Effects of E2F1-cyclinE1-E2 circuit down-regulation in hepatocellular carcinoma cells

Background: No effective therapy is available for hepatocellular carcinoma. To identify novel therapeutic strategies, we explored the effects of the depletion of E2F1, cyclin E1–E2 whose inter-relationships in hepatocellular carcinoma cell proliferation have never been defined. Methods: siRNA-mediated depletion of the targets was studied in the hepatocellular carcinoma cells HepG2, HuH7 and JHH6 characterized by high, medium and low hepatocyte differentiation grade, respectively; a model of normal human hepatocytes was also considered. Results: The depletion of each target mRNA reduced the levels of the other two mRNAs, thus demonstrating a close regulatory control, also confirmed by over-expression experiments. At the protein level, an exception to this trend was observed for cyclinE1 whose amount increased upon cyclin E2 (HepG2, HuH7, JHH6) and E2F1 (HepG2) depletion. In HepG2, reduced cyclinE1 proteolysis accounted for this observation. Additionally, cyclin E1–E2–E2F1 targeting decreased the levels of cyclin A2 mRNA and of the hyper-phosphorylated form of pRb thus leading to an S-phase cell decrease; migration was impaired as well. Finally, the model of human hepatocytes used was clearly less affected by target mRNAs depletion than hepatocellular carcinoma cells. Conclusion: Our data provide novel mutual relationships amongst cyclin E1–E2–E2F1 and indicate their role in sustaining hepatocellular carcinoma cell proliferation/migration, validating the concept of an anti-cyclin E1–E2–E2F1 therapeutic approach for hepatocellular carcinoma.