LAUSR

Introduction The transcription factor STAT6 is strongly expressed in various tumours with highest expression in malignant lymphomas and pancreatic, colorectal, prostate and breast cancers. STAT6 expression in colorectal cancer is associated with an increased malignancy and a poor prognosis. Incidence of colorectal cancer is approximately 1,361,000 patients per annum worldwide and approximately 60% show STAT6 expression. Patients with colorectal cancer expressing STAT6 also show poor survival rates. The 5 year relative survival rate for patients with stage IIIC and IV colon cancer is about 53% and 11% respectively. Techniques aimed at reducing or blocking STAT6 expression may be useful in treating those cancers with STAT6 expression. Material and methods Celixir owns a patent covering four STAT6 specific small interfering RNA (siRNA) sequences. We tested these four sequences in vitro using the human colon adenocarcinoma cell line HT-29. The four sequences were introduced individually and in combination into HT-29 cells by cationic lipid-mediated transfer at different concentrations (10 to 200 nM). STAT6 mRNA and protein levels were analysed to confirm the transfection was successful. The proliferation of cells was analysed using cell counting at various times (using a NucleoCounter) and the apoptosis of cells was analysed using Annexin V and propidium iodide (PI) staining. Results and discussions 100 nM siRNA concentration was the most effective and all four individual sequences knocked-down STAT6 mRNA and protein by more than 50%. All individual sequences were capable of significantly inhibiting cell proliferation, with sequences #1 and #4 reducing the number of cells to ~50% compared with control 7 days after treatment. STAT6 silencing also significantly induced late apoptotic events. Despite using several combinations of all four sequences at the same time and serially, the best results were obtained with sequences #1 and #4 individually. Conclusion These in vitro results demonstrate that the STAT6 siRNA sequences are capable of inhibiting theproliferation of and inducing late apoptosis of HT-29 colon cancer cells and,in some instances, halving the number of cancer cells. We propose to repeatthese experiments in vivousing immunocompromised mice containing human colon cancer xenografts and totest the siRNA sequences in other cancers in which STAT6 is expressed.