, RNase protection assays, RT-PCR, or a combination of them. The IGF-IR gene is constitutively expressed at each and every developmental stage, although its levels may vary over a wide range. IGF binding and IGF-IR mRNA can be detected as early as the oocyte stage.4 In preimplantation mouse embryos the receptor mediates preferentially the effects of IGF-II, since no insulin or IGF-I transcripts can be detected at this early stage.5 Following implantation, the IGF-IR is expressed by virtually every cell. The widespread distribution of the IGF-IR underscores its fundamental role as a survival factor required by most cells during ontogeny.6,7 Further evidence for a survival role for the IGF-IR is provided by the fact that the majority of cultured cells constitutively express the IGF-IR gene during their proliferative stages. In sharp contrast to the universal expression of the IGF-IR gene, the IGF ligands are expressed during ontogeny following distinct spatial and temporal patterns, suggesting that IGF-I and IGF-II have different roles during embryonic development. It appears, however, that the IGF-IR is able to mediate the endocrine effects of circulating IGFs (present both in the bloodstream and in the cerebrospinal fluid) as well as the effects of locally produced IGFs. Late developmental stages, characterized by a reduction in the number of rapidly-proliferating cells and by an increment in the proportion of postmitotic, terminally differentiated cells, are associated with a generalized reduction in the levels of IGF-IR mRNA and binding in most organs.8 These findings are consistent with the results of experiments demonstrating that cultured cells induced to differentiate exhibit a reduction in the levels of IGF-IR. Dedifferentiation states associated with malignancy, similar to early developmental stages, exhibit high levels of IGF-IR, consistent with the increased proliferative activity of the transformed cell.9,10 Furthermore, augmented receptor levels correlate with a large reduction in apoptosis. The implication of these observations is that activation of the IGF-IR may rescue from apoptosis cell populations that are otherwise tagged for elimination. Induction of apoptosis, on the other hand, seems to be the common theme of a number of approaches that are aimed at targeting the IGF-IR as a potential anticancer therapy. An interesting novel paradigm of IGF-IR-independent proliferation has been recently described in metastatic prostate and breast cancer cells.11,12 The implications of these observations, suggesting that the IGF-IR is required during the early stages of transformation, but not at the metastatic stage, are discussed in this book by C.T. Roberts. The Role of the IGF-IR in the Transformation Process Because the IGF-IR is expressed at very high levels in most naturally-occurring cancers, as well as in experimentally-induced tumors, it is important to examine its role in different cellular systems. Artificial overexpression of the IGF-IR in fibroblasts results in a ligand-dependent, highly transformed phenotype, which includes the formation of tumors in nude mice.13 On the other hand, abrogation of the IGF-IR signaling pathway using specific anti-IGF-IR antibodies resulted in a drastic reduction in cellular proliferation of melanoma,14 breast,15 hematopoietic,16 colorectal,17 ©2003 Copyright Landes Bioscience. Not for Distribution. Table 1. IGF-IR in human cancers Insulin-Like Growth Factors neuroblastoma18 and Wilms’ tumor cells.19 Likewise, inhibition of IGF-I-mediated growth and clonogenicity in soft agar of human T98G and rat C6 glioblastoma cells was achieved by introducing antisense oligodeoxynucleotides against the IGF-IR mRNA, or by transfection with plasmids encoding antisense cDNA fragments.20,21 The central role of the IGF-IR in the transformation process is further illustrated by the results of experiments showing that fibroblast cell lines established from mouse embryos in which the IGF-IR was disrupted by homologous recombination (R-) cannot be transformed by any of a number of oncogenes (including the SV40 large T antigen, activated ras, bovine papillomavirus E5 protein, and others).22-23 Reintroduction of a functional receptor renders R- cells susceptible to the transforming activities of these oncogenes. However, certain exceptions to this general paradigm have been reported. For instance, stable transfection of the GTPase-deficient mutant human Gα13 resulted in transformation of R- cells, as