The IGF-I receptor in cell growth, transformation and apoptosis

Biochimica et Biophysica Acta 1332 Ž1997. F105–F126

The IGF-I receptor in cell growth, transformation and apoptosis Renato Baserga a

a,)

, Atsushi Hongo a , Michele Rubini b, Marco Prisco a , Barbara Valentinis

Kimmel Cancer Center, Thomas Jefferson UniÕersity, 233 South 10th Street, Bluemle Life Sciences Building, Philadelphia, PA 19107-5541, USA b Istituto di Genetica Medica, UniÕersita` di Ferrara, Ferrara, Italy Received 13 December 1996; accepted 6 February 1997

Contents 1.

Introduction to the IGF system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F106

2.

The receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F106

3.

The ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F107

4.

The IGF binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F108

5.

Signal transducing pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F108 5.1. The immediate substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F108 5.2. The downstream pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F109

6.

Functions of the IGF-I receptor . . . . . . . . . 6.1. Mitogenicity . . . . . . . . . . . . . . . . . . 6.2. Transformation . . . . . . . . . . . . . . . . 6.3. Anti-apoptotic signaling . . . . . . . . . . . 6.4. Receptor number and mitogenic response 6.5. The IGF system and the brain . . . . . . .

7.

Mutational analysis of the IGF-I receptor and itssubstrates . . . . . . . . . . . . . . . . . . . . . . F115

8.

Oncogenes, p53, tumor suppressors and the IGF-Ireceptor . . . . . . . . . . . . . . . . . . . . . . F116

9.

The IGF-I receptor and tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F118 9.1. In animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F118 9.2. In humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F118

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10. The immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F119 11. Uniqueness of the IGF-I receptor targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F119

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Corresponding author. Fax: q1 215 9230249; E-mail: [email protected]

0304-419Xr97r$32.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 9 X Ž 9 7 . 0 0 0 0 7 - 3

a

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R. Baserga et al.r Biochimica et Biophysica Acta 1332 (1997) F105–F126 12. Questions without answers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F120 13. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F121 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F121

1. Introduction to the IGF system The following brief summary is based on a recent review by Rubin and Baserga w1x, and the reader is referred to that review for extended references. A simplified representation of the IGF system includes 3 receptors, 3 ligands and 6 IGF binding proteins ŽIGFBPs.. The ligands are the mature IGF-I Ž 70 aminoacids. , the mature IGF-II Ž 67 aminoacids. and insulin. The receptors are the type I insulin-like growth factor receptor Ž IGF-IR. , the insulin receptor ŽIR. and the IGF-II receptor Ž IGF-IIR. . In terms of cellular proliferation, the IGF-IR is the most active of the 3 receptors Žsee below.: it is activated by all 3 ligands, and, although it has several functional features in common with the IR w2x, the b subunit of the IGF-IR is 10 times more mitogenic than the beta subunit of the IR w3x. Insulin at supraphysiological concentrations also activates the IGF-IR; this is an important point to remember, because when insulin is used at m g concentrations, it exerts its mitogenic stimulation through the IGF-IR w4x. Under appropriate conditions, the IR is mitogenic. The IGF-IIR, per se, does not stimulate cell proliferation: in fact, deletion of the IGF-IIR genes causes an increase in body weight of mice Ž see below.. It is now considered as a down-regulator of IGF-II, i.e. a way for the organism and the cells to regulate the availability of IGF-II. The availability of IGF-I and IGF-II is also regulated by the six IGFBPs, that can serve as a storage site for these ligands Žinsulin does not bind to IGFBPs.. There are other ligands and receptors in the IGF system that have to be mentioned. The insulin receptor-related receptor ŽIRRR. , has substantial homology to the IGF-I and insulin receptors w5x. In chimeric constructs with the alpha subunits of the insulin or IGF-I receptors, its tyrosine kinase domain can be shown to be mitogenic, but the ligand that activates the IRRR has not yet been identified. Neither insulin

nor the mature IGFs, and not even serum, can activate the IRRR w6x. This suggests that the IRRR may be activated by an unknown ligand through a strictly autocrine mechanism, There is another receptor, c-ros w7x, that also displays homologies to the other two receptors, but its function has not yet been clarified, nor do we know whether it may be redundant for the IGF-I and insulin receptors or not. There are also several other ligands, which will be discussed below, in Section 3.

2. The receptors The IGF-IR, or type 1 IGF receptor, belongs to the family of tyrosine kinase receptors w8x, and its aminoacid sequence is 70% homologous to that of the IR w9x. The human IGF-IR gene also has a striking homology to the IR gene in overall size Žapprox. 100 kb. and in the number Ž 21. and size of individual exons w10x. The IGF-IR is synthesized as a single precursor polypeptide of 1367 aminoacids, with the structure: NH 2-signal peptide, a subunit, b subunit-COOH. It is customary to number the aminoacid residues of the IGF-IR from the first aminoacid of the mature peptide wafter removal of the signal peptide. , up to 1,337 w9x, and we will follow this numbering in this review. After removal of the signal peptide, the pro-receptor is cleaved after residue 706, to form the a and b subunits, linked by disulfide bonds w11x. The a subunit of 706 aminoacids, required for ligand binding w12x, is entirely extracellular, forms a dimer with another a subunit, and it is in this form that it is active. The b subunit has an extracellular, a transmembrane and an intracellular region ŽFig. 1.. The intracellular region of the IGF-IR has a binding site for two of the major substrates of both the IGF-I- and insulin receptors, IRS-1 and Shc, at residue Y950, an

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puzzle. As mentioned above, it down-regulates IGF-II levels Žsee also below., but it also has a 43 aminoacid sequence that is homologous to the type II region of fibronectin, suggesting a possible role in the attachment of cells to their substrates.

3. The ligands

Fig. 1. Diagram of the IGF-I receptor. The receptor is active as a dimer. Some of the important residues are indicated: Y950 is the binding site for IRS-1 and Shc; lysine 1003 is the ATP-binding site; the Y cluster at 1131, 1135 and 1136 is the tyrosine kinase domain; the C-terminus domain, with Ys at 1250 and 1251, is involved in transformation Žsee text..

ATP-binding site at lysine 1003 and a tyrosine kinase domain, whose activity is centered around tyrosines 1131, 1135 and 1136. It is in the tyrosine kinase domain that the IR and the IGF-IR have the highest homology Ž84%., while the lowest homologies are in the cysteine-rich regions of the a subunit Ž48%., which presumably defines the affinities of the ligands, and in the C-terminus Ž 44%., where some important activities of the two receptors can be mapped Žsee below.. The IGF-IR. like the IR, undergoes extensive post-translational modifications, which include serine and tyrosine phosphorylation, and glycosylation w13x. The result is that the receptors’ mobility in gels is substantially slower than it could be predicted from their aminoacid composition. The IGF-IR, like the IR, has anabolic functions, but, in terms of growth, it has three properties that distinguish it: it is mitogenic, it is required for the establishment and maintenance of the transformed phenotype, at least in many cell types, and it protects cells from apoptosis, both in vitro and in vivo. The IGF-IIR w14x is also known as the cation-dependent mannose-6-phosphate receptor. It has a very large extracellular domain, and a short intracellular C-terminal cytoplasmic domain. It has no tyrosine kinase activity, and its biological function remains a

