The type 1 insulin-like growth factor receptor (IGF-1R) is a tyrosine kinase receptor with a 70% homology to the insulin receptor (IR). It was originally considered a redundant receptor used by cells only when signaling from the IR was absent or defective. In the past few years, however, the IGF-1R has emerged as a receptor with unique characteristics that differentiate it sharply from the IR.1, 2 It is especially in cancer biology that the IGF-1R seems to have assumed an important role, distinct from the IR. To simplify the differences between the 2 receptors (there is some overlapping between the 2 receptors3), one can say that the IGF-1R is the predominant receptor in mitogenesis, transformation and protection from apoptosis. All these characteristics add up to sustained cell proliferation, which is, of course, a characteristic of cancer cells. The IGF-1R also plays a role in cell adhesion4 and in longevity.5 This mini review will focus on 3 aspects dealing with the role of the IGF-1R in cancer: its importance in the establishment and maintenance of the transformed phenotype, its role in apoptosis and the mechanism(s) by which it controls cell size and cell proliferation.
The type 1 insulin-like growth factor receptor (IGF-1R) plays an important role in the establishment and maintenance of the transformed phenotype. It also has a strong antiapoptotic activity and has a significant influence on the control of cell and body size. Downregulation of the IGF-1R leads to massive apoptosis of cancer cells. These characteristics make it an attractive target for anticancer therapy. © 2003 Wiley-Liss, Inc.
ROLE OF IGF-1R IN TRANSFORMATION
The importance of the IGF-1R in the establishment and maintenance of transformation was discovered by chance when Sell et al.6, 7 found that R-cells could not be transformed by a number of viral and cellular oncogenes. R-cells are 3T3-like fibroblasts derived from mouse embryos with a targeted disruption of the IGF-1R genes.8 R-cells do not express IGF-1 receptors, but they express the A-isoform of the insulin receptor, and the transducing signals downstream from this receptor seem to be intact.9 R-cells grow in 10% serum, but do not respond to IGF-1 and most purified growth factors, such as platelet-derived growth factor and epidermal growth factor. They do respond, however, to progranulin,10 a little known growth factor that seems to play an important role in wound healing.11 The observation that R-cells are refractory to transformation by viral and cellular oncogenes is remarkable because mouse embryo fibroblasts (MEFs) are notorious for their propensity to transform spontaneously in culture. Reintroduction of the IGF-1R in R-cells promptly restored their ability to transform.7 These findings have been confirmed and extended in other laboratories,5 more recently by Morrison et al.12 We now know that R-cells can transform spontaneously, but at a rate 3 logarithms lower than MEFs expressing the receptor. We also know that R-cells can be transformed by v-src (but not by c-src13) and by Galpha13.14 It should not be surprising that there are exceptions to the rule. Any gain-of-function mutation downstream from the IGF-1R would bypass it. However, the concept that cells that do not express the IGF-1R are resistant to transformation remains substantially valid. Despite the fact that this observation was made more than 10 years ago, we do not know yet the mechanism by which the absence of an IGF-1R impairs the ability of viral and cellular oncogenes to transform cells. There is tantalizing evidence that the insulin receptor substrate-1 (IRS-1) may play a role in it. IRS-1 is a docking protein for the IGF-1R15 and is known to interact with the SV40 T antigen.16 This is an interesting possibility which, for the moment, remains only a hypothesis.