tested using the soft agar assay. These results demonstrate that Gα13 can induce cellular transformation through pathways apparently independent of the IGF-IR.24 Cooperation between oncogenes, in addition, may activate additional survival pathways which may potentially result in the transformation of IGF-IR-null cells. Thus, while human papillomavirus-16 E6 and E7 proteins were unable to induce colony formation in R- cells when transfected separately, combined transfection of both oncogenes resulted in a transformed phenotype.25 The transforming activity of the IGF-IR depends, to a large extent, on its strong antiapoptotic activity. The ability of the IGF-IR to protect cells from apoptosis (thus conferring them an increased survivability) has been demonstrated in many different cell types, including fibroblasts, neural-derived, hematopoietic, and others.26-29 Furthermore, a highly significant correlation was established between the number of cell surface IGF-IRs and the in vivo and in vitro survival capacity of the cell.30 Mapping of Receptor Domains Involved in Transformation Early studies indicated that an intact tyrosine kinase domain is required for the transduction of the proliferative actions of the IGF-IR.31 Specifically, mutations in the ATP binding site and triple tyrosine residues at positions 1250, 1251, and 1316, either individually or in combination, totally abrogated tumor formation.32 Essential roles in IGF-I-mediated mitogenesis were also associated with tyrosine residues 1131, 1135, and 1136.33 Transfection of rat-1 fibroblasts with a truncated β-subunit mutant (952 STOP) resulted in cells which were unable to grow in soft agar and to induce tumors in athymic mice.34 Furthermore, mutation of a series of four serine residues at the C-terminal domain which are involved in specific binding to the 14-3-3 protein, a potential substrate of IGF-IR action, similarly affected tumorigenesis.35 Mapping of functional domains in the cytoplasmic portion of the IGF-IR revealed that the domains required for its antiapoptotic function are distinct from those required for its proliferative or transforming activities.36 Furthermore, the domains of the receptor required for inhibition of apoptosis are necessary but not sufficient for transformation. While most research focused on elucidating the structure-function relationship of the cytoplasmic portion of the receptor, an important modulatory role has been ascribed to the 36-amino acid extracellular segment of the IGF-IR β-subunit. Using N-terminally truncated IGF-IR fused to avian sarcoma virus UR2 gag p19 it was shown that the 20 residues located immediately upstream of the transmembrane domain have an inhibitory effect on the transforming and tumorigenic potential of the fusion protein, whereas N-linked glycosylation within this region had a positive effect.37,38 Phosphorylation of the IGF-IR in Malignancy As previously indicated, the presence of a functional IGF-IR is an essential prerequisite for oncogenic transformation. Furthermore, an intact tyrosine kinase domain is fundamental in order for the receptor to exert its potent mitogenic, antiapoptotic, and transforming activities. The mechanisms of action of a number of oncogenic agents depend, in fact, on their ability to efficiently phosphorylate the receptor. Thus, transformation by pp60src, the protein encoded by the src oncogene of Rous sarcoma virus, results in the constitutive tyrosine phosphorylation of the IGF-IR β subunit.39,40 It has been estimated that between 10-50% of the receptors are phosphorylated in the unstimulated src-transformed cell. Addition of IGF-I synergistically increased the extent of phosphorylation of the receptor. These results raise the possibility that pp60src alters growth regulation by ©2003 Copyright Landes Bioscience. Not for Distribution. 