Antonino Belfiore

In mammals, the insulin receptor (IR) gene has acquired an additional exon, exon 11. This exon may be skipped in a developmental and tissue-specific manner. The IR, therefore, occurs in two isoforms (exon 11 minus IR-A and exon 11 plus IR-B). The most relevant functional difference between these two isoforms is the high affinity of IR-A for IGF-II. IR-A is predominantly expressed during prenatal life. It enhances the effects of IGF-II during embryogenesis and fetal development. It is also significantly expressed in adult tissues, especially in the brain. Conversely, IR-B is predominantly expressed in adult, well-differentiated tissues, including the liver, where it enhances the metabolic effects of insulin. Dysregulation of IR splicing in insulin target tissues may occur in patients with insulin resistance; however, its role in type 2 diabetes is unclear. IR-A is often aberrantly expressed in cancer cells, thus increasing their responsiveness to IGF-II and to insulin and explaining the cancer-promoting effect of hyperinsulinemia observed in obese and type 2 diabetic patients. Aberrant IR-A expression may favor cancer resistance to both conventional and targeted therapies by a variety of mechanisms. Finally, IR isoforms form heterodimers, IR-A/IR-B, and hybrid IR/IGF-IR receptors (HR-A and HR-B). The functional characteristics of such hybrid receptors and their role in physiology, in diabetes, and in malignant cells are not yet fully understood. These receptors seem to enhance cell responsiveness to IGFs.

Insulin-like growth factor II (IGF-II) is a peptide growth factor that is homologous to both insulin-like growth factor I (IGF-I) and insulin and plays an important role in embryonic development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type 1 insulin-like growth factor receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to the alternative splicing of exon 11. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These differences in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal growth and cancer biology.

The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.

There is evidence, both in vitro and in vivo, that receptor tyrosine kinases play a key role in the formation and progression of human cancer. In particular, the insulin-like growth factor receptor (IGF-IR), a tyrosine kinase receptor for IGF-I and IGF-II, has been well documented in cell culture, animal studies, and humans to play a role in malignant transformation, progression, protection from apoptosis, and metastasis. In addition, the hormone insulin (which is very closely related to the IGFs) and its tyrosine kinase receptor (the IR, which is very closely related to the IGR-IR) have been documented both in vitro and in vivo to play a key role in cancer biology. Indeed, several epidemiological studies have shown that insulin resistance status, characterized by hyperinsulinaemia, is associated with an increased risk for a number of malignancies, including carcinomas of the breast, prostate, colon and kidney. Recent data have elucidated some molecular mechanisms by which IR is involved in cancer. IR is over-expressed in several human malignancies. Interestingly, one of the two IR isoform (IR-A) is especially over-expressed in cancer. IR-A is the IR foetal isoform and has the peculiar characteristic to bind not only insulin but also IGF-II. In addition, the IR contributes to formation of hybrid receptors with the IGF-IR (HR). By binding to hybrid receptors, insulin may stimulate specific IGF-IR signalling pathways. Over-expression of IR-A is, therefore, a major mechanism of IGF system over-activation in cancer. In this respect, IR-A isoform and hybrid receptors should be regarded as potential molecular targets, in addition to IGF-IR, for novel anti-cancer therapy. These findings may have important implications for both the prevention and treatment of common human malignancies. They underline the concept that hyperinsulinaemia, associated with insulin resistance and obesity, should be treated by changes in life style and/or pharmacological approaches to avoid an increased risk for cancer. Moreover, native insulin and insulin analogue administration should be carefully evaluated in terms of the possible increase in cancer risk.