IGF-1R IN APOPTOSIS
The logical corollary to the resistance of R-cells to transformation is that downregulation of the IGF-1R should reverse the transformed phenotype in cancer cells. We tested this corollary several years ago by transfecting glioblastoma cells (and other cancer cells in culture) with a plasmid expressing an antisense RNA to a 5′ sequence of the IGF-1R. The results were more dramatic than anticipated.17 Cells expressing the antisense to the IGF-1R underwent massive apoptosis when grown in rats or mice or in anchorage-independent conditions.18 Antisense oligodeoxynucleotides had the same effect,19 and so did the introduction into cancer cells of dominant negative mutants of the IGF-1R.20 More recently, downregulation of the IGF-1R has been achieved with the use of specific antibodies.21 The effect of IGF-1R targeting is more accentuated on metastatic tumors than on primary tumors.22, 23 In the past few years, reports have been accumulating consistently showing that downregulation of the IGF-1R causes apoptosis and growth inhibition of cancer cells.5 A list of tumors or tumor cells affected by IGF-1R targeting includes glioblastoma,19 melanoma,18, 24 neuroblastoma,25 prostate cancer,20 colon cancer,26 rhabdomyosarcoma,27, 28 lung cancer,29 Ewing's sarcoma,30 medulloblastoma31 and others. An extensive review of the role of the IGF-1R and its ligands in human tumors can be found in Khandwala et al.32 An illustration of the effect of IGF-1R targeting on the growth of human tumor xenotransplants is shown in Figure 1. DU 145 human prostatic cancer cells were injected into the subcutaneous tissue of nude mice. The animals were then treated with intraperitoneal injections of antisense oligodeoxynucleotides to the IGF-1R.18 Scrambled oligodeoxynucleotides and phosphate-buffered saline were used as control injections. After 3 weeks of treatment, the mice were allowed to live out for another 10 weeks. Figure 1 gives both the tumor burden (all mice) and the maximum tumor size for the 3 groups (both in mg). Clearly, the antisense treatment effectively inhibits the growth of DU 145 cells in nude mice by more than 90%. Three mice treated with antisense had no tumors at all, which could be considered as cures. The other 2 mice had small nodules that, histologically, showed very little residual tumor (data not shown).
This, by itself, is not particularly exciting. Many agents and many targets cause death of cancer cells. The problem is that many chemotherapeutic agents kill normal cells as effectively, or almost as effectively, as cancer cells. The IGF-1R, however, has an interesting characteristic. Downregulation of the receptor has a very modest effect on cells in monolayer cultures, about 10–15% inhibition of growth. The same treatment on the same cells incubated in soft agar or injected into mice or rats causes extensive apoptosis and tumor regression.18, 19 An illustration of this differential effect is given in Figure 2. Human melanoma cells were engineered to express either a sense or an antisense partial RNA to the IGF-1R. Only in the antisense expressors was the level of receptor markedly reduced. The cell lines were then tested in monolayer cultures or in soft agar (wild-type untransfected cells were also included). The antisense plasmid had little or no effect on the growth of cells in monolayers, but it strongly inhibited colony formation in soft agar. Tumor growth in nude mice was also strongly inhibited.18 In other words, the IGF-1R is not an absolute requirement for growth in monolayer cultures, but is a quasiabsolute requirement for growth in anchorage-independent conditions.
Other findings corroborate these observations. In mice carrying xenotransplants, tumor regression occurs with antisense oligodeoxynucleotides at a concentration of 200 mg/kg. The same antisense oligodexynucleotide is nontoxic to normal mice up to a dose of 1,280 mg/kg (data not shown). The preferential effect of IGF-1R targeting on cells in anchorage independence explains its increased effect on experimental metastases compared to primary transplantable tumors.22, 23, 33 Metastases (and local recurrences) usually start from small clusters of sparse cells, which can be thought to be in anchorage-independent conditions. Whatever is the explanation, we and others have repeatedly shown that targeting of the IGF-1R has very little effect on cells in monolayers (after all, R-cells grow normally in 10% serum, despite the fact that they do not have IGF-1 receptors). The same drugs that are almost ineffective on cells in monolayer have a spectacular effect when tested on cells in soft agar or seeded on polyHEMA plates or on tumor cells growing in mice. Testing a candidate inhibitor of the IGF-1R on cells in monolayer may actually miss the best candidates for antitumor activity. At present, very little is known about the basis of the differential effect of the IGF-1R on cells in monolayers versus cells in anchorage independence. The fact that the IGF-1R is not an absolute requirement for normal growth may offer some suggestions, but it is not an explanation. We will return to it in a later section.