348 349 rendering the cells constitutively subject to a mitogenic signal. Moreover, they suggest that the IGF-IR kinase is more active as an autokinase in transformed than in nontransformed cells. Likewise, the EWS-FLI-1 chimeric protein that results from the reciprocal translocation t(11;22)(q24;q12) and that is characteristic of the Ewings family of tumors, requires the presence of an intact IGF-IR for transformation. Fibroblasts which were stably transfected with the fusion protein exhibited a larger degree of IGF-I-stimulated IRS-1 phosphorylation, suggesting that expression of the EWS-FLI-1 oncogene may sensitize the IGF-IR signaling pathway to the action of IGF-I.41 Transcriptional Regulation of the IGF-IR Gene Transcriptional regulation of the IGF-IR gene constitutes one of the most important mechanisms employed by the cell to control expression of this receptor during normal development and in response to physiological and pathological stimuli. Cloning and characterization of the IGF-IR promoter region revealed a number of features that are shared by a family of genes which are constitutively expressed by most cells and that are referred to as housekeeping genes. The IGF-IR regulatory region is very rich in G and C nucleotides and lacks TATA or CAAT motifs, two regulatory elements that are required for efficient transcription initiation of most eukaryotic genes. Accurate transcription of the IGF-IR gene is directed from an “initiator” sequence, a control element that is present in the promoters of genes that are highly regulated during differentiation and development, and which is able to assemble a functional transcription complex in the absence of a TATA box.42-45 Similar to other widely expressed genes, the promoter region of the IGF-IR gene contains a number of GC boxes (GGGCGG), which are putative binding sites for members of the Sp1 family of transcription factors.46 Sp1 is an ubiquitous zinc-finger nuclear protein that stimulates transcription from a group of RNA polymerase II-dependent promoters. Using transient cotransfections, electrophoretic mobility shift assays (EMSA), and DNaseI footprinting experiments it was demonstrated that Sp1 is a potent transactivator of the IGF-IR promoter. The capacity of Sp1 to transactivate this gene is consistent with its ability to bind with high affinity to consensus sites present in the promoter region.47 Although ubiquitously expressed, levels of Sp1 fluctuate during development.48 Similar to the IGF-IR gene, lowest Sp1 mRNA and protein levels are seen in terminally differentiated cells, suggesting that Sp1 constitutes a positive activator of IGF-IR transcription in most physiological states. In addition, the mechanism of action by which certain tumor suppressors, including p53, inhibit transcription of the IGF-IR gene involves specific interaction with Sp1.49 This point will be discussed below in more detail. Regulation of the IGF-IR Gene by Oncogenes Cellular and viral oncogenes can induce transformation by “recruiting” and activating the IGF signaling pathway. In a previous section I described the mechanism of action of the src oncogene of the Rous sarcoma virus, and showed that it involves the ligand-independent phosphorylation of the IGF-IR β subunit. Additional oncogenes were shown to directly transactivate the IGF-IR promoter. An example of this class of oncogenes is c-myb (the cellular equivalent of the viral transforming oncogene v-myb). Overexpression of c-myb in Balb/c-3T3 cells induced an increase in the levels of both the IGF-IR and IGF-I ligand transcripts.50,51 This event resulted in abrogation of the requirement for IGF-I in the growing media, which in itself constitutes one of the distinctive hallmarks of a malignantly-transformed cell. An additional oncoprotein shown to stimulate the transcription of the IGF-IR gene is the hepatitis B virus X (HBx) gene product. In hepatocellular carcinoma-derived cell lines containing HBx protein, the endogenous levels of IGF-IR mRNA were ~5-fold higher than in cells that do not express HBx transcripts. Similarly, transfection of HepG2 cells with an expression vector encoding the HBx cDNA induced an ~2-3-fold increase in the levels of IGF-IR promoter activity, mRNA, and IGF binding.52 These findings clearly demonstrate that the mechanism of action of oncogene HBx in the pathophysiology of hepatocellular carcinoma involves the transactivation of the IGF-IR gene. In summary, the requirement for a functional IGF-IR in order for a cell to undergo oncogenic transformation can be explained, at a molecular level, by the fact that many oncogenes “adopt” the IGF-IR signaling pathway as their mechanism of transformation. Certain oncogenes achieve this goal by directly transactivating the IGF-IR promoter and thus drastically increasing the concentration ©2003 Copyright Landes Bioscience. Not for Distribution. The Molecular Basis of IGF-I Receptor Gene Expression in Human Cancer 350 Insulin-Like Growth Factors of receptors in the preneoplastic cell. Additional oncogenes induce a large increase in the level of phosphorylation of the IGF-IR β subunit. Regardless of the molecular