This brief summary of the ligands involved in the IGF system is based on reviews by Foyt and Roberts w15x, Rechler w16x and Rubin and Baserga w1x, to which the reader is referred for the long list of individual references, and for the structure of the respective genes. The 3 major ligands are insulin, and the mature IGF-I and IGF-II. There is substantial structural homology among the three ligands, and IGF-I and IGF-II have a 70% sequence homology. IGF-I is synthesized as preproreceptor, from which the signal peptide at the NH2-terminus and E peptides at the C-terminus are cleaved, to generate the mature IGF-I, 70 aminoacids long. The sequence of the mature IGF-I is highly conserved in species as different as the salmon and humans. IGF-II is also synthesized as a preproreceptor, from which the mature IGF-II Ž67 residues. is generated. However, whereas IGF-I is found usually only in its mature form, IGF-II is also found in the proreceptor form, i.e. without the signal peptide, but with the variants of the E peptides at the C-terminus. Thus, because of alternative splicing and cleavage, IGF-II can also come in molecular weights of 10–15 kDa, besides the mature form. These ‘big’ IGF-IIs. as they are sometimes called w17x, are mitogenic, indeed, according to some authors, as mitogenic as the mature polypeptide w17–20x. There are also reports that the E peptides of IGF-I are also mitogenic in their own right w21x. A truncated version of IGF-I has also been described, lacking the first three N-terminal aminoacids, designated as des-IGF-I w22x. This IGF-I does not bind to IGF binding proteins, and is abundant in the central nervous system Ž see below.. All these ligands are mitogenic, under appropriate circumstances, and their mitogenicity is largely based on the activation of the IGF-IR. As mentioned above, the IR can also transmit a mitogenic signal, but it is a

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weaker signal than that of the IGF-IR w3x and the IGF-IIR is not mitogenic at all. Insulin, to activate the IGF-IR, has to be used in m g concentrations, i.e. several orders of magnitude higher than its plasma levels. Its mitogenicity in vivo is therefore somewhat doubtful; in vitro, it can be mitogenic in cells overexpressing the IR. In cell cultures, insulin as a growth factor is often used in m g concentrations, which activate the IGF-IR w4x. This is unfortunate, because some investigators report insulin as a requirement for the growth of specific cells, when, in reality, in the concentrations used, it simply replaces IGF-I.

4. The IGF binding proteins Six members of the IGF binding proteins have been identified w1,23x. Their function is still ill-defined. In some cases, the presence of IGF binding proteins stimulates IGF-mediate cell proliferation, in other cases, they are inhibitory. This may be also due to the possibility that different IGFBPs may have different functions. It seems reasonable to assume, though, that they act as a buffer system for IGFs, i.e. by limiting their availability to cells. The IGFs in their various forms Žincluding ‘big’ IGF-II. are present in human plasma at concentrations of 700–750 ngrml, vastly exceeding their mitogenic threshold. IGFBPs and the IGF-IIR probably act by down regulating the IGF pool, with the former one also functioning as a storage system, from which IGFs can be released when needed. However, the IGFBPs may also participate in the regulation of cell proliferation, independently of their binding capacity for IGF-I or IGF-II. Thus, Valentinis et al. w24x showed that the IGFBP-3 can inhibit the proliferation of R y cells, which are 3T3-like cells completely devoid of IGFIRs. IGFBPs have also a non-biological function, and that is the ability of causing havoc with Scatchard analyses for the IGF-IR, because they bind both IGF-I and IGF-II with high affinity Ž insulin does not bind at all. . Thus, in certain human tumor cell lines, as much as 90% of IGF-I binding in Scatchard analyses is due to the presence of large amounts of IGFBPs ŽMichael Steller, personal communication.. Although IGFBP can be eliminated from the binding

mixture by appropriate manipulations, the easiest way to avoid their contributions is to carry out the analysis with radioactive des-IGF-I, which, as mentioned above, does not bind to IGFBPs.

5. Signal transducing pathways 5.1. The immediate substrates When activated by its ligands, the IGF-IR transmits a signal to its major substrates, IRS-1, IRS-2 and Shc w25–30x. A fourth substrate of the IGF-IR, Grb10, has recently been described w31x. A similar substrate, a splice variant of Grb-IR, which is a homolog of Grb10, has also been identified w32x, but, at least at the moment of writing, it is not clear whether these Grb10 molecules are redundant or have different functions. The protein 14.3.3 also binds directly to the IGF-IR in the yeast two hybrid system ŽCraparo and Gustafson, personal communication. . From these substrates, signals originate, which are subsequently transduced via signal transducing pathways. One of these pathways, for both the IR and the IGF-IR, as well as other growth factor receptors Žfor instance, the PDGF and EGF receptors. is the ras pathway w33x. It is well established that IRS-1 plays an essential role in the mitogenicity of both the IR and the IGF-IR in cells in culture w34–37x. Less clear are the roles of the other substrates, although the Shc proteins may also be involved in the mitogenic response w35x. For the moment, very little is known of the role of IRS-2 and Grb10 Ž and its variants. , in IGF-IRmediated mitogenesis and transformation. There are contradictory indications for the Grb10 family. An overexpressed Grb10 seems to inhibit IGF-I-mediated mitogenesis and transformation Žunpublished data by Morrione et al... However. a positive role of Grb10 in mitogenesis is suggested by that fact that a dominant negative of another form of Grb10, inhibits IGF-I-mediated DNA synthesis w32x. IRS-1, originally described as a docking protein, specific for the insulin and IGF-I receptors w38x, has turned out to be at the cross-road of several signaling pathways. It is essential for IL-4 stimulation of hemopoietic cells w39,40x, for signaling by the growth hormone receptor and interferon-gamma w41x, it is

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involved in interactions with the JAK family of transducing molecules w42–44x, with certain types of integrins w45x, with the Syp phosphatase w46x and the SV40 T antigen w47x. In additiion, it is tyrosyl phosphorylated by several cytokines w43x, and it interacts with G-protein coupled receptors w48x. It seems therefore that IRS-1 and Ž probably. its homolog IRS-2 are at the crossroads of signaling from different pathways, and may play a broader role than it was hitherto suspected, in both cellular proliferation and the regulation of gene expression. 5.2. The downstream pathways The ras pathway w49x, reviewed extensively in several recent papers, involves, among others, PI-3 kinase, Grb2, Sos and other transducing molecules w28,50–52x. Blenis w53x gives a list of the proteins involved in the signal transduction cascade, from ras to raf to MEKK, MEK, ERK, p70 RSK and finally transcription factors, w52x. This pathway is shared by several other growth factors, especially the three growth factors, PDGF, EGF and IGF-I, that, together, sustain the growth of wild type mouse embryo cells, like 3T3 cells. However, overexpressed PDGF and EGF receptors cannot sustain ligand-dependent growth of R y cells w54,55x, R y cells being 3T3-like cells w56x originating from mouse embryos with a targeted disruption of the IGF-IR genes Ž see below. . On the contrary, an overexpressed IGF-IR can sustain IGF-I-dependent cell proliferation, in the complete absence of other growth factors w57x. Although the evidence is overwhelming that ras activation is required for optimal cell proliferation and transformation w58,59x, these and other findings indicate that it is not sufficient. Indeed, an overexpressed and constitutively activated ras fails to induce growth of R y cells in serum-free medium supplemented with individual growth factors w60x. One has therefore to postulate a pathway, that is not shared with the PDGF and EGF receptors, but is peculiar to the IGF-IR. Ras-independent pathways for growth Žand transformation, see below. have also been suggested independently by others w61–64x. A good candidate for a ras-independent pathway originating from the IGF-IR could be src w65x, especially in view of the fact that v-src can tyrosyl phosphorylate both the IGF-IR and IRS-1 w66x.