The approaches used to target the IGF-1R can be summarized quickly. Antisense strategies were the first to be used successfully in vitro and in vivo.5 The problems with antisense strategies are well known and will not be discussed in this brief review. Antibodies have also been successful in inducing apoptosis of cancer cells, and their usefulness is further supported by the observation that antibodies to the IGF-1R, like antisense strategies, downregulate the receptor.21 In theory, a specific inhibitor of the receptor's tyrosine kinase activity would be the best solution. The problem is that this type of inhibitor will have to distinguish the tyrosine kinase domain of the IGF-1R from the one of the insulin receptor. The 2 domains are highly homologous, but there are small differences that could be exploited. Reducing the levels of the ligands (IGF-1 and IGF-1I) is not a good choice. This approach has given good results in mice, which express only IGF-1 in adult life.34 But in adult humans, IGF-1 and IGF-1I are both expressed, and both of them would have to be targeted. It is easier to target the binding domains for both ligands in the extracellular subunit of the receptor. Finally, because of its strong antiapoptotic activity, downregulation of the IGF-1R could be used in combination with other anticancer therapies that cause apoptosis of cancer cells.24, 35
IGF-1R AND CELL GROWTH
In the last analysis, cell proliferation depends on both the cell cycle program and on the regulation of cell size. Without doubling of cell size from G/1 to G/2, cell division does not occur.36 Cell size is essentially controlled by ribosome biogenesis37 and ribosome biogenesis is limited by the activity of RNA polymerase I,38 the enzyme that controls rRNA synthesis. Among the several proteins involved in regulating RNA polymerase I activity is the upstream binding factor (UBF39), which is localized, like rRNA synthesis, in the nucleolus.40 A connection between cell and/or body size and the IGF axis was already known through the efforts of Efstratiadis,41 who first showed that deletion of the IGF-1R genes causes a 50% reduction in the size of mouse embryos. The connection goes much further. The IGF-1R signals through several pathways, one of which is dependent on IRS-1. There are 4 IRS proteins in mammalian cells,15 but IRS-1 and IRS-2 are the most prominent in transmitting signals from either the IGF-1R or the IR. Deletion of the IRS-1 genes gives birth to mice that, interestingly, are also 50% in size when compared to wild-type littermates.42Drosophila, instead of 4 IRS proteins, has a single one, called chico; deletion of chico causes a 50% reduction in size of the adult flies.43 Both the size and the number of cells are reduced.43 Other transducing molecules downstream of IRS-1 have a similar effect: deletion of S6K144, 45 or Akt46, 47 in mice or flies results in a 50% reduction in size. Thus, it seems reasonable to conclude that the IGF-1R/IRS-1 axis controls about 50% of cell and body growth (in all these examples, we have used a 50% figure, although the size reductions hover on either side of that percentage). These findings support our previous statement on the differential effect of IGF-1R targeting on normal and tumor cells. It is true that the 50% IGF-1-dependent growth cannot be replaced by other growth factors, but it is still 50% and allows a measure of growth, albeit subnormal. When cells are placed in anchorage-independent conditions instead, and the IGF-1R is downregulated, the cells simply die. Our hypothesis is that there is a function of the IGF-1R in anchorage-independent growth that is nonredundant, except in very few instances. This function is frankly not known, although a possible explanation resides in the fact that the IGF-1R and IRS-1 have a marked effect on cell-to-cell adhesion,4 which reduces the stress of anchorage independence.
There are 2 questions at this point: Where does the other 50% growth come from? And why do we keep saying IGF-1R and not IR, whose original substrate was actually IRS-1? The answer to the second question is superficially simple. Mouse embryos with deletion of the IR genes are of normal size at birth,48 although they die promptly of keto-acidosis. We said “superficially simple,” because the IR (especially the A-isoform) has also a role in growth,41 although it may not be so dramatic as the role of the IGF-1R. The answer to the first question is more elusive. Other growth factors and other pathways must be involved, but for the moment any candidate would only be conjectural. Efstratiadis41 points out that the IGF-1R is the only growth factor receptor with a growth phenotype in mouse embryos. For the moment, we will limit ourselves to say that 50% of growth is IGF-1R-independent, and that fraction, wherever it comes from, must be activating RNA polymerase I, as this enzyme ultimately controls cell and body size.