mechanism employed, the transformed cell displays essentially identical phenotypes, including the ability to proliferate in the absence of exogenous IGFs. Given the strong proliferative action of the IGF-IR, it may be asked how does the adult, terminally differentiated cell succeed in remaining out of the cell cycle. One potential mechanism that may be responsible of keeping IGF-IR levels below a certain threshold involves its transcriptional suppression by a family of negative growth regulators, collectively referred to as tumor suppressors. We may predict that beneath these receptor concentrations, the cell will remain at the G0 stage and will not engage in any mitogenic activity. Mutation, deletion, or chromosomal rearrangement of tumor suppressor genes in transformed cells may lead to transcriptional derepression of the IGF-IR gene. P53 is a tumor suppressor that, in its hyperphosphorylated state, blocks progression of cells through the cell cycle.53,54 P53 is involved in the etiology of many human tumors, and mutations of the p53 gene are the most frequent mutations in human cancers.55,56 The p53 protein functions as a transcription factor that binds specifically to DNA sequences in various promoters and stimulates their transcriptional activity. It can also function as a transcriptional repressor of many growth-regulated genes. Transient expression of wild type (wt) p53 in osteosarcoma- and rhabdomyosarcomaderived cell lines suppressed the activity of cotransfected IGF-IR promoter constructs by 75-90 %. On the other hand, cotransfection of tumor-derived, mutant versions of p53 (encoding point mutations at codons 143, 248, and 273) stimulated promoter activity by ~2.3- to 4-fold of control values.57 In addition, wt p53 decreased the IGF-I-induced tyrosine phosphorylation of the IGF-IR and of IRS-1, whereas mutant p53 stimulated their phosphorylation.49 Although the mechanism/s for transcriptional suppression by wt p53 are not fully understood, results of EMSA assays suggest that wt p53 can bind to the TATA-binding protein (TBP), thus preventing this protein from binding to the initiator region of the IGF-IR gene and assembling a functional initiation complex. An additional potential mechanism of action of p53 involves its interaction with Sp1. Wild-type and mutant forms of p53 were shown to physically interact with Sp1, which counteracted the inhibitory effect of wt p53 in a dose-dependent manner. Further support for a physiological role for the IGF-IR gene as a target for p53 action was provided by experiments performed in murine hemopoietic cells using a temperature-sensitive mutant of p53. Expression of p53 in its wt conformation reduced the number of IGF-IRs in cells in which the transfected receptor was under the control of the IGF-IR, but not the cytomegalovirus, promoter.58 Taken together, these results suggest that, at least part of, the effects of wt p53 on apoptosis and cell cycle arrest are mediated via suppression of the IGF-IR promoter. Lack of inhibition by mutant p53 may accelerate tumor growth and inhibit apoptosis, thus providing an increased survival capacity to malignant cell populations (Fig. 1). In addition to controlling the activity of the IGF-IR gene, p53 has been shown to modulate additional components of the IGF signaling system. Thus, the expression of IGF-II transcripts is reduced by wt p53.59 On the other hand, the activity of the IGFBP3 gene is stimulated by wt, but not mutant, p53.60 Because IGFBP3 is an inhibitor of mitogenic signaling by IGFs, it may be inferred that p53 can regulate the IGF system both at the level of availability of IGF ligands, and at the level of activity of the IGF-IR promoter.61 A similar paradigm of tumor suppressor modulation of IGF-IR gene expression was reported for BRCA1, the breast and ovarian cancer susceptibility gene.62 The BRCA1 gene encodes a 220-kDa phosphorylated protein that functions as a transcription factor with tumor suppressor activity.63 Mutations at the brca1 locus are linked to a large proportion of familial breast and/or ovarian cancer. Transient expression of a BRCA1 expression vector in a number of cell lines resulted in the dose-dependent suppression of cotransfected IGF-IR promoter constructs.64 Although the molecular targets of BRCA1 have not yet been identified, it is possible that part of the proapoptotic activity of BRCA1 is achieved via suppression of the strongly antiapoptotic IGF-IR gene. Mutant versions of BRCA1 lacking