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6. Functions of the IGF-I receptor 6.1. Mitogenicity 6.1.1. In Õitro The growth of cells in culture is strictly regulated by growth factors, and it usually requires more than one growth factor. As an example, 3T3 cells need for optimal growth both PDGF Žor EGF. and IGF-I w67–70x. Singly, these growth factors do not support growth of 3T3 cells or other growth-regulated cells in monolayer. It is clear, though, that the IGF-IR activated by its ligands Ž IGF-I, IGF-II and insulin at supraphysiological concentrations. plays a substantial role in the control of cell proliferation in mammalian cells w11,71–74x, both in vivo and in vitro. Many cell types in culture require IGF-I for optimal growth Žsee review by Goldring and Goldring, w75x., and these cell types include human diploid fibroblasts w76x, epithelial cells, smooth muscle cells, endothelial cells, T lymphocytes, myeloid cells, chondrocytes, osteoblasts as well as the stem cells of the bone marrow w77x. The requirement for a functional IGF-IR for growth in serum-free medium supplemented by purified growth factors, has been formally demonstrated by the use of R y cells, generated by a 3T3-like protocol from mouse embryos with a targeted disruption of the IGF-IR genes w78,79x. R y cells do not grow in SFM supplemented by the growth factors that sustain the growth of mouse embryo cells derived from wild type littermates or of other 3T3 cells w56,60x. Although R y cells grow in 10% serum, they do so at a reduced rate in comparison to wild type cells, in a remarkable agreement with the growth rate of mouse embryos null for the IGF-IR. It should be noted that cells in 10% serum always grow much better than in SFM, regardless of the supplementation with a variety of growth factors, indicating the existence in serum of growth factorŽs. that have not yet been identified. The importance of the IGF-I receptor in the control of cell proliferation in vitro is also supported by other evidence: for instance, interference with the function of the IGF-I receptor leads to inhibition of cell growth. This has been demonstrated by using antisense expression vectors or antisense oligodeoxynucleotides to the IGF-I receptor RNA: the antisense strategy was successful in inhibiting

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cellular proliferation in fibroblasts w57,80,81x, in interleukin-2 stimulated T lymphocytes and in HL-60 cells w82x, in glioblastoma cells lines w56x and in ovarian carcinoma cells w83x. Growth can also be inhibited by using peptide analogues of IGF-I w84,85x, or by the stable transfection of a plasmid expressing an antisense RNA to the IGF-I RNA w86,87x.

6.1.2. In ÕiÕo In children and adolescents, IGF-I plasma levels are the best predictor of adolescent body growth w88x. By contrast, low IGF-I plasma levels are found in pygmies, and in Laron type dwarfs w1,71x. In the latter syndrome, a genetic defect in the growth hormone receptor leads to reduced plasma levels of IGF-I, and replacement therapy with IGF-I has actually been used in some clinical situations w89x. The correlation between IGF-I levels and body size extends also to dogs and mice. For instance, large Standard Poodles have plasma IGF-I concentrations 6-fold higher than Toy Poodles, and the Newfoundland, a giant breed, has significantly higher IGF-I levels than the German Shepherd, which is considered a large, but not giant, breed w90x. Transgenic mice overexpressing the IGF-I gene confirm the importance of the IGF system in body growth. Transgenic mice overexpressing the growth hormone grow to larger size than control mice w91x, a phenomenon to be ascribed to the fact that growth hormone is a potent inducer of IGF-I expression in the liver, causing a marked increase in IGF-I plasma levels. D’Ercole and co-workers w92x reported that transgenic mice overexpressing IGF-I have an increased body weight. In a transgenic mouse line, in which the IGF-I construct was engineered to be exclusively expressed in the heart, the overproduction was sufficient to cause an 85% increase in IGF-I plasma levels and a significant increase in body weight w93x. The essential role of the IGF system in development has been elucidated by the elegant experiments of Efstratiadis and co-workers w78,79x. These investigators have shown that a targeted disruption of the IGF-II gene results in progeny, which, at birth, have a body weight that is 60% the body weight of wild type littermates. When both the IGF-II and the IGF-IR genes are disrupted by homologous recombination,

the homozygous mutant embryos Žno IGF-II and no IGF-IR genes. at birth have a body weight that is only 30% the weight of wild type littermates. Since IGF-II is the predominant ligand of the IGF-IR in mouse embryos Ž which express negligible amounts of IGF-I. , it can be stated that the activated IGF-IR accounts for 70% of embryonal murine growth. Subsequent experiments by Efstratiadis and co-workers have yielded even more interesting results. Mouse mutants with a targeted disruption of the imprinted Igf2r gene have increased serum and tissue levels of IGF-II and, at birth, exhibit overgrowth of 135% in respect to wild type littermates. However, these mutant mice die perinatally, possibly because, in the absence of IGF-IIR-mediated turnover of IGF-II, the IGF-IR is overstimulated w94x. Consistent with this hypothesis are the findings that Igf2r mutants have high plasma levels of IGF-II Ž4 times the normal levels., and are rescued Ž normal size, viable. , when they carry a second mutation, eliminating either the IGF-II or the IGF-IR genes. Triple mutants lacking IGF-IR, IGF-IIR and IGF-II are non-viable dwarfs w94x, 30% in size, like the double mutants lacking IGF-II and IGF-IR w78,79x. It suggests that IGF-II may signal also through an unknown or unidentified receptor, for the moment designated as XR, the X receptor. 6.2. Transformation Overexpression andror constitutive activation of IGF-IR in a variety of cell types leads to ligand-dependent growth in serum-free medium and to the establishment of a transformed phenotype; i.e., ability to form colonies in soft agar andror to produce

Table 1 Transformation by overexpressed growth factor receptors Receptor type

Ref.

PDGF EGF FGF IGF-I Insulin CSF-1 c-met

w99,100x w57,101,102x w103,104x w96,96x w105x w106x w107x

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tumors in mice w54,60,80,95–98x. However, a great number of overexpressed gene products can transform cells, including proto-oncogenes, activated cellular oncogenes, signal transducing molecules, glycolytic enzymes, in fact, transformation can be considered as a common outcome of the overexpression of gene products. Growth factor receptors are no exception, and Table 1 gives a partial list of those receptors, whose overexpression is known to induce transformation. What makes IGF-IR different from other growth factor receptors are two crucial findings: the first is that R y cells Ži.e. mouse embryo cells with a targeted disruption of the IGF-IR genes. are refractory to transformation by certain viral and cellular oncogenes that readily transform mouse embryo cells with a physiological number of IGF-IRs, like 3T3 cells of various derivations. The list of oncogenes that fail to transform R y cells include the SV40 large T antigen w56x, an activated ras or a combination of T antigen and ras w60x, the bovine papilloma virus E5 protein w108x, and overexpressed growth factor receptors, such as the EGF receptor w54x, the PDGF b receptor w55x and the IR w109x. R y cells do transform spontaneously, but at a lower rate than other 3T3 cells. The fact that R y cells can occasionally transform spontaneously should not be not surprising, because mouse embryo cells have a propensity to chromosomal rearrangements and mutations. Any gain-of-function mutation in a signal transducing molecule downstream of the IGF-IR would by-pass the IGF-IR requirement for transformation. The second crucial finding is that the transformed phenotype can be reversed to a non-transformed phenotype in a variety of tumor cells by decreasing the number of IGF-IRs, or by interfering with its function. Different approaches have been used, including antisense expression plasmids or antisense oligodeoxynucleotides against either IGF-II w110x, or IGF-I w86,87x, antisense strategies against the IGF-IR w56,111–116x, antibodies to the IGF-IR w117–119x, and dominant negative mutants of the IGF-IR w120– 123x. All these procedures can reverse the transformed phenotype, andror inhibit tumorigenesis, and induce loss of the metastatic phenotype w124x. These findings are summarized in Table 2. The list in incomplete, but it clearly shows that targeting of the IGF-IR can reverse the transformed phenotype in

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Table 2 Reversal of the transformed phenotype by targeting of the IGF-I receptor cell type

Method

Ref.

Human melanoma ŽA, T. Breast carcinoma ŽT. Lung carcinoma ŽT. Ovarian carcinoma ŽT. Rhabdomyosarcoma ŽT. Rodents: Rat glioblastoma ŽA,T. Rat rhabdomyosarcoma ŽT. Murine melanoma ŽT. Murine pancreatic ca. Murine leukemia ŽT. Rat 1 cells ŽT. Mouse embryo cells ŽA. Hamster mesothelioma ŽT.

