Downregulation of type I IGF receptor increases insulin sensitivity of breast cancer cells

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The type I insulin-like growth factor (IGF) receptor (IGF1R) is an attractive target for breast cancer intervention. Due to the sequence and structure similarity of IGF1R to its homologous receptor, insulin receptor (IR), great effort has been given to discover inhibitors that only target IGF1R, but not IR. However, since IGF1R and IR form hybrid receptors, we hypothesize that inhibiting IGF1R alone in breast cancer may affect insulin receptor signaling as well. Using ribonuclease protection assay and Western blotting method, we examined 24 breast tumors and found that all breast tumors express both IGF1R and IR. The presence of both IGF1R and IR suggests that IGF1R inhibition might affect IR function in breast cancer. In several breast cancer cell lines, the presence of IGF1R and IR were assayed and the relative ratios of IGF1R vs IR mRNA were calculated. Of the several cell lines studied, while MCF-7 expressed about 10 fold excess of IGF1R compared with IR, LCC6, MDA-MB 231-BO, MDA-MA 231 had equivalent levels of IGF1R and IR mRNA. Therefore, those three cell lines were chosen for further analysis. siRNA sequences that specifically targeting IGF1R or IR were designed. IGF1R and IR expression was specifically decreased by 78% and 97% by the appropriate siRNAs without affecting the protein level of the other receptor. After transfecting cells with IGF1R or IR siRNA for 48 hours, cells were treated with insulin or IGF-I, and phosphorylation of Akt and MAPK was measured to examine downstream signaling events. As expected, decreased levels of IR resulted in diminished activation of MAPK and PI-3K by insulin. siRNA downregulation of IGF1R greatly decreased the ability of IGF-I to activate MAPK and PI-3K pathways. Interestingly, downregulation of IGF1R enhanced the ability of insulin to activate MAPK and PI-3K pathways. Accordingly, the glucose uptake of breast cancer cells was enhanced when IGF1R was downregulated. Using flow cytometry analysis, we found out that insulin binding was increased when IGF1R level was downregulated, suggesting downregulation of IGF1R may cause the disruption of IGF1R/IR hybrid receptor, forcing the balance of more IR homodimer formation. In conclusion, our data suggest that specific targeting of IGF1R alone in breast cancer may increase insulin sensitivity of breast cancer cells. Since IR also activates signaling pathways in breast cancer cells, agents targeting both receptors may be necessary to disrupt the malignant phenotype regulated by this growth factor system.