transactivational activity can potentially derepress the IGF-IR promoter, resulting in augmented levels of IGF-IR mRNA and IGF-I binding in breast cancer. A link between radiosensitivity and the IGF-IR gene has been recently reported.65 Ataxia telangiectasia (AT) cells, displaying a mutant ATM gene, express low levels of IGF-IR and show ©2003 Copyright Landes Bioscience. Not for Distribution. Regulation of the IGF-IR Gene by Tumor Suppressor p53 351 Figure 1. Regulation of IGF-IR gene expression by wild-type and mutant p53. The expression of the IGF-IR gene in the normal cell appears to be under inhibitory regulation by wt p53. As a result of this negative control, cellular proliferation is reduced and apoptosis is increased. Loss-of-function mutation of p53 in malignant cells can derepress the IGF-IR promoter, with ensuing increases in the levels of cell surface receptors. The mechanism of action of p53 involves interaction with transcription factor Sp1 and with the TATA-binding protein, TBP. decreased IGF-IR promoter activity compared with wild type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and protein levels. These results suggest that reduced expression of the IGF-IR may contribute to the radiosensitivity of AT cells. Regulation of the IGF-IR Gene by Tumor Suppressor WT1 An additional tumor suppressor whose mechanism of action has been well characterized is the Wilms’ tumor suppressor, WT1. Wilms’ tumor is a pediatric kidney cancer that arises from metanephric blastema cells and whose etiology is associated with deletion or mutation of the WT1 gene. The WT1 gene encodes a 52-54 kDa protein that contains four zinc fingers of the C2-H2 class in its C-terminus.66 WT1, via its zinc finger domain, binds to target DNAs containing versions of the consensus sequence GCGGGGGCG. Among other promoters, this specific motif is present in the regulatory regions of the IGF-II and IGF-IR genes.67,68 During normal kidney development, WT1 functions as a transcription factor with important roles in the differentiation of the metanephric blastema to renal epithelium.69 IGF-II and the IGF-IR are also involved in kidney development, being their expressions negatively regulated by the WT1 gene product. In addition, the IGF-II-IGF-IR axis has an important role in Wilms’ tumor progression, as illustrated by the fact that administration of anti-IGF-IR antibodies to nude mice bearing Wilms’ tumor heterotransplants prevented tumor growth and resulted in partial tumor remission.19 Furthermore, the levels of IGF-II and IGF-IR mRNAs are significantly elevated in the tumors in comparison to normal adjacent tissue.70,71 The molecular mechanisms responsible for transcriptional repression of the IGF-IR gene by WT1 were revealed by means of transient and stable transfections, EMSA and DNase footprinting assays. WT1 represses IGF-IR promoter activity in a dose-dependent manner, binding to sites in both the 5'-flanking and 5'-untranslated regions.68 The DNA-binding capacity of WT1 is critical ©2003 Copyright Landes Bioscience. Not for Distribution. The Molecular Basis of IGF-I Receptor Gene Expression in Human Cancer Insulin-Like Growth Factors for maximal repression of the IGF-IR promoter, but some effects may be mediated through protein-protein interactions involving the N-terminal domain.72 Stable expression of the WT1 cDNA in kidney tumor-derived G401 cells resulted in a decreased rate of cellular proliferation, decreased levels of IGF-IR mRNA and binding, and reduced activity of a transfected IGF-IR promoter. In addition, WT1-expressing cells exhibited a reduction in IGF-I-stimulated proliferation, thymidine incorporation, and anchorage-independent growth.73 In summary, Wilms’ tumors and other nephropathies are cases of aberrant dedifferentiation that are characterized, in many instances, by underexpression, deletion, or mutation of the WT1 gene. Loss of WT1 activity may result in derepression of the IGF-IR and IGF-II promoters. Increased transcription and expression of both ligand and receptor genes may induce a mitogenic event with important consequences in tumor progression. Regulation of the IGF-IR Gene by Disrupted Transcription Factors As mentioned above, certain human malignancies are characterized by recurrent chromosomal translocations, frequently resulting in the fusion of genes. These fusion gene products (or chimeras) often comprise domains derived from unrelated transcription factors and nucleic acid-binding proteins. Furthermore, the