Antisense Antibody Antisense Dominant neg. Antisense

w112x w117,118x w115x w83x w113x

Antisense Antisense Antisense Antisense Antisense Dominant neg. Gene deletion Antisense

w111x w125x w125x w110x w126x w120x w56x w116x

The capital letters in parentheses stand for colony formation in soft agar ŽA. or tumorigenesis ŽT. in either nude or syngeneic animals.

several types of tumor cells, from humans and rodents. Targeting the IGF-IR is more efficient than targeting its ligands. The adult rodent has negligible plasma levels of IGF-II, but other adult animals, including humans, have substantial circulating concentrations of both IGFs w127x. Targeting one ligand leaves the other one free to activate the IGF-IR. 6.3. Anti-apoptotic signaling A historical review of apoptosis and other forms of cell death, including the morphological aspects, can be found in Majno and Joris w128x, while Steller w129x gives a lucid discussion of the genetic basis of cellular suicide. Cleveland and Ihle w130x have summarized the biochemical pathway of one of the most common forms of apoptosis, whereas Fisher w131x and Thompson w132x have pointed out its possible therapeutic implications. In this review, we shall limit ourselves to the role of the IGF-IR in the apoptotic process. 6.3.1. EÕidence for an anti-apoptotic effect of the actiÕated IGF-I receptor The protective effect of the activated IGF-IR on cell survival has been known for some years, espe-

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cially in the central nervous system ŽCNS.. For instance, Gluckman et al. w133x reported a protective effect of IGF-I in ischemic injuries of the CNS, and D’Mello et al. w134x found that IGF-I inhibited low potassium-induced apoptosis of cerebellar granule neurons. Other reports have confirmed that IGF-I protects neurons from a variety of injurious agents w135–138x. IGF-I also inhibits apoptosis caused by IL-3 withdrawal in hemopoietic cells w96,139x, and Harrington et al. w140x found that IGF-I Žand, to a lesser extent, PDGF, but not EGF or FGF. exerted a protective effect on c-myc induced apoptosis w141,142x. All these reports dealt with the protective effects of the ligand, but it soon became apparent that the receptor was the limiting factor. Thus, an overexpressed IGF-IR protects cells in vitro from apoptosis induced by etoposide w143x, tumor necrosis factor w144x, IL-3 withdrawal in hemopoietic cells w145x, and several injurious agents in neuroblastoma cells w146x. Even more dramatic are the in vivo data. A decrease in IGF-IR levels below normal levels w125,147x, or the use of a dominant negative of the IGF-IR w123x causes massive apoptosis of tumor cells in vivo. These in vivo experiments are to be detailed in the next section. The role of the IGF-IR in apoptosis, and the possibility that it may discriminate between normal and tumor cells, have been discussed in two recent reviews by Baserga w74,126x. However, an important point is that the protective effect of an activated IGF-IR on apoptosis is apparently dependent on its ability to inhibit ICE and ICE-like proteins w148x, thus indicating that the signaling pathway from the IGF-IR dovetails with the ICE pathway, which is the most commonly used pathway in the apoptotic process w149,150x. 6.3.2. Assay for apoptosis in ÕiÕo The fact that tumor cells with an impaired IGF-IR function fail to produce tumors in animals Žsee above. implies that they must have died when injected into rodents. The complete absence of tumor development suggests that the effect of targeting the IGF-IR is not simply to slow down growth, but actually to kill cells, either by apoptosis or some other mechanism of cell death. It is technically difficult to demonstrate cell death in general w151x, or apoptosis in particular, in vivo, when cells are injected subcutaneously into rodents. If the cells die, one finds very little at the

site of injection, certainly not enough to make a quantitative Ž or even a qualitative. assessment of apoptosis. To demonstrate and quantitate the extent of cell death Žand apoptosis. , in tumor cells in vivo w112x, we use a diffusion chamber w152x that allows the passage of proteins, nutrients, antibodies, etc., but not of cells w153x. In this method, the cells are loaded into a sterilized diffusion chamber, which is then inserted into the subcutaneous tissue of rats or mice, where it is left for the desired length of time. Cells from several transplantable tumors of human or rodent origin, placed in diffusion chambers, double in number in 24 h, during in vivo incubation, indicating that the conditions are optimal for their survival and proliferation. Indeed, it should be clear that cells in the diffusion chamber behave precisely as they do when injected subcutaneously into animals. The diffusion chamber simply offers three advantages: Ž1. it allows an accurate determination of the number of cells that die, a measurement that would be impossible to obtain from cells injected subcutaneously; Ž2. because of the rapidity with which cells die Ž less than 24 h., it gives a very quick answer, while a tumorigenesis experiment in nude mice always requires several weeks for a complete evaluation; and Ž 3. again because of the rapidity with which apoptosis takes place, non-syngeneic tumors can also be studied, since there is no time to marshall an immune response in 24 h While several rodent and human cell lines from transplantable tumors grow quite well in diffusion chambers, the same cells stably transfected with a plasmid expressing an antisense RNA to the IGF-IR RNA or treated with antisense oligodeoxynucleotides ŽODN. to the IGF-IR RNA undergo massive apoptosis w125,147x. Apoptosis begins almost immediately, is far advanced in 3–4 h, and is all but complete by 24 h w125x. The targeting of the IGF-IRs is much more effective in inducing apoptosis in vivo than in monolayer cell cultures. The same conditions that in vitro, in monolayer cultures, result only in inhibition of IGFI-mediated growth, often very modest, will cause massive apoptosis in vivo w125,147x. Colony formation in soft agar is somewhat intermediate: reduction in the number of colonies in soft agar is more pronounced than growth inhibition in monolayer cultures, but not as dramatic as the extent of apoptosis in

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vivo. Indeed, in some cases, it is possible to demonstrate that certain dominant negatives of the IGF-IR can cause inhibition of colony formation in soft agar, but no induction of apoptosis in vivo, while other dominant negatives can do both w122,123x. These findings, repeated on several cell types, suggest an explanation, i.e. that soft agar growth or growth in the subcutaneous tissue of animals requires anchorage-independence, a property which is not tested in monolayers. This hypothesis is supported by the fact that the activated IGF-IR is not an absolute requirement for growth in monolayer ŽR y cells grow, albeit slowly, in 10% serum., but is quasi-obligatory for the establishment and maintenance of the transformed phenotype Žof which anchorage independence is one of the earliest characteristics. .

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6.4. Receptor number and mitogenic response

Fig. 2. Effect of PDGF stimulation on the number of IGF-I receptors. The cells used were 3T3 cells expressing a human IGF-I receptor cDNA under the control of a rat IGF-I receptor promoter w165x. Scatchard analysis was carried out in cells in serum-free medium ŽI. and in cells stimulated with PDGF Ž10 ngrml for 16 h, l..

It has been suggested that receptor density or density of signal transducing molecules may be the determinant factor in the response of cells to growth factors w154x. Cells overexpressing the IGF-IR, or the PDGFR or the EGFR, or other receptors grow in SFM supplemented solely by the respective ligands Žsee Table 1.. One could argue that all that is needed for stimulation of growth is a sufficient number of receptors per cell, regardless of receptor type. We had already observed, however, that the EGF-mediated growth of cells overexpressing the EGFR, was inhibited by an antisense ODN to the IGF-IR RNA, while the IGF-I-mediated growth of cells overexpressing the IGF-IR was not inhibited by an ODN to the EGFR w57x. The dependence of the EGFR on a functional IGF system was subsequently confirmed by Steller and co-workers in human cervical cancer cells w155,156x. Also suggesting that the density of receptors or signal transducing molecules per psoae may not be sufficient for growth in SFM supplemented by growth factors is our finding that R y cells, overexpressing an activated, mutant raze, do not grow in SFM supplemented by PDGF and EGF w60x. It should be noted that in these and other experiments, the re-introduction into R y cells of a functional IGF-R promptly abrogated the growth deficiencies w56,60,157x. It is still possible, though, that the number of

IGF-I receptors may play a determinant role in the mitogenic response. As mentioned before, it has been repeatedly shown that a markedly increased number of IGF-I receptors allow cells to grow in SFM supplemented solely with IGF-I w57,80,81,96x. Conversely, a decrease in the number of IGF-IR results in inhibition of cell proliferation, both in vitro and in vivo Žsee Section 6.1.. Other observations correlate receptor number and IGF-I mediated growth. In the first place, Go mouse embryo cells Žunresponsive to IGF-I only. do have functional IGF-I receptors, that can be autophosphorylated and can transmit a signal that is not mitogenic, but induces growth in size w158x and the expression of several genes w159–161x. Secondly, PDGF induces an increase in the number of IGF-I binding sites w162–164x, a finding more recently confirmed by other methods. A typical example is shown in Fig. 2, where the number of IGF-I binding sites is clearly increased Ž by a factor of 2. after stimulation with PDGF. In fact, PDGF activates the IGF-IR promoter w165x. In mammary gland MCF-7 carcinoma cells, IGF-I by itself is not mitogenic, but it becomes so after the number of IGF-IRs has been increased by estrogen treatment w166x. Finally, the number of IGF-IRs is also increased by other growth factors, for instance bFGF w167x. All these increases in IGF-IR number make cells responsive to IGF-I.

