Insulin-Like Growth Factor Type-I Receptor Signaling
By Jeff A. Toretsky, MD
Insulin-like growth factor type-i (igf-i) is a cytokine that enhances cell survival both in neoplastic and non-neoplastic cells. Neurologists are studying how increasing IGF-I could enhance neuronal survival following cerebral-vascular accidents. Oncologists seek to reduce IGF-I levels to enhance the effectiveness of chemotherapy. IGF-I initiates multiple signaling cascades upon activation of its cognate receptor. Most investigators consider the phosphoinositol 3’-kinase (PI 3-K)/Akt axis the prime survival pathway initiated by IGF-I. Some evidence supports the hypothesis that IGF-I inhibition of the stress-activated protein kinase/jun N-terminal kinase (SAPK/JNK) may contribute to cell survival.
Insulin-Like Growth Factor-I Receptor
The signaling pathway initiated by the ligand IGF-I is of pivotal importance in human carcinogenesis. The IGF-I receptor (IGF-IR) functions as the chief arbiter of growth-stimulatory signals from IGF-I. It structurally resembles and has significant homology to the insulin receptor. The IGF-IR is a tetrameric receptor consisting of two a-subunits and two transmembrane b-subunits.1 The a-subunits are extracellular and bind a molecule of IGF-I, while each transmembrane b-subunit contains an intracellular tyrosine kinase domain.1 Mice lacking the IGF-IR are 45% the size of heterozygote littermates and die shortly after birth due to diaphragm hypoplasia.2 Cell lines were derived from the knock-out mice fibroblasts and the wild-type littermates, and are designated as R- and W-, respectively. These cell lines have been used to demonstrate that the IGF-IR is necessary for transformation by SV-40, activated RAS, and many cytokine receptors.3-5 Only an activated G-protein does not require the IGF-IR for transformation.6
IGF-I binding causes IGF-IR autophosphorylation on a series of tyrosine residues, thereby initiating several discrete signaling pathways. (See Figure 1.) Following autophosphorylation, the IGF-IR phosphorylates insulin receptor substrate-1 (IRS-1), a 180 kD scaffolding protein. Mitogenesis is transduced through the linker molecule Grb2, which is a bipolar molecule with src-homology-2 (SH2) domains on each end. The Grb2 SH2 domain binds phosphorylated IRS-1/2 and activates mitogenic proteins via the Ras-Raf pathway. A second pathway prevents cells from undergoing apoptosis as long as ligand is present. This pathway is transduced by SH2 binding of the p85 sub-unit of PI 3-K to phosphorylated IRS-1/2. Specific PI 3-K pathway inhibitors have been shown to induce apoptosis in cell lines over-expressing the IGF-IR. The level of ligand, IGF-I, is the primary determinant of IGF-IR signaling, although qualitative receptor changes have been reported.7 Increasing IGF-IR per cell correlates with increased tumorigenicity and decreased apoptosis.8-10 Interruption of IGF-IR signaling using antisense RNA, antisense oligodeoxynucleotides, and dominant negative constructs targeting the receptor were found to lead to cell death by apoptosis.11,12
Apoptosis
Apoptosis is a genetically defined cell death that involves activation of a core enzymatic machine consisting of cysteine proteases, called caspases. The activation of caspases in most cases represents irreversible progression to cell death. The process of apoptosis occurs during normal growth and differentiation to eliminate cells that are no longer necessary for tissue function. Since tumor cells are believed to contain a block in differentiation, they may be rendered more susceptible than normal cells to the induction of apoptosis, as embryonal cells would be if they did not differentiate. Therefore, tumor cells are selected because of genetic lesions that inhibit apoptosis and promote survival through pathways such as IGF-IR.13 The ability to tip the balance of cancer cell proliferation toward cellular suicide rather than increased growth would radically alter and improve the outcomes of cancer therapy.
There are two major components of the apoptosis pathway. First, the "central death" signal is activated by an insult to the cell; an example is chemotherapy-induced DNA damage. The Bcl-2 family of proteins can act either as positive or negative regulators of the "central death" signal.14 Following the activation of the "central death" signal, caspases, a family of proteolytic enzymes, are activated. The caspase cascade leads to activation of those target enzymes that produce morphologic changes of apoptosis, such as nuclear fragmentation and ultimate cell death. The activation of caspases in most cases represents irreversible progression to cell death.