chimeras usually display altered transcriptional activities that confer upon them a gain-of-function type of action. Desmoplastic small round cell tumor (DSRCT) is an aggressive primitive pediatric tumor associated with the recurrent translocation t(11;22)(p13;q12). This rearrangement joins the N-terminal (activation) domain of the Ewings’ sarcoma gene, EWS (an ubiquitously expressed RNA-binding protein), to the C-terminal (DNA-binding) domain of WT1, including 3 out of 4 zinc fingers.74,75 The fusion protein, EWS-WT1, is capable of binding consensus WT1 sites in the IGF-IR promoter region with an affinity comparable to that of native WT1, and of transactivating the IGF-IR promoter in transient transfection assays.76 Hence, fusion of EWS to WT1 abrogates the tumor suppressor function of WT1 and the RNA-binding capacity of EWS, and generates an oncogene capable of binding and transactivating the IGF-IR promoter (Fig. 2). Augmented levels of IGF-IR may constitute an important prerequisite in progression of DSRCT. A similar paradigm of transactivation of the IGF-IR gene by a disrupted transcription factor was recently reported for the PAX3-FKHR oncoprotein.77 This chimeric protein results from the recurrent translocation t(2;13)(q35;q14), the cytogenetic event characteristic of alveolar rhabdomyosarcoma (ARMS).78 PAX3-FKHR includes the N-terminal domain of PAX3 (a developmentallyregulated transcription factor that contains a paired-box and an homeodomain DNA-binding motifs) fused in-frame to the C-terminal domain of FKHR (a member of the forkhead family of transcription factors). Transfection of sarcoma-derived cell lines with expression vectors encoding PAX3-FKHR resulted in transactivation of a cotransfected IGF-IR promoter construct, whereas PAX3 exhibited a reduced potency in comparison to the chimera. These results can be interpreted to suggest that the IGF-IR gene constitutes a molecular target for aberrant transcription factor PAX3-FKHR. Increased levels of IGF-IR are potentially critical in the etiology of ARMS and other pediatric sarcomas. Regulation of the IGF-IR Gene by Growth Factors, Cytokines and Steroid Hormones In addition to cellular factors such as oncogenes and tumor suppressors, the expression of the IGF-IR gene can be also modulated by various secreted factors, including peptide and steroid hormones, growth factors, and cytokines. Humoral regulation of the IGF-IR gene is important in many physiological processes. For example, cell cycle progression occurs only in the presence of two families of growth factors: competence factors (such as PDGF and FGF) and progression factors (such as IGF-I). It has been postulated that the main role of competence factors is to induce the production of sufficient amounts of progression factors and their receptors that will allow the cell to engage in mitogenesis.79 In fact, both FGF and PDGF have been shown to stimulate transcription of the IGF-IR gene.80,81 PDGF increased the activity of the IGF-IR promoter via an ~100-bp promoter fragment located immediately upstream of the initiator element that includes a consensus c-myc binding site.82 On the other hand, IGF-I negatively regulates the expression of the IGF-IR gene. The expression of the IGF-IR gene depends also on the ambient concentration of steroid hormones. Estrogens, for instance, were shown to stimulate the levels of IGF-IR mRNA and binding in ©2003 Copyright Landes Bioscience. Not for Distribution. 352 353 Figure 2. Regulation of the IGF-IR gene by disrupted transcription factor EWS-WT1. Desmoplastic small round cell tumor is characterized by the recurrent chromosomal translocation t(11;22)(p13;q12), that fuses the N-terminal domain of the Ewings’ gene product, EWS, to the C-terminal, DNA-binding domain, of WT1. This event abrogates the tumor suppressor activity of WT1 and generates a chimeric oncoprotein, EWS-WT1, whose mechanism of action involves binding to, and transactivation of, the IGF-IR promoter. WT1 binding elements (denoted as black boxes) are located both upstream and downstream of the IGF-IR gene transcription start site. MCF7 cells by ~7-fold, suggesting that a potential mechanism by which estrogens stimulate breast cancer proliferation involves sensitization to the mitogenic effects of IGFs by augmenting receptor concentration.83 Progestins, on the other hand, induced a reduction in the levels of IGF-IR mRNA and binding in estrogen-responsive breast cancer cell lines. It appears that this effect is