Role of the insulin receptor in IGF-system signaling and biology.

Abstract Abstract #4055 The role of the type I insulin-like growth factor (IGF) receptor (IGF1R) in breast cancer has been extensively studied, and has led to the development of several IGF1R inhibitors currently in clinical trials. The closely related insulin receptor (IR) has been shown to contribute to IGF signaling through the formation of IGF1R/IR-hybrid receptors and through a homodimer of an alternatively spliced form of the IR (IR-A). Understanding the role of the IR in cancer biology will help determine which receptor(s) in the IGF system should be targeted to efficiently inhibit IGF signaling. Our previous work has shown that selective downregulation of IGF1R by siRNA resulted in enhanced sensitivity to insulin through increasing the numbers of insulin receptor homodimers. To determine how downregulation of IR affected IGF1R signaling, we used T47D cells, a breast cancer cell line expressing both the IR and IGF1R, as our model system. We transiently downregulated IR levels using siRNA and evaluated signaling. 5 nM of insulin activated downstream signaling molecules, such as insulin receptor substrate 1(IRS-1) and Akt in control cells, but this response was diminished in the IR-downregulated cells. In contrast, 5 nM of IGF-I did not result in differences in signaling between IR-downregulated cells and control cells. Using IR shRNA, we created stable clones with IR levels reduced by more than 72%. IR shRNA clones demonstrated decreased insulin binding, as measured by 125I-insulin binding assay. Additionally, clones showed decreased response to insulin signaling. 5 nM of insulin activated IRS-1 and Akt in parental T47D cells, but this response was attenuated in the IR-downregulated clones. Proliferation was also decreased in response to 5 nM insulin in the clones compared to parental cells. In contrast, IGF-I mediated signaling was not altered in clones when compared to vector control cells. Further, no change in proliferation was observed in response to IGF-I between wild-type and IR shRNA clones. We also generated stable IR shRNA clones using LCC6 cells. Insulin signaling was reduced in LCC6 clones when compared to wild-type LCC6 cells. However, in contrast to the T47D clones, IGF-I signaling was increased in LCC6 IR shRNA clones. Thus, the role of the insulin receptor in cancer biology may be dependent upon the ratio of IR, IGF1R, and the hybrid receptor. If cells contain mostly homodimers of IGF1R and IR, then downregulation of IR may affect only insulin response. However, if there are high levels of hybrid receptor, then downregulation of IR could affect, and even augment, IGF1R signaling. Our data suggest that the ratio and confirmation states of both IR and IGF1R should be understood to predict the effect of receptor downregulation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4055.