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These increases are almost invariably small, in the order of 2-3-fold. Since a cell doubles its size and its protein amount from the beginning of G1 to the end of G2 , a doubling in the number of receptors could simply be part of the doubling in mass of the cell, perhaps even important, but not determinant. The various possibilities have been discussed in a review by Baserga w168x. Since there is a correlation between IGF-IR mitogenicity and receptors number, a crucial question that must be settled is whether this increase is necessary or coincidental. The fact that cells grossly overexpressing the IGF-IR grow in IGF-I only, without activation of the PDGF and EGF receptors w57,80x does not really give us an answer, since gross overexpression may activate pathways that are not activated under physiological conditions. To answer this question, Rubini et al. w169x took advantage of R y cells Ž that have no endogenous IGF-IRs. and transfected them with an IGF-IR cDNA under the control of its own promoter, obtaining a large number of clones with different numbers of IGF-IRs. The crucial clones turned out to be those with receptors’ numbers ranging between 3 = 10 3 and 30 = 10 3 receptors per cells, and the results with these clones are shown in Table 3. However, many clones were tested, and the data can be summarized as follows: 1. all clones Ž at least 8. with less than 15 = 10 3 receptors per cells did not grow in SFM supplemented solely with IGF-I, or with IGF-I and PDGF ŽR12 is selected as an example of this group in

Table 3 Effect of IGF-I receptor number on mitogenesis and transformation Cell line

Receptor number P10 3

Mitogenesis

Transformation

R12 R12qPDGF R503 R503qPDGF R508 R600

3 7 15 23 22 30

y y y q q q

y y y y q q

Receptor number increases in each cell line after treatment with PDGF. Mitogenesis means ability to grow in SFM supplemented with IGF-I only Žor, in the case of R12 and R503, in IGF-I plus PDGF.. Transformation is the ability of cell lines to form colonies in soft agar, in 10% serum, with IGF-I supplementation. From w169x.

Table 3.; 2. all clones with 22 = 10 3 or more receptors per cell, grew in IGF-I only and, of course, in IGF-I plus PDGF; 3. a clone, R508, with 15 = 10 3 receptors per cell did not grow in IGF-I only, but grew in IGF-I plus PDGF, when its receptor number had increased to 23 = 10 3; and 4. 22 = 10 3 receptors per cell are sufficient for growth in soft agar, although only after supplementation of 10% serum with IGF-I. These results are important in two ways: 1. they show that small increments in IGF-IR number can change the mitogenic response to IGF-I from negative to positive; and 2. PDGF will not cooperate with IGF-I in inducing mitogenesis, unless a certain number of IGF-IRs are present. 6.5. The IGF system and the brain While the activation of the IGF-IR plays an important role in many cell types, its role in the brain is of special interest, because it may function there in ways that are not manifested in other tissues. The IGF system does play a role in cellular proliferation in the CNS, especially during development w136x, but it may also be involved in differentiation of neuronal cells w170x, in apoptosis Ž see above. , and even in behavior. Bondy and co-workers w171x have carried out extensive studies on the localization of the IGF-IR and its ligands in the brain; their results indicate that the IGF system is very active in the CNS during development, both embryonal and postnatal. In mature brain, the highest levels of IGF-I binding sites and IGF-IR mRNA are found in the choroid plexus and circumventricular organs. Both IGF-I binding sites and IGFIR mRNA colocalize also in different parts of the mature brain, especially the suprachiasmatic, medial geniculate and superior olivary nuclei. In the cerebellar cortex, the IGF-IR is found in granule cells, and the fact that IGF-I protects these cells from low potassium-induced apoptosis w134x is certainly not coincidental. As mentioned above, transgenic mice overexpressing IGF-I have an increased body weight w92x and they also display an increase in brain weight, which is actually slightly larger than the increase in total body weight. In a transgenic mouse line, with an 85% increase in IGF-I plasma levels, there was also a significant increase in brain weight w93x. We have already mentioned above the reports of

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protection of neuronal cells from apoptosis from diverse injurious agents by IGF-I Žsee Section 6.3. , to which other reports can be added w171x. It seems that the activated IGF-IR, important as a mitogenic agent in the CNS during development, loses this function in the mature animal Žwhere cell proliferation in the CNS is at a minimum. and replaces it with a protective function, sparing neurons from cell death, and helping them in maintaining their differentiated state. However, this does not seem to be the case with tumors of the CNS, that have abundant IGF-IRs and secrete both IGF-I and IGF-II w172x. The presence of a hyperactive IGF system has been shown not only in aggressive glioblastomas w86,87,111x, but also in neuroblastoma cells w173x, in astrocytomas and in meningiomas w174x. A unique aspect of the IGF system in the brain is its possible role in behaviour. To begin with, there is the startling observation by Sara et al. w175x that the truncated IGF-I Ž missing the first 3 aminoacids. is abundant in the brain, and that the 3 aminoacids cleaved from the mature IGF-I, form a tripeptide, glycyl-prolyl-glutamate, which is also found in the brain. Apart from the fact that the truncated IGF-I Ž des-IGF-I. does not bind IGF binding proteins and is therefore more active, the released tripeptide has a distinct biological action as a neurotransmitter, for instance, modulating the release of dopamine. More recently, Baker et al. w176x reported their studies on mice with a targeted disruption of the IGF-I genes. Both sexes are infertile dwarfs. The males have small testes, levels of testosterone are about 18% of normal, and the animals do not exhibit aggressive behavior when caged with other males. Conversely, the transgenic mice studied by Reiss et al. w93x, with increased levels of IGF-I, displayed considerable more aggressiveness than the parental mouse strain. The decrease in testosterone levels caused by the absence of IGF-I is a reasonable explanation of their decreased aggressiveness, but IGF-I cannot be the only component in an endocrine profile of violence. Some of the giant breeds of dogs, with very high plasma levels of IGF-I Žsee above. are no more aggressive than some small breeds, as anyone familiar with Saint Bernard’s and Toy Poodles can easily attest. But, despite the obvious multi-component aspect of behavior, it is clear that the IGF system is playing a role in it, that could be interesting.