Bcl-2 family members regulate apoptosis through homo- and heterodimerization. Bcl-xL protein levels, described above as an anti-apoptotic member of the Bcl-2 family, are increased by IGF-I,13 and its homodimerization leads to inhibition of the "central death" signal. (See Figure 2.) Heterodimerization of Bcl-xL with other Bcl-2 family members permits the "central death" signal to proceed and leads to apoptosis. One member of the Bcl-2 family shown to heterodimerize with Bcl-xL is Bad. In order to prevent apoptosis, the cell needs to prevent Bad from heterodimerizing with Bcl-xL. This has been shown to occur through phosphorylation of Bad.15 Akt phosphorylates Bad, which no longer binds Bcl-xL, thus preventing apoptosis.16
When the Bcl-2 family members are permissive to apoptosis, the mitochondria releases cytochrome c into the cytosol. Cytochrome c binds to the CED-4 homologue Apaf-1, which binds Pro-caspase-9. Pro-caspase-9 then undergoes proteolytic cleavage to caspase-9 and initiates a caspase cascade. Caspase-9 then cleaves caspase-3.17 Akt phosphorylates both pro-caspase-9 and caspase-9 to inactivate these molecules and prevent progression of the caspase cascade to apoptosis.17
Akt is a Connection Between IGF-IR Signaling and Apoptosis
Akt is activated by signaling pathways from cytokines or intracellular oncogenes, such as activated Ras. All these, including IGF-IR, ultimately lead to the activation of PI 3-K, which adds a third phosphate to PI(3,4)P2 to produce PIP3. (See Figure 1.) PIP3 binds Akt, a serine-threonine protein kinase, changes its conformation, and translocates Akt to cytosolic membrane.16,18 Akt then requires dual phosphorylation, one of which is accomplished by PDK1, to become activated.19,20 Activated Akt then phosphorylates survival-mediating targets, including Bcl-2 family member Bad and Caspase-9.16-18 We have shown that a constitutively activated Akt augments the survival of Ewing’s sarcoma family of tumors in the face of chemotherapy.21
Could IGF-I Act Through Other Pathways in Apoptosis Prevention?
MAP kinase is a well characterized pathway activated by many growth signals that leads to transcription factor activation, DNA replication, and cell proliferation. (See Figure 1.) Evidence using a pheochromocytoma model (PC-12 cells) implicates IGF-IR stimulation of the MAP kinase pathway in addition to PI-3 kinase as an alternative pathway for anti-apoptosis.22 However, these cells were differentiated with nerve growth factor prior to the experiments so they may not reflect the response of a malignant cell.
While the IGF-IR-Akt pathway is well described both in terms of its activation and downstream targets, IGF-I may enhance survival through other signaling pathways, including stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK). (See Figure 1.) The SAPK/JNK is a MAP-kinase homologue, but rather than inducing proliferation, induces apoptosis in response to chemical stimuli, including chemotherapeutic agents. The induction of apoptosis by SAPK/JNK is not regulated by Bcl family proteins.23 IGF-I was shown to inhibit the activation of SAPK/JNK by protecting HEK293 cells from anisomycin induced apoptosis. This protection was eliminated in the presence of a PI 3-K inhibitor and diminished in the presence of a dominant negative Akt molecule, suggesting that the IGF-I regulation of SAPK/JNK is distal to Akt.24 To enhance apoptosis and chemosensitivity, SAPK/JNK would need to be activated, thus opposing the survival nature of IGF-I.
Protein kinase C-d is necessary for the transformation of fibroblasts with IGF-IR.25 Protein kinase C is thought to activate NF-kB, and has been shown to do so in response to chemotherapeutic agents.26 NF-kB activation occurs in cells following treatment with TNF-a, daunorubicin, and ionizing radiation, and has been shown to be anti-apoptotic. The association of NF-kB with activation following injury and inhibition of apoptosis led investigators to classify this as a survival signal. Inhibition of NF-kB using a dominant negative construct however, failed to increase the sensitivity of cancer cells to chemotherapy.27
Based on the above evidence, it is likely that IGF-I-mediated survival is enacted by alterations in signal transduction. Inhibition of IGF-I mediated pathways are likely to play a role in the future of cancer therapy. These inhibitory strategies are taking the form of binding proteins, tyrosine kinase inhibitors, and competitive antibodies. As more is learned about this critical pathway, the clinical applications are likely to emerge in many tumor model systems. These include pancreas, lung, breast, colon, and prostate carcinomas.28-32 (Dr. Toretsky is an Assistant Professor, Department of Pediatrics, Program in Oncology, Molecular and Cellular Biology, University of Maryland, Baltimore, MD.)
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The insulin-like growth factor-I receptor is:
a. dimeric and cytosolic.
b. dimeric and transmembrane.
c. tetrameric and cytosolic.
d. tetrameric and transmembrane.
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