Mechanisms of cytokine-induced IGF-I insensitivity in cancer cells

Cell growth is a balance between proliferation and cell death, and cellular signaling pathways in response to both extracellular and intracellular stimuli control it. The data presented in this dissertation are aimed at understanding regulatory links between the endocrine and immune systems, focusing on how proinflammatory cytokines impair hormone actions on cellular growth. The prototypical proinflammatory cytokines, tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) and interleukin-6 (IL-6), inhibit IGF-I-promoted DNA synthesis in human MCF-7 breast adenocarcinoma cells, T-47D breast ductal carcinoma cells, murine FDCP progenitor myeloid cells and human promyeloid HL-60 cells. Flow cytometry confirmed that all three cytokines suppress IGF-I-induced DNA synthesis by preventing cells from entering the S phase of the cell cycle, leading to G₁ arrest in MCF-7 cells. TNFα and IL-1β act by inhibiting the ability of IGF-I to tyrosine phosphorylate insulin receptor substrate-1 (IRS-1), to induce accumulation of both E2F-1 and cyclin A, to hyperphosphorylate retinoblastoma (RB), and to induce enzymatic activity and association of Cdk2 with cyclin A. The cytostatic property of these cytokines was also shown by their ability to block IGF-I-stimulated luciferase activity of a cyclin A promoter reporter. Deletion of an E2F recognition site from this reporter eliminates the regulatory effects of both IGF-I and TNFα/IL-1β on cyclin A transcription, indicating an essential role for E2F-1 in mediating this cross talk. Moreover, knockdown experiments using E2F-1 siRNA abrogate IGF-I-induced cyclin A accumulation and RB hyperphosphorylation. These finding demonstrates that IGF-I stimulation of E2F-1 synthesis triggers G₁-S progression by inducing cyclin A and inactivating RB. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, but not the mammalian target of rapamycin (mTOR) or mitogen-activated protein kinase (MAPK) pathways, is required for IGF-I to hyperphosphorylate RB and to induce both E2F-1 and cyclin A accumulation. Prolonged Akt phosphorylation serves as a convergent target for cytokines to antagonize IGF-I promoted G₁-S progression. Collectively, experiments in this dissertation have identified critical growth-promoting proteins that are targeted by both IGF-I and proinflammatory cytokines, which offers a new model by which cytokines suppress cell growth by impairing signals from the IGF receptor, leading to IGF-I insensitivity.