mediated by IGF-II, whose secretion is stimulated by progestins and which, in turn, down-regulates the IGF-IR gene.84 Finally, certain cytokines were also shown to control the activity of the IGF-IR gene. Tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) are multifunctional cytokines which are produced mainly by activated macrophages and lymphocytes, respectively, although TNF-α is also synthesized by a number of non-hematopoietic cells. TNF-α controls cellular proliferation by inducing apoptosis, alone or in combination with other cytokines. Furthermore, TNF-α and IFN-γ reportedly inhibited a number of IGF-mediated biological actions, as well as the expression of the IGF-I and IGF-II genes. It has been recently demonstrated that both cytokines suppressed the activity of the IGF-IR promoter, resulting in a drastic reduction in the levels of IGF-IR mRNA and protein.85 TNF-α, in addition, decreased the stability of mRNA molecules. Regulation of IGF-IR gene expression at both transcriptional and posttranscriptional levels may constitute a potential mechanism by which TNF-α and IFN-γ (and probably other cytokines) affect cellular proliferation. Conclusions The IGF-IR plays a critical role in normal and pathological growth processes. Controlling the expression of this gene appears to be an important mechanism that allows the cell to “decide” whether to adopt proliferative or apoptotic pathways. The expression of this gene can be tightly regulated by secreted factors of endocrine or local (autocrine/paracrine) origin that can either stimulate or inhibit the synthesis of the IGF-IR. In addition, a number of nuclear proteins displaying either oncogenic ©2003 Copyright Landes Bioscience. Not for Distribution. The Molecular Basis of IGF-I Receptor Gene Expression in Human Cancer Insulin-Like Growth Factors or antioncogenic activities have been also shown to regulate the activity of the IGF-IR gene at the transcriptional level. Transcription factors with tumor suppressor activity, such as p53 and WT1, negatively regulate the expression of the IGF-IR gene. The etiology of neoplasias associated with loss-of-function mutation of tumor suppressors is linked to the inability of the mutant forms to suppress the activity of their molecular targets, including the IGF-IR gene. On the other hand, gain-of-function mutations of oncogenes are associated with increased transactivational activity of the IGF-IR promoter and/or augmented phosphorylation of the cytoplasmic domain of the receptor. Interactions between stimulatory and inhibitory factors may ultimately determine the level of expression of the IGF-IR gene. Acknowledgments The work in the authors' laboratory is supported by grants from The Israel Cancer Association, The Israel Science Foundation, The U.S.-Israel Binational Science Foundation, and The Fogarty International Center. References 1. Werner H, LeRoith D. The role of the insulin-like growth factor system in human cancer. Adv Cancer Res 1996; 68:183-223. 2. Almeida A, Muleris M, Dutrillaux B et al. The insulin-like growth factor I receptor gene is the target for the 15q26 amplicon in breast cancer. Genes Chrom Cancer 1994; 11:63-65. 3. Zhang J, Trent JM, Meltzer PS. Rapid isolation and characterization of amplified DNA by chromosome microdissection: Identification of IGF1R amplification in malignant melanoma. Oncogene 1993; 8:2827-2831. 4. Scavo L, Shuldiner AR, Serrano J et al. Genes encoding receptors for insulin and insulin-like growth factor I are expressed in Xenopus oocytes and embryos. Proc Natl Acad Sci USA 1991; 88:6214-6218. 5. Schultz GA, Hahnel A, Arcellana-Panlilio M et al. Expression of IGF ligand and receptor genes during preimplantation mammalian development. Mol Reprod Dev 1993; 35:414-420. 6. Bondy CA, Werner H, Roberts Jr CT et al. Cellular pattern of insulin-like growth factor I (IGF-I) and type I IGF receptor gene expression in early organogenesis: comparison with IGF-II gene expression. Mol Endocrinol 1990; 4:1386-1398. 7. Bondy CA, Werner H, Roberts CT Jr et al. 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. Neuroscience 1992; 46:909-923. 8. Werner H, Woloschak M, Adamo M et al. Developmental regulation of the rat insulin-like growth factor I receptor gene. Proc Natl Acad Sci USA 1989; 86:7451-7455. 9. Werner H , LeRoith D. The insulin-like growth factor-I receptor signaling pathways are important for tumorigenesis and inhibition of apoptosis. 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