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7. Mutational analysis of the IGF-I receptor and its substrates Mutations in the various domains of the IGF-IR can affect its various functions. A single point mutation at lysine 1003 Ž the ATP-binding site. results in an IGF-IR that has essentially lost most of its functions w54,177x. A triple mutation of the three Ys Ž1131, 1135, 1136. in the tyrosine kinase domain results in a markedly reduced level of autophoshorylation and in the abolition of both mitogenicity and transforming activity w178,179x. Other mutations affect only selective functions of the receptor. For instance, a human IGF-IR mutated at Y950 is neither mitogenic nor transforming, but it still protects murine hemopoietic cells from apoptosis induced by IL-3 withdrawal w145 x. When the C-terminal 108 aminoacids of the human IGF-IR are deleted, the truncated receptor can still transmit an IGF-I-mediated mitogenic signal, but is no longer capable of transforming activity w180x. Other mutants of the IGF-IR w121,181,182x have been tested, and the results are summarized in Table 4. In these experiments, mutant receptors were transfected into Ry cells to generate stable cell lines with no other IGF-IR but the transfected one. This is a very important point, because it is now clear that receptors, especially when overexpressed, can form hybrid receptors or can transactivate each other, confounding the experimental data w183–187x. The conclusions from the many mutants and the several experiments can be summarized as follows: Ž1. mitogenicity and transforming activity of the IGFIR can be localized on separate domains Žw121,180– 182x and Table 4. , clearly indicating that there is at least one pathway for transformation that is additional to and distinct from the mitogenic pathways, and is very likely an unidentified ras-independent pathway w60x; Ž2. the transforming domain of the IGF-IR can be localized between residues 1245 and 1310 Ž182 and Table 4.; and Ž3. Y1251 and the serine quartet at 1280–1283 are very important for transformation Žw121,181x and Table 4. with the region around 1293 probably playing a weaker role in it. When these residues are mutated, the transforming activity of the IGF-IR is impaired, whereas its mitogenic activity in response to IGF-I, is perfectly normal.

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Table 4 Summary of the mitogenic, transforming and anti-apoptotic activities of the IGF-I receptor and its mutants Receptor

Mitogenicity Transformation Anti-apoptosis

wt d 1229 d1245 d1270 d1293 d1310 K 1003 Y950F Y3F Y1250F Y1251F S1280r1283A 1293r1294ŽFL. Y1316F

qq q q ND q q y y y q q q q q

q yy q q? q y y y q y y q q

q q q q q y q y q y ? y q

These data are summarized from w182x for mitogenicity and transforming activity, and from w145x for protection from apoptosis. Mitogenicity is the ability to make cells grow in SFM supplemented solely with IGF-I; transforming activity is the ability to make cells form colonies in soft agar; and protection from apoptosis as protection of FL5.12 cells from apoptosis induced by IL-3 withdrawal. NDs not done. d, truncation of the receptor at the indicated residues. Y, tyrosine; F, phenylalanine; S, serine; K, lysine; A, alanine; Y3F, mutations at Y 1131, 1135 and 1136.

Table 4 also shows that the domainŽs. for protection from apoptosis can be separated from the mitogenic and transforming domains. The serine mutant, for instance w121x is mitogenic, non-transforming and protects from apoptosis. The C-terminal truncated receptors behave in a similar manner, while the mitogenic and transforming 1293r1294 mutant does not protect cells from apoptosis. One has therefore to conclude that the 3 functions of the IGF-IR that relate to cell proliferation are spatially separated on the receptor, presumably generating distinct pathways. It is not surprising that the transforming pathway is additional to and distinct from the mitogenic pathways, since transformed cells are anchorage-independent, and this characteristic is not required by cells growing in monolayers. The finding that transforming activity and protection from apoptosis can also be separated from each other was unexpected, since many investigators have assumed that apoptosis and

transformation are mutually exclusive and that they are the two sides of the same coin. Clearly, the situation is more complicated. But, more important, these observations raise the possibility of inhibiting one pathway without affecting the others, a possibility with potential for practical applications. Finally, we can summarize the mitogenic, transforming and anti-apoptotic properties of the IGF-IR and its major substrates as follows w188x: the wild type IGF-IR, by itself, is fully transforming ŽRq and p6 cells. both of which grossly overexpress a human IGF-IR., as already reported by several laboratories Žsee above. , while its two major substrates, IRS-1 and Shc, singly or in combination, are not transforming in R y cells. However, overexpression of IRS-1 Žbut not Shc. can transform cells with a normal number of wild type IGF-IRs. In addition, an antisense plasmid against the IRS-1 RNA inhibits transformation w37x. It seems therefore that IRS-1 is required, but is not sufficient, for transformation, its transforming activity being conditioned by the presence of a functional wild type IGF-IR.

8. Oncogenes, p53, tumor suppressors and the IGF-I receptor If the IGF-IR plays a crucial role in transformation and in protection from apoptosis, the obvious corollary is that oncogenes will upregulate the system, while tumor suppressor genes will down-regulate it. Several reports have shown that the IGF autocrine or paracrine loop is upregulated by other growth factors, like PDGF and EGF Žsee Section 6.4., hormones, for instance, growth hormone and estrogens w166x, and oncogenes like SV40 T antigen w81x, c-myb w189x, the Ewing’s sarcoma-Wilms’ tumor 1 fusion protein, responsible for the desmoplastic small round cell tumor w190x and the hepatitis B virus w191x. Conversely, tumor suppressor genes like WT1 w192x, and p53 w193x, as well as interferon w194x cause a decrease in IGF-IR levels, through a repression of transcription from the IGF-IR promoter. p53 has been connected to the IGF system through other findings: it represses transcription from the IGF-II w195x and the insulin receptor w196x promoters and it induces the expression of the IGF-binding protein 3 w197x that

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Fig. 3. Cloning efficiency of cells transfected with a plasmid expressing the tsp53. R508 cells ŽA and B. and Rq cells ŽC and D. were transfected with plasmids p53cG and pPDV6q and selected with puromycin. Half of the plates were incubated at 328C ŽA and C., the other half at 378C ŽB and D.. The plates were stained after 3 weeks in culture. R508 cells have 15 000 receptors per cell, the receptor cDNA being under the control of its own promoter. In Rq cells Žwith 1P10 6 receptors per cell., the cDNA in under the control of a viral promoter, insensitive to p53.

antagonizes the effects of IGF-I and IGF-II. It would be interesting to know whether this mechanism applies also to other tumor suppressor genes and to other growth factor receptors w74,198x. The fact that cells without IGF-IRs Ž by targeted disruption of the IGF-IR genes. are refractory to transformation by several oncogenes Ž see above., seems to confirm an essential role of this receptor in transformation and, therefore, a contrary role to tumor suppressor genes. The effect of p53 is illustrated in the experiment of Fig. 3. The cells used in this experiment were both derived from R y cells w60x. R508 cells have 15 = 10 3 receptors per cells, the cDNA being under the control of the rat IGF-IR promoter w165,199,200x, which is repressed by wild type p53 w193x. In R q cells, the receptor is under the control of a viral promoter w60x, which is insensitive to wild type or mutant p53. Because the viral promoter is stronger than the IGFIIR promoter, R q cells have 20 times more receptors per cell than R508 cells. These cells were co-trans-

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fected with a plasmid expressing the puromycin resistance gene and a plasmid expressing a temperaturesensitive p53, wild type at 32 C, and mutant at 37–39 C w201x, and selected either at 328C or 378C Žthe cells would detach from the plates if cultivated at 398C in the presence of puromycin. . Fig. 3 shows representative plates, stained after three weeks of growth. An overexpressed IGF-IR clearly makes a difference in clonogenicity, even at 378C Žcompare B and D plates. . But Fig. 3 also shows that, at 328C Žwild type p53., the number of colonies markedly decreases in cells expressing the IGF-IR under the control of the rat IGF-IR promoter Ž plates A and B. ; when the receptor is under the control of the SV40 promoter, incubation at 328C does not decrease clonogenicity in respect to 378C Žplates C and D. . While the difference may also be explained by the overexpressed IGF-IR overwhelming the p53-mediated cell cycle arrest, the results by Werner et al. w193x seem to favor an effect of p53 on the IGF-IR. In the light of these results, we determined the levels of IGF-IR in p53 yry cells Ži.e. cells null for the p53 gene. , and in wild type cells Žboth cell lines kindly provided by Tyler Jacks, MIT, Cambridge, MA.. Contrary to our expectations, the levels of the IGF-I binding sites were essentially the same in both cell lines Žnot shown., perhaps reflecting the possibility that IGF-IR levels may be regulated by several other factors besides p53. Curiously, both cell lines had a very high number of IGF-I binding sites, close to 10 5 receptors per cell, which is way above what one usually finds in 3T3-like mouse embryo fibroblasts Ž 15-20= 10 3 receptors per cell.. The importance of these reports should not be underestimated. The fact that oncogenes upregulate the IGF system and tumor suppressor genes downregulate it, is only part of a rapidly increasing evidence that oncogenes and anti-oncogenes may act through the regulation of growth factors and their receptors. This has been discussed in detail in a review by Baserga w74x, where other examples with other receptors and other oncogenes are also listed. But it should be remembered that the reports of induction of IGF-I by c-myb w189x and SV40 T antigen w81x, were the first ones to show that the decrease in growth factor requirements by transformed cells is due to the fact that oncogenes stimulate endogenous growth factor production.

