Investigating The Molecular Mechanisms Of Curcumin Against Colorectal Cancer Stem-Like Cells

Background: Curcumin inhibits the proliferation of cancer stem-like cells (CSCs) obtained from human colorectal cancer (CRC) tissues and adenomas. In NOD/SCID mice bearing xenografts from patient derived CRC CSCs, curcumin significantly decreased tumour growth and improved survival. Using an affinity pull-down assay with curcumin-coupled beads, curcumin pulls Nanog from cell lysate and directly binds the recombinant protein. In addition, individual Nanog domains were used to demonstrate binding of curcumin to the homeodomain (Nanog93-160). The transcription factor Nanog is crucial for the selfrenewal of CSCs. Nanog expression in CRC tissue correlates with lymph node metastasis and poor prognosis. Since Nanog is not expressed in most tissues, including normal adult stem cells, it represents a therapeutic target specific to cancer cells. The aim of this thesis was to further characterise the curcumin-Nanog interaction. Method: Patient-derived tissues were collected and profiled for CSC markers including Nanog (n=90). The curcumin-Nanog interaction in the CSC population was investigated using Caco-2 cells and Nanog overexpressing cells (HCT116GFP/Nanog) with matched control cell line (HCT116GFP). Specifically, the effect of curcumin on transcriptional activity, protein expression and gene expression of Nanog and downstream targets (BMI1 and FAK) was assessed by luciferase reporter assay, western blot and RT-PCR. The effects of curcumin on CSC vs non-CSC populations (defined by aldehyde dehydrogenase) were also assessed using these methods. In addition, flow cytometry was used to assess Nanog+ expression, proliferation and apoptosis. Subsequently, patient tissues were explanted and treated for 24 hours with curcumin. Explant tissues were then processed and analysed using flow cytometry (n=20). The effect of curcumin on Nanog+expression and proliferation was assessed. Results: Nanog expression was significantly higher in adenoma (6.85% n=6) and CRC tissues (3.47% n=46) compared to normal tissues (0.88% n=38). Curcumin significantly decreased the transcriptional activity of Nanog. In addition Nanog, BMI and FAK protein expression reduced following treatment. No change in Nanog gene expression was observed, although concurrent increases in BMI and FAK gene expression were detected. Further, a decrease in Nanog+ and Nanog+Ki67+ expression was seen following curcumin treatment. A decrease in Nanog, BMI and FAK protein expression was detected following curcumin treatment in ALDHhigh cells. In patient explant tissues, no change was detected in Nanog+ expression following curcumin treatment, however, there was a significant reduction in Nanog+Ki67+ expression. Conclusion: Data suggest Nanog+Ki67+ is targeted by curcumin in adenoma and CRC tissues. Ongoing studies to identify molecular and histological features that delineate responders from non-responders are underway. Nanog may serve as a biomarker in clinical trials to identify individuals most amenable to curcumin treatment alone or in combination for the prevention and/or treatment of CRC.