Visiting Fellows Papers

Aim: Preoperative chemoradiation is recommended for locally advanced rectal cancer. However, response is variable among patients, and greater response is associated with improved survival and reduced local recurrence rates. We have shown that taking HMG-CoA reductase inhibitors, commonly known as statins, during chemoradiation is associated with improved response, suggesting a potential radiationsensitizing role for these drugs. The purpose of this study was to investigate the effects of simvastatin on colorectal (CRC) cancer cells in vitro and explore the underlying mechanisms. Method: The sensitivity of CRC cell lines HRT18 and SW480 to simvastatin, radiation or combination therapy was assessed using colony formation and ATPbased cell viability assays. To test the dependence of any effects on isoprenylation pathways downstream of HMG-CoA, a rescue arm was added wherein the isoprenoid geranylgeranyl diphospate (GGPP) was included in the media. Protein lysates were obtained and tested for expression and phosphorylation status of proteins downstream of Epidermal Growth Factor Receptor (AKT, RAS, MEK, ERK1/2), since these have been reported as a potential target of statins. The effects of simvastatin were also tested on patient-derived CRC stem cells established from surgical specimens, a subpopulation of tumor cells characterized by radiation resistance. Results: CRC cells lines (HRT18 and SW480) were sensitive to clinically relevant doses of simvastatin (Inhibitory Concentration [IC] 50: 1.8 and 2 lM, respectively). The combination of radiation and simvastatin yielded decreased cell growth and viability compared to either treatment alone in both lines, in effect halving the radiation dose necessary to achieve comparable inhibition (Figure). These effects of simvastatin on cell growth were rescued by the addition of GGPP to the media, suggesting its depletion drives these effects. Protein analysis of cells treated with simvastatin compared to control cells demonstrated a significant decrease in ERK1/2 phosphorylation, while AKT phosphorylation levels remained constant. Two different patient-derived CRC stem cell lines were radiation-resistant at baseline, but resistance was overcome by simvastatin treatment (IC50: 0.06–0.2 lM), and by combination treatment of simvastatin and radiation. Conclusion: In accordance with our prior clinical observations, treatment with simvastatin enhanced the sensitivity of CRC cells to radiation in vitro. The dependence of these effects on GGPP depletion and the associated decrease in ERK1/2 phosphorylation suggest a prominent role for the EGFR-RAS-ERK1/2 axis. In addition, simvastatin effectively targets and kills the radioresistant subpopulation of CRC stem cells which are known to contribute to tumor resistance and recurrence. Mechanistic and in vivo studies are under development, laying the foundation for the ultimate goal of a clinical trial.