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9. The IGF-I receptor and tumorigenesis

Table 5 Tumorigenesis in nude mice

9.1. In animals

Treatment

% recovery

Expected delay

Palpable tumors

Unfortunately, the IGF-IR knock-out mice of Efstratiadis and co-workers Ž see above. cannot help us in elucidating the role of the IGF-IR in tumorigenesis, because these mice die immediately after birth w79x. There are sporadic indications that high levels of IGF-I may increase the incidence of tumors in animals: for instance, in the Irish Wolfhound, with very high IGF-I plasma levels, there is a high incidence of tumors, especially osteosarcomas, much above the incidence of tumors in smaller breeds of dogs w202x with low IGF-I plasma levels. The best evidence for a role of the IGF-IR in tumorigenesis comes from experiments in which the IGF-IR function was impaired or abrogated by a variety of approaches, including antisense strategies, antibodies against the receptor, and dominant negatives. Some of these results have already been summarized in Section 6.2, Table 2. The best results have actually been obtained with dominant negatives, specifically with a dominant negative truncated at residue 952 w120x and a second one truncated at residue 486 w123x. When cells are transfected with plasmids expressing these two dominant negatives, there is actual complete abrogation of tumorigenesis in nude mice, which is the result of massive apoptosis w123x. Indeed, Resnicoff et al. w147x, using antisense ODN, have shown that there is a correlation between concentration of antisense ODN, the decrease in IGF-IR number, the extent of apoptosis, and growth in nude mice Ž Table 5.. In control cells Žuntreated or treated with a random ODN at very high concentrations. , the number of receptors remains at normal levels, the cells in the diffusion chambers double in number in 24 h, give tumors in nude mice palpable after only 4 days, and give tumors in rats with an intact immune system, tumors that continuously increase in size and eventually kill the animals. Treatment with antisense ODN against the IGF-IR causes a concentration-dependent decrease in receptor number, which is mirrored by a concentration-dependent increase in the extent of apoptosis in vivo. In nude mice, delay in tumor growth is proportional to the extent of apoptosis, indicating a close relationship between the ability to induce apoptosis in vivo and the retardation of tumor

None or random ODN) AS 0.15 m M AS 1.50 m M AS 6.50 m M AS 16.00 m M

200 54 35 0.5 0.010

4 6 7 13 19

4 6 11 17 24

C6 cells were incubated in vitro for 24 h with the indicated ODN ŽAS stands for antisense. before injection into the subcutaneous tissue of nude mice. The concentration of AS ODN is in m M. Recovery expressed as percentage of cells recovered from the diffusion chamber after 24 h Expected delay is the number of days after injection before the tumors become palpable, which is 4 days with untreated cells. The other expected delays are based on percentage of cells recovered Žsee Column 1.. The last column gives the actual number of days after injection, when tumors became palpable. ŽReproduced, with modifications, from w147x..

growth. In rats with an intact immune system, tumors developed at the lowest concentrations of antisense ODN, but the tumors eventually regressed and never recurred. At higher ODN concentrations, the tumors do not even appear w147x. The difference between nude mice and animals with an intact immune system clearly indicate that there is also a host response, which we will discuss later. Finally, we should point out that antisense ODN to the IGF-IR RNA are remarkably non-toxic. Unpublished data, using the same ODN of Table 5, have shown little or no toxicity in mice at doses of 1,280 mgrkg; a single injection of 5 mg antisense ODN Ž200 mgrkg. killed 99.5% of the tumor cells in the diffusion chamber. 9.2. In humans Macaulay w203x has marshalled the available evidence for the role of the IGF-I receptor and its ligands in human cancers. There are numerous references indicating abnormalities in the expression of the receptor and its ligands in various forms of human cancer. The list includes breast cancer, thyroid carcinoma, lung cancer, hepatoma, colon cancer, sarcomas, brain tumors, neuroendocrine tumors, cancers of the pancreas and of the kidney, and others. The evidence is largely anecdotal, i.e. the abnormalities are seen in some but not all tumors, a problem that is

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not unique to the IGF-IR and its ligands. It is striking, however, that all reported abnormalities are of the upward regulation type, i.e. an increased number of IGF-IRs, or ectopic expression or overexpression of either IGF-I and IGF-II. This is compatible with the evidence gathered in experimental animals and in cells in culture. While an overexpressed IGF-IR is transforming, the crucial observation is that a functional impairment of the receptor reverses the transformed phenotype, best exemplified by the finding Ždiscussed above. that deletion of the IGF-IR renders mouse embryo fibroblasts refractory to transformation. Extrapolated to human tumors, this finding suggests that we should not observe human tumors with deletions or crippling mutations of the IGF-IR genes, since transformation would be inhibited in cells with an absent or non-functional receptor. Of course, this is not an absolute rule, since there are cells, like B lymphocytes, that do not have IGFIRs, or have them in extremely low numbers. Most B cell lymphomas are devoid of IGF-IRs w204x. So, there are alternative pathways for transformation of cells, to be sure, but the IGF-IR certainly occupies an important role at least for several types of cells w126x. Finally, Bengtsson w205x has reported an increase in the incidence of certain types of tumors in acromegalics, who have high IGF-I plasma levels. It would be interesting to know whether tumor incidence is decreased instead in Laron dwarfs Žsee above. .

10. The immune response This host response is a peculiar one, and merits some discussion. Briefly, rats Žor mice. previously injected with, or carrying in a diffusion chamber, tumor cells with a targeted IGF-IR Ž antisense strategies or dominant negatives. become totally resistant to a subsequent challenge with wild type C6 rat glioblastoma cells w111,188x. In fact, injection of C6 cells expressing an antisense RNA to the IGF-IR RNA Žnon-tumorigenic. can cause complete regression of established wild type C6 tumors w111x. Untreated wild type cells do not confer this resistance. This would not be particularly surprising, except that the animals become resistant to challenge with wild type C6 cells, even when they are pre-treated with unrelated tumor cells, indeed even tumor cells of

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different species w188x. In these experiments, the ‘immunizing’ tumor cells are loaded in a diffusion chamber, which is then inserted into the subcutaneous tissue of a rat for 24 h The chamber is then removed, and from one week to 6 weeks later, the rats are challenged with wild type C6 cells. Provided the ‘immunizing’ cells had a decrease in the number or a functional impairment of the IGF-IR, they invariably elicited a response that made the rats resistant to subsequent challenge with C6 cells. We have observed this host response whether the ‘immunizing’ cells express an antisense RNA to the IGF-IR RNA w111x, or are treated with antisense oligodeoxynucleotides against the IGF-IR RNA w125x, or are expressing a dominant negative of the IGF-IR w123x. Several indications suggest that this host response, despite its unorthodox behavior, has the characteristics of an immune response, that is not MHCrestricted. We really do not have an explanation for this host response. The reports that the IGF-IR and IR co-precipitate with MHC complexes in immunoprecipitates w206–213x could be an exciting clue or a false lead.