We have seen that IRS-1 controls 50% of cell size in mice, flies and cells in culture.49 A logical question to ask is by which mechanism. Until recently, IRS-1 was considered, reasonably enough, as a cytoplasmic protein, in close contact with the transmembranous IGF-1R and IR. But in the past year, several laboratories have reported the presence of IRS-1 in the nuclei of cells.50, 51, 52, 53, 54, 55 Nuclear IRS-1 in found in cells stimulated by IGF-1 or in association with viral T antigens, but it is also found in hepatocytes that are very large cells but not given to active proliferation (unless a partial hepatectomy is performed). IRS-1 also localizes to the nucleoli and binds UBF in coimmunoprecipitates.52, 54 Since UBF is involved in the regulation of RNA polymerase I activity, this finding establishes the molecular link between IRS-1 and cell growth. IRS-1 activates UBF and in so doing increases rRNA synthesis52, 54 and therefore cell size.
The next question is the relationship of IRS-1/cell size to cancer. An obvious but not very informative answer is that cells, to divide, have to increase in size, and IRS-1 could provide a mechanism for cell growth. However, there is more direct evidence. 32D murine hemopoietic cells expressing the human IGF-1R do not undergo apoptosis upon shifting from IL-3 to IGF-1, but, after a burst of cellular proliferation, they differentiate along the granulocytic pathway.56 32D cells do not express IRS-1, a characteristic they share with other cell types prone to differentiation.5 Ectopic expression of IRS-1 in 32D IGF-1R cells inhibits differentiation, the cells become larger, they are permanently IL-3-independent and even form tumors in mice.49 IRS-1, a known regulator of cell size, can therefore play a role in transformation.
IGF-1 and IGF-1I were originally discovered as mitogens.1, 2 Their mitogenic effect has been discussed at length in more than one review1, 2, 17 and will not be further examined in this mini review. For sustained cell proliferation, cells need to implement the cell cycle program as well as to increase in size.57 The crucial question at this point is how the cell coordinates the 2 programs under physiological conditions. There are reports of proteins involved in both the cell cycle program and ribosome biogenesis (cell size). They include pescadillo (also called PES-1), involved in DNA replication and ribosome biogenesis58; TAF II/250, which is required for G/1 progression59 and binds to UBF60; TIID, involved in both RNA polymerase II and RNA polymerase I transcription61; and cdk4-cyclin D1 and cdk2-cyclin E, which phosphorylate UBF on serine 484 and activate it.62 There are probably other connections between the 350-plus proteins identified in the nucleolus,63, 64 but the ones cited above are the first connections between the 2 programs. They suggest that the cell that has started the cell cycle program makes sure, before mitosis, that the size increase is properly coordinated with cell division. Since IGF-1 by itself is mitogenic in certain cells, one would like to hypothesize that one of these molecules with the double function of controlling cell size and the cell cycle program may be regulated by the activated IGF-1R.
It is obvious that the IGF-1R plays a nontrivial role in the control of cell and body growth, largely through one of its docking proteins, IRS-1. More than that, the IGF-1R also plays an important role in the establishment and maintenance of the transformed phenotype. There is a reasonable explanation for the role of the IGF-1R in regulating cell and body size. The IGF-1R/IRS-1 axis connects through UBF with the rDNA transcriptional machinery, which essentially controls cell size. There is yet no satisfactory information on the mechanism by which the IGF-1R plays such a crucial role in anchorage-independent growth. It makes sense to attribute the important role of the IGF-1R in anchorage independence to its role in cell adhesion,4 but the precise mechanism has not been elucidated. The interaction of the IGF-1R with RACK1 suggests a possible candidate for this function.65 Nevertheless, this characteristic can be exploited to distinguish, at least in theory, between tumor and normal cells. As such, the IGF-1R constitutes a promising target for anticancer therapies, either by itself or in combination with other anticancer agents.