11. Uniqueness of the IGF-I receptor targeting The fact that targeting of the IGF-IR results in tumor cell death, inhibition of tumorigenesis Žsee above. and prevention of metastases w124x, is not, in itself, extraordinary, since these effects are shared with many other agents and modalities. There is, however, something unique about the IGF-IR, which we would like to point out. 1. Targeting of the IGF-IR does not simply decrease the growth rate of transplantable tumors. It causes massive apoptosis and, in some cases, complete inhibition of tumorigenesis Žsee Section 9.1.; 2. It has been w74,126x that targeting of the IGF-IR will cause massive apoptosis of tumor cells, while being much less effective on normal cells. Three separate pieces of evidence suggest that normal cells are more resistant to apoptosis than tumor cells: a. in mouse embryos with a targeted disruption of the IGF-IR genes Žsee above. , there is no evidence of increased apoptosis; b. human diploid fibroblasts are more resistant to apoptosis induced by serum deprivation than transformed cells w214x; and c. WI-38 hu-

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man diploid fibroblasts do not undergo apoptosis in vivo even when treated with high concentrations of antisense oligodeoxynucleotides to the IGF-IR RNA w188x, that readily kill tumor cells. 3. Targeting the IGF-IR induces a host response that gives an additional means to control tumor growth Žsee above..

12. Questions without answers There are, as usual, more questions left than answers, and we would like to mention here some of these unanswered questions, in the hope that other investigators may become interested in them. 1. A small increment in the number of IGF-IRs Žfrom 15 P 10 3 to 22 = 10 3, w169x. makes an all-ornone difference in the IGF-I-mediated mitogenic response Žsee also Table 3.. What causes this difference? Does an increased number of receptors result in a prolonged stimulation of the same pathways, or are new pathways activated? 2. The two next questions are simpler, if one can find the answer. Which is the ligand for the IRRR? and which is the XR, the postulated 4th receptor for the IGF system? An answer to the latter question may soon come from genetics experiments that are going on in the laboratory of Argyris Efstratiadis, but the ligand for the IRRR remains a mystery. It is not IGF-I or IGF-II, it is not in serum Žthat never-ending source of growth factors. , and yet it must have an important function, since its cytoplasmic domain is highly mitogenic. At present, the chances are that the IRRR is involved in a tight autocrine loop, with an unidentified ligand. 3. Seemingly related to the last two questions is the finding by Efstratiadis and co-workers w94x that overproduction of IGF-II is toxic to the animal, and is toxic through the activation of the IGF-IR Ž see Section 6.1.. The question here is that, in vitro, an overexpressed IGF-IR, activated by excess ligand, is not toxic at all. In fact, cells grow faster under these conditions. So, why is the excess activation of the IGF-IR toxic in vivo, but not in vitro? 4. We have mentioned above that IGF-I protects cells from apoptosis by inhibiting the activation of ICE proteins w148x. Which is the pathway leading from the IGF-IR to ICE? One possible clue is given

by the finding that tumor necrosis factor ŽTNF. induces serine phosphorylation of IRS-1 and the IR w215x. Indeed, Hotamisligil et al. w216x have reported that IRS-1 serine phosphorylation precedes the inhibition of IR tyrosylphosphorylation and function. These findings suggest that serine phosphorylation of IRS-1 causes inhibition of the IR, the implication being that this may be a mechanism involved in TNF-induced apoptosis. This is the most compelling evidence that serine phosphorylation of IRS-1 may result in inhibition of IR Ž or IGF-IR. function, but there are also anecdotal observations suggesting that serine phosphorylation may have an inhibitory effect on cell proliferation and a pro-apoptotic effect. The best example, of course, is the TGF-b receptor, which is a serinerthreonine kinase w217x, and usually has an inhibitory effect on the growth of fibroblasts in culture, and can induce, under certain circumstances, apoptosis. All the above mentioned experiments were done using the IR as the target of the serine-phosphorylated IRS-1, but, because of the extensive homology between the IR and the IGF-IR Ž70%, with peaks of 84% in the tyrosine kinase domain and the binding region for IRS-1., it is reasonable to expect that the same observation may also apply to the IGF-IR. Indeed, Fig. 4 shows that serine phosphorylation of IRS-1 occurs also after treatment with okadaic acid, a potent inducer of apoptosis w218x. Serine-phosphorylation of IRS-1 causes a shift in mobility w219x, and a decrease in the extent of tyrosyl phosphorylation, caused by stimulation with IGF-I Žsee Fig. 4.. Interestingly, Wu et al. w144x have already reported that overexpression of the IGF-IR protects cells from TNF a-induced apoptosis. Another possibility that should be explored is the ability of the IGF-IR to activate the transcription factor NFkB. The recent discovery that TNF alpha induced apoptosis is suppressed by activation of NFkB w220–223x, and the protective effect of an overexpressed IGF-IR against several forms of apoptosis, including TNF a-induced apoptosis w144x, call for an investigation of the relationship between the IGF-IR and NFkB. Indeed, Bertrand et al. w224x have already reported that both insulin and IGF-I activate NFkB. 5. Another fascinating question is why overexpression of receptors Žor other cellular gene products. causes transformation. Is it just a question of prolonged stimulation of the same pathways, or does it

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Fig. 4. Serine phosphorylation of IRS-1 by okadaic acid. Okadaic acid, an inhibitor of serinerthreonine phosphatases, is a potent inducer of apoptosis. Serine phosphorylation of IRS-1 causes a shift in mobility, which is quite evident in lanes 5 and 6, where the lysates were from cells treated with 2 m M okadaic acid. In panel A, lysates were immunoprecipitated with an antibody to IRS-1, and blotted with an anti-phosphotyrosine antibody; panel B is an immunoblot with an anti-IRS-1 antibody, on the same membrane after stripping. Lanes: 1, serum-free medium; 2–4, stimulated with IGF-I Ž20 ngrml., respectively for 10, 30 and 60 min; 5, okadaic acid for 40 min; 6, okadaic acid for 40 min. and IGF-I for 10 min; 7, same as lane 2. Note the decrease in tyrosine phosphorylation in cells treated with okadaic acid.

activate unknown pathways? In the case of the IGFIR, it is clear that a modest increment in receptor number Ž22–30 P 10 3 ceceptors per cell. is sufficient to confer the transformed phenotype to mouse embryo fibroblasts. But numbers is not the only thing, because a grossly overexpressed truncated receptor, lacking the C-terminus, is fully mitogenic but no longer transforming w97,180,182x. This is strongly suggestive of a new pathway, but which one? 6. Finally, the role of the IGF-IR in the CNS and in behavior has only begun. With neurobiology coming of age, this topic may very well be the most important one in the future story of the IGF-IR and its functions.

13. Conclusions From the point of view of basic research, the developments of the past few years in the field of IGF-IR function, have clearly established this receptor as a very important growth factor receptor, in terms of mitogenicity, transforming activity and ability to protect cells from apoptosis. One could say that

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the IGF-IR is exiting from the area of endocrinology, where it had slept for several years, overshadowed by its cousin, the IR, and has taken its rightful place in the field of normal and abnormal growth. The mutational analysis of this receptor has disclosed very interesting features, especially the possibility of the spatial separation on the same receptor of distinct properties, such as mitogenicity and transforming ability. One of its pathways Ž the ras pathway. has very well described, but, clearly, this pathway Ž which the IGF-IR shares with other growth factor receptors. is only one of its pathways. A challenge for the future will be to discover the additional pathways that make this receptor somewhat different from other growth factor receptors. Still in the area of basic research, it is possible that the study of the IGF-IR in the CNS may yield a veritable gold mine, whether in terms of its effect on differentiation, or growth or even as a modifier of behavior. The IGF-IR may also become important in the future as a target for inhibition of abnormal growth. As any experienced investigator in cancer research knows all too well, results in experimental animals can rarely be extrapolated in their entireties to human tumors. But in animals, the results with the targeting of the IGF-IR, whether with antisense strategies or dominant negatives, have been dramatic, and it is expected that targeting of this receptor will soon undergo the test of clinical trials. But even if these were to turn out like many other clinical trials, the IGF-IR is and will remain an exceptionally rich source of basic information on the mechanisms of cell